diff --git a/book.bib b/book.bib index 957b6bb..a3ad5af 100644 --- a/book.bib +++ b/book.bib @@ -168,11 +168,14 @@ @article{armitageExtractionIdentification1975 } @article{aronHalfUDG2020, - title = {Half-{{UDG}} Treated Double-Stranded Ancient {{DNA}} Library Preparation for Illumina Sequencing v1 [{{Data}} Set]}, - author = {Aron, F and Neumann, GU and Brandt, G}, + title = {Half-{{UDG}} Treated Double-Stranded Ancient {{DNA}} Library Preparation for {{Illumina}} Sequencing}, + author = {Aron, Franziska and Neumann, Gunnar and Brandt, Guido}, year = {2020}, - journal = {protocols. io}, - publisher = {{ZappyLab, Inc Los Gatos, CA}} + month = dec, + journal = {protocols.io}, + doi = {10.17504/protocols.io.bmh6k39e}, + abstract = {Protocol for the preparation of double-stranded genomic libraries for Illumina sequencing, optimised for ancient DNA (aDNA) with partial USER enzyme treatment. This protocol gen...}, + langid = {english} } @article{asscherAtomicDisorder2011, @@ -4312,11 +4315,14 @@ @article{srdjenovicSimultaneousHPLC2008 } @article{stahlDoublestrandedIndexing2019, - title = {Illumina Double-Stranded {{DNA}} Dual Indexing for Ancient {{DNA}} v1 [{{Data}} Set]}, - author = {Stahl, R and Warinner, C and Velsko, I and Orfanou, E and Aron, F and Brandt, G}, - year = {2019}, - journal = {protocols. io}, - publisher = {{ZappyLab, Inc Los Gatos, CA}} + title = {Illumina Double-Stranded {{DNA}} Dual Indexing for Ancient {{DNA}}}, + author = {Stahl, Raphaela and Warinner, Christina and Velsko, Irina and Orfanou, Eleftheria and Aron, Franziska and Brandt, Guido}, + year = {2020}, + month = dec, + journal = {protocols.io}, + doi = {10.17504/protocols.io.4r3l287x3l1y/v3}, + abstract = {This protocol converts partially completed double-stranded DNA libraries e.g. from: Non-UDG treated double-stranded ancient DNA library preparation for Illumina sequencing (dx....}, + langid = {english} } @article{Standards1994, diff --git a/endgame.tex b/endgame.tex index bb0f3ce..3c1a39e 100644 --- a/endgame.tex +++ b/endgame.tex @@ -6,7 +6,6 @@ b5paper, ]{book} \usepackage{amsmath,amssymb} -\usepackage{lmodern} \usepackage{setspace} \usepackage{iftex} \ifPDFTeX @@ -14,10 +13,14 @@ \usepackage[utf8]{inputenc} \usepackage{textcomp} % provide euro and other symbols \else % if luatex or xetex - \usepackage{unicode-math} + \usepackage{unicode-math} % this also loads fontspec \defaultfontfeatures{Scale=MatchLowercase} \defaultfontfeatures[\rmfamily]{Ligatures=TeX,Scale=1} - \setmainfont[Path=./font/ttf/,Extension=.ttf,UprightFont=*-Regular,BoldFont=*-Bold,ItalicFont=*-Italic,BoldItalicFont=*-BoldItalic]{AtkinsonHyperlegible} +\fi +\usepackage{lmodern} +\ifPDFTeX\else + % xetex/luatex font selection + \setmainfont[Path=./font/ttf/,Extension=.ttf,UprightFont=*-Regular,BoldFont=*-Bold,ItalicFont=*-Italic,BoldItalicFont=*-BoldItalic]{AtkinsonHyperlegible} \fi % Use upquote if available, for straight quotes in verbatim environments \IfFileExists{upquote.sty}{\usepackage{upquote}}{} @@ -59,32 +62,43 @@ \makeatletter \def\fps@figure{htbp} \makeatother +\usepackage{svg} \setlength{\emergencystretch}{3em} % prevent overfull lines \providecommand{\tightlist}{% \setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}} \setcounter{secnumdepth}{5} +% definitions for citeproc citations +\NewDocumentCommand\citeproctext{}{} +\NewDocumentCommand\citeproc{mm}{% + \begingroup\def\citeproctext{#2}\cite{#1}\endgroup} +\makeatletter + % allow citations to break across lines + \let\@cite@ofmt\@firstofone + % avoid brackets around text for \cite: + \def\@biblabel#1{} + \def\@cite#1#2{{#1\if@tempswa , #2\fi}} +\makeatother \newlength{\cslhangindent} \setlength{\cslhangindent}{1.5em} \newlength{\csllabelwidth} \setlength{\csllabelwidth}{3em} -\newlength{\cslentryspacingunit} % times entry-spacing -\setlength{\cslentryspacingunit}{\parskip} -\newenvironment{CSLReferences}[2] % #1 hanging-ident, #2 entry spacing - {% don't indent paragraphs - \setlength{\parindent}{0pt} +\newenvironment{CSLReferences}[2] % #1 hanging-indent, #2 entry-spacing + {\begin{list}{}{% + \setlength{\itemindent}{0pt} + \setlength{\leftmargin}{0pt} + \setlength{\parsep}{0pt} % turn on hanging indent if param 1 is 1 \ifodd #1 - \let\oldpar\par - \def\par{\hangindent=\cslhangindent\oldpar} + \setlength{\leftmargin}{\cslhangindent} + \setlength{\itemindent}{-1\cslhangindent} \fi % set entry spacing - \setlength{\parskip}{#2\cslentryspacingunit} - }% - {} + \setlength{\itemsep}{#2\baselineskip}}} + {\end{list}} \usepackage{calc} -\newcommand{\CSLBlock}[1]{#1\hfill\break} -\newcommand{\CSLLeftMargin}[1]{\parbox[t]{\csllabelwidth}{#1}} -\newcommand{\CSLRightInline}[1]{\parbox[t]{\linewidth - \csllabelwidth}{#1}\break} +\newcommand{\CSLBlock}[1]{\hfill\break\parbox[t]{\linewidth}{\strut\ignorespaces#1\strut}} +\newcommand{\CSLLeftMargin}[1]{\parbox[t]{\csllabelwidth}{\strut#1\strut}} +\newcommand{\CSLRightInline}[1]{\parbox[t]{\linewidth - \csllabelwidth}{\strut#1\strut}} \newcommand{\CSLIndent}[1]{\hspace{\cslhangindent}#1} \usepackage{booktabs} \usepackage{amsthm} @@ -104,6 +118,54 @@ \definecolor{gray75}{gray}{0.75} \newcommand{\hsp}{\hspace{20pt}} \titleformat{\chapter}[hang]{\Huge\bfseries}{\thechapter\hsp\textcolor{gray75}{|}\hsp}{0pt}{\Huge\bfseries} + +% chapter and section names in header +\renewcommand{\chaptermark}[1]{\markboth{\MakeLowercase{\chaptername\ \thechapter\ -\ #1}}{}} +\renewcommand*{\chaptermark}[1]{\markboth{\MakeLowercase{\chaptername\ \thechapter\ -\ #1}}{}} +\renewcommand{\sectionmark}[1]{\markright{\MakeLowercase{#1}}{} } + +% fix header names for toc, lof, and lot +\usepackage{etoolbox} +\patchcmd{\tableofcontents}{\MakeUppercase\contentsname}{\MakeLowercase\contentsname}{}{} +\patchcmd{\tableofcontents}{\MakeUppercase\contentsname}{\MakeLowercase\contentsname}{}{} % twice to remove on both sides of header +\patchcmd{\listoffigures}{\MakeUppercase\listfigurename}{\MakeLowercase\listfigurename}{}{} +\patchcmd{\listoffigures}{\MakeUppercase\listfigurename}{\MakeLowercase\listfigurename}{}{} % twice to remove on both sides of header +\patchcmd{\listoftables}{\MakeUppercase\listtablename}{\MakeLowercase\listtablename}{}{} +\patchcmd{\listoftables}{\MakeUppercase\listtablename}{\MakeLowercase\listtablename}{}{} % twice to remove on both sides of header + + + +% modify captions for figures and tables +\usepackage[labelfont=bf, + labelsep=endash, + singlelinecheck=off, + format=plain, + margin=1.2cm, + aboveskip=12pt, + belowskip=12pt, + font={footnotesize,stretch=1}, + justification=justified]{caption} + +% make table font smaller +\BeforeBeginEnvironment{longtable}{\begin{center}\footnotesize} +\AfterEndEnvironment{longtable}{\end{center}} + +\raggedbottom +\sloppy +\usepackage{booktabs} +\usepackage{longtable} +\usepackage{array} +\usepackage{multirow} +\usepackage{wrapfig} +\usepackage{float} +\usepackage{colortbl} +\usepackage{pdflscape} +\usepackage{tabu} +\usepackage{threeparttable} +\usepackage{threeparttablex} +\usepackage[normalem]{ulem} +\usepackage{makecell} +\usepackage{xcolor} \makeatletter \@ifpackageloaded{tcolorbox}{}{\usepackage[skins,breakable]{tcolorbox}} \@ifpackageloaded{fontawesome5}{}{\usepackage{fontawesome5}} @@ -121,8 +183,6 @@ \definecolor{quarto-callout-caution-color-frame}{HTML}{fd7e14} \makeatother \makeatletter -\makeatother -\makeatletter \@ifpackageloaded{bookmark}{}{\usepackage{bookmark}} \makeatother \makeatletter @@ -161,25 +221,17 @@ \newcommand*\listoflistings{\listof{codelisting}{List of Listings}} \makeatother \makeatletter -\@ifpackageloaded{caption}{}{\usepackage{caption}} -\@ifpackageloaded{subcaption}{}{\usepackage{subcaption}} -\makeatother -\makeatletter -\@ifpackageloaded{tcolorbox}{}{\usepackage[skins,breakable]{tcolorbox}} -\makeatother -\makeatletter -\@ifundefined{shadecolor}{\definecolor{shadecolor}{rgb}{.97, .97, .97}} -\makeatother -\makeatletter \makeatother \makeatletter +\@ifpackageloaded{caption}{}{\usepackage{caption}} +\@ifpackageloaded{subcaption}{}{\usepackage{subcaption}} \makeatother \ifLuaTeX \usepackage{selnolig} % disable illegal ligatures \fi -\IfFileExists{bookmark.sty}{\usepackage{bookmark}}{\usepackage{hyperref}} +\usepackage{bookmark} \IfFileExists{xurl.sty}{\usepackage{xurl}}{} % add URL line breaks if available -\urlstyle{same} % disable monospaced font for URLs +\urlstyle{same} \hypersetup{ pdftitle={Putting Dental Calculus Under the Microscope}, pdfauthor={Bjørn Peare Bartholdy}, @@ -208,7 +260,7 @@ op gezag van Rector Magnificus prof.dr.ir. H. Bijl, \\ volgens besluit van het College voor Promoties \\ te verdedigen op Donderdag 30 Mei 2024 \\ - klokke uur \\[1.5cm] + klokke 11.15 uur \\[1.5cm] door} \\[1.5cm] \Large{Bjørn Peare Bartholdy}\par \end{center} @@ -221,21 +273,29 @@ \noindent\begin{tabular}{p{8em} l} \large \textbf{Promotor} & \large Dr.~Amanda G. Henry \\ - \rule{0pt}{4ex}\large\textbf{Co-Promotor} & \large Dr.~Annelou van -Gijn \\ + \rule{0pt}{4ex}\large\textbf{Second} \\ \large\textbf{Promotor} & \large Prof.dr. +Annelou van Gijn \\ \large - \rule{0pt}{8ex}\textbf{Committee} & \rule{0pt}{4ex}\large Dr.~Patrick -Degryse \\ - & \indent\textit{Leiden UniversityKatholieke Universiteit -Leuven} \\ & \rule{0pt}{4ex}\large Dr.~Matthew James Collins \\ - & \indent\textit{University of CopenhagenUniversity of -Cambridge} \\ & \rule{0pt}{4ex}\large Dr.~Alison Crowther \\ + \rule{0pt}{8ex}\textbf{Committee} & \rule{0pt}{4ex}\large Prof.dr. +Patrick Degryse + \\[0.2mm] + & \indent\textit{Leiden University} \\[0.2mm] + & \indent\textit{Katholieke Universiteit +Leuven} \\ & \rule{0pt}{4ex}\large Prof.dr. Matthew James Collins + \\[0.2mm] + & \indent\textit{University of Copenhagen} \\[0.2mm] + & \indent\textit{University of +Cambridge} \\ & \rule{0pt}{4ex}\large Dr.~Alison Crowther + \\[0.2mm] & \indent\textit{University of -Queensland} \\ & \rule{0pt}{4ex}\large Dr.~Carla Lancelotti \\ +Queensland} \\ & \rule{0pt}{4ex}\large Prof.dr. Carla Lancelotti + \\[0.2mm] & \indent\textit{Universitat Pompeu Fabra and -ICREA} \\ & \rule{0pt}{4ex}\large Dr.~Christina Warinner \\ +ICREA} \\ & \rule{0pt}{4ex}\large Dr.~Christina Warinner + \\[0.2mm] & \indent\textit{Max Planck Institute for Evolutionary -AnthropologyHarvard University} \\ +Anthropology} \\[0.2mm] + & \indent\textit{Harvard University} \\ \end{tabular} \begingroup @@ -243,13 +303,13 @@ \vfill \begin{flushleft} \line(1,0){225} \\ %%%% Change colour of line to match chapters %%%% -\textbf{Front cover image:} Design by Krijn Boom and image by Petra +\textbf{Cover:} Design by Krijn Boom and image by Petra Korlevic \\[0.4cm] \textbf{Funding:} This research has received funding from the European Research Council under the European Union's Horizon 2020 research and innovation program, grant agreement number STG--677576 (``HARVEST''). \\[0.4cm] -\textbf{Print version:} \href{https://github.com/}{2024.XX.X} +\textbf{Print version:} \href{https://doi.org/}{2024.04.0} \end{flushleft} \endgroup %% End: promotor and committee page %% @@ -257,8 +317,6 @@ \frontmatter %%%\maketitle % -\ifdefined\Shaded\renewenvironment{Shaded}{\begin{tcolorbox}[borderline west={3pt}{0pt}{shadecolor}, interior hidden, sharp corners, frame hidden, boxrule=0pt, breakable, enhanced]}{\end{tcolorbox}}\fi - \renewcommand*\contentsname{Table of contents} { \setcounter{tocdepth}{2} @@ -268,7 +326,7 @@ \listoftables \setstretch{1.24} \mainmatter -\hypertarget{preface}{} +\phantomsection\label{preface} \bookmarksetup{startatroot} \chapter*{Preface} @@ -284,8 +342,8 @@ \chapter*{Preface} the scientific content, which is why you won't see the phrase `dental calculus' here. Oh, shoot\ldots{} -Feel free to jump directly to \protect\hyperlink{chap-intro}{Chapter 1} -if you don't want to read this. +Feel free to jump directly to \href{01-intro.qmd}{Chapter 1} if you +don't want to read this. When I started my PhD research I had no intentions of shaking things up. I was going to put my head down and do my research, publish my articles @@ -349,10 +407,10 @@ \chapter*{Preface} journal. We need to ask ourselves why we are doing science, and for whom we are doing it. +\phantomsection\label{acknowledgements} \bookmarksetup{startatroot} -\hypertarget{acknowledgements}{% -\chapter*{Acknowledgements}\label{acknowledgements}} +\chapter*{Acknowledgements} \addcontentsline{toc}{chapter}{Acknowledgements} \markboth{Acknowledgements}{Acknowledgements} @@ -397,7 +455,7 @@ \chapter*{Acknowledgements}\label{acknowledgements}} whole process. I couldn't have done it without you. I only wish you could have been here to see me finish it. -\hypertarget{open-science-statement}{} +\phantomsection\label{open-science-statement} \bookmarksetup{startatroot} \chapter*{Open Science Statement} @@ -427,8 +485,7 @@ \chapter*{Open Science Statement} \bookmarksetup{startatroot} -\hypertarget{chap-intro}{% -\chapter{Introduction}\label{chap-intro}} +\chapter{Introduction}\label{chap-intro} Dental calculus is becoming a popular substance in research on the behaviour and biology of people in the past. You may also know it as @@ -446,63 +503,59 @@ \chapter{Introduction}\label{chap-intro}} I will briefly describe the formation of dental calculus here, but for a more thorough review of the entire process I refer you to -\protect\hyperlink{chap-background}{Chapter 2}. Dental calculus is -formed from dental plaque, a substance that grows on your teeth and -consists mainly of bacteria and a surrounding structure called the -extracellular matrix. When the local environment within and around the -plaque reaches a favourable alkaline pH, both the extracellular matrix -and bacteria within will calcify -(\protect\hyperlink{ref-jinSupragingivalCalculus2002}{Jin \& Yip, 2002}; -\protect\hyperlink{ref-whiteDentalCalculus1997}{D. J. White, 1997}). The -alkaline pH causes minerals (especially calcium and phosphate) from +\hyperref[chap-background]{Chapter 2}. Dental calculus is formed from +dental plaque, a substance that grows on your teeth and consists mainly +of bacteria and a surrounding structure called the extracellular matrix. +When the local environment within and around the plaque reaches a +favourable alkaline pH, both the extracellular matrix and bacteria +within will calcify (\citeproc{ref-jinSupragingivalCalculus2002}{Jin \& +Yip, 2002}; \citeproc{ref-whiteDentalCalculus1997}{D. J. White, 1997}). +The alkaline pH causes minerals (especially calcium and phosphate) from saliva to enter the plaque, causing the extracellular matrix and eventually also the bacteria to harden, resulting in a concrete-like deposit on the surface of the teeth. The process repeats itself when new bacteria colonise the surface of the newly formed dental calculus, creating a layered structure, though somewhat disorganised -(\protect\hyperlink{ref-akcaliDentalCalculus2018}{Akcalı \& Lang, 2018}; -\protect\hyperlink{ref-jepsenCalculusRemoval2011}{Jepsen et al., 2011}). -Dental plaque can accumulate more easily on teeth (and dental calculus) -because they are a hard, non-shedding surface. Most of the surfaces in -our mouth are covered by a layer of cells called the oral epithelium. -These cells are continuously renewed as new cells are formed and dead -cells fall off (\protect\hyperlink{ref-squierOralMucosa1998}{Squier \& -Finkelstein, 1998}). This constant turnover means that it is difficult -for bacteria to build the communities they require for producing -biofilms. Enamel, the white substance that covers the crown of your -teeth, behaves differently. It stops growing when the tooth has fully -formed. After that, there is no renewal. This allows bacteria to -continue to grow and develop communities if there is no intervention -from you (or your dentist). Dental plaque can trap a variety of -different microparticles, including bacteria, human proteins, and small -debris from the food we eat -(\protect\hyperlink{ref-delafuenteDNAHuman2013}{De La Fuente et al., -2013}; \protect\hyperlink{ref-hendyProteomicCalculus2018}{Hendy et al., -2018}; \protect\hyperlink{ref-henryCalculusSyria2008}{Henry \& Piperno, -2008}). When the plaque mineralises, it can preserve these -microparticles over long periods of time, even after the person whose -teeth provided a home for the calculus has died. Also, the main crystal -structures in calculus strongly bind DNA, making calculus a fantastic -source of ancient DNA (aDNA) from the mouth -(\protect\hyperlink{ref-warinnerNewEra2015}{Warinner et al., 2015}). -Another advantage of dental calculus is that it represents a more recent -and direct source of diet than teeth or other bones. While bones and -teeth can take years to remodel and incorporate a dietary signal, -calculus forms on a much smaller timescale and is in direct contact with -the dietary material. Calculus can form within weeks at any point during -an individual's life and may, therefore, indicate a recent and direct -consumption of food, while bone can take years to show a (indirect) -dietary signal, following food molecules entering the bloodstream, and -finally entering the bone from there. Further, enamel stops forming -after the crown of the last tooth has developed---third molars, or -'wisdom teeth---at around 16 years of age, and the turnover of dentin is -very limited -(\protect\hyperlink{ref-hillsonDentalAnthropology1996}{Hillson, 1996}). -These properties are probably why archaeologists have become -increasingly interested in dental calculus. - -\hypertarget{intro-arch}{% -\section{Dental calculus in archaeology}\label{intro-arch}} +(\citeproc{ref-akcaliDentalCalculus2018}{Akcalı \& Lang, 2018}; +\citeproc{ref-jepsenCalculusRemoval2011}{Jepsen et al., 2011}). Dental +plaque can accumulate more easily on teeth (and dental calculus) because +they are a hard, non-shedding surface. Most of the surfaces in our mouth +are covered by a layer of cells called the oral epithelium. These cells +are continuously renewed as new cells are formed and dead cells fall off +(\citeproc{ref-squierOralMucosa1998}{Squier \& Finkelstein, 1998}). This +constant turnover means that it is difficult for bacteria to build the +communities they require for producing biofilms. Enamel, the white +substance that covers the crown of your teeth, behaves differently. It +stops growing when the tooth has fully formed. After that, there is no +renewal. This allows bacteria to continue to grow and develop +communities if there is no intervention from you (or your dentist). +Dental plaque can trap a variety of different microparticles, including +bacteria, human proteins, and small debris from the food we eat +(\citeproc{ref-delafuenteDNAHuman2013}{De La Fuente et al., 2013}; +\citeproc{ref-hendyProteomicCalculus2018}{Hendy et al., 2018}; +\citeproc{ref-henryCalculusSyria2008}{Henry \& Piperno, 2008}). When the +plaque mineralises, it can preserve these microparticles over long +periods of time, even after the person whose teeth provided a home for +the calculus has died. Also, the main crystal structures in calculus +strongly bind DNA, making calculus a fantastic source of ancient DNA +(aDNA) from the mouth (\citeproc{ref-warinnerNewEra2015}{Warinner et +al., 2015}). Another advantage of dental calculus is that it represents +a more recent and direct source of diet than teeth or other bones. While +bones and teeth can take years to remodel and incorporate a dietary +signal, calculus forms on a much smaller timescale and is in direct +contact with the dietary material. Calculus can form within weeks at any +point during an individual's life and may, therefore, indicate a recent +and direct consumption of food, while bone can take years to show a +(indirect) dietary signal, following food molecules entering the +bloodstream, and finally entering the bone from there. Further, enamel +stops forming after the crown of the last tooth has developed---third +molars, or 'wisdom teeth---at around 16 years of age, and the turnover +of dentin is very limited +(\citeproc{ref-hillsonDentalAnthropology1996}{Hillson, 1996}). These +properties are probably why archaeologists have become increasingly +interested in dental calculus. + +\section{Dental calculus in archaeology}\label{intro-arch} The main archaeological interest in dental calculus is to explore research questions involving diet and the evolution of the oral biome @@ -510,30 +563,30 @@ \section{Dental calculus in archaeology}\label{intro-arch}} such a small, seemingly insignificant material. This relates to its ability to retain and preserve a wide variety of different materials, from the food we eat to the bacteria that make their home in our mouths -(\protect\hyperlink{ref-adlerSequencingAncient2013}{Adler et al., 2013}; -\protect\hyperlink{ref-yatesOralMicrobiome2021}{Fellows Yates et al., -2021}; \protect\hyperlink{ref-henryCalculusSyria2008}{Henry \& Piperno, -2008}; \protect\hyperlink{ref-warinnerPathogensHost2014}{Warinner, -Rodrigues, et al., 2014}; -\protect\hyperlink{ref-warinnerEvidenceMilk2014}{Warinner, Hendy, et -al., 2014}). The goal of current studies targeting archaeological dental +(\citeproc{ref-adlerSequencingAncient2013}{Adler et al., 2013}; +\citeproc{ref-yatesOralMicrobiome2021}{Fellows Yates et al., 2021}; +\citeproc{ref-henryCalculusSyria2008}{Henry \& Piperno, 2008}; +\citeproc{ref-warinnerPathogensHost2014}{Warinner, Rodrigues, et al., +2014}; \citeproc{ref-warinnerEvidenceMilk2014}{Warinner, Hendy, et al., +2014}). The goal of current studies targeting archaeological dental calculus have not changed much since the early uses of dental calculus in archaeological research, but the methods certainly have, allowing us to unearth information that was previously not considered possible. By my count, archaeological dental calculus has now been subject to various -forms of microscopy -(\protect\hyperlink{ref-charlierSEMCalculus2010}{Charlier et al., 2010}; -\protect\hyperlink{ref-middletonOpalPhytoliths1994}{Middleton \& Rovner, -1994}; \protect\hyperlink{ref-powerSynchrotronRadiationbased2022}{Robert +forms of microscopy (\citeproc{ref-charlierSEMCalculus2010}{Charlier et +al., 2010}; \citeproc{ref-middletonOpalPhytoliths1994}{Middleton \& +Rovner, 1994}; \citeproc{ref-powerSynchrotronRadiationbased2022}{Robert C. Power et al., 2022}); extractions of biomolecules including DNA, proteins, and metabolites -(\protect\hyperlink{ref-adlerSequencingAncient2013}{Adler et al., 2013}; -\protect\hyperlink{ref-warinnerEvidenceMilk2014}{Warinner, Hendy, et -al., 2014}); and stable isotope analyses. +(\citeproc{ref-adlerSequencingAncient2013}{Adler et al., 2013}; +\citeproc{ref-warinnerEvidenceMilk2014}{Warinner, Hendy, et al., 2014}); +and stable isotope analyses. \begin{figure} -{\centering \includegraphics{01-intro_files/figure-pdf/fig-plot-and-wordclouds-1.pdf} +\centering{ + +\includegraphics{01-intro_files/figure-pdf/fig-plot-and-wordclouds-1.pdf} } @@ -541,7 +594,7 @@ \section{Dental calculus in archaeology}\label{intro-arch}} per year in bioarchaeology and clinical dentistry with the term `dental calculus' in the title.} -\end{figure} +\end{figure}% Perhaps the most common use of dental calculus is to recreate the diet of past people and populations (Figure~\ref{fig-plot-and-wordclouds}B). @@ -552,150 +605,140 @@ \section{Dental calculus in archaeology}\label{intro-arch}} destroying the plant fragments when releasing them from the calculus. As far as I can tell, the first attempt at this was the extraction of phytoliths (silicified plant remains) from the teeth of cows, sheep, and -horses -(\protect\hyperlink{ref-armitageExtractionIdentification1975}{Armitage, +horses (\citeproc{ref-armitageExtractionIdentification1975}{Armitage, 1975}). This was a somewhat isolated use-case, and the method didn't really catch on until the 1990s -(\protect\hyperlink{ref-ciochonOpalPhytoliths1990}{Ciochon et al., -1990}; Middleton 1990, in -\protect\hyperlink{ref-middletonOpalPhytoliths1994}{Middleton \& Rovner, -1994}). The first extractions from human teeth followed shortly -(\protect\hyperlink{ref-foxPhytolithCalculus1996}{Fox et al., 1996}), -and there are now studies using plant microremains (especially starch +(\citeproc{ref-ciochonOpalPhytoliths1990}{Ciochon et al., 1990}; +Middleton 1990, in \citeproc{ref-middletonOpalPhytoliths1994}{Middleton +\& Rovner, 1994}). The first extractions from human teeth followed +shortly (\citeproc{ref-foxPhytolithCalculus1996}{Fox et al., 1996}), and +there are now studies using plant microremains (especially starch granules and phytoliths) from dental calculus to infer diet in past peoples from across the world, including Pacific Islands -(\protect\hyperlink{ref-dudgeonDietGeography2014}{Dudgeon \& Tromp, -2014}), China (\protect\hyperlink{ref-chenStarchGrains2021}{Chen et al., -2021}), Europe (\protect\hyperlink{ref-fiorinCombiningDental2021}{Fiorin -et al., 2021}), and more -(\protect\hyperlink{ref-buckleyDentalCalculus2014}{Buckley et al., -2014}; \protect\hyperlink{ref-henryCalculusSyria2008}{Henry \& Piperno, -2008}; \protect\hyperlink{ref-mickleburghNewInsights2012}{Mickleburgh \& -Pagán-Jiménez, 2012}). The durable nature of dental calculus also means -that microremains within it can survive for millennia, allowing us to -look at the diets of early humans and other hominins -(\protect\hyperlink{ref-buckleyDentalCalculus2014}{Buckley et al., -2014}; \protect\hyperlink{ref-chenStarchGrains2021}{Chen et al., 2021}; -\protect\hyperlink{ref-hardyStarchGranules2009}{Hardy et al., 2009}; -\protect\hyperlink{ref-hardyNeanderthalMedics2012}{Hardy et al., 2012}; -\protect\hyperlink{ref-henryDietAustralopithecus2012}{Henry et al., -2012}, \protect\hyperlink{ref-henryNeanderthalCalculus2014}{2014}; -\protect\hyperlink{ref-henryCalculusSyria2008}{Henry \& Piperno, 2008}; -\protect\hyperlink{ref-pipernoStarchGrains2008}{Piperno \& Dillehay, -2008}). +(\citeproc{ref-dudgeonDietGeography2014}{Dudgeon \& Tromp, 2014}), China +(\citeproc{ref-chenStarchGrains2021}{Chen et al., 2021}), Europe +(\citeproc{ref-fiorinCombiningDental2021}{Fiorin et al., 2021}), and +more (\citeproc{ref-buckleyDentalCalculus2014}{Buckley et al., 2014}; +\citeproc{ref-henryCalculusSyria2008}{Henry \& Piperno, 2008}; +\citeproc{ref-mickleburghNewInsights2012}{Mickleburgh \& Pagán-Jiménez, +2012}). The durable nature of dental calculus also means that +microremains within it can survive for millennia, allowing us to look at +the diets of early humans and other hominins +(\citeproc{ref-buckleyDentalCalculus2014}{Buckley et al., 2014}; +\citeproc{ref-chenStarchGrains2021}{Chen et al., 2021}; +\citeproc{ref-hardyStarchGranules2009}{Hardy et al., 2009}; +\citeproc{ref-hardyNeanderthalMedics2012}{Hardy et al., 2012}; +\citeproc{ref-henryDietAustralopithecus2012}{Henry et al., 2012}, +\citeproc{ref-henryNeanderthalCalculus2014}{2014}; +\citeproc{ref-henryCalculusSyria2008}{Henry \& Piperno, 2008}; +\citeproc{ref-pipernoStarchGrains2008}{Piperno \& Dillehay, 2008}). That bacteria can become trapped within calculus has been known to archaeologists for a while -(\protect\hyperlink{ref-brothwellDiggingBones1981}{Brothwell, 1981}, ; -\protect\hyperlink{ref-vandermeerschMiddlePaleolithic1994}{Vandermeersch -et al., 1994}), but it wasn't used in archaeological research until DNA +(\citeproc{ref-brothwellDiggingBones1981}{Brothwell, 1981}, ; +\citeproc{ref-vandermeerschMiddlePaleolithic1994}{Vandermeersch et al., +1994}), but it wasn't used in archaeological research until DNA extraction started to become more accessible -(\protect\hyperlink{ref-delafuenteDNAHuman2013}{De La Fuente et al., -2013}). Dental calculus then became part of the third scientific -revolution in archaeology. The early studies focused on oral health in -the past (\protect\hyperlink{ref-adlerSequencingAncient2013}{Adler et -al., 2013}; \protect\hyperlink{ref-delafuenteDNAHuman2013}{De La Fuente -et al., 2013}; -\protect\hyperlink{ref-warinnerPathogensHost2014}{Warinner, Rodrigues, -et al., 2014}). Bacteria have shorter lifespans than humans which makes -them useful when studying the evolution of bacteria in the human mouth -(\protect\hyperlink{ref-delafuenteDNAHuman2013}{De La Fuente et al., -2013}; \protect\hyperlink{ref-yatesOralMicrobiome2021}{Fellows Yates et -al., 2021}). Diet has also been a focus of paleogenetic research. This -has mainly been addressed by considering how long-term changes in the -patterns of bacteria within the mouths of our ancestors have changed -that could be related to changes in diet. Just like we adapt to deal -with various diseases, climates, etc., we also adapt to changes in our -diet (\protect\hyperlink{ref-adlerSequencingAncient2013}{Adler et al., -2013}; \protect\hyperlink{ref-yatesOralMicrobiome2021}{Fellows Yates et -al., 2021}). Directly identifying genetic markers of plants and animals -within dental calculus is difficult, but not impossible (see Warinner, -Hendy, et al. (\protect\hyperlink{ref-warinnerEvidenceMilk2014}{2014})). -Most of the DNA within dental calculus will be oral bacteria, and this -will overwhelm the small signal from plant DNA, which makes species -identifications problematic -(\protect\hyperlink{ref-fagernasMicrobialBiogeography2022}{Fagernäs et -al., 2022}). A newer field of biomolecular archaeology, paleoproteomics, -may be able to address this issue by targeting plant proteins, along -with a range of other dietary protein sources. Hendy and coauthors were -able to identify a number of these in dental calculus, as well as -proteins from cereals, and milk proteins from different sources -(\protect\hyperlink{ref-hendyProteomicCalculus2018}{Hendy et al., -2018}). Dental calculus has also become a target for extracting other -biomolecules that may be related to diet, such as alkaloids, fatty -acids, and carbohydrates -(\protect\hyperlink{ref-gismondiMultidisciplinaryApproach2020}{Gismondi -et al., 2020}; \protect\hyperlink{ref-velskoDentalCalculus2017}{Velsko -et al., 2017}). The methods used for this have also proven to be useful -in detecting compounds that are related to other activities and -ceremonies, such as nicotine -(\protect\hyperlink{ref-eerkensDentalCalculus2018}{Eerkens et al., +(\citeproc{ref-delafuenteDNAHuman2013}{De La Fuente et al., 2013}). +Dental calculus then became part of the third scientific revolution in +archaeology. The early studies focused on oral health in the past +(\citeproc{ref-adlerSequencingAncient2013}{Adler et al., 2013}; +\citeproc{ref-delafuenteDNAHuman2013}{De La Fuente et al., 2013}; +\citeproc{ref-warinnerPathogensHost2014}{Warinner, Rodrigues, et al., +2014}). Bacteria have shorter lifespans than humans which makes them +useful when studying the evolution of bacteria in the human mouth +(\citeproc{ref-delafuenteDNAHuman2013}{De La Fuente et al., 2013}; +\citeproc{ref-yatesOralMicrobiome2021}{Fellows Yates et al., 2021}). +Diet has also been a focus of paleogenetic research. This has mainly +been addressed by considering how long-term changes in the patterns of +bacteria within the mouths of our ancestors have changed that could be +related to changes in diet. Just like we adapt to deal with various +diseases, climates, etc., we also adapt to changes in our diet +(\citeproc{ref-adlerSequencingAncient2013}{Adler et al., 2013}; +\citeproc{ref-yatesOralMicrobiome2021}{Fellows Yates et al., 2021}). +Directly identifying genetic markers of plants and animals within dental +calculus is difficult, but not impossible (see Warinner, Hendy, et al. +(\citeproc{ref-warinnerEvidenceMilk2014}{2014})). Most of the DNA within +dental calculus will be oral bacteria, and this will overwhelm the small +signal from plant DNA, which makes species identifications problematic +(\citeproc{ref-fagernasMicrobialBiogeography2022}{Fagernäs et al., +2022}). A newer field of biomolecular archaeology, paleoproteomics, may +be able to address this issue by targeting plant proteins, along with a +range of other dietary protein sources. Hendy and coauthors were able to +identify a number of these in dental calculus, as well as proteins from +cereals, and milk proteins from different sources +(\citeproc{ref-hendyProteomicCalculus2018}{Hendy et al., 2018}). Dental +calculus has also become a target for extracting other biomolecules that +may be related to diet, such as alkaloids, fatty acids, and +carbohydrates +(\citeproc{ref-gismondiMultidisciplinaryApproach2020}{Gismondi et al., +2020}; \citeproc{ref-velskoDentalCalculus2017}{Velsko et al., 2017}). +The methods used for this have also proven to be useful in detecting +compounds that are related to other activities and ceremonies, such as +nicotine (\citeproc{ref-eerkensDentalCalculus2018}{Eerkens et al., 2018}), and may provide some evidence of medicinal practices -(\protect\hyperlink{ref-gismondiMultidisciplinaryApproach2020}{Gismondi -et al., 2020}). +(\citeproc{ref-gismondiMultidisciplinaryApproach2020}{Gismondi et al., +2020}). To a lesser extent, the presence and amount of dental calculus on teeth has been used as an indicator of dental health -(\protect\hyperlink{ref-drewettExcavationOval1975}{Drewett, 1975}; -\protect\hyperlink{ref-lieverseDentalHealth2007}{Lieverse et al., 2007}; -\protect\hyperlink{ref-sagneStudiesPeriodontal1977}{Sagne \& Olsson, -1977}; \protect\hyperlink{ref-zhangDentalDisease1982}{Zhang, 1982}). -Pilloud \& Fancher -(\protect\hyperlink{ref-pilloudOutliningDefinition2019}{2019}) explored -the terms associated with a number publications on dental or oral -health, dental calculus came up as one of them; albeit not the most -common, which was (unsurprisingly) dental caries -(Figure~\ref{fig-dental-terms}). +(\citeproc{ref-drewettExcavationOval1975}{Drewett, 1975}; +\citeproc{ref-lieverseDentalHealth2007}{Lieverse et al., 2007}; +\citeproc{ref-sagneStudiesPeriodontal1977}{Sagne \& Olsson, 1977}; +\citeproc{ref-zhangDentalDisease1982}{Zhang, 1982}). Pilloud \& Fancher +(\citeproc{ref-pilloudOutliningDefinition2019}{2019}) explored the terms +associated with a number publications on dental or oral health, dental +calculus came up as one of them; albeit not the most common, which was +(unsurprisingly) dental caries (Figure~\ref{fig-dental-terms}). To a lesser, lesser extent, it has also provided some interesting insights on non-dietary activities, such as occupations and smoking habits. In a rare find, blue particles were detected in the dental calculus of a Medieval German woman. These blue particles originated from lapis lazuli, an exotic stone often ground into pigments and used -to illuminate manuscripts -(\protect\hyperlink{ref-radiniMedievalWomen2019}{Radini et al., 2019}). -Nicotine was detected in dental calculus of pre-colonisation individuals -from California using Ultra-Performance Liquid Chromatography Mass -Spectrometry (UPLC-MS), showing direct consumption of tobacco and -providing more detailed insights on the demographics of consumption in a -way that no other human-adjacent archaeological materials can. +to illuminate manuscripts (\citeproc{ref-radiniMedievalWomen2019}{Radini +et al., 2019}). Nicotine was detected in dental calculus of +pre-colonisation individuals from California using Ultra-Performance +Liquid Chromatography Mass Spectrometry (UPLC-MS), showing direct +consumption of tobacco and providing more detailed insights on the +demographics of consumption in a way that no other human-adjacent +archaeological materials can. \begin{figure} -{\centering \includegraphics{figures/wordcloud.png} +\centering{ + +\includegraphics{figures/wordcloud.png} } \caption{\label{fig-dental-terms}Word cloud of most common dental terms in articles. Figure is from Pilloud \& Fancher -(\protect\hyperlink{ref-pilloudOutliningDefinition2019}{2019}), Figure -1.} +(\citeproc{ref-pilloudOutliningDefinition2019}{2019}), Figure 1.} -\end{figure} +\end{figure}% It wasn't always appreciated for the wealth of information hidden within its hardened shell. Until roughly 20 years ago, archaeologists who encountered calculus had limited use for this material. Some researchers quantified it using a simple four-stage scoring method that was developed for recording deposits on archaeological dental calculus -(\protect\hyperlink{ref-brothwellDiggingBones1981}{Brothwell, 1981}), -similar to a common clinical scoring system -(\protect\hyperlink{ref-greeneSimplifiedOral1964}{J. G. Greene \& -Vermillion, 1964}). The four-stage system is probably still the most -widely used among archaeologists. More detailed methods are also -available (\protect\hyperlink{ref-dobneyMethodEvaluating1987}{Dobney \& -Brothwell, 1987}; -\protect\hyperlink{ref-greeneQuantifyingCalculus2005}{T. R. Greene et -al., 2005}), but the original method is generally preferred for its +(\citeproc{ref-brothwellDiggingBones1981}{Brothwell, 1981}), similar to +a common clinical scoring system +(\citeproc{ref-greeneSimplifiedOral1964}{J. G. Greene \& Vermillion, +1964}). The four-stage system is probably still the most widely used +among archaeologists. More detailed methods are also available +(\citeproc{ref-dobneyMethodEvaluating1987}{Dobney \& Brothwell, 1987}; +\citeproc{ref-greeneQuantifyingCalculus2005}{T. R. Greene et al., +2005}), but the original method is generally preferred for its simplicity. Unfortunately, knowing the size of a calculus deposit is not as valuable as being able to analyse the deposit itself, and the deposits were often removed because they obscured tooth and root -morphology (\protect\hyperlink{ref-scottBriefHistory2015}{Scott, 2015}). -This had made a lot of people very angry and been widely regarded as a -bad move (\protect\hyperlink{ref-adamsRestaurantEnd2002}{Adams, 2002, p. -1}). Hindsight being what it is, it's hard to blame anyone. A lot of -dental research mainly focuses on the prevention and removal of dental -calculus. +morphology (\citeproc{ref-scottBriefHistory2015}{Scott, 2015}). This had +made a lot of people very angry and been widely regarded as a bad move +(\citeproc{ref-adamsRestaurantEnd2002}{Adams, 2002, p. 1}). Hindsight +being what it is, it's hard to blame anyone. A lot of dental research +mainly focuses on the prevention and removal of dental calculus. The wide range of applications for dental calculus that we know about today, and the fact that it's pretty much ubiquitous in the past thanks @@ -709,31 +752,30 @@ \section{Dental calculus in archaeology}\label{intro-arch}} how its growth and structure affect the reliability of these methods and potentially distort our interpretations of the past? -\hypertarget{intro-what}{% -\section{What is dental calculus?}\label{intro-what}} +\section{What is dental calculus?}\label{intro-what} To answer these questions, we must first answer a single, surprisingly difficult question: What is dental calculus? I'm not referring to its formation or composition, which I briefly described -\protect\hyperlink{chap-intro}{above}. How do we categorise it? Is it a -dental disease? An oral health condition? A byproduct of oral -conditions? We start by exploring various definitions of oral health. -Definitions in an introduction are a little cliché and tedious, but -often necessary. Since oral health is a complex topic, definitions of -oral health are often purposefully (and confusingly) broad, and they -extend beyond physical well-being and into the realms of emotional and -social comfort. The World Dental Federation (FDI) defines oral health as -the ability to perform mouth- and face-related functions with confidence -and without pain (including smiling, speaking, eating, etc.) -(\protect\hyperlink{ref-fdiOralHealth}{{``{FDI}'s Definition of Oral -Health \textbar{} {FDI},''} n.d.}) +\hyperref[chap-intro]{above}. How do we categorise it? Is it a dental +disease? An oral health condition? A byproduct of oral conditions? We +start by exploring various definitions of oral health. Definitions in an +introduction are a little cliché and tedious, but often necessary. Since +oral health is a complex topic, definitions of oral health are often +purposefully (and confusingly) broad, and they extend beyond physical +well-being and into the realms of emotional and social comfort. The +World Dental Federation (FDI) defines oral health as the ability to +perform mouth- and face-related functions with confidence and without +pain (including smiling, speaking, eating, etc.) +(\citeproc{ref-fdiOralHealth}{{``{FDI}'s Definition of Oral Health +\textbar{} {FDI},''} n.d.}) (\url{https://www.fdiworlddental.org/fdis-definition-oral-health}). Both the World Health Organisation (WHO) and FDI take a similar approach to defining oral conditions, giving a list of conditions that cause discomfort, pain, disfigurement, or death. The list includes the dental conditions tooth decay (caries), gum disease (periodontal disease), and dental trauma, but not dental calculus -(\protect\hyperlink{ref-whoOralHealth}{{``Oral Health,''} n.d.}) +(\citeproc{ref-whoOralHealth}{{``Oral Health,''} n.d.}) (\url{https://www.who.int/news-room/fact-sheets/detail/oral-health}). While these are not likely to cause death, they are often the source of physical and emotional discomfort, and may cause further health @@ -742,85 +784,80 @@ \section{What is dental calculus?}\label{intro-what}} Dental calculus and dental plaque are not considered oral conditions according to WHO. In fact, dental plaque is part of the normal functioning of our oral biome -(\protect\hyperlink{ref-marshDentalPlaque2006}{Marsh, 2006}). When -plaque reaches a certain level of acidity over a prolonged period of -time, the normal functioning of the bacteria within the plaque may shift -towards a disease-causing function. The biofilm will cause the surface -of the enamel to demineralise, eventually resulting in a cavity (or -caries). Dental caries are unequivocally considered a dental disease. -If, instead, the biofilm calcifies, dental calculus is the result. Its +(\citeproc{ref-marshDentalPlaque2006}{Marsh, 2006}). When plaque reaches +a certain level of acidity over a prolonged period of time, the normal +functioning of the bacteria within the plaque may shift towards a +disease-causing function. The biofilm will cause the surface of the +enamel to demineralise, eventually resulting in a cavity (or caries). +Dental caries are unequivocally considered a dental disease. If, +instead, the biofilm calcifies, dental calculus is the result. Its status in oral health is questionable. Dental calculus is not known to be painful, nor does it affect the ability to perform the functions listed above. However, with continued accumulation, it may affect the confidence of the person performing -these tasks (\protect\hyperlink{ref-collinsHomelessDental2007}{Collins -\& Freeman, 2007}), and in extreme cases it can affect function -(\protect\hyperlink{ref-balajiUnusualPresentation2019}{Balaji et al., -2019}). Most of the virulence and disease-causing potential is lost when -the bacteria within dental plaque calcify -(\protect\hyperlink{ref-akcaliDentalCalculus2018}{Akcalı \& Lang, -2018}). It has been shown to contain pockets of living bacteria that can -be detrimental to oral and dental health -(\protect\hyperlink{ref-tanCalculusUltrastructure2004}{Tan, Gillam, et -al., 2004}; \protect\hyperlink{ref-tanBacterialViability2004}{Tan, -Mordan, et al., 2004}). The rough, porous surface of dental calculus is -also a great place for bacteria to attach more easily and develop a new -layer of plaque on the surface of the calculus. This is likely why there -is often a correlation (NOT causation) between dental calculus and -periodontitis, especially subgingival calculus -(\protect\hyperlink{ref-jepsenCalculusRemoval2011}{Jepsen et al., 2011}; -\protect\hyperlink{ref-whiteDentalCalculus1997}{D. J. White, 1997}). -Since it seems to fulfill some of the criteria of an oral condition, it -should be considered as such, at least under the definitions provided by -WHO and FDI. Whether or not dental calculus can be considered an oral -disease is more questionable. While it does grow on the surface of -teeth, it doesn't seem to affect the underlying enamel. And while there -is a relationship with periodontal disease (which has been defined as a +these tasks (\citeproc{ref-collinsHomelessDental2007}{Collins \& +Freeman, 2007}), and in extreme cases it can affect function +(\citeproc{ref-balajiUnusualPresentation2019}{Balaji et al., 2019}). +Most of the virulence and disease-causing potential is lost when the +bacteria within dental plaque calcify +(\citeproc{ref-akcaliDentalCalculus2018}{Akcalı \& Lang, 2018}). It has +been shown to contain pockets of living bacteria that can be detrimental +to oral and dental health +(\citeproc{ref-tanCalculusUltrastructure2004}{Tan, Gillam, et al., +2004}; \citeproc{ref-tanBacterialViability2004}{Tan, Mordan, et al., +2004}). The rough, porous surface of dental calculus is also a great +place for bacteria to attach more easily and develop a new layer of +plaque on the surface of the calculus. This is likely why there is often +a correlation (NOT causation) between dental calculus and periodontitis, +especially subgingival calculus +(\citeproc{ref-jepsenCalculusRemoval2011}{Jepsen et al., 2011}; +\citeproc{ref-whiteDentalCalculus1997}{D. J. White, 1997}). Since it +seems to fulfill some of the criteria of an oral condition, it should be +considered as such, at least under the definitions provided by WHO and +FDI. Whether or not dental calculus can be considered an oral disease is +more questionable. While it does grow on the surface of teeth, it +doesn't seem to affect the underlying enamel. And while there is a +relationship with periodontal disease (which has been defined as a dental disease), the nature of this relationship is still under debate, with calculus likely being a secondary contributor -(\protect\hyperlink{ref-jepsenCalculusRemoval2011}{Jepsen et al., -2011}). As such, we can probably limit the definition to an oral -condition and not necessarily a dental disease -(\protect\hyperlink{ref-pilloudOutliningDefinition2019}{Pilloud \& -Fancher, 2019}). In fact, dental calculus is quite hard, so a layer of -dental calculus on a tooth can actually protect it from wearing down -(although there are better options). +(\citeproc{ref-jepsenCalculusRemoval2011}{Jepsen et al., 2011}). As +such, we can probably limit the definition to an oral condition and not +necessarily a dental disease +(\citeproc{ref-pilloudOutliningDefinition2019}{Pilloud \& Fancher, +2019}). In fact, dental calculus is quite hard, so a layer of dental +calculus on a tooth can actually protect it from wearing down (although +there are better options). -\hypertarget{intro-study}{% -\section{The study of dental calculus}\label{intro-study}} +\section{The study of dental calculus}\label{intro-study} It seems that the researchers who are studying dental calculus approach it from a wide range of different fields and backgrounds, including genetics, proteomics, botany, and (bio)archaeology. The paleogeneticists mine it for the wealth of information it contains on oral health and -disease in the past -(\protect\hyperlink{ref-yatesOralMicrobiome2021}{Fellows Yates et al., -2021}; \protect\hyperlink{ref-warinnerPathogensHost2014}{Warinner, +disease in the past (\citeproc{ref-yatesOralMicrobiome2021}{Fellows +Yates et al., 2021}; \citeproc{ref-warinnerPathogensHost2014}{Warinner, Rodrigues, et al., 2014}). Paleodiet researchers extract microremains -and residues from food -(\protect\hyperlink{ref-henryCalculusSyria2008}{Henry \& Piperno, 2008}; -\protect\hyperlink{ref-mickleburghNewInsights2012}{Mickleburgh \& +and residues from food (\citeproc{ref-henryCalculusSyria2008}{Henry \& +Piperno, 2008}; \citeproc{ref-mickleburghNewInsights2012}{Mickleburgh \& Pagán-Jiménez, 2012}) to infer dietary practices. Bioarchaeologists use its presence and amount to broadly infer diet, and dental and overall health in a given population -(\protect\hyperlink{ref-belcastroContinuityDiscontinuity2007}{Belcastro -et al., 2007}; \protect\hyperlink{ref-lieverseDentalHealth2007}{Lieverse -et al., 2007}; \protect\hyperlink{ref-novakDentalHealth2015}{Novak, -2015}; \protect\hyperlink{ref-slausDentalHealth2011}{Šlaus et al., -2011}; \protect\hyperlink{ref-yaussyCalculusSurvivorship2019}{Yaussy \& -DeWitte, 2019}). This leaves research output from studies of calculus -scattered across multiple venues, with no clear gathering point. I think -it's fair to say that dental calculus should be included in discussions -of pathological oral conditions, even if its role is secondary. But who -is currently studying dental calculus as a substance in its own right? -And why do we need to learn more about it if we're just interested in -what's inside? Related discussions have started to take place in recent -years (\protect\hyperlink{ref-bucchiComparisonsMethods2019}{Bucchi et -al., 2019}; \protect\hyperlink{ref-radiniDirtyTeeth2022}{Radini \& -Nikita, 2022}; -\protect\hyperlink{ref-wrightAdvancingRefining2021}{Wright et al., -2021}). +(\citeproc{ref-belcastroContinuityDiscontinuity2007}{Belcastro et al., +2007}; \citeproc{ref-lieverseDentalHealth2007}{Lieverse et al., 2007}; +\citeproc{ref-novakDentalHealth2015}{Novak, 2015}; +\citeproc{ref-slausDentalHealth2011}{Šlaus et al., 2011}; +\citeproc{ref-yaussyCalculusSurvivorship2019}{Yaussy \& DeWitte, 2019}). +This leaves research output from studies of calculus scattered across +multiple venues, with no clear gathering point. I think it's fair to say +that dental calculus should be included in discussions of pathological +oral conditions, even if its role is secondary. But who is currently +studying dental calculus as a substance in its own right? And why do we +need to learn more about it if we're just interested in what's inside? +Related discussions have started to take place in recent years +(\citeproc{ref-bucchiComparisonsMethods2019}{Bucchi et al., 2019}; +\citeproc{ref-radiniDirtyTeeth2022}{Radini \& Nikita, 2022}; +\citeproc{ref-wrightAdvancingRefining2021}{Wright et al., 2021}). The lack of a specific field of study for dental calculus to belong may be related to how it's taught to students (and if it's taught at all). @@ -830,31 +867,29 @@ \section{The study of dental calculus}\label{intro-study}} mention dental calculus as more of a footnote than anything else. A couple of lines describing what it is (usually `mineralised plaque') and that it can contain food debris and bacteria T. D. White et al. -(\protect\hyperlink{ref-whiteHumanOsteology2011}{2011}). They're not -wrong. Diseases that manifest themselves in the skeleton as lesions on -the bones have a very clear home in paleopathology. No one questions -whether or not the degeneration of vertebrae from tuberculosis should be +(\citeproc{ref-whiteHumanOsteology2011}{2011}). They're not wrong. +Diseases that manifest themselves in the skeleton as lesions on the +bones have a very clear home in paleopathology. No one questions whether +or not the degeneration of vertebrae from tuberculosis should be included in the paleopathology textbooks (at least not as far as I'm aware). These textbooks often include chapters on dental disease, where more detailed descriptions of dental calculus are usually found (e.g. -\protect\hyperlink{ref-robertsDentalDisease2007}{Roberts \& Manchester, -2007}; \protect\hyperlink{ref-waldronPalaeopathology2020}{Waldron, -2020}). Dental caries, calculus' more famous sibling, will often get a -few pages. In some cases, dental calculus may even be hidden within a -section on periodontal disease or plaque -(\protect\hyperlink{ref-aufderheidePaleopathology1998}{Aufderheide et -al., 1998}; e.g. -\protect\hyperlink{ref-ortnerIdentificationPathological2003}{Ortner, -2003}). The focus of these (sub)sections is varied, with some simply -describing what it is, and others giving brief discussion on the -relationship between calculus and periodontal disease. A more detailed -section was dedicated to dental calculus in \emph{Ortner's -Identification of Pathological Conditions in Human Skeletal Remains}, -with a detailed description of formation, structure, and application in -(biomolecular) archaeology -(\protect\hyperlink{ref-kinastonOrtnerDentition2019}{Kinaston et al., +\citeproc{ref-robertsDentalDisease2007}{Roberts \& Manchester, 2007}; +\citeproc{ref-waldronPalaeopathology2020}{Waldron, 2020}). Dental +caries, calculus' more famous sibling, will often get a few pages. In +some cases, dental calculus may even be hidden within a section on +periodontal disease or plaque +(\citeproc{ref-aufderheidePaleopathology1998}{Aufderheide et al., 1998}; +e.g. \citeproc{ref-ortnerIdentificationPathological2003}{Ortner, 2003}). +The focus of these (sub)sections is varied, with some simply describing +what it is, and others giving brief discussion on the relationship +between calculus and periodontal disease. A more detailed section was +dedicated to dental calculus in \emph{Ortner's Identification of +Pathological Conditions in Human Skeletal Remains}, with a detailed +description of formation, structure, and application in (biomolecular) +archaeology (\citeproc{ref-kinastonOrtnerDentition2019}{Kinaston et al., 2019}). The description extends well beyond any (paleo)pathological significance of dental calculus. Can we fault the authors/editors for not giving it more attention? After all, it's not a dental disease, and @@ -868,20 +903,19 @@ \section{The study of dental calculus}\label{intro-study}} Companion to Dental Anthropology}, an otherwise great resource on studying archaeological teeth. The editors briefly acknowledge the valuable information gained from calculus and that it holds a lot of -potential; but that's it -(\protect\hyperlink{ref-scottBriefHistory2015}{Scott, 2015}). Other -notable absences include textbooks such as \emph{Technique and -Application in Dental Anthropology} and \emph{New Direction in Dental -Anthropology} -(\protect\hyperlink{ref-townsendDentalAnthropology2012}{Townsend et al., -2012}), both of which dedicate considerable attention to dental caries. +potential; but that's it (\citeproc{ref-scottBriefHistory2015}{Scott, +2015}). Other notable absences include textbooks such as \emph{Technique +and Application in Dental Anthropology} and \emph{New Direction in +Dental Anthropology} +(\citeproc{ref-townsendDentalAnthropology2012}{Townsend et al., 2012}), +both of which dedicate considerable attention to dental caries. Hillson's \emph{Dental Anthropology}, a book that I consider to be the `bible' for dental anthropology, has a section on dental calculus in the Dental Disease chapter. It covers a basic description, the composition, microscopic structure, methods used for recording archaeological calculus, and the distribution in the dentition (i.e.~which teeth are more prone to calculus buildup) -(\protect\hyperlink{ref-hillsonDentalAnthropology1996}{Hillson, 1996}). +(\citeproc{ref-hillsonDentalAnthropology1996}{Hillson, 1996}). Considering these are entire books devoted to the dentition, it seems odd that there is often only a few paragraphs (if that) on dental calculus. Granted, the only function teeth serve in the growth of dental @@ -897,50 +931,47 @@ \section{The study of dental calculus}\label{intro-study}} the introduction section. These are quite variable and are often limited by the word count of the journal. Despite this, the descriptions will often be as long, if not longer, than the sections in textbooks devoted -to dental calculus -(\protect\hyperlink{ref-velskoMicrobialDifferences2019}{Velsko et al., -2019}). The focus of these paragraphs are generally the same. They -describe the formation and mineral composition of dental calculus, and -provide some examples of how dental calculus has been used in related -studies (not unlike the beginning of this chapter). The contribution of -dental calculus to archaeology has been significant, so it is likely to -receive more and more attention going forward. In fact, an entire -chapter was recently devoted to dental calculus in the second edition of -\emph{Handbook of Archaeological Sciences} -(\protect\hyperlink{ref-fagernasDentalCalculus2023}{Fagernäs \& -Warinner, 2023}). Take that, dental caries! - -\hypertarget{the-challenges-of-studying-dental-calculus}{% +to dental calculus (\citeproc{ref-velskoMicrobialDifferences2019}{Velsko +et al., 2019}). The focus of these paragraphs are generally the same. +They describe the formation and mineral composition of dental calculus, +and provide some examples of how dental calculus has been used in +related studies (not unlike the beginning of this chapter). The +contribution of dental calculus to archaeology has been significant, so +it is likely to receive more and more attention going forward. In fact, +an entire chapter was recently devoted to dental calculus in the second +edition of \emph{Handbook of Archaeological Sciences} +(\citeproc{ref-fagernasDentalCalculus2023}{Fagernäs \& Warinner, 2023}). +Take that, dental caries! + \section{The challenges of studying dental -calculus}\label{the-challenges-of-studying-dental-calculus}} +calculus}\label{the-challenges-of-studying-dental-calculus} What we know about dental calculus and the influence of diet was reviewed in an article aimed at (bio)archaeologists. The overall conclusion reached in the article: it's still pretty unclear -(\protect\hyperlink{ref-lieverseDietAetiology1999}{Lieverse, 1999}). -Now, 20-some years later, there has been limited progress on this point. -High-protein diets are linked to an increase of urea, which is linked to -an increase in oral pH, which is linked to mineral deposition -(\protect\hyperlink{ref-dibdinOralUrea1998}{Dibdin \& Dawes, 1998}; -\protect\hyperlink{ref-wongCalciumPhosphate2002}{Wong et al., 2002}). -BUT, protein may also inhibit crystalisation -(\protect\hyperlink{ref-hidakaDietCalculus2007}{S. Hidaka \& Oishi, -2007}). Starch consumption has been linked to increased rates of caries -in early farming populations -(\protect\hyperlink{ref-storeyPaleopathologyOrigins1986}{Storey, 1986}). -This is consistent with \emph{in vitro} testing, at least for starches -high in amylose content. So a high-starch diet causes caries, not -calculus, right? Well, starches with a high amylopectin content are -linked to increased calcification -(\protect\hyperlink{ref-hidakaDietCalculus2007}{S. Hidaka \& Oishi, -2007}). It likely depends on what is consumed along with the starch -(\protect\hyperlink{ref-hidakaStarchRole2008}{Saburo Hidaka et al., +(\citeproc{ref-lieverseDietAetiology1999}{Lieverse, 1999}). Now, 20-some +years later, there has been limited progress on this point. High-protein +diets are linked to an increase of urea, which is linked to an increase +in oral pH, which is linked to mineral deposition +(\citeproc{ref-dibdinOralUrea1998}{Dibdin \& Dawes, 1998}; +\citeproc{ref-wongCalciumPhosphate2002}{Wong et al., 2002}). BUT, +protein may also inhibit crystalisation +(\citeproc{ref-hidakaDietCalculus2007}{S. Hidaka \& Oishi, 2007}). +Starch consumption has been linked to increased rates of caries in early +farming populations +(\citeproc{ref-storeyPaleopathologyOrigins1986}{Storey, 1986}). This is +consistent with \emph{in vitro} testing, at least for starches high in +amylose content. So a high-starch diet causes caries, not calculus, +right? Well, starches with a high amylopectin content are linked to +increased calcification (\citeproc{ref-hidakaDietCalculus2007}{S. Hidaka +\& Oishi, 2007}). It likely depends on what is consumed along with the +starch (\citeproc{ref-hidakaStarchRole2008}{Saburo Hidaka et al., 2008}). There is also some (\emph{in vitro}) evidence to suggest that silica may promote dental calculus formation by promoting mineral precipitation, i.e.~the transfer of minerals from saliva to the biofilm -(\protect\hyperlink{ref-damenSilicicAcid1989}{Damen \& Ten Cate, 1989}). -Overall, this is an understudied area in both clinical and -archaeological contexts. +(\citeproc{ref-damenSilicicAcid1989}{Damen \& Ten Cate, 1989}). Overall, +this is an understudied area in both clinical and archaeological +contexts. Another aspect of diet and dental calculus where we are still looking for answers, is the process that causes fragments of food and other @@ -950,41 +981,37 @@ \section{The challenges of studying dental exactly how this happens, and herein lies the potential for bias. Efforts have been made to understand how much of the consumed food makes it into the calculus. These include studies on modern humans -(\protect\hyperlink{ref-leonardPlantMicroremains2015}{Leonard et al., -2015}) and non-human primates -(\protect\hyperlink{ref-powerChimpCalculus2015}{R. C. Power et al., -2015}; \protect\hyperlink{ref-powerRepresentativenessDental2021}{Robert -C. Power et al., 2021}), where food intake is meticulously documented, -and calculus subsequently analysed. These studies have common findings; -the amount of the diet that becomes trapped in the dental calculus of -any one person has no clear relationship to the amount of food that was +(\citeproc{ref-leonardPlantMicroremains2015}{Leonard et al., 2015}) and +non-human primates (\citeproc{ref-powerChimpCalculus2015}{R. C. Power et +al., 2015}; \citeproc{ref-powerRepresentativenessDental2021}{Robert C. +Power et al., 2021}), where food intake is meticulously documented, and +calculus subsequently analysed. These studies have common findings; the +amount of the diet that becomes trapped in the dental calculus of any +one person has no clear relationship to the amount of food that was consumed. The most likely reason is that the formation of dental calculus differs between people -(\protect\hyperlink{ref-powerChimpCalculus2015}{R. C. Power et al., -2015}). So, it's not a great way to study the diet of a single person, -but generally suitable to study patterns in the diet of a population. -The more people you study, the more likely you are to gain a complete -picture of the diet in a population. The fact that we can still see (in -some cases, literally) remains that were consumed thousands of years ago -is pretty cool. We just need a better understanding of why the record of -diet from dental calculus differs from the actual intake of food. This -will allow us to make more robust interpretations about past dietary -practices. Something that may influence the dietary record that we get -from calculus is the method we use to extract the dietary remains from +(\citeproc{ref-powerChimpCalculus2015}{R. C. Power et al., 2015}). So, +it's not a great way to study the diet of a single person, but generally +suitable to study patterns in the diet of a population. The more people +you study, the more likely you are to gain a complete picture of the +diet in a population. The fact that we can still see (in some cases, +literally) remains that were consumed thousands of years ago is pretty +cool. We just need a better understanding of why the record of diet from +dental calculus differs from the actual intake of food. This will allow +us to make more robust interpretations about past dietary practices. +Something that may influence the dietary record that we get from +calculus is the method we use to extract the dietary remains from calculus. Our understanding of dental calculus extraction methods is improving, with studies looking at the effect of various acids used to dissolve calculus (commonly EDTA or HCl) -(\protect\hyperlink{ref-bucchiComparisonsMethods2019}{Bucchi et al., -2019}; \protect\hyperlink{ref-palmerComparingUse2021}{Palmer et al., -2021}; -\protect\hyperlink{ref-sotoCharacterizationDecontamination2019}{Soto et -al., 2019}; \protect\hyperlink{ref-trompEDTACalculus2017}{Tromp et al., -2017}); as is our understanding of how the choice of tooth may affect -our results -(\protect\hyperlink{ref-fagernasMicrobialBiogeography2021}{Fagernäs et -al., 2021}), and that not all we see is related to deliberate -consumption (\protect\hyperlink{ref-delaneyMoreWhat2023}{Delaney et al., -2023}). +(\citeproc{ref-bucchiComparisonsMethods2019}{Bucchi et al., 2019}; +\citeproc{ref-palmerComparingUse2021}{Palmer et al., 2021}; +\citeproc{ref-sotoCharacterizationDecontamination2019}{Soto et al., +2019}; \citeproc{ref-trompEDTACalculus2017}{Tromp et al., 2017}); as is +our understanding of how the choice of tooth may affect our results +(\citeproc{ref-fagernasMicrobialBiogeography2021}{Fagernäs et al., +2021}), and that not all we see is related to deliberate consumption +(\citeproc{ref-delaneyMoreWhat2023}{Delaney et al., 2023}). These studies provide valuable insights into potential biases of our sampling methods and the representation of diet within dental calculus, @@ -1020,10 +1047,9 @@ \section{The challenges of studying dental more interest in preventing dental calculus from forming in the first place, so most studies focus on short-duration models to explore anti-microbial treatments and inhibition of biofilm formation and plaque -buildup (\protect\hyperlink{ref-extercateAAA2010}{Exterkate et al., -2010}). As shown in a previous study, calculus and plaque have distinct -microbial profiles -(\protect\hyperlink{ref-velskoMicrobialDifferences2019}{Velsko et al., +buildup (\citeproc{ref-extercateAAA2010}{Exterkate et al., 2010}). As +shown in a previous study, calculus and plaque have distinct microbial +profiles (\citeproc{ref-velskoMicrobialDifferences2019}{Velsko et al., 2019}), so the applicability of short-term models to explore archaeological questions on dental calculus are limited, since plaque is rarely (if ever) preserved. Archaeologists are more interested in @@ -1037,8 +1063,7 @@ \section{The challenges of studying dental certainly aren't interested in how food debris becomes trapped inside our calculus. Dental calculus has also become less of a problem with the use of modern dental hygiene practices and regular visits to the dentist -(\protect\hyperlink{ref-velskoMicrobialDifferences2019}{Velsko et al., -2019}). +(\citeproc{ref-velskoMicrobialDifferences2019}{Velsko et al., 2019}). To summarise: Bioarchaeologists are interested in how dental calculus relates to dental and general health; paleodietary researchers are @@ -1052,8 +1077,7 @@ \section{The challenges of studying dental from the past becomes trapped inside. To summarise the summary: we need to ask more basic questions about dental calculus. -\hypertarget{intro-aims}{% -\section{Aims}\label{intro-aims}} +\section{Aims}\label{intro-aims} This dissertation is a contribution to a dental-calculus-centric body of knowledge, and addresses a gap in the fundamental research on dental @@ -1094,50 +1118,48 @@ \section{Aims}\label{intro-aims}} better understanding of how dietary intake relates to the record of diet we extract from archaeological dental calculus? -\hypertarget{thesis-outline-and-structure}{% \section{Thesis outline and -structure}\label{thesis-outline-and-structure}} +structure}\label{thesis-outline-and-structure} If you have made it to this point, you have probably read most of -\protect\hyperlink{chap-intro}{\textbf{Chapter 1}}, in which I provide -some context to the study of dental calculus in archaeology and identify -some areas that could benefit from further investigation. -\protect\hyperlink{chap-background}{\textbf{Chapter 2}} provides some -background information on oral biofilms and oral biofilm models in more -detail than I can do in the research articles included in Chapters 3 and -4. So if you're already well-versed in oral microbiology, feel free to -skip to Chapter 3. If not, I recommend picking up a textbook written by -actual experts in the field of oral microbiology. If, for some reason, -you can't access one of these, feel free to read -\protect\hyperlink{chap-background}{\textbf{Chapter 2}}. I suppose there -are worse options than something written by a PhD student in -archaeology. The chapter reflects the current knowledge of biofilms and -the oral microbiome (as best I could summarise) at the time of writing, -and no warranty is given for the inevitable new developments that will -change what we now believe to be true. +\hyperref[chap-intro]{\textbf{Chapter 1}}, in which I provide some +context to the study of dental calculus in archaeology and identify some +areas that could benefit from further investigation. +\hyperref[chap-background]{\textbf{Chapter 2}} provides some background +information on oral biofilms and oral biofilm models in more detail than +I can do in the research articles included in Chapters 3 and 4. So if +you're already well-versed in oral microbiology, feel free to skip to +Chapter 3. If not, I recommend picking up a textbook written by actual +experts in the field of oral microbiology. If, for some reason, you +can't access one of these, feel free to read +\hyperref[chap-background]{\textbf{Chapter 2}}. I suppose there are +worse options than something written by a PhD student in archaeology. +The chapter reflects the current knowledge of biofilms and the oral +microbiome (as best I could summarise) at the time of writing, and no +warranty is given for the inevitable new developments that will change +what we now believe to be true. To address the aims of the dissertation outlined -\protect\hyperlink{intro-aims}{above}, I developed a protocol to grow -dental calculus in a lab on plastic tubes instead of looking at the real -stuff you normally find inside your mouth. The reason for using -lab-grown biofilms instead of humans is that the \emph{in vitro} lab -model offers more control over all the factors that go into the growth -of dental calculus, at least in theory. The real world is messy, and -sometimes you need to remove things from the real world to break it down -and really get into the nitty gritty of how it works. There are many -different kinds of biofilm models, including single species of bacteria, -select species determined by the researchers (defined consortium), and -multiple species from some natural source (the human mouth, for -example). I will cover the different types of models in more detail in -\protect\hyperlink{background}{\textbf{Chapter 2}}. Since there are many -biofilm models to choose from, developing a new protocol may seem +\hyperref[intro-aims]{above}, I developed a protocol to grow dental +calculus in a lab on plastic tubes instead of looking at the real stuff +you normally find inside your mouth. The reason for using lab-grown +biofilms instead of humans is that the \emph{in vitro} lab model offers +more control over all the factors that go into the growth of dental +calculus, at least in theory. The real world is messy, and sometimes you +need to remove things from the real world to break it down and really +get into the nitty gritty of how it works. There are many different +kinds of biofilm models, including single species of bacteria, select +species determined by the researchers (defined consortium), and multiple +species from some natural source (the human mouth, for example). I will +cover the different types of models in more detail in +\hyperref[background]{\textbf{Chapter 2}}. Since there are many biofilm +models to choose from, developing a new protocol may seem counter-productive; however, few are developed for long-term growth and even fewer with the purpose of mineralising the biofilm to form dental calculus. One of the exceptions involves a highly complex setup that is unlikely to be supported by budgets and facilities available to most archaeological laboratories -(\protect\hyperlink{ref-sissonsMultistationPlaque1991}{Sissons et al., -1991}). +(\citeproc{ref-sissonsMultistationPlaque1991}{Sissons et al., 1991}). After developing a working protocol, the next step was to determine if the stuff I grew in the lab is actually dental calculus. Or at least @@ -1145,49 +1167,48 @@ \section{Thesis outline and questions. To do this, we (myself and coauthors) determined the mineral and bacterial composition of our model using Fourier Transform Infrared (FTIR) spectroscopy and metagenomic classification -\protect\hyperlink{byoc-valid}{\textbf{Chapter 3}}. We then compared the -results of these analyses to naturally grown dental calculus, both -modern and archaeological. +\hyperref[byoc-valid]{\textbf{Chapter 3}}. We then compared the results +of these analyses to naturally grown dental calculus, both modern and +archaeological. Being confident that our model looks and behaves like human dental calculus, we then set out to test some very basic behaviours of starch -grains within dental calculus. -\protect\hyperlink{byoc-starch}{\textbf{Chapter 4}} is a research -article where we `fed' the biofilm with a known quantity of starch -granules during the growth period to see if the input quantity/ratio -matched the extracted quantity (or output). Those who are familiar with -dental calculus research will not be surprised that it did not. The more -interesting outcome of the study is the more detailed explanation of how -the input and output starch quantities were mismatched. - -\protect\hyperlink{mb11CalculusPilot}{\textbf{Chapter 5}} is a separate -article, in the sense that it doesn't involve the biofilm model in any -way. Rather, it addresses the theme of the overall utility of dental -calculus in archaeological research. We look at possible medicinal -compounds in the dental calculus of a Post-medieval Dutch population. We -employed Ultra High Performance Liquid Chromatography coupled with -tandem Mass Spectrometry (UHPLC-MS/MS) to identify various compounds in -dental calculus, including alkaloids and other compounds. It shows the +grains within dental calculus. \hyperref[byoc-starch]{\textbf{Chapter +4}} is a research article where we `fed' the biofilm with a known +quantity of starch granules during the growth period to see if the input +quantity/ratio matched the extracted quantity (or output). Those who are +familiar with dental calculus research will not be surprised that it did +not. The more interesting outcome of the study is the more detailed +explanation of how the input and output starch quantities were +mismatched. + +\hyperref[mb11CalculusPilot]{\textbf{Chapter 5}} is a separate article, +in the sense that it doesn't involve the biofilm model in any way. +Rather, it addresses the theme of the overall utility of dental calculus +in archaeological research. We look at possible medicinal compounds in +the dental calculus of a Post-medieval Dutch population. We employed +Ultra High Performance Liquid Chromatography coupled with tandem Mass +Spectrometry (UHPLC-MS/MS) to identify various compounds in dental +calculus, including alkaloids and other compounds. It shows the potential of dental calculus to inform about past practices, but also highlights some of the limitations we are currently experiencing in the -field. \protect\hyperlink{chap-discussion}{\textbf{Chapter 6}} is a -discussion on the limitations and future potential of dental calculus in -the field of archaeology, and what biofilm models can contribute to our -understanding of past diet. +field. \hyperref[chap-discussion]{\textbf{Chapter 6}} is a discussion on +the limitations and future potential of dental calculus in the field of +archaeology, and what biofilm models can contribute to our understanding +of past diet. -\hypertarget{references-cited}{% -\section*{References cited}\label{references-cited}} +\section*{References cited}\label{references-cited} \addcontentsline{toc}{section}{References cited} \markright{References cited} -\hypertarget{refs-1}{} +\phantomsection\label{refs-1} \begin{CSLReferences}{1}{0} -\leavevmode\vadjust pre{\hypertarget{ref-adamsRestaurantEnd2002}{}}% +\bibitem[\citeproctext]{ref-adamsRestaurantEnd2002} Adams, D. (2002). \emph{The {Restaurant} at the {End} of the {Universe}}. {Picador}. -\leavevmode\vadjust pre{\hypertarget{ref-adlerSequencingAncient2013}{}}% +\bibitem[\citeproctext]{ref-adlerSequencingAncient2013} Adler, C. J., Dobney, K., Weyrich, L. S., Kaidonis, J., Walker, A. W., Haak, W., Bradshaw, C. J., Townsend, G., Sołtysiak, A., Alt, K. W., Parkhill, J., \& Cooper, A. (2013). Sequencing ancient calcified dental @@ -1195,28 +1216,28 @@ \section*{References cited}\label{references-cited}} {Neolithic} and {Industrial} revolutions. \emph{Nature Genetics}, \emph{45}(4), 450--455, 455e1. \url{https://doi.org/10.1038/ng.2536} -\leavevmode\vadjust pre{\hypertarget{ref-akcaliDentalCalculus2018}{}}% +\bibitem[\citeproctext]{ref-akcaliDentalCalculus2018} Akcalı, A., \& Lang, N. P. (2018). Dental calculus: The calcified biofilm and its role in disease development. \emph{Periodontology 2000}, \emph{76}(1), 109--115. \url{https://doi.org/10.1111/prd.12151} -\leavevmode\vadjust pre{\hypertarget{ref-armitageExtractionIdentification1975}{}}% +\bibitem[\citeproctext]{ref-armitageExtractionIdentification1975} Armitage, P. L. (1975). The {Extraction} and {Identification} of {Opal Phytoliths} from the {Teeth} of {Ungulates}. \emph{Journal of Archaeological Science}, \emph{2}, 187--197. -\leavevmode\vadjust pre{\hypertarget{ref-aufderheidePaleopathology1998}{}}% +\bibitem[\citeproctext]{ref-aufderheidePaleopathology1998} Aufderheide, A. C., Rodriguez-Martin, C., \& Langsjoen, O. (1998). \emph{The {Cambridge} encyclopedia of human paleopathology} (Vol. 478). {Cambridge University Press Cambridge}. -\leavevmode\vadjust pre{\hypertarget{ref-balajiUnusualPresentation2019}{}}% +\bibitem[\citeproctext]{ref-balajiUnusualPresentation2019} Balaji, V. R., Niazi, T. M., \& Dhanasekaran, M. (2019). An unusual presentation of dental calculus. \emph{Journal of Indian Society of Periodontology}, \emph{23}(5), 484--486. \url{https://doi.org/10.4103/jisp.jisp_680_18} -\leavevmode\vadjust pre{\hypertarget{ref-belcastroContinuityDiscontinuity2007}{}}% +\bibitem[\citeproctext]{ref-belcastroContinuityDiscontinuity2007} Belcastro, G., Rastelli, E., Mariotti, V., Consiglio, C., Facchini, F., \& Bonfiglioli, B. (2007). Continuity or discontinuity of the life-style in central {Italy} during the {Roman} imperial age-early middle ages @@ -1224,12 +1245,12 @@ \section*{References cited}\label{references-cited}} Physical Anthropology}, \emph{132}(3), 381--394. \url{https://doi.org/10.1002/ajpa.20530} -\leavevmode\vadjust pre{\hypertarget{ref-brothwellDiggingBones1981}{}}% +\bibitem[\citeproctext]{ref-brothwellDiggingBones1981} Brothwell, D. (1981). \emph{Digging up {Bones}: {The} excavation, treatment and study of human skeletal remains} (3rd ed.). {British Museum (Natural History)}. -\leavevmode\vadjust pre{\hypertarget{ref-bucchiComparisonsMethods2019}{}}% +\bibitem[\citeproctext]{ref-bucchiComparisonsMethods2019} Bucchi, A., Burguet-Coca, A., Expósito, I., Aceituno Bocanegra, F. J., \& Lozano, M. (2019). Comparisons between methods for analyzing dental calculus samples from {El Mirador} cave ({Sierra} de {Atapuerca}, @@ -1237,13 +1258,13 @@ \section*{References cited}\label{references-cited}} \emph{11}(11), 6305--6314. \url{https://doi.org/10.1007/s12520-019-00919-z} -\leavevmode\vadjust pre{\hypertarget{ref-buckleyDentalCalculus2014}{}}% +\bibitem[\citeproctext]{ref-buckleyDentalCalculus2014} Buckley, S., Usai, D., Jakob, T., Radini, A., \& Hardy, K. (2014). Dental {Calculus Reveals Unique Insights} into {Food Items}, {Cooking} and {Plant Processing} in {Prehistoric Central Sudan}. \emph{PLOS ONE}, \emph{9}(7), e100808. \url{https://doi.org/10.1371/journal.pone.0100808} -\leavevmode\vadjust pre{\hypertarget{ref-charlierSEMCalculus2010}{}}% +\bibitem[\citeproctext]{ref-charlierSEMCalculus2010} Charlier, P., Huynh-Charlier, I., Munoz, O., Billard, M., Brun, L., \& Grandmaison, G. L. de la. (2010). The microscopic (optical and {SEM}) examination of dental calculus deposits ({DCD}). {Potential} interest in @@ -1251,69 +1272,69 @@ \section*{References cited}\label{references-cited}} Medicine}, \emph{12}(4), 163--171. \url{https://doi.org/10.1016/j.legalmed.2010.03.003} -\leavevmode\vadjust pre{\hypertarget{ref-chenStarchGrains2021}{}}% +\bibitem[\citeproctext]{ref-chenStarchGrains2021} Chen, T., Hou, L., Jiang, H., Wu, Y., \& Henry, A. G. (2021). Starch grains from human teeth reveal the plant consumption of proto-{Shang} -people (c. 2000{\textendash}1600 {BC}) from {Nancheng} site, {Hebei}, +people (c. 2000\textendash 1600 {BC}) from {Nancheng} site, {Hebei}, {China}. \emph{Archaeological and Anthropological Sciences}, \emph{13}(9), 153. \url{https://doi.org/10.1007/s12520-021-01416-y} -\leavevmode\vadjust pre{\hypertarget{ref-ciochonOpalPhytoliths1990}{}}% +\bibitem[\citeproctext]{ref-ciochonOpalPhytoliths1990} Ciochon, R. L., Piperno, D. R., \& Thompson, R. G. (1990). Opal phytoliths found on the teeth of the extinct ape {Gigantopithecus} blacki: Implications for paleodietary studies. \emph{Proceedings of the National Academy of Sciences}, \emph{87}(20), 8120--8124. \url{https://doi.org/10.1073/pnas.87.20.8120} -\leavevmode\vadjust pre{\hypertarget{ref-collinsHomelessDental2007}{}}% +\bibitem[\citeproctext]{ref-collinsHomelessDental2007} Collins, J., \& Freeman, R. (2007). Homeless in {North} and {West Belfast}: An oral health needs assessment. \emph{British Dental Journal}, \emph{202}(12), E31--E31. \url{https://doi.org/10.1038/bdj.2007.473} -\leavevmode\vadjust pre{\hypertarget{ref-damenSilicicAcid1989}{}}% +\bibitem[\citeproctext]{ref-damenSilicicAcid1989} Damen, J. J. M., \& Ten Cate, J. M. (1989). The {Effect} of {Silicic Acid} on {Calcium Phosphate Precipitation}. \emph{Journal of Dental Research}, \emph{68}(9), 1355--1359. \url{https://doi.org/10.1177/00220345890680091301} -\leavevmode\vadjust pre{\hypertarget{ref-delafuenteDNAHuman2013}{}}% +\bibitem[\citeproctext]{ref-delafuenteDNAHuman2013} De La Fuente, C., Flores, S., \& Moraga, M. (2013). {DNA From Human Ancient Bacteria}: {A} novel source of genetic evidence from archaeological dental calculus. \emph{Archaeometry}, \emph{55}(4), 767--778. \url{https://doi.org/10.1111/j.1475-4754.2012.00707.x} -\leavevmode\vadjust pre{\hypertarget{ref-delaneyMoreWhat2023}{}}% +\bibitem[\citeproctext]{ref-delaneyMoreWhat2023} Delaney, S., Alexander, M., \& Radini, A. (2023). More than what we eat: {Investigating} an alternative pathway for intact starch granules in dental calculus using {Experimental Archaeology}. \emph{Quaternary International}, \emph{653--654}, 19--32. \url{https://doi.org/10.1016/j.quaint.2022.03.004} -\leavevmode\vadjust pre{\hypertarget{ref-dibdinOralUrea1998}{}}% +\bibitem[\citeproctext]{ref-dibdinOralUrea1998} Dibdin, G. H., \& Dawes, C. (1998). A {Mathematical Model} of the {Influence} of {Salivary Urea} on the {pH} of {Fasted Dental Plaque} and on the {Changes Occurring} during a {Cariogenic Challenge}. \emph{Caries Research}, \emph{32}(1), 70--74. \url{https://doi.org/10.1159/000016432} -\leavevmode\vadjust pre{\hypertarget{ref-dobneyMethodEvaluating1987}{}}% +\bibitem[\citeproctext]{ref-dobneyMethodEvaluating1987} Dobney, K., \& Brothwell, D. (1987). A method for evaluating the amount of dental calculus on teeth from archaeological sites. \emph{Journal of Archaeological Science}, \emph{14}(4), 343--351. \url{https://doi.org/10.1016/0305-4403(87)90024-0} -\leavevmode\vadjust pre{\hypertarget{ref-drewettExcavationOval1975}{}}% +\bibitem[\citeproctext]{ref-drewettExcavationOval1975} Drewett, P. (1975). \emph{The {Excavation} of an {Oval Burial Mound} of the {Third Millennium} be at {Alfriston}, {East Sussex}, 1974}. 38. -\leavevmode\vadjust pre{\hypertarget{ref-dudgeonDietGeography2014}{}}% +\bibitem[\citeproctext]{ref-dudgeonDietGeography2014} Dudgeon, J. V., \& Tromp, M. (2014). Diet, {Geography} and {Drinking Water} in {Polynesia}: {Microfossil Research} from {Archaeological Human Dental Calculus}, {Rapa Nui} ({Easter Island}). \emph{International Journal of Osteoarchaeology}, \emph{24}(5), 634--648. \url{https://doi.org/10.1002/oa.2249} -\leavevmode\vadjust pre{\hypertarget{ref-eerkensDentalCalculus2018}{}}% +\bibitem[\citeproctext]{ref-eerkensDentalCalculus2018} Eerkens, J. W., Tushingham, S., Brownstein, K. J., Garibay, R., Perez, K., Murga, E., Kaijankoski, P., Rosenthal, J. S., \& Gang, D. R. (2018). Dental calculus as a source of ancient alkaloids: {Detection} of @@ -1321,21 +1342,21 @@ \section*{References cited}\label{references-cited}} \emph{Journal of Archaeological Science: Reports}, \emph{18}, 509--515. \url{https://doi.org/10.1016/j.jasrep.2018.02.004} -\leavevmode\vadjust pre{\hypertarget{ref-extercateAAA2010}{}}% +\bibitem[\citeproctext]{ref-extercateAAA2010} Exterkate, R. A. M., Crielaard, W., \& Ten Cate, J. M. (2010). Different {Response} to {Amine Fluoride} by {Streptococcus} mutans and {Polymicrobial Biofilms} in a {Novel High-Throughput Active Attachment Model}. \emph{Caries Research}, \emph{44}(4), 372--379. \url{https://doi.org/10.1159/000316541} -\leavevmode\vadjust pre{\hypertarget{ref-fagernasMicrobialBiogeography2021}{}}% +\bibitem[\citeproctext]{ref-fagernasMicrobialBiogeography2021} Fagernäs, Z., Salazar-García, D. C., Avilés, A., Haber, M., Henry, A., Maurandi, J. L., Ozga, A., Velsko, I. M., \& Warinner, C. (2021). Understanding the microbial biogeography of ancient human dentitions to guide study design and interpretation. \emph{bioRxiv}, 2021.08.16.456492. \url{https://doi.org/10.1101/2021.08.16.456492} -\leavevmode\vadjust pre{\hypertarget{ref-fagernasMicrobialBiogeography2022}{}}% +\bibitem[\citeproctext]{ref-fagernasMicrobialBiogeography2022} Fagernäs, Z., Salazar-García, D. C., Haber Uriarte, M., Avilés Fernández, A., Henry, A. G., Lomba Maurandi, J., Ozga, A. T., Velsko, I. M., \& Warinner, C. (2022). Understanding the microbial biogeography of @@ -1343,17 +1364,17 @@ \section*{References cited}\label{references-cited}} \emph{FEMS Microbes}, \emph{3}, xtac006. \url{https://doi.org/10.1093/femsmc/xtac006} -\leavevmode\vadjust pre{\hypertarget{ref-fagernasDentalCalculus2023}{}}% +\bibitem[\citeproctext]{ref-fagernasDentalCalculus2023} Fagernäs, Z., \& Warinner, C. (2023). Dental {Calculus}. In A. M. Pollard, R. A. Armitage, \& C. Makarevicz (Eds.), \emph{Handbook of {Archaeological Sciences}} (Second edition). -\leavevmode\vadjust pre{\hypertarget{ref-fdiOralHealth}{}}% +\bibitem[\citeproctext]{ref-fdiOralHealth} {FDI}'s definition of oral health \textbar{} {FDI}. (n.d.). In \emph{FDI World Dental Federation}. https://www.fdiworlddental.org/fdis-definition-oral-health. -\leavevmode\vadjust pre{\hypertarget{ref-yatesOralMicrobiome2021}{}}% +\bibitem[\citeproctext]{ref-yatesOralMicrobiome2021} Fellows Yates, J. A., Velsko, I. M., Aron, F., Posth, C., Hofman, C. A., Austin, R. M., Parker, C. E., Mann, A. E., Nägele, K., Arthur, K. W., Arthur, J. W., Bauer, C. C., Crevecoeur, I., Cupillard, C., Curtis, M. @@ -1363,21 +1384,21 @@ \section*{References cited}\label{references-cited}} National Academy of Sciences}, \emph{118}(20). \url{https://doi.org/10.1073/pnas.2021655118} -\leavevmode\vadjust pre{\hypertarget{ref-fiorinCombiningDental2021}{}}% +\bibitem[\citeproctext]{ref-fiorinCombiningDental2021} Fiorin, E., Moore, J., Montgomery, J., Lippi, M. M., Nowell, G., \& Forlin, P. (2021). Combining dental calculus with isotope analysis in the {Alps}: {New} evidence from the {Roman} and medieval cemeteries of {Lamon}, northern {Italy}. \emph{Quaternary International}. \url{https://doi.org/10.1016/j.quaint.2021.11.022} -\leavevmode\vadjust pre{\hypertarget{ref-foxPhytolithCalculus1996}{}}% +\bibitem[\citeproctext]{ref-foxPhytolithCalculus1996} Fox, C. L., Juan, J., \& Albert, R. M. (1996). Phytolith analysis on dental calculus, enamel surface, and burial soil: {Information} about diet and paleoenvironment. \emph{American Journal of Physical Anthropology}, \emph{101}(1), 101--113. \url{https://doi.org/10.1002/(SICI)1096-8644(199609)101:1\%3C101::AID-AJPA7\%3E3.0.CO;2-Y} -\leavevmode\vadjust pre{\hypertarget{ref-gismondiMultidisciplinaryApproach2020}{}}% +\bibitem[\citeproctext]{ref-gismondiMultidisciplinaryApproach2020} Gismondi, A., Baldoni, M., Gnes, M., Scorrano, G., D'Agostino, A., Marco, G. D., Calabria, G., Petrucci, M., Müldner, G., Tersch, M. V., Nardi, A., Enei, F., Canini, A., Rickards, O., Alexander, M., \& @@ -1387,26 +1408,26 @@ \section*{References cited}\label{references-cited}} ONE}, \emph{15}(1), e0227433. \url{https://doi.org/10.1371/journal.pone.0227433} -\leavevmode\vadjust pre{\hypertarget{ref-greeneSimplifiedOral1964}{}}% +\bibitem[\citeproctext]{ref-greeneSimplifiedOral1964} Greene, J. G., \& Vermillion, J. R. (1964). The {Simplified Oral Hygiene Index}. \emph{The Journal of the American Dental Association}, \emph{68}(1), 7--13. \url{https://doi.org/10.14219/jada.archive.1964.0034} -\leavevmode\vadjust pre{\hypertarget{ref-greeneQuantifyingCalculus2005}{}}% +\bibitem[\citeproctext]{ref-greeneQuantifyingCalculus2005} Greene, T. R., Kuba, C. L., \& Irish, J. D. (2005). Quantifying calculus: {A} suggested new approach for recording an important indicator of diet and dental health. \emph{HOMO - Journal of Comparative Human Biology}, \emph{56}(2), 119--132. \url{https://doi.org/10.1016/j.jchb.2005.02.002} -\leavevmode\vadjust pre{\hypertarget{ref-hardyStarchGranules2009}{}}% +\bibitem[\citeproctext]{ref-hardyStarchGranules2009} Hardy, K., Blakeney, T., Copeland, L., Kirkham, J., Wrangham, R., \& Collins, M. (2009). Starch granules, dental calculus and new perspectives on ancient diet. \emph{Journal of Archaeological Science}, \emph{36}(2), 248--255. \url{https://doi.org/10.1016/j.jas.2008.09.015} -\leavevmode\vadjust pre{\hypertarget{ref-hardyNeanderthalMedics2012}{}}% +\bibitem[\citeproctext]{ref-hardyNeanderthalMedics2012} Hardy, K., Buckley, S., Collins, M. J., Estalrrich, A., Brothwell, D., Copeland, L., García-Tabernero, A., García-Vargas, S., de la Rasilla, M., Lalueza-Fox, C., Huguet, R., Bastir, M., Santamaría, D., Madella, @@ -1415,7 +1436,7 @@ \section*{References cited}\label{references-cited}} calculus. \emph{Naturwissenschaften}, \emph{99}(8), 617--626. \url{https://doi.org/10.1007/s00114-012-0942-0} -\leavevmode\vadjust pre{\hypertarget{ref-hendyProteomicCalculus2018}{}}% +\bibitem[\citeproctext]{ref-hendyProteomicCalculus2018} Hendy, J., Warinner, C., Bouwman, A., Collins, M. J., Fiddyment, S., Fischer, R., Hagan, R., Hofman, C. A., Holst, M., Chaves, E., Klaus, L., Larson, G., Mackie, M., McGrath, K., Mundorff, A. Z., Radini, A., Rao, @@ -1424,32 +1445,32 @@ \section*{References cited}\label{references-cited}} \emph{Proceedings. Biological Sciences}, \emph{285}(1883), 20180977. \url{https://doi.org/10.1098/rspb.2018.0977} -\leavevmode\vadjust pre{\hypertarget{ref-henryNeanderthalCalculus2014}{}}% +\bibitem[\citeproctext]{ref-henryNeanderthalCalculus2014} Henry, A. G., Brooks, A. S., \& Piperno, D. R. (2014). Plant foods and the dietary ecology of {Neanderthals} and early modern humans. \emph{Journal of Human Evolution}, \emph{69}, 44--54. \url{https://doi.org/10.1016/j.jhevol.2013.12.014} -\leavevmode\vadjust pre{\hypertarget{ref-henryCalculusSyria2008}{}}% +\bibitem[\citeproctext]{ref-henryCalculusSyria2008} Henry, A. G., \& Piperno, D. R. (2008). Using plant microfossils from dental calculus to recover human diet: A case study from {Tell} -al-{Raq{ā}}'i, {Syria}. \emph{Journal of Archaeological Science}, +al-{Raqā}'i, {Syria}. \emph{Journal of Archaeological Science}, \emph{35}(7), 1943--1950. \url{https://doi.org/10.1016/j.jas.2007.12.005} -\leavevmode\vadjust pre{\hypertarget{ref-henryDietAustralopithecus2012}{}}% +\bibitem[\citeproctext]{ref-henryDietAustralopithecus2012} Henry, A. G., Ungar, P. S., Passey, B. H., Sponheimer, M., Rossouw, L., Bamford, M., Sandberg, P., de Ruiter, D. J., \& Berger, L. (2012). The diet of {Australopithecus} sediba. \emph{Nature}, \emph{487}(7405), 90--93. \url{https://doi.org/10.1038/nature11185} -\leavevmode\vadjust pre{\hypertarget{ref-hidakaDietCalculus2007}{}}% +\bibitem[\citeproctext]{ref-hidakaDietCalculus2007} Hidaka, S., \& Oishi, A. (2007). An in vitro study of the effect of some dietary components on calculus formation: Regulation of calcium phosphate precipitation. \emph{Oral Diseases}, \emph{13}(3), 296--302. \url{https://doi.org/10.1111/j.1601-0825.2006.01283.x} -\leavevmode\vadjust pre{\hypertarget{ref-hidakaStarchRole2008}{}}% +\bibitem[\citeproctext]{ref-hidakaStarchRole2008} Hidaka, Saburo, Okamoto, Y., Tsukamoto, S., \& Oishi, A. (2008). The {Possible Role} of {Starch} in {Oral Calcification}: {The In Vitro Formation} of {Hydroxyapatite} is {Regulated} by a {Combination} of @@ -1457,22 +1478,22 @@ \section*{References cited}\label{references-cited}} Open Food Science Journal}, \emph{2}(1), 10--22. \url{https://doi.org/10.2174/1874256400802010010} -\leavevmode\vadjust pre{\hypertarget{ref-hillsonDentalAnthropology1996}{}}% +\bibitem[\citeproctext]{ref-hillsonDentalAnthropology1996} Hillson, S. (1996). \emph{Dental {Anthropology}}. {Cambridge University Press}. -\leavevmode\vadjust pre{\hypertarget{ref-jepsenCalculusRemoval2011}{}}% +\bibitem[\citeproctext]{ref-jepsenCalculusRemoval2011} Jepsen, S., Deschner, J., Braun, A., Schwarz, F., \& Eberhard, J. (2011). Calculus removal and the prevention of its formation. \emph{Periodontology 2000}, \emph{55}(1), 167--188. \url{https://doi.org/10.1111/j.1600-0757.2010.00382.x} -\leavevmode\vadjust pre{\hypertarget{ref-jinSupragingivalCalculus2002}{}}% +\bibitem[\citeproctext]{ref-jinSupragingivalCalculus2002} Jin, Y., \& Yip, H.-K. (2002). Supragingival {Calculus}: {Formation} and {Control}. \emph{Critical Reviews in Oral Biology \& Medicine}. \url{https://doi.org/10.1177/154411130201300506} -\leavevmode\vadjust pre{\hypertarget{ref-kinastonOrtnerDentition2019}{}}% +\bibitem[\citeproctext]{ref-kinastonOrtnerDentition2019} Kinaston, R., Willis, A., Miszkiewicz, J. J., Tromp, M., \& Oxenham, M. F. (2019). The {Dentition}: {Development}, {Disturbances}, {Disease}, {Diet}, and {Chemistry}. In J. E. Buikstra (Ed.), \emph{Ortner's @@ -1480,78 +1501,77 @@ \section*{References cited}\label{references-cited}} Remains} ({Third Edition})} (pp. 749--797). {Academic Press}. \url{https://doi.org/10.1016/B978-0-12-809738-0.00021-1} -\leavevmode\vadjust pre{\hypertarget{ref-leonardPlantMicroremains2015}{}}% +\bibitem[\citeproctext]{ref-leonardPlantMicroremains2015} Leonard, C., Vashro, L., O'Connell, J. F., \& Henry, A. G. (2015). Plant microremains in dental calculus as a record of plant consumption: {A} test with {Twe} forager-horticulturalists. \emph{Journal of Archaeological Science: Reports}, \emph{2}, 449--457. \url{https://doi.org/10.1016/j.jasrep.2015.03.009} -\leavevmode\vadjust pre{\hypertarget{ref-lieverseDietAetiology1999}{}}% +\bibitem[\citeproctext]{ref-lieverseDietAetiology1999} Lieverse, A. R. (1999). Diet and the aetiology of dental calculus. \emph{International Journal of Osteoarchaeology}, \emph{9}(4), 219--232. \url{https://doi.org/10.1002/(SICI)1099-1212(199907/08)9:4\%3C219::AID-OA475\%3E3.0.CO;2-V} -\leavevmode\vadjust pre{\hypertarget{ref-lieverseDentalHealth2007}{}}% +\bibitem[\citeproctext]{ref-lieverseDentalHealth2007} Lieverse, A. R., Link, D. W., Bazaliiskiy, V. I., Goriunova, O. I., \& Weber, A. W. (2007). Dental health indicators of -hunter{\textendash}gatherer adaptation and cultural change in -{Siberia}'s {Cis-Baikal}. \emph{American Journal of Physical -Anthropology}, \emph{134}(3), 323--339. -\url{https://doi.org/10.1002/ajpa.20672} +hunter\textendash gatherer adaptation and cultural change in {Siberia}'s +{Cis-Baikal}. \emph{American Journal of Physical Anthropology}, +\emph{134}(3), 323--339. \url{https://doi.org/10.1002/ajpa.20672} -\leavevmode\vadjust pre{\hypertarget{ref-marshDentalPlaque2006}{}}% +\bibitem[\citeproctext]{ref-marshDentalPlaque2006} Marsh, P. D. (2006). Dental plaque as a biofilm and a microbial -community {\textendash} implications for health and disease. \emph{BMC +community \textendash{} implications for health and disease. \emph{BMC Oral Health}, \emph{6}(S1), S14. \url{https://doi.org/10.1186/1472-6831-6-S1-S14} -\leavevmode\vadjust pre{\hypertarget{ref-mickleburghNewInsights2012}{}}% +\bibitem[\citeproctext]{ref-mickleburghNewInsights2012} Mickleburgh, H. L., \& Pagán-Jiménez, J. R. (2012). New insights into the consumption of maize and other food plants in the pre-{Columbian Caribbean} from starch grains trapped in human dental calculus. \emph{Journal of Archaeological Science}, \emph{39}(7), 2468--2478. \url{https://doi.org/10.1016/j.jas.2012.02.020} -\leavevmode\vadjust pre{\hypertarget{ref-middletonOpalPhytoliths1994}{}}% +\bibitem[\citeproctext]{ref-middletonOpalPhytoliths1994} Middleton, W. D., \& Rovner, I. (1994). Extraction of {Opal Phytoliths} from {Herbivore Dental Calculus}. \emph{Journal of Archaeological Science}, \emph{21}(4), 469--473. \url{https://doi.org/10.1006/jasc.1994.1046} -\leavevmode\vadjust pre{\hypertarget{ref-novakDentalHealth2015}{}}% +\bibitem[\citeproctext]{ref-novakDentalHealth2015} Novak, M. (2015). Dental health and diet in early medieval {Ireland}. \emph{Archives of Oral Biology}, \emph{60}(9), 1299--1309. \url{https://doi.org/10.1016/j.archoralbio.2015.06.004} -\leavevmode\vadjust pre{\hypertarget{ref-whoOralHealth}{}}% +\bibitem[\citeproctext]{ref-whoOralHealth} Oral health. (n.d.). In \emph{World Health Organization}. https://www.who.int/news-room/fact-sheets/detail/oral-health. -\leavevmode\vadjust pre{\hypertarget{ref-ortnerIdentificationPathological2003}{}}% +\bibitem[\citeproctext]{ref-ortnerIdentificationPathological2003} Ortner, D. J. (2003). \emph{Identification of {Pathological Conditions} in {Human Skeletal Remains}}. {Academic Press}. -\leavevmode\vadjust pre{\hypertarget{ref-palmerComparingUse2021}{}}% +\bibitem[\citeproctext]{ref-palmerComparingUse2021} Palmer, K. S., Makarewicz, C. A., Tishkin, A. A., Tur, S. S., Chunag, A., Diimajav, E., Jamsranjav, B., \& Buckley, M. (2021). Comparing the {Use} of {Magnetic Beads} with {Ultrafiltration} for {Ancient Dental Calculus Proteomics}. \emph{Journal of Proteome Research}, \emph{20}(3), 1689--1704. \url{https://doi.org/10.1021/acs.jproteome.0c00862} -\leavevmode\vadjust pre{\hypertarget{ref-pilloudOutliningDefinition2019}{}}% +\bibitem[\citeproctext]{ref-pilloudOutliningDefinition2019} Pilloud, M. A., \& Fancher, J. P. (2019). Outlining a {Definition} of {Oral Health} within the {Study} of {Human Skeletal Remains}: {Defining Oral Health}. \emph{Dental Anthropology Journal}, \emph{32}(2), 3--11. \url{https://doi.org/10.26575/daj.v32i2.297} -\leavevmode\vadjust pre{\hypertarget{ref-pipernoStarchGrains2008}{}}% +\bibitem[\citeproctext]{ref-pipernoStarchGrains2008} Piperno, D. R., \& Dillehay, T. D. (2008). Starch grains on human teeth reveal early broad crop diet in northern {Peru}. \emph{Proceedings of the National Academy of Sciences}, \emph{105}(50), 19622--19627. \url{https://doi.org/10.1073/pnas.0808752105} -\leavevmode\vadjust pre{\hypertarget{ref-powerSynchrotronRadiationbased2022}{}}% +\bibitem[\citeproctext]{ref-powerSynchrotronRadiationbased2022} Power, Robert C., Henry, A. G., Moosmann, J., Beckmann, F., Temming, H., Roberts, A., \& Cabec, A. L. (2022). Synchrotron radiation-based phase-contrast microtomography of human dental calculus allows @@ -1559,26 +1579,26 @@ \section*{References cited}\label{references-cited}} samples. \emph{Journal of Medical Imaging}, \emph{9}(3), 031505. \url{https://doi.org/10.1117/1.JMI.9.3.031505} -\leavevmode\vadjust pre{\hypertarget{ref-powerChimpCalculus2015}{}}% +\bibitem[\citeproctext]{ref-powerChimpCalculus2015} Power, R. C., Salazar-Garcia, D. C., Wittig, R. M., Freiberg, M., \& Henry, A. G. (2015). Dental calculus evidence of {Tai Forest Chimpanzee} plant consumption and life history transitions. \emph{Scientific Reports}, \emph{5}, 15161. \url{https://doi.org/10.1038/srep15161} -\leavevmode\vadjust pre{\hypertarget{ref-powerRepresentativenessDental2021}{}}% +\bibitem[\citeproctext]{ref-powerRepresentativenessDental2021} Power, Robert C., Wittig, R. M., Stone, J. R., Kupczik, K., \& Schulz-Kornas, E. (2021). The representativeness of the dental calculus -dietary record: Insights from {Ta{ï}} chimpanzee faecal phytoliths. +dietary record: Insights from {Taï} chimpanzee faecal phytoliths. \emph{Archaeological and Anthropological Sciences}, \emph{13}(6), 104. \url{https://doi.org/10.1007/s12520-021-01342-z} -\leavevmode\vadjust pre{\hypertarget{ref-radiniDirtyTeeth2022}{}}% +\bibitem[\citeproctext]{ref-radiniDirtyTeeth2022} Radini, A., \& Nikita, E. (2022). Beyond dirty teeth: {Integrating} dental calculus studies with osteoarchaeological parameters. \emph{Quaternary International}. \url{https://doi.org/10.1016/j.quaint.2022.03.003} -\leavevmode\vadjust pre{\hypertarget{ref-radiniMedievalWomen2019}{}}% +\bibitem[\citeproctext]{ref-radiniMedievalWomen2019} Radini, A., Tromp, M., Beach, A., Tong, E., Speller, C., McCormick, M., Dudgeon, J. V., Collins, M. J., Rühli, F., Kröger, R., \& Warinner, C. (2019). Medieval women's early involvement in manuscript production @@ -1586,37 +1606,37 @@ \section*{References cited}\label{references-cited}} \emph{Science Advances}, \emph{5}(1), eaau7126. \url{https://doi.org/10.1126/sciadv.aau7126} -\leavevmode\vadjust pre{\hypertarget{ref-robertsDentalDisease2007}{}}% +\bibitem[\citeproctext]{ref-robertsDentalDisease2007} Roberts, C. A., \& Manchester, K. (2007). Dental {Disease}. In \emph{The {Archaeology} of {Disease}} (3rd Edition, pp. 63--83). {Cornell University Press}. -\leavevmode\vadjust pre{\hypertarget{ref-sagneStudiesPeriodontal1977}{}}% +\bibitem[\citeproctext]{ref-sagneStudiesPeriodontal1977} Sagne, S., \& Olsson, G. (1977). Studies of the {Periodontal Status} of a {Medieval Population}. \emph{Dentomaxillofacial Radiology}, \emph{6}(1), 46--52. \url{https://doi.org/10.1259/dmfr.1977.0006} -\leavevmode\vadjust pre{\hypertarget{ref-scottBriefHistory2015}{}}% +\bibitem[\citeproctext]{ref-scottBriefHistory2015} Scott, G. R. (2015). A {Brief History} of {Dental Anthropology}. In J. D. Irish \& G. R. Scott (Eds.), \emph{A {Companion} to {Dental Anthropology}} (pp. 7--17). {John Wiley \& Sons, Ltd}. \url{https://doi.org/10.1002/9781118845486.ch18} -\leavevmode\vadjust pre{\hypertarget{ref-sissonsMultistationPlaque1991}{}}% +\bibitem[\citeproctext]{ref-sissonsMultistationPlaque1991} Sissons, C. H., Cutress, T. W., Hoffman, M. P., \& Wakefield, J. S. J. (1991). A {Multi-station Dental Plaque Microcosm} ({Artificial Mouth}) for the {Study} of {Plaque Growth}, {Metabolism}, {pH}, and {Mineralization}: \emph{Journal of Dental Research}. \url{https://doi.org/10.1177/00220345910700110301} -\leavevmode\vadjust pre{\hypertarget{ref-slausDentalHealth2011}{}}% +\bibitem[\citeproctext]{ref-slausDentalHealth2011} Šlaus, M., Bedić, Ž., Rajić Šikanjić, P., Vodanović, M., \& Domić Kunić, A. (2011). Dental health at the transition from the {Late Antique} to the early {Medieval} period on {Croatia}'s eastern {Adriatic} coast. \emph{International Journal of Osteoarchaeology}, \emph{21}(5), 577--590. \url{https://doi.org/10.1002/oa.1163} -\leavevmode\vadjust pre{\hypertarget{ref-sotoCharacterizationDecontamination2019}{}}% +\bibitem[\citeproctext]{ref-sotoCharacterizationDecontamination2019} Soto, M., Inwood, J., Clarke, S., Crowther, A., Covelli, D., Favreau, J., Itambu, M., Larter, S., Lee, P., Lozano, M., Maley, J., Mwambwiga, A., Patalano, R., Sammynaiken, R., Vergès, J. M., Zhu, J., \& Mercader, @@ -1625,51 +1645,51 @@ \section*{References cited}\label{references-cited}} Anthropological Sciences}, \emph{11}(9), 4847--4872. \url{https://doi.org/10.1007/s12520-019-00830-7} -\leavevmode\vadjust pre{\hypertarget{ref-squierOralMucosa1998}{}}% +\bibitem[\citeproctext]{ref-squierOralMucosa1998} Squier, C. A., \& Finkelstein, M. W. (1998). Oral {Mucosa}. In A. R. Ten Cate (Ed.), \emph{Oral {Histology}: {Development}, {Structure}, and -{Function}} (5th ed., pp. 345--385). {Mosby}. +{Function}} (Fifth, pp. 345--385). {Mosby}. -\leavevmode\vadjust pre{\hypertarget{ref-storeyPaleopathologyOrigins1986}{}}% +\bibitem[\citeproctext]{ref-storeyPaleopathologyOrigins1986} Storey, R. (1986). Paleopathology at the {Origins} of {Agriculture}. {Mark Nathan Cohen} and {George J}. {Armelagos}, editors. {Academic Press}, {Inc}., {Orlando}, 1984. Xx + 615 pp., figures, tables, references, index. \$59.00 (cloth). \emph{American Antiquity}, \emph{51}(3), 661--662. \url{https://doi.org/10.2307/281762} -\leavevmode\vadjust pre{\hypertarget{ref-tanCalculusUltrastructure2004}{}}% +\bibitem[\citeproctext]{ref-tanCalculusUltrastructure2004} Tan, B. T. K., Gillam, D. G., Mordan, N. J., \& Galgut, P. N. (2004). A preliminary investigation into the ultrastructure of dental calculus and associated bacteria. \emph{Journal of Clinical Periodontology}, \emph{31}(5), 364--369. \url{https://doi.org/10.1111/j.1600-051X.2004.00484.x} -\leavevmode\vadjust pre{\hypertarget{ref-tanBacterialViability2004}{}}% +\bibitem[\citeproctext]{ref-tanBacterialViability2004} Tan, B. T. K., Mordan, N. J., Embleton, J., Pratten, J., \& Galgut, P. N. (2004). Study of {Bacterial Viability} within {Human Supragingival Dental Calculus}. \emph{Journal of Periodontology}, \emph{75}(1), 23--29. \url{https://doi.org/10.1902/jop.2004.75.1.23} -\leavevmode\vadjust pre{\hypertarget{ref-townsendDentalAnthropology2012}{}}% +\bibitem[\citeproctext]{ref-townsendDentalAnthropology2012} Townsend, G., Kanazawa, E., \& Takayama, H. (Eds.). (2012). \emph{New {Directions} in {Dental Anthropology}: {Paradigms}, {Methodologies} and {Outcomes}}. {The University of Adelaide Press}. \url{https://doi.org/10.1017/9780987171870} -\leavevmode\vadjust pre{\hypertarget{ref-trompEDTACalculus2017}{}}% +\bibitem[\citeproctext]{ref-trompEDTACalculus2017} Tromp, M., Buckley, H., Geber, J., \& Matisoo-Smith, E. (2017). {EDTA} decalcification of dental calculus as an alternate means of microparticle extraction from archaeological samples. \emph{Journal of Archaeological Science: Reports}, \emph{14}, 461--466. \url{https://doi.org/10.1016/j.jasrep.2017.06.035} -\leavevmode\vadjust pre{\hypertarget{ref-vandermeerschMiddlePaleolithic1994}{}}% +\bibitem[\citeproctext]{ref-vandermeerschMiddlePaleolithic1994} Vandermeersch, B., Arensburg, B., Tillier, A. M., Rak, Y., Weiner, S., Spiers, M., \& Aspillaga, E. (1994). Middle {Paleolithic Dental Bacteria From Kebara}, {Israel}. \emph{Comptes Rendus De L Academie Des Sciences Serie Ii}, \emph{319}(6), 727--731. -\leavevmode\vadjust pre{\hypertarget{ref-velskoMicrobialDifferences2019}{}}% +\bibitem[\citeproctext]{ref-velskoMicrobialDifferences2019} Velsko, I. M., Fellows Yates, J. A., Aron, F., Hagan, R. W., Frantz, L. A. F., Loe, L., Martinez, J. B. R., Chaves, E., Gosden, C., Larson, G., \& Warinner, C. (2019). Microbial differences between dental plaque and @@ -1677,7 +1697,7 @@ \section*{References cited}\label{references-cited}} \emph{Microbiome}, \emph{7}(1), 102. \url{https://doi.org/10.1186/s40168-019-0717-3} -\leavevmode\vadjust pre{\hypertarget{ref-velskoDentalCalculus2017}{}}% +\bibitem[\citeproctext]{ref-velskoDentalCalculus2017} Velsko, I. M., Overmyer, K. A., Speller, C., Klaus, L., Collins, M. J., Loe, L., Frantz, L. A. F., Sankaranarayanan, K., Lewis, C. M., Martinez, J. B. R., Chaves, E., Coon, J. J., Larson, G., \& Warinner, C. (2017). @@ -1685,11 +1705,11 @@ \section*{References cited}\label{references-cited}} \emph{Metabolomics}, \emph{13}(11), 134. \url{https://doi.org/10.1007/s11306-017-1270-3} -\leavevmode\vadjust pre{\hypertarget{ref-waldronPalaeopathology2020}{}}% +\bibitem[\citeproctext]{ref-waldronPalaeopathology2020} Waldron, T. (2020). \emph{Palaeopathology}. {Cambridge University Press}. -\leavevmode\vadjust pre{\hypertarget{ref-warinnerEvidenceMilk2014}{}}% +\bibitem[\citeproctext]{ref-warinnerEvidenceMilk2014} Warinner, C., Hendy, J., Speller, C., Cappellini, E., Fischer, R., Trachsel, C., Arneborg, J., Lynnerup, N., Craig, O. E., Swallow, D. M., Fotakis, A., Christensen, R. J., Olsen, J. V., Liebert, A., Montalva, @@ -1698,7 +1718,7 @@ \section*{References cited}\label{references-cited}} human dental calculus. \emph{Scientific Reports}, \emph{4}, 7104. \url{https://doi.org/10.1038/srep07104} -\leavevmode\vadjust pre{\hypertarget{ref-warinnerPathogensHost2014}{}}% +\bibitem[\citeproctext]{ref-warinnerPathogensHost2014} Warinner, C., Rodrigues, J. F., Vyas, R., Trachsel, C., Shved, N., Grossmann, J., Radini, A., Hancock, Y., Tito, R. Y., Fiddyment, S., Speller, C., Hendy, J., Charlton, S., Luder, H. U., Salazar-Garcia, D. @@ -1707,50 +1727,50 @@ \section*{References cited}\label{references-cited}} oral cavity. \emph{Nature Genetics}, \emph{46}(4), 336--344. \url{https://doi.org/10.1038/ng.2906} -\leavevmode\vadjust pre{\hypertarget{ref-warinnerNewEra2015}{}}% +\bibitem[\citeproctext]{ref-warinnerNewEra2015} Warinner, C., Speller, C., \& Collins, M. J. (2015). A new era in palaeomicrobiology: Prospects for ancient dental calculus as a long-term record of the human oral microbiome. \emph{Philosophical Transactions of the Royal Society B: Biological Sciences}, \emph{370}(1660), 20130376. \url{https://doi.org/10.1098/rstb.2013.0376} -\leavevmode\vadjust pre{\hypertarget{ref-whiteDentalCalculus1997}{}}% +\bibitem[\citeproctext]{ref-whiteDentalCalculus1997} White, D. J. (1997). Dental calculus: Recent insights into occurrence, formation, prevention, removal and oral health effects of supragingival and subgingival deposits. \emph{European Journal of Oral Sciences}, \emph{105}(5), 508--522. \url{https://doi.org/10.1111/j.1600-0722.1997.tb00238.x} -\leavevmode\vadjust pre{\hypertarget{ref-whiteHumanOsteology2011}{}}% +\bibitem[\citeproctext]{ref-whiteHumanOsteology2011} White, T. D., Black, M. T., \& Folkens, P. A. (2011). \emph{Human {Osteology}} (3rd edition). {Academic Press}. -\leavevmode\vadjust pre{\hypertarget{ref-whiteBoneManual2005}{}}% +\bibitem[\citeproctext]{ref-whiteBoneManual2005} White, T. D., \& Folkens, P. A. (2005). \emph{The {Human Bone Manual}} (1st edition). {Academic Press}. -\leavevmode\vadjust pre{\hypertarget{ref-wongCalciumPhosphate2002}{}}% +\bibitem[\citeproctext]{ref-wongCalciumPhosphate2002} Wong, L., Sissons, C. H., Pearce, E. I. F., \& Cutress, T. W. (2002). Calcium phosphate deposition in human dental plaque microcosm biofilms induced by a ureolytic {pH-rise} procedure. \emph{Archives of Oral Biology}, \emph{47}(11), 779--790. \url{https://doi.org/10.1016/S0003-9969(02)00114-0} -\leavevmode\vadjust pre{\hypertarget{ref-wrightAdvancingRefining2021}{}}% +\bibitem[\citeproctext]{ref-wrightAdvancingRefining2021} Wright, S. L., Dobney, K., \& Weyrich, L. S. (2021). Advancing and refining archaeological dental calculus research using multiomic frameworks. \emph{STAR: Science \& Technology of Archaeological Research}, \emph{7}(1), 13--30. \url{https://doi.org/10.1080/20548923.2021.1882122} -\leavevmode\vadjust pre{\hypertarget{ref-yaussyCalculusSurvivorship2019}{}}% +\bibitem[\citeproctext]{ref-yaussyCalculusSurvivorship2019} Yaussy, S. L., \& DeWitte, S. N. (2019). Calculus and survivorship in medieval {London}: {The} association between dental disease and a demographic measure of general health. \emph{American Journal of Physical Anthropology}, \emph{168}(3), 552--565. \url{https://doi.org/10.1002/ajpa.23772} -\leavevmode\vadjust pre{\hypertarget{ref-zhangDentalDisease1982}{}}% +\bibitem[\citeproctext]{ref-zhangDentalDisease1982} Zhang, Y. (1982). Dental disease of neolithic age skulls excavated in shaanxi province. \emph{Chinese Medical Journal}, \emph{95}(06), 391--396. \url{https://doi.org/10.5555/cmj.0366-6999.95.06.p391.01} @@ -1759,8 +1779,7 @@ \section*{References cited}\label{references-cited}} \bookmarksetup{startatroot} -\hypertarget{chap-background}{% -\chapter{Background}\label{chap-background}} +\chapter{Background}\label{chap-background} The human mouth, or oral cavity, contains many different types of surfaces on which bacteria can attach and grow. These surfaces are both @@ -1790,14 +1809,11 @@ \chapter{Background}\label{chap-background}} reconstructing past diets. In any case, we all have mouths, so on some level I'm sure this knowledge will be relevant. -\hypertarget{background-biofilms}{% -\section{Oral biofilms}\label{background-biofilms}} +\section{Oral biofilms}\label{background-biofilms} The concept of biofilms represents a recent paradigm shift in -microbiology -(\protect\hyperlink{ref-costertonBacterialBiofilms1987}{Costerton et -al., 1987}, -\protect\hyperlink{ref-costertonMicrobialBiofilms1995}{1995}). +microbiology (\citeproc{ref-costertonBacterialBiofilms1987}{Costerton et +al., 1987}, \citeproc{ref-costertonMicrobialBiofilms1995}{1995}). Previously, researchers believed that you could isolate the organism of interest and learn about its growth, metabolism, etc. They assumed bacteria would behave the same as a free-floating organism in a lab test @@ -1811,27 +1827,24 @@ \section{Oral biofilms}\label{background-biofilms}} free-floating (planktonic) organisms. It equips them with resistance to both antimicrobials (such as antibiotic medication) and immune responses from the host that would normally be detrimental to their ability to -survive (\protect\hyperlink{ref-marshDentalPlaque2005}{Marsh, 2005}; -\protect\hyperlink{ref-marshPhysiologicalApproaches1997}{Marsh \& -Bradshaw, 1997}). Resistance to varying conditions is especially -important in the oral cavity, which is a site of frequent fluctuations -in temperature, pH, and oxygen availability. The viscoelastic nature of -the biofilm provides some protection against mechanical destruction and -dislodgement caused by, for example, the tongue and dental hygiene -practices -(\protect\hyperlink{ref-petersonViscoelasticityBiofilms2015}{Peterson et -al., 2015}). It also allows them to acquire nutrients from outside the +survive (\citeproc{ref-marshDentalPlaque2005}{Marsh, 2005}; +\citeproc{ref-marshPhysiologicalApproaches1997}{Marsh \& Bradshaw, +1997}). Resistance to varying conditions is especially important in the +oral cavity, which is a site of frequent fluctuations in temperature, +pH, and oxygen availability. The viscoelastic nature of the biofilm +provides some protection against mechanical destruction and dislodgement +caused by, for example, the tongue and dental hygiene practices +(\citeproc{ref-petersonViscoelasticityBiofilms2015}{Peterson et al., +2015}). It also allows them to acquire nutrients from outside the biofilm, as well as generate and distribute nutrients within the biofilm to the various communities of bacteria residing inside -(\protect\hyperlink{ref-flemmingBiofilmsEmergent2016}{Flemming et al., -2016}). Biofilms are quite persistent structures, and very few surfaces -exist that can completely prevent bacterial colonisation and biofilm -formation -(\protect\hyperlink{ref-rennerPhysicochemicalRegulation2011}{Renner \& -Weibel, 2011}). +(\citeproc{ref-flemmingBiofilmsEmergent2016}{Flemming et al., 2016}). +Biofilms are quite persistent structures, and very few surfaces exist +that can completely prevent bacterial colonisation and biofilm formation +(\citeproc{ref-rennerPhysicochemicalRegulation2011}{Renner \& Weibel, +2011}). -\hypertarget{dental-plaque}{% -\subsection{Dental plaque}\label{dental-plaque}} +\subsection{Dental plaque}\label{dental-plaque} Dental calculus forms from a specific oral biofilm known as dental plaque. After we clean our teeth, our saliva coats the surface of our @@ -1839,27 +1852,29 @@ \subsection{Dental plaque}\label{dental-plaque}} acquired enamel pellicle). The pellicle is a film that protects our teeth from both mechanical wear and chemical decay, but in doing so, provides a viable surface for microorganisms to attach and initiate -biofilm growth (\protect\hyperlink{ref-yaoIdentificationProtein2003}{Yao -et al., 2003}). Biofilm formation goes through several, often -arbitrarily defined, stages of growth. They are arbitrary because they -are defined by the researchers who study them, but are also necessary as -a foundation to explain the development of a biofilm. Rather than -thinking about the stages as occurring sequentially, you should think of -them as occurring concurrently across different areas of the tooth -surface. Biofilm formation is a very dynamic process, and is often +biofilm growth (\citeproc{ref-yaoIdentificationProtein2003}{Yao et al., +2003}). Biofilm formation goes through several, often arbitrarily +defined, stages of growth. They are arbitrary because they are defined +by the researchers who study them, but are also necessary as a +foundation to explain the development of a biofilm. Rather than thinking +about the stages as occurring sequentially, you should think of them as +occurring concurrently across different areas of the tooth surface. +Biofilm formation is a very dynamic process, and is often over-simplified in visualisations (not unlike Figure~\ref{fig-biofilm-form}). \begin{figure} -{\centering \includegraphics{./figures/biofilm_formation.png} +\centering{ + +\includegraphics{./figures/biofilm_formation.png} } \caption{\label{fig-biofilm-form}A simplified overview of biofilm formation stages. Created with BioRender.com.} -\end{figure} +\end{figure}% The pellicle contains molecules (known as adhesins) that enable specific bacteria to attach to complementary receptors on the pellicle, in a @@ -1867,54 +1882,55 @@ \subsection{Dental plaque}\label{dental-plaque}} difference being that it simply attaches to the surface of the tooth rather than being sucked into the tooth. When the pellicle adheres to the tooth, it becomes a surface for bacterial attachment -(\protect\hyperlink{ref-yaoIdentificationProtein2003}{Yao et al., -2003}). The first bacteria to attach are known as early coloniser -bacteria (or pioneer colonisers) and include \emph{Streptococcus} -species (spp.), \emph{Actinomyces} spp., and \emph{Haemophilus} spp. -(\protect\hyperlink{ref-uzelMicrobialShifts2011}{Uzel et al., 2011}; -\protect\hyperlink{ref-zijngeBiofilmArchitecture2010}{Zijnge et al., -2010}). The initial attachment occurs when the random movement of -bacteria and the flow of saliva brings them close enough to the pellicle -to attach. Some bacteria have a limited, often random, ability to move -if they have long tail-like structures known as flagella, but most are -brought to the surface by saliva. +(\citeproc{ref-yaoIdentificationProtein2003}{Yao et al., 2003}). The +first bacteria to attach are known as early coloniser bacteria (or +pioneer colonisers) and include \emph{Streptococcus} species (spp.), +\emph{Actinomyces} spp., and \emph{Haemophilus} spp. +(\citeproc{ref-uzelMicrobialShifts2011}{Uzel et al., 2011}; +\citeproc{ref-zijngeBiofilmArchitecture2010}{Zijnge et al., 2010}). The +initial attachment occurs when the random movement of bacteria and the +flow of saliva brings them close enough to the pellicle to attach. Some +bacteria have a limited, often random, ability to move if they have long +tail-like structures known as flagella, but most are brought to the +surface by saliva. As bacteria approach the pellicle-coated surface of a tooth, there are both attractive and repulsive forces at work. Repulsion because both the bacteria and pellicle proteins have a net negative charge -(\protect\hyperlink{ref-songEffectsMaterial2015}{Song et al., 2015}), -causing electrostatic repulsive force; and attraction from van der Waals -forces. Bacteria may be more or less likely to attach depending on the -distance from the bacteria to the surface. If the bacteria come too -close to the surface, the initial attraction (primary maximum) will most -likely be overcome by repulsion (primary maximum). Bacteria are more -likely to attach when they encounter attractive forces at a further -distance (secondary minimum), ultimately leading to a game of +(\citeproc{ref-songEffectsMaterial2015}{Song et al., 2015}), causing +electrostatic repulsive force; and attraction from van der Waals forces. +Bacteria may be more or less likely to attach depending on the distance +from the bacteria to the surface. If the bacteria come too close to the +surface, the initial attraction (primary maximum) will most likely be +overcome by repulsion (primary maximum). Bacteria are more likely to +attach when they encounter attractive forces at a further distance +(secondary minimum), ultimately leading to a game of `will-they-won't-they' between the bacteria and pellicle. This initial attachment is a weak physicochemical long-distance (10--20 nm; it's a long distance for bacteria) attraction; therefore, attachment is initially reversible, as bacteria can become detached by salivary flow or shearing action by the tongue -(\protect\hyperlink{ref-marshDentalPlaque2016}{Marsh et al., 2016}). -This model of bacterial attachment, also known as the DLVO theory, can -partially explain the aspects involved in microbial adhesion. Further -explanation includes hydrodynamic forces, where hydrophobic components -of the pellicle and cell surface interact -(\protect\hyperlink{ref-bosPhysicochemistryInitial1999}{Bos, 1999}; -\protect\hyperlink{ref-vigeantReversibleIrreversible2002}{Vigeant et -al., 2002}). Overcoming the repulsive forces may be in part facilitated -by motility in some organisms. The aforementioned flagellum, for -example, may give the necessary `push' to reach a region of net -attractive forces -(\protect\hyperlink{ref-jinSupragingivalCalculus2002}{Jin \& Yip, -2002}). Additionally, the ionic strength of saliva may play a role in -reducing electrostatic repulsion with increasing ionic strength -(\protect\hyperlink{ref-rennerPhysicochemicalRegulation2011}{Renner \& -Weibel, 2011}). +(\citeproc{ref-marshDentalPlaque2016}{Marsh et al., 2016}). This model +of bacterial attachment, also known as the DLVO theory, can partially +explain the aspects involved in microbial adhesion. Further explanation +includes hydrodynamic forces, where hydrophobic components of the +pellicle and cell surface interact +(\citeproc{ref-bosPhysicochemistryInitial1999}{Bos, 1999}; +\citeproc{ref-vigeantReversibleIrreversible2002}{Vigeant et al., 2002}). +Overcoming the repulsive forces may be in part facilitated by motility +in some organisms. The aforementioned flagellum, for example, may give +the necessary `push' to reach a region of net attractive forces +(\citeproc{ref-jinSupragingivalCalculus2002}{Jin \& Yip, 2002}). +Additionally, the ionic strength of saliva may play a role in reducing +electrostatic repulsion with increasing ionic strength +(\citeproc{ref-rennerPhysicochemicalRegulation2011}{Renner \& Weibel, +2011}). \begin{figure} -{\centering \includegraphics{figures/bacterial-structure.png} +\centering{ + +\includegraphics{figures/bacterial-structure.png} } @@ -1923,7 +1939,7 @@ \subsection{Dental plaque}\label{dental-plaque}} features of gram-positive bacteria on the right. Created with BioRender.com.} -\end{figure} +\end{figure}% Attachment becomes stronger and colonisation becomes more solidified at a shorter distance, as surface molecules on the bacteria interact with @@ -1933,20 +1949,18 @@ \subsection{Dental plaque}\label{dental-plaque}} components on the dental pellicle (adhesin-receptor interactions). These attachments are very specific because only certain bacteria have the right molecules on their surface -(\protect\hyperlink{ref-jinSupragingivalCalculus2002}{Jin \& Yip, -2002}). These receptors are often carbohydrates formed by the host, -meaning us. Early colonisers are also able to attach to proteins and -enzymes present in saliva, as well as onto the surface of other bacteria -already attached to the pellicle -(\protect\hyperlink{ref-jinSupragingivalCalculus2002}{Jin \& Yip, 2002}; -\protect\hyperlink{ref-nikitkovaStarchBiofilms2013}{Nikitkova et al., +(\citeproc{ref-jinSupragingivalCalculus2002}{Jin \& Yip, 2002}). These +receptors are often carbohydrates formed by the host, meaning us. Early +colonisers are also able to attach to proteins and enzymes present in +saliva, as well as onto the surface of other bacteria already attached +to the pellicle (\citeproc{ref-jinSupragingivalCalculus2002}{Jin \& Yip, +2002}; \citeproc{ref-nikitkovaStarchBiofilms2013}{Nikitkova et al., 2013}). When bacteria come within a shorter distance of the pellicle they may also attach directly to the surface with other hair-like structures (fimbriae) that are present on the surface of some bacteria. These hair-like structures attach to matching receptors that are present -in the pellicle -(\protect\hyperlink{ref-nobbsStreptococcusAdherence2009}{Nobbs et al., -2009}). +in the pellicle (\citeproc{ref-nobbsStreptococcusAdherence2009}{Nobbs et +al., 2009}). While some bacteria specialise in attaching to surfaces, not all of them possess this ability. However, once the specialists have attached, they @@ -1954,26 +1968,24 @@ \subsection{Dental plaque}\label{dental-plaque}} allowing them to attach to their surface (coadhesion) rather than directly to the pellicle. For example, \emph{Streptococcus gordonii} can attach to the pellicle and facilitate coadhesion with \emph{Actinomyces -naeslundii} -(\protect\hyperlink{ref-palmerCoaggregationInteractions2003}{Palmer et -al., 2003}). Not all attachments involve proteins. They can also involve -carbohydrates, enzymes, and various appendages on the surface of the -bacteria, although these appendages often consist of proteins in their -structure, for example the already mentioned pili and fimbriae -(\protect\hyperlink{ref-nobbsStreptococcusAdherence2009}{Nobbs et al., -2009}). This can occur on a large scale, causing the number and types of +naeslundii} (\citeproc{ref-palmerCoaggregationInteractions2003}{Palmer +et al., 2003}). Not all attachments involve proteins. They can also +involve carbohydrates, enzymes, and various appendages on the surface of +the bacteria, although these appendages often consist of proteins in +their structure, for example the already mentioned pili and fimbriae +(\citeproc{ref-nobbsStreptococcusAdherence2009}{Nobbs et al., 2009}). +This can occur on a large scale, causing the number and types of bacteria on the tooth surface to grow, due to the ability of different species to attach to one another (coaggregation) -(\protect\hyperlink{ref-jinSupragingivalCalculus2002}{Jin \& Yip, 2002}; -\protect\hyperlink{ref-marshDentalPlaque2006}{Marsh, 2006}). -Coaggregation and coadhesion are important parts of the growing oral -biofilm. Most taxa don't have the necessary morphology to attach -directly to a substrate, however most oral taxa CAN coaggregate with -other species through cell-cell interactions, usually involving -polysaccharides on the bacterial-cell surfaces -(\protect\hyperlink{ref-kolenbranderOralMultispecies2010}{Kolenbrander -et al., 2010}; -\protect\hyperlink{ref-palmerInterbacterialAdhesion2017}{Palmer et al., +(\citeproc{ref-jinSupragingivalCalculus2002}{Jin \& Yip, 2002}; +\citeproc{ref-marshDentalPlaque2006}{Marsh, 2006}). Coaggregation and +coadhesion are important parts of the growing oral biofilm. Most taxa +don't have the necessary morphology to attach directly to a substrate, +however most oral taxa CAN coaggregate with other species through +cell-cell interactions, usually involving polysaccharides on the +bacterial-cell surfaces +(\citeproc{ref-kolenbranderOralMultispecies2010}{Kolenbrander et al., +2010}; \citeproc{ref-palmerInterbacterialAdhesion2017}{Palmer et al., 2017}). As the biofilm formed by early colonisers grows through continued @@ -1981,48 +1993,47 @@ \subsection{Dental plaque}\label{dental-plaque}} biofilm increases. The proportion of early-colonising streptococci gradually decreases while there is an increase of \emph{Tannerella forsythia}, \emph{Actinomyces} spp., and \emph{Fusobacterium nucleatum} -(\protect\hyperlink{ref-zijngeBiofilmArchitecture2010}{Zijnge et al., -2010}). \emph{F. nucleatum} is a bacterium also known as the `bridging -species', as it's believed to play an important part in linking together -early and late coloniser species---including \emph{Prevotella} spp., -\emph{S. gordonii}, and \emph{Porphyromonas gingivalis}--- which might -not otherwise be able to coaggregate -(\protect\hyperlink{ref-kolenbranderOralMultispecies2010}{Kolenbrander -et al., 2010}; -\protect\hyperlink{ref-kolenbranderAdhereToday1993}{Kolenbrander \& +(\citeproc{ref-zijngeBiofilmArchitecture2010}{Zijnge et al., 2010}). +\emph{F. nucleatum} is a bacterium also known as the `bridging species', +as it's believed to play an important part in linking together early and +late coloniser species---including \emph{Prevotella} spp., \emph{S. +gordonii}, and \emph{Porphyromonas gingivalis}--- which might not +otherwise be able to coaggregate +(\citeproc{ref-kolenbranderOralMultispecies2010}{Kolenbrander et al., +2010}; \citeproc{ref-kolenbranderAdhereToday1993}{Kolenbrander \& London, 1993}). The increasing diversity of bacteria adhering to a surface results in communities of bacteria with the ability to communicate with each other, distribute nutrients, and alter the local environment for more favourable conditions. This is made possible by the presence of an extracellular matrix, formed by the production of polymers by certain bacterial species -(\protect\hyperlink{ref-marshMicrobiologyDental2010}{Marsh, 2010}). +(\citeproc{ref-marshMicrobiologyDental2010}{Marsh, 2010}). Microenvironmental changes can allow species to survive in otherwise unfavourable environments; for example, the survival of many obligate anaerobes in an environment which is largely aerobic (oxygen continuously enters the oral cavity as we breathe). Bacteria with the ability to consume oxygen and produce carbon dioxide allow bacteria with a lower oxygen tolerance to thrive -(\protect\hyperlink{ref-marshDentalPlaque2005}{Marsh, 2005}). In fact, -dental plaque predominantly consists of obligate and facultative -anaerobes and is especially true for periodontitis-associated biofilms, -which tend to be dominated by more species with a lower oxygen tolerance -than their non-periodontitis counterparts -(\protect\hyperlink{ref-curtisRoleMicrobiota2020}{Curtis et al., 2020}). -A pH balance may be maintained by species that are able to consume -acidic metabolic products produced by other species, and convert them to -weaker acids. \emph{Veillonella} spp. especially -(\protect\hyperlink{ref-marshDentalPlaque2005}{Marsh, 2005}). Metabolic -products of some bacteria are used by others as nutrients. By-products -of urea metabolism can be used by some organisms, who further break down -the by-products, which can be used by yet other organisms -(\protect\hyperlink{ref-flemmingBiofilmsEmergent2016}{Flemming et al., -2016}). Working as a community can increase survivability in the harsh -and dynamic environment of the oral cavity, with rapid changes in pH, +(\citeproc{ref-marshDentalPlaque2005}{Marsh, 2005}). In fact, dental +plaque predominantly consists of obligate and facultative anaerobes and +is especially true for periodontitis-associated biofilms, which tend to +be dominated by more species with a lower oxygen tolerance than their +non-periodontitis counterparts +(\citeproc{ref-curtisRoleMicrobiota2020}{Curtis et al., 2020}). A pH +balance may be maintained by species that are able to consume acidic +metabolic products produced by other species, and convert them to weaker +acids. \emph{Veillonella} spp. especially +(\citeproc{ref-marshDentalPlaque2005}{Marsh, 2005}). Metabolic products +of some bacteria are used by others as nutrients. By-products of urea +metabolism can be used by some organisms, who further break down the +by-products, which can be used by yet other organisms +(\citeproc{ref-flemmingBiofilmsEmergent2016}{Flemming et al., 2016}). +Working as a community can increase survivability in the harsh and +dynamic environment of the oral cavity, with rapid changes in pH, oxygen, nutrient availability, etc; though, extended fluctuations in environmental conditions can alter the composition of biofilms -(\protect\hyperlink{ref-huangFactorsAssociated2012}{Huang et al., 2012}, -\protect\hyperlink{ref-huangEffectArginine2017}{2017}). +(\citeproc{ref-huangFactorsAssociated2012}{Huang et al., 2012}, +\citeproc{ref-huangEffectArginine2017}{2017}). Perhaps ironically, an important part of the maturation of a biofilm is the removal of bacteria from the biofilm itself. Removal can occur @@ -2036,21 +2047,18 @@ \subsection{Dental plaque}\label{dental-plaque}} active decision to `peace out'. Dispersion of bacteria from a biofilm requires production of matrix-degrading enzymes, and, as such, not all bacteria can actively disperse from a biofilm -(\protect\hyperlink{ref-petrovaEscapingBiofilm2016}{Petrova \& Sauer, -2016}). The detached bacteria then colonise other parts of the biofilm, -making the biofilm a highly dynamic structure undergoing continuous -remodelling -(\protect\hyperlink{ref-flemmingBiofilmsEmergent2016}{Flemming et al., -2016}). +(\citeproc{ref-petrovaEscapingBiofilm2016}{Petrova \& Sauer, 2016}). The +detached bacteria then colonise other parts of the biofilm, making the +biofilm a highly dynamic structure undergoing continuous remodelling +(\citeproc{ref-flemmingBiofilmsEmergent2016}{Flemming et al., 2016}). So far, the picture of biofilm formation is one of peaceful coexsistence, collaboration, and even neighbourly interspecies actions. A basis for this cooperation is increased overall benefits to the -communities -(\protect\hyperlink{ref-renduelesMechanismsCompetition2015}{Rendueles \& -Ghigo, 2015}). However, competition between bacteria still exists within -the biofilm. The metabolic by-products produced by some bacteria may be -toxic for others, allowing the producers to gain a competitive +communities (\citeproc{ref-renduelesMechanismsCompetition2015}{Rendueles +\& Ghigo, 2015}). However, competition between bacteria still exists +within the biofilm. The metabolic by-products produced by some bacteria +may be toxic for others, allowing the producers to gain a competitive advantage. The aforementioned acid-production by some bacteria can cause unfavourable conditions for species that prefer more neutral pH environments, particularly in the absence of the secondary feeders that @@ -2058,33 +2066,31 @@ \subsection{Dental plaque}\label{dental-plaque}} bacterial competition is the ability of bacteria to produce substances that are toxic to other bacteria. These are often proteins or peptides termed bacteriocins, and can either inhibit or even kill other bacteria -(\protect\hyperlink{ref-dawBacteriocinsNature1996}{Daw \& Falkiner, -1996}; \protect\hyperlink{ref-grahamEnterococcusFaecalis2017}{Graham et -al., 2017}). \emph{S. sanguinis} and \emph{S. gordonii} can produce +(\citeproc{ref-dawBacteriocinsNature1996}{Daw \& Falkiner, 1996}; +\citeproc{ref-grahamEnterococcusFaecalis2017}{Graham et al., 2017}). +\emph{S. sanguinis} and \emph{S. gordonii} can produce H\textsubscript{2}O\textsubscript{2} that is toxic to \emph{S. mutans}, a member of their own genus. \emph{S. mutans} can, in turn, produce mutacin, which inhibits the growth of \emph{S. sorbrinus}. There is no love lost among these close relatives -(\protect\hyperlink{ref-chenSpecificGenes1999}{Chen et al., 1999}). In -addition to H\textsubscript{2}O\textsubscript{2}, oral streptococci can -produce lactate by consuming carbohydrates, giving them a competitive -advantage over acid-sensitive species by altering the local environment. -Some species are resistant to specific metabolic by-products that others +(\citeproc{ref-chenSpecificGenes1999}{Chen et al., 1999}). In addition +to H\textsubscript{2}O\textsubscript{2}, oral streptococci can produce +lactate by consuming carbohydrates, giving them a competitive advantage +over acid-sensitive species by altering the local environment. Some +species are resistant to specific metabolic by-products that others consider toxic, and may even consider them a delicacy (so to speak). \emph{Veillonella} spp. are an example of organisms that thrive under these conditions, allowing both streptococci and \emph{Veillonella} spp. to accumulate in the biofilm and create a favourable environment to -select species -(\protect\hyperlink{ref-edlundUncoveringComplex2018}{Edlund et al., -2018}). These are simplistic examples, and often competition involves -more interactions between multiple species taking on various roles of -`sensing', `mediating', and `killing' -(\protect\hyperlink{ref-renduelesMechanismsCompetition2015}{Rendueles \& -Ghigo, 2015}). Competition between and within species will ultimately -shape the wider biofilm communities. - -\hypertarget{dental-calculus}{% -\subsection{Dental calculus}\label{dental-calculus}} +select species (\citeproc{ref-edlundUncoveringComplex2018}{Edlund et +al., 2018}). These are simplistic examples, and often competition +involves more interactions between multiple species taking on various +roles of `sensing', `mediating', and `killing' +(\citeproc{ref-renduelesMechanismsCompetition2015}{Rendueles \& Ghigo, +2015}). Competition between and within species will ultimately shape the +wider biofilm communities. + +\subsection{Dental calculus}\label{dental-calculus} The exact mechanism of dental calculus formation is not fully understood, but involves processes of biomineralisation and crystal @@ -2104,14 +2110,14 @@ \subsection{Dental calculus}\label{dental-calculus}} spontaneous (or homogenous) nucleation, as it's unclear whether mineral concentrations are sufficient to cause spontaneous nucleation, or whether other biochemical processes act as a catalyst -(\protect\hyperlink{ref-omelonReviewPhosphate2013}{Omelon et al., -2013}). That it's a chemical process can be shown by the ability to -produce calculus deposits in germ-free rats -(\protect\hyperlink{ref-glasBiophysicalStudies1962}{Glas \& Krasse, -1962}; \protect\hyperlink{ref-theiladeGermfreeCalculus1964}{Theilade et -al., 1964}). However, it's unclear how the germ-free calculus compares -to conventional calculus, and, to my knowledge there have only been -studies on rats. Just because calculus growth can be induced in sterile +(\citeproc{ref-omelonReviewPhosphate2013}{Omelon et al., 2013}). That +it's a chemical process can be shown by the ability to produce calculus +deposits in germ-free rats +(\citeproc{ref-glasBiophysicalStudies1962}{Glas \& Krasse, 1962}; +\citeproc{ref-theiladeGermfreeCalculus1964}{Theilade et al., 1964}). +However, it's unclear how the germ-free calculus compares to +conventional calculus, and, to my knowledge there have only been studies +on rats. Just because calculus growth can be induced in sterile conditions doesn't mean bacteria are not an essential part of the process. Bacteria are inevitably part of the scaffolding of dental calculus in humans, since, as I mentioned in the beginning of this @@ -2119,15 +2125,14 @@ \subsection{Dental calculus}\label{dental-calculus}} essentially built by bacteria. Mineralisation does seem to start in the biofilm matrix between microorganisms, but they are eventually also mineralised along with the biofilm matrix -(\protect\hyperlink{ref-friskoppUltrastructureNondecalcified1983}{Friskopp, +(\citeproc{ref-friskoppUltrastructureNondecalcified1983}{Friskopp, 1983}). There are pockets of living bacteria within dental calculus. These pockets and the layer of plaque that covers the surface of dental calculus are likely what cause the correlation between calculus presence -and periodontal disease -(\protect\hyperlink{ref-tanBacterialViability2004}{B. T. K. Tan et al., -2004}). While the process can be explained by chemistry, the conditions -leading up to and surrounding the process are both chemical and -biological in nature, and certainly involve bacteria. +and periodontal disease (\citeproc{ref-tanBacterialViability2004}{B. T. +K. Tan et al., 2004}). While the process can be explained by chemistry, +the conditions leading up to and surrounding the process are both +chemical and biological in nature, and certainly involve bacteria. The main source of minerals in the oral cavity is saliva, which enters the mouth through salivary glands. The three main paired glands are the @@ -2135,58 +2140,54 @@ \subsection{Dental calculus}\label{dental-calculus}} under the tongue, and under the lower jaw bone, respectively. Saliva contains sodium (Na), potassium (K), calcium (Ca), chlorine (Cl), bicarbonate (buffer), and inorganic phosphate (Pi) -(\protect\hyperlink{ref-dawesEffectsDiet1970}{Dawes, 1970}; -\protect\hyperlink{ref-doddsHealthBenefits2005}{Dodds et al., 2005}), -and the locations of the glands contribute to the pattern of dental -calculus deposits within the mouth, which commonly grow on the buccal -portion of maxillary (upper) molars and the lingual portion of -mandibular (lower) incisors -(\protect\hyperlink{ref-jinSupragingivalCalculus2002}{Jin \& Yip, 2002}; -\protect\hyperlink{ref-whiteDentalCalculus1997}{White, 1997}). Salivary -pH also affects saturation of salts, which in turn is influenced by -salivary flow rates. Increased flow rate of saliva will increase -salivary pH, which reduces dissolution and increases precipitation of -calcium and phosphate. This is an important mechanism that protects our -teeth against demineralisation of the enamel caused by caries. -Protection is provided by the exchange of calcium and phosphate from -saliva to enamel -(\protect\hyperlink{ref-dahlenMicrobiologicalStudy2010}{Dahlén et al., -2010}). Saliva further acts as a buffer for the oral cavity, reducing -the impact of short-term drops in pH caused by metabolic byproducts of -acid-producing bacteria -(\protect\hyperlink{ref-doddsHealthBenefits2005}{Dodds et al., 2005}; -\protect\hyperlink{ref-jinSupragingivalCalculus2002}{Jin \& Yip, 2002}). -Higher rates of salivary flow are also likely to contribute to an -increase in calcium and phosphate secretion in addition to pH, all +(\citeproc{ref-dawesEffectsDiet1970}{Dawes, 1970}; +\citeproc{ref-doddsHealthBenefits2005}{Dodds et al., 2005}), and the +locations of the glands contribute to the pattern of dental calculus +deposits within the mouth, which commonly grow on the buccal portion of +maxillary (upper) molars and the lingual portion of mandibular (lower) +incisors (\citeproc{ref-jinSupragingivalCalculus2002}{Jin \& Yip, 2002}; +\citeproc{ref-whiteDentalCalculus1997}{White, 1997}). Salivary pH also +affects saturation of salts, which in turn is influenced by salivary +flow rates. Increased flow rate of saliva will increase salivary pH, +which reduces dissolution and increases precipitation of calcium and +phosphate. This is an important mechanism that protects our teeth +against demineralisation of the enamel caused by caries. Protection is +provided by the exchange of calcium and phosphate from saliva to enamel +(\citeproc{ref-dahlenMicrobiologicalStudy2010}{Dahlén et al., 2010}). +Saliva further acts as a buffer for the oral cavity, reducing the impact +of short-term drops in pH caused by metabolic byproducts of +acid-producing bacteria (\citeproc{ref-doddsHealthBenefits2005}{Dodds et +al., 2005}; \citeproc{ref-jinSupragingivalCalculus2002}{Jin \& Yip, +2002}). Higher rates of salivary flow are also likely to contribute to +an increase in calcium and phosphate secretion in addition to pH, all contributing to an environment favouring plaque mineralisation. Metabolic byproducts produced by bacteria can also affect local pH, both pushing towards alkaline conditions as well as acidic. A major cause of acidic pH is metabolism of overabundant dietary sugars and starch, especially the metabolic activity of \emph{Streptococcus mutans}, known to be one of the main culprits behind dental caries -(\protect\hyperlink{ref-bowenOralBiofilms2018}{Bowen et al., 2018}; -\protect\hyperlink{ref-duarteInfluencesStarch2008}{Duarte et al., 2008}; -\protect\hyperlink{ref-extercateAAA2010}{Exterkate et al., 2010}). +(\citeproc{ref-bowenOralBiofilms2018}{Bowen et al., 2018}; +\citeproc{ref-duarteInfluencesStarch2008}{Duarte et al., 2008}; +\citeproc{ref-extercateAAA2010}{Exterkate et al., 2010}). Conversely, alkaline conditions can be generated by metabolism of various products that can either be directly or indirectly linked to diet. One such product is urea. Urea is present in saliva, and its concentration depends on multiple factors. One of these factors is a high-protein diet, which increases levels of urea in serum and saliva -(\protect\hyperlink{ref-lieverseDietAetiology1999}{Lieverse, 1999}). -Hydrolysis of urea produces ammonia and causes a rise in pH. Bacteria -possess the ability to produce ammonia from urea, which is further used -by ammonia-oxidising organisms and converted to nitrite -(\protect\hyperlink{ref-flemmingBiofilmsEmergent2016}{Flemming et al., -2016}; \protect\hyperlink{ref-sissonsPHResponse1994}{Sissons et al., -1994}; \protect\hyperlink{ref-wongCalciumPhosphate2002}{Wong et al., -2002}). In a similar way, arginine can be broken down to ammonia and -increase in pH. Another pathway to alkalinity is through enzymatic -activity. Saliva contains proteases which specialise in breaking down -proteins into smaller components such as ammonia, and increased protease -activity in saliva may therefore cause an increase in calculus -production (\protect\hyperlink{ref-jinSupragingivalCalculus2002}{Jin \& -Yip, 2002}). +(\citeproc{ref-lieverseDietAetiology1999}{Lieverse, 1999}). Hydrolysis +of urea produces ammonia and causes a rise in pH. Bacteria possess the +ability to produce ammonia from urea, which is further used by +ammonia-oxidising organisms and converted to nitrite +(\citeproc{ref-flemmingBiofilmsEmergent2016}{Flemming et al., 2016}; +\citeproc{ref-sissonsPHResponse1994}{Sissons et al., 1994}; +\citeproc{ref-wongCalciumPhosphate2002}{Wong et al., 2002}). In a +similar way, arginine can be broken down to ammonia and increase in pH. +Another pathway to alkalinity is through enzymatic activity. Saliva +contains proteases which specialise in breaking down proteins into +smaller components such as ammonia, and increased protease activity in +saliva may therefore cause an increase in calculus production +(\citeproc{ref-jinSupragingivalCalculus2002}{Jin \& Yip, 2002}). There are also a number of inhibitors and promoters of mineralisation present in the oral cavity, originating both from saliva and bacteria. @@ -2196,34 +2197,31 @@ \subsection{Dental calculus}\label{dental-calculus}} (formerly \emph{Bacterionema matruchotii}) accumulates calcium within its cell structure, and has therefore received a lot of attention in biomineralisation studies Ennever \& Creamer -(\protect\hyperlink{ref-enneverMicrobiologicCalcification1967}{1967}). +(\citeproc{ref-enneverMicrobiologicCalcification1967}{1967}). Biomineralisation is not a feature unique to \emph{Corynebacterium matruchotii}. Even species associated with caries may induce calcification under the right conditions and after cell death -(\protect\hyperlink{ref-moorerCalcificationCariogenic1993}{Moorer et -al., 1993}; -\protect\hyperlink{ref-sidawayMicrobiologicalStudy1978a}{Sidaway, -1978}). Inhibitors of biomineralisation include salivary proline-rich +(\citeproc{ref-moorerCalcificationCariogenic1993}{Moorer et al., 1993}; +\citeproc{ref-sidawayMicrobiologicalStudy1978a}{Sidaway, 1978}). +Inhibitors of biomineralisation include salivary proline-rich polypeptides, small amino acids important for the immune system; and statherin, a protein that controls the precipitation of calcium -phosphate in saliva -(\protect\hyperlink{ref-jinSupragingivalCalculus2002}{Jin \& Yip, -2002}). +phosphate in saliva (\citeproc{ref-jinSupragingivalCalculus2002}{Jin \& +Yip, 2002}). It's likely that multiple biomineralisation events occur under various conditions, resulting in a heterogeneous calculus composition with crystals of various stages of growth -(\protect\hyperlink{ref-friskoppUltrastructureNondecalcified1983}{Friskopp, -1983}; \protect\hyperlink{ref-friskoppComparativeScanning1980}{Friskopp -\& Hammarström, 1980}). The differing susceptibility of bacteria to +(\citeproc{ref-friskoppUltrastructureNondecalcified1983}{Friskopp, +1983}; \citeproc{ref-friskoppComparativeScanning1980}{Friskopp \& +Hammarström, 1980}). The differing susceptibility of bacteria to calcification is also a contributor to the heterogeneous composition. Overall, plaque mineralisation is a complex interaction between conditions in the local environment, availability of minerals, the equilibrium between precipitation and dissolution, balance between nucleation promoters and inhibitors. -\hypertarget{background-biofilm-models}{% -\section{Oral biofilm models}\label{background-biofilm-models}} +\section{Oral biofilm models}\label{background-biofilm-models} Biofilm models are a way of studying the growth and development of biofilms. By creating models that replicate the conditions and @@ -2246,45 +2244,45 @@ \section{Oral biofilm models}\label{background-biofilm-models}} and 98-well plates) with a substratum, usually glass cover-slips or hydroxyapatite discs, placed at the bottom of the well. Similar models suspend the substrata from a lid to promote active attachment of -bacteria to the substrata -(\protect\hyperlink{ref-extercateAAA2010}{Exterkate et al., 2010}). When -the substrata are attached to a lid instead of the multiwell plates, it -allows samples to be periodically transferred between solutions/media if -necessary, adding more flexibility to the experimental setup. +bacteria to the substrata (\citeproc{ref-extercateAAA2010}{Exterkate et +al., 2010}). When the substrata are attached to a lid instead of the +multiwell plates, it allows samples to be periodically transferred +between solutions/media if necessary, adding more flexibility to the +experimental setup. Next, an inoculate is chosen. This can be anything from a single species of bacterium (pure culture), to multiple select species (defined consortium), to all organisms occurring naturally within a system -(microcosm) (\protect\hyperlink{ref-mcbainBiofilmModels2009}{McBain, -2009}). The purpose of the inoculate is to initiate biofilm formation by -allowing the bacteria to adsorb to the substrata, ideally in the -presence of a conditioning film, such as saliva. For pure cultures and -defined consortia, the inoculate may come from saliva or another oral -site, such as dental plaque. The bacteria of interest are then isolated -using selective media, essentially providing ideal growing conditions to +(microcosm) (\citeproc{ref-mcbainBiofilmModels2009}{McBain, 2009}). The +purpose of the inoculate is to initiate biofilm formation by allowing +the bacteria to adsorb to the substrata, ideally in the presence of a +conditioning film, such as saliva. For pure cultures and defined +consortia, the inoculate may come from saliva or another oral site, such +as dental plaque. The bacteria of interest are then isolated using +selective media, essentially providing ideal growing conditions to certain types of bacteria, promoting their growth and eliminating others -(e.g. \protect\hyperlink{ref-bassonEstablishmentCommunity1996}{Basson \& -van Wyk, 1996}). Alternatively, the bacteria can be acquired directly -from companies like the American Type Culture Collection (ATCC). For +(e.g. \citeproc{ref-bassonEstablishmentCommunity1996}{Basson \& van Wyk, +1996}). Alternatively, the bacteria can be acquired directly from +companies like the American Type Culture Collection (ATCC). For microcosms, the inoculate is often the saliva itself, or dental plaque, in its (mostly) raw form. The inoculate is added to the wells to initiate biofilm formation on the substrata as described -\protect\hyperlink{dental-plaque}{above}. As such, the content of the -inoculate influences the complexity of the biofilm microbiome as well as -the interactions between the communities within the biofilm -(\protect\hyperlink{ref-roderStudyingBacterial2016}{Røder et al., -2016}). It's not always possible to use donated saliva as a growth -medium for the duration of the experiment, especially if the experiment -lasts more than a few days. Media with salivary components can be -created as a substitute for long lasting experiments. There are many -different recipes for media floating around out there, but most of them -are generally a mixture containing mucin, proteins, minerals commonly -found in saliva, and a buffer to maintain pH -(\protect\hyperlink{ref-extercateAAA2010}{Exterkate et al., 2010}; -\protect\hyperlink{ref-prattenVitroStudies1998}{Pratten et al., 1998}; -\protect\hyperlink{ref-shellisSyntheticSaliva1978}{Shellis, 1978}; -\protect\hyperlink{ref-sissonsMultistationPlaque1991}{Sissons et al., -1991}; \protect\hyperlink{ref-tianUsingDGGE2010}{Tian et al., 2010}). +\hyperref[dental-plaque]{above}. As such, the content of the inoculate +influences the complexity of the biofilm microbiome as well as the +interactions between the communities within the biofilm +(\citeproc{ref-roderStudyingBacterial2016}{Røder et al., 2016}). It's +not always possible to use donated saliva as a growth medium for the +duration of the experiment, especially if the experiment lasts more than +a few days. Media with salivary components can be created as a +substitute for long lasting experiments. There are many different +recipes for media floating around out there, but most of them are +generally a mixture containing mucin, proteins, minerals commonly found +in saliva, and a buffer to maintain pH +(\citeproc{ref-extercateAAA2010}{Exterkate et al., 2010}; +\citeproc{ref-prattenVitroStudies1998}{Pratten et al., 1998}; +\citeproc{ref-shellisSyntheticSaliva1978}{Shellis, 1978}; +\citeproc{ref-sissonsMultistationPlaque1991}{Sissons et al., 1991}; +\citeproc{ref-tianUsingDGGE2010}{Tian et al., 2010}). More complicated models make use of increasingly sophisticated equipment to mimic the oral environment. Another level of model complexity can be @@ -2294,55 +2292,52 @@ \section{Oral biofilm models}\label{background-biofilm-models}} providing a finite amount of nutrients in a closed system. An example of a batch culture model is a biofilm grown on an agar plate, which has a finite amount of resources -(\protect\hyperlink{ref-kearnsMasterRegulator2005}{Kearns et al., -2005}). Once the nutrients in the agar have been depleted, that's it. At -the other end of the spectrum is a system with a pump attached to a -reservoir that can continuously supply the biofilm with growth medium, -similar to salivary flow. In between the former options is the -semi-continuous supply of nutrients. This can, for example, be the -multiwell plate model with a lid, where the samples can be periodically -transferred to new plates containing fresh growth medium -(\protect\hyperlink{ref-extercateAAA2010}{Exterkate et al., 2010}). -Other parameters that can be controlled to more closely simulate -conditions in the oral cavity are pH and gas phase, as can be done with -the multistation artificial mouth. This system gives researchers control +(\citeproc{ref-kearnsMasterRegulator2005}{Kearns et al., 2005}). Once +the nutrients in the agar have been depleted, that's it. At the other +end of the spectrum is a system with a pump attached to a reservoir that +can continuously supply the biofilm with growth medium, similar to +salivary flow. In between the former options is the semi-continuous +supply of nutrients. This can, for example, be the multiwell plate model +with a lid, where the samples can be periodically transferred to new +plates containing fresh growth medium +(\citeproc{ref-extercateAAA2010}{Exterkate et al., 2010}). Other +parameters that can be controlled to more closely simulate conditions in +the oral cavity are pH and gas phase, as can be done with the +multistation artificial mouth. This system gives researchers control over a large number of parameters using multiple chambers with complete control over the flow of treatment and/or nutrient conditions---environmental conditions such as pH, temperature, and gas phase---and access to real-time measurements -(\protect\hyperlink{ref-sissonsArtificialPlaque1997}{Sissons, 1997}). +(\citeproc{ref-sissonsArtificialPlaque1997}{Sissons, 1997}). The duration of an experiment depends on the scope of the study. If the purpose is to learn more about initial biofilm formation and prevention, it may only be necessary to grow the biofilms for a few hours to 48 -hours (\protect\hyperlink{ref-dibdinDiffusionSugars1981}{Dibdin, 1981}; -\protect\hyperlink{ref-extercateAAA2010}{Exterkate et al., 2010}). If, -instead, the goal is to learn more about biofilm maturation and -calcification, the experiments can run for days or even weeks -(\protect\hyperlink{ref-filocheFluorescenceAssay2007}{Filoche et al., -2007}; \protect\hyperlink{ref-sissonsMultistationPlaque1991}{Sissons et -al., 1991}; \protect\hyperlink{ref-wongCalciumPhosphate2002}{Wong et -al., 2002}). +hours (\citeproc{ref-dibdinDiffusionSugars1981}{Dibdin, 1981}; +\citeproc{ref-extercateAAA2010}{Exterkate et al., 2010}). If, instead, +the goal is to learn more about biofilm maturation and calcification, +the experiments can run for days or even weeks +(\citeproc{ref-filocheFluorescenceAssay2007}{Filoche et al., 2007}; +\citeproc{ref-sissonsMultistationPlaque1991}{Sissons et al., 1991}; +\citeproc{ref-wongCalciumPhosphate2002}{Wong et al., 2002}). Models developed for studying oral biofilms include, in increasing complexity, the ACTA active attachment model -(\protect\hyperlink{ref-extercateAAA2010}{Exterkate et al., 2010}), -Calgary biofilm device -(\protect\hyperlink{ref-ceriCalgaryBiofilm1999}{Ceri et al., 1999}), -modified Robbins device -(\protect\hyperlink{ref-honraetModifiedRobbins2006}{Honraet \& Nelis, -2006}), constant depth film-fermenter -(\protect\hyperlink{ref-petersConstantDepth1988}{Peters \& Wimpenny, -1988}), and the multistation artificial mouth -(\protect\hyperlink{ref-sissonsMultistationPlaque1991}{Sissons et al., -1991}) representing the upper echelon of complexity. Summaries of -biofilm models, including benefits and limitations of the various types, -can be found in reviews by McBain -McBain -(\protect\hyperlink{ref-mcbainBiofilmModels2009}{2009}), Tan and -colleagues -C. H. Tan et al. -(\protect\hyperlink{ref-tanAllTogether2017}{2017}), and Røder and +(\citeproc{ref-extercateAAA2010}{Exterkate et al., 2010}), Calgary +biofilm device (\citeproc{ref-ceriCalgaryBiofilm1999}{Ceri et al., +1999}), modified Robbins device +(\citeproc{ref-honraetModifiedRobbins2006}{Honraet \& Nelis, 2006}), +constant depth film-fermenter +(\citeproc{ref-petersConstantDepth1988}{Peters \& Wimpenny, 1988}), and +the multistation artificial mouth +(\citeproc{ref-sissonsMultistationPlaque1991}{Sissons et al., 1991}) +representing the upper echelon of complexity. Summaries of biofilm +models, including benefits and limitations of the various types, can be +found in reviews by McBain -McBain +(\citeproc{ref-mcbainBiofilmModels2009}{2009}), Tan and colleagues -C. +H. Tan et al. (\citeproc{ref-tanAllTogether2017}{2017}), and Røder and colleagues -Røder et al. -(\protect\hyperlink{ref-roderStudyingBacterial2016}{2016}). +(\citeproc{ref-roderStudyingBacterial2016}{2016}). It might be tempting to think that the goal should always be to mimic the oral environment as closely as possible. However, there are benefits @@ -2352,326 +2347,323 @@ \section{Oral biofilm models}\label{background-biofilm-models}} physiological and factors and making it easier to take various measurements. Microcosms have the benefit of more closely mimicking the complexity of the organisms' natural environment -(\protect\hyperlink{ref-mcbainBiofilmModels2009}{McBain, 2009}). -However, even microcosms can be limited in their ability to recreate the -complexity and diversity of the oral microbiome -(\protect\hyperlink{ref-tianUsingDGGE2010}{Tian et al., 2010}). -Alternatives to \emph{in vitro} models are \emph{in situ} models which -usually involve growing plaque on a removable surface inside the mouth -of a willing participant. These models add a level of realism, as they -are grown inside an actual oral cavity, and can reflect biogeographical +(\citeproc{ref-mcbainBiofilmModels2009}{McBain, 2009}). However, even +microcosms can be limited in their ability to recreate the complexity +and diversity of the oral microbiome +(\citeproc{ref-tianUsingDGGE2010}{Tian et al., 2010}). Alternatives to +\emph{in vitro} models are \emph{in situ} models which usually involve +growing plaque on a removable surface inside the mouth of a willing +participant. These models add a level of realism, as they are grown +inside an actual oral cavity, and can reflect biogeographical differences in biofilm composition caused by differing conditions across the oral cavity. They also come with additional difficulties and reduced control over experimental parameters -(\protect\hyperlink{ref-marshRoleMicrobiology1995}{Marsh, 1995}; -\protect\hyperlink{ref-zeroSituCaries1995}{Zero, 1995}). +(\citeproc{ref-marshRoleMicrobiology1995}{Marsh, 1995}; +\citeproc{ref-zeroSituCaries1995}{Zero, 1995}). -Reiterating a point made in the -\protect\hyperlink{chap-intro}{Introduction}, and -\protect\hyperlink{chap-discussion}{Discussion}, and probably somewhere -in the articles as well, the benefit of using an oral biofilm model over +Reiterating a point made in the \hyperref[chap-intro]{Introduction}, and +\hyperref[chap-discussion]{Discussion}, and probably somewhere in the +articles as well, the benefit of using an oral biofilm model over naturally occurring dental calculus in the mouth of a research participant, is the control that it provides to tweak every aspect of the system, from the quantity and quality of nutrients available, to the amount of enzymes and bacterial species present. Plus, the added ethical benefit of not needing to ask someone to give up their oral hygiene regime for a few weeks. The following chapters, -\protect\hyperlink{byoc-valid}{Chapter 3} and -\protect\hyperlink{byoc-starch}{Chapter 4}, provide a small glimpse of -what a model looks like, and how it might be used to inform -archaeological research. +\hyperref[byoc-valid]{Chapter 3} and \hyperref[byoc-starch]{Chapter 4}, +provide a small glimpse of what a model looks like, and how it might be +used to inform archaeological research. -\hypertarget{references-cited-1}{% -\section*{References cited}\label{references-cited-1}} +\section*{References cited}\label{references-cited-1} \addcontentsline{toc}{section}{References cited} \markright{References cited} -\hypertarget{refs-2}{} +\phantomsection\label{refs-2} \begin{CSLReferences}{1}{0} -\leavevmode\vadjust pre{\hypertarget{ref-bassonEstablishmentCommunity1996}{}}% +\bibitem[\citeproctext]{ref-bassonEstablishmentCommunity1996} Basson, N. J., \& van Wyk, C. W. (1996). The establishment of a community of oral bacteria that controls the growth of {Candida} albicans in a chemostat. \emph{Oral Microbiology and Immunology}, \emph{11}(3), 199--202. \url{https://doi.org/10.1111/j.1399-302X.1996.tb00358.x} -\leavevmode\vadjust pre{\hypertarget{ref-bosPhysicochemistryInitial1999}{}}% +\bibitem[\citeproctext]{ref-bosPhysicochemistryInitial1999} Bos, R. (1999). Physico-chemistry of initial microbial adhesive -interactions {\textendash} its mechanisms and methods for study. +interactions \textendash{} its mechanisms and methods for study. \emph{FEMS Microbiology Reviews}, \emph{23}(2), 179--229. \url{https://doi.org/10.1016/S0168-6445(99)00004-2} -\leavevmode\vadjust pre{\hypertarget{ref-bowenOralBiofilms2018}{}}% +\bibitem[\citeproctext]{ref-bowenOralBiofilms2018} Bowen, W. H., Burne, R. A., Wu, H., \& Koo, H. (2018). Oral {Biofilms}: {Pathogens}, {Matrix} and {Polymicrobial Interactions} in {Microenvironments}. \emph{Trends in Microbiology}, \emph{26}(3), 229--242. \url{https://doi.org/10.1016/j.tim.2017.09.008} -\leavevmode\vadjust pre{\hypertarget{ref-boyan-salyersRelationshipProteolipids1980}{}}% +\bibitem[\citeproctext]{ref-boyan-salyersRelationshipProteolipids1980} Boyan-Salyers, B. D., \& Boskey, A. L. (1980). Relationship between proteolipids and calcium-phospholipid-phosphate complexes {inBacterionema} matruchotii calcification. \emph{Calcified Tissue International}, \emph{30}(1), 167--174. \url{https://doi.org/10.1007/BF02408622} -\leavevmode\vadjust pre{\hypertarget{ref-ceriCalgaryBiofilm1999}{}}% +\bibitem[\citeproctext]{ref-ceriCalgaryBiofilm1999} Ceri, H., Olson, M. E., Stremick, C., Read, R. R., Morck, D., \& Buret, A. (1999). The {Calgary Biofilm Device}: {New Technology} for {Rapid Determination} of {Antibiotic Susceptibilities} of {Bacterial Biofilms}. \emph{Journal of Clinical Microbiology}, \emph{37}(6), 1771--1776. \url{https://doi.org/10.1128/JCM.37.6.1771-1776.1999} -\leavevmode\vadjust pre{\hypertarget{ref-chenSpecificGenes1999}{}}% +\bibitem[\citeproctext]{ref-chenSpecificGenes1999} Chen, P., Qi, F., Novak, J., \& Caufield, P. W. (1999). \href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC91190}{The {Specific Genes} for {Lantibiotic Mutacin II Biosynthesis} in {Streptococcus} mutans {T8 Are Clustered} and {Can Be} {Transferred En Bloc}}. \emph{Applied and Environmental Microbiology}, \emph{65}(3), 1356--1360. -\leavevmode\vadjust pre{\hypertarget{ref-costertonBacterialBiofilms1987}{}}% +\bibitem[\citeproctext]{ref-costertonBacterialBiofilms1987} Costerton, J. W., Cheng, K. J., Geesey, G. G., Ladd, T. I., Nickel, J. C., Dasgupta, M., \& Marrie, T. J. (1987). Bacterial {Biofilms} in {Nature} and {Disease}. \emph{Annual Review of Microbiology}, \emph{41}(1), 435--464. \url{https://doi.org/10.1146/annurev.mi.41.100187.002251} -\leavevmode\vadjust pre{\hypertarget{ref-costertonMicrobialBiofilms1995}{}}% +\bibitem[\citeproctext]{ref-costertonMicrobialBiofilms1995} Costerton, J. W., Lewandowski, Z., Caldwell, D. E., Korber, D. R., \& Lappin-Scott, H. M. (1995). Microbial {Biofilms}. \emph{Annual Review of Microbiology}, \emph{49}(1), 711--745. \url{https://doi.org/10.1146/annurev.mi.49.100195.003431} -\leavevmode\vadjust pre{\hypertarget{ref-curtisRoleMicrobiota2020}{}}% +\bibitem[\citeproctext]{ref-curtisRoleMicrobiota2020} Curtis, M. A., Diaz, P. I., \& Dyke, T. E. V. (2020). The role of the microbiota in periodontal disease. \emph{Periodontology 2000}, \emph{83}(1), 14--25. \url{https://doi.org/10.1111/prd.12296} -\leavevmode\vadjust pre{\hypertarget{ref-dahlenMicrobiologicalStudy2010}{}}% +\bibitem[\citeproctext]{ref-dahlenMicrobiologicalStudy2010} Dahlén, G., Konradsson, K., Eriksson, S., Teanpaisan, R., Piwat, S., \& Carlén, A. (2010). A microbiological study in relation to the presence of caries and calculus. \emph{Acta Odontologica Scandinavica}, \emph{68}(4), 199--206. \url{https://doi.org/10.3109/00016351003745514} -\leavevmode\vadjust pre{\hypertarget{ref-dawBacteriocinsNature1996}{}}% +\bibitem[\citeproctext]{ref-dawBacteriocinsNature1996} Daw, M. A., \& Falkiner, F. R. (1996). Bacteriocins: Nature, function and structure. \emph{Micron (Oxford, England: 1993)}, \emph{27}(6), 467--479. \url{https://doi.org/10.1016/s0968-4328(96)00028-5} -\leavevmode\vadjust pre{\hypertarget{ref-dawesEffectsDiet1970}{}}% +\bibitem[\citeproctext]{ref-dawesEffectsDiet1970} Dawes, C. (1970). Effects of {Diet} on {Salivary Secretion} and {Composition}. \emph{Journal of Dental Research}, \emph{49}, 1263--1272. -\leavevmode\vadjust pre{\hypertarget{ref-dibdinDiffusionSugars1981}{}}% +\bibitem[\citeproctext]{ref-dibdinDiffusionSugars1981} Dibdin, G. H. (1981). Diffusion of sugars and carboxylic acids through human dental plaque in vitro. \emph{Archives of Oral Biology}, \emph{26}(6), 515--523. \url{https://doi.org/10.1016/0003-9969(81)90010-8} -\leavevmode\vadjust pre{\hypertarget{ref-doddsHealthBenefits2005}{}}% +\bibitem[\citeproctext]{ref-doddsHealthBenefits2005} Dodds, M. W. J., Johnson, D. A., \& Yeh, C.-K. (2005). Health benefits of saliva: A review. \emph{Journal of Dentistry}, \emph{33}(3), 223--233. \url{https://doi.org/10.1016/j.jdent.2004.10.009} -\leavevmode\vadjust pre{\hypertarget{ref-duarteInfluencesStarch2008}{}}% +\bibitem[\citeproctext]{ref-duarteInfluencesStarch2008} Duarte, S., Klein, M. I., Aires, C. P., Cury, J. A., Bowen, W. H., \& Koo, H. (2008). Influences of starch and sucrose on {Streptococcus} mutans biofilms. \emph{Oral Microbiology and Immunology}, \emph{23}(3), 206--212. \url{https://doi.org/10.1111/j.1399-302X.2007.00412.x} -\leavevmode\vadjust pre{\hypertarget{ref-edlundUncoveringComplex2018}{}}% +\bibitem[\citeproctext]{ref-edlundUncoveringComplex2018} Edlund, A., Yang, Y., Yooseph, S., He, X., Shi, W., \& McLean, J. S. (2018). Uncovering complex microbiome activities via metatranscriptomics during 24 hours of oral biofilm assembly and maturation. \emph{Microbiome}, \emph{6}(1), 217. \url{https://doi.org/10.1186/s40168-018-0591-4} -\leavevmode\vadjust pre{\hypertarget{ref-enneverIntracellularCalcification1960}{}}% +\bibitem[\citeproctext]{ref-enneverIntracellularCalcification1960} Ennever, J. (1960). Intracellular {Calcification} by {Oral Filamentous Microorganisms}. \emph{The Journal of Periodontology}, \emph{31}(4), 304--307. \url{https://doi.org/10.1902/jop.1960.31.4.304} -\leavevmode\vadjust pre{\hypertarget{ref-enneverMicrobiologicCalcification1967}{}}% +\bibitem[\citeproctext]{ref-enneverMicrobiologicCalcification1967} Ennever, J., \& Creamer, H. (1967). Microbiologic calcification: {Bone} mineral and bacteria. \emph{Calcified Tissue Research}, \emph{1}(1), 87--93. \url{https://doi.org/10.1007/BF02008078} -\leavevmode\vadjust pre{\hypertarget{ref-extercateAAA2010}{}}% +\bibitem[\citeproctext]{ref-extercateAAA2010} Exterkate, R. A. M., Crielaard, W., \& Ten Cate, J. M. (2010). Different {Response} to {Amine Fluoride} by {Streptococcus} mutans and {Polymicrobial Biofilms} in a {Novel High-Throughput Active Attachment Model}. \emph{Caries Research}, \emph{44}(4), 372--379. \url{https://doi.org/10.1159/000316541} -\leavevmode\vadjust pre{\hypertarget{ref-filocheFluorescenceAssay2007}{}}% +\bibitem[\citeproctext]{ref-filocheFluorescenceAssay2007} Filoche, S. K., Coleman, M. J., Angker, L., \& Sissons, C. H. (2007). A fluorescence assay to determine the viable biomass of microcosm dental plaque biofilms. \emph{Journal of Microbiological Methods}, \emph{69}(3), 489--496. \url{https://doi.org/10.1016/j.mimet.2007.02.015} -\leavevmode\vadjust pre{\hypertarget{ref-flemmingBiofilmsEmergent2016}{}}% +\bibitem[\citeproctext]{ref-flemmingBiofilmsEmergent2016} Flemming, H.-C., Wingender, J., Szewzyk, U., Steinberg, P., Rice, S. A., \& Kjelleberg, S. (2016). Biofilms: An emergent form of bacterial life. \emph{Nature Reviews Microbiology}, \emph{14}(9), 563--575. \url{https://doi.org/10.1038/nrmicro.2016.94} -\leavevmode\vadjust pre{\hypertarget{ref-friskoppUltrastructureNondecalcified1983}{}}% +\bibitem[\citeproctext]{ref-friskoppUltrastructureNondecalcified1983} Friskopp, J. (1983). Ultrastructure of {Nondecalcified Supragingival} and {Subgingival Calculus}. \emph{Journal of Periodontology}, \emph{54}(9), 542--550. \url{https://doi.org/10.1902/jop.1983.54.9.542} -\leavevmode\vadjust pre{\hypertarget{ref-friskoppComparativeScanning1980}{}}% +\bibitem[\citeproctext]{ref-friskoppComparativeScanning1980} Friskopp, J., \& Hammarström, L. (1980). A {Comparative}, {Scanning Electron Microscopic Study} of {Supragingival} and {Subgingival Calculus}. \emph{Journal of Periodontology}, \emph{51}(10), 553--562. \url{https://doi.org/10.1902/jop.1980.51.10.553} -\leavevmode\vadjust pre{\hypertarget{ref-glasBiophysicalStudies1962}{}}% +\bibitem[\citeproctext]{ref-glasBiophysicalStudies1962} Glas, J.-E., \& Krasse, B. (1962). Biophysical {Studies} on {Dental Calculus} from {Germfree} and {Conventional Rats}. \emph{Acta Odontologica Scandinavica}, \emph{20}(2), 127--134. \url{https://doi.org/10.3109/00016356209026100} -\leavevmode\vadjust pre{\hypertarget{ref-grahamEnterococcusFaecalis2017}{}}% +\bibitem[\citeproctext]{ref-grahamEnterococcusFaecalis2017} Graham, C. E., Cruz, M. R., Garsin, D. A., \& Lorenz, M. C. (2017). Enterococcus faecalis bacteriocin {EntV} inhibits hyphal morphogenesis, biofilm formation, and virulence of {Candida} albicans. \emph{Proceedings of the National Academy of Sciences}, \emph{114}(17), 4507--4512. \url{https://doi.org/10.1073/pnas.1620432114} -\leavevmode\vadjust pre{\hypertarget{ref-honraetModifiedRobbins2006}{}}% +\bibitem[\citeproctext]{ref-honraetModifiedRobbins2006} Honraet, K., \& Nelis, H. J. (2006). Use of the modified robbins device and fluorescent staining to screen plant extracts for the inhibition of {S}. Mutans biofilm formation. \emph{Journal of Microbiological Methods}, \emph{64}(2), 217--224. \url{https://doi.org/10.1016/j.mimet.2005.05.005} -\leavevmode\vadjust pre{\hypertarget{ref-huangFactorsAssociated2012}{}}% +\bibitem[\citeproctext]{ref-huangFactorsAssociated2012} Huang, X., Exterkate, R. A. M., \& ten Cate, J. M. (2012). Factors {Associated} with {Alkali Production} from {Arginine} in {Dental Biofilms}. \emph{Journal of Dental Research}, \emph{91}(12), 1130--1134. \url{https://doi.org/10.1177/0022034512461652} -\leavevmode\vadjust pre{\hypertarget{ref-huangEffectArginine2017}{}}% +\bibitem[\citeproctext]{ref-huangEffectArginine2017} Huang, X., Zhang, K., Deng, M., Exterkate, R. A. M., Liu, C., Zhou, X., Cheng, L., \& ten Cate, J. M. (2017). Effect of arginine on the growth and biofilm formation of oral bacteria. \emph{Archives of Oral Biology}, \emph{82}, 256--262. \url{https://doi.org/10.1016/j.archoralbio.2017.06.026} -\leavevmode\vadjust pre{\hypertarget{ref-jinSupragingivalCalculus2002}{}}% +\bibitem[\citeproctext]{ref-jinSupragingivalCalculus2002} Jin, Y., \& Yip, H.-K. (2002). Supragingival {Calculus}: {Formation} and {Control}. \emph{Critical Reviews in Oral Biology \& Medicine}. \url{https://doi.org/10.1177/154411130201300506} -\leavevmode\vadjust pre{\hypertarget{ref-kearnsMasterRegulator2005}{}}% +\bibitem[\citeproctext]{ref-kearnsMasterRegulator2005} Kearns, D. B., Chu, F., Branda, S. S., Kolter, R., \& Losick, R. (2005). A master regulator for biofilm formation by {Bacillus} subtilis. \emph{Molecular Microbiology}, \emph{55}(3), 739--749. \url{https://doi.org/10.1111/j.1365-2958.2004.04440.x} -\leavevmode\vadjust pre{\hypertarget{ref-kolenbranderAdhereToday1993}{}}% +\bibitem[\citeproctext]{ref-kolenbranderAdhereToday1993} Kolenbrander, P. E., \& London, J. (1993). Adhere today, here tomorrow: Oral bacterial adherence. \emph{Journal of Bacteriology}, \emph{175}(11), 3247--3252. \url{https://doi.org/10.1128/jb.175.11.3247-3252.1993} -\leavevmode\vadjust pre{\hypertarget{ref-kolenbranderOralMultispecies2010}{}}% +\bibitem[\citeproctext]{ref-kolenbranderOralMultispecies2010} Kolenbrander, P. E., Palmer, R. J., Periasamy, S., \& Jakubovics, N. S. (2010). Oral multispecies biofilm development and the key role of -cell{\textendash}cell distance. \emph{Nature Reviews Microbiology}, +cell\textendash cell distance. \emph{Nature Reviews Microbiology}, \emph{8}(7), 471--480. \url{https://doi.org/10.1038/nrmicro2381} -\leavevmode\vadjust pre{\hypertarget{ref-lieverseDietAetiology1999}{}}% +\bibitem[\citeproctext]{ref-lieverseDietAetiology1999} Lieverse, A. R. (1999). Diet and the aetiology of dental calculus. \emph{International Journal of Osteoarchaeology}, \emph{9}(4), 219--232. \url{https://doi.org/10.1002/(SICI)1099-1212(199907/08)9:4\%3C219::AID-OA475\%3E3.0.CO;2-V} -\leavevmode\vadjust pre{\hypertarget{ref-marshRoleMicrobiology1995}{}}% +\bibitem[\citeproctext]{ref-marshRoleMicrobiology1995} Marsh, P. D. (1995). The {Role} of {Microbiology} in {Models} of {Dental Caries}. \emph{Advances in Dental Research}, \emph{9}(3), 244--254. \url{https://doi.org/10.1177/08959374950090030901} -\leavevmode\vadjust pre{\hypertarget{ref-marshDentalPlaque2005}{}}% +\bibitem[\citeproctext]{ref-marshDentalPlaque2005} Marsh, P. D. (2005). Dental plaque: Biological significance of a biofilm and community life-style. \emph{Journal of Clinical Periodontology}, \emph{32}(s6), 7--15. \url{https://doi.org/10.1111/j.1600-051X.2005.00790.x} -\leavevmode\vadjust pre{\hypertarget{ref-marshDentalPlaque2006}{}}% +\bibitem[\citeproctext]{ref-marshDentalPlaque2006} Marsh, P. D. (2006). Dental plaque as a biofilm and a microbial -community {\textendash} implications for health and disease. \emph{BMC +community \textendash{} implications for health and disease. \emph{BMC Oral Health}, \emph{6}(S1), S14. \url{https://doi.org/10.1186/1472-6831-6-S1-S14} -\leavevmode\vadjust pre{\hypertarget{ref-marshMicrobiologyDental2010}{}}% +\bibitem[\citeproctext]{ref-marshMicrobiologyDental2010} Marsh, P. D. (2010). Microbiology of {Dental Plaque Biofilms} and {Their Role} in {Oral Health} and {Caries}. \emph{Dental Clinics of North America}, \emph{54}(3), 441--454. \url{https://doi.org/10.1016/j.cden.2010.03.002} -\leavevmode\vadjust pre{\hypertarget{ref-marshPhysiologicalApproaches1997}{}}% +\bibitem[\citeproctext]{ref-marshPhysiologicalApproaches1997} Marsh, P. D., \& Bradshaw, D. J. (1997). Physiological {Approaches} to the {Control} of {Oral Biofilms}. \emph{Advances in Dental Research}, \emph{11}(1), 176--185. \url{https://doi.org/10.1177/08959374970110010901} -\leavevmode\vadjust pre{\hypertarget{ref-marshDentalPlaque2016}{}}% +\bibitem[\citeproctext]{ref-marshDentalPlaque2016} Marsh, P. D., Lewis, M. A. O., Rogers, H., Williams, D. W., \& Wilson, M. (2016). Dental {Plaque}. In \emph{Marsh and {Martin}'s {Oral Microbiology}} (6th Edition, pp. 81--111). {Elsevier Health Sciences}. -\leavevmode\vadjust pre{\hypertarget{ref-mcbainBiofilmModels2009}{}}% +\bibitem[\citeproctext]{ref-mcbainBiofilmModels2009} McBain, A. J. (2009). In {Vitro Biofilm Models}: {An Overview}. In \emph{Advances in {Applied Microbiology}} (Vol. 69, pp. 99--132). {Academic Press}. \url{https://doi.org/10.1016/S0065-2164(09)69004-3} -\leavevmode\vadjust pre{\hypertarget{ref-moorerCalcificationCariogenic1993}{}}% +\bibitem[\citeproctext]{ref-moorerCalcificationCariogenic1993} Moorer, W. R., Ten Cate, J. M., \& Buijs, J. F. (1993). Calcification of a {Cariogenic Streptococcus} and of {Corynebacterium} ({Bacterionema}) matruchotii. \emph{Journal of Dental Research}, \emph{72}(6), 1021--1026. \url{https://doi.org/10.1177/00220345930720060501} -\leavevmode\vadjust pre{\hypertarget{ref-nikitkovaStarchBiofilms2013}{}}% +\bibitem[\citeproctext]{ref-nikitkovaStarchBiofilms2013} Nikitkova, A. E., Haase, E. M., \& Scannapieco, F. A. (2013). Taking the {Starch} out of {Oral Biofilm Formation}: {Molecular Basis} and {Functional Significance} of {Salivary} {\(\alpha\)}-{Amylase Binding} to {Oral Streptococci}. \emph{Applied and Environmental Microbiology}, \emph{79}(2), 416--423. \url{https://doi.org/10.1128/AEM.02581-12} -\leavevmode\vadjust pre{\hypertarget{ref-nobbsStreptococcusAdherence2009}{}}% +\bibitem[\citeproctext]{ref-nobbsStreptococcusAdherence2009} Nobbs, A. H., Lamont, R. J., \& Jenkinson, H. F. (2009). Streptococcus {Adherence} and {Colonization}. \emph{Microbiology and Molecular Biology Reviews}, \emph{73}(3), 407--450. \url{https://doi.org/10.1128/MMBR.00014-09} -\leavevmode\vadjust pre{\hypertarget{ref-omelonReviewPhosphate2013}{}}% +\bibitem[\citeproctext]{ref-omelonReviewPhosphate2013} Omelon, S., Ariganello, M., Bonucci, E., Grynpas, M., \& Nanci, A. (2013). A {Review} of {Phosphate Mineral Nucleation} in {Biology} and {Geobiology}. \emph{Calcified Tissue International}, \emph{93}(4), 382--396. \url{https://doi.org/10.1007/s00223-013-9784-9} -\leavevmode\vadjust pre{\hypertarget{ref-palmerCoaggregationInteractions2003}{}}% +\bibitem[\citeproctext]{ref-palmerCoaggregationInteractions2003} Palmer, R. J., Gordon, S. M., Cisar, J. O., \& Kolenbrander, P. E. (2003). Coaggregation-{Mediated Interactions} of {Streptococci} and {Actinomyces Detected} in {Initial Human Dental Plaque}. \emph{Journal of Bacteriology}, \emph{185}(11), 3400--3409. \url{https://doi.org/10.1128/JB.185.11.3400-3409.2003} -\leavevmode\vadjust pre{\hypertarget{ref-palmerInterbacterialAdhesion2017}{}}% +\bibitem[\citeproctext]{ref-palmerInterbacterialAdhesion2017} Palmer, R. J., Shah, N., Valm, A., Paster, B., Dewhirst, F., Inui, T., \& Cisar, J. O. (2017). Interbacterial {Adhesion Networks} within {Early Oral Biofilms} of {Single Human Hosts}. \emph{Applied and Environmental Microbiology}, \emph{83}(11), e00407--17. \url{https://doi.org/10.1128/AEM.00407-17} -\leavevmode\vadjust pre{\hypertarget{ref-petersConstantDepth1988}{}}% +\bibitem[\citeproctext]{ref-petersConstantDepth1988} Peters, A., \& Wimpenny, J. W. T. (1988). A {Constant-Depth Laboratory Model Film Fermenter}. In \emph{{CRC Handbook} of {Laboratory Model Systems} for {Microbial Ecosystems}}. {CRC Press}. -\leavevmode\vadjust pre{\hypertarget{ref-petersonViscoelasticityBiofilms2015}{}}% +\bibitem[\citeproctext]{ref-petersonViscoelasticityBiofilms2015} Peterson, B. W., He, Y., Ren, Y., Zerdoum, A., Libera, M. R., Sharma, P. K., van Winkelhoff, A.-J., Neut, D., Stoodley, P., van der Mei, H. C., \& Busscher, H. J. (2015). Viscoelasticity of biofilms and their @@ -2679,102 +2671,102 @@ \section*{References cited}\label{references-cited-1}} Microbiology Reviews}, \emph{39}(2), 234--245. \url{https://doi.org/10.1093/femsre/fuu008} -\leavevmode\vadjust pre{\hypertarget{ref-petrovaEscapingBiofilm2016}{}}% +\bibitem[\citeproctext]{ref-petrovaEscapingBiofilm2016} Petrova, O. E., \& Sauer, K. (2016). Escaping the biofilm in more than one way: Desorption, detachment or dispersion. \emph{Current Opinion in Microbiology}, \emph{30}, 67--78. \url{https://doi.org/10.1016/j.mib.2016.01.004} -\leavevmode\vadjust pre{\hypertarget{ref-prattenVitroStudies1998}{}}% +\bibitem[\citeproctext]{ref-prattenVitroStudies1998} Pratten, Wills, Barnett, \& Wilson. (1998). In vitro studies of the effect of antiseptic-containing mouthwashes on the formation and viability of {Streptococcus} sanguis biofilms. \emph{Journal of Applied Microbiology}, \emph{84}(6), 1149--1155. \url{https://doi.org/10.1046/j.1365-2672.1998.00462.x} -\leavevmode\vadjust pre{\hypertarget{ref-renduelesMechanismsCompetition2015}{}}% +\bibitem[\citeproctext]{ref-renduelesMechanismsCompetition2015} Rendueles, O., \& Ghigo, J.-M. (2015). Mechanisms of {Competition} in {Biofilm Communities}. \emph{Microbiology Spectrum}, \emph{3}(3), 3.3.28. \url{https://doi.org/10.1128/microbiolspec.MB-0009-2014} -\leavevmode\vadjust pre{\hypertarget{ref-rennerPhysicochemicalRegulation2011}{}}% +\bibitem[\citeproctext]{ref-rennerPhysicochemicalRegulation2011} Renner, L. D., \& Weibel, D. B. (2011). Physicochemical regulation of biofilm formation. \emph{MRS Bulletin}, \emph{36}(5), 347--355. \url{https://doi.org/10.1557/mrs.2011.65} -\leavevmode\vadjust pre{\hypertarget{ref-roderStudyingBacterial2016}{}}% +\bibitem[\citeproctext]{ref-roderStudyingBacterial2016} Røder, H. L., Sørensen, S. J., \& Burmølle, M. (2016). Studying {Bacterial Multispecies Biofilms}: {Where} to {Start}? \emph{Trends in Microbiology}, \emph{24}(6), 503--513. \url{https://doi.org/10.1016/j.tim.2016.02.019} -\leavevmode\vadjust pre{\hypertarget{ref-shellisSyntheticSaliva1978}{}}% +\bibitem[\citeproctext]{ref-shellisSyntheticSaliva1978} Shellis, R. P. (1978). A synthetic saliva for cultural studies of dental plaque. \emph{Archives of Oral Biology}, \emph{23}(6), 485--489. \url{https://doi.org/10.1016/0003-9969(78)90081-X} -\leavevmode\vadjust pre{\hypertarget{ref-sidawayMicrobiologicalStudy1978a}{}}% +\bibitem[\citeproctext]{ref-sidawayMicrobiologicalStudy1978a} Sidaway, D. A. (1978). A microbiological study of dental calculus. \emph{Journal of Periodontal Research}, \emph{13}(4), 360--366. \url{https://doi.org/10.1111/j.1600-0765.1978.tb00190.x} -\leavevmode\vadjust pre{\hypertarget{ref-sissonsArtificialPlaque1997}{}}% +\bibitem[\citeproctext]{ref-sissonsArtificialPlaque1997} Sissons, C. H. (1997). Artificial {Dental Plaque Biofilm Model Systems}. \emph{Advances in Dental Research}, \emph{11}(1), 110--126. \url{https://doi.org/10.1177/08959374970110010201} -\leavevmode\vadjust pre{\hypertarget{ref-sissonsMultistationPlaque1991}{}}% +\bibitem[\citeproctext]{ref-sissonsMultistationPlaque1991} Sissons, C. H., Cutress, T. W., Hoffman, M. P., \& Wakefield, J. S. J. (1991). A {Multi-station Dental Plaque Microcosm} ({Artificial Mouth}) for the {Study} of {Plaque Growth}, {Metabolism}, {pH}, and {Mineralization}: \emph{Journal of Dental Research}. \url{https://doi.org/10.1177/00220345910700110301} -\leavevmode\vadjust pre{\hypertarget{ref-sissonsPHResponse1994}{}}% +\bibitem[\citeproctext]{ref-sissonsPHResponse1994} Sissons, C. H., Wong, L., Hancock, E. M., \& Cutress, T. W. (1994). The {pH} response to urea and the effect of liquid flow in {``artificial mouth''} microcosm plaques. \emph{Archives of Oral Biology}, \emph{39}(6), 497--505. \url{https://doi.org/10.1016/0003-9969(94)90146-5} -\leavevmode\vadjust pre{\hypertarget{ref-songEffectsMaterial2015}{}}% +\bibitem[\citeproctext]{ref-songEffectsMaterial2015} Song, F., Koo, H., \& Ren, D. (2015). Effects of {Material Properties} on {Bacterial Adhesion} and {Biofilm Formation}. \emph{Journal of Dental Research}, \emph{94}(8), 1027--1034. \url{https://doi.org/10.1177/0022034515587690} -\leavevmode\vadjust pre{\hypertarget{ref-takazoeCalciumHydroxyapatite1970}{}}% +\bibitem[\citeproctext]{ref-takazoeCalciumHydroxyapatite1970} Takazoe, I., Vogel, J., \& Ennever, J. (1970). Calcium {Hydroxyapatite Nucleation} by {Lipid Extract} of {Bacterionema} matruchotii. \emph{Journal of Dental Research}, \emph{49}(2), 395--398. \url{https://doi.org/10.1177/00220345700490023301} -\leavevmode\vadjust pre{\hypertarget{ref-tanBacterialViability2004}{}}% +\bibitem[\citeproctext]{ref-tanBacterialViability2004} Tan, B. T. K., Mordan, N. J., Embleton, J., Pratten, J., \& Galgut, P. N. (2004). Study of {Bacterial Viability} within {Human Supragingival Dental Calculus}. \emph{Journal of Periodontology}, \emph{75}(1), 23--29. \url{https://doi.org/10.1902/jop.2004.75.1.23} -\leavevmode\vadjust pre{\hypertarget{ref-tanAllTogether2017}{}}% +\bibitem[\citeproctext]{ref-tanAllTogether2017} Tan, C. H., Lee, K. W. K., Burmølle, M., Kjelleberg, S., \& Rice, S. A. (2017). All together now: Experimental multispecies biofilm model systems. \emph{Environmental Microbiology}, \emph{19}(1), 42--53. \url{https://doi.org/10.1111/1462-2920.13594} -\leavevmode\vadjust pre{\hypertarget{ref-theiladeGermfreeCalculus1964}{}}% +\bibitem[\citeproctext]{ref-theiladeGermfreeCalculus1964} Theilade, J., Fitzgerald, R. J., Scott, D. B., \& Nylen, M. U. (1964). Electron microscopic observations of dental calculus in germfree and conventional rats. \emph{Archives of Oral Biology}, \emph{9}(1), 97--IN17. \url{https://doi.org/10.1016/0003-9969(64)90051-2} -\leavevmode\vadjust pre{\hypertarget{ref-tianUsingDGGE2010}{}}% +\bibitem[\citeproctext]{ref-tianUsingDGGE2010} Tian, Y., He, X., Torralba, M., Yooseph, S., Nelson, K. e., Lux, R., McLean, J. s., Yu, G., \& Shi, W. (2010). Using {DGGE} profiling to develop a novel culture medium suitable for oral microbial communities. \emph{Molecular Oral Microbiology}, \emph{25}(5), 357--367. \url{https://doi.org/10.1111/j.2041-1014.2010.00585.x} -\leavevmode\vadjust pre{\hypertarget{ref-uzelMicrobialShifts2011}{}}% +\bibitem[\citeproctext]{ref-uzelMicrobialShifts2011} Uzel, N. G., Teles, F. R., Teles, R. P., Song, X. Q., Torresyap, G., Socransky, S. S., \& Haffajee, A. D. (2011). Microbial shifts during dental biofilm re-development in the absence of oral hygiene in @@ -2782,7 +2774,7 @@ \section*{References cited}\label{references-cited-1}} Periodontology}, \emph{38}(7), 612--620. \url{https://doi.org/10.1111/j.1600-051X.2011.01730.x} -\leavevmode\vadjust pre{\hypertarget{ref-vigeantReversibleIrreversible2002}{}}% +\bibitem[\citeproctext]{ref-vigeantReversibleIrreversible2002} Vigeant, M. A.-S., Ford, R. M., Wagner, M., \& Tamm, L. K. (2002). Reversible and {Irreversible Adhesion} of {Motile Escherichia} coli {Cells Analyzed} by {Total Internal Reflection Aqueous Fluorescence @@ -2790,33 +2782,33 @@ \section*{References cited}\label{references-cited-1}} \emph{68}(6), 2794--2801. \url{https://doi.org/10.1128/AEM.68.6.2794-2801.2002} -\leavevmode\vadjust pre{\hypertarget{ref-whiteDentalCalculus1997}{}}% +\bibitem[\citeproctext]{ref-whiteDentalCalculus1997} White, D. J. (1997). Dental calculus: Recent insights into occurrence, formation, prevention, removal and oral health effects of supragingival and subgingival deposits. \emph{European Journal of Oral Sciences}, \emph{105}(5), 508--522. \url{https://doi.org/10.1111/j.1600-0722.1997.tb00238.x} -\leavevmode\vadjust pre{\hypertarget{ref-wongCalciumPhosphate2002}{}}% +\bibitem[\citeproctext]{ref-wongCalciumPhosphate2002} Wong, L., Sissons, C. H., Pearce, E. I. F., \& Cutress, T. W. (2002). Calcium phosphate deposition in human dental plaque microcosm biofilms induced by a ureolytic {pH-rise} procedure. \emph{Archives of Oral Biology}, \emph{47}(11), 779--790. \url{https://doi.org/10.1016/S0003-9969(02)00114-0} -\leavevmode\vadjust pre{\hypertarget{ref-yaoIdentificationProtein2003}{}}% +\bibitem[\citeproctext]{ref-yaoIdentificationProtein2003} Yao, Y., Berg, E. A., Costello, C. E., Troxler, R. F., \& Oppenheim, F. G. (2003). Identification of protein components in human acquired enamel pellicle and whole saliva using novel proteomics approaches. \emph{J Biol Chem}, \emph{278}(7), 5300--5308. \url{https://doi.org/10.1074/jbc.M206333200} -\leavevmode\vadjust pre{\hypertarget{ref-zeroSituCaries1995}{}}% +\bibitem[\citeproctext]{ref-zeroSituCaries1995} Zero, D. T. (1995). In {Situ Caries Models}. \emph{Advances in Dental Research}, \emph{9}(3), 214--230. \url{https://doi.org/10.1177/08959374950090030501} -\leavevmode\vadjust pre{\hypertarget{ref-zijngeBiofilmArchitecture2010}{}}% +\bibitem[\citeproctext]{ref-zijngeBiofilmArchitecture2010} Zijnge, V., van Leeuwen, M. B. M., Degener, J. E., Abbas, F., Thurnheer, T., Gmür, R., \& M. Harmsen, H. J. (2010). Oral {Biofilm Architecture} on {Natural Teeth}. \emph{PLoS ONE}, \emph{5}(2), e9321. @@ -2826,8 +2818,7 @@ \section*{References cited}\label{references-cited-1}} \bookmarksetup{startatroot} -\hypertarget{byoc-valid}{% -\chapter{Article 1}\label{byoc-valid}} +\chapter{Article 1}\label{byoc-valid} Assessing the validity of a calcifying oral biofilm model as a suitable proxy for dental calculus @@ -2873,129 +2864,120 @@ \chapter{Article 1}\label{byoc-valid}} \newpage{} -\hypertarget{introduction}{% -\section{Introduction}\label{introduction}} +\section{Introduction}\label{introduction} Dental calculus is becoming an increasingly popular substance for exploring health and diet in past populations -(\protect\hyperlink{ref-warinnerNewEra2015}{Warinner et al., 2015}). -During life, dental plaque undergoes periodic mineralisation, trapping -biomolecules and microfossils that are embedded within the dental plaque -biofilm in the newly-formed dental calculus. This process is repeated as -new plaque is deposited and subsequently mineralises, resulting in a -layered structure representing a temporal record of biofilm growth and -development (\protect\hyperlink{ref-warinnerPathogensHost2014}{Warinner -et al., 2014}). The calculus serves as a protective casing for the -entrapped biomolecules and microfossils, preserving them for thousands -of years after death and burial -(\protect\hyperlink{ref-yatesOralMicrobiome2021}{Fellows Yates et al., -2021}). Studies using archaeological dental calculus span a wide range -of topics in different regions and time periods. These include -characterisation of the oral microbiome and its evolution in past -populations (\protect\hyperlink{ref-adlerSequencingAncient2013}{Adler et -al., 2013}; \protect\hyperlink{ref-yatesOralMicrobiome2021}{Fellows -Yates et al., 2021}; -\protect\hyperlink{ref-kazarinaPostmedievalMicrobial2021}{Kazarina et -al., 2021}; -\protect\hyperlink{ref-velskoMicrobialDifferences2019}{Velsko et al., -2019}; \protect\hyperlink{ref-warinnerPathogensHost2014}{Warinner et -al., 2014}), as well as extraction of microbotanical remains -(\protect\hyperlink{ref-hardyStarchGranules2009}{Hardy et al., 2009}; -\protect\hyperlink{ref-henryCalculusSyria2008}{Henry \& Piperno, 2008}; -\protect\hyperlink{ref-maHumanDiet2022}{Ma et al., 2022}; -\protect\hyperlink{ref-mickleburghNewInsights2012}{Mickleburgh \& -Pagán-Jiménez, 2012}) and other residues to infer dietary patterns and -nicotine use -(\protect\hyperlink{ref-bartholdyMultiproxyAnalysis2023}{Bartholdy et -al., 2023}; \protect\hyperlink{ref-buckleyDentalCalculus2014}{Buckley et -al., 2014}; \protect\hyperlink{ref-eerkensDentalCalculus2018}{Eerkens et -al., 2018}; \protect\hyperlink{ref-hendyProteomicCalculus2018}{Hendy et -al., 2018}; \protect\hyperlink{ref-velskoDentalCalculus2017}{Velsko, -Overmyer, et al., 2017}). Dental calculus has already provided a unique -and valuable insight into the past, but the exact mechanism of the -incorporation, retention, and preservation of microfossils and -biomolecules exogenous to the microbial biofilm is largely unknown; even -the process of plaque mineralisation is not fully understood -(\protect\hyperlink{ref-jinSupragingivalCalculus2002}{Jin \& Yip, 2002}; -\protect\hyperlink{ref-omelonReviewPhosphate2013}{Omelon et al., 2013}). -This means that there may be hidden biases affecting our interpretations -of dietary/activity patterns extrapolated from ancient dental calculus. +(\citeproc{ref-warinnerNewEra2015}{Warinner et al., 2015}). During life, +dental plaque undergoes periodic mineralisation, trapping biomolecules +and microfossils that are embedded within the dental plaque biofilm in +the newly-formed dental calculus. This process is repeated as new plaque +is deposited and subsequently mineralises, resulting in a layered +structure representing a temporal record of biofilm growth and +development (\citeproc{ref-warinnerPathogensHost2014}{Warinner et al., +2014}). The calculus serves as a protective casing for the entrapped +biomolecules and microfossils, preserving them for thousands of years +after death and burial (\citeproc{ref-yatesOralMicrobiome2021}{Fellows +Yates et al., 2021}). Studies using archaeological dental calculus span +a wide range of topics in different regions and time periods. These +include characterisation of the oral microbiome and its evolution in +past populations (\citeproc{ref-adlerSequencingAncient2013}{Adler et +al., 2013}; \citeproc{ref-yatesOralMicrobiome2021}{Fellows Yates et al., +2021}; \citeproc{ref-kazarinaPostmedievalMicrobial2021}{Kazarina et al., +2021}; \citeproc{ref-velskoMicrobialDifferences2019}{Velsko et al., +2019}; \citeproc{ref-warinnerPathogensHost2014}{Warinner et al., 2014}), +as well as extraction of microbotanical remains +(\citeproc{ref-hardyStarchGranules2009}{Hardy et al., 2009}; +\citeproc{ref-henryCalculusSyria2008}{Henry \& Piperno, 2008}; +\citeproc{ref-maHumanDiet2022}{Ma et al., 2022}; +\citeproc{ref-mickleburghNewInsights2012}{Mickleburgh \& Pagán-Jiménez, +2012}) and other residues to infer dietary patterns and nicotine use +(\citeproc{ref-bartholdyMultiproxyAnalysis2023}{Bartholdy et al., 2023}; +\citeproc{ref-buckleyDentalCalculus2014}{Buckley et al., 2014}; +\citeproc{ref-eerkensDentalCalculus2018}{Eerkens et al., 2018}; +\citeproc{ref-hendyProteomicCalculus2018}{Hendy et al., 2018}; +\citeproc{ref-velskoDentalCalculus2017}{Velsko, Overmyer, et al., +2017}). Dental calculus has already provided a unique and valuable +insight into the past, but the exact mechanism of the incorporation, +retention, and preservation of microfossils and biomolecules exogenous +to the microbial biofilm is largely unknown; even the process of plaque +mineralisation is not fully understood +(\citeproc{ref-jinSupragingivalCalculus2002}{Jin \& Yip, 2002}; +\citeproc{ref-omelonReviewPhosphate2013}{Omelon et al., 2013}). This +means that there may be hidden biases affecting our interpretations of +dietary/activity patterns extrapolated from ancient dental calculus. These biases have been explored archaeologically -(\protect\hyperlink{ref-fagernasMicrobialBiogeography2022}{Fagernäs et -al., 2022}; \protect\hyperlink{ref-trompEDTACalculus2017}{Tromp et al., -2017}) as well as in contemporary humans -(\protect\hyperlink{ref-leonardPlantMicroremains2015}{Leonard et al., -2015}) and non-human primates -(\protect\hyperlink{ref-powerChimpCalculus2015}{Power et al., 2015}), -but not experimentally. +(\citeproc{ref-fagernasMicrobialBiogeography2022}{Fagernäs et al., +2022}; \citeproc{ref-trompEDTACalculus2017}{Tromp et al., 2017}) as well +as in contemporary humans +(\citeproc{ref-leonardPlantMicroremains2015}{Leonard et al., 2015}) and +non-human primates (\citeproc{ref-powerChimpCalculus2015}{Power et al., +2015}), but not experimentally. Dental plaque is an oral biofilm and is part of the normal state of the oral cavity. However, when left unchecked, plaque can lead to infections, such as dental caries and periodontitis, and/or -mineralisation (\protect\hyperlink{ref-marshDentalPlaque2006}{Marsh, -2006}). The dental plaque biofilm grows in a well-characterized manner -before mineralisation, in a process that repeats regularly to build up -dental calculus. Shortly after teeth are cleaned (whether mechanically -or otherwise), salivary components adsorb to the crown or root and form -the acquired dental pellicle. The pellicle provides a viable surface for +mineralisation (\citeproc{ref-marshDentalPlaque2006}{Marsh, 2006}). The +dental plaque biofilm grows in a well-characterized manner before +mineralisation, in a process that repeats regularly to build up dental +calculus. Shortly after teeth are cleaned (whether mechanically or +otherwise), salivary components adsorb to the crown or root and form the +acquired dental pellicle. The pellicle provides a viable surface for bacteria to attach, especially early-coloniser species within the genera \emph{Streptococcus} and \emph{Actinomyces} -(\protect\hyperlink{ref-marshDentalPlaque2006}{Marsh, 2006}). Once the -tooth surface has been populated by specialists in surface-attachment, -other species of bacteria can attach to the adherent cells, increasing -the biofilm density and diversity. The bacterial species secrete +(\citeproc{ref-marshDentalPlaque2006}{Marsh, 2006}). Once the tooth +surface has been populated by specialists in surface-attachment, other +species of bacteria can attach to the adherent cells, increasing the +biofilm density and diversity. The bacterial species secrete polysaccharides, proteins, lipids, and nucleic acids, into their immediate environment to form a matrix that provides structural support, nutrition, and allows for environmental niche partitioning -(\protect\hyperlink{ref-flemmingBiofilmsEmergent2016}{Flemming et al., -2016}). +(\citeproc{ref-flemmingBiofilmsEmergent2016}{Flemming et al., 2016}). Biofilms can become susceptible to calcification under certain microenvironmental conditions, including an increased concentration of salts and a decrease in statherin and proline-rich proteins in saliva, rises in local plaque pH, and increased hydrolysis of urea -(\protect\hyperlink{ref-whiteDentalCalculus1997}{White, 1997}; -\protect\hyperlink{ref-wongCalciumPhosphate2002}{Wong et al., 2002}). -These conditions can cause increased precipitation and decreased -dissolution of calcium phosphate salts within saliva and the plaque -biofilm. The resulting supersaturation of calcium phosphate salts is the -main driver of biofilm mineralisation -(\protect\hyperlink{ref-jinSupragingivalCalculus2002}{Jin \& Yip, -2002}). The primary minerals in dental calculus are hydroxyapatite, -octacalcium phosphate, whitlockite, and brushite. During initial -mineralisation the main mineral component is brushite, which shifts to -hydroxyapatite in more mature dental calculus -(\protect\hyperlink{ref-hayashizakiSiteSpecific2008}{Hayashizaki et al., -2008}; \protect\hyperlink{ref-jinSupragingivalCalculus2002}{Jin \& Yip, -2002}). The exact elemental composition of dental calculus varies among -individuals due to various factors, including diet -(\protect\hyperlink{ref-hayashizakiSiteSpecific2008}{Hayashizaki et al., -2008}; \protect\hyperlink{ref-jiFluorideMagnesium2000}{Ji et al., -2000}). +(\citeproc{ref-whiteDentalCalculus1997}{White, 1997}; +\citeproc{ref-wongCalciumPhosphate2002}{Wong et al., 2002}). These +conditions can cause increased precipitation and decreased dissolution +of calcium phosphate salts within saliva and the plaque biofilm. The +resulting supersaturation of calcium phosphate salts is the main driver +of biofilm mineralisation +(\citeproc{ref-jinSupragingivalCalculus2002}{Jin \& Yip, 2002}). The +primary minerals in dental calculus are hydroxyapatite, octacalcium +phosphate, whitlockite, and brushite. During initial mineralisation the +main mineral component is brushite, which shifts to hydroxyapatite in +more mature dental calculus +(\citeproc{ref-hayashizakiSiteSpecific2008}{Hayashizaki et al., 2008}; +\citeproc{ref-jinSupragingivalCalculus2002}{Jin \& Yip, 2002}). The +exact elemental composition of dental calculus varies among individuals +due to various factors, including diet +(\citeproc{ref-hayashizakiSiteSpecific2008}{Hayashizaki et al., 2008}; +\citeproc{ref-jiFluorideMagnesium2000}{Ji et al., 2000}). Dental plaque can also be grown \emph{in vitro}, and these oral biofilm models are commonly used in dental research to assess the efficacy of certain treatments on dental pathogens -(\protect\hyperlink{ref-extercateAAA2010}{Exterkate et al., 2010}; -\protect\hyperlink{ref-filochePlaqueMicrocosm2007}{Filoche et al., -2007}) without the ethical issues of inducing plaque accumulation in -study participants and the complexity of access and sampling in humans -or animals. Oral biofilm models are often short-term models grown over a -few days, but longer term models also exist (up to six weeks) which are -used to develop mature plaque or dental calculus -(\protect\hyperlink{ref-middletonVitroCalculus1965}{Middleton, 1965}; -\protect\hyperlink{ref-sissonsMultistationPlaque1991}{Sissons et al., -1991}; \protect\hyperlink{ref-velskoConsistentReproducible2018}{Velsko -\& Shaddox, 2018}; \protect\hyperlink{ref-wongCalciumPhosphate2002}{Wong -et al., 2002}). A well-known limitation of biofilm models is the -difficulty in capturing the diversity and complexity of bacterial -communities and metabolic dependencies, micro-environments, nutrient -availability, and host immune-responses in the natural oral biome -(\protect\hyperlink{ref-bjarnsholtVivoBiofilm2013}{Bjarnsholt et al., -2013}; \protect\hyperlink{ref-edlundUncoveringComplex2018}{Edlund et -al., 2018}; \protect\hyperlink{ref-velskoCytokineResponse2017}{Velsko, -Cruz-Almeida, et al., 2017}; -\protect\hyperlink{ref-velskoConsistentReproducible2018}{Velsko \& +(\citeproc{ref-extercateAAA2010}{Exterkate et al., 2010}; +\citeproc{ref-filochePlaqueMicrocosm2007}{Filoche et al., 2007}) without +the ethical issues of inducing plaque accumulation in study participants +and the complexity of access and sampling in humans or animals. Oral +biofilm models are often short-term models grown over a few days, but +longer term models also exist (up to six weeks) which are used to +develop mature plaque or dental calculus +(\citeproc{ref-middletonVitroCalculus1965}{Middleton, 1965}; +\citeproc{ref-sissonsMultistationPlaque1991}{Sissons et al., 1991}; +\citeproc{ref-velskoConsistentReproducible2018}{Velsko \& Shaddox, +2018}; \citeproc{ref-wongCalciumPhosphate2002}{Wong et al., 2002}). A +well-known limitation of biofilm models is the difficulty in capturing +the diversity and complexity of bacterial communities and metabolic +dependencies, micro-environments, nutrient availability, and host +immune-responses in the natural oral biome +(\citeproc{ref-bjarnsholtVivoBiofilm2013}{Bjarnsholt et al., 2013}; +\citeproc{ref-edlundUncoveringComplex2018}{Edlund et al., 2018}; +\citeproc{ref-velskoCytokineResponse2017}{Velsko, Cruz-Almeida, et al., +2017}; \citeproc{ref-velskoConsistentReproducible2018}{Velsko \& Shaddox, 2018}). These limitations can be overcome by complex experimental setups, but at the cost of lower throughput and increased requirements for laboratory facilities. @@ -3011,7 +2993,7 @@ \section{Introduction}\label{introduction}} inadvertently bias the interpretations. This type of research has, so far, been limited, but has the potential to greatly benefit archaeological research on past diet -(\protect\hyperlink{ref-radiniDirtyTeeth2022}{Radini \& Nikita, 2022}). +(\citeproc{ref-radiniDirtyTeeth2022}{Radini \& Nikita, 2022}). We present an oral biofilm model that can serve as a viable proxy for dental calculus for archaeology-oriented research questions. It is a @@ -3041,8 +3023,7 @@ \section{Introduction}\label{introduction}} archaeological material, when working within the limitations of an oral biofilm model. -\hypertarget{materials-and-methods}{% -\section{Materials and methods}\label{materials-and-methods}} +\section{Materials and methods}\label{materials-and-methods} Our biofilm setup consists of whole saliva as the inoculate to approximate natural microbial communities within the human oral cavity, @@ -3053,12 +3034,14 @@ \section{Materials and methods}\label{materials-and-methods}} wheat starch solutions were added during the biofilm growth to explore the biases involved in their incorporation and extraction from dental calculus. These results are presented in a separate article -(\protect\hyperlink{ref-bartholdyInvestigatingBiases2022}{Bartholdy \& -Henry, 2022}). +(\citeproc{ref-bartholdyInvestigatingBiases2022}{Bartholdy \& Henry, +2022}). \begin{figure} -{\centering \includegraphics{figures/Exp_protocol.png} +\centering{ + +\includegraphics{figures/Exp_protocol.png} } @@ -3069,7 +3052,7 @@ \section{Materials and methods}\label{materials-and-methods}} spectroscopy, and saliva (S), artificial saliva (M), and calculus samples were used for metagenomic analysis.} -\end{figure} +\end{figure}% To determine the composition of microbial communities, we sampled the medium from the biofilm wells over the course of the experiment. We @@ -3082,34 +3065,32 @@ \section{Materials and methods}\label{materials-and-methods}} starch treatments, but differences between these samples were not explored in this study. -\hypertarget{biofilm-growth}{% -\subsection{Biofilm growth}\label{biofilm-growth}} +\subsection{Biofilm growth}\label{biofilm-growth} We employ a multispecies oral biofilm model following a modified protocol from Sissons and colleagues -(\protect\hyperlink{ref-sissonsMultistationPlaque1991}{1991}) and -Shellis (\protect\hyperlink{ref-shellisSyntheticSaliva1978}{1978}). The -setup comprises a polypropylene 24 deepwell PCR plate (KingFisher -97003510) with a lid containing 24 pegs (substrata), which are -autoclaved at 120\(^{\circ}\)C, 1 bar overpressure, for 20 mins. +(\citeproc{ref-sissonsMultistationPlaque1991}{1991}) and Shellis +(\citeproc{ref-shellisSyntheticSaliva1978}{1978}). The setup comprises a +polypropylene 24 deepwell PCR plate (KingFisher 97003510) with a lid +containing 24 pegs (substrata), which are autoclaved at +120\(^{\circ}\)C, 1 bar overpressure, for 20 mins. The artificial saliva (hereafter referred to as medium) is a modified version of the basal medium mucin (BMM) described by Sissons and -colleagues -(\protect\hyperlink{ref-sissonsMultistationPlaque1991}{1991}). It is a +colleagues (\citeproc{ref-sissonsMultistationPlaque1991}{1991}). It is a complex medium containing 2.5 g/l partially purified mucin from porcine stomach (Type III, Sigma M1778), 5 g/l trypticase peptone (Roth 2363.1), 10 g/l proteose peptone (Oxoid LP0085), 5 g/l yeast extract (BD 211921), 2.5 g/l KCl, 0.35 g/l NaCl, 1.8 mmol/l CaCl\textsubscript{2}, 5.2 mmol/l Na\textsubscript{2}HPO\textsubscript{4} -(\protect\hyperlink{ref-sissonsMultistationPlaque1991}{Sissons et al., -1991}), 6.4 mmol/l NaHCO\textsubscript{3} -(\protect\hyperlink{ref-shellisSyntheticSaliva1978}{Shellis, 1978}), 2.5 -mg/l haemin. This is subsequently adjusted to pH 7 with NaOH pellets and +(\citeproc{ref-sissonsMultistationPlaque1991}{Sissons et al., 1991}), +6.4 mmol/l NaHCO\textsubscript{3} +(\citeproc{ref-shellisSyntheticSaliva1978}{Shellis, 1978}), 2.5 mg/l +haemin. This is subsequently adjusted to pH 7 with NaOH pellets and stirring, autoclaved (15 min, 120\(^{\circ}\)C, 1 bar overpressure), and supplemented with 5.8 (mu)mol/l menadione, 5 mmol/l urea, and 1 mmol/l -arginine (\protect\hyperlink{ref-sissonsMultistationPlaque1991}{Sissons -et al., 1991}). +arginine (\citeproc{ref-sissonsMultistationPlaque1991}{Sissons et al., +1991}). Fresh whole saliva (WS) for inoculation was provided by a 31-year-old male donor with no history of caries, who abstained from oral hygiene @@ -3151,17 +3132,16 @@ \subsection{Biofilm growth}\label{biofilm-growth}} that there was no host salivary \(\alpha\)-amylase activity in the system. The results of the starch incorporation and \(\alpha\)-amylase activity assay have been reported in a separate article -(\protect\hyperlink{ref-bartholdyInvestigatingBiases2022}{Bartholdy \& -Henry, 2022}). +(\citeproc{ref-bartholdyInvestigatingBiases2022}{Bartholdy \& Henry, +2022}). After 15 days, mineralisation was encouraged with a calcium phosphate monofluorophosphate urea (CPMU) solution containing 20 mmol/l CaCl\textsubscript{2}, 12 mmol/l NaH\textsubscript{2}PO\textsubscript{4}, 5 mmol/l Na\textsubscript{2}PO\textsubscript{3}F, 500 mmol/l urea -(\protect\hyperlink{ref-pearceConcomitantDeposition1987}{Pearce \& -Sissons, 1987}; -\protect\hyperlink{ref-sissonsMultistationPlaque1991}{Sissons et al., +(\citeproc{ref-pearceConcomitantDeposition1987}{Pearce \& Sissons, +1987}; \citeproc{ref-sissonsMultistationPlaque1991}{Sissons et al., 1991}), and 0.04 g/l MgCl. The substrata were submerged in 1 ml/well CPMU five times daily, every two hours, for six minutes, at 30 rpm. During the mineralisation period, starch treatments were reduced to once @@ -3175,17 +3155,15 @@ \subsection{Biofilm growth}\label{biofilm-growth}} control samples that were only fed sucrose were included to detect starch contamination. -\hypertarget{metagenomics}{% -\subsection{Metagenomics}\label{metagenomics}} +\subsection{Metagenomics}\label{metagenomics} -\hypertarget{tbl-dna-samples}{} \begin{longtable}[]{@{}lrr@{}} + \caption{\label{tbl-dna-samples}Number of samples taken during the -experiment, separated by sampling day and sample type.}\tabularnewline -\toprule\noalign{} -Sample type & Sampling day & n \\ -\midrule\noalign{} -\endfirsthead +experiment, separated by sampling day and sample type.} + +\tabularnewline + \toprule\noalign{} Sample type & Sampling day & n \\ \midrule\noalign{} @@ -3204,6 +3182,7 @@ \subsection{Metagenomics}\label{metagenomics}} medium & 21 & 2 \\ medium & 24 & 2 \\ model\_calculus & 24 & 16 \\ + \end{longtable} A total of 35 samples were taken during the experiment from the donated @@ -3215,100 +3194,92 @@ \subsection{Metagenomics}\label{metagenomics}} The DNA was sheared to 500bp through sonication with a Covaris M220 Focused-ultrasonicator. Double-stranded libraries were prepared -(\protect\hyperlink{ref-aronHalfUDG2020}{Aron et al., 2020}) and dual -indexed (\protect\hyperlink{ref-stahlDoublestrandedIndexing2019}{Stahl -et al., 2019}), with the indexing protocol being adapted for longer DNA -fragments. Briefly, the modifications consisted of adding 3 μl of DMSO -to the indexing reaction, and extending the amplification cycles to +(\citeproc{ref-aronHalfUDG2020}{Aron et al., 2020}) and dual indexed +(\citeproc{ref-stahlDoublestrandedIndexing2019}{Stahl et al., 2020}), +with the indexing protocol being adapted for longer DNA fragments. +Briefly, the modifications consisted of adding 3 μl of DMSO to the +indexing reaction, and extending the amplification cycles to 95\(^{\circ}\)C for 60 s, 58\(^{\circ}\)C for 60 s, and 72\(^{\circ}\)C for 90 s. The libraries were paired-end sequenced on a NextSeq 500 to 150bp, and demultiplexed by an in-house script. -\hypertarget{preprocessing}{% -\subsubsection{Preprocessing}\label{preprocessing}} +\subsubsection{Preprocessing}\label{preprocessing} The raw DNA reads were preprocessed using the nf-core/eager, v2.4.4 -pipeline (\protect\hyperlink{ref-yatesEAGER2020}{Fellows Yates et al., -2020}). The pipeline included adapter removal and read merging using -AdapterRemoval, v2.3.2 -(\protect\hyperlink{ref-AdapterRemovalv2}{Schubert et al., 2016}). -Merged reads were mapped to the human reference genome (GRCh38) using -BWA, v0.7.17-r1188 (\protect\hyperlink{ref-BWA}{Li \& Durbin, 2009}) (-n +pipeline (\citeproc{ref-yatesEAGER2020}{Fellows Yates et al., 2020}). +The pipeline included adapter removal and read merging using +AdapterRemoval, v2.3.2 (\citeproc{ref-AdapterRemovalv2}{Schubert et al., +2016}). Merged reads were mapped to the human reference genome (GRCh38) +using BWA, v0.7.17-r1188 (\citeproc{ref-BWA}{Li \& Durbin, 2009}) (-n 0.01; -l 32), and unmapped reads were extracted using Samtools, v1.12. The final step of the pipeline, metagenomic classification, was -conducted in kraken, v2.1.2 (\protect\hyperlink{ref-kraken2}{Wood et -al., 2019}) using the Standard 60GB database +conducted in kraken, v2.1.2 (\citeproc{ref-kraken2}{Wood et al., 2019}) +using the Standard 60GB database (\url{https://genome-idx.s3.amazonaws.com/kraken/k2_standard_20220926.tar.gz}). Environmental reference samples were downloaded directly from ENA and from NCBI using the SRA Toolkit. Oral reference samples were downloaded from the Human Metagenome Project (HMP), and modern calculus samples -from Velsko et al. -(\protect\hyperlink{ref-velskoDentalCalculus2017}{2017}). From the HMP -data, only paired reads were processed, singletons were removed. +from Velsko et al. (\citeproc{ref-velskoDentalCalculus2017}{2017}). From +the HMP data, only paired reads were processed, singletons were removed. \emph{In vitro} biofilm model samples from Edlund et al. -(\protect\hyperlink{ref-edlundUncoveringComplex2018}{2018}) were used as -a reference. Links to the specific sequences are included in the -metadata. Human-filtered reads produced in this study were uploaded to -ENA under accession number PRJEB61886. +(\citeproc{ref-edlundUncoveringComplex2018}{2018}) were used as a +reference. Links to the specific sequences are included in the metadata. +Human-filtered reads produced in this study were uploaded to ENA under +accession number PRJEB61886. -\hypertarget{authentication}{% -\subsubsection{Authentication}\label{authentication}} +\subsubsection{Authentication}\label{authentication} Species with lower than 0.001\% relative abundance across all samples were removed from the species table. SourceTracker2 -(\protect\hyperlink{ref-knightsSourceTracker2011}{Knights et al., 2011}) -was used to estimate source composition of the abundance-filtered oral -biofilm model samples using a Bayesian framework, and samples falling -below 70\% oral source were removed from downstream analyses. -Well-preserved abundance-filtered samples were compared to oral and -environmental controls to detect potential external contamination. The R -package decontam v1.20.0 (\protect\hyperlink{ref-Rdecontam}{Davis et -al., 2018}) was used to identify potential contaminants in the -abundance-filtered table using DNA concentrations with a probability -threshold of 0.95 and negative controls with a probability threshold of -0.05. Putative contaminant species were filtered out of the OTU tables -for all downstream analyses. - -\hypertarget{community-composition}{% -\subsubsection{Community composition}\label{community-composition}} +(\citeproc{ref-knightsSourceTracker2011}{Knights et al., 2011}) was used +to estimate source composition of the abundance-filtered oral biofilm +model samples using a Bayesian framework, and samples falling below 70\% +oral source were removed from downstream analyses. Well-preserved +abundance-filtered samples were compared to oral and environmental +controls to detect potential external contamination. The R package +decontam v1.22.0 (\citeproc{ref-Rdecontam}{Davis et al., 2018}) was used +to identify potential contaminants in the abundance-filtered table using +DNA concentrations with a probability threshold of 0.95 and negative +controls with a probability threshold of 0.05. Putative contaminant +species were filtered out of the OTU tables for all downstream analyses. + +\subsubsection{Community composition}\label{community-composition} Relative abundances of communities were calculated at the species- and genus-level, as recommended for compositional data -(\protect\hyperlink{ref-gloorMicrobiomeDatasets2017}{Gloor et al., -2017}). Shannon index and Pileou's evenness index were calculated on +(\citeproc{ref-gloorMicrobiomeDatasets2017}{Gloor et al., 2017}). +Shannon index and Pileou's evenness index were calculated on species-level OTU tables of all model and oral reference samples using -the vegan v2.6.4 R package (\protect\hyperlink{ref-Rvegan}{Oksanen et -al., 2022}). Shannon index was calculated for all experimental samples -to see if there is an overall loss or gain in diversity and richness -across the experiment. Sparse principal component analysis (sPCA) was -performed on model biofilm samples to assess differences in microbial -composition between samples within the experiment, and a separate sPCA -analysis was performed on model calculus and oral reference samples. The -sPCA analysis was conducted using the mixOmics v 6.24.0 R package -(\protect\hyperlink{ref-RmixOmics}{Rohart et al., 2017}). +the vegan v2.6.4 R package (\citeproc{ref-Rvegan}{Oksanen et al., +2022}). Shannon index was calculated for all experimental samples to see +if there is an overall loss or gain in diversity and richness across the +experiment. Sparse principal component analysis (sPCA) was performed on +model biofilm samples to assess differences in microbial composition +between samples within the experiment, and a separate sPCA analysis was +performed on model calculus and oral reference samples. The sPCA +analysis was conducted using the mixOmics v 6.26.0 R package +(\citeproc{ref-RmixOmics}{Rohart et al., 2017}). The core microbiome was calculated by taking the mean genus-level relative abundance within each sample type for model calculus, modern reference calculus, sub- and supragingival plaque. Genera present at lower than 5\% relative abundance were grouped into the category `other'. Information on the oxygen tolerance of bacterial species was -downloaded from BacDive -(\protect\hyperlink{ref-reimerBacDive2022}{Reimer et al., 2022}) and all -variations of the major categories anaerobe, facultative anaerobe, and -aerobe were combined into the appropriate major category. At the time of -writing, 55.7\% species were missing aerotolerance values. This was -mitigated by aggregating genus-level tolerances to species with missing -values, and may have some errors (although unlikely to make any -significant difference). - -\hypertarget{differential-abundance}{% -\subsubsection{Differential abundance}\label{differential-abundance}} +downloaded from BacDive (\citeproc{ref-reimerBacDive2022}{Reimer et al., +2022}) and all variations of the major categories anaerobe, facultative +anaerobe, and aerobe were combined into the appropriate major category. +At the time of writing, 55.7\% species were missing aerotolerance +values. This was mitigated by aggregating genus-level tolerances to +species with missing values, and may have some errors (although unlikely +to make any significant difference). + +\subsubsection{Differential abundance}\label{differential-abundance} Differential abundance of species was calculated using the Analysis of Compositions of Microbiomes with Bias Correction (ANCOM-BC) method from -the ANCOMBC R package v2.2.2 (\protect\hyperlink{ref-linANCOMBC2020}{Lin -\& Peddada, 2020}), with a species-level OTU table as input. Results are +the ANCOMBC R package v2.4.0 (\citeproc{ref-linANCOMBC2020}{Lin \& +Peddada, 2020}), with a species-level OTU table as input. Results are presented as the log fold change of species between paired sample types with 95\% confidence intervals. P-values are adjusted using the false discovery rate (FDR) method. Samples are grouped by sample type @@ -3318,35 +3289,32 @@ \subsubsection{Differential abundance}\label{differential-abundance}} which species are enriched in the different samples and causing clustering in the sPCA. -\hypertarget{ftir}{% -\subsection{FTIR}\label{ftir}} +\subsection{FTIR}\label{ftir} To determine the mineral composition and level of crystallisation of the model dental calculus samples, we used Fourier Transform Infrared (FTIR) spectroscopy. We compared the spectra of model dental calculus with spectra of archaeological and modern dental calculus and used a built-in Omnic search library for mineral identification -(\protect\hyperlink{ref-mentzerDistributionAuthigenic2014}{Mentzer et -al., 2014}; -\protect\hyperlink{ref-weinerInfraredSpectroscopy2010}{Weiner, 2010b}). -The archaeological dental calculus was sampled from an isolated -permanent tooth from Middenbeemster, a rural, 19th century Dutch site -(\protect\hyperlink{ref-lemmersMiddenbeemster2013}{Lemmers et al., -2013}). Samples were analysed at the Laboratory for Sedimentary -Archaeology, Haifa University. The analysis was conducted with a Thermo -Scientific Nicolet is5 spectrometer in transmission, at 4 cm\(^{-1}\) -resolution, with an average of 32 scans between 4000 and 400 cm\(^{-1}\) +(\citeproc{ref-mentzerDistributionAuthigenic2014}{Mentzer et al., 2014}; +\citeproc{ref-weinerInfraredSpectroscopy2010}{Weiner, 2010b}). The +archaeological dental calculus was sampled from an isolated permanent +tooth from Middenbeemster, a rural, 19th century Dutch site +(\citeproc{ref-lemmersMiddenbeemster2013}{Lemmers et al., 2013}). +Samples were analysed at the Laboratory for Sedimentary Archaeology, +Haifa University. The analysis was conducted with a Thermo Scientific +Nicolet is5 spectrometer in transmission, at 4 cm\(^{-1}\) resolution, +with an average of 32 scans between 4000 and 400 cm\(^{-1}\) wavenumbers. -\hypertarget{tbl-ftir-byoc}{} \begin{longtable}[]{@{}lrrr@{}} + \caption{\label{tbl-ftir-byoc}Summary of samples used in FTIR analysis, including type of sample, sampling day, number of samples (n), and mean -weight in mg.}\tabularnewline -\toprule\noalign{} -Sample type & Sampling day & n & Weight (mg) \\ -\midrule\noalign{} -\endfirsthead +weight in mg.} + +\tabularnewline + \toprule\noalign{} Sample type & Sampling day & n & Weight (mg) \\ \midrule\noalign{} @@ -3358,6 +3326,7 @@ \subsection{FTIR}\label{ftir}} biofilm & 16 & 7 & 2.00 \\ biofilm & 20 & 6 & 3.50 \\ model\_calculus & 24 & 8 & 3.87 \\ + \end{longtable} Analysis was conducted on 26 model calculus samples from days 7, 12, 16, @@ -3367,98 +3336,89 @@ \subsection{FTIR}\label{ftir}} from the samples sequenced for DNA, but following the same setup and protocol (as described above). Samples were analysed following the method presented in Asscher, Regev, et al. -(\protect\hyperlink{ref-asscherAtomicDisorder2011}{2011}) and Asscher, -Weiner, et al. -(\protect\hyperlink{ref-asscherVariationsAtomic2011}{2011}). A few -\(\mu\)g of each sample were repeatedly ground together with KBr and -pressed in a 7 mm die under two tons of pressure using a Specac -mini-pellet press (Specac Ltd., GS01152). Repeated measurements of the -splitting factor (SF) of the absorbance bands at 605 and 567 cm−1 -wavenumbers were taken after each grind, and a grind curve was produced -following Asscher, Regev, et al. -(\protect\hyperlink{ref-asscherAtomicDisorder2011}{2011}) to try and -detect changes in the hydroxyapatite crystallinity over time. Samples -were ground and analysed up to six times (sample suffix a-f) for the -grinding curve. Grinding curves were prepared for samples from days 16, -20, and 24. No grind curves were produced for samples from days 7 and -12. These were largely composed of organics and proteins, and did not -form enough mineral (hydroxyapatite) for analysis. The splitting factor -of carbonate hydroxyapatite was calculated using a macro script, +(\citeproc{ref-asscherAtomicDisorder2011}{2011}) and Asscher, Weiner, et +al. (\citeproc{ref-asscherVariationsAtomic2011}{2011}). A few \(\mu\)g +of each sample were repeatedly ground together with KBr and pressed in a +7 mm die under two tons of pressure using a Specac mini-pellet press +(Specac Ltd., GS01152). Repeated measurements of the splitting factor +(SF) of the absorbance bands at 605 and 567 cm−1 wavenumbers were taken +after each grind, and a grind curve was produced following Asscher, +Regev, et al. (\citeproc{ref-asscherAtomicDisorder2011}{2011}) to try +and detect changes in the hydroxyapatite crystallinity over time. +Samples were ground and analysed up to six times (sample suffix a-f) for +the grinding curve. Grinding curves were prepared for samples from days +16, 20, and 24. No grind curves were produced for samples from days 7 +and 12. These were largely composed of organics and proteins, and did +not form enough mineral (hydroxyapatite) for analysis. The splitting +factor of carbonate hydroxyapatite was calculated using a macro script, following Weiner \& Bar-Yosef -(\protect\hyperlink{ref-weinerStatesPreservation1990}{1990}). The -calculation involves dividing the sum of the height of the absorptions -at 603 cm\(^{-1}\) and 567 cm\(^{-1}\) by the height of the valley -between them. Following Asscher, Regev, et al. -(\protect\hyperlink{ref-asscherAtomicDisorder2011}{2011}) and Asscher, -Weiner, et al. -(\protect\hyperlink{ref-asscherVariationsAtomic2011}{2011}), we plotted -the splitting factor against the full width at half maximum (FWHM) of -the main absorption at 1035-1043 cm\(^{-1}\) to explore crystallinity +(\citeproc{ref-weinerStatesPreservation1990}{1990}). The calculation +involves dividing the sum of the height of the absorptions at 603 +cm\(^{-1}\) and 567 cm\(^{-1}\) by the height of the valley between +them. Following Asscher, Regev, et al. +(\citeproc{ref-asscherAtomicDisorder2011}{2011}) and Asscher, Weiner, et +al. (\citeproc{ref-asscherVariationsAtomic2011}{2011}), we plotted the +splitting factor against the full width at half maximum (FWHM) of the +main absorption at 1035-1043 cm\(^{-1}\) to explore crystallinity (crystal size) and the order and disorder of hydroxyapatite. We then compared our grinding curve slopes and FWHM to the ones produced by Asscher, Weiner, et al. -(\protect\hyperlink{ref-asscherVariationsAtomic2011}{2011}). Asscher, -Weiner, et al. -(\protect\hyperlink{ref-asscherVariationsAtomic2011}{2011}) and Asscher, -Regev, et al. (\protect\hyperlink{ref-asscherAtomicDisorder2011}{2011}) +(\citeproc{ref-asscherVariationsAtomic2011}{2011}). Asscher, Weiner, et +al. (\citeproc{ref-asscherVariationsAtomic2011}{2011}) and Asscher, +Regev, et al. (\citeproc{ref-asscherAtomicDisorder2011}{2011}) demonstrated that while the decrease in FWHM of each grinding in the curve reflects a decrease in particle size due to grinding, the location of the curves within a plot of the FWHM against the splitting factor expresses the disorder effect. Thus the curves with steeper slopes, higher splitting factor, and lower FWHM represent lower levels of disorder in the mineral (Figure 2 in -\protect\hyperlink{ref-asscherVariationsAtomic2011}{Asscher, Weiner, et -al., 2011}). +\citeproc{ref-asscherVariationsAtomic2011}{Asscher, Weiner, et al., +2011}). -\hypertarget{statistics}{% -\subsection{Statistics}\label{statistics}} +\subsection{Statistics}\label{statistics} -Statistical analysis was conducted in R version 4.3.2 (2023-10-31) (Eye -Holes) (\protect\hyperlink{ref-Rbase}{R Core Team, 2020}). Data cleaning -and wrangling performed with packages from tidyverse -(\protect\hyperlink{ref-tidyverse2019}{Wickham et al., 2019}). Plots -were created using ggplot2 v3.4.4 -(\protect\hyperlink{ref-ggplot2}{Wickham, 2016}). +Statistical analysis was conducted in R version 4.3.3 (2024-02-29) +(Angel Food Cake) (\citeproc{ref-Rbase}{R Core Team, 2020}). Data +cleaning and wrangling performed with packages from tidyverse +(\citeproc{ref-tidyverse2019}{Wickham et al., 2019}). Plots were created +using ggplot2 v3.4.4 (\citeproc{ref-ggplot2}{Wickham, 2016}). -\hypertarget{results}{% -\section{Results}\label{results}} +\section{Results}\label{results} -\hypertarget{metagenomic-analysis}{% -\subsection{Metagenomic analysis}\label{metagenomic-analysis}} +\subsection{Metagenomic analysis}\label{metagenomic-analysis} -\hypertarget{sample-authentication}{% -\subsubsection{Sample authentication}\label{sample-authentication}} +\subsubsection{Sample authentication}\label{sample-authentication} To determine the extent of contamination in our samples, we performed a source-tracking analysis using SourceTracker2 -(\protect\hyperlink{ref-knightsSourceTracker2011}{Knights et al., -2011}). Results suggest that the majority of taxa across samples have an -oral microbial signature, and therefore our samples are minimally -affected by external contamination (Figure S1). We compared -SourceTracker2 results to a database of oral taxa from the cuperdec -v1.1.0 R package -(\protect\hyperlink{ref-yatesOralMicrobiome2021}{Fellows Yates et al., -2021}) to prevent removal of samples where oral taxa were assigned to a -non-oral source (Figure S2), as some taxa with a signature from multiple -sources are often classified as ``Unknown'' -(\protect\hyperlink{ref-velskoMicrobialDifferences2019}{Velsko et al., -2019}). We included several oral sources, which may increase the risk of -this occurring. Samples containing a large proportion (\textgreater70\%) -of environmental contamination were removed. The removed samples were +(\citeproc{ref-knightsSourceTracker2011}{Knights et al., 2011}). Results +suggest that the majority of taxa across samples have an oral microbial +signature, and therefore our samples are minimally affected by external +contamination (Figure S1). We compared SourceTracker2 results to a +database of oral taxa from the cuperdec v1.1.0 R package +(\citeproc{ref-yatesOralMicrobiome2021}{Fellows Yates et al., 2021}) to +prevent removal of samples where oral taxa were assigned to a non-oral +source (Figure S2), as some taxa with a signature from multiple sources +are often classified as ``Unknown'' +(\citeproc{ref-velskoMicrobialDifferences2019}{Velsko et al., 2019}). We +included several oral sources, which may increase the risk of this +occurring. Samples containing a large proportion (\textgreater70\%) of +environmental contamination were removed. The removed samples were predominantly medium samples from later in the experiment, and a few model calculus samples. After contaminated samples were removed, suspected contaminant-species were removed from the remaining samples -using the decontam R package (\protect\hyperlink{ref-Rdecontam}{Davis et -al., 2018}). After contamination removal, samples consisted of between -88 and 284 species with a mean of 182. +using the decontam R package (\citeproc{ref-Rdecontam}{Davis et al., +2018}). After contamination removal, samples consisted of between 88 and +284 species with a mean of 182. -\hypertarget{decrease-in-community-diversity-across-experiment}{% \subsubsection{Decrease in community diversity across -experiment}\label{decrease-in-community-diversity-across-experiment}} +experiment}\label{decrease-in-community-diversity-across-experiment} \begin{figure} -{\centering \includegraphics{figures/byoc-valid-fig-diversity-byoc-1.pdf} +\centering{ + +\includegraphics{figures/byoc-valid-fig-diversity-byoc-1.pdf} } @@ -3467,7 +3427,7 @@ \subsubsection{Decrease in community diversity across by sampling time. inoc = samples from days 0-5; treatm = samples from days 6-23; model = model calculus samples from day 24.} -\end{figure} +\end{figure}% To monitor the development of microbial communities over the course of the experiment, we used the Shannon Index to assess the species @@ -3481,13 +3441,14 @@ \subsubsection{Decrease in community diversity across species increased between the treatment period and the final model calculus (Figure~\ref{fig-diversity-byoc}). -\hypertarget{medium-and-model-calculus-samples-are-distinct-from-the-inoculate}{% \subsubsection{Medium and model calculus samples are distinct from the -inoculate}\label{medium-and-model-calculus-samples-are-distinct-from-the-inoculate}} +inoculate}\label{medium-and-model-calculus-samples-are-distinct-from-the-inoculate} \begin{figure} -{\centering \includegraphics{figures/byoc-valid-fig-spca-byoc-1.pdf} +\centering{ + +\includegraphics{figures/byoc-valid-fig-spca-byoc-1.pdf} } @@ -3495,7 +3456,7 @@ \subsubsection{Medium and model calculus samples are distinct from the tolerance in samples from this study only. Figure shows the main sPCA plot (A), species loadings on PC2 (B), and species loadings on PC1 (C).} -\end{figure} +\end{figure}% We next examined whether there is a change in the species composition over time in our samples by assessing the beta-diversity in a PCA. The @@ -3517,7 +3478,9 @@ \subsubsection{Medium and model calculus samples are distinct from the \begin{figure} -{\centering \includegraphics{figures/byoc-valid-fig-diffabund-byoc-1.pdf} +\centering{ + +\includegraphics{figures/byoc-valid-fig-diffabund-byoc-1.pdf} } @@ -3527,25 +3490,26 @@ \subsubsection{Medium and model calculus samples are distinct from the are standard error. Plot shows the top 30 absolute log-fold changes between model calculus and saliva.} -\end{figure} +\end{figure}% We determined whether there are species that are differentially abundant between our sample types using the ANCOMBC R package -(\protect\hyperlink{ref-linANCOMBC2020}{Lin \& Peddada, 2020}), giving -us an idea of how the biofilm develops under our experimental -conditions. Species enriched in saliva compared to model calculus are -largely aerobic or facultatively anaerobic, while species enriched in -model calculus compared to saliva are mainly anaerobes. The differences -between saliva and calculus are more pronounced than between medium and -model calculus, which is expected (Figure~\ref{fig-diffabund-byoc}). - -\hypertarget{lower-diversity-in-artificial-samples-than-oral-references}{% +(\citeproc{ref-linANCOMBC2020}{Lin \& Peddada, 2020}), giving us an idea +of how the biofilm develops under our experimental conditions. Species +enriched in saliva compared to model calculus are largely aerobic or +facultatively anaerobic, while species enriched in model calculus +compared to saliva are mainly anaerobes. The differences between saliva +and calculus are more pronounced than between medium and model calculus, +which is expected (Figure~\ref{fig-diffabund-byoc}). + \subsubsection{Lower diversity in artificial samples than oral -references}\label{lower-diversity-in-artificial-samples-than-oral-references}} +references}\label{lower-diversity-in-artificial-samples-than-oral-references} \begin{figure} -{\centering \includegraphics{figures/byoc-valid-fig-shannon-compar-1.pdf} +\centering{ + +\includegraphics{figures/byoc-valid-fig-shannon-compar-1.pdf} } @@ -3553,7 +3517,7 @@ \subsubsection{Lower diversity in artificial samples than oral medium samples, as well as oral reference samples and comparative \emph{in vitro} study.} -\end{figure} +\end{figure}% We used the Shannon Index to compare alpha-diversity in our model to oral reference samples. The mean Shannon Index of model @@ -3568,14 +3532,15 @@ \subsubsection{Lower diversity in artificial samples than oral compared to reference samples. The number of species follows the same trend. -\hypertarget{model-calculus-is-distinct-from-dental-calculus-and-other-oral-samples}{% \subsubsection{Model calculus is distinct from dental calculus and other oral -samples}\label{model-calculus-is-distinct-from-dental-calculus-and-other-oral-samples}} +samples}\label{model-calculus-is-distinct-from-dental-calculus-and-other-oral-samples} \begin{figure} -{\centering \includegraphics{figures/byoc-valid-fig-core-genera-1.pdf} +\centering{ + +\includegraphics{figures/byoc-valid-fig-core-genera-1.pdf} } @@ -3583,7 +3548,7 @@ \subsubsection{Model calculus is distinct from dental calculus and other of samples represented as mean relative abundances at the genus level. Other = other genera present in lower than 5\% relative abundance.} -\end{figure} +\end{figure}% We calculated the mean relative abundances of the genera in each sample to compare the core genera of model calculus with oral reference @@ -3599,12 +3564,13 @@ \subsubsection{Model calculus is distinct from dental calculus and other represented by fastidious early-coloniser species like \emph{Capnocytophaga} and \emph{Neisseria} spp., which require an environment with at least 5\% carbon dioxide to thrive -(\protect\hyperlink{ref-tonjumNeisseria2017}{Tønjum \& van Putten, -2017}). +(\citeproc{ref-tonjumNeisseria2017}{Tønjum \& van Putten, 2017}). \begin{figure} -{\centering \includegraphics{figures/byoc-valid-fig-spca-compar-1.pdf} +\centering{ + +\includegraphics{figures/byoc-valid-fig-spca-compar-1.pdf} } @@ -3612,7 +3578,7 @@ \subsubsection{Model calculus is distinct from dental calculus and other calculus and reference samples. Figure shows (A) the main sPCA plot, (B) the species loadings from PC2, and (C) species loadings on PC1.} -\end{figure} +\end{figure}% To directly compare the beta-diversity of our model calculus with oral reference samples, including modern dental calculus, we used an sPCA @@ -3633,7 +3599,9 @@ \subsubsection{Model calculus is distinct from dental calculus and other \begin{figure} -{\centering \includegraphics{figures/byoc-valid-fig-diffabund-comp-1.pdf} +\centering{ + +\includegraphics{figures/byoc-valid-fig-diffabund-comp-1.pdf} } @@ -3645,7 +3613,7 @@ \subsubsection{Model calculus is distinct from dental calculus and other sample types, ordered by decreasing log-fold change. Bars represent standard error.} -\end{figure} +\end{figure}% To investigate which species are enriched in different sample types, and compare the final product of our model with naturally occurring plaque @@ -3665,10 +3633,9 @@ \subsubsection{Model calculus is distinct from dental calculus and other \emph{Cryptobacterium curtum}, \emph{Eggerthella lenta}, and \emph{Mogibacterium diversum} (Figure~\ref{fig-diffabund-comp}B). -\hypertarget{samples-show-an-increased-mineralisation-over-the-course-of-the-experiment}{% \subsection{Samples show an increased mineralisation over the course of the -experiment}\label{samples-show-an-increased-mineralisation-over-the-course-of-the-experiment}} +experiment}\label{samples-show-an-increased-mineralisation-over-the-course-of-the-experiment} To determine whether the model dental calculus is comparable to natural dental calculus, both modern and archaeological dental calculus were @@ -3710,9 +3677,8 @@ \subsection{Samples show an increased mineralisation over the course of between the spectra, with most spectra exhibiting a higher phosphate-to-protein/lipid ratio (Figure~\ref{fig-ftir-spectra}C and D). -\hypertarget{model-calculus-has-a-similar-mineral-composition-to-natural-calculus}{% \subsection{Model calculus has a similar mineral composition to natural -calculus}\label{model-calculus-has-a-similar-mineral-composition-to-natural-calculus}} +calculus}\label{model-calculus-has-a-similar-mineral-composition-to-natural-calculus} Archaeological and modern reference spectra are largely indistinguishable and consist of a broad O--H absorbance band (3400 @@ -3723,13 +3689,14 @@ \subsection{Model calculus has a similar mineral composition to natural (Figure~\ref{fig-ftir-spectra}E) which, together with the hydroxyl and the carbonate, can be identified as derived from carbonate hydroxyapatite, the main mineral found in mature dental calculus -(\protect\hyperlink{ref-hayashizakiSiteSpecific2008}{Hayashizaki et al., -2008}; \protect\hyperlink{ref-jinSupragingivalCalculus2002}{Jin \& Yip, -2002}). +(\citeproc{ref-hayashizakiSiteSpecific2008}{Hayashizaki et al., 2008}; +\citeproc{ref-jinSupragingivalCalculus2002}{Jin \& Yip, 2002}). \begin{figure} -{\centering \includegraphics{figures/byoc-valid-fig-ftir-spectra-1.pdf} +\centering{ + +\includegraphics{figures/byoc-valid-fig-ftir-spectra-1.pdf} } @@ -3739,32 +3706,31 @@ \subsection{Model calculus has a similar mineral composition to natural hydroxyl group. Analysis ID for model samples is constructed as: F{[}day sampled{]}.{[}well sampled{]}\_{[}grind sample{]}.} -\end{figure} +\end{figure}% -\hypertarget{samples-show-similar-crystallinity-and-order-to-reference-calculus}{% \subsection{Samples show similar crystallinity and order to reference -calculus}\label{samples-show-similar-crystallinity-and-order-to-reference-calculus}} +calculus}\label{samples-show-similar-crystallinity-and-order-to-reference-calculus} We determined the level of crystallinity and order of the carbonate hydroxyapatite in our samples as an indication for its maturity by using the grinding curves method presented by Asscher, Regev, et al. -(\protect\hyperlink{ref-asscherAtomicDisorder2011}{2011}) and Asscher, -Weiner, et al. -(\protect\hyperlink{ref-asscherVariationsAtomic2011}{2011}).\\ +(\citeproc{ref-asscherAtomicDisorder2011}{2011}) and Asscher, Weiner, et +al. (\citeproc{ref-asscherVariationsAtomic2011}{2011}).\\ Samples were compared to published trendlines for archaeological and -modern enamel -(\protect\hyperlink{ref-asscherAtomicDisorder2011}{Asscher, Regev, et -al., 2011}). We see no appreciable differences between days 16, 20, and -24. The archaeological dental calculus shows a slightly increased slope -compared to model calculus from the three sampling days used in the -grind curve (Figure~\ref{fig-grind-curve}), possibly indicating larger -crystal size due to more complete crystalisation. The steeper slope of -enamel samples is consistent with a more ordered structure in enamel -compared to dental calculus. +modern enamel (\citeproc{ref-asscherAtomicDisorder2011}{Asscher, Regev, +et al., 2011}). We see no appreciable differences between days 16, 20, +and 24. The archaeological dental calculus shows a slightly increased +slope compared to model calculus from the three sampling days used in +the grind curve (Figure~\ref{fig-grind-curve}), possibly indicating +larger crystal size due to more complete crystalisation. The steeper +slope of enamel samples is consistent with a more ordered structure in +enamel compared to dental calculus. \begin{figure} -{\centering \includegraphics{figures/byoc-valid-fig-grind-curve-1.pdf} +\centering{ + +\includegraphics{figures/byoc-valid-fig-grind-curve-1.pdf} } @@ -3772,10 +3738,9 @@ \subsection{Samples show similar crystallinity and order to reference calculus compared to published trendlines (dashed light grey lines) for archaeological (dotted line) and modern (dashed line) enamel.} -\end{figure} +\end{figure}% -\hypertarget{discussion}{% -\section{Discussion}\label{discussion}} +\section{Discussion}\label{discussion} In this study we present a calcifying oral biofilm model to produce artificial dental calculus. Our proposed use of the model is to address @@ -3791,23 +3756,22 @@ \section{Discussion}\label{discussion}} and FTIR analysis to explore the bacterial and mineral composition, and compare with oral reference samples. -\hypertarget{microbiome}{% -\subsection{Microbiome}\label{microbiome}} +\subsection{Microbiome}\label{microbiome} Model calculus has lower species diversity than inocula saliva and oral reference samples, which is a common limitation in biofilm models -(\protect\hyperlink{ref-bjarnsholtVivoBiofilm2013}{Bjarnsholt et al., -2013}; \protect\hyperlink{ref-edlundBiofilmModel2013}{Edlund et al., -2013}). The donated saliva for the experiment had a lower diversity than -the reference saliva samples, and may have contributed to a lower -diversity in experimental samples. Consequently, there is also a lower -diversity and richness when compared to other modern oral reference -samples, including oral mucosa, saliva, plaque, and calculus. Samples of -the medium from early in the experiment have similar species profiles to -the donated saliva, but gradually diverge over the course of the -experiment. This may be caused by experimental setup not sufficiently -mimicking the oral environment, allowing species to thrive that do not -normally thrive in the natural oral environment. +(\citeproc{ref-bjarnsholtVivoBiofilm2013}{Bjarnsholt et al., 2013}; +\citeproc{ref-edlundBiofilmModel2013}{Edlund et al., 2013}). The donated +saliva for the experiment had a lower diversity than the reference +saliva samples, and may have contributed to a lower diversity in +experimental samples. Consequently, there is also a lower diversity and +richness when compared to other modern oral reference samples, including +oral mucosa, saliva, plaque, and calculus. Samples of the medium from +early in the experiment have similar species profiles to the donated +saliva, but gradually diverge over the course of the experiment. This +may be caused by experimental setup not sufficiently mimicking the oral +environment, allowing species to thrive that do not normally thrive in +the natural oral environment. Oral reference samples have a relative abundance of streptococci similar to our model, but a more diverse representation from other genera and an @@ -3821,9 +3785,8 @@ \subsection{Microbiome}\label{microbiome}} facultative anaerobes. This is likely a result of communities of bacteria within the biofilm creating favourable microenvironments facilitated by the protective properties of the biofilm matrix -(\protect\hyperlink{ref-edlundUncoveringComplex2018}{Edlund et al., -2018}; \protect\hyperlink{ref-flemmingBiofilmsEmergent2016}{Flemming et -al., 2016}). +(\citeproc{ref-edlundUncoveringComplex2018}{Edlund et al., 2018}; +\citeproc{ref-flemmingBiofilmsEmergent2016}{Flemming et al., 2016}). Overall, the majority of model calculus samples contained a distinctly oral signature, providing a promising starting point for the use of the @@ -3832,15 +3795,14 @@ \subsection{Microbiome}\label{microbiome}} can be large differences in the oral microbiome of two individuals at the species level due to variations in age, sex, and other demographic factors, as well as how and when saliva samples were collected -(\protect\hyperlink{ref-burchamPatternsOral2020}{Burcham et al., 2020}; -\protect\hyperlink{ref-nearingAssessingVariation2020}{Nearing et al., -2020}). Whether or not distinct microbial profiles, and the -extracellular matrix they produce, will affect the retention of dietary -particles in plaque remains to be seen, but is an important question to -address in the future. +(\citeproc{ref-burchamPatternsOral2020}{Burcham et al., 2020}; +\citeproc{ref-nearingAssessingVariation2020}{Nearing et al., 2020}). +Whether or not distinct microbial profiles, and the extracellular matrix +they produce, will affect the retention of dietary particles in plaque +remains to be seen, but is an important question to address in the +future. -\hypertarget{mineralisation}{% -\subsection{Mineralisation}\label{mineralisation}} +\subsection{Mineralisation}\label{mineralisation} FTIR analysis allowed us to address the mineralisation process of the model, which showed an increasing mineral composition over the course of @@ -3848,10 +3810,10 @@ \subsection{Mineralisation}\label{mineralisation}} content of early samples was replaced by inorganic content in the form of carbonated hydroxyapatite, consistent with a shift from a high presence of bacterial cells in a matrix of extracellular polysaccharides -(\protect\hyperlink{ref-jainIsolationCharacterization2013}{Jain et al., -2013}; \protect\hyperlink{ref-sutherlandBiofilmMatrix2001}{Sutherland, -2001}; \protect\hyperlink{ref-zhangMeasurementPolysaccharides1998}{Zhang -et al., 1998}) to a predominantly mineral content. +(\citeproc{ref-jainIsolationCharacterization2013}{Jain et al., 2013}; +\citeproc{ref-sutherlandBiofilmMatrix2001}{Sutherland, 2001}; +\citeproc{ref-zhangMeasurementPolysaccharides1998}{Zhang et al., 1998}) +to a predominantly mineral content. The model calculus samples resemble both the modern reference calculus and the archaeological calculus in mineral composition and @@ -3861,36 +3823,33 @@ \subsection{Mineralisation}\label{mineralisation}} possible explanation is that the inorganic crystals within archaeological calculus have had more time to grow into the space left by degraded organic matter -(\protect\hyperlink{ref-weinerBiologicalMaterials2010}{Weiner, 2010a}); -however, we only analysed one archaeological sample and cannot -definitively address this. The short duration of model calculus growth -may also have affected the results, compared to the longer-term growth -and mineralisation of natural calculus. The constant disruptions in -growth of \emph{in vivo} dental plaque/calculus, due to oral hygiene and -other external pressures on biofilm growth, may lead to multiple stages -of calcium phosphates, whereas our model has more stable growth -conditions. +(\citeproc{ref-weinerBiologicalMaterials2010}{Weiner, 2010a}); however, +we only analysed one archaeological sample and cannot definitively +address this. The short duration of model calculus growth may also have +affected the results, compared to the longer-term growth and +mineralisation of natural calculus. The constant disruptions in growth +of \emph{in vivo} dental plaque/calculus, due to oral hygiene and other +external pressures on biofilm growth, may lead to multiple stages of +calcium phosphates, whereas our model has more stable growth conditions. One of the most well-known biomineralisers, \emph{Corynebacterium matruchotii} -(\protect\hyperlink{ref-enneverCharacterizationBacterionema1978}{Ennever -et al., 1978}; -\protect\hyperlink{ref-takazoeCalciumHydroxyapatite1970}{Takazoe et al., +(\citeproc{ref-enneverCharacterizationBacterionema1978}{Ennever et al., +1978}; \citeproc{ref-takazoeCalciumHydroxyapatite1970}{Takazoe et al., 1970}), exhibited a lower abundance in our model calculus compared to modern reference calculus. However, the mineral composition of the end results were similar, reinforcing the idea that, under the right circumstances, biofilms with a range of microbial profiles can facilitate mineralisation -(\protect\hyperlink{ref-moorerCalcificationCariogenic1993}{Moorer et -al., 1993}). Bacteria and their ability to secrete an extracellular -matrix are integral in the formation of dental calculus, and inevitably -serve as part of the structure that dental calculus is built upon -(\protect\hyperlink{ref-rohanizadehUltrastructuralStudy2005}{Rohanizadeh -\& LeGeros, 2005}), while the exact species composition of the biofilm +(\citeproc{ref-moorerCalcificationCariogenic1993}{Moorer et al., 1993}). +Bacteria and their ability to secrete an extracellular matrix are +integral in the formation of dental calculus, and inevitably serve as +part of the structure that dental calculus is built upon +(\citeproc{ref-rohanizadehUltrastructuralStudy2005}{Rohanizadeh \& +LeGeros, 2005}), while the exact species composition of the biofilm communities may be less important. -\hypertarget{replicability}{% -\subsection{Replicability}\label{replicability}} +\subsection{Replicability}\label{replicability} Model calculus displayed similar species diversity and microbial profiles across all samples, indicating a high level of replicability @@ -3899,16 +3858,15 @@ \subsection{Replicability}\label{replicability}} between-experiment replicability in our model, though others have already shown that replicability in long-term models is possible when using the same inocula -(\protect\hyperlink{ref-velskoConsistentReproducible2018}{Velsko \& -Shaddox, 2018}). The variation in mineral composition in our model was -initially high, but samples from day 24 were largely similar in -composition as observed in the FTIR spectra. The use of a simple -multiwell plate setup allows us to submit many samples to the same -conditions, increasing replicability between samples -(\protect\hyperlink{ref-extercateAAA2010}{Exterkate et al., 2010}). +(\citeproc{ref-velskoConsistentReproducible2018}{Velsko \& Shaddox, +2018}). The variation in mineral composition in our model was initially +high, but samples from day 24 were largely similar in composition as +observed in the FTIR spectra. The use of a simple multiwell plate setup +allows us to submit many samples to the same conditions, increasing +replicability between samples (\citeproc{ref-extercateAAA2010}{Exterkate +et al., 2010}). -\hypertarget{limitations}{% -\subsection{Limitations}\label{limitations}} +\subsection{Limitations}\label{limitations} While our in vitro model calculus system provides reproducible and consistent artificial dental calculus for archaeological research, as @@ -3920,14 +3878,12 @@ \subsection{Limitations}\label{limitations}} donor instead of pooling saliva from multiple individuals. However, having a single inoculum donor allows us to maintain the integrity of a native oral microbiome which may be lost when samples are pooled -(\protect\hyperlink{ref-edlundBiofilmModel2013}{Edlund et al., 2013}). -It is also possible that the diversity was affected by the collection -and storage methods we used. This has been shown to have minimal effect -on microbial profiles at the genus level -(\protect\hyperlink{ref-limSalivaMicrobiome2017}{Lim et al., 2017}), but -some effect on beta diversity calculations -(\protect\hyperlink{ref-omoriComparativeEvaluation2021}{Omori et al., -2021}). +(\citeproc{ref-edlundBiofilmModel2013}{Edlund et al., 2013}). It is also +possible that the diversity was affected by the collection and storage +methods we used. This has been shown to have minimal effect on microbial +profiles at the genus level (\citeproc{ref-limSalivaMicrobiome2017}{Lim +et al., 2017}), but some effect on beta diversity calculations +(\citeproc{ref-omoriComparativeEvaluation2021}{Omori et al., 2021}). Some samples were grown with starch-sucrose solutions as nutrients, while controls were grown with sucrose only. Due to the financial cost, @@ -3957,8 +3913,7 @@ \subsection{Limitations}\label{limitations}} the biofilm itself. Going forward we recommend sampling from the actual biofilm, as this is the sample type under investigation. -\hypertarget{future-work}{% -\subsection{Future work}\label{future-work}} +\subsection{Future work}\label{future-work} Further protocol optimisation will also be necessary to address some of the limitations of our current model, such as reducing the frequency of @@ -3966,20 +3921,19 @@ \subsection{Future work}\label{future-work}} growth of slow-growing fastidious organisms and limit generalists such as enterococci, and supplementing it with serum to provide additional nutrients and biofilm stability -(\protect\hyperlink{ref-ammannZurichBiofilm2012}{Ammann et al., 2012}; -\protect\hyperlink{ref-tianUsingDGGE2010}{Tian et al., 2010}). More -infrequent medium replacement would facilitate slow-growing bacteria in +(\citeproc{ref-ammannZurichBiofilm2012}{Ammann et al., 2012}; +\citeproc{ref-tianUsingDGGE2010}{Tian et al., 2010}). More infrequent +medium replacement would facilitate slow-growing bacteria in establishing their metabolic relationships, allowing the byproducts of some species to become abundant enough for others that depend on these -to grow (\protect\hyperlink{ref-marshDentalPlaque2005}{Marsh, 2005}). +to grow (\citeproc{ref-marshDentalPlaque2005}{Marsh, 2005}). Our goals for additional validation measures involve functional profiles of bacteria, to see if metabolic behaviour of bacteria is consistent with \emph{in vivo} conditions, and whether this is affected by the presence/absence of amylase and starch treatments. The absence of host salivary \(\alpha\)-amylase activity in our model (as shown in Bartholdy -\& Henry -(\protect\hyperlink{ref-bartholdyInvestigatingBiases2022}{2022})) +\& Henry (\citeproc{ref-bartholdyInvestigatingBiases2022}{2022})) provides an opportunity to explore the effect of various amylase levels on biofilm growth and composition, as well as the incorporation of dietary compounds in dental calculus. @@ -3990,8 +3944,7 @@ \subsection{Future work}\label{future-work}} dietary components of interest, such as cooked starches, whole plant extracts, and various proteins. -\hypertarget{conclusions}{% -\section{Conclusions}\label{conclusions}} +\section{Conclusions}\label{conclusions} The bacterial profile of our model calculus is not an exact match to the natural modern or archaeological reference calculus, but species @@ -4019,12 +3972,11 @@ \section{Conclusions}\label{conclusions}} rather to balance limitations of each method and serve as a complementary approach to expand our toolkit. -\hypertarget{references-cited-2}{% -\section{References cited}\label{references-cited-2}} +\section{References cited}\label{references-cited-2} -\hypertarget{refs-3}{} +\phantomsection\label{refs-3} \begin{CSLReferences}{1}{0} -\leavevmode\vadjust pre{\hypertarget{ref-adlerSequencingAncient2013}{}}% +\bibitem[\citeproctext]{ref-adlerSequencingAncient2013} Adler, C. J., Dobney, K., Weyrich, L. S., Kaidonis, J., Walker, A. W., Haak, W., Bradshaw, C. J., Townsend, G., Sołtysiak, A., Alt, K. W., Parkhill, J., \& Cooper, A. (2013). Sequencing ancient calcified dental @@ -4032,57 +3984,58 @@ \section{References cited}\label{references-cited-2}} {Neolithic} and {Industrial} revolutions. \emph{Nature Genetics}, \emph{45}(4), 450--455, 455e1. \url{https://doi.org/10.1038/ng.2536} -\leavevmode\vadjust pre{\hypertarget{ref-ammannZurichBiofilm2012}{}}% +\bibitem[\citeproctext]{ref-ammannZurichBiofilm2012} Ammann, T. W., Gmür, R., \& Thurnheer, T. (2012). Advancement of the 10-species subgingival {Zurich} biofilm model by examining different nutritional conditions and defining the structure of the in vitro biofilms. \emph{BMC Microbiology}, \emph{12}, 227. \url{https://doi.org/10.1186/1471-2180-12-227} -\leavevmode\vadjust pre{\hypertarget{ref-aronHalfUDG2020}{}}% +\bibitem[\citeproctext]{ref-aronHalfUDG2020} Aron, F., Neumann, G., \& Brandt, G. (2020). Half-{UDG} treated -double-stranded ancient {DNA} library preparation for illumina -sequencing v1 {[}{Data} set{]}. \emph{Protocols. Io}. +double-stranded ancient {DNA} library preparation for {Illumina} +sequencing. \emph{Protocols.io}. +\url{https://doi.org/10.17504/protocols.io.bmh6k39e} -\leavevmode\vadjust pre{\hypertarget{ref-asscherAtomicDisorder2011}{}}% +\bibitem[\citeproctext]{ref-asscherAtomicDisorder2011} Asscher, Y., Regev, L., Weiner, S., \& Boaretto, E. (2011). Atomic {Disorder} in {Fossil Tooth} and {Bone Mineral}: {An FTIR Study Using} the {Grinding Curve Method}. \emph{ArcheoSciences. Revue -d'arch{é}om{é}trie}, \emph{35}, 135--141. +d'archéométrie}, \emph{35}, 135--141. \url{https://doi.org/10.4000/archeosciences.3062} -\leavevmode\vadjust pre{\hypertarget{ref-asscherVariationsAtomic2011}{}}% +\bibitem[\citeproctext]{ref-asscherVariationsAtomic2011} Asscher, Y., Weiner, S., \& Boaretto, E. (2011). Variations in {Atomic Disorder} in {Biogenic Carbonate Hydroxyapatite Using} the {Infrared Spectrum Grinding Curve Method}. \emph{Advanced Functional Materials}, \emph{21}(17), 3308--3313. \url{https://doi.org/10.1002/adfm.201100266} -\leavevmode\vadjust pre{\hypertarget{ref-bartholdyMultiproxyAnalysis2023}{}}% +\bibitem[\citeproctext]{ref-bartholdyMultiproxyAnalysis2023} Bartholdy, B. P., Hasselstrøm, J. B., Sørensen, L. K., Casna, M., Hoogland, M., Beemster, H. G., \& Henry, A. G. (2023). \emph{Multiproxy analysis exploring patterns of diet and disease in dental calculus and skeletal remains from a 19th century {Dutch} population}. {Zenodo}. \url{https://doi.org/10.5281/zenodo.7649151} -\leavevmode\vadjust pre{\hypertarget{ref-bartholdyInvestigatingBiases2022}{}}% +\bibitem[\citeproctext]{ref-bartholdyInvestigatingBiases2022} Bartholdy, B. P., \& Henry, A. G. (2022). Investigating {Biases Associated With Dietary Starch Incorporation} and {Retention With} an {Oral Biofilm Model}. \emph{Frontiers in Earth Science}, \emph{10}. \url{https://doi.org/10.3389/feart.2022.886512} -\leavevmode\vadjust pre{\hypertarget{ref-bjarnsholtVivoBiofilm2013}{}}% +\bibitem[\citeproctext]{ref-bjarnsholtVivoBiofilm2013} Bjarnsholt, T., Alhede, M., Alhede, M., Eickhardt-Sørensen, S. R., Moser, C., Kühl, M., Jensen, P. Ø., \& Høiby, N. (2013). The in vivo biofilm. \emph{Trends in Microbiology}, \emph{21}(9), 466--474. \url{https://doi.org/10.1016/j.tim.2013.06.002} -\leavevmode\vadjust pre{\hypertarget{ref-buckleyDentalCalculus2014}{}}% +\bibitem[\citeproctext]{ref-buckleyDentalCalculus2014} Buckley, S., Usai, D., Jakob, T., Radini, A., \& Hardy, K. (2014). Dental {Calculus Reveals Unique Insights} into {Food Items}, {Cooking} and {Plant Processing} in {Prehistoric Central Sudan}. \emph{PLOS ONE}, \emph{9}(7), e100808. \url{https://doi.org/10.1371/journal.pone.0100808} -\leavevmode\vadjust pre{\hypertarget{ref-burchamPatternsOral2020}{}}% +\bibitem[\citeproctext]{ref-burchamPatternsOral2020} Burcham, Z. M., Garneau, N. L., Comstock, S. S., Tucker, R. M., Knight, R., Metcalf, J. L., Genetics of Taste Lab Citizen Scientists, Miranda, A., Reinhart, B., Meyers, D., Woltkamp, D., Boxer, E., Hutchens, J., @@ -4092,14 +4045,14 @@ \section{References cited}\label{references-cited-2}} \emph{Scientific Reports}, \emph{10}(1), 2133. \url{https://doi.org/10.1038/s41598-020-59016-0} -\leavevmode\vadjust pre{\hypertarget{ref-Rdecontam}{}}% +\bibitem[\citeproctext]{ref-Rdecontam} Davis, N. M., Proctor, D. M., Holmes, S. P., Relman, D. A., \& Callahan, B. J. (2018). Simple statistical identification and removal of contaminant sequences in marker-gene and metagenomics data. \emph{Microbiome}, \emph{6}(1), 226. \url{https://doi.org/10.1186/s40168-018-0605-2} -\leavevmode\vadjust pre{\hypertarget{ref-edlundBiofilmModel2013}{}}% +\bibitem[\citeproctext]{ref-edlundBiofilmModel2013} Edlund, A., Yang, Y., Hall, A. P., Guo, L., Lux, R., He, X., Nelson, K. E., Nealson, K. H., Yooseph, S., Shi, W., \& McLean, J. S. (2013). An in vitrobiofilm model system maintaining a highly reproducible species and @@ -4107,14 +4060,14 @@ \section{References cited}\label{references-cited-2}} \emph{Microbiome}, \emph{1}(1), 25. \url{https://doi.org/10.1186/2049-2618-1-25} -\leavevmode\vadjust pre{\hypertarget{ref-edlundUncoveringComplex2018}{}}% +\bibitem[\citeproctext]{ref-edlundUncoveringComplex2018} Edlund, A., Yang, Y., Yooseph, S., He, X., Shi, W., \& McLean, J. S. (2018). Uncovering complex microbiome activities via metatranscriptomics during 24 hours of oral biofilm assembly and maturation. \emph{Microbiome}, \emph{6}(1), 217. \url{https://doi.org/10.1186/s40168-018-0591-4} -\leavevmode\vadjust pre{\hypertarget{ref-eerkensDentalCalculus2018}{}}% +\bibitem[\citeproctext]{ref-eerkensDentalCalculus2018} Eerkens, J. W., Tushingham, S., Brownstein, K. J., Garibay, R., Perez, K., Murga, E., Kaijankoski, P., Rosenthal, J. S., \& Gang, D. R. (2018). Dental calculus as a source of ancient alkaloids: {Detection} of @@ -4122,20 +4075,20 @@ \section{References cited}\label{references-cited-2}} \emph{Journal of Archaeological Science: Reports}, \emph{18}, 509--515. \url{https://doi.org/10.1016/j.jasrep.2018.02.004} -\leavevmode\vadjust pre{\hypertarget{ref-enneverCharacterizationBacterionema1978}{}}% +\bibitem[\citeproctext]{ref-enneverCharacterizationBacterionema1978} Ennever, J., Riggan, L. J., Vogel, J. J., \& Boyan-Salyers, B. (1978). Characterization of {Bacterionema} matruchotii {Calcification Nucleator}. \emph{Journal of Dental Research}, \emph{57}(4), 637--642. \url{https://doi.org/10.1177/00220345780570041901} -\leavevmode\vadjust pre{\hypertarget{ref-extercateAAA2010}{}}% +\bibitem[\citeproctext]{ref-extercateAAA2010} Exterkate, R. A. M., Crielaard, W., \& Ten Cate, J. M. (2010). Different {Response} to {Amine Fluoride} by {Streptococcus} mutans and {Polymicrobial Biofilms} in a {Novel High-Throughput Active Attachment Model}. \emph{Caries Research}, \emph{44}(4), 372--379. \url{https://doi.org/10.1159/000316541} -\leavevmode\vadjust pre{\hypertarget{ref-fagernasMicrobialBiogeography2022}{}}% +\bibitem[\citeproctext]{ref-fagernasMicrobialBiogeography2022} Fagernäs, Z., Salazar-García, D. C., Haber Uriarte, M., Avilés Fernández, A., Henry, A. G., Lomba Maurandi, J., Ozga, A. T., Velsko, I. M., \& Warinner, C. (2022). Understanding the microbial biogeography of @@ -4143,14 +4096,14 @@ \section{References cited}\label{references-cited-2}} \emph{FEMS Microbes}, \emph{3}, xtac006. \url{https://doi.org/10.1093/femsmc/xtac006} -\leavevmode\vadjust pre{\hypertarget{ref-yatesEAGER2020}{}}% +\bibitem[\citeproctext]{ref-yatesEAGER2020} Fellows Yates, J. A., Lamnidis, T. C., Borry, M., Valtueña, A. A., Fagernäs, Z., Clayton, S., Garcia, M. U., Neukamm, J., \& Peltzer, A. (2020). Reproducible, portable, and efficient ancient genome reconstruction with nf-core/eager. \emph{bioRxiv}, 2020.06.11.145615. \url{https://doi.org/10.1101/2020.06.11.145615} -\leavevmode\vadjust pre{\hypertarget{ref-yatesOralMicrobiome2021}{}}% +\bibitem[\citeproctext]{ref-yatesOralMicrobiome2021} Fellows Yates, J. A., Velsko, I. M., Aron, F., Posth, C., Hofman, C. A., Austin, R. M., Parker, C. E., Mann, A. E., Nägele, K., Arthur, K. W., Arthur, J. W., Bauer, C. C., Crevecoeur, I., Cupillard, C., Curtis, M. @@ -4160,38 +4113,38 @@ \section{References cited}\label{references-cited-2}} National Academy of Sciences}, \emph{118}(20). \url{https://doi.org/10.1073/pnas.2021655118} -\leavevmode\vadjust pre{\hypertarget{ref-filochePlaqueMicrocosm2007}{}}% +\bibitem[\citeproctext]{ref-filochePlaqueMicrocosm2007} Filoche, S. K., Soma, K. J., \& Sissons, C. H. (2007). Caries-related plaque microcosm biofilms developed in microplates. \emph{Oral Microbiology and Immunology}, \emph{22}(2), 73--79. \url{https://doi.org/10.1111/j.1399-302X.2007.00323.x} -\leavevmode\vadjust pre{\hypertarget{ref-flemmingBiofilmsEmergent2016}{}}% +\bibitem[\citeproctext]{ref-flemmingBiofilmsEmergent2016} Flemming, H.-C., Wingender, J., Szewzyk, U., Steinberg, P., Rice, S. A., \& Kjelleberg, S. (2016). Biofilms: An emergent form of bacterial life. \emph{Nature Reviews Microbiology}, \emph{14}(9), 563--575. \url{https://doi.org/10.1038/nrmicro.2016.94} -\leavevmode\vadjust pre{\hypertarget{ref-gloorMicrobiomeDatasets2017}{}}% +\bibitem[\citeproctext]{ref-gloorMicrobiomeDatasets2017} Gloor, G. B., Macklaim, J. M., Pawlowsky-Glahn, V., \& Egozcue, J. J. (2017). Microbiome {Datasets Are Compositional}: {And This Is Not Optional}. \emph{Frontiers in Microbiology}, \emph{8}, 2224. \url{https://doi.org/10.3389/fmicb.2017.02224} -\leavevmode\vadjust pre{\hypertarget{ref-hardyStarchGranules2009}{}}% +\bibitem[\citeproctext]{ref-hardyStarchGranules2009} Hardy, K., Blakeney, T., Copeland, L., Kirkham, J., Wrangham, R., \& Collins, M. (2009). Starch granules, dental calculus and new perspectives on ancient diet. \emph{Journal of Archaeological Science}, \emph{36}(2), 248--255. \url{https://doi.org/10.1016/j.jas.2008.09.015} -\leavevmode\vadjust pre{\hypertarget{ref-hayashizakiSiteSpecific2008}{}}% +\bibitem[\citeproctext]{ref-hayashizakiSiteSpecific2008} Hayashizaki, J., Ban, S., Nakagaki, H., Okumura, A., Yoshii, S., \& Robinson, C. (2008). Site specific mineral composition and microstructure of human supra-gingival dental calculus. \emph{Archives of Oral Biology}, \emph{53}(2), 168--174. \url{https://doi.org/10.1016/j.archoralbio.2007.09.003} -\leavevmode\vadjust pre{\hypertarget{ref-hendyProteomicCalculus2018}{}}% +\bibitem[\citeproctext]{ref-hendyProteomicCalculus2018} Hendy, J., Warinner, C., Bouwman, A., Collins, M. J., Fiddyment, S., Fischer, R., Hagan, R., Hofman, C. A., Holst, M., Chaves, E., Klaus, L., Larson, G., Mackie, M., McGrath, K., Mundorff, A. Z., Radini, A., Rao, @@ -4200,21 +4153,21 @@ \section{References cited}\label{references-cited-2}} \emph{Proceedings. Biological Sciences}, \emph{285}(1883), 20180977. \url{https://doi.org/10.1098/rspb.2018.0977} -\leavevmode\vadjust pre{\hypertarget{ref-henryCalculusSyria2008}{}}% +\bibitem[\citeproctext]{ref-henryCalculusSyria2008} Henry, A. G., \& Piperno, D. R. (2008). Using plant microfossils from dental calculus to recover human diet: A case study from {Tell} -al-{Raq{ā}}'i, {Syria}. \emph{Journal of Archaeological Science}, +al-{Raqā}'i, {Syria}. \emph{Journal of Archaeological Science}, \emph{35}(7), 1943--1950. \url{https://doi.org/10.1016/j.jas.2007.12.005} -\leavevmode\vadjust pre{\hypertarget{ref-jainIsolationCharacterization2013}{}}% +\bibitem[\citeproctext]{ref-jainIsolationCharacterization2013} Jain, K., Parida, S., Mangwani, N., Dash, H. R., \& Das, S. (2013). Isolation and characterization of biofilm-forming bacteria and associated extracellular polymeric substances from oral cavity. \emph{Annals of Microbiology}, \emph{63}(4), 1553--1562. \url{https://doi.org/10.1007/s13213-013-0618-9} -\leavevmode\vadjust pre{\hypertarget{ref-jiFluorideMagnesium2000}{}}% +\bibitem[\citeproctext]{ref-jiFluorideMagnesium2000} Ji, H., Nakagaki, H., Hayashizaki, J., Tsuboi, S., Kato, K., Toyama, A., Arai, K., Thuy, T. T., Ha, N. T. T., Kameyama, Y., Kirkham, J., \& Robinson, C. (2000). Fluoride and magnesium concentrations in human @@ -4222,117 +4175,117 @@ \section{References cited}\label{references-cited-2}} \emph{Archives of Oral Biology}, \emph{45}(7), 611--615. \url{https://doi.org/10.1016/S0003-9969(00)00021-2} -\leavevmode\vadjust pre{\hypertarget{ref-jinSupragingivalCalculus2002}{}}% +\bibitem[\citeproctext]{ref-jinSupragingivalCalculus2002} Jin, Y., \& Yip, H.-K. (2002). Supragingival {Calculus}: {Formation} and {Control}. \emph{Critical Reviews in Oral Biology \& Medicine}. \url{https://doi.org/10.1177/154411130201300506} -\leavevmode\vadjust pre{\hypertarget{ref-kazarinaPostmedievalMicrobial2021}{}}% +\bibitem[\citeproctext]{ref-kazarinaPostmedievalMicrobial2021} Kazarina, A., Petersone-Gordina, E., Kimsis, J., Kuzmicka, J., Zayakin, P., Griškjans, Ž., Gerhards, G., \& Ranka, R. (2021). The {Postmedieval Latvian Oral Microbiome} in the {Context} of {Modern Dental Calculus} and {Modern Dental Plaque Microbial Profiles}. \emph{Genes}, \emph{12}(2), 309. \url{https://doi.org/10.3390/genes12020309} -\leavevmode\vadjust pre{\hypertarget{ref-knightsSourceTracker2011}{}}% +\bibitem[\citeproctext]{ref-knightsSourceTracker2011} Knights, D., Kuczynski, J., Charlson, E. S., Zaneveld, J., Mozer, M. C., Collman, R. G., Bushman, F. D., Knight, R., \& Kelley, S. T. (2011). Bayesian community-wide culture-independent microbial source tracking. \emph{Nature Methods}, \emph{8}(9), 761--763. \url{https://doi.org/10.1038/nmeth.1650} -\leavevmode\vadjust pre{\hypertarget{ref-lemmersMiddenbeemster2013}{}}% +\bibitem[\citeproctext]{ref-lemmersMiddenbeemster2013} Lemmers, S. A. M., Schats, R., Hoogland, M. L. P., \& Waters-Rist, A. (2013). {Fysisch antropologische analyse Middenbeemster}. In \emph{{De begravingen bij de Keyserkerk te Middenbeemster}} (pp. 35--60). -\leavevmode\vadjust pre{\hypertarget{ref-leonardPlantMicroremains2015}{}}% +\bibitem[\citeproctext]{ref-leonardPlantMicroremains2015} Leonard, C., Vashro, L., O'Connell, J. F., \& Henry, A. G. (2015). Plant microremains in dental calculus as a record of plant consumption: {A} test with {Twe} forager-horticulturalists. \emph{Journal of Archaeological Science: Reports}, \emph{2}, 449--457. \url{https://doi.org/10.1016/j.jasrep.2015.03.009} -\leavevmode\vadjust pre{\hypertarget{ref-BWA}{}}% +\bibitem[\citeproctext]{ref-BWA} Li, H., \& Durbin, R. (2009). Fast and accurate short read alignment -with {Burrows}{\textendash}{Wheeler} transform. \emph{Bioinformatics}, +with {Burrows}\textendash{{Wheeler}} transform. \emph{Bioinformatics}, \emph{25}(14), 1754--1760. \url{https://doi.org/10.1093/bioinformatics/btp324} -\leavevmode\vadjust pre{\hypertarget{ref-limSalivaMicrobiome2017}{}}% +\bibitem[\citeproctext]{ref-limSalivaMicrobiome2017} Lim, Y., Totsika, M., Morrison, M., \& Punyadeera, C. (2017). The saliva microbiome profiles are minimally affected by collection method or {DNA} extraction protocols. \emph{Scientific Reports}, \emph{7}(1), 8523. \url{https://doi.org/10.1038/s41598-017-07885-3} -\leavevmode\vadjust pre{\hypertarget{ref-linANCOMBC2020}{}}% +\bibitem[\citeproctext]{ref-linANCOMBC2020} Lin, H., \& Peddada, S. D. (2020). Analysis of compositions of microbiomes with bias correction. \emph{Nature Communications}, \emph{11}(1), 3514. \url{https://doi.org/10.1038/s41467-020-17041-7} -\leavevmode\vadjust pre{\hypertarget{ref-maHumanDiet2022}{}}% +\bibitem[\citeproctext]{ref-maHumanDiet2022} Ma, Z., Liu, S., Li, Z., Ye, M., \& Huan, X. (2022). Human {Diet Patterns During} the {Qijia Cultural Period}: {Integrated Evidence} of {Stable Isotopes} and {Plant Micro-remains From} the {Lajia Site}, {Northwest China}. \emph{Frontiers in Earth Science}, \emph{10}. -\leavevmode\vadjust pre{\hypertarget{ref-marshDentalPlaque2005}{}}% +\bibitem[\citeproctext]{ref-marshDentalPlaque2005} Marsh, P. D. (2005). Dental plaque: Biological significance of a biofilm and community life-style. \emph{Journal of Clinical Periodontology}, \emph{32}(s6), 7--15. \url{https://doi.org/10.1111/j.1600-051X.2005.00790.x} -\leavevmode\vadjust pre{\hypertarget{ref-marshDentalPlaque2006}{}}% +\bibitem[\citeproctext]{ref-marshDentalPlaque2006} Marsh, P. D. (2006). Dental plaque as a biofilm and a microbial -community {\textendash} implications for health and disease. \emph{BMC +community \textendash{} implications for health and disease. \emph{BMC Oral Health}, \emph{6}(S1), S14. \url{https://doi.org/10.1186/1472-6831-6-S1-S14} -\leavevmode\vadjust pre{\hypertarget{ref-mentzerDistributionAuthigenic2014}{}}% +\bibitem[\citeproctext]{ref-mentzerDistributionAuthigenic2014} Mentzer, S. M., Miller, C. E., Kloos, P., Wadley, L., \& Conard, N. J. (2014). The distribution of authigenic minerals in the {Middle Stone Age} deposits of {Sibudu} ({South Africa}), and implications for the preservation of archaeological features. \emph{European Society for the Study of Human Evolution, {4thAnnual} Meeting, Florence, Italy}. -\leavevmode\vadjust pre{\hypertarget{ref-mickleburghNewInsights2012}{}}% +\bibitem[\citeproctext]{ref-mickleburghNewInsights2012} Mickleburgh, H. L., \& Pagán-Jiménez, J. R. (2012). New insights into the consumption of maize and other food plants in the pre-{Columbian Caribbean} from starch grains trapped in human dental calculus. \emph{Journal of Archaeological Science}, \emph{39}(7), 2468--2478. \url{https://doi.org/10.1016/j.jas.2012.02.020} -\leavevmode\vadjust pre{\hypertarget{ref-middletonVitroCalculus1965}{}}% +\bibitem[\citeproctext]{ref-middletonVitroCalculus1965} Middleton, J. D. (1965). Human salivary proteins and artificial calculus formation in vitro. \emph{Archives of Oral Biology}, \emph{10}(2), 227--235. \url{https://doi.org/10.1016/0003-9969(65)90024-5} -\leavevmode\vadjust pre{\hypertarget{ref-moorerCalcificationCariogenic1993}{}}% +\bibitem[\citeproctext]{ref-moorerCalcificationCariogenic1993} Moorer, W. R., Ten Cate, J. M., \& Buijs, J. F. (1993). Calcification of a {Cariogenic Streptococcus} and of {Corynebacterium} ({Bacterionema}) matruchotii. \emph{Journal of Dental Research}, \emph{72}(6), 1021--1026. \url{https://doi.org/10.1177/00220345930720060501} -\leavevmode\vadjust pre{\hypertarget{ref-nearingAssessingVariation2020}{}}% +\bibitem[\citeproctext]{ref-nearingAssessingVariation2020} Nearing, J. T., DeClercq, V., Van Limbergen, J., \& Langille, M. G. I. (2020). Assessing the {Variation} within the {Oral Microbiome} of {Healthy Adults}. \emph{mSphere}, \emph{5}(5), e00451--20. \url{https://doi.org/10.1128/mSphere.00451-20} -\leavevmode\vadjust pre{\hypertarget{ref-Rvegan}{}}% +\bibitem[\citeproctext]{ref-Rvegan} Oksanen, J., Simpson, G. L., Blanchet, F. G., Kindt, R., Legendre, P., Minchin, P. R., O'Hara, R. B., Solymos, P., Stevens, M. H. H., Szoecs, E., Wagner, H., Barbour, M., Bedward, M., Bolker, B., Borcard, D., Carvalho, G., Chirico, M., De Caceres, M., Durand, S., \ldots{} Weedon, J. (2022). \emph{Vegan: {Community} ecology package} {[}Manual{]}. -\leavevmode\vadjust pre{\hypertarget{ref-omelonReviewPhosphate2013}{}}% +\bibitem[\citeproctext]{ref-omelonReviewPhosphate2013} Omelon, S., Ariganello, M., Bonucci, E., Grynpas, M., \& Nanci, A. (2013). A {Review} of {Phosphate Mineral Nucleation} in {Biology} and {Geobiology}. \emph{Calcified Tissue International}, \emph{93}(4), 382--396. \url{https://doi.org/10.1007/s00223-013-9784-9} -\leavevmode\vadjust pre{\hypertarget{ref-omoriComparativeEvaluation2021}{}}% +\bibitem[\citeproctext]{ref-omoriComparativeEvaluation2021} Omori, M., Kato-Kogoe, N., Sakaguchi, S., Fukui, N., Yamamoto, K., Nakajima, Y., Inoue, K., Nakano, H., Motooka, D., Nakano, T., Nakamura, S., \& Ueno, T. (2021). Comparative evaluation of microbial profiles of @@ -4340,109 +4293,110 @@ \section{References cited}\label{references-cited-2}} different methods. \emph{Clinical Oral Investigations}, \emph{25}(5), 2779--2789. \url{https://doi.org/10.1007/s00784-020-03592-y} -\leavevmode\vadjust pre{\hypertarget{ref-pearceConcomitantDeposition1987}{}}% +\bibitem[\citeproctext]{ref-pearceConcomitantDeposition1987} Pearce, E. I. F., \& Sissons, C. H. (1987). The {Concomitant Deposition} of {Strontium} and {Fluoride} in {Dental Plaque}. \emph{Journal of Dental Research}, \emph{66}(10), 1518--1522. \url{https://doi.org/10.1177/00220345870660100101} -\leavevmode\vadjust pre{\hypertarget{ref-powerChimpCalculus2015}{}}% +\bibitem[\citeproctext]{ref-powerChimpCalculus2015} Power, R. C., Salazar-Garcia, D. C., Wittig, R. M., Freiberg, M., \& Henry, A. G. (2015). Dental calculus evidence of {Tai Forest Chimpanzee} plant consumption and life history transitions. \emph{Scientific Reports}, \emph{5}, 15161. \url{https://doi.org/10.1038/srep15161} -\leavevmode\vadjust pre{\hypertarget{ref-Rbase}{}}% +\bibitem[\citeproctext]{ref-Rbase} R Core Team. (2020). \emph{R: {A} language and environment for statistical computing} {[}Manual{]}. {R Foundation for Statistical -Computing}; {R Foundation for Statistical Computing}. +Computing}. -\leavevmode\vadjust pre{\hypertarget{ref-radiniDirtyTeeth2022}{}}% +\bibitem[\citeproctext]{ref-radiniDirtyTeeth2022} Radini, A., \& Nikita, E. (2022). Beyond dirty teeth: {Integrating} dental calculus studies with osteoarchaeological parameters. \emph{Quaternary International}. \url{https://doi.org/10.1016/j.quaint.2022.03.003} -\leavevmode\vadjust pre{\hypertarget{ref-reimerBacDive2022}{}}% +\bibitem[\citeproctext]{ref-reimerBacDive2022} Reimer, L. C., Sardà Carbasse, J., Koblitz, J., Ebeling, C., Podstawka, A., \& Overmann, J. (2022). {BacDive} in 2022: The knowledge base for standardized bacterial and archaeal data. \emph{Nucleic Acids Research}, \emph{50}(D1), D741--D746. \url{https://doi.org/10.1093/nar/gkab961} -\leavevmode\vadjust pre{\hypertarget{ref-rohanizadehUltrastructuralStudy2005}{}}% +\bibitem[\citeproctext]{ref-rohanizadehUltrastructuralStudy2005} Rohanizadeh, R., \& LeGeros, R. Z. (2005). Ultrastructural study of -calculus{\textendash}enamel and calculus{\textendash}root interfaces. +calculus\textendash enamel and calculus\textendash root interfaces. \emph{Archives of Oral Biology}, \emph{50}(1), 89--96. \url{https://doi.org/10.1016/j.archoralbio.2004.07.001} -\leavevmode\vadjust pre{\hypertarget{ref-RmixOmics}{}}% +\bibitem[\citeproctext]{ref-RmixOmics} Rohart, F., Gautier, B., Singh, A., \& Le Cao, K.-A. (2017). {mixOmics}: {An R} package for 'omics feature selection and multiple data integration. \emph{PLoS Computational Biology}, \emph{13}(11), e1005752. -\leavevmode\vadjust pre{\hypertarget{ref-AdapterRemovalv2}{}}% +\bibitem[\citeproctext]{ref-AdapterRemovalv2} Schubert, M., Lindgreen, S., \& Orlando, L. (2016). {AdapterRemoval} v2: Rapid adapter trimming, identification, and read merging. \emph{BMC Research Notes}, \emph{9}, 88. \url{https://doi.org/10.1186/s13104-016-1900-2} -\leavevmode\vadjust pre{\hypertarget{ref-shellisSyntheticSaliva1978}{}}% +\bibitem[\citeproctext]{ref-shellisSyntheticSaliva1978} Shellis, R. P. (1978). A synthetic saliva for cultural studies of dental plaque. \emph{Archives of Oral Biology}, \emph{23}(6), 485--489. \url{https://doi.org/10.1016/0003-9969(78)90081-X} -\leavevmode\vadjust pre{\hypertarget{ref-sissonsMultistationPlaque1991}{}}% +\bibitem[\citeproctext]{ref-sissonsMultistationPlaque1991} Sissons, C. H., Cutress, T. W., Hoffman, M. P., \& Wakefield, J. S. J. (1991). A {Multi-station Dental Plaque Microcosm} ({Artificial Mouth}) for the {Study} of {Plaque Growth}, {Metabolism}, {pH}, and {Mineralization}: \emph{Journal of Dental Research}. \url{https://doi.org/10.1177/00220345910700110301} -\leavevmode\vadjust pre{\hypertarget{ref-stahlDoublestrandedIndexing2019}{}}% +\bibitem[\citeproctext]{ref-stahlDoublestrandedIndexing2019} Stahl, R., Warinner, C., Velsko, I., Orfanou, E., Aron, F., \& Brandt, -G. (2019). Illumina double-stranded {DNA} dual indexing for ancient -{DNA} v1 {[}{Data} set{]}. \emph{Protocols. Io}. +G. (2020). Illumina double-stranded {DNA} dual indexing for ancient +{DNA}. \emph{Protocols.io}. +\url{https://doi.org/10.17504/protocols.io.4r3l287x3l1y/v3} -\leavevmode\vadjust pre{\hypertarget{ref-sutherlandBiofilmMatrix2001}{}}% -Sutherland, I. W. (2001). The biofilm matrix {\textendash} an +\bibitem[\citeproctext]{ref-sutherlandBiofilmMatrix2001} +Sutherland, I. W. (2001). The biofilm matrix \textendash{} an immobilized but dynamic microbial environment. \emph{Trends in Microbiology}, \emph{9}(5), 222--227. \url{https://doi.org/10.1016/S0966-842X(01)02012-1} -\leavevmode\vadjust pre{\hypertarget{ref-takazoeCalciumHydroxyapatite1970}{}}% +\bibitem[\citeproctext]{ref-takazoeCalciumHydroxyapatite1970} Takazoe, I., Vogel, J., \& Ennever, J. (1970). Calcium {Hydroxyapatite Nucleation} by {Lipid Extract} of {Bacterionema} matruchotii. \emph{Journal of Dental Research}, \emph{49}(2), 395--398. \url{https://doi.org/10.1177/00220345700490023301} -\leavevmode\vadjust pre{\hypertarget{ref-tianUsingDGGE2010}{}}% +\bibitem[\citeproctext]{ref-tianUsingDGGE2010} Tian, Y., He, X., Torralba, M., Yooseph, S., Nelson, K. e., Lux, R., McLean, J. s., Yu, G., \& Shi, W. (2010). Using {DGGE} profiling to develop a novel culture medium suitable for oral microbial communities. \emph{Molecular Oral Microbiology}, \emph{25}(5), 357--367. \url{https://doi.org/10.1111/j.2041-1014.2010.00585.x} -\leavevmode\vadjust pre{\hypertarget{ref-tonjumNeisseria2017}{}}% +\bibitem[\citeproctext]{ref-tonjumNeisseria2017} Tønjum, T., \& van Putten, J. (2017). 179 - {Neisseria}. In J. Cohen, W. G. Powderly, \& S. M. Opal (Eds.), \emph{Infectious {Diseases} ({Fourth Edition})} (pp. 1553--1564.e1). {Elsevier}. \url{https://doi.org/10.1016/B978-0-7020-6285-8.00179-9} -\leavevmode\vadjust pre{\hypertarget{ref-trompEDTACalculus2017}{}}% +\bibitem[\citeproctext]{ref-trompEDTACalculus2017} Tromp, M., Buckley, H., Geber, J., \& Matisoo-Smith, E. (2017). {EDTA} decalcification of dental calculus as an alternate means of microparticle extraction from archaeological samples. \emph{Journal of Archaeological Science: Reports}, \emph{14}, 461--466. \url{https://doi.org/10.1016/j.jasrep.2017.06.035} -\leavevmode\vadjust pre{\hypertarget{ref-velskoCytokineResponse2017}{}}% +\bibitem[\citeproctext]{ref-velskoCytokineResponse2017} Velsko, I. M., Cruz-Almeida, Y., Huang, H., Wallet, S. M., \& Shaddox, L. M. (2017). Cytokine response patterns to complex biofilms by mononuclear cells discriminate patient disease status and biofilm dysbiosis. \emph{Journal of Oral Microbiology}, \emph{9}(1), 1330645. \url{https://doi.org/10.1080/20002297.2017.1330645} -\leavevmode\vadjust pre{\hypertarget{ref-velskoMicrobialDifferences2019}{}}% +\bibitem[\citeproctext]{ref-velskoMicrobialDifferences2019} Velsko, I. M., Fellows Yates, J. A., Aron, F., Hagan, R. W., Frantz, L. A. F., Loe, L., Martinez, J. B. R., Chaves, E., Gosden, C., Larson, G., \& Warinner, C. (2019). Microbial differences between dental plaque and @@ -4450,7 +4404,7 @@ \section{References cited}\label{references-cited-2}} \emph{Microbiome}, \emph{7}(1), 102. \url{https://doi.org/10.1186/s40168-019-0717-3} -\leavevmode\vadjust pre{\hypertarget{ref-velskoDentalCalculus2017}{}}% +\bibitem[\citeproctext]{ref-velskoDentalCalculus2017} Velsko, I. M., Overmyer, K. A., Speller, C., Klaus, L., Collins, M. J., Loe, L., Frantz, L. A. F., Sankaranarayanan, K., Lewis, C. M., Martinez, J. B. R., Chaves, E., Coon, J. J., Larson, G., \& Warinner, C. (2017). @@ -4458,13 +4412,13 @@ \section{References cited}\label{references-cited-2}} \emph{Metabolomics}, \emph{13}(11), 134. \url{https://doi.org/10.1007/s11306-017-1270-3} -\leavevmode\vadjust pre{\hypertarget{ref-velskoConsistentReproducible2018}{}}% +\bibitem[\citeproctext]{ref-velskoConsistentReproducible2018} Velsko, I. M., \& Shaddox, L. M. (2018). Consistent and reproducible long-term in vitro growth of health and disease-associated oral subgingival biofilms. \emph{BMC Microbiology}, \emph{18}(1), 70. \url{https://doi.org/10.1186/s12866-018-1212-x} -\leavevmode\vadjust pre{\hypertarget{ref-warinnerPathogensHost2014}{}}% +\bibitem[\citeproctext]{ref-warinnerPathogensHost2014} Warinner, C., Rodrigues, J. F., Vyas, R., Trachsel, C., Shved, N., Grossmann, J., Radini, A., Hancock, Y., Tito, R. Y., Fiddyment, S., Speller, C., Hendy, J., Charlton, S., Luder, H. U., Salazar-Garcia, D. @@ -4473,42 +4427,42 @@ \section{References cited}\label{references-cited-2}} oral cavity. \emph{Nature Genetics}, \emph{46}(4), 336--344. \url{https://doi.org/10.1038/ng.2906} -\leavevmode\vadjust pre{\hypertarget{ref-warinnerNewEra2015}{}}% +\bibitem[\citeproctext]{ref-warinnerNewEra2015} Warinner, C., Speller, C., \& Collins, M. J. (2015). A new era in palaeomicrobiology: Prospects for ancient dental calculus as a long-term record of the human oral microbiome. \emph{Philosophical Transactions of the Royal Society B: Biological Sciences}, \emph{370}(1660), 20130376. \url{https://doi.org/10.1098/rstb.2013.0376} -\leavevmode\vadjust pre{\hypertarget{ref-weinerBiologicalMaterials2010}{}}% +\bibitem[\citeproctext]{ref-weinerBiologicalMaterials2010} Weiner, S. (2010a). Biological {Materials}: {Bones} and {Teeth}. In \emph{Microarchaeology: {Beyond} the {Visible Archaeological Record}} (pp. 99--134). {Cambridge University Press}. -\leavevmode\vadjust pre{\hypertarget{ref-weinerInfraredSpectroscopy2010}{}}% +\bibitem[\citeproctext]{ref-weinerInfraredSpectroscopy2010} Weiner, S. (2010b). Infrared {Spectroscopy} in {Archaeology}. In \emph{Microarchaeology: {Beyond} the {Visible Archaeological Record}} -(1st ed., pp. 275--316). {Cambridge University Press}. +(First, pp. 275--316). {Cambridge University Press}. \url{https://doi.org/10.1017/CBO9780511811210} -\leavevmode\vadjust pre{\hypertarget{ref-weinerStatesPreservation1990}{}}% +\bibitem[\citeproctext]{ref-weinerStatesPreservation1990} Weiner, S., \& Bar-Yosef, O. (1990). States of preservation of bones from prehistoric sites in the {Near East}: {A} survey. \emph{Journal of Archaeological Science}, \emph{17}(2), 187--196. \url{https://doi.org/10.1016/0305-4403(90)90058-D} -\leavevmode\vadjust pre{\hypertarget{ref-whiteDentalCalculus1997}{}}% +\bibitem[\citeproctext]{ref-whiteDentalCalculus1997} White, D. J. (1997). Dental calculus: Recent insights into occurrence, formation, prevention, removal and oral health effects of supragingival and subgingival deposits. \emph{European Journal of Oral Sciences}, \emph{105}(5), 508--522. \url{https://doi.org/10.1111/j.1600-0722.1997.tb00238.x} -\leavevmode\vadjust pre{\hypertarget{ref-ggplot2}{}}% +\bibitem[\citeproctext]{ref-ggplot2} Wickham, H. (2016). \emph{Ggplot2: {Elegant Graphics} for {Data Analysis}}. {Springer-Verlag}. -\leavevmode\vadjust pre{\hypertarget{ref-tidyverse2019}{}}% +\bibitem[\citeproctext]{ref-tidyverse2019} Wickham, H., Averick, M., Bryan, J., Chang, W., McGowan, L. D., François, R., Grolemund, G., Hayes, A., Henry, L., Hester, J., Kuhn, M., Pedersen, T. L., Miller, E., Bache, S. M., Müller, K., Ooms, J., @@ -4516,19 +4470,19 @@ \section{References cited}\label{references-cited-2}} Welcome to the {tidyverse}. \emph{Journal of Open Source Software}, \emph{4}(43), 1686. \url{https://doi.org/10.21105/joss.01686} -\leavevmode\vadjust pre{\hypertarget{ref-wongCalciumPhosphate2002}{}}% +\bibitem[\citeproctext]{ref-wongCalciumPhosphate2002} Wong, L., Sissons, C. H., Pearce, E. I. F., \& Cutress, T. W. (2002). Calcium phosphate deposition in human dental plaque microcosm biofilms induced by a ureolytic {pH-rise} procedure. \emph{Archives of Oral Biology}, \emph{47}(11), 779--790. \url{https://doi.org/10.1016/S0003-9969(02)00114-0} -\leavevmode\vadjust pre{\hypertarget{ref-kraken2}{}}% +\bibitem[\citeproctext]{ref-kraken2} Wood, D. E., Lu, J., \& Langmead, B. (2019). Improved metagenomic analysis with {Kraken} 2. \emph{Genome Biology}, \emph{20}(1), 257. \url{https://doi.org/10.1186/s13059-019-1891-0} -\leavevmode\vadjust pre{\hypertarget{ref-zhangMeasurementPolysaccharides1998}{}}% +\bibitem[\citeproctext]{ref-zhangMeasurementPolysaccharides1998} Zhang, X., Bishop, P. L., \& Kupferle, M. J. (1998). Measurement of polysaccharides and proteins in biofilm extracellular polymers. \emph{Water Science and Technology}, \emph{37}(4), 345--348. @@ -4538,8 +4492,7 @@ \section{References cited}\label{references-cited-2}} \bookmarksetup{startatroot} -\hypertarget{article-2}{% -\chapter{Article 2}\label{article-2}} +\chapter{Article 2}\label{article-2} Investigating Biases Associated with Dietary Starch Incorporation and Retention with an Oral Biofilm Model @@ -4573,108 +4526,96 @@ \chapter{Article 2}\label{article-2}} \newpage{} -\hypertarget{byocstarch-intro}{% -\section{Introduction}\label{byocstarch-intro}} +\section{Introduction}\label{byocstarch-intro} Dental calculus has proven to contain a wealth of dietary information in the form of plant microfossils -(\protect\hyperlink{ref-hardyStarchGranules2009}{Hardy et al., 2009}; -\protect\hyperlink{ref-henryCalculusSyria2008}{Henry \& Piperno, 2008}), -proteins (\protect\hyperlink{ref-hendyProteomicCalculus2018}{Hendy et -al., 2018}; \protect\hyperlink{ref-warinnerEvidenceMilk2014}{Warinner, -Hendy, et al., 2014}), and other organic residues -(\protect\hyperlink{ref-buckleyDentalCalculus2014}{Buckley et al., -2014}). This dietary information can be preserved within the mineralised -dental plaque over many millennia, providing a unique window into the +(\citeproc{ref-hardyStarchGranules2009}{Hardy et al., 2009}; +\citeproc{ref-henryCalculusSyria2008}{Henry \& Piperno, 2008}), proteins +(\citeproc{ref-hendyProteomicCalculus2018}{Hendy et al., 2018}; +\citeproc{ref-warinnerEvidenceMilk2014}{Warinner, Hendy, et al., 2014}), +and other organic residues +(\citeproc{ref-buckleyDentalCalculus2014}{Buckley et al., 2014}). This +dietary information can be preserved within the mineralised dental +plaque over many millennia, providing a unique window into the food-related behaviours of past populations -(\protect\hyperlink{ref-henryCalculusSyria2008}{Henry \& Piperno, 2008}; -\protect\hyperlink{ref-jovanovicNeolithicCalculus2021}{Jovanović et al., -2021}; \protect\hyperlink{ref-taoWheatCalculus2020}{Tao et al., 2020}) -and extinct species -(\protect\hyperlink{ref-hardyNeanderthalMedics2012}{Hardy et al., 2012}; -\protect\hyperlink{ref-henryNeanderthalCalculus2014}{Henry et al., -2014}). +(\citeproc{ref-henryCalculusSyria2008}{Henry \& Piperno, 2008}; +\citeproc{ref-jovanovicNeolithicCalculus2021}{Jovanović et al., 2021}; +\citeproc{ref-taoWheatCalculus2020}{Tao et al., 2020}) and extinct +species (\citeproc{ref-hardyNeanderthalMedics2012}{Hardy et al., 2012}; +\citeproc{ref-henryNeanderthalCalculus2014}{Henry et al., 2014}). Until recently, only a few studies directly investigated the presence of plant microremains in the dental calculus of archaeological remains. The ability to extract phytoliths from the dental calculus of archaeological fauna to investigate diet was first noted by Armitage -(\protect\hyperlink{ref-armitageExtractionIdentification1975}{1975}), -and later by Middleton and Rovner -(\protect\hyperlink{ref-middletonOpalPhytoliths1994}{1994}), and Fox and -colleagues (\protect\hyperlink{ref-foxPhytolithCalculus1996}{1996}). -Starches and phytoliths were extracted from human dental calculus by -Cummings and Magennis -(\protect\hyperlink{ref-cummingsMayanCalculus1997}{1997}).\\ +(\citeproc{ref-armitageExtractionIdentification1975}{1975}), and later +by Middleton and Rovner +(\citeproc{ref-middletonOpalPhytoliths1994}{1994}), and Fox and +colleagues (\citeproc{ref-foxPhytolithCalculus1996}{1996}). Starches and +phytoliths were extracted from human dental calculus by Cummings and +Magennis (\citeproc{ref-cummingsMayanCalculus1997}{1997}).\\ In more recent years, the study of dental calculus has increased exponentially, and the wealth of information contained within the mineralised matrix has largely been acknowledged. The use of dental calculus spans a wide variety of archaeological research areas, such as oral microbiome characterisation (including pathogens) through the analysis of DNA and proteins -(\protect\hyperlink{ref-adlerSequencingAncient2013}{Adler et al., 2013}; -\protect\hyperlink{ref-warinnerPathogensHost2014}{Warinner, Rodrigues, -et al., 2014}), microbotanical remains -(\protect\hyperlink{ref-hardyStarchGranules2009}{Hardy et al., 2009}; -\protect\hyperlink{ref-henryCalculusSyria2008}{Henry \& Piperno, 2008}; -\protect\hyperlink{ref-mickleburghNewInsights2012}{Mickleburgh \& -Pagán-Jiménez, 2012}), other organic residues and proteins from dietary -compounds (\protect\hyperlink{ref-buckleyDentalCalculus2014}{Buckley et -al., 2014}; \protect\hyperlink{ref-hendyProteomicCalculus2018}{Hendy et -al., 2018}), and nicotine use -(\protect\hyperlink{ref-eerkensDentalCalculus2018}{Eerkens et al., +(\citeproc{ref-adlerSequencingAncient2013}{Adler et al., 2013}; +\citeproc{ref-warinnerPathogensHost2014}{Warinner, Rodrigues, et al., +2014}), microbotanical remains +(\citeproc{ref-hardyStarchGranules2009}{Hardy et al., 2009}; +\citeproc{ref-henryCalculusSyria2008}{Henry \& Piperno, 2008}; +\citeproc{ref-mickleburghNewInsights2012}{Mickleburgh \& Pagán-Jiménez, +2012}), other organic residues and proteins from dietary compounds +(\citeproc{ref-buckleyDentalCalculus2014}{Buckley et al., 2014}; +\citeproc{ref-hendyProteomicCalculus2018}{Hendy et al., 2018}), and +nicotine use (\citeproc{ref-eerkensDentalCalculus2018}{Eerkens et al., 2018}). Especially the extraction of starch granules has become a rich source of dietary information, as starch granules have proven to preserve well within dental calculus over a variety of geographical and -temporal ranges -(\protect\hyperlink{ref-henryNeanderthalCalculus2014}{Henry et al., -2014}; \protect\hyperlink{ref-jovanovicNeolithicCalculus2021}{Jovanović -et al., 2021}; \protect\hyperlink{ref-pipernoStarchGrains2008}{Piperno -\& Dillehay, 2008}; \protect\hyperlink{ref-taoWheatCalculus2020}{Tao et -al., 2020}). +temporal ranges (\citeproc{ref-henryNeanderthalCalculus2014}{Henry et +al., 2014}; \citeproc{ref-jovanovicNeolithicCalculus2021}{Jovanović et +al., 2021}; \citeproc{ref-pipernoStarchGrains2008}{Piperno \& Dillehay, +2008}; \citeproc{ref-taoWheatCalculus2020}{Tao et al., 2020}). Despite this, our knowledge of dental calculus and the incorporation pathways of the various markers is limited -(\protect\hyperlink{ref-radiniFoodPathways2017}{Radini et al., 2017}), -as is our knowledge of information-loss caused by these pathways. -Additionally, the methods we use to extract and analyse dental calculus, -and make inferences on past diets represent another potential source of -bias. Studies on both archaeological and modern individuals have -explored these biases in more detail. Extraction methods were tested by -Tromp and colleagues -(\protect\hyperlink{ref-trompEDTACalculus2017}{2017}), specifically +(\citeproc{ref-radiniFoodPathways2017}{Radini et al., 2017}), as is our +knowledge of information-loss caused by these pathways. Additionally, +the methods we use to extract and analyse dental calculus, and make +inferences on past diets represent another potential source of bias. +Studies on both archaeological and modern individuals have explored +these biases in more detail. Extraction methods were tested by Tromp and +colleagues (\citeproc{ref-trompEDTACalculus2017}{2017}), specifically regarding decalcification using HCl or EDTA. The authors found significantly more starches with the EDTA extraction method than the HCl extraction method; however, as noted by the authors, comparisons involving archaeological calculus are problematic due to variability between and within individuals. Studies conducted on modern humans -(\protect\hyperlink{ref-leonardPlantMicroremains2015}{Leonard et al., -2015}) and non-human primates -(\protect\hyperlink{ref-powerChimpCalculus2015}{R. C. Power et al., -2015}; \protect\hyperlink{ref-powerRepresentativenessDental2021}{Robert -C. Power et al., 2021}) have explored how well microremains (phytoliths -and starches) extracted from dental calculus represent the actual -dietary intake. These studies are justifiably limited, despite -meticulous documentation and observation, due to unknown variables and -uncertainty involved in this kind of \emph{in vivo} research. Dental -calculus is a complex oral biofilm with a multifactorial aetiology and -variable formation rates both within and between individuals -(\protect\hyperlink{ref-haffajeeBiofilmPosition2009}{Haffajee et al., -2009}; \protect\hyperlink{ref-jepsenCalculusRemoval2011}{Jepsen et al., -2011}), contributing to the stochasticity of starch representation being +(\citeproc{ref-leonardPlantMicroremains2015}{Leonard et al., 2015}) and +non-human primates (\citeproc{ref-powerChimpCalculus2015}{R. C. Power et +al., 2015}; \citeproc{ref-powerRepresentativenessDental2021}{Robert C. +Power et al., 2021}) have explored how well microremains (phytoliths and +starches) extracted from dental calculus represent the actual dietary +intake. These studies are justifiably limited, despite meticulous +documentation and observation, due to unknown variables and uncertainty +involved in this kind of \emph{in vivo} research. Dental calculus is a +complex oral biofilm with a multifactorial aetiology and variable +formation rates both within and between individuals +(\citeproc{ref-haffajeeBiofilmPosition2009}{Haffajee et al., 2009}; +\citeproc{ref-jepsenCalculusRemoval2011}{Jepsen et al., 2011}), +contributing to the stochasticity of starch representation being observed in numerous studies. Additionally, the concentration of oral \(\alpha\)-amylase differs both between and within individuals -(\protect\hyperlink{ref-froehlichEffectOral1987}{Froehlich et al., -1987}; \protect\hyperlink{ref-naterHumanAmylase2005}{Nater et al., -2005}), causing different rates of hydrolysis of the starch granules -present in the oral cavity. Add to this the effects of the many -different methods of starch processing -(\protect\hyperlink{ref-hardyRecoveringInformation2018}{Hardy et al., +(\citeproc{ref-froehlichEffectOral1987}{Froehlich et al., 1987}; +\citeproc{ref-naterHumanAmylase2005}{Nater et al., 2005}), causing +different rates of hydrolysis of the starch granules present in the oral +cavity. Add to this the effects of the many different methods of starch +processing (\citeproc{ref-hardyRecoveringInformation2018}{Hardy et al., 2018}), as well as post-depositional processes that are still being -explored -(\protect\hyperlink{ref-graneroStarchTaphonomy2020}{García-Granero, -2020}; -\protect\hyperlink{ref-mercaderExaggeratedExpectations2018}{Mercader et +explored (\citeproc{ref-graneroStarchTaphonomy2020}{García-Granero, +2020}; \citeproc{ref-mercaderExaggeratedExpectations2018}{Mercader et al., 2018}), and it becomes clear that using dental calculus to reconstruct diet is a highly unpredictable process. @@ -4694,45 +4635,42 @@ \section{Introduction}\label{byocstarch-intro}} number of incorporated starch granules increases as the size of the calculus deposit increases. -\hypertarget{materials-and-methods-1}{% -\section{Materials and Methods}\label{materials-and-methods-1}} +\section{Materials and Methods}\label{materials-and-methods-1} -\hypertarget{biofilm-formation}{% -\subsection{Biofilm formation}\label{biofilm-formation}} +\subsection{Biofilm formation}\label{biofilm-formation} In this study we employ a multispecies oral biofilm model following a modified protocol from Sissons and colleagues -(\protect\hyperlink{ref-sissonsMultistationPlaque1991}{1991}) and -Shellis (\protect\hyperlink{ref-shellisSyntheticSaliva1978}{1978}). In -brief, a biofilm inoculated with whole saliva was grown on a substrate -suspended in artificial saliva, and fed with sugar (sucrose). After -several days of growth, the biofilm was exposed to starch solutions. -Mineralisation of the biofilm was aided by exposure to a calcium -phosphate solution. After 25 days of growth, the mineralised biofilm was -collected for further analysis. The setup comprises a polypropylene 24 -deepwell PCR plate (KingFisher 97003510) with a lid containing 24 pegs, -which is autoclaved at 120°C, 1 bar overpressure, for 20 mins. The -individual pegs were the substrata on which the calculus grew. Using -this system allowed for easy transfer of the growing biofilm between -saliva, feeding solutions, and mineral solutions. +(\citeproc{ref-sissonsMultistationPlaque1991}{1991}) and Shellis +(\citeproc{ref-shellisSyntheticSaliva1978}{1978}). In brief, a biofilm +inoculated with whole saliva was grown on a substrate suspended in +artificial saliva, and fed with sugar (sucrose). After several days of +growth, the biofilm was exposed to starch solutions. Mineralisation of +the biofilm was aided by exposure to a calcium phosphate solution. After +25 days of growth, the mineralised biofilm was collected for further +analysis. The setup comprises a polypropylene 24 deepwell PCR plate +(KingFisher 97003510) with a lid containing 24 pegs, which is autoclaved +at 120°C, 1 bar overpressure, for 20 mins. The individual pegs were the +substrata on which the calculus grew. Using this system allowed for easy +transfer of the growing biofilm between saliva, feeding solutions, and +mineral solutions. The artificial saliva (AS) is a modified version of the basal medium mucin (BMM) described by Sissons and colleagues -(\protect\hyperlink{ref-sissonsMultistationPlaque1991}{1991}). It -contains 2.5 g/l partially purified mucin from porcine stomach (Type -III, Sigma M1778), 5 g/l trypticase peptone (Roth 2363.1), 10 g/l -proteose peptone (Oxoid LP0085), 5 g/l yeast extract (BD 211921), 2.5 -g/l KCl, 0.35 g/l NaCl, 1.8 mmol/l CaCl\textsubscript{2}, 5.2 mmol/l +(\citeproc{ref-sissonsMultistationPlaque1991}{1991}). It contains 2.5 +g/l partially purified mucin from porcine stomach (Type III, Sigma +M1778), 5 g/l trypticase peptone (Roth 2363.1), 10 g/l proteose peptone +(Oxoid LP0085), 5 g/l yeast extract (BD 211921), 2.5 g/l KCl, 0.35 g/l +NaCl, 1.8 mmol/l CaCl\textsubscript{2}, 5.2 mmol/l Na\textsubscript{2}HPO\textsubscript{4} -(\protect\hyperlink{ref-sissonsMultistationPlaque1991}{Sissons et al., -1991}), 6.4 mmol/l NaHCO\textsubscript{3} -(\protect\hyperlink{ref-shellisSyntheticSaliva1978}{Shellis, 1978}), 2.5 -mg/l haemin. This is subsequently adjusted to pH 7 with NaOH pellets and +(\citeproc{ref-sissonsMultistationPlaque1991}{Sissons et al., 1991}), +6.4 mmol/l NaHCO\textsubscript{3} +(\citeproc{ref-shellisSyntheticSaliva1978}{Shellis, 1978}), 2.5 mg/l +haemin. This is subsequently adjusted to pH 7 with NaOH pellets and stirring, autoclaved (15 min, 120°C, 1 bar overpressure), and supplemented with 5.8 \(\mu\)mol/l menadione, 5 mmol/l urea, and 1 -mmol/l arginine -(\protect\hyperlink{ref-sissonsMultistationPlaque1991}{Sissons et al., -1991}). +mmol/l arginine (\citeproc{ref-sissonsMultistationPlaque1991}{Sissons et +al., 1991}). Fresh whole saliva (WS) for inoculation was provided by a 31-year-old male donor with no history of caries, who abstained from oral hygiene @@ -4774,9 +4712,8 @@ \subsection{Biofilm formation}\label{biofilm-formation}} CaCl\textsubscript{2}, 12 mmol/l NaH\textsubscript{2}PO\textsubscript{4}, 5 mmol/l Na\textsubscript{2}PO\textsubscript{3}F, 500 mmol/l urea, and (0.04 g/l -MgCl) (\protect\hyperlink{ref-pearceConcomitantDeposition1987}{Pearce \& -Sissons, 1987}; -\protect\hyperlink{ref-sissonsMultistationPlaque1991}{Sissons et al., +MgCl) (\citeproc{ref-pearceConcomitantDeposition1987}{Pearce \& Sissons, +1987}; \citeproc{ref-sissonsMultistationPlaque1991}{Sissons et al., 1991}). The substrata were submerged in 1 ml/well CPMU for six minutes, five times daily, in a two-hour cycle. During the mineralisation period, starch treatments were reduced to once per day after the five CPMU @@ -4786,14 +4723,16 @@ \subsection{Biofilm formation}\label{biofilm-formation}} \begin{figure}[H] -{\centering \includegraphics[width=4.27in,height=\textheight]{figures/protocol_overview.png} +\centering{ + +\includegraphics[width=4.27in,height=\textheight]{figures/protocol_overview.png} } \caption{\label{fig-protocol}Overview of experiment protocol including the plate setup.} -\end{figure} +\end{figure}% All laboratory work was conducted in sterile conditions under a laminar flow hood to prevent starch and bacterial contamination. Control samples @@ -4801,9 +4740,8 @@ \subsection{Biofilm formation}\label{biofilm-formation}} contamination from the environment or cross-contamination from other wells in the same plate. -\hypertarget{amylase-activity-detection}{% \subsection{Amylase activity -detection}\label{amylase-activity-detection}} +detection}\label{amylase-activity-detection} An \(\alpha\)-amylase (EC 3.2.1.1) activity assay was conducted on artificial saliva samples collected from the plate wells on days 3, 6, @@ -4819,15 +4757,13 @@ \subsection{Amylase activity (Thermo Scientific 51119000). The protocol is a modified version of an Enzymatic Assay of \(\alpha\)-Amylase (\url{https://www.sigmaaldrich.com/NL/en/technical-documents/protocol/protein-biology/enzyme-activity-assays/enzymatic-assay-of-a-amylase}) -(\protect\hyperlink{ref-bernfeldAmylase1955}{Bernfeld, 1955}), which -measures the amount of maltose released from starch by -\(\alpha\)-amylase activity. Results are reported in units (U) per mL -enzyme, where 1 U releases 1 \(\mu\)mole of maltose in 6 minutes. The -detailed protocol can be found here: -\url{https://www.protocols.io/view/amylase-activity-bw8jphun}. +(\citeproc{ref-bernfeldAmylase1955}{Bernfeld, 1955}), which measures the +amount of maltose released from starch by \(\alpha\)-amylase activity. +Results are reported in units (U) per mL enzyme, where 1 U releases 1 +\(\mu\)mole of maltose in 6 minutes. The detailed protocol can be found +here: \url{https://www.protocols.io/view/amylase-activity-bw8jphun}. -\hypertarget{treatment-solutions}{% -\subsection{Treatment solutions}\label{treatment-solutions}} +\subsection{Treatment solutions}\label{treatment-solutions} A 1 ml aliquot of each starch solution was taken, from which 10 \(\mu\)l was mounted on a microscope slide with an 18 x 18 mm coverslip, and @@ -4838,36 +4774,33 @@ \subsection{Treatment solutions}\label{treatment-solutions}} treatments (see Supplementary Material for more details). For potato treatment samples, the whole slide was counted. -\hypertarget{extraction-method}{% -\subsection{Extraction method}\label{extraction-method}} +\subsection{Extraction method}\label{extraction-method} Extraction of starches from the calculus samples was performed by dissolving the calculus in 0.5 \(\tiny{M}\) ethylenediaminetetraacetic -acid (EDTA) (\protect\hyperlink{ref-lemoyneCalculusPretreatments2021}{Le -Moyne \& Crowther, 2021}; -\protect\hyperlink{ref-modiCalculusMethodologies2020}{Modi et al., -2020}; \protect\hyperlink{ref-trompEDTACalculus2017}{Tromp et al., -2017}), and vortexing for 3 days until the sample was completely -dissolved. Twenty \(\mu\)l of sample was mounted onto a slide with an -18x18 mm coverslip. When transferring the sample to the slide, the -sample was homogenised using the pipette to ensure that the counted -transects were representative of the whole slide. The count from the -slide was extrapolated to the whole sample (see Supplementary Material -for more detail). +acid (EDTA) (\citeproc{ref-lemoyneCalculusPretreatments2021}{Le Moyne \& +Crowther, 2021}; \citeproc{ref-modiCalculusMethodologies2020}{Modi et +al., 2020}; \citeproc{ref-trompEDTACalculus2017}{Tromp et al., 2017}), +and vortexing for 3 days until the sample was completely dissolved. +Twenty \(\mu\)l of sample was mounted onto a slide with an 18x18 mm +coverslip. When transferring the sample to the slide, the sample was +homogenised using the pipette to ensure that the counted transects were +representative of the whole slide. The count from the slide was +extrapolated to the whole sample (see Supplementary Material for more +detail). Both wheat and potato granules were divided into three size categories: small (\textless10 \(\mu\)m), medium (10 -- 20 \(\mu\)m), and large (\textgreater20 \(\mu\)m). -\hypertarget{statistical-analysis}{% -\subsection{Statistical analysis}\label{statistical-analysis}} +\subsection{Statistical analysis}\label{statistical-analysis} -Statistical analysis was conducted in R version 4.3.2 (2023-10-31) -(\protect\hyperlink{ref-Rbase}{R Core Team, 2020}) and the following -packages: tidyverse (\protect\hyperlink{ref-tidyverse2019}{Wickham et -al., 2019}), broom (\protect\hyperlink{ref-Rbroom}{Robinson et al., -2021}), here (\protect\hyperlink{ref-Rhere}{Müller, 2020}), and -patchwork (\protect\hyperlink{ref-Rpatchwork}{Pedersen, 2020}). +Statistical analysis was conducted in R version 4.3.3 (2024-02-29) +(\citeproc{ref-Rbase}{R Core Team, 2020}) and the following packages: +tidyverse (\citeproc{ref-tidyverse2019}{Wickham et al., 2019}), broom +(\citeproc{ref-Rbroom}{Robinson et al., 2021}), here +(\citeproc{ref-Rhere}{Müller, 2020}), and patchwork +(\citeproc{ref-Rpatchwork}{Pedersen, 2020}). To see if biofilm growth was differently affected by starch treatments, a one-way ANOVA with sample weight as the dependent variable (DV) and @@ -4883,11 +4816,10 @@ \subsection{Statistical analysis}\label{statistical-analysis}} and wheat counts. This was applied to total biofilm weight and starch count per mg calculus (also z-score standardised) to account for differences in starch concentration in the calculus (as per -\protect\hyperlink{ref-wesolowskiEvaluatingMicrofossil2010}{Wesolowski -et al., 2010}). +\citeproc{ref-wesolowskiEvaluatingMicrofossil2010}{Wesolowski et al., +2010}). -\hypertarget{results-1}{% -\section{Results}\label{results-1}} +\section{Results}\label{results-1} All samples yielded sufficient biofilm growth and starch incorporation to be included in the analysis (Figure~\ref{fig-microscope}), resulting @@ -4898,79 +4830,26 @@ \section{Results}\label{results-1}} \begin{figure} -\begin{minipage}[t]{0.50\linewidth} - -{\centering - -\begin{figure}[H] - -{\centering \includegraphics[width=4.63in,height=\textheight]{figures/starches_w_bar.jpg} - -} - -\end{figure} - -} - -\end{minipage}% +\begin{minipage}{0.50\linewidth} +\includegraphics{figures/starches_w_bar.jpg}\end{minipage}% % -\begin{minipage}[t]{0.50\linewidth} - -{\centering - -\begin{figure}[H] - -{\centering \includegraphics[width=4.63in,height=\textheight]{figures/st2C3.2-mix.jpg} - -} - -\end{figure} - -} - -\end{minipage}% +\begin{minipage}{0.50\linewidth} +\includegraphics{figures/st2C3.2-mix.jpg}\end{minipage}% \newline -\begin{minipage}[t]{0.50\linewidth} - -{\centering - -\begin{figure}[H] - -{\centering \includegraphics[width=4.63in,height=\textheight]{figures/st1B4-wheat.jpg} - -} - -\end{figure} - -} - -\end{minipage}% +\begin{minipage}{0.50\linewidth} +\includegraphics{figures/st1B4-wheat.jpg}\end{minipage}% % -\begin{minipage}[t]{0.50\linewidth} - -{\centering - -\begin{figure}[H] - -{\centering \includegraphics[width=4.63in,height=\textheight]{figures/2D2-potato.jpg} - -} - -\end{figure} - -} - -\end{minipage}% +\begin{minipage}{0.50\linewidth} +\includegraphics{figures/2D2-potato.jpg}\end{minipage}% \caption{\label{fig-microscope}Microscope images of biofilm samples that were exposed to the starch solutions. Starch granules can be seen within bacterial communities and isolated. Scale bar = 20 μm.} -\end{figure} +\end{figure}% -\hypertarget{no-amylase-activity-detected-in-the-model}{% \subsection{No amylase activity detected in the -model}\label{no-amylase-activity-detected-in-the-model}} +model}\label{no-amylase-activity-detected-in-the-model} No \(\alpha\)-amylase activity was detected in any of the artificial saliva samples from any of the days that were sampled. Only positive @@ -4981,9 +4860,8 @@ \subsection{No amylase activity detected in the as the unit definition may differ; however, they are sufficient to show that there is no activity in the model. -\hypertarget{treatment-type-had-minimal-effect-on-biofilm-growth}{% \subsection{Treatment type had minimal effect on biofilm -growth}\label{treatment-type-had-minimal-effect-on-biofilm-growth}} +growth}\label{treatment-type-had-minimal-effect-on-biofilm-growth} A one-way ANOVA suggests that the type of starch used during the biofilm growth period had a minimal effect on the growth of the biofilm @@ -4991,14 +4869,13 @@ \subsection{Treatment type had minimal effect on biofilm 0.335. A summary of sample weights is available in Table~\ref{tbl-anova}. -\hypertarget{tbl-anova}{} \begin{longtable}[]{@{}lrrrr@{}} + \caption{\label{tbl-anova}Summary statistics for biofilm dry-weights (in -mg) by treatment.}\tabularnewline -\toprule\noalign{} -Treatment & Mean & SD & Min & Max \\ -\midrule\noalign{} -\endfirsthead +mg) by treatment.} + +\tabularnewline + \toprule\noalign{} Treatment & Mean & SD & Min & Max \\ \midrule\noalign{} @@ -5009,10 +4886,10 @@ \subsection{Treatment type had minimal effect on biofilm mix & 4.28 & 1.95 & 1.50 & 8.44 \\ potato & 6.25 & 2.07 & 2.54 & 8.92 \\ wheat & 5.53 & 3.45 & 0.56 & 9.80 \\ + \end{longtable} -\hypertarget{starch-counts}{% -\subsection{Starch counts}\label{starch-counts}} +\subsection{Starch counts}\label{starch-counts} It was not possible to differentiate between potato and wheat starches smaller than ca. 10 \(\mu\)m. These were counted as wheat, as we assumed @@ -5032,7 +4909,6 @@ \subsection{Starch counts}\label{starch-counts}} (\ensuremath{3.02\times 10^{6}}) and in the biofilm samples (4850) (Table~\ref{tbl-solution-count} and Table~\ref{tbl-sample-count}). -\hypertarget{tbl-solution-count}{} \begin{longtable}[]{@{} >{\raggedright\arraybackslash}p{(\columnwidth - 10\tabcolsep) * \real{0.1084}} >{\raggedright\arraybackslash}p{(\columnwidth - 10\tabcolsep) * \real{0.0843}} @@ -5040,25 +4916,12 @@ \subsection{Starch counts}\label{starch-counts}} >{\raggedright\arraybackslash}p{(\columnwidth - 10\tabcolsep) * \real{0.1928}} >{\raggedright\arraybackslash}p{(\columnwidth - 10\tabcolsep) * \real{0.1928}} >{\raggedright\arraybackslash}p{(\columnwidth - 10\tabcolsep) * \real{0.2169}}@{}} + \caption{\label{tbl-solution-count}Mean starch counts from solutions, -including the proportional makeup of the different sizes of -granules.}\tabularnewline -\toprule\noalign{} -\begin{minipage}[b]{\linewidth}\raggedright -Solution -\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedright -Starch -\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedright -Small (\%) -\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedright -Medium (\%) -\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedright -Large (\%) -\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedright -Total (\%) -\end{minipage} \\ -\midrule\noalign{} -\endfirsthead +including the proportional makeup of the different sizes of granules.} + +\tabularnewline + \toprule\noalign{} \begin{minipage}[b]{\linewidth}\raggedright Solution @@ -5087,9 +4950,9 @@ \subsection{Starch counts}\label{starch-counts}} 3016000 (100.0\%) \\ wheat & wheat & 16139467 (63.5\%) & 6434133 (25.3\%) & 2830400 (11.1\%) & 25404000 (100.0\%) \\ + \end{longtable} -\hypertarget{tbl-sample-count}{} \begin{longtable}[]{@{} >{\raggedright\arraybackslash}p{(\columnwidth - 18\tabcolsep) * \real{0.1053}} >{\raggedright\arraybackslash}p{(\columnwidth - 18\tabcolsep) * \real{0.0737}} @@ -5101,33 +4964,13 @@ \subsection{Starch counts}\label{starch-counts}} >{\raggedleft\arraybackslash}p{(\columnwidth - 18\tabcolsep) * \real{0.0526}} >{\raggedright\arraybackslash}p{(\columnwidth - 18\tabcolsep) * \real{0.1368}} >{\raggedleft\arraybackslash}p{(\columnwidth - 18\tabcolsep) * \real{0.0632}}@{}} + \caption{\label{tbl-sample-count}Mean starch counts extracted from samples with standard deviation (SD), including the proportion of -granule sizes of the total count.}\tabularnewline -\toprule\noalign{} -\begin{minipage}[b]{\linewidth}\raggedright -Treatment -\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedright -Starch -\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedright -Small (\%) -\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedleft -SD -\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedright -Medium (\%) -\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedleft -SD -\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedright -Large (\%) -\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedleft -SD -\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedright -Total (\%) -\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedleft -SD -\end{minipage} \\ -\midrule\noalign{} -\endfirsthead +granule sizes of the total count.} + +\tabularnewline + \toprule\noalign{} \begin{minipage}[b]{\linewidth}\raggedright Treatment @@ -5164,11 +5007,11 @@ \subsection{Starch counts}\label{starch-counts}} (19.20\%) & 929 & 4846 (100\%) & 3316 \\ wheat & wheat & 15235 (55.00\%) & 11944 & 12148 (43.9\%) & 11052 & 1953 (7.06\%) & 2016 & 27680 (100\%) & 23554 \\ + \end{longtable} -\hypertarget{proportion-of-available-starches-incorporated-in-samples}{% \subsubsection{Proportion of available starches incorporated in -samples}\label{proportion-of-available-starches-incorporated-in-samples}} +samples}\label{proportion-of-available-starches-incorporated-in-samples} The proportion of total starches from the solutions that were incorporated into the samples ranged from 0.06\% to 0.16\%, with potato @@ -5180,14 +5023,13 @@ \subsubsection{Proportion of available starches incorporated in solutions, but the highest proportional incorporation, and vice versa for the mixed treatment. -\hypertarget{tbl-sample-prop}{} \begin{longtable}[]{@{}llllll@{}} + \caption{\label{tbl-sample-prop}The mean percentage of starches from the -solutions that were incorporated into the samples.}\tabularnewline -\toprule\noalign{} -Treatment & Starch & Small & Medium & Large & Total \\ -\midrule\noalign{} -\endfirsthead +solutions that were incorporated into the samples.} + +\tabularnewline + \toprule\noalign{} Treatment & Starch & Small & Medium & Large & Total \\ \midrule\noalign{} @@ -5199,15 +5041,15 @@ \subsubsection{Proportion of available starches incorporated in mix & both & 0.05\% & 0.11\% & 0.07\% & 0.07\% \\ potato & potato & 0.28\% & 0.27\% & 0.06\% & 0.16\% \\ wheat & wheat & 0.09\% & 0.19\% & 0.07\% & 0.12\% \\ + \end{longtable} Wheat incorporation was most affected in the mixed-treatment samples, with only 0.06\% of the total available starches being incorporated into the sample, compared to 0.16\% in the separated wheat treatment. -\hypertarget{size-ratios-differ-between-solutions-and-samples}{% \subsubsection{Size ratios differ between solutions and -samples}\label{size-ratios-differ-between-solutions-and-samples}} +samples}\label{size-ratios-differ-between-solutions-and-samples} Overall, medium starch granules had a higher mean rate of incorporation (0.171\%) than small (0.120\%) and large (0.066\%) starch granules @@ -5225,7 +5067,9 @@ \subsubsection{Size ratios differ between solutions and \begin{figure}[H] -{\centering \includegraphics{figures/byoc-starch-fig-ratio-plots-1.pdf} +\centering{ + +\includegraphics{figures/byoc-starch-fig-ratio-plots-1.pdf} } @@ -5234,12 +5078,11 @@ \subsubsection{Size ratios differ between solutions and separated wheat (A) and potato (B) treatments, and mixed wheat (C) and potato (D) treatments.} -\end{figure} +\end{figure}% -\hypertarget{biofilm-weight-correlated-positively-with-extracted-starch-counts}{% \subsubsection{Biofilm weight correlated positively with extracted starch -counts}\label{biofilm-weight-correlated-positively-with-extracted-starch-counts}} +counts}\label{biofilm-weight-correlated-positively-with-extracted-starch-counts} Pearson's \emph{r} suggests a strong positive correlation between the total weight of the biofilms and the total starch count (standardised by @@ -5249,7 +5092,9 @@ \subsubsection{Biofilm weight correlated positively with extracted \begin{figure}[H] -{\centering \includegraphics{figures/byoc-starch-fig-cor-plot-1.pdf} +\centering{ + +\includegraphics{figures/byoc-starch-fig-cor-plot-1.pdf} } @@ -5258,15 +5103,14 @@ \subsubsection{Biofilm weight correlated positively with extracted (B) sample weight in mg and standardised count of starch grains per mg calculus.} -\end{figure} +\end{figure}% The same test was applied to total biofilm weight and starch count per mg calculus (also standardised by z-score), resulting in a weak positive correlation, \emph{r} = 0.3, 90\%CI{[}0.0618, 0.506{]}, p = 0.0403 (Figure~\ref{fig-cor-plot}B). -\hypertarget{discussion-1}{% -\section{Discussion}\label{discussion-1}} +\section{Discussion}\label{discussion-1} Here, we have provided a method for exploring the incorporation of dietary starches into the mineral matrix of a dental calculus biofilm @@ -5289,29 +5133,27 @@ \section{Discussion}\label{discussion-1}} proportion of granules recovered from the model calculus (0.06\% to 0.16\%), the absolute counts were still substantially greater than counts recovered from archaeological remains -(\protect\hyperlink{ref-trompEDTACalculus2017}{Tromp et al., 2017}; -\protect\hyperlink{ref-trompDietaryNondietary2015}{Tromp \& Dudgeon, -2015}; -\protect\hyperlink{ref-wesolowskiEvaluatingMicrofossil2010}{Wesolowski -et al., 2010}), which could in part be due to the lack of oral amylase -activity in our model. Previous research conducted on dental calculus -from contemporary humans and non-human primates suggest a high level of +(\citeproc{ref-trompEDTACalculus2017}{Tromp et al., 2017}; +\citeproc{ref-trompDietaryNondietary2015}{Tromp \& Dudgeon, 2015}; +\citeproc{ref-wesolowskiEvaluatingMicrofossil2010}{Wesolowski et al., +2010}), which could in part be due to the lack of oral amylase activity +in our model. Previous research conducted on dental calculus from +contemporary humans and non-human primates suggest a high level of stochasticity involved in the retention of starch granules in dental calculus, and that starch granules extracted from dental calculus are underrepresented with regard to actual starch intake, which is consistent with our findings (illustrated by high standard deviations and low proportional incorporation). Leonard and colleagues -(\protect\hyperlink{ref-leonardPlantMicroremains2015}{2015}) found -individual calculus samples to be a poor predictor of diet in a -population, as many of the consumed plants were missing from some -individual samples, but were present in others.\\ -Power and colleagues -(\protect\hyperlink{ref-powerChimpCalculus2015}{2015}) presented similar -findings in non-human primates, where phytoliths were more -representative of individual diets than starch granules. The size bias -is also consistent with the findings by Power and colleagues -(\protect\hyperlink{ref-powerChimpCalculus2015}{2015}), who found that -plants producing starches 10--20 \(\mu\)m in size were over-represented; +(\citeproc{ref-leonardPlantMicroremains2015}{2015}) found individual +calculus samples to be a poor predictor of diet in a population, as many +of the consumed plants were missing from some individual samples, but +were present in others.\\ +Power and colleagues (\citeproc{ref-powerChimpCalculus2015}{2015}) +presented similar findings in non-human primates, where phytoliths were +more representative of individual diets than starch granules. The size +bias is also consistent with the findings by Power and colleagues +(\citeproc{ref-powerChimpCalculus2015}{2015}), who found that plants +producing starches 10--20 \(\mu\)m in size were over-represented; however, the representation of granules larger than 20 \(\mu\)m in their study is unclear. @@ -5324,38 +5166,34 @@ \section{Discussion}\label{discussion-1}} Figure~\ref{fig-ratio-plots}). Large potato granules were most affected, potentially because of the greater size-range. They can reach up to 100 \(\mu\)m in maximum length, whereas wheat granules generally only reach -up to 35 \(\mu\)m -(\protect\hyperlink{ref-gismondiStarchGranules2019}{Gismondi et al., -2019}; \protect\hyperlink{ref-haslamDecompositionStarch2004}{Haslam, -2004}; \protect\hyperlink{ref-seidemannStarchAtlas1966}{Seidemann, 1966, -pp. 174--176}). Granule morphology may also play a role. Large wheat -granules are lenticular and have a larger surface area compared to -volume, whereas large potato granules are ovoid and have a larger volume -compared to surface area -(\protect\hyperlink{ref-janeAnthologyStarch1994}{Jane et al., 1994}; -\protect\hyperlink{ref-reichertStarchBible1913b}{Reichert, 1913, pp. -364--365}; \protect\hyperlink{ref-seidemannStarchAtlas1966}{Seidemann, -1966, pp. 174--176}; -\protect\hyperlink{ref-vandeveldeStarchMorphology2002}{van de Velde et -al., 2002}). Another potentially important factor from our results is -the size of the calculus deposit. We found a strong positive correlation +up to 35 \(\mu\)m (\citeproc{ref-gismondiStarchGranules2019}{Gismondi et +al., 2019}; \citeproc{ref-haslamDecompositionStarch2004}{Haslam, 2004}; +\citeproc{ref-seidemannStarchAtlas1966}{Seidemann, 1966, pp. 174--176}). +Granule morphology may also play a role. Large wheat granules are +lenticular and have a larger surface area compared to volume, whereas +large potato granules are ovoid and have a larger volume compared to +surface area (\citeproc{ref-janeAnthologyStarch1994}{Jane et al., 1994}; +\citeproc{ref-reichertStarchBible1913b}{Reichert, 1913, pp. 364--365}; +\citeproc{ref-seidemannStarchAtlas1966}{Seidemann, 1966, pp. 174--176}; +\citeproc{ref-vandeveldeStarchMorphology2002}{van de Velde et al., +2002}). Another potentially important factor from our results is the +size of the calculus deposit. We found a strong positive correlation between size of biofilm deposit and retained starch granules (Figure~\ref{fig-cor-plot}A), meaning larger calculus deposits contain a higher quantity of granules; a result that contradicts findings from -archaeological contexts -(\protect\hyperlink{ref-dudgeonDietGeography2014}{Dudgeon \& Tromp, -2014}; -\protect\hyperlink{ref-wesolowskiEvaluatingMicrofossil2010}{Wesolowski -et al., 2010}). When the concentration of starch granules per mg -calculus is considered, the correlation is weaker, but still present +archaeological contexts (\citeproc{ref-dudgeonDietGeography2014}{Dudgeon +\& Tromp, 2014}; +\citeproc{ref-wesolowskiEvaluatingMicrofossil2010}{Wesolowski et al., +2010}). When the concentration of starch granules per mg calculus is +considered, the correlation is weaker, but still present (Figure~\ref{fig-cor-plot}B). While the larger deposits contain a higher absolute count, our findings also suggest that they contain a slightly higher concentration of starches. This may also explain the lower mean retention of starch granules in mixed treatments compared to wheat treatments. Wheat treatment samples (mean = 5.53 mg) were on average -larger than mixed treatment samples (mean = 4.28 mg) (Table -@ref(tab:anova-tbl)); and while mixed treatment solutions contained the -highest mean overall granule counts, wheat treatment samples had the +larger than mixed treatment samples (mean = 4.28 mg) +(Table~\ref{tbl-anova}); and while mixed treatment solutions contained +the highest mean overall granule counts, wheat treatment samples had the highest mean starch retention. Further research is needed to determine why this differs from previous archaeological findings. @@ -5364,17 +5202,16 @@ \section{Discussion}\label{discussion-1}} investigated potential mechanisms. We know that a proportion of the starch granules entering the mouth can become trapped in the plaque/calculus, and can be recovered from archaeological samples of -considerable age -(\protect\hyperlink{ref-buckleyDentalCalculus2014}{Buckley et al., -2014}; \protect\hyperlink{ref-henryNeanderthalCalculus2014}{Henry et -al., 2014}; \protect\hyperlink{ref-wuDietEarliest2021}{Wu et al., -2021}). Studies have also shown that not all starch granules come from a -dietary source. Other pathways include cross-contamination from plant -interactions in soil, such as palm phytoliths adhering to the skin of -sweet potatoes (\protect\hyperlink{ref-trompDietaryNondietary2015}{Tromp -\& Dudgeon, 2015}), or accidental ingestion not related to food -consumption (\protect\hyperlink{ref-radiniFoodPathways2017}{Radini et -al., 2017}, \protect\hyperlink{ref-radiniMedievalWomen2019}{2019}).\\ +considerable age (\citeproc{ref-buckleyDentalCalculus2014}{Buckley et +al., 2014}; \citeproc{ref-henryNeanderthalCalculus2014}{Henry et al., +2014}; \citeproc{ref-wuDietEarliest2021}{Wu et al., 2021}). Studies have +also shown that not all starch granules come from a dietary source. +Other pathways include cross-contamination from plant interactions in +soil, such as palm phytoliths adhering to the skin of sweet potatoes +(\citeproc{ref-trompDietaryNondietary2015}{Tromp \& Dudgeon, 2015}), or +accidental ingestion not related to food consumption +(\citeproc{ref-radiniFoodPathways2017}{Radini et al., 2017}, +\citeproc{ref-radiniMedievalWomen2019}{2019}).\\ When starch granules enter the mouth, whether through ingestion of food or accidental intake, they immediately encounter multiple obstacles. It is likely that the bulk of starch granules are swallowed along with the @@ -5382,105 +5219,97 @@ \section{Discussion}\label{discussion-1}} that are broken off during mastication may be retained in the dentition through attachment to tooth surfaces (including plaque and dental calculus) and mucous membranes -(\protect\hyperlink{ref-doddsCarbohydrateRetention1988}{Dodds \& Edgar, -1988}; \protect\hyperlink{ref-kashketFoodRetention1991}{S. Kashket et -al., 1991}). Bacteria also have the ability to adhere to starch granules -(\protect\hyperlink{ref-toppingResistantStarch2003}{Topping et al., -2003}), which would allow starches to attach to bacterial communities -within the biofilm. These granules are then susceptible to mechanical -removal by the tongue, salivary clearance, and hydrolysis by -\(\alpha\)-amylase (\protect\hyperlink{ref-kashketFoodParticles1996}{S. -Kashket et al., 1996}). The susceptibility of granules to hydrolysis -depends on the crystallinity and size of the starch granule, as well as -the mode of processing. Smaller and pre-processed (e.g., cooked) starch -granules are more susceptible to enzymatic degradation, while dehydrated -starches will have a reduced susceptibility -(\protect\hyperlink{ref-bjorckStarchProcessing1984}{Björck et al., -1984}; \protect\hyperlink{ref-francoStarchDegradation1992}{Franco et -al., 1992}; -\protect\hyperlink{ref-haslamDecompositionStarch2004}{Haslam, 2004}; -\protect\hyperlink{ref-henryCookingStarch2009}{Henry et al., 2009}; -\protect\hyperlink{ref-lingstromStarchyFood1994}{Lingstrom et al., -1994}). Cracks on the surface of the dental calculus, as well as -unmineralised islands and channels may also be able to contain starch -granules (\protect\hyperlink{ref-charlierSEMCalculus2010}{Charlier et -al., 2010}; \protect\hyperlink{ref-powerSEMCalculus2014}{R. C. Power et -al., 2014}; \protect\hyperlink{ref-tanCalculusUltrastructure2004}{Tan, -Gillam, et al., 2004}). Starch granules that are trapped in these -pockets are (at least to some extent) protected from aforementioned -clearance mechanisms, especially once a new layer of plaque has covered -the surface of the plaque/calculus. The size bias against large granules -(\textgreater20 \(\mu\)m) from both wheat and potato -(Table~\ref{tbl-sample-prop}) may give further credence to this -incorporation pathway, as the smaller starch granules have an advantage -over larger granules, and can be stored in larger quantities. This was -also suggested by Power and colleagues -(\protect\hyperlink{ref-powerSEMCalculus2014}{2014}), who observed +(\citeproc{ref-doddsCarbohydrateRetention1988}{Dodds \& Edgar, 1988}; +\citeproc{ref-kashketFoodRetention1991}{S. Kashket et al., 1991}). +Bacteria also have the ability to adhere to starch granules +(\citeproc{ref-toppingResistantStarch2003}{Topping et al., 2003}), which +would allow starches to attach to bacterial communities within the +biofilm. These granules are then susceptible to mechanical removal by +the tongue, salivary clearance, and hydrolysis by \(\alpha\)-amylase +(\citeproc{ref-kashketFoodParticles1996}{S. Kashket et al., 1996}). The +susceptibility of granules to hydrolysis depends on the crystallinity +and size of the starch granule, as well as the mode of processing. +Smaller and pre-processed (e.g., cooked) starch granules are more +susceptible to enzymatic degradation, while dehydrated starches will +have a reduced susceptibility +(\citeproc{ref-bjorckStarchProcessing1984}{Björck et al., 1984}; +\citeproc{ref-francoStarchDegradation1992}{Franco et al., 1992}; +\citeproc{ref-haslamDecompositionStarch2004}{Haslam, 2004}; +\citeproc{ref-henryCookingStarch2009}{Henry et al., 2009}; +\citeproc{ref-lingstromStarchyFood1994}{Lingstrom et al., 1994}). Cracks +on the surface of the dental calculus, as well as unmineralised islands +and channels may also be able to contain starch granules +(\citeproc{ref-charlierSEMCalculus2010}{Charlier et al., 2010}; +\citeproc{ref-powerSEMCalculus2014}{R. C. Power et al., 2014}; +\citeproc{ref-tanCalculusUltrastructure2004}{Tan, Gillam, et al., +2004}). Starch granules that are trapped in these pockets are (at least +to some extent) protected from aforementioned clearance mechanisms, +especially once a new layer of plaque has covered the surface of the +plaque/calculus. The size bias against large granules (\textgreater20 +\(\mu\)m) from both wheat and potato (Table~\ref{tbl-sample-prop}) may +give further credence to this incorporation pathway, as the smaller +starch granules have an advantage over larger granules, and can be +stored in larger quantities. This was also suggested by Power and +colleagues (\citeproc{ref-powerSEMCalculus2014}{2014}), who observed clusters of starches within dental calculus, rather than an even distribution across the surface of the dental calculus. Granules trapped in plaque/calculus may still be susceptible to hydrolysis, as \(\alpha\)-amylase has the ability to bind to both tooth enamel and bacteria within a biofilm and retain a portion of its hydrolytic -activity (\protect\hyperlink{ref-nikitkovaStarchBiofilms2013}{Nikitkova -et al., 2013}; -\protect\hyperlink{ref-scannapiecoSalivaryAmylase1993}{Scannapieco et -al., 1993}; \protect\hyperlink{ref-tanBacterialViability2004}{Tan, -Mordan, et al., 2004}; -\protect\hyperlink{ref-tanCalculusUltrastructure2004}{Tan, Gillam, et -al., 2004}). After the death of an individual, starches within dental +activity (\citeproc{ref-nikitkovaStarchBiofilms2013}{Nikitkova et al., +2013}; \citeproc{ref-scannapiecoSalivaryAmylase1993}{Scannapieco et al., +1993}; \citeproc{ref-tanBacterialViability2004}{Tan, Mordan, et al., +2004}; \citeproc{ref-tanCalculusUltrastructure2004}{Tan, Gillam, et al., +2004}). After the death of an individual, starches within dental calculus are susceptible to further degradation by post-depositional processes, depending on burial environment (pH, temperature, moisture content, microorganisms) -(\protect\hyperlink{ref-francoStarchDegradation1992}{Franco et al., -1992}; -\protect\hyperlink{ref-graneroStarchTaphonomy2020}{García-Granero, -2020}; \protect\hyperlink{ref-haslamDecompositionStarch2004}{Haslam, -2004}; \protect\hyperlink{ref-henryCookingStarch2009}{Henry et al., -2009}). Future study should explore how burial affects the recovery of -starch from the biofilm model. +(\citeproc{ref-francoStarchDegradation1992}{Franco et al., 1992}; +\citeproc{ref-graneroStarchTaphonomy2020}{García-Granero, 2020}; +\citeproc{ref-haslamDecompositionStarch2004}{Haslam, 2004}; +\citeproc{ref-henryCookingStarch2009}{Henry et al., 2009}). Future study +should explore how burial affects the recovery of starch from the +biofilm model. The absence of \(\alpha\)-amylase in the model is a limitation of this study, as the total granule counts were not subject to hydrolysis. This would likely have reduced and affected the size ratios, as smaller starches may be more susceptible to hydrolysis -(\protect\hyperlink{ref-francoStarchDegradation1992}{Franco et al., -1992}; \protect\hyperlink{ref-haslamDecompositionStarch2004}{Haslam, -2004}). The absence may also affect biofilm growth due to the lack of -amylase-bacterium interactions -(\protect\hyperlink{ref-nikitkovaStarchBiofilms2013}{Nikitkova et al., -2013}). Conversely, the model may benefit from the absence of +(\citeproc{ref-francoStarchDegradation1992}{Franco et al., 1992}; +\citeproc{ref-haslamDecompositionStarch2004}{Haslam, 2004}). The absence +may also affect biofilm growth due to the lack of amylase-bacterium +interactions (\citeproc{ref-nikitkovaStarchBiofilms2013}{Nikitkova et +al., 2013}). Conversely, the model may benefit from the absence of \(\alpha\)-amylase, because it can allow us to directly explore its effect on starch counts in future experiments, where \(\alpha\)-amylase can be added to the model in concentrations similar to those found in the oral cavity -(\protect\hyperlink{ref-scannapiecoSalivaryAmylase1993}{Scannapieco et -al., 1993}). We are able to show how absolute counts in the treatments -cause a difference in incorporation. However, this was merely a -side-effect of the difference in the number of granules in potato and -wheat solutions of the same concentration (w/v). Further research should -test multiple differing concentrations of the same starch type. The use -of EDTA may also have affected counts. While previous studies have shown -negligible morphological changes caused by exposure to EDTA -(\protect\hyperlink{ref-lemoyneCalculusPretreatments2021}{Le Moyne \& -Crowther, 2021}; -\protect\hyperlink{ref-modiCalculusMethodologies2020}{Modi et al., -2020}; \protect\hyperlink{ref-trompEDTACalculus2017}{Tromp et al., -2017}), these studies have not considered changes to separate size -categories within starch types, and whether shifts in size ratios occur -due to exposure to the pre-treatment chemicals. The total number of -granules on a slide often exceeded a number that was feasible to count -in a reasonable time period, so we calculated the total counts by -extrapolating from three slide transects. Thus, we reasonably assume -that the three transects are a good representation of the entire slide, -and that the distribution of all granules on the slide is relatively -homogeneous.\\ +(\citeproc{ref-scannapiecoSalivaryAmylase1993}{Scannapieco et al., +1993}). We are able to show how absolute counts in the treatments cause +a difference in incorporation. However, this was merely a side-effect of +the difference in the number of granules in potato and wheat solutions +of the same concentration (w/v). Further research should test multiple +differing concentrations of the same starch type. The use of EDTA may +also have affected counts. While previous studies have shown negligible +morphological changes caused by exposure to EDTA +(\citeproc{ref-lemoyneCalculusPretreatments2021}{Le Moyne \& Crowther, +2021}; \citeproc{ref-modiCalculusMethodologies2020}{Modi et al., 2020}; +\citeproc{ref-trompEDTACalculus2017}{Tromp et al., 2017}), these studies +have not considered changes to separate size categories within starch +types, and whether shifts in size ratios occur due to exposure to the +pre-treatment chemicals. The total number of granules on a slide often +exceeded a number that was feasible to count in a reasonable time +period, so we calculated the total counts by extrapolating from three +slide transects. Thus, we reasonably assume that the three transects are +a good representation of the entire slide, and that the distribution of +all granules on the slide is relatively homogeneous.\\ Finally, we only used native starches in the experimental procedure and the results will likely differ for processed starches -(\protect\hyperlink{ref-graneroStarchTaphonomy2020}{García-Granero, -2020}). Based on the comparatively low counts obtained by Leonard and -colleagues (\protect\hyperlink{ref-leonardPlantMicroremains2015}{2015}, -Supplement 2), processing and amylase may have a substantial effect on -starch granule retention in the oral cavity. +(\citeproc{ref-graneroStarchTaphonomy2020}{García-Granero, 2020}). Based +on the comparatively low counts obtained by Leonard and colleagues +(\citeproc{ref-leonardPlantMicroremains2015}{2015}, Supplement 2), +processing and amylase may have a substantial effect on starch granule +retention in the oral cavity. While we are unable to sufficiently address the mechanism(s) of starch incorporation with the data obtained in this study, the dental calculus @@ -5488,33 +5317,30 @@ \section{Discussion}\label{discussion-1}} may improve interpretations of dietary practices in past populations. Further analyses using this model can address the call for more baseline testing of biases associated with dietary research conducted on dental -calculus (\protect\hyperlink{ref-lemoyneCalculusPretreatments2021}{Le -Moyne \& Crowther, 2021}). Our high-throughput experimental setup allows -us a higher degree of control over the factors that influence starch +calculus (\citeproc{ref-lemoyneCalculusPretreatments2021}{Le Moyne \& +Crowther, 2021}). Our high-throughput experimental setup allows us a +higher degree of control over the factors that influence starch incorporation and retention, such as dietary intake, differential survivability of starches, and inter- and intra-individual variation in plaque accumulation and mineralisation. The latter is especially difficult to control \emph{in vivo} as it is influenced by numerous factors including genetics, diet, salivary flow, and tooth position and -morphology -(\protect\hyperlink{ref-fagernasMicrobialBiogeography2021}{Fagernäs et -al., 2021}; \protect\hyperlink{ref-haffajeeBiofilmPosition2009}{Haffajee -et al., 2009}; \protect\hyperlink{ref-jepsenCalculusRemoval2011}{Jepsen -et al., 2011}; -\protect\hyperlink{ref-proctorSpatialGradient2018}{Proctor et al., -2018}; \protect\hyperlink{ref-simonsoroOralGeography2013}{Simón-Soro et -al., 2013}), as well as evolutionary differences -(\protect\hyperlink{ref-yatesOralMicrobiome2021}{Fellows Yates et al., -2021}). The set of limitations for our model differ from \emph{in vivo} -methods and, as such, we expect our model to complement the results and +morphology (\citeproc{ref-fagernasMicrobialBiogeography2021}{Fagernäs et +al., 2021}; \citeproc{ref-haffajeeBiofilmPosition2009}{Haffajee et al., +2009}; \citeproc{ref-jepsenCalculusRemoval2011}{Jepsen et al., 2011}; +\citeproc{ref-proctorSpatialGradient2018}{Proctor et al., 2018}; +\citeproc{ref-simonsoroOralGeography2013}{Simón-Soro et al., 2013}), as +well as evolutionary differences +(\citeproc{ref-yatesOralMicrobiome2021}{Fellows Yates et al., 2021}). +The set of limitations for our model differ from \emph{in vivo} methods +and, as such, we expect our model to complement the results and interpretations of existing and new \emph{in vivo} studies. It can also facilitate training of students and researchers on methods of dental calculus analysis, such as starch and phytolith extraction and identification, where it can replace the use of finite archaeological resources. -\hypertarget{conclusion}{% -\section{Conclusion}\label{conclusion}} +\section{Conclusion}\label{conclusion} This preliminary study shows that a very small proportion of the input starch granules are retained in a dental calculus model. This and @@ -5546,15 +5372,14 @@ \section{Conclusion}\label{conclusion}} of \emph{in vivo} studies, and unearth the potential biases associated with dietary research conducted on archaeological dental calculus. -\hypertarget{references-cited-3}{% -\section*{References cited}\label{references-cited-3}} +\section*{References cited}\label{references-cited-3} \addcontentsline{toc}{section}{References cited} \markright{References cited} -\hypertarget{refs-4}{} +\phantomsection\label{refs-4} \begin{CSLReferences}{1}{0} -\leavevmode\vadjust pre{\hypertarget{ref-adlerSequencingAncient2013}{}}% +\bibitem[\citeproctext]{ref-adlerSequencingAncient2013} Adler, C. J., Dobney, K., Weyrich, L. S., Kaidonis, J., Walker, A. W., Haak, W., Bradshaw, C. J., Townsend, G., Sołtysiak, A., Alt, K. W., Parkhill, J., \& Cooper, A. (2013). Sequencing ancient calcified dental @@ -5562,30 +5387,30 @@ \section*{References cited}\label{references-cited-3}} {Neolithic} and {Industrial} revolutions. \emph{Nature Genetics}, \emph{45}(4), 450--455, 455e1. \url{https://doi.org/10.1038/ng.2536} -\leavevmode\vadjust pre{\hypertarget{ref-armitageExtractionIdentification1975}{}}% +\bibitem[\citeproctext]{ref-armitageExtractionIdentification1975} Armitage, P. L. (1975). The {Extraction} and {Identification} of {Opal Phytoliths} from the {Teeth} of {Ungulates}. \emph{Journal of Archaeological Science}, \emph{2}, 187--197. -\leavevmode\vadjust pre{\hypertarget{ref-bernfeldAmylase1955}{}}% +\bibitem[\citeproctext]{ref-bernfeldAmylase1955} Bernfeld, P. (1955). Amylases, {\(\alpha\)} and {\(\beta\)}. In \emph{Methods in {Enzymology}} (Vol. 1, pp. 149--158). {Academic Press}. \url{https://doi.org/10.1016/0076-6879(55)01021-5} -\leavevmode\vadjust pre{\hypertarget{ref-bjorckStarchProcessing1984}{}}% +\bibitem[\citeproctext]{ref-bjorckStarchProcessing1984} Björck, I., Asp, N.-G., Birkhed, D., Eliasson, A.-C., Sjöberg, L.-B., \& Lundquist, I. (1984). Effects of processing on starch availability {In} vitro and {In} vivo. {II}. {Drum-drying} of wheat flour. \emph{Journal of Cereal Science}, \emph{2}(3), 165--178. \url{https://doi.org/10.1016/S0733-5210(84)80030-2} -\leavevmode\vadjust pre{\hypertarget{ref-buckleyDentalCalculus2014}{}}% +\bibitem[\citeproctext]{ref-buckleyDentalCalculus2014} Buckley, S., Usai, D., Jakob, T., Radini, A., \& Hardy, K. (2014). Dental {Calculus Reveals Unique Insights} into {Food Items}, {Cooking} and {Plant Processing} in {Prehistoric Central Sudan}. \emph{PLOS ONE}, \emph{9}(7), e100808. \url{https://doi.org/10.1371/journal.pone.0100808} -\leavevmode\vadjust pre{\hypertarget{ref-charlierSEMCalculus2010}{}}% +\bibitem[\citeproctext]{ref-charlierSEMCalculus2010} Charlier, P., Huynh-Charlier, I., Munoz, O., Billard, M., Brun, L., \& Grandmaison, G. L. de la. (2010). The microscopic (optical and {SEM}) examination of dental calculus deposits ({DCD}). {Potential} interest in @@ -5593,21 +5418,21 @@ \section*{References cited}\label{references-cited-3}} Medicine}, \emph{12}(4), 163--171. \url{https://doi.org/10.1016/j.legalmed.2010.03.003} -\leavevmode\vadjust pre{\hypertarget{ref-doddsCarbohydrateRetention1988}{}}% +\bibitem[\citeproctext]{ref-doddsCarbohydrateRetention1988} Dodds, M. W. J., \& Edgar, W. M. (1988). The {Relationship Between Plaque pH}, {Plaque Acid Anion Profiles}, and {Oral Carbohydrate Retention After Ingestion} of {Several} '{Reference Foods}' by {Human Subjects}. \emph{Journal of Dental Research}, \emph{67}(5), 861--865. \url{https://doi.org/10.1177/00220345880670051301} -\leavevmode\vadjust pre{\hypertarget{ref-dudgeonDietGeography2014}{}}% +\bibitem[\citeproctext]{ref-dudgeonDietGeography2014} Dudgeon, J. V., \& Tromp, M. (2014). Diet, {Geography} and {Drinking Water} in {Polynesia}: {Microfossil Research} from {Archaeological Human Dental Calculus}, {Rapa Nui} ({Easter Island}). \emph{International Journal of Osteoarchaeology}, \emph{24}(5), 634--648. \url{https://doi.org/10.1002/oa.2249} -\leavevmode\vadjust pre{\hypertarget{ref-eerkensDentalCalculus2018}{}}% +\bibitem[\citeproctext]{ref-eerkensDentalCalculus2018} Eerkens, J. W., Tushingham, S., Brownstein, K. J., Garibay, R., Perez, K., Murga, E., Kaijankoski, P., Rosenthal, J. S., \& Gang, D. R. (2018). Dental calculus as a source of ancient alkaloids: {Detection} of @@ -5615,14 +5440,14 @@ \section*{References cited}\label{references-cited-3}} \emph{Journal of Archaeological Science: Reports}, \emph{18}, 509--515. \url{https://doi.org/10.1016/j.jasrep.2018.02.004} -\leavevmode\vadjust pre{\hypertarget{ref-fagernasMicrobialBiogeography2021}{}}% +\bibitem[\citeproctext]{ref-fagernasMicrobialBiogeography2021} Fagernäs, Z., Salazar-García, D. C., Avilés, A., Haber, M., Henry, A., Maurandi, J. L., Ozga, A., Velsko, I. M., \& Warinner, C. (2021). Understanding the microbial biogeography of ancient human dentitions to guide study design and interpretation. \emph{bioRxiv}, 2021.08.16.456492. \url{https://doi.org/10.1101/2021.08.16.456492} -\leavevmode\vadjust pre{\hypertarget{ref-yatesOralMicrobiome2021}{}}% +\bibitem[\citeproctext]{ref-yatesOralMicrobiome2021} Fellows Yates, J. A., Velsko, I. M., Aron, F., Posth, C., Hofman, C. A., Austin, R. M., Parker, C. E., Mann, A. E., Nägele, K., Arthur, K. W., Arthur, J. W., Bauer, C. C., Crevecoeur, I., Cupillard, C., Curtis, M. @@ -5632,53 +5457,53 @@ \section*{References cited}\label{references-cited-3}} National Academy of Sciences}, \emph{118}(20). \url{https://doi.org/10.1073/pnas.2021655118} -\leavevmode\vadjust pre{\hypertarget{ref-foxPhytolithCalculus1996}{}}% +\bibitem[\citeproctext]{ref-foxPhytolithCalculus1996} Fox, C. L., Juan, J., \& Albert, R. M. (1996). Phytolith analysis on dental calculus, enamel surface, and burial soil: {Information} about diet and paleoenvironment. \emph{American Journal of Physical Anthropology}, \emph{101}(1), 101--113. \url{https://doi.org/10.1002/(SICI)1096-8644(199609)101:1\%3C101::AID-AJPA7\%3E3.0.CO;2-Y} -\leavevmode\vadjust pre{\hypertarget{ref-francoStarchDegradation1992}{}}% +\bibitem[\citeproctext]{ref-francoStarchDegradation1992} Franco, C. M. L., Preto, S. J. do R., \& Ciacco, C. F. (1992). Factors that {Affect} the {Enzymatic Degradation} of {Natural Starch Granules} --{Effect} of the {Size} of the {Granules}. \emph{Starch - St{ä}rke}, +-{Effect} of the {Size} of the {Granules}. \emph{Starch - Stärke}, \emph{44}(11), 422--426. \url{https://doi.org/10.1002/star.19920441106} -\leavevmode\vadjust pre{\hypertarget{ref-froehlichEffectOral1987}{}}% +\bibitem[\citeproctext]{ref-froehlichEffectOral1987} Froehlich, D. A., Pangborn, R. M., \& Whitaker, J. R. (1987). The effect of oral stimulation on human parotid salivary flow rate and alpha-amylase secretion. \emph{Physiology \& Behavior}, \emph{41}(3), 209--217. \url{https://doi.org/10.1016/0031-9384(87)90355-6} -\leavevmode\vadjust pre{\hypertarget{ref-graneroStarchTaphonomy2020}{}}% +\bibitem[\citeproctext]{ref-graneroStarchTaphonomy2020} García-Granero, J. J. (2020). Starch taphonomy, equifinality and the importance of context: {Some} notes on the identification of food processing through starch grain analysis. \emph{Journal of Archaeological Science}, \emph{124}, 105267. \url{https://doi.org/10.1016/j.jas.2020.105267} -\leavevmode\vadjust pre{\hypertarget{ref-gismondiStarchGranules2019}{}}% +\bibitem[\citeproctext]{ref-gismondiStarchGranules2019} Gismondi, A., D'Agostino, A., Canuti, L., Di Marco, G., Basoli, F., \& Canini, A. (2019). Starch granules: A data collection of 40 food species. \emph{Plant Biosystems - An International Journal Dealing with All Aspects of Plant Biology}, \emph{153}(2), 273--279. \url{https://doi.org/10.1080/11263504.2018.1473523} -\leavevmode\vadjust pre{\hypertarget{ref-haffajeeBiofilmPosition2009}{}}% +\bibitem[\citeproctext]{ref-haffajeeBiofilmPosition2009} Haffajee, A. D., Teles, R. P., Patel, M. R., Song, X., Yaskell, T., \& Socransky, S. S. (2009). Factors affecting human supragingival biofilm composition. {II}. {Tooth} position. \emph{Journal of Periodontal Research}, \emph{44}(4), 520--528. \url{https://doi.org/10.1111/j.1600-0765.2008.01155.x} -\leavevmode\vadjust pre{\hypertarget{ref-hardyStarchGranules2009}{}}% +\bibitem[\citeproctext]{ref-hardyStarchGranules2009} Hardy, K., Blakeney, T., Copeland, L., Kirkham, J., Wrangham, R., \& Collins, M. (2009). Starch granules, dental calculus and new perspectives on ancient diet. \emph{Journal of Archaeological Science}, \emph{36}(2), 248--255. \url{https://doi.org/10.1016/j.jas.2008.09.015} -\leavevmode\vadjust pre{\hypertarget{ref-hardyNeanderthalMedics2012}{}}% +\bibitem[\citeproctext]{ref-hardyNeanderthalMedics2012} Hardy, K., Buckley, S., Collins, M. J., Estalrrich, A., Brothwell, D., Copeland, L., García-Tabernero, A., García-Vargas, S., de la Rasilla, M., Lalueza-Fox, C., Huguet, R., Bastir, M., Santamaría, D., Madella, @@ -5687,20 +5512,20 @@ \section*{References cited}\label{references-cited-3}} calculus. \emph{Naturwissenschaften}, \emph{99}(8), 617--626. \url{https://doi.org/10.1007/s00114-012-0942-0} -\leavevmode\vadjust pre{\hypertarget{ref-hardyRecoveringInformation2018}{}}% +\bibitem[\citeproctext]{ref-hardyRecoveringInformation2018} Hardy, K., Buckley, S., \& Copeland, L. (2018). Pleistocene dental calculus: {Recovering} information on {Paleolithic} food items, medicines, paleoenvironment and microbes. \emph{Evolutionary Anthropology: Issues, News, and Reviews}, \emph{27}(5), 234--246. \url{https://doi.org/10.1002/evan.21718} -\leavevmode\vadjust pre{\hypertarget{ref-haslamDecompositionStarch2004}{}}% +\bibitem[\citeproctext]{ref-haslamDecompositionStarch2004} Haslam, M. (2004). The decomposition of starch grains in soils: Implications for archaeological residue analyses. \emph{Journal of Archaeological Science}, \emph{31}(12), 1715--1734. \url{https://doi.org/10.1016/j.jas.2004.05.006} -\leavevmode\vadjust pre{\hypertarget{ref-hendyProteomicCalculus2018}{}}% +\bibitem[\citeproctext]{ref-hendyProteomicCalculus2018} Hendy, J., Warinner, C., Bouwman, A., Collins, M. J., Fiddyment, S., Fischer, R., Hagan, R., Hofman, C. A., Holst, M., Chaves, E., Klaus, L., Larson, G., Mackie, M., McGrath, K., Mundorff, A. Z., Radini, A., Rao, @@ -5709,38 +5534,38 @@ \section*{References cited}\label{references-cited-3}} \emph{Proceedings. Biological Sciences}, \emph{285}(1883), 20180977. \url{https://doi.org/10.1098/rspb.2018.0977} -\leavevmode\vadjust pre{\hypertarget{ref-henryNeanderthalCalculus2014}{}}% +\bibitem[\citeproctext]{ref-henryNeanderthalCalculus2014} Henry, A. G., Brooks, A. S., \& Piperno, D. R. (2014). Plant foods and the dietary ecology of {Neanderthals} and early modern humans. \emph{Journal of Human Evolution}, \emph{69}, 44--54. \url{https://doi.org/10.1016/j.jhevol.2013.12.014} -\leavevmode\vadjust pre{\hypertarget{ref-henryCookingStarch2009}{}}% +\bibitem[\citeproctext]{ref-henryCookingStarch2009} Henry, A. G., Hudson, H. F., \& Piperno, D. R. (2009). Changes in starch grain morphologies from cooking. \emph{Journal of Archaeological Science}, \emph{36}(3), 915--922. \url{https://doi.org/10.1016/j.jas.2008.11.008} -\leavevmode\vadjust pre{\hypertarget{ref-henryCalculusSyria2008}{}}% +\bibitem[\citeproctext]{ref-henryCalculusSyria2008} Henry, A. G., \& Piperno, D. R. (2008). Using plant microfossils from dental calculus to recover human diet: A case study from {Tell} -al-{Raq{ā}}'i, {Syria}. \emph{Journal of Archaeological Science}, +al-{Raqā}'i, {Syria}. \emph{Journal of Archaeological Science}, \emph{35}(7), 1943--1950. \url{https://doi.org/10.1016/j.jas.2007.12.005} -\leavevmode\vadjust pre{\hypertarget{ref-janeAnthologyStarch1994}{}}% +\bibitem[\citeproctext]{ref-janeAnthologyStarch1994} Jane, J.-L., Kasemsuwan, T., Leas, S., Zobel, H., \& Robyt, J. F. (1994). Anthology of {Starch Granule Morphology} by {Scanning Electron -Microscopy}. \emph{Starch - St{ä}rke}, \emph{46}(4), 121--129. +Microscopy}. \emph{Starch - Stärke}, \emph{46}(4), 121--129. \url{https://doi.org/10.1002/star.19940460402} -\leavevmode\vadjust pre{\hypertarget{ref-jepsenCalculusRemoval2011}{}}% +\bibitem[\citeproctext]{ref-jepsenCalculusRemoval2011} Jepsen, S., Deschner, J., Braun, A., Schwarz, F., \& Eberhard, J. (2011). Calculus removal and the prevention of its formation. \emph{Periodontology 2000}, \emph{55}(1), 167--188. \url{https://doi.org/10.1111/j.1600-0757.2010.00382.x} -\leavevmode\vadjust pre{\hypertarget{ref-jovanovicNeolithicCalculus2021}{}}% +\bibitem[\citeproctext]{ref-jovanovicNeolithicCalculus2021} Jovanović, J., Power, R. C., de Becdelièvre, C., Goude, G., \& Stefanović, S. (2021). Microbotanical evidence for the spread of cereal use during the {Mesolithic-Neolithic} transition in the {Southeastern @@ -5748,41 +5573,41 @@ \section*{References cited}\label{references-cited-3}} \emph{Journal of Archaeological Science}, \emph{125}, 105288. \url{https://doi.org/10.1016/j.jas.2020.105288} -\leavevmode\vadjust pre{\hypertarget{ref-kashketFoodRetention1991}{}}% +\bibitem[\citeproctext]{ref-kashketFoodRetention1991} Kashket, S., Van Houte, J., Lopez, L. R., \& Stocks, S. (1991). Lack of {Correlation Between Food Retention} on the {Human Dentition} and {Consumer Perception} of {Food Stickiness}. \emph{Journal of Dental Research}, \emph{70}(10), 1314--1319. \url{https://doi.org/10.1177/00220345910700100101} -\leavevmode\vadjust pre{\hypertarget{ref-kashketFoodParticles1996}{}}% +\bibitem[\citeproctext]{ref-kashketFoodParticles1996} Kashket, S., Zhang, J., \& Houte, J. V. (1996). Accumulation of {Fermentable Sugars} and {Metabolic Acids} in {Food Particles} that {Become Entrapped} on the {Dentition}. \emph{Journal of Dental Research}, 8. -\leavevmode\vadjust pre{\hypertarget{ref-lemoyneCalculusPretreatments2021}{}}% +\bibitem[\citeproctext]{ref-lemoyneCalculusPretreatments2021} Le Moyne, C., \& Crowther, A. (2021). Effects of chemical pre-treatments on modified starch granules: {Recommendations} for dental calculus decalcification for ancient starch research. \emph{Journal of Archaeological Science: Reports}, \emph{35}, 102762. \url{https://doi.org/10.1016/j.jasrep.2020.102762} -\leavevmode\vadjust pre{\hypertarget{ref-leonardPlantMicroremains2015}{}}% +\bibitem[\citeproctext]{ref-leonardPlantMicroremains2015} Leonard, C., Vashro, L., O'Connell, J. F., \& Henry, A. G. (2015). Plant microremains in dental calculus as a record of plant consumption: {A} test with {Twe} forager-horticulturalists. \emph{Journal of Archaeological Science: Reports}, \emph{2}, 449--457. \url{https://doi.org/10.1016/j.jasrep.2015.03.009} -\leavevmode\vadjust pre{\hypertarget{ref-lingstromStarchyFood1994}{}}% +\bibitem[\citeproctext]{ref-lingstromStarchyFood1994} Lingstrom, P., Birkhed, D., Ruben, J., \& Arends, J. (1994). Effect of {Frequent Consumption} of {Starchy Food Items} on {Enamel} and {Dentin Demineralization} and on {Plaque pH} in situ. \emph{Journal of Dental Research}, \emph{73}(3), 652--660. \url{https://doi.org/10.1177/00220345940730031101} -\leavevmode\vadjust pre{\hypertarget{ref-mercaderExaggeratedExpectations2018}{}}% +\bibitem[\citeproctext]{ref-mercaderExaggeratedExpectations2018} Mercader, J., Akeju, T., Brown, M., Bundala, M., Collins, M. J., Copeland, L., Crowther, A., Dunfield, P., Henry, A., Inwood, J., Itambu, M., Kim, J.-J., Larter, S., Longo, L., Oldenburg, T., Patalano, R., @@ -5791,20 +5616,20 @@ \section*{References cited}\label{references-cited-3}} taphonomic and authenticity criteria. \emph{FACETS}, \emph{3}(1), 777--798. \url{https://doi.org/10.1139/facets-2017-0126} -\leavevmode\vadjust pre{\hypertarget{ref-mickleburghNewInsights2012}{}}% +\bibitem[\citeproctext]{ref-mickleburghNewInsights2012} Mickleburgh, H. L., \& Pagán-Jiménez, J. R. (2012). New insights into the consumption of maize and other food plants in the pre-{Columbian Caribbean} from starch grains trapped in human dental calculus. \emph{Journal of Archaeological Science}, \emph{39}(7), 2468--2478. \url{https://doi.org/10.1016/j.jas.2012.02.020} -\leavevmode\vadjust pre{\hypertarget{ref-middletonOpalPhytoliths1994}{}}% +\bibitem[\citeproctext]{ref-middletonOpalPhytoliths1994} Middleton, W. D., \& Rovner, I. (1994). Extraction of {Opal Phytoliths} from {Herbivore Dental Calculus}. \emph{Journal of Archaeological Science}, \emph{21}(4), 469--473. \url{https://doi.org/10.1006/jasc.1994.1046} -\leavevmode\vadjust pre{\hypertarget{ref-modiCalculusMethodologies2020}{}}% +\bibitem[\citeproctext]{ref-modiCalculusMethodologies2020} Modi, A., Pisaneschi, L., Zaro, V., Vai, S., Vergata, C., Casalone, E., Caramelli, D., Moggi-Cecchi, J., Mariotti Lippi, M., \& Lari, M. (2020). Combined methodologies for gaining much information from ancient dental @@ -5813,80 +5638,80 @@ \section*{References cited}\label{references-cited-3}} Sciences}, \emph{12}(1), 10. \url{https://doi.org/10.1007/s12520-019-00983-5} -\leavevmode\vadjust pre{\hypertarget{ref-Rhere}{}}% +\bibitem[\citeproctext]{ref-Rhere} Müller, K. (2020). \emph{Here: {A} simpler way to find your files} {[}Manual{]}. -\leavevmode\vadjust pre{\hypertarget{ref-naterHumanAmylase2005}{}}% +\bibitem[\citeproctext]{ref-naterHumanAmylase2005} Nater, U. M., Rohleder, N., Gaab, J., Berger, S., Jud, A., Kirschbaum, C., \& Ehlert, U. (2005). Human salivary alpha-amylase reactivity in a psychosocial stress paradigm. \emph{International Journal of Psychophysiology}, \emph{55}(3), 333--342. \url{https://doi.org/10.1016/j.ijpsycho.2004.09.009} -\leavevmode\vadjust pre{\hypertarget{ref-nikitkovaStarchBiofilms2013}{}}% +\bibitem[\citeproctext]{ref-nikitkovaStarchBiofilms2013} Nikitkova, A. E., Haase, E. M., \& Scannapieco, F. A. (2013). Taking the {Starch} out of {Oral Biofilm Formation}: {Molecular Basis} and {Functional Significance} of {Salivary} {\(\alpha\)}-{Amylase Binding} to {Oral Streptococci}. \emph{Applied and Environmental Microbiology}, \emph{79}(2), 416--423. \url{https://doi.org/10.1128/AEM.02581-12} -\leavevmode\vadjust pre{\hypertarget{ref-pearceConcomitantDeposition1987}{}}% +\bibitem[\citeproctext]{ref-pearceConcomitantDeposition1987} Pearce, E. I. F., \& Sissons, C. H. (1987). The {Concomitant Deposition} of {Strontium} and {Fluoride} in {Dental Plaque}. \emph{Journal of Dental Research}, \emph{66}(10), 1518--1522. \url{https://doi.org/10.1177/00220345870660100101} -\leavevmode\vadjust pre{\hypertarget{ref-Rpatchwork}{}}% +\bibitem[\citeproctext]{ref-Rpatchwork} Pedersen, T. L. (2020). \emph{Patchwork: {The} composer of plots} {[}Manual{]}. -\leavevmode\vadjust pre{\hypertarget{ref-pipernoStarchGrains2008}{}}% +\bibitem[\citeproctext]{ref-pipernoStarchGrains2008} Piperno, D. R., \& Dillehay, T. D. (2008). Starch grains on human teeth reveal early broad crop diet in northern {Peru}. \emph{Proceedings of the National Academy of Sciences}, \emph{105}(50), 19622--19627. \url{https://doi.org/10.1073/pnas.0808752105} -\leavevmode\vadjust pre{\hypertarget{ref-powerChimpCalculus2015}{}}% +\bibitem[\citeproctext]{ref-powerChimpCalculus2015} Power, R. C., Salazar-Garcia, D. C., Wittig, R. M., Freiberg, M., \& Henry, A. G. (2015). Dental calculus evidence of {Tai Forest Chimpanzee} plant consumption and life history transitions. \emph{Scientific Reports}, \emph{5}, 15161. \url{https://doi.org/10.1038/srep15161} -\leavevmode\vadjust pre{\hypertarget{ref-powerSEMCalculus2014}{}}% +\bibitem[\citeproctext]{ref-powerSEMCalculus2014} Power, R. C., Salazar-García, D. C., Wittig, R. M., \& Henry, A. G. (2014). Assessing use and suitability of scanning electron microscopy in the analysis of micro remains in dental calculus. \emph{Journal of Archaeological Science}, \emph{49}, 160--169. \url{https://doi.org/10.1016/j.jas.2014.04.016} -\leavevmode\vadjust pre{\hypertarget{ref-powerRepresentativenessDental2021}{}}% +\bibitem[\citeproctext]{ref-powerRepresentativenessDental2021} Power, Robert C., Wittig, R. M., Stone, J. R., Kupczik, K., \& Schulz-Kornas, E. (2021). The representativeness of the dental calculus -dietary record: Insights from {Ta{ï}} chimpanzee faecal phytoliths. +dietary record: Insights from {Taï} chimpanzee faecal phytoliths. \emph{Archaeological and Anthropological Sciences}, \emph{13}(6), 104. \url{https://doi.org/10.1007/s12520-021-01342-z} -\leavevmode\vadjust pre{\hypertarget{ref-proctorSpatialGradient2018}{}}% +\bibitem[\citeproctext]{ref-proctorSpatialGradient2018} Proctor, D. M., Fukuyama, J. A., Loomer, P. M., Armitage, G. C., Lee, S. A., Davis, N. M., Ryder, M. I., Holmes, S. P., \& Relman, D. A. (2018). A spatial gradient of bacterial diversity in the human oral cavity shaped by salivary flow. \emph{Nature Communications}, \emph{9}(1), 681. \url{https://doi.org/10.1038/s41467-018-02900-1} -\leavevmode\vadjust pre{\hypertarget{ref-Rbase}{}}% +\bibitem[\citeproctext]{ref-Rbase} R Core Team. (2020). \emph{R: {A} language and environment for statistical computing} {[}Manual{]}. {R Foundation for Statistical -Computing}; {R Foundation for Statistical Computing}. +Computing}. -\leavevmode\vadjust pre{\hypertarget{ref-radiniFoodPathways2017}{}}% +\bibitem[\citeproctext]{ref-radiniFoodPathways2017} Radini, A., Nikita, E., Buckley, S., Copeland, L., \& Hardy, K. (2017). Beyond food: {The} multiple pathways for inclusion of materials into ancient dental calculus. \emph{American Journal of Physical Anthropology}, \emph{162}, 71--83. \url{https://doi.org/10.1002/ajpa.23147} -\leavevmode\vadjust pre{\hypertarget{ref-radiniMedievalWomen2019}{}}% +\bibitem[\citeproctext]{ref-radiniMedievalWomen2019} Radini, A., Tromp, M., Beach, A., Tong, E., Speller, C., McCormick, M., Dudgeon, J. V., Collins, M. J., Rühli, F., Kröger, R., \& Warinner, C. (2019). Medieval women's early involvement in manuscript production @@ -5894,67 +5719,67 @@ \section*{References cited}\label{references-cited-3}} \emph{Science Advances}, \emph{5}(1), eaau7126. \url{https://doi.org/10.1126/sciadv.aau7126} -\leavevmode\vadjust pre{\hypertarget{ref-reichertStarchBible1913b}{}}% +\bibitem[\citeproctext]{ref-reichertStarchBible1913b} Reichert, E. T. (1913). \emph{The differentiation and specificity of starches in relation to genera, species, etc: Stereochemistry applied to protoplasmic processes and products, and as a strictly scientific basis for the classification of plants and animals} (Vol. 2). {Carnegie institution of Washington}. -\leavevmode\vadjust pre{\hypertarget{ref-Rbroom}{}}% +\bibitem[\citeproctext]{ref-Rbroom} Robinson, D., Hayes, A., \& Couch, S. (2021). \emph{Broom: {Convert} statistical objects into tidy tibbles} {[}Manual{]}. -\leavevmode\vadjust pre{\hypertarget{ref-scannapiecoSalivaryAmylase1993}{}}% +\bibitem[\citeproctext]{ref-scannapiecoSalivaryAmylase1993} Scannapieco, F. A., Torres, G., \& Levine, M. J. (1993). Salivary {\(\alpha\)}-amylase: Role in dental plaque and caries formation. \emph{Critical Reviews in Oral Biology \& Medicine}, \emph{4}(3), 301--307. -\leavevmode\vadjust pre{\hypertarget{ref-cummingsMayanCalculus1997}{}}% +\bibitem[\citeproctext]{ref-cummingsMayanCalculus1997} Scott Cummings, L., \& Magennis, A. (1997). A phytolith and starch record of food and grit in {Mayan} human tooth tartar. In A. Pinilla, J. Juan-Tresserras, \& M. J. Machado (Eds.), \emph{The {State-of-the-Art} of {Phytoliths} in {Soils} and {Plants}}. {CSIC Press}. -\leavevmode\vadjust pre{\hypertarget{ref-seidemannStarchAtlas1966}{}}% -Seidemann, J. (1966). \emph{St\{{\textbackslash{}}"a\}rke-{Atlas}: -{Grundlagen} der {St}\{{\textbackslash{}}"a\}rke-{Mikroskopie} und -{Beschreibung} der wichtigsten {St}\{{\textbackslash{}}"a\}rkearten}. +\bibitem[\citeproctext]{ref-seidemannStarchAtlas1966} +Seidemann, J. (1966). \emph{St\{\textbackslash"a\}rke-{Atlas}: +{Grundlagen} der {St}\{\textbackslash"a\}rke-{Mikroskopie} und +{Beschreibung} der wichtigsten {St}\{\textbackslash"a\}rkearten}. {Parey}. -\leavevmode\vadjust pre{\hypertarget{ref-shellisSyntheticSaliva1978}{}}% +\bibitem[\citeproctext]{ref-shellisSyntheticSaliva1978} Shellis, R. P. (1978). A synthetic saliva for cultural studies of dental plaque. \emph{Archives of Oral Biology}, \emph{23}(6), 485--489. \url{https://doi.org/10.1016/0003-9969(78)90081-X} -\leavevmode\vadjust pre{\hypertarget{ref-simonsoroOralGeography2013}{}}% +\bibitem[\citeproctext]{ref-simonsoroOralGeography2013} Simón-Soro, A., Tomás, I., Cabrera-Rubio, R., Catalan, M. D., Nyvad, B., \& Mira, A. (2013). Microbial geography of the oral cavity. \emph{Journal of Dental Research}, \emph{92}(7), 616--621. \url{https://doi.org/10.1177/0022034513488119} -\leavevmode\vadjust pre{\hypertarget{ref-sissonsMultistationPlaque1991}{}}% +\bibitem[\citeproctext]{ref-sissonsMultistationPlaque1991} Sissons, C. H., Cutress, T. W., Hoffman, M. P., \& Wakefield, J. S. J. (1991). A {Multi-station Dental Plaque Microcosm} ({Artificial Mouth}) for the {Study} of {Plaque Growth}, {Metabolism}, {pH}, and {Mineralization}: \emph{Journal of Dental Research}. \url{https://doi.org/10.1177/00220345910700110301} -\leavevmode\vadjust pre{\hypertarget{ref-tanCalculusUltrastructure2004}{}}% +\bibitem[\citeproctext]{ref-tanCalculusUltrastructure2004} Tan, B. T. K., Gillam, D. G., Mordan, N. J., \& Galgut, P. N. (2004). A preliminary investigation into the ultrastructure of dental calculus and associated bacteria. \emph{Journal of Clinical Periodontology}, \emph{31}(5), 364--369. \url{https://doi.org/10.1111/j.1600-051X.2004.00484.x} -\leavevmode\vadjust pre{\hypertarget{ref-tanBacterialViability2004}{}}% +\bibitem[\citeproctext]{ref-tanBacterialViability2004} Tan, B. T. K., Mordan, N. J., Embleton, J., Pratten, J., \& Galgut, P. N. (2004). Study of {Bacterial Viability} within {Human Supragingival Dental Calculus}. \emph{Journal of Periodontology}, \emph{75}(1), 23--29. \url{https://doi.org/10.1902/jop.2004.75.1.23} -\leavevmode\vadjust pre{\hypertarget{ref-taoWheatCalculus2020}{}}% +\bibitem[\citeproctext]{ref-taoWheatCalculus2020} Tao, D., Zhang, G., Zhou, Y., \& Zhao, H. (2020). Investigating wheat consumption based on multiple evidences: {Stable} isotope analysis on human bone and starch grain analysis on dental calculus of humans from @@ -5962,33 +5787,33 @@ \section*{References cited}\label{references-cited-3}} \emph{International Journal of Osteoarchaeology}, \emph{30}(5), 594--606. \url{https://doi.org/10.1002/oa.2884} -\leavevmode\vadjust pre{\hypertarget{ref-toppingResistantStarch2003}{}}% +\bibitem[\citeproctext]{ref-toppingResistantStarch2003} Topping, D. L., Fukushima, M., \& Bird, A. R. (2003). Resistant starch as a prebiotic and synbiotic: State of the art. \emph{Proceedings of the Nutrition Society}, \emph{62}(1), 171--176. \url{https://doi.org/10.1079/PNS2002224} -\leavevmode\vadjust pre{\hypertarget{ref-trompEDTACalculus2017}{}}% +\bibitem[\citeproctext]{ref-trompEDTACalculus2017} Tromp, M., Buckley, H., Geber, J., \& Matisoo-Smith, E. (2017). {EDTA} decalcification of dental calculus as an alternate means of microparticle extraction from archaeological samples. \emph{Journal of Archaeological Science: Reports}, \emph{14}, 461--466. \url{https://doi.org/10.1016/j.jasrep.2017.06.035} -\leavevmode\vadjust pre{\hypertarget{ref-trompDietaryNondietary2015}{}}% +\bibitem[\citeproctext]{ref-trompDietaryNondietary2015} Tromp, M., \& Dudgeon, J. V. (2015). Differentiating dietary and non-dietary microfossils extracted from human dental calculus: The importance of sweet potato to ancient diet on {Rapa Nui}. \emph{Journal of Archaeological Science}, \emph{54}, 54--63. \url{https://doi.org/10.1016/j.jas.2014.11.024} -\leavevmode\vadjust pre{\hypertarget{ref-vandeveldeStarchMorphology2002}{}}% +\bibitem[\citeproctext]{ref-vandeveldeStarchMorphology2002} van de Velde, F., van Riel, J., \& Tromp, R. H. (2002). Visualisation of starch granule morphologies using confocal scanning laser microscopy ({CSLM}). \emph{Journal of the Science of Food and Agriculture}, \emph{82}(13), 1528--1536. \url{https://doi.org/10.1002/jsfa.1165} -\leavevmode\vadjust pre{\hypertarget{ref-warinnerEvidenceMilk2014}{}}% +\bibitem[\citeproctext]{ref-warinnerEvidenceMilk2014} Warinner, C., Hendy, J., Speller, C., Cappellini, E., Fischer, R., Trachsel, C., Arneborg, J., Lynnerup, N., Craig, O. E., Swallow, D. M., Fotakis, A., Christensen, R. J., Olsen, J. V., Liebert, A., Montalva, @@ -5997,7 +5822,7 @@ \section*{References cited}\label{references-cited-3}} human dental calculus. \emph{Scientific Reports}, \emph{4}, 7104. \url{https://doi.org/10.1038/srep07104} -\leavevmode\vadjust pre{\hypertarget{ref-warinnerPathogensHost2014}{}}% +\bibitem[\citeproctext]{ref-warinnerPathogensHost2014} Warinner, C., Rodrigues, J. F., Vyas, R., Trachsel, C., Shved, N., Grossmann, J., Radini, A., Hancock, Y., Tito, R. Y., Fiddyment, S., Speller, C., Hendy, J., Charlton, S., Luder, H. U., Salazar-Garcia, D. @@ -6006,14 +5831,14 @@ \section*{References cited}\label{references-cited-3}} oral cavity. \emph{Nature Genetics}, \emph{46}(4), 336--344. \url{https://doi.org/10.1038/ng.2906} -\leavevmode\vadjust pre{\hypertarget{ref-wesolowskiEvaluatingMicrofossil2010}{}}% +\bibitem[\citeproctext]{ref-wesolowskiEvaluatingMicrofossil2010} Wesolowski, V., Ferraz Mendonça de Souza, S. M., Reinhard, K. J., \& Ceccantini, G. (2010). Evaluating microfossil content of dental calculus from {Brazilian} sambaquis. \emph{Journal of Archaeological Science}, \emph{37}(6), 1326--1338. \url{https://doi.org/10.1016/j.jas.2009.12.037} -\leavevmode\vadjust pre{\hypertarget{ref-tidyverse2019}{}}% +\bibitem[\citeproctext]{ref-tidyverse2019} Wickham, H., Averick, M., Bryan, J., Chang, W., McGowan, L. D., François, R., Grolemund, G., Hayes, A., Henry, L., Hester, J., Kuhn, M., Pedersen, T. L., Miller, E., Bache, S. M., Müller, K., Ooms, J., @@ -6021,7 +5846,7 @@ \section*{References cited}\label{references-cited-3}} Welcome to the {tidyverse}. \emph{Journal of Open Source Software}, \emph{4}(43), 1686. \url{https://doi.org/10.21105/joss.01686} -\leavevmode\vadjust pre{\hypertarget{ref-wuDietEarliest2021}{}}% +\bibitem[\citeproctext]{ref-wuDietEarliest2021} Wu, Y., Tao, D., Wu, X., \& Liu, W. (2021). \emph{Diet of the earliest modern humans in {East Asia}} {[}Preprint{]}. {In Review}. \url{https://doi.org/10.21203/rs.3.rs-442096/v1} @@ -6030,8 +5855,7 @@ \section*{References cited}\label{references-cited-3}} \bookmarksetup{startatroot} -\hypertarget{article-3}{% -\chapter{Article 3}\label{article-3}} +\chapter{Article 3}\label{article-3} Multiproxy analysis exploring patterns of diet and disease in dental calculus and skeletal remains from a 19th century Dutch population @@ -6069,106 +5893,103 @@ \chapter{Article 3}\label{article-3}} \textbf{Cite as:} Bartholdy, B. P., Hasselstrøm, J. B., Sørensen, L. K., Casna, M., -Hoogland, M., Beemster, H. G., \& Henry, A. G. (2023). Multiproxy +Hoogland, M., Beemster, H. G., \& Henry, A. G. (2024). Multiproxy analysis exploring patterns of diet and disease in dental calculus and -skeletal remains from a 19th century Dutch population. Zenodo. -https://doi.org/10.5281/zenodo.7649151 +skeletal remains from a 19th century Dutch population. Peer Community +Journal, 4. https://doi.org/10.24072/pcjournal.414 \normalsize \newpage{} -\hypertarget{mb11CalculusPilot}{% -\section{Introduction}\label{mb11CalculusPilot}} +\section{Introduction}\label{mb11CalculusPilot} Dental calculus has proven to be an excellent source of a wide variety of information about our past. The increased accessibility and advancement of methods in aDNA, paleoproteomics, and mass spectrometry, has expanded our ability to identify biomarkers of diet and disease on an increasingly large scale -(\protect\hyperlink{ref-gismondiMultidisciplinaryApproach2020}{Gismondi -et al., 2020}; \protect\hyperlink{ref-velskoDentalCalculus2017}{Velsko -et al., 2017}; \protect\hyperlink{ref-warinnerEvidenceMilk2014}{Warinner -et al., 2014}). +(\citeproc{ref-gismondiMultidisciplinaryApproach2020}{Gismondi et al., +2020}; \citeproc{ref-velskoDentalCalculus2017}{Velsko et al., 2017}; +\citeproc{ref-warinnerEvidenceMilk2014}{Warinner et al., 2014}). One such collection of biomarkers is alkaloids, a plant-derived group of compounds. Many alkaloids have important medicinal and psychoactive effects in humans, and their direct detection, or detection of their metabolites, is of great interest to archaeologists. Previous studies have successfully recovered alkaloids in archaeological contexts, -including ceramics -(\protect\hyperlink{ref-smithDetectionOpium2018}{Smith et al., 2018}), -pipes (\protect\hyperlink{ref-raffertyCurrentResearch2012}{Rafferty et +including ceramics (\citeproc{ref-smithDetectionOpium2018}{Smith et al., +2018}), pipes (\citeproc{ref-raffertyCurrentResearch2012}{Rafferty et al., 2012}), human hair -(\protect\hyperlink{ref-echeverriaNicotineHair2013}{Echeverría \& -Niemeyer, 2013}; -\protect\hyperlink{ref-ogaldeIdentificationPsychoactive2009}{Ogalde et +(\citeproc{ref-echeverriaNicotineHair2013}{Echeverría \& Niemeyer, +2013}; \citeproc{ref-ogaldeIdentificationPsychoactive2009}{Ogalde et al., 2009}), and even dental calculus employing both targeted -(\protect\hyperlink{ref-eerkensDentalCalculus2018}{Eerkens et al., -2018}) and untargeted approaches -(\protect\hyperlink{ref-buckleyDentalCalculus2014}{Buckley et al., -2014}; -\protect\hyperlink{ref-gismondiMultidisciplinaryApproach2020}{Gismondi -et al., 2020}). Especially nicotine, the principal alkaloid in tobacco -leaves, has been widely studied in the archaeological record due to its -apparent stability and ability to survive over long periods of time -(\protect\hyperlink{ref-eerkensDentalCalculus2018}{Eerkens et al., -2018}; \protect\hyperlink{ref-raffertyCurrentResearch2012}{Rafferty et -al., 2012}; -\protect\hyperlink{ref-tushinghamHuntergathererTobacco2013}{Tushingham -et al., 2013}). +(\citeproc{ref-eerkensDentalCalculus2018}{Eerkens et al., 2018}) and +untargeted approaches (\citeproc{ref-buckleyDentalCalculus2014}{Buckley +et al., 2014}; +\citeproc{ref-gismondiMultidisciplinaryApproach2020}{Gismondi et al., +2020}). Especially nicotine, the principal alkaloid in tobacco leaves, +has been widely studied in the archaeological record due to its apparent +stability and ability to survive over long periods of time +(\citeproc{ref-eerkensDentalCalculus2018}{Eerkens et al., 2018}; +\citeproc{ref-raffertyCurrentResearch2012}{Rafferty et al., 2012}; +\citeproc{ref-tushinghamHuntergathererTobacco2013}{Tushingham et al., +2013}). Alkaloids may enter the oral cavity via two pathways: (1) direct -incorporation through oral consumption of alkaloid-containing plants, -whether deliberate or accidental; and (2) passive diffusion as alkaloids -and other compounds are transferred from plasma to saliva, and then into -the oral cavity through the salivary glands in the hours to days -following consumption -(\protect\hyperlink{ref-coneInterpretationOral2007}{Cone \& Huestis, -2007}). The relation to plasma is why there is often a close correlation -between presence (not concentration) of drugs in oral fluid and blood -(\protect\hyperlink{ref-coneInterpretationOral2007}{Cone \& Huestis, -2007}; \protect\hyperlink{ref-milmanOralFluid2011}{Milman et al., 2011}; -\protect\hyperlink{ref-willeRelationshipOral2009}{Wille et al., 2009}). -The second pathway allows the identification of parent compounds that -are not consumed orally, as long as they, or their metabolites, are -excreted through saliva. . +incorporation through ingestion of alkaloid-containing plants, whether +deliberate or accidental; and (2) passive diffusion as alkaloids and +other compounds are transferred from plasma to saliva, and then +gradually secreted into the oral cavity through the salivary glands in +the hours-to-days following ingestion +(\citeproc{ref-coneInterpretationOral2007}{Cone \& Huestis, 2007}). The +second pathway allows the identification of parent compounds that do not +enter the mouth (e.g.~injection), as long as they, or their metabolites, +are excreted through the saliva, thus eventually entering the oral +cavity. Many of the components involved in the formation and growth of dental calculus originate from oral fluid. Proteins, bacteria, salts and other compounds are transferred from saliva to biofilms on the tooth surface -(\protect\hyperlink{ref-jinSupragingivalCalculus2002}{Jin \& Yip, 2002}; -\protect\hyperlink{ref-whiteDentalCalculus1997}{White, 1997}). This may -also allow various alkaloids of dietary and medicinal origin to become +(\citeproc{ref-jinSupragingivalCalculus2002}{Jin \& Yip, 2002}; +\citeproc{ref-whiteDentalCalculus1997}{White, 1997}). This may also +allow various alkaloids of dietary and medicinal origin to become incorporated in dental plaque. Dental plaque undergoes frequent mineralisation events, ultimately causing the entrapped alkaloids and their metabolites to become preserved within the dental calculus. Barring intentional or accidental removal of the calculus during life, burial, excavation, and post-excavation cleaning, the alkaloids can then be detected by various methods to show a record of consumption during -life. +life. Because drugs may be transferred from plasma to saliva, there is +often a close correlation between drugs detected in oral fluid and +blood, though there are differences in detected concentrations +(\citeproc{ref-coneInterpretationOral2007}{Cone \& Huestis, 2007}; +\citeproc{ref-milmanOralFluid2011}{Milman et al., 2011}; +\citeproc{ref-willeRelationshipOral2009}{Wille et al., 2009}). This was +also shown to be true for dental calculus and blood +(\citeproc{ref-sorensenDrugsCalculus2021}{Sørensen et al., 2021}), +making dental calculus a potentially useful substance for detecting +ancient alkaloids and other dietary compounds. In this study we use a ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method that was developed in a previous study on dental calculus from cadavers and validated by comparing the results to compounds detected in the blood of -the same individuals -(\protect\hyperlink{ref-sorensenDrugsCalculus2021}{Sørensen et al., -2021}). All compounds that were detected in the blood were also detected -in dental calculus, with additional compounds present in dental calculus -that were not present in blood, suggesting that dental calculus +the same individuals (\citeproc{ref-sorensenDrugsCalculus2021}{Sørensen +et al., 2021}). All compounds that were detected in the blood were also +detected in dental calculus, with additional compounds present in dental +calculus that were not present in blood, suggesting that dental calculus represents a comprehensive history of consumption over a long period of -time (\protect\hyperlink{ref-sorensenDrugsCalculus2021}{Sørensen et al., -2021}). We were able to detect both parent compounds and metabolites, -including caffeine, nicotine, theophylline, and cotinine, in the dental -calculus of individuals from a 19th century Dutch population from -Middenbeemster. By detecting these compounds we are able to show the -consumption of tea and coffee and smoking of tobacco on an individual -scale, which is also confirmed by historic documentation and -identification of pipe notches in the dentition. - -\hypertarget{mb11CalculusPilot-mat}{% -\section{Materials}\label{mb11CalculusPilot-mat}} +time (\citeproc{ref-sorensenDrugsCalculus2021}{Sørensen et al., 2021}). +We were able to detect both parent compounds and metabolites, including +caffeine, nicotine, theophylline, and cotinine, in the dental calculus +of individuals from a 19th century Dutch population from Middenbeemster. +By detecting these compounds we are able to show the consumption of tea +and coffee and smoking of tobacco on an individual scale, which is also +confirmed by historic documentation and identification of pipe notches +in the dentition. + +\section{Materials}\label{mb11CalculusPilot-mat} The sample consists of 41 individuals from Middenbeemster, a 19th century rural Dutch site. The village of Middenbeemster and the @@ -6179,27 +6000,34 @@ \section{Materials}\label{mb11CalculusPilot-mat}} (Hakvoort 2013). The excavated cemetery is associated with the Keyserkerk church, where the inhabitants of the Middenbeemster village and the surrounding Beemsterpolder were buried between AD 1615 and 1866 -(\protect\hyperlink{ref-lemmersMiddenbeemster2013}{Lemmers et al., -2013}). Archival documents are available for those buried between AD -1829 and 1866, when the majority of individuals were interred -(\protect\hyperlink{ref-palmerActivityReconstruction2016}{Palmer et al., -2016}). The main occupation of the inhabitants was dairy farming, -consisting largely of manual labour prior to the industrial revolution -(\protect\hyperlink{ref-aten400Jaar2012}{Aten et al., 2012}; -\protect\hyperlink{ref-palmerActivityReconstruction2016}{Palmer et al., -2016}). - -To reduce the number of potentially confounding factors to account for -in the analysis, we preferentially selected males from the middle adult -age category (35-49 years). The sample consists of 27 males, 11 probable -males, 2 probable females, and 1 female -(Figure~\ref{fig-sample-demography}). We selected males due to a higher -occurrence of pipe notches and dental calculus deposits than females -(unpublished observation). +(\citeproc{ref-lemmersMiddenbeemster2013}{Lemmers et al., 2013}). +Archival documents are available for those buried between AD 1829 and +1866, when the majority of individuals were interred. The main +occupation of the inhabitants was dairy farming, consisting largely of +manual labour prior to the industrial revolution +(\citeproc{ref-aten400Jaar2012}{Aten et al., 2012}; +\citeproc{ref-palmerActivityReconstruction2016}{Palmer et al., 2016}). + +For our sample, we preferentially selected males from the middle adult +age category (35-49 years) to minimise the effect of confounding +cultural and biological factors. Previous research on Middenbeemster has +shown a gendered division of labour +(\citeproc{ref-palmerActivityReconstruction2016}{Palmer et al., 2016}), +and there are biological differences in dental calculus formation and +drug metabolism that are related to age and sex +(\citeproc{ref-huangDecipheringGenetic2023}{Huang et al., 2023}; +\citeproc{ref-unoSexAgedependent2017}{Uno et al., 2017}; +\citeproc{ref-whiteDentalCalculus1997}{White, 1997}). The sample +consists of 27 males, 11 probable males, 2 probable females, and 1 +female (Figure~\ref{fig-sample-demography}). We selected males due to a +higher occurrence of pipe notches and dental calculus deposits than +females (unpublished observation). \begin{figure} -{\centering \includegraphics{05-article_files/figure-pdf/fig-sample-demography-1.pdf} +\centering{ + +\includegraphics{05-article_files/figure-pdf/fig-sample-demography-1.pdf} } @@ -6210,35 +6038,34 @@ \section{Materials}\label{mb11CalculusPilot-mat}} (35-49 years); old = old adult (50+ years). Male? = probable male; Female? = probable female.} -\end{figure} +\end{figure}% -\hypertarget{methods}{% -\section{Methods}\label{methods}} +\section{Methods}\label{methods} -\hypertarget{skeletal-analysis}{% -\subsection{Skeletal analysis}\label{skeletal-analysis}} +\subsection{Skeletal analysis}\label{skeletal-analysis} Demographic and pathological analyses were conducted in the Laboratory for Human Osteoarchaeology at Leiden University. Sex was estimated using cranial and pelvic morphological traits -(\protect\hyperlink{ref-Standards1994}{Buikstra \& Ubelaker, 1994}). -Age-at-death was estimated using dental wear, auricular and pubic -surface appearance, cranial suture closure, and epiphyseal fusion -(\protect\hyperlink{ref-SucheyBrooks1990}{Brooks \& Suchey, 1990}; -\protect\hyperlink{ref-buckberryAuricular2002}{Buckberry \& Chamberlain, -2002}; \protect\hyperlink{ref-Standards1994}{Buikstra \& Ubelaker, -1994}; \protect\hyperlink{ref-lovejoyAuricular1985}{Lovejoy et al., -1985}; \protect\hyperlink{ref-meindlSutureClosure1985}{Meindl \& -Lovejoy, 1985}), and divided into the following categories: early young -adult (18-24 years), late young adult (25-34 years), middle adult ( -35-49 years), old adult (50+ years). - -\hypertarget{paleopathology}{% -\subsubsection{Paleopathology}\label{paleopathology}} +(\citeproc{ref-Standards1994}{Buikstra \& Ubelaker, 1994}). Age-at-death +was estimated using dental wear, auricular and pubic surface appearance, +cranial suture closure, and epiphyseal fusion +(\citeproc{ref-SucheyBrooks1990}{Brooks \& Suchey, 1990}; +\citeproc{ref-buckberryAuricular2002}{Buckberry \& Chamberlain, 2002}; +\citeproc{ref-Standards1994}{Buikstra \& Ubelaker, 1994}; +\citeproc{ref-lovejoyAuricular1985}{Lovejoy et al., 1985}; +\citeproc{ref-meindlSutureClosure1985}{Meindl \& Lovejoy, 1985}), and +divided into the following categories: early young adult (18-24 years), +late young adult (25-34 years), middle adult (35-49 years), old adult +(50+ years). Preservation was visually scored on a four-stage scale +(excellent, good, fair, poor) based on the surface condition of the +bones and the extent of taphonomic degradation. + +\subsubsection{Paleopathology}\label{paleopathology} Pathological conditions and lesions that occur frequently in the population were included in the analysis. Data were dichotomised to -presence/absence to allow statistical analysis. Osteoarthritis was +presence/absence to allow for statistical analysis. Osteoarthritis was considered present in cases where eburnation was visible on one or more joint surfaces. Vertebral osteophytosis is identified by marginal lipping and/or osteophyte formation on the margin of the superior and @@ -6247,50 +6074,46 @@ \subsubsection{Paleopathology}\label{paleopathology}} No distinction was made between active or healing lesions. Degenerative disc disease, or spondylosis, is identified as a large diffuse depression of the superior and/or inferior surfaces of the vertebral -body (\protect\hyperlink{ref-rogersPalaeopathologyJoint2000}{Rogers, -2000}). Schmorl's nodes are identified as any cortical depressions on -the surface of the vertebral body. Data on chronic maxillary sinusitis -from Casna et al. -(\protect\hyperlink{ref-casnaUrbanizationRespiratory2021}{2021}) were -included in this study to assess the relationship between upper +body (\citeproc{ref-rogersPalaeopathologyJoint2000}{Rogers, 2000}). +Schmorl's nodes are identified as any cortical depressions on the +surface of the vertebral body. Data on chronic maxillary sinusitis from +Casna et al. (\citeproc{ref-casnaUrbanizationRespiratory2021}{2021}) +were included in this study to assess the relationship between upper respiratory diseases with environmental factors (i.e.~tobacco smoke, caffeine consumption). Lesions associated with chronic maxillary sinusitis as defined by Boocock et al. -(\protect\hyperlink{ref-boocockMaxillarySinusitis1995}{1995}) were -recorded for each individual and classified as ``pitting'', -``spicule-type bone formation'', ``remodeled spicules'', or ``white -pitted bone''. chronic maxillary sinusitis was scored as absent when the -sinus presented smooth surfaces with little or no associated pitting. +(\citeproc{ref-boocockMaxillarySinusitis1995}{1995}) were recorded for +each individual and classified as ``pitting'', ``spicule-type bone +formation'', ``remodeled spicules'', or ``white pitted bone''. chronic +maxillary sinusitis was scored as absent when the sinus presented smooth +surfaces with little or no associated pitting. -\hypertarget{dental-pathology}{% -\subsubsection{Dental pathology}\label{dental-pathology}} +\subsubsection{Dental pathology}\label{dental-pathology} Caries ratios were calculated by dividing the number of lesions by the number of teeth scored, resulting in a single caries ratio per individual. If the surface where the lesion originated is not visible, i.e.~if the lesion covered multiple surfaces, this was scored as ``crown''. Calculus indices were calculated according to Greene and -colleagues -(\protect\hyperlink{ref-greeneQuantifyingCalculus2005}{2005}). Calculus -was scored with a four-stage scoring system (0-3) to score absent, -slight, moderate, and heavy calculus deposits -(\protect\hyperlink{ref-brothwellDiggingBones1981}{Brothwell, 1981}) on -the lingual, buccal (and labial), and interproximal surfaces of each -tooth. Only one score was used for the combined interproximal surfaces, +colleagues (\citeproc{ref-greeneQuantifyingCalculus2005}{2005}). +Calculus was scored with a four-stage scoring system (0-3) to score +absent, slight, moderate, and heavy calculus deposits +(\citeproc{ref-brothwellDiggingBones1981}{Brothwell, 1981}) on the +lingual, buccal (and labial), and interproximal surfaces of each tooth. +Only one score was used for the combined interproximal surfaces, resulting in three scores per tooth (when surfaces are intact), and four calculus indices per individual; upper anterior, upper posterior, lower anterior, lower posterior. Each index was calculated by dividing the sum of calculus scores for each surface by the total number of surfaces scored in each quadrant. If a tooth could not be scored on all three surfaces, the tooth was not included -(\protect\hyperlink{ref-greeneQuantifyingCalculus2005}{Greene et al., -2005}). Periodontitis was scored on a visual four-stage (0-3) scoring -system according to distance from cemento-enamel junction of each tooth -to alveolar bone (\protect\hyperlink{ref-maatManualPhysical2005}{Maat \& -Mastwijk, 2005}). +(\citeproc{ref-greeneQuantifyingCalculus2005}{Greene et al., 2005}). +Periodontitis was scored on a visual four-stage (0-3) scoring system +according to distance from cemento-enamel junction of each tooth to +alveolar bone (\citeproc{ref-maatManualPhysical2005}{Maat \& Mastwijk, +2005}). -\hypertarget{calculus-sampling}{% -\subsection{Calculus sampling}\label{calculus-sampling}} +\subsection{Calculus sampling}\label{calculus-sampling} Where possible, we used material that had already been sampled for a previous study to prevent unnecessary repeated sampling of individuals. @@ -6298,11 +6121,11 @@ \subsection{Calculus sampling}\label{calculus-sampling}} laboratory at the Laboratories of Molecular Anthropology and Microbiome Research in Norman, Oklahoma, U.S.A, using established ancient DNA protocols. More details on the methods can be found in the published -articles (\protect\hyperlink{ref-ziesemer16SChallenges2015}{Ziesemer et -al., 2015}, \protect\hyperlink{ref-ziesemerGenomeCalculus2018}{2018}). -Of the 41 individuals that were originally included in our sample, 29 -were replicated in a separate analysis only using calculus from the -previous study.\\ +articles (\citeproc{ref-ziesemer16SChallenges2015}{Ziesemer et al., +2015}, \citeproc{ref-ziesemerGenomeCalculus2018}{2018}). Of the 41 +individuals that were originally included in our sample, 29 were +replicated in a separate analysis only using calculus from the previous +study.\\ New dental calculus samples were taken under sterile conditions in a positive pressure laminar flow hood in a dedicated dental calculus lab at Leiden University. The surface of the tooth was lightly brushed with @@ -6313,16 +6136,14 @@ \subsection{Calculus sampling}\label{calculus-sampling}} Aarhus University for ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. -\hypertarget{uhplc-msms}{% -\subsection{UHPLC-MS/MS}\label{uhplc-msms}} +\subsection{UHPLC-MS/MS}\label{uhplc-msms} The list of targeted compounds included both naturally occurring compounds known to have been used in the past, as well as synthetic modern drugs that did not exist at the time (e.g.~Fentanyl, MDMA, Amphetamine). These were part of the toxicology screening for the -original method -(\protect\hyperlink{ref-sorensenDrugsCalculus2021}{Sørensen et al., -2021}), developed on cadavers. In our study they serve as an +original method (\citeproc{ref-sorensenDrugsCalculus2021}{Sørensen et +al., 2021}), developed on cadavers. In our study they serve as an authentication step, as their presence in archaeological samples could only be the result of contamination. @@ -6339,10 +6160,9 @@ \subsection{UHPLC-MS/MS}\label{uhplc-msms}} biphenyl column for chromatography. To obtain quantitative results, isotope dilution was applied. For more details about the method and validation, see the original study by Sørensen and colleagues -(\protect\hyperlink{ref-sorensenDrugsCalculus2021}{2021}). +(\citeproc{ref-sorensenDrugsCalculus2021}{2021}). -\hypertarget{statistical-analysis-1}{% -\subsection{Statistical analysis}\label{statistical-analysis-1}} +\subsection{Statistical analysis}\label{statistical-analysis-1} All compounds and pathological conditions/lesions were converted to a presence/absence score. Pearson product-moment correlation was applied @@ -6355,17 +6175,15 @@ \subsection{Statistical analysis}\label{statistical-analysis-1}} by using quartiles. Polychoric correlation was applied to the paired dichotomous variables and dichotomous-ordinal variables. -All statistical analysis was conducted in R version 4.3.2 (2023-10-31), -Eye Holes, (\protect\hyperlink{ref-Rbase}{R Core Team, 2020}). Data +All statistical analysis was conducted in R version 4.3.3 (2024-02-29), +Angel Food Cake, (\citeproc{ref-Rbase}{R Core Team, 2020}). Data wrangling was conducted with the \textbf{tidyverse} -(\protect\hyperlink{ref-tidyverse2019}{Wickham et al., 2019}) and -visualisations were created using \textbf{ggplot2} -(\protect\hyperlink{ref-ggplot2}{Wickham, 2016}). Polychoric -correlations were calculated with the \textbf{psych} package -(\protect\hyperlink{ref-Rpsych}{Revelle, 2022}). +(\citeproc{ref-tidyverse2019}{Wickham et al., 2019}) and visualisations +were created using \textbf{ggplot2} (\citeproc{ref-ggplot2}{Wickham, +2016}). Polychoric correlations were calculated with the \textbf{psych} +package (\citeproc{ref-Rpsych}{Revelle, 2022}). -\hypertarget{results-2}{% -\section{Results}\label{results-2}} +\section{Results}\label{results-2} Multiple compounds were detected in the dental calculus samples. Compounds detected at a lower concentration than the lower limit of @@ -6374,17 +6192,16 @@ \section{Results}\label{results-2}} (Table~\ref{tbl-compound-detect}). For a full list of targeted compounds, see Supplementary Material. -\hypertarget{tbl-compound-detect}{} \begin{longtable}[]{@{}lllr@{}} + \caption{\label{tbl-compound-detect}Target compound including whether it was detected (TRUE) or not (FALSE) in each batch, as well as the lower limit of quantitation (LLOQ) in ng. CBD = cannabidiol; CBN = cannabinol; THC = tetrahydrocannabinol; THCA-A = tetrahydrocannabinolic acid A; -THCVA = tetrahydrocannabivarin acid.}\tabularnewline -\toprule\noalign{} -Compound & Batch 1 & Batch 2 & LLOQ \\ -\midrule\noalign{} -\endfirsthead +THCVA = tetrahydrocannabivarin acid.} + +\tabularnewline + \toprule\noalign{} Compound & Batch 1 & Batch 2 & LLOQ \\ \midrule\noalign{} @@ -6402,6 +6219,7 @@ \section{Results}\label{results-2}} THCA-A & TRUE & FALSE & 0.025 \\ THCVA & TRUE & FALSE & 0.010 \\ Theophylline & TRUE & TRUE & 0.010 \\ + \end{longtable} The pattern we expect to see in authentic compounds representing @@ -6419,11 +6237,13 @@ \section{Results}\label{results-2}} and 2. Nicotine and cotinine have the same relative quantities in the samples, i.e., the sample with the highest extracted quantity of nicotine also had the highest extracted quantity of cotinine -Figure~\ref{fig-auth-plot-batch2}. +(Figure~\ref{fig-auth-plot-batch2}). \begin{figure} -{\centering \includegraphics{05-article_files/figure-pdf/fig-auth-plot-batch2-1.pdf} +\centering{ + +\includegraphics{05-article_files/figure-pdf/fig-auth-plot-batch2-1.pdf} } @@ -6436,7 +6256,7 @@ \section{Results}\label{results-2}} tetrahydrocannabinol; THCA-A = tetrahydrocannabinolic acid A; THCVA = tetrahydrocannabivarin acid.} -\end{figure} +\end{figure}% To see if preservation of the skeletal remains had any effect on the detection of compounds, we compare extracted quantities of compounds to @@ -6450,7 +6270,9 @@ \section{Results}\label{results-2}} \begin{figure} -{\centering \includegraphics{05-article_files/figure-pdf/fig-detection-preservation-1.pdf} +\centering{ + +\includegraphics{05-article_files/figure-pdf/fig-detection-preservation-1.pdf} } @@ -6458,9 +6280,10 @@ \section{Results}\label{results-2}} box plots depicting the distribution of extracted quantities of each compound from batch 2 separated by state of preservation of the skeleton. (B) Extracted quantity (ng) of compound plotted against -weights of the calculus samples from batch 2.} +weights of the calculus samples from batch 2. r = Pearson correlation +coefficient.} -\end{figure} +\end{figure}% The presence of pipe notch(es) in an individual and concurrent detection of nicotine and/or cotinine is used as a crude indicator of the accuracy @@ -6475,54 +6298,35 @@ \section{Results}\label{results-2}} One individual---an old adult, probable female---was positive for both nicotine and cotinine, and had no signs of a pipe notch. -\hypertarget{correlations-between-detected-alkaloids-and-diseases}{% \subsection{Correlations between detected alkaloids and -diseases}\label{correlations-between-detected-alkaloids-and-diseases}} +diseases}\label{correlations-between-detected-alkaloids-and-diseases} For further statistical analyses, only the UHPLC-MS/MS results from batch 2 were used, as batch 1 had multiple compounds that were not detected in batch 2 and may have been contaminated. -\hypertarget{tbl-pearson}{} \begin{longtable}[]{@{} - >{\raggedright\arraybackslash}p{(\columnwidth - 16\tabcolsep) * \real{0.1548}} - >{\raggedright\arraybackslash}p{(\columnwidth - 16\tabcolsep) * \real{0.0952}} - >{\raggedright\arraybackslash}p{(\columnwidth - 16\tabcolsep) * \real{0.1071}} - >{\raggedright\arraybackslash}p{(\columnwidth - 16\tabcolsep) * \real{0.0833}} - >{\raggedright\arraybackslash}p{(\columnwidth - 16\tabcolsep) * \real{0.1071}} - >{\raggedright\arraybackslash}p{(\columnwidth - 16\tabcolsep) * \real{0.0833}} - >{\raggedright\arraybackslash}p{(\columnwidth - 16\tabcolsep) * \real{0.1548}} - >{\raggedright\arraybackslash}p{(\columnwidth - 16\tabcolsep) * \real{0.1071}} - >{\raggedright\arraybackslash}p{(\columnwidth - 16\tabcolsep) * \real{0.1071}}@{}} + >{\raggedright\arraybackslash}p{(\columnwidth - 16\tabcolsep) * \real{0.1500}} + >{\raggedright\arraybackslash}p{(\columnwidth - 16\tabcolsep) * \real{0.1000}} + >{\raggedright\arraybackslash}p{(\columnwidth - 16\tabcolsep) * \real{0.1000}} + >{\raggedright\arraybackslash}p{(\columnwidth - 16\tabcolsep) * \real{0.1000}} + >{\raggedright\arraybackslash}p{(\columnwidth - 16\tabcolsep) * \real{0.1000}} + >{\raggedright\arraybackslash}p{(\columnwidth - 16\tabcolsep) * \real{0.1000}} + >{\raggedright\arraybackslash}p{(\columnwidth - 16\tabcolsep) * \real{0.1500}} + >{\raggedright\arraybackslash}p{(\columnwidth - 16\tabcolsep) * \real{0.1000}} + >{\raggedright\arraybackslash}p{(\columnwidth - 16\tabcolsep) * \real{0.1000}}@{}} + \caption{\label{tbl-pearson}Pearson correlation (\emph{r}) on dichotomous skeletal lesions and compound concentrations (ng/mg) from the second batch. Correlations between pairs of dichotomous variables -are removed due to incompatibility with a Pearson correlation. OA = -osteoarthritis; VOP = vertebral osteophytosis; SN = Schmorl's nodes; DDD -= degenerative disc disease; CO = cribra orbitalia; CMS = chronic -maxillary sinusitis; SA = salicylic acid; PN = pipe -notches.}\tabularnewline -\toprule\noalign{} -\begin{minipage}[b]{\linewidth}\raggedright -\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedright -Caries -\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedright -Nicotine -\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedright -SA -\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedright -Calculus -\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedright -PN -\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedright -Theophylline -\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedright -Caffeine -\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedright -Cotinine -\end{minipage} \\ -\midrule\noalign{} -\endfirsthead +are removed due to incompatibility with a Pearson correlation. Moderate +and strong correlations in \textbf{bold}. OA = osteoarthritis; VOP = +vertebral osteophytosis; SN = Schmorl's nodes; DDD = degenerative disc +disease; CO = cribra orbitalia; CMS = chronic maxillary sinusitis; SA = +salicylic acid; PN = pipe notches.} + +\tabularnewline + \toprule\noalign{} \begin{minipage}[b]{\linewidth}\raggedright \end{minipage} & \begin{minipage}[b]{\linewidth}\raggedright @@ -6546,48 +6350,51 @@ \subsection{Correlations between detected alkaloids and \endhead \bottomrule\noalign{} \endlastfoot -OA & -0.12 & -0.074 & 0.21 & 0.07 & 0.14 & 0.28 & 0.00098 & -0.067 \\ -VOP & -0.088 & -0.16 & 0.34 & 0.061 & 0.25 & -0.06 & 0.013 & -0.13 \\ -SN & -0.24 & 0.16 & 0.095 & 0.089 & 0.17 & 0.24 & 0.16 & 0.093 \\ -DDD & -0.0038 & 0.0037 & 0.19 & -0.39 & -0.077 & 0.31 & 0.06 & --0.0086 \\ -CO & 0.064 & -0.051 & 0.2 & 0.14 & -0.2 & -0.11 & 0.19 & -0.065 \\ -CMS & -0.19 & 0.28 & 0.0017 & -0.27 & 0.032 & 0.19 & 0.36 & 0.22 \\ -Caries & & -0.2 & -0.36 & -0.15 & -0.17 & -0.21 & -0.0045 & -0.22 \\ -Nicotine & & & -0.21 & 0.01 & -0.014 & 0.43 & 0.14 & 0.98 \\ -SA & & & & 0.14 & 0.37 & 0.038 & 0.17 & -0.17 \\ -Calculus & & & & & 0.13 & -0.15 & -0.13 & 0.031 \\ -PN & & & & & & -0.16 & 0.18 & -0.0068 \\ -Theophylline & & & & & & & 0.51 & 0.36 \\ -Caffeine & & & & & & & & 0.078 \\ +OA & -0.12 & -0.07 & 0.21 & 0.07 & 0.14 & 0.28 & 0 & -0.07 \\ +VOP & -0.09 & -0.16 & 0.34 & 0.06 & 0.25 & -0.06 & 0.01 & -0.13 \\ +SN & -0.24 & 0.16 & 0.09 & 0.09 & 0.17 & 0.24 & 0.16 & 0.09 \\ +DDD & 0 & 0 & 0.19 & -0.39 & -0.08 & 0.31 & 0.06 & -0.01 \\ +CO & 0.06 & -0.05 & 0.2 & 0.14 & -0.2 & -0.11 & 0.19 & -0.06 \\ +CMS & -0.19 & 0.28 & 0 & -0.27 & 0.03 & 0.19 & 0.36 & 0.22 \\ +Caries & & -0.2 & -0.36 & -0.15 & -0.17 & -0.21 & 0 & -0.22 \\ +Nicotine & & & -0.21 & 0.01 & -0.01 & \textbf{0.43} & 0.14 & +\textbf{0.98} \\ +SA & & & & 0.14 & 0.37 & 0.04 & 0.17 & -0.17 \\ +Calculus & & & & & 0.13 & -0.15 & -0.13 & 0.03 \\ +PN & & & & & & -0.16 & 0.18 & -0.01 \\ +Theophylline & & & & & & & \textbf{0.51} & 0.36 \\ +Caffeine & & & & & & & & 0.08 \\ + \end{longtable} Point-biserial correlation was conducted on paired continuous and dichotomous variables, to see if any relationships exist between extracted concentrations and other variables. The strongest point-biserial (Pearson) correlation correlations were a near-perfect -positive correlation between cotinine and nicotine (0.982), and moderate -correlations between theophylline and nicotine (0.432), caffeine and -theophylline (0.507) (Table~\ref{tbl-pearson}). +positive correlation between cotinine and nicotine (0.98), and moderate +correlations between theophylline and nicotine (0.43), caffeine and +theophylline (0.51) (Table~\ref{tbl-pearson}). Polychoric correlation was conducted on the dichotomised compounds and pathological conditions, as well as the discretised dental diseases. Salicylic acid was removed due to its ubiquitous presence in the sample, and is likely to cause spurious correlations. Strong correlations were -found between cotinine and nicotine (0.847). Moderate correlations were -found between OA and DDD (0.47), VOP and periodontitis (0.487), SN and -cotinine (0.559), DDD and calculus (-0.416), CMS and caffeine (0.53), -caries and periodontitis (0.523), periodontitis and VOP (0.487), -periodontitis and age-at-death (0.407), nicotine and SN (0.53), calculus -and DDD (-0.416), age-at-death and theophylline (-0.45), theophylline -and age-at-death (-0.45), caffeine and periodontitis (0.494), cotinine -and CMS (0.427). Remaining correlations were weak or absent +found between cotinine and nicotine (0.85). Moderate correlations were +found between OA and DDD (0.47), VOP and periodontitis (0.49), SN and +cotinine (0.56), DDD and calculus (-0.42), CMS and caffeine (0.53), +caries and periodontitis (0.52), periodontitis and VOP (0.49), +periodontitis and age-at-death (0.41), nicotine and SN (0.53), calculus +and DDD (-0.42), age-at-death and theophylline (-0.45), theophylline and +age-at-death (-0.45), caffeine and periodontitis (0.49), cotinine and +CMS (0.43). Remaining correlations were weak or absent (Figure~\ref{fig-polycorr}). Correlations with age will be depressed because age was largely controlled for in the sample selection. \begin{figure} -{\centering \includegraphics{05-article_files/figure-pdf/fig-polycorr-1.pdf} +\centering{ + +\includegraphics{05-article_files/figure-pdf/fig-polycorr-1.pdf} } @@ -6598,49 +6405,47 @@ \subsection{Correlations between detected alkaloids and = cribra orbitalia; CMS = chronic maxillary sinusitis; SA = salicylic acid.} -\end{figure} +\end{figure}% -\hypertarget{discussion-2}{% -\section{Discussion}\label{discussion-2}} +\section{Discussion}\label{discussion-2} In this study we were able to extract and identify multiple alkaloids and salicylic acid from the dental calculus of individuals from Middenbeemster, a 19th century Dutch archaeological site. We applied ultra-high-performance liquid chromatography-tandem mass spectrometry -(UHPLC-MS/MS), a method that was validated by co-occurrence of drugs and -metabolites in dental calculus and blood -(\protect\hyperlink{ref-sorensenDrugsCalculus2021}{Sørensen et al., -2021}). Here we have shown that the method can also be successfully -applied to archaeological dental calculus. We extend findings from -previous studies on alkaloids in archaeological samples by extracting -multiple different alkaloids from dental calculus, including nicotine, -cotinine, caffeine, theophylline, and salicylic acid in multiple -individuals. The detection of these compounds was solidified in a -replication analysis on different samples from the same individuals. -Cocaine and multiple cannabinoids were also detected during the first -analysis, but were not replicated. We discuss the implications of these -findings in light of historical and archaeological evidence for the -consumption of these drugs. +(UHPLC-MS/MS) using a method that was validated by co-occurrence of +drugs and metabolites in dental calculus and blood +(\citeproc{ref-sorensenDrugsCalculus2021}{Sørensen et al., 2021}). Here +we have shown that the method can also be successfully applied to +archaeological dental calculus. We extend findings from previous studies +on alkaloids in archaeological samples by detecting multiple different +alkaloids in dental calculus, including nicotine, cotinine, caffeine, +theophylline, and salicylic acid. The detection of these compounds was +solidified in a replication analysis on different samples from the same +individuals. Cocaine and multiple cannabinoids were also detected during +the first analysis, but were not replicated. We contextualize these +findings within the historical and archaeological evidence for +consumption of these drugs and dietary compounds. Nicotine and its principal/main metabolite, cotinine, were strongly positively correlated, both in concentration and presence/absence in individuals (Table~\ref{tbl-pearson} and Figure~\ref{fig-polycorr}). The detection of nicotine and cotinine is not surprising, as pipe-smoking in the Beemsterpolder is well-documented in the literature -(\protect\hyperlink{ref-aten400Jaar2012}{Aten et al., 2012}; -\protect\hyperlink{ref-boumanBegravenis2017}{Bouman, 2017}), and visible -on the skeletal remains as pipe notches -(\protect\hyperlink{ref-lemmersMiddenbeemster2013}{Lemmers et al., -2013}). There is also documented medicinal use of nicotine in the -Beemsterpolder, where a tobacco-smoke enema was used for headaches, -respiratory problems, colds, and drowsiness from around 1780 to 1830 -(\protect\hyperlink{ref-aten400Jaar2012}{Aten et al., 2012}). In our -sample, we also detected nicotine and cotinine (replicated) in an old -adult, probable female individual. In this particular case it is -unlikely that the compounds entered the dental calculus through -pipe-smoking, as the individual had no visible pipe notches; more likely -the tobacco entered through an alternate mode of consumption, secondhand -smoke, or the aforementioned tobacco-smoke enema. +(\citeproc{ref-aten400Jaar2012}{Aten et al., 2012}; +\citeproc{ref-boumanBegravenis2017}{Bouman, 2017}), and visible on the +skeletal remains as pipe notches +(\citeproc{ref-lemmersMiddenbeemster2013}{Lemmers et al., 2013}). There +is also documented medicinal use of nicotine in the Beemsterpolder, +where a tobacco-smoke enema was used for headaches, respiratory +problems, colds, and drowsiness from around 1780 to 1830 +(\citeproc{ref-aten400Jaar2012}{Aten et al., 2012}). In our sample, we +also detected nicotine and cotinine (replicated) in an old adult, +probable female individual. In this particular case it is unlikely that +the compounds entered the dental calculus through pipe-smoking, as the +individual had no visible pipe notches; more likely the tobacco entered +through an alternate mode of consumption, secondhand smoke, or the +aforementioned tobacco-smoke enema. Theophylline and caffeine were positively correlated in our samples, though to a lesser extent than nicotine and cotinine, so we are unable @@ -6651,109 +6456,108 @@ \section{Discussion}\label{discussion-2}} allowing us to interpret the ratio and correlations between the compounds. Caffeine is present in coffee, tea, and cocoa beans, with concentrations slightly higher in coffee -(\protect\hyperlink{ref-bispoSimultaneousDetermination2002}{Bispo et -al., 2002}; \protect\hyperlink{ref-chinCaffeineContent2008}{Chin et al., -2008}; \protect\hyperlink{ref-srdjenovicSimultaneousHPLC2008}{Srdjenovic -et al., 2008}; -\protect\hyperlink{ref-stavricVariabilityCaffeine1988}{Stavric et al., -1988}). Theophylline is present in both coffee beans and tea leaves, but -in negligible quantities -(\protect\hyperlink{ref-stavricVariabilityCaffeine1988}{Stavric et al., -1988}). It is also a primary metabolite of caffeine produced by the -liver. Given the low correlation, there are likely multiple sources of -caffeine and theophylline in the population, with tea and coffee being -the most obvious.\\ +(\citeproc{ref-bispoSimultaneousDetermination2002}{Bispo et al., 2002}; +\citeproc{ref-chinCaffeineContent2008}{Chin et al., 2008}; +\citeproc{ref-srdjenovicSimultaneousHPLC2008}{Srdjenovic et al., 2008}; +\citeproc{ref-stavricVariabilityCaffeine1988}{Stavric et al., 1988}). +Theophylline is present in both coffee beans and tea leaves, but in +negligible quantities +(\citeproc{ref-stavricVariabilityCaffeine1988}{Stavric et al., 1988}). +It is also a primary metabolite of caffeine produced by the liver. Given +the low correlation, there are likely multiple sources of caffeine and +theophylline in the population, with tea and coffee being the most +obvious.\\ Tea consumption had become widespread in the Netherlands by 1820, reaching all parts of society -(\protect\hyperlink{ref-nierstraszTeaTrade2015}{Nierstrasz, 2015, p. -91}). Historically, we also know that both tea and coffee were consumed -in the Beemsterpolder during the 19th century. `Theegasten' (teatime) -was a special occasion occurring from 15.00-20.00 hours, where tea was -served along with the evening bread -(\protect\hyperlink{ref-schuijtemakerTeTheegasten2011}{Schuijtemaker, -2011}). Many households also owned at least one coffee pot and tea pot -(\protect\hyperlink{ref-boumanBegravenis2017}{Bouman, 2017}). -Distinguishing between tea, coffee, and chocolate may be possible by -also including theobromine and comparing ratios of the compounds, as -theobromine is present in higher quantities in chocolate compared to -caffeine and theophylline -(\protect\hyperlink{ref-alanonAssessmentFlavanol2016}{Alañón et al., -2016}; \protect\hyperlink{ref-bispoSimultaneousDetermination2002}{Bispo -et al., 2002}; -\protect\hyperlink{ref-stavricVariabilityCaffeine1988}{Stavric et al., +(\citeproc{ref-nierstraszTeaTrade2015}{Nierstrasz, 2015, p. 91}). +Historically, we also know that both tea and coffee were consumed in the +Beemsterpolder during the 19th century. `Theegasten' (teatime) was a +special occasion occurring from 15.00-20.00 hours, where tea was served +along with the evening bread +(\citeproc{ref-schuijtemakerTeTheegasten2011}{Schuijtemaker, 2011}). +Many households also owned at least one coffee pot and tea pot +(\citeproc{ref-boumanBegravenis2017}{Bouman, 2017}). Distinguishing +between tea, coffee, and chocolate may be possible by also including +theobromine and comparing ratios of the compounds, as theobromine is +present in higher quantities in chocolate compared to caffeine and +theophylline (\citeproc{ref-alanonAssessmentFlavanol2016}{Alañón et al., +2016}; \citeproc{ref-bispoSimultaneousDetermination2002}{Bispo et al., +2002}; \citeproc{ref-stavricVariabilityCaffeine1988}{Stavric et al., 1988}). However, In addition to oral factors affecting alkaloid uptake in dental calculus, there is some indication that theobromine does not preserve well in the archaeological record -(\protect\hyperlink{ref-velskoDentalCalculus2017}{Velsko et al., 2017}), -and frequent consumption of all three items would be difficult to parse. +(\citeproc{ref-velskoDentalCalculus2017}{Velsko et al., 2017}), and +frequent consumption of all three items would be difficult to parse. +Additionally, we do not understand well enough the effect of the burial +on these specific compounds, and the original concentration of the +compounds in plants can be quite variable +(\citeproc{ref-kingCautionaryTales2017}{King et al., 2017}). Salicylic acid was found in all but one individual in our sample. It can be extracted from the bark of willow trees, \emph{Salix alba}, and has long been used for its pain-relieving properties -(\protect\hyperlink{ref-bruinsmaBijdragenTot1872}{Bruinsma, 1872, p. -119}). It is also present in many plant-based foods -(\protect\hyperlink{ref-duthieNaturalSalicylates2011}{Duthie \& Wood, -2011}; \protect\hyperlink{ref-malakarNaturallyOccurring2017}{Malakar et -al., 2017}), including potatoes, which were a staple of the -Beemsterpolder diet (\protect\hyperlink{ref-aten400Jaar2012}{Aten et -al., 2012}). The extracted quantity from our samples decreased over the -three washes, followed by a sharp increase in the final calculus -extraction, which is what we would expect to see if the salicylic acid -was incorporated during life Figure~\ref{fig-auth-plot-batch2}. However, -it has been shown that salicilyc acid is a very mobile organic acid and -the ubiquitous presence may be due to environmental contamination, which -would also explain the high quantity in the washes -(\protect\hyperlink{ref-badriRegulationFunction2009}{Badri \& Vivanco, -2009}; \protect\hyperlink{ref-chenCa2Dependent2001}{Chen et al., 2001}). -Given the multiple plausible sources of this residue, it will be -necessary to explore the extent to which salicylic acid can leach into -the dental calculus from the soil, and what the rate of degradation is -for salicylic acid when trapped in dental calculus. +(\citeproc{ref-bruinsmaBijdragenTot1872}{Bruinsma, 1872, p. 119}). It is +also present in many plant-based foods +(\citeproc{ref-duthieNaturalSalicylates2011}{Duthie \& Wood, 2011}; +\citeproc{ref-malakarNaturallyOccurring2017}{Malakar et al., 2017}), +including potatoes, which were a staple of the Beemsterpolder diet +(\citeproc{ref-aten400Jaar2012}{Aten et al., 2012}). The extracted +quantity from our samples decreased over the three washes, followed by a +sharp increase in the final calculus extraction, which is what we would +expect to see if the salicylic acid was incorporated during life +(Figure~\ref{fig-auth-plot-batch2}). It is important to note that, +especially with salicylic acid, there is a possibility for the compound +to enter the calculus through contact with the surrounding soil. +Salicylic acid is a very mobile organic acid +(\citeproc{ref-badriRegulationFunction2009}{Badri \& Vivanco, 2009}; +\citeproc{ref-chenCa2Dependent2001}{Chen et al., 2001}) and the +ubiqutous presence in our samples may be explained by the compound +leching into the dental calculus from the burial environment. We can +therefore not confidently rule out environmental contamination without +analysing samples from the surrounding soil. Cannabinoids---specifically THC, THCA-A, THCVA, CBD, CBN---were found in the first batch, but none were replicated in the second batch. Medicinal use of cannabinoids has been well-established in Europe since Medieval-times, and it was also grown in the Netherlands -(\protect\hyperlink{ref-bruinsmaBijdragenTot1872}{Bruinsma, 1872}). +(\citeproc{ref-bruinsmaBijdragenTot1872}{Bruinsma, 1872}). Administration was most common in the form of concoctions containing various portions of the cannabis plant for ingestion; not until the late 19th century did it become recommended to smoke it for more immediate -effects (\protect\hyperlink{ref-clarkeCannabisEvolution2013}{Clarke, -2013}). A Dutch medicinal use of hemp involved an emulsion prepared from -the seeds of the plants to treat pain and various stomach ailments. -Another preparation involving the roots of the plants was used for -inflammation, gout, and joint pains -(\protect\hyperlink{ref-clarkeCannabisEvolution2013}{Clarke, 2013}). The -ability to detect cannabinoids in calculus may be limited by their -reduced ability to diffuse from serum to salivary glands due to an -affinity for protein-binding, -(\protect\hyperlink{ref-coneInterpretationOral2007}{Cone \& Huestis, -2007}), meaning detection would rely on oral consumption. Even then, the -overall instability of some cannabinoids could also affect detection -(\protect\hyperlink{ref-lindholstLongTerm2010}{Lindholst, 2010}; -\protect\hyperlink{ref-sorensenEffectAntioxidants2018}{Sørensen \& -Hasselstrøm, 2018}). However, given the lack of replication, we cannot -with security confirm that cannabis was used by the Beemster population. +effects (\citeproc{ref-clarkeCannabisEvolution2013}{Clarke, 2013}). +Dutch medicinal preparations were used to treat a variety of ailments +and symptoms, including pain, inflammation, various stomach ailments, +gout, and joint pains +(\citeproc{ref-clarkeCannabisEvolution2013}{Clarke, 2013}). Because +cannabinoids have an affinity for protein-binding, they are less likely +to diffuse from serum to saliva +(\citeproc{ref-coneInterpretationOral2007}{Cone \& Huestis, 2007}). This +may make them difficult to detect in dental calculus unless the +cannibinoids were consumed orally; even then, the overall instability of +some cannabinoids could also limit detection +(\citeproc{ref-lindholstLongTerm2010}{Lindholst, 2010}; +\citeproc{ref-sorensenEffectAntioxidants2018}{Sørensen \& Hasselstrøm, +2018}). Given the lack of replication, we cannot with security confirm +that cannabis was used by the Beemster population. Despite many of our sampled individuals having lived during the height of the opium era in the Netherlands -(\protect\hyperlink{ref-machtHistoryOpium1915}{Macht, 1915}), none of -the targeted opioids (morphine, codeine, thebaine, papaverine, -norcodeine, noscapine) were detected. The absence of opioids could be a -result of the people ascribing more to the ``traditional'' rather than +(\citeproc{ref-machtHistoryOpium1915}{Macht, 1915}), none of the +targeted opioids (morphine, codeine, thebaine, papaverine, norcodeine, +noscapine) were detected. The absence of opioids could be a result of +the people ascribing more to the ``traditional'' rather than ``scientific'' medicine, although laudanum and another opium containing concoction was part of the ``traditional'' medicine in the Netherlands -(\protect\hyperlink{ref-leuwProhibitionLegalization1994}{Leuw \& -Marshall, 1994}), including Middenbeemster -(\protect\hyperlink{ref-aten400Jaar2012}{Aten et al., 2012}). It was -also generally considered a drug of the upper class -(\protect\hyperlink{ref-scheltemaOpiumTrade1907}{Scheltema, 1907}), and -may have been more common in urban centers. The absence could also be -attributed to postmortem degradation. It has been shown that, while -abundant in opium, morphine degrades rapidly, while thebaine and -papaverine are more resistant to various ageing processes -(\protect\hyperlink{ref-chovanecOpiumMasses2012}{Chovanec et al., -2012}). The latter were also absent from our samples. +(\citeproc{ref-leuwProhibitionLegalization1994}{Leuw \& Marshall, +1994}), including Middenbeemster (\citeproc{ref-aten400Jaar2012}{Aten et +al., 2012}). It was also generally considered a drug of the upper class +(\citeproc{ref-scheltemaOpiumTrade1907}{Scheltema, 1907}), and may have +been more common in urban centers. The absence could also be attributed +to postmortem degradation. It has been shown that, while morphine is +abundant in opium, it degrades rapidly. Thebaine and papaverine are more +resistant to various ageing processes +(\citeproc{ref-chovanecOpiumMasses2012}{Chovanec et al., 2012}), +however, these were also absent from our samples. The only strictly modern compound (at least in a European context) detected in the sample was cocaine, which was detected in the first @@ -6762,17 +6566,12 @@ \section{Discussion}\label{discussion-2}} entered popular medical practice in 1884. Coca arrived in Europe as early as 1771, but as botanical specimens rather than for consumption, and there were also issues importing enough viable specimens of coca for -cocaine extraction (\protect\hyperlink{ref-abucaCocaTrade2019}{Abduca, -2019, p. 108}; \protect\hyperlink{ref-mortimerHistoryCoca1901}{Mortimer, -1901, p. 179}). We considered it possible that it would be present in a -sample with most individuals originating from the early- to mid-19th -century. If corroborated, this would have been the first case of -coca-leaf-consumption in Europe. In our replication batch, we included -all of the individuals who had been cocaine-positive in the first batch. -We were unable to replicate any of the cocaine results, and we were -unable to detect the principal metabolite, benzoylecgonine, in either -batch. We suspect that the original detection of cocaine was a result of -lab contamination during analysis. +cocaine extraction (\citeproc{ref-abucaCocaTrade2019}{Abduca, 2019, p. +108}; \citeproc{ref-mortimerHistoryCoca1901}{Mortimer, 1901, p. 179}). +This would have been the first case of coca-leaf-consumption in Europe; +however, we were unable to replicate any of the cocaine results in the +second batch. We suspect that the original detection of cocaine was a +result of lab contamination during analysis. We explored the relationship between detected compounds and various skeletal indicators, such as pathological and dental lesions, @@ -6784,43 +6583,40 @@ \section{Discussion}\label{discussion-2}} indicative of the impact tobacco smoking had on the respiratory health of the Beemster inhabitants. Tobacco smoke may play a significant role in diseases of the upper respiratory tract, including chronic maxillary -sinusities (\protect\hyperlink{ref-rehImpactTobacco2012}{Reh et al., -2012}). Although the mechanisms by which smoking increases the risk of +sinusitis (\citeproc{ref-rehImpactTobacco2012}{Reh et al., 2012}). +Although the mechanisms by which smoking increases the risk of infections is not fully understood, solid evidence has been presented linking tobacco smoke to increased mucosal permeability and impairment of mucociliary clearance -(\protect\hyperlink{ref-arcaviCigaretteSmoking2004}{Arcavi \& Benowitz, -2004}). Such changes, together with an altered immunologic response, are -thought to predispose to the development of chronic maxillary sinusitis -(\protect\hyperlink{ref-slavinDiagnosisManagement2005}{Slavin et al., -2005}).\\ +(\citeproc{ref-arcaviCigaretteSmoking2004}{Arcavi \& Benowitz, 2004}). +Such changes, together with an altered immunologic response, are thought +to predispose to the development of chronic maxillary sinusitis +(\citeproc{ref-slavinDiagnosisManagement2005}{Slavin et al., 2005}).\\ We also observed a moderate positive correlation between chronic maxillary sinusitis and caffeine which contradicts previous research linking chronic coffee consumption with a positive effect on the respiratory system, suggesting a preventive association between caffeine -intake and pneumonia (e.g. -\protect\hyperlink{ref-alfaroChronicCoffee2018}{Alfaro et al., 2018}; -\protect\hyperlink{ref-kondoAssociationCoffee2021}{Kondo et al., 2021}). -However, while the lower respiratory tract seems to benefit from chronic -coffee consumption, it is possible that elevated caffeine intake impacts -mucosal moisture due to its dehydrating effect -(\protect\hyperlink{ref-maughanCaffeineIngestion2003}{Maughan \& -Griffin, 2003}), thereby exposing individuals to greater risk of sinus -infection. +intake and pneumonia (e.g. \citeproc{ref-alfaroChronicCoffee2018}{Alfaro +et al., 2018}; \citeproc{ref-kondoAssociationCoffee2021}{Kondo et al., +2021}). However, while the lower respiratory tract seems to benefit from +chronic coffee consumption, it is possible that elevated caffeine intake +impacts mucosal moisture due to its dehydrating effect +(\citeproc{ref-maughanCaffeineIngestion2003}{Maughan \& Griffin, 2003}), +thereby exposing individuals to greater risk of sinus infection. The detection of nicotine in dental calculus has previously been presented by Eerkens and colleagues -(\protect\hyperlink{ref-eerkensDentalCalculus2018}{2018}) in two -individuals from pre-contact California. They also targeted caffeine, -cotinine, and theophylline in their samples, but were unable to detect -any of them. It remains to be seen whether this is due to differences in -methods used, or due to our samples being more recent. They also suggest -that the choice of tooth for sampling may impact the detection of -certain compounds, as the incorporation in dental calculus may depend on -the mode of consumption. Tobacco smokers may have more nicotine present -in calculus on incisors, whereas tobacco chewers may have more on molars -(\protect\hyperlink{ref-eerkensDentalCalculus2018}{Eerkens et al., -2018}). However, sampling may not be limited to mode of consumption. The +(\citeproc{ref-eerkensDentalCalculus2018}{2018}) in two individuals from +pre-contact California. They also targeted caffeine, cotinine, and +theophylline in their samples, but were unable to detect any of them. It +remains to be seen whether this is due to differences in methods used, +or due to our samples being more recent. They also suggest that the +choice of tooth for sampling may impact the detection of certain +compounds, as the incorporation in dental calculus may depend on the +mode of consumption. Tobacco smokers may have more nicotine present in +calculus on incisors, whereas tobacco chewers may have more on molars +(\citeproc{ref-eerkensDentalCalculus2018}{Eerkens et al., 2018}). +However, sampling may not be limited to mode of consumption. The presence of cotinine suggests that the excretion of a compound after being metabolised in the body is also a source of deposition, and that deposition of alkaloids in dental calculus can occur both on the way @@ -6828,9 +6624,9 @@ \section{Discussion}\label{discussion-2}} i.e.~disposal of waste products via saliva secretion into the mouth. Especially mucin-rich saliva from the sublingual and submandibular glands preferentially binds toxins -(\protect\hyperlink{ref-doddsHealthBenefits2005}{Dodds et al., 2005}), -and since these glands are located closest to the lower incisors, they -may be the most effective target for these studies. This has yet to be +(\citeproc{ref-doddsHealthBenefits2005}{Dodds et al., 2005}), and since +these glands are located closest to the lower incisors, they may be the +most effective target for these studies. This has yet to be systematically tested in archaeological dental calculus. Because we homogenised samples from multiple teeth of an individual, we were unable to test the effect of oral biogeography. It is also possible that @@ -6839,68 +6635,60 @@ \section{Discussion}\label{discussion-2}} ingestion can be explained by this pathway. However, the literature on biofilm biodegradation of alkaloids is limited, and \emph{in vitro} studies have only found minimal contributions by certain oral bacteria -in isolation (\protect\hyperlink{ref-cogoVitroEvaluation2008}{Cogo et -al., 2008}; \protect\hyperlink{ref-sunMetabolomicsEvaluation2016}{Sun et -al., 2016}); it is possible that a larger role is played by oral -bacteria within larger, more metabolically active communities, -e.g.~biofilms -(\protect\hyperlink{ref-takahashiOralMicrobiome2015}{Takahashi, 2015}). - -Because we targeted individuals with moderate-to-large calculus -deposits, it is likely a biased sample. The presence of calculus may -increase the risk of premature death -(\protect\hyperlink{ref-yaussyCalculusSurvivorship2019}{Yaussy \& -DeWitte, 2019}), and periodontal disease (which may or may not be -associated with dental calculus build-up) is a risk-factor for -respiratory diseases, if periodontal and respiratory pathogens enter the -bloodstream -(\protect\hyperlink{ref-azarpazhoohSystematicReview2006}{Azarpazhooh \& -Leake, 2006}; -\protect\hyperlink{ref-scannapiecoRoleOral1999}{Scannapieco, 1999}; -\protect\hyperlink{ref-scannapiecoPotentialAssociations2001}{Scannapieco -\& Ho, 2001}). In our sample, the percentage of chronic maxillary -sinusitis (37.0\%) is lower than in another (more representative) male -sample (44.1\%) -(\protect\hyperlink{ref-casnaUrbanizationRespiratory2021}{Casna et al., +in isolation (\citeproc{ref-cogoVitroEvaluation2008}{Cogo et al., 2008}; +\citeproc{ref-sunMetabolomicsEvaluation2016}{Sun et al., 2016}); it is +possible that a larger role is played by oral bacteria within larger, +more metabolically active communities, e.g.~biofilms +(\citeproc{ref-takahashiOralMicrobiome2015}{Takahashi, 2015}). + +Targeting individuals with moderate-to-large calculus deposits likely +biased our sample, as the presence of calculus may increase the risk of +premature death (\citeproc{ref-yaussyCalculusSurvivorship2019}{Yaussy \& +DeWitte, 2019}). Additionally, periodontal disease (often linked to the +presence of calculus) is a risk-factor for respiratory diseases, if +periodontal and respiratory pathogens enter the bloodstream +(\citeproc{ref-azarpazhoohSystematicReview2006}{Azarpazhooh \& Leake, +2006}; \citeproc{ref-scannapiecoRoleOral1999}{Scannapieco, 1999}; +\citeproc{ref-scannapiecoPotentialAssociations2001}{Scannapieco \& Ho, +2001}). In our sample, the percentage of chronic maxillary sinusitis +(37.0\%) is lower than in another (more representative) male sample +(44.1\%) (\citeproc{ref-casnaUrbanizationRespiratory2021}{Casna et al., 2021}), and the caries percentage is similarly lower in our sample (17.6\%) than a more representative sample (22.9\%) -(\protect\hyperlink{ref-lemmersMiddenbeemster2013}{Lemmers et al., -2013}).\\ +(\citeproc{ref-lemmersMiddenbeemster2013}{Lemmers et al., 2013}).\\ We used the presence/absence of a pipe notch and concurrent detection of tobacco as a crude estimate of the accuracy of the method, which we found to be around 59.3\%. This is a very rough estimate, as the presence of a pipe notch is likely not a perfect indicator of whether or not someone consumed tobacco. Dental calculus is also more transient -than for example bone, as it can be mechanically removed, intentionally -or unintentionally, during life, eliminating all trace of the alkaloids +than for example bone, as it can become dislodged during life, +intentionally or unintentionally, eliminating all trace of the alkaloids consumed prior to its removal.\\ -Quantitation of the detected compounds may have limited value in -archaeological samples due to degradation, and will greatly affect our -correlations related to concentration. Following burial, compound -stability over time will play a large role, as will microbial -degradation of compounds by bacteria and fungi in soil -(\protect\hyperlink{ref-liuNicotinedegradingMicroorganisms2015}{Liu et -al., 2015}), as well as the soil environment, such as temperature, pH, -and oxygen availability -(\protect\hyperlink{ref-lindholstLongTerm2010}{Lindholst, 2010}; -\protect\hyperlink{ref-mackiePreservationMetaproteome2017}{Mackie et -al., 2017}).\\ -The detected quantity of a compound will also depend on the quantity in -dental calculus during life, which is largely controlled by quantity of -consumption, how often the calculus was disrupted/removed, metabolic -breakdown of the compound, and inter- and intra-individual factors -related to stages of biofilm formation, maturation, and mineralisation -(\protect\hyperlink{ref-lustmannScanningElectron1976}{Lustmann et al., -1976}; \protect\hyperlink{ref-velskoMicrobialDifferences2019}{Velsko et -al., 2019}; \protect\hyperlink{ref-zijngeBiofilmArchitecture2010}{Zijnge -et al., 2010}). In short, this means it is not really possible to detect -the absence of a compound. The absence of a compound is not evidence of -absence of consumption. This complicates the interpretation of our -results. We have attempted to minimise errors occurring due to this -limitation by including a relatively large sample of individuals and -replicating our analysis. Although given the relatively low detection -rate seen in tobacco, this remains a major limitation, and will likely -be compounded by increasing antiquity of the samples. +Following burial, compound stability over time will play a large role, +as will microbial degradation of compounds by bacteria and fungi in soil +(\citeproc{ref-liuNicotinedegradingMicroorganisms2015}{Liu et al., +2015}), as well as the soil environment, such as temperature, pH, and +oxygen availability (\citeproc{ref-lindholstLongTerm2010}{Lindholst, +2010}; \citeproc{ref-mackiePreservationMetaproteome2017}{Mackie et al., +2017}).\\ +Due to this, quantitation of the detected compounds may have limited +value in archaeological samples due to degradation, and will greatly +affect our correlations related to concentration. The detected quantity +of a compound will also depend on the quantity in dental calculus during +life, which is largely controlled by the quantity consumed, how often +the calculus was disrupted/removed, metabolic breakdown of the compound, +and inter- and intra-individual factors related to stages of biofilm +maturation (\citeproc{ref-lustmannScanningElectron1976}{Lustmann et al., +1976}; \citeproc{ref-velskoMicrobialDifferences2019}{Velsko et al., +2019}; \citeproc{ref-zijngeBiofilmArchitecture2010}{Zijnge et al., +2010}). In short, this means it is not really possible to equate the +absence of a compound as evidence for the absence of consumption, which +complicates the interpretation of our results. We have attempted to +minimise errors occurring due to this limitation by including a +relatively large sample of individuals and replicating our analysis. +Although, given the relatively low detection rate seen in tobacco, this +remains a major limitation, and will likely be compounded by increasing +antiquity of the samples. Future studies should explore how sampling from various types of teeth and their position in the mouth affects the probability of a compound @@ -6918,14 +6706,20 @@ \section{Discussion}\label{discussion-2}} There is an increasing interest in using oral fluid as a means of detecting alkaloids in living individuals due to the non-invasive nature of the testing compared to blood and urine sampling -(\protect\hyperlink{ref-coneSalivaTesting1993}{Cone, 1993}; -\protect\hyperlink{ref-valenDetermination212017}{Valen et al., 2017}). -These \emph{in vivo} studies are a valuable source of method validation -and can help determine the feasibility of detecting certain alkaloids in +(\citeproc{ref-coneSalivaTesting1993}{Cone, 1993}; +\citeproc{ref-valenDetermination212017}{Valen et al., 2017}). These +\emph{in vivo} studies are a valuable source of method validation and +can help determine the feasibility of detecting certain alkaloids in oral fluid and, subsequently, dental calculus. Archaeologists, though, -will likely be responsible for exploring dental calculus specific +will likely be responsible for exploring dental-calculus-specific incorporation and retention of alkaloids, as well as their long-term -preservation in the burial environment. +preservation in the burial environment. Finally, following our +experience with salicylic acid, we encourage all future studies to +ensure that a control sample is taken from the soil, either from the +soil surrounding the individual, or, ideally, directly from the skeletal +remains. This should preferably happen before cleaning, but there will +often be soil left over in cavities (e.g.~nasal cavity, orbit, auditory +meatus). While a major limitation is the uncertainty surrounding whether or not a compound is actually absent, the power of the method lies in the ability @@ -6935,19 +6729,18 @@ \section{Discussion}\label{discussion-2}} useful way to identify tobacco consumption, pipe smoking was not the only mode of tobacco consumption, with others including chewing, drinking, cigars, and snuff -(\protect\hyperlink{ref-goodmanTobaccoHistory1994}{Goodman, 1994, p. -67}). Pipe-smoking was mainly practised by males -(\protect\hyperlink{ref-eerkensDentalCalculus2018}{Eerkens et al., -2018}; \protect\hyperlink{ref-lemmersMiddenbeemster2013}{Lemmers et al., -2013}), so methods like the one presented here are suitable for -exploring tobacco consumption in an entire society, rather than a -trivial subset of past populations. Combined with other methods, it can -also give us a more complete picture of dietary patterns and -medicinal/recreational plant-use in the past by capturing multiple -possible incorporation pathways of dietary (and other) compounds. - -\hypertarget{conclusion-1}{% -\section{Conclusion}\label{conclusion-1}} +(\citeproc{ref-goodmanTobaccoHistory1994}{Goodman, 1994, p. 67}). +Pipe-smoking was mainly practised by males +(\citeproc{ref-eerkensDentalCalculus2018}{Eerkens et al., 2018}; +\citeproc{ref-lemmersMiddenbeemster2013}{Lemmers et al., 2013}), so +methods like the one presented here are suitable for exploring tobacco +consumption in an entire society, rather than a trivial subset of past +populations. Combined with other methods, it can also give us a more +complete picture of dietary patterns and medicinal/recreational +plant-use in the past by capturing multiple possible incorporation +pathways of dietary (and other) compounds. + +\section{Conclusion}\label{conclusion-1} This preliminary study outlines the benefits of using calculus to target a variety of compounds that could have been consumed as medicine or @@ -6968,54 +6761,53 @@ \section{Conclusion}\label{conclusion-1}} reservoir of information regarding the consumption of various alkaloids, whether dietary, medicinal, recreational, or otherwise. -\hypertarget{references-cited-4}{% -\section*{References cited}\label{references-cited-4}} +\section*{References cited}\label{references-cited-4} \addcontentsline{toc}{section}{References cited} \markright{References cited} -\hypertarget{refs-5}{} +\phantomsection\label{refs-5} \begin{CSLReferences}{1}{0} -\leavevmode\vadjust pre{\hypertarget{ref-abucaCocaTrade2019}{}}% +\bibitem[\citeproctext]{ref-abucaCocaTrade2019} Abduca, R. (2019). Coca leaf transfers to {Europe}. {Effects} on the consumption of coca in {North-western Argentina}. In M. Kaller \& F. Jacob (Eds.), \emph{Transatlantic {Trade} and {Global Cultural Transfers Since} 1492: {More} than {Commodities}}. {Routledge}. -\leavevmode\vadjust pre{\hypertarget{ref-alanonAssessmentFlavanol2016}{}}% +\bibitem[\citeproctext]{ref-alanonAssessmentFlavanol2016} Alañón, M. E., Castle, S. M., Siswanto, P. J., Cifuentes-Gómez, T., \& Spencer, J. P. E. (2016). Assessment of flavanol stereoisomers and caffeine and theobromine content in commercial chocolates. \emph{Food Chemistry}, \emph{208}, 177--184. \url{https://doi.org/10.1016/j.foodchem.2016.03.116} -\leavevmode\vadjust pre{\hypertarget{ref-alfaroChronicCoffee2018}{}}% +\bibitem[\citeproctext]{ref-alfaroChronicCoffee2018} Alfaro, T. M., Monteiro, R. A., Cunha, R. A., \& Cordeiro, C. R. (2018). Chronic coffee consumption and respiratory disease: {A} systematic review. \emph{The Clinical Respiratory Journal}, \emph{12}(3), 1283--1294. \url{https://doi.org/10.1111/crj.12662} -\leavevmode\vadjust pre{\hypertarget{ref-arcaviCigaretteSmoking2004}{}}% +\bibitem[\citeproctext]{ref-arcaviCigaretteSmoking2004} Arcavi, L., \& Benowitz, N. L. (2004). Cigarette {Smoking} and {Infection}. \emph{Archives of Internal Medicine}, \emph{164}(20), 2206--2216. \url{https://doi.org/10.1001/archinte.164.20.2206} -\leavevmode\vadjust pre{\hypertarget{ref-aten400Jaar2012}{}}% +\bibitem[\citeproctext]{ref-aten400Jaar2012} Aten, D., Bossaers, K. W. J. M., \& Misset, C. (2012). \emph{{400 jaar Beemster: 1612-2012}}. {Stichting Uitgeverij Noord-Holland}. -\leavevmode\vadjust pre{\hypertarget{ref-azarpazhoohSystematicReview2006}{}}% +\bibitem[\citeproctext]{ref-azarpazhoohSystematicReview2006} Azarpazhooh, A., \& Leake, J. L. (2006). Systematic {Review} of the {Association Between Respiratory Diseases} and {Oral Health}. \emph{Journal of Periodontology}, \emph{77}(9), 1465--1482. \url{https://doi.org/10.1902/jop.2006.060010} -\leavevmode\vadjust pre{\hypertarget{ref-badriRegulationFunction2009}{}}% +\bibitem[\citeproctext]{ref-badriRegulationFunction2009} Badri, D. V., \& Vivanco, J. M. (2009). Regulation and function of root exudates. \emph{Plant, Cell \& Environment}, \emph{32}(6), 666--681. \url{https://doi.org/10.1111/j.1365-3040.2009.01926.x} -\leavevmode\vadjust pre{\hypertarget{ref-bispoSimultaneousDetermination2002}{}}% +\bibitem[\citeproctext]{ref-bispoSimultaneousDetermination2002} Bispo, M. S., Veloso, M. C. C., Pinheiro, H. L. C., De Oliveira, R. F. S., Reis, J. O. N., \& De Andrade, J. B. (2002). Simultaneous {Determination} of {Caffeine}, {Theobromine}, and {Theophylline} by @@ -7023,114 +6815,114 @@ \section*{References cited}\label{references-cited-4}} Chromatographic Science}, \emph{40}(1), 45--48. \url{https://doi.org/10.1093/chromsci/40.1.45} -\leavevmode\vadjust pre{\hypertarget{ref-boocockMaxillarySinusitis1995}{}}% +\bibitem[\citeproctext]{ref-boocockMaxillarySinusitis1995} Boocock, P., Roberts, C. A., \& Manchester, K. (1995). Maxillary sinusitis in {Medieval Chichester}, {England}. \emph{American Journal of Physical Anthropology}, \emph{98}(4), 483--495. \url{https://doi.org/10.1002/ajpa.1330980408} -\leavevmode\vadjust pre{\hypertarget{ref-boumanBegravenis2017}{}}% +\bibitem[\citeproctext]{ref-boumanBegravenis2017} Bouman, J. (2017). {De Begravenis}. \emph{De Nieuwe Schouwschuit}, \emph{15}, 11--15. -\leavevmode\vadjust pre{\hypertarget{ref-SucheyBrooks1990}{}}% +\bibitem[\citeproctext]{ref-SucheyBrooks1990} Brooks, S., \& Suchey, J. M. (1990). Skeletal age determination based on -the os pubis: {A} comparison of the {Acs{á}di-Nemesk{é}ri} and +the os pubis: {A} comparison of the {Acsádi-Nemeskéri} and {Suchey-Brooks} methods. \emph{Human Evolution}, \emph{5}(3), 227--238. \url{https://doi.org/10.1007/BF02437238} -\leavevmode\vadjust pre{\hypertarget{ref-brothwellDiggingBones1981}{}}% +\bibitem[\citeproctext]{ref-brothwellDiggingBones1981} Brothwell, D. (1981). \emph{Digging up {Bones}: {The} excavation, treatment and study of human skeletal remains} (3rd ed.). {British Museum (Natural History)}. -\leavevmode\vadjust pre{\hypertarget{ref-bruinsmaBijdragenTot1872}{}}% +\bibitem[\citeproctext]{ref-bruinsmaBijdragenTot1872} Bruinsma, J. J. (1872). \emph{Bijdragen tot de {Geneeskundige Plaatsbeschrijving} van {Nederland}}. {Van Weelden en Mingelen}. -\leavevmode\vadjust pre{\hypertarget{ref-buckberryAuricular2002}{}}% +\bibitem[\citeproctext]{ref-buckberryAuricular2002} Buckberry, J. L., \& Chamberlain, A. T. (2002). {Age estimation from the auricular surface of the ilium: A revised method}. \emph{American Journal of Physical Anthropology}, \emph{119}(3), 231--239. \url{https://doi.org/10.1002/ajpa.10130} -\leavevmode\vadjust pre{\hypertarget{ref-buckleyDentalCalculus2014}{}}% +\bibitem[\citeproctext]{ref-buckleyDentalCalculus2014} Buckley, S., Usai, D., Jakob, T., Radini, A., \& Hardy, K. (2014). Dental {Calculus Reveals Unique Insights} into {Food Items}, {Cooking} and {Plant Processing} in {Prehistoric Central Sudan}. \emph{PLOS ONE}, \emph{9}(7), e100808. \url{https://doi.org/10.1371/journal.pone.0100808} -\leavevmode\vadjust pre{\hypertarget{ref-Standards1994}{}}% +\bibitem[\citeproctext]{ref-Standards1994} Buikstra, J. E., \& Ubelaker, D. H. (1994). Standards for data collection from human skeletal remains: {Proceedings} of a seminar at the {Field Museum} of {Natural History} ({Arkansas Archaeology Research Series} 44). \emph{Fayetteville Arkansas Archaeological Survey}. -\leavevmode\vadjust pre{\hypertarget{ref-casnaUrbanizationRespiratory2021}{}}% +\bibitem[\citeproctext]{ref-casnaUrbanizationRespiratory2021} Casna, M., Burrell, C. L., Schats, R., Hoogland, M. L. P., \& Schrader, S. A. (2021). Urbanization and respiratory stress in the {Northern Low Countries}: {A} comparative study of chronic maxillary sinusitis in two -early modern sites from the {Netherlands} ({AD} 1626{\textendash}1866). +early modern sites from the {Netherlands} ({AD} 1626\textendash 1866). \emph{International Journal of Osteoarchaeology}, \emph{31}(5), 891--901. \url{https://doi.org/10.1002/oa.3006} -\leavevmode\vadjust pre{\hypertarget{ref-chenCa2Dependent2001}{}}% +\bibitem[\citeproctext]{ref-chenCa2Dependent2001} Chen, H.-J., Hou, W.-C., Kuć, J., \& Lin, Y.-H. (2001). Ca2+-dependent and {Ca2}+-independent excretion modes of salicylic acid in tobacco cell suspension culture. \emph{Journal of Experimental Botany}, \emph{52}(359), 1219--1226. \url{https://doi.org/10.1093/jexbot/52.359.1219} -\leavevmode\vadjust pre{\hypertarget{ref-chinCaffeineContent2008}{}}% +\bibitem[\citeproctext]{ref-chinCaffeineContent2008} Chin, J. M., Merves, M. L., Goldberger, B. A., Sampson-Cone, A., \& Cone, E. J. (2008). Caffeine {Content} of {Brewed Teas}. \emph{Journal of Analytical Toxicology}, \emph{32}(8), 702--704. \url{https://doi.org/10.1093/jat/32.8.702} -\leavevmode\vadjust pre{\hypertarget{ref-chovanecOpiumMasses2012}{}}% +\bibitem[\citeproctext]{ref-chovanecOpiumMasses2012} Chovanec, Z., Rafferty, S., \& Swiny, S. (2012). Opium for the {Masses}. \emph{Ethnoarchaeology}, \emph{4}(1), 5--36. \url{https://doi.org/10.1179/eth.2012.4.1.5} -\leavevmode\vadjust pre{\hypertarget{ref-clarkeCannabisEvolution2013}{}}% +\bibitem[\citeproctext]{ref-clarkeCannabisEvolution2013} Clarke, R. (2013). \emph{Cannabis : {Evolution} and {Ethnobotany}}. {University of California Press}. -\leavevmode\vadjust pre{\hypertarget{ref-cogoVitroEvaluation2008}{}}% +\bibitem[\citeproctext]{ref-cogoVitroEvaluation2008} Cogo, K., Montan, M. F., Bergamaschi, C. de C., D. Andrade, E., Rosalen, P. L., \& Groppo, F. C. (2008). In vitro evaluation of the effect of nicotine, cotinine, and caffeine on oral microorganisms. \emph{Canadian Journal of Microbiology}, \emph{54}(6), 501--508. \url{https://doi.org/10.1139/W08-032} -\leavevmode\vadjust pre{\hypertarget{ref-coneSalivaTesting1993}{}}% +\bibitem[\citeproctext]{ref-coneSalivaTesting1993} Cone, E. J. (1993). Saliva {Testing} for {Drugs} of {Abuse}. \emph{Annals of the New York Academy of Sciences}, \emph{694}(1), 91--127. \url{https://doi.org/10.1111/j.1749-6632.1993.tb18346.x} -\leavevmode\vadjust pre{\hypertarget{ref-coneInterpretationOral2007}{}}% +\bibitem[\citeproctext]{ref-coneInterpretationOral2007} Cone, E. J., \& Huestis, M. A. (2007). Interpretation of {Oral Fluid Tests} for {Drugs} of {Abuse}. \emph{Annals of the New York Academy of Sciences}, \emph{1098}, 51--103. \url{https://doi.org/10.1196/annals.1384.037} -\leavevmode\vadjust pre{\hypertarget{ref-doddsHealthBenefits2005}{}}% +\bibitem[\citeproctext]{ref-doddsHealthBenefits2005} Dodds, M. W. J., Johnson, D. A., \& Yeh, C.-K. (2005). Health benefits of saliva: A review. \emph{Journal of Dentistry}, \emph{33}(3), 223--233. \url{https://doi.org/10.1016/j.jdent.2004.10.009} -\leavevmode\vadjust pre{\hypertarget{ref-duthieNaturalSalicylates2011}{}}% +\bibitem[\citeproctext]{ref-duthieNaturalSalicylates2011} Duthie, G. G., \& Wood, A. D. (2011). Natural salicylates: Foods , functions and disease prevention. \emph{Food \& Function}, \emph{2}(9), 515--520. \url{https://doi.org/10.1039/C1FO10128E} -\leavevmode\vadjust pre{\hypertarget{ref-echeverriaNicotineHair2013}{}}% +\bibitem[\citeproctext]{ref-echeverriaNicotineHair2013} Echeverría, J., \& Niemeyer, H. M. (2013). Nicotine in the hair of mummies from {San Pedro} de {Atacama} ({Northern Chile}). \emph{Journal of Archaeological Science}, \emph{40}(10), 3561--3568. \url{https://doi.org/10.1016/j.jas.2013.04.030} -\leavevmode\vadjust pre{\hypertarget{ref-eerkensDentalCalculus2018}{}}% +\bibitem[\citeproctext]{ref-eerkensDentalCalculus2018} Eerkens, J. W., Tushingham, S., Brownstein, K. J., Garibay, R., Perez, K., Murga, E., Kaijankoski, P., Rosenthal, J. S., \& Gang, D. R. (2018). Dental calculus as a source of ancient alkaloids: {Detection} of @@ -7138,7 +6930,7 @@ \section*{References cited}\label{references-cited-4}} \emph{Journal of Archaeological Science: Reports}, \emph{18}, 509--515. \url{https://doi.org/10.1016/j.jasrep.2018.02.004} -\leavevmode\vadjust pre{\hypertarget{ref-gismondiMultidisciplinaryApproach2020}{}}% +\bibitem[\citeproctext]{ref-gismondiMultidisciplinaryApproach2020} Gismondi, A., Baldoni, M., Gnes, M., Scorrano, G., D'Agostino, A., Marco, G. D., Calabria, G., Petrucci, M., Müldner, G., Tersch, M. V., Nardi, A., Enei, F., Canini, A., Rickards, O., Alexander, M., \& @@ -7148,23 +6940,36 @@ \section*{References cited}\label{references-cited-4}} ONE}, \emph{15}(1), e0227433. \url{https://doi.org/10.1371/journal.pone.0227433} -\leavevmode\vadjust pre{\hypertarget{ref-goodmanTobaccoHistory1994}{}}% +\bibitem[\citeproctext]{ref-goodmanTobaccoHistory1994} Goodman, J. (1994). \emph{Tobacco in history: The cultures of dependence}. {Routledge}. -\leavevmode\vadjust pre{\hypertarget{ref-greeneQuantifyingCalculus2005}{}}% +\bibitem[\citeproctext]{ref-greeneQuantifyingCalculus2005} Greene, T. R., Kuba, C. L., \& Irish, J. D. (2005). Quantifying calculus: {A} suggested new approach for recording an important indicator of diet and dental health. \emph{HOMO - Journal of Comparative Human Biology}, \emph{56}(2), 119--132. \url{https://doi.org/10.1016/j.jchb.2005.02.002} -\leavevmode\vadjust pre{\hypertarget{ref-jinSupragingivalCalculus2002}{}}% +\bibitem[\citeproctext]{ref-huangDecipheringGenetic2023} +Huang, Y., Shan, Y., Zhang, W., Lee, A. M., Li, F., Stranger, B. E., \& +Huang, R. S. (2023). Deciphering genetic causes for sex differences in +human health through drug metabolism and transporter genes. \emph{Nature +Communications}, \emph{14}(1), 175. +\url{https://doi.org/10.1038/s41467-023-35808-6} + +\bibitem[\citeproctext]{ref-jinSupragingivalCalculus2002} Jin, Y., \& Yip, H.-K. (2002). Supragingival {Calculus}: {Formation} and {Control}. \emph{Critical Reviews in Oral Biology \& Medicine}. \url{https://doi.org/10.1177/154411130201300506} -\leavevmode\vadjust pre{\hypertarget{ref-kondoAssociationCoffee2021}{}}% +\bibitem[\citeproctext]{ref-kingCautionaryTales2017} +King, A., Powis, T. G., Cheong, K. F., \& Gaikwad, N. W. (2017). +Cautionary tales on the identification of caffeinated beverages in +{North America}. \emph{Journal of Archaeological Science}, \emph{85}, +30--40. \url{https://doi.org/10.1016/j.jas.2017.06.006} + +\bibitem[\citeproctext]{ref-kondoAssociationCoffee2021} Kondo, K., Suzuki, K., Washio, M., Ohfuji, S., Adachi, S., Kan, S., Imai, S., Yoshimura, K., Miyashita, N., Fujisawa, N., Maeda, A., Fukushima, W., \& Hirota, Y. (2021). Association between coffee and @@ -7172,77 +6977,77 @@ \section*{References cited}\label{references-cited-4}} case-control study. \emph{Scientific Reports}, \emph{11}(1), 5570. \url{https://doi.org/10.1038/s41598-021-84348-w} -\leavevmode\vadjust pre{\hypertarget{ref-lemmersMiddenbeemster2013}{}}% +\bibitem[\citeproctext]{ref-lemmersMiddenbeemster2013} Lemmers, S. A. M., Schats, R., Hoogland, M. L. P., \& Waters-Rist, A. (2013). {Fysisch antropologische analyse Middenbeemster}. In \emph{{De begravingen bij de Keyserkerk te Middenbeemster}} (pp. 35--60). -\leavevmode\vadjust pre{\hypertarget{ref-leuwProhibitionLegalization1994}{}}% +\bibitem[\citeproctext]{ref-leuwProhibitionLegalization1994} Leuw, E., \& Marshall, I. H. (1994). \emph{Between {Prohibition} and {Legalization}: {The Dutch Experiment} in {Drug Policy}}. {Kugler Publications}. -\leavevmode\vadjust pre{\hypertarget{ref-lindholstLongTerm2010}{}}% +\bibitem[\citeproctext]{ref-lindholstLongTerm2010} Lindholst, C. (2010). Long term stability of cannabis resin and cannabis extracts. \emph{Australian Journal of Forensic Sciences}, \emph{42}(3), 181--190. \url{https://doi.org/10.1080/00450610903258144} -\leavevmode\vadjust pre{\hypertarget{ref-liuNicotinedegradingMicroorganisms2015}{}}% +\bibitem[\citeproctext]{ref-liuNicotinedegradingMicroorganisms2015} Liu, J., Ma, G., Chen, T., Hou, Y., Yang, S., Zhang, K.-Q., \& Yang, J. (2015). Nicotine-degrading microorganisms and their potential applications. \emph{Applied Microbiology and Biotechnology}, \emph{99}(9), 3775--3785. \url{https://doi.org/10.1007/s00253-015-6525-1} -\leavevmode\vadjust pre{\hypertarget{ref-lovejoyAuricular1985}{}}% +\bibitem[\citeproctext]{ref-lovejoyAuricular1985} Lovejoy, C. O., Meindl, R. S., Pryzbeck, T. R., \& Mensforth, R. P. (1985). Chronological metamorphosis of the auricular surface of the ilium: {A} new method for the determination of adult skeletal age at death. \emph{American Journal of Physical Anthropology}, \emph{68}(1), 15--28. \url{https://doi.org/10.1002/ajpa.1330680103} -\leavevmode\vadjust pre{\hypertarget{ref-lustmannScanningElectron1976}{}}% +\bibitem[\citeproctext]{ref-lustmannScanningElectron1976} Lustmann, J., Lewin-Epstein, J., \& Shteyer, A. (1976). Scanning electron microscopy of dental calculus. \emph{Calcified Tissue Research}, \emph{21}(1), 47--55. \url{https://doi.org/10.1007/BF02547382} -\leavevmode\vadjust pre{\hypertarget{ref-maatManualPhysical2005}{}}% +\bibitem[\citeproctext]{ref-maatManualPhysical2005} Maat, G., \& Mastwijk, R. (2005). Manual for the physical anthropological report. \emph{Barge's Anthropologica}, \emph{6}. -\leavevmode\vadjust pre{\hypertarget{ref-machtHistoryOpium1915}{}}% +\bibitem[\citeproctext]{ref-machtHistoryOpium1915} Macht, D. I. (1915). The history of opium and some of its preparations and alkaloids. \emph{The Journal of the American Medical Association}, \emph{LXIV}(6), 5. -\leavevmode\vadjust pre{\hypertarget{ref-mackiePreservationMetaproteome2017}{}}% +\bibitem[\citeproctext]{ref-mackiePreservationMetaproteome2017} Mackie, M., Hendy, J., Lowe, A. D., Sperduti, A., Holst, M., Collins, M. J., \& Speller, C. F. (2017). Preservation of the metaproteome: Variability of protein preservation in ancient dental calculus. \emph{STAR: Science \& Technology of Archaeological Research}, \emph{3}(1), 58--70. \url{https://doi.org/10.1080/20548923.2017.1361629} -\leavevmode\vadjust pre{\hypertarget{ref-malakarNaturallyOccurring2017}{}}% +\bibitem[\citeproctext]{ref-malakarNaturallyOccurring2017} Malakar, S., Gibson, P. R., Barrett, J. S., \& Muir, J. G. (2017). Naturally occurring dietary salicylates: {A} closer look at common {Australian} foods. \emph{Journal of Food Composition and Analysis}, \emph{57}, 31--39. \url{https://doi.org/10.1016/j.jfca.2016.12.008} -\leavevmode\vadjust pre{\hypertarget{ref-maughanCaffeineIngestion2003}{}}% +\bibitem[\citeproctext]{ref-maughanCaffeineIngestion2003} Maughan, R. J., \& Griffin, J. (2003). Caffeine ingestion and fluid balance: A review. \emph{Journal of Human Nutrition and Dietetics}, \emph{16}(6), 411--420. \url{https://doi.org/10.1046/j.1365-277X.2003.00477.x} -\leavevmode\vadjust pre{\hypertarget{ref-meindlSutureClosure1985}{}}% +\bibitem[\citeproctext]{ref-meindlSutureClosure1985} Meindl, R. S., \& Lovejoy, C. O. (1985). Ectocranial suture closure: {A} revised method for the determination of skeletal age at death based on the lateral-anterior sutures. \emph{American Journal of Physical Anthropology}, \emph{68}(1), 57--66. \url{https://doi.org/10.1002/ajpa.1330680106} -\leavevmode\vadjust pre{\hypertarget{ref-milmanOralFluid2011}{}}% +\bibitem[\citeproctext]{ref-milmanOralFluid2011} Milman, G., Schwope, D. M., Schwilke, E. W., Darwin, W. D., Kelly, D. L., Goodwin, R. S., Gorelick, D. A., \& Huestis, M. A. (2011). Oral {Fluid} and {Plasma Cannabinoid Ratios} after {Around-the-Clock @@ -7250,78 +7055,78 @@ \section*{References cited}\label{references-cited-4}} \emph{Clinical Chemistry}, \emph{57}(11), 1597--1606. \url{https://doi.org/10.1373/clinchem.2011.169490} -\leavevmode\vadjust pre{\hypertarget{ref-mortimerHistoryCoca1901}{}}% +\bibitem[\citeproctext]{ref-mortimerHistoryCoca1901} Mortimer, W. G. (1901). \emph{Peru. {History} of coca, "the divine plant" of the {Incas}; with an introductory account of the {Incas}, and of the {Andean Indians} of to-day}. {New York, J. H. Vail \& Company}. -\leavevmode\vadjust pre{\hypertarget{ref-nierstraszTeaTrade2015}{}}% +\bibitem[\citeproctext]{ref-nierstraszTeaTrade2015} Nierstrasz, C. (2015). \emph{Rivalry for {Trade} in {Tea} and {Textiles}: {The English} and {Dutch East India} companies -(1700{\textendash}1800)}. {Springer}. +(1700\textendash 1800)}. {Springer}. -\leavevmode\vadjust pre{\hypertarget{ref-ogaldeIdentificationPsychoactive2009}{}}% +\bibitem[\citeproctext]{ref-ogaldeIdentificationPsychoactive2009} Ogalde, J. P., Arriaza, B. T., \& Soto, E. C. (2009). Identification of psychoactive alkaloids in ancient {Andean} human hair by gas chromatography/mass spectrometry. \emph{Journal of Archaeological Science}, \emph{36}(2), 467--472. \url{https://doi.org/10.1016/j.jas.2008.09.036} -\leavevmode\vadjust pre{\hypertarget{ref-palmerActivityReconstruction2016}{}}% +\bibitem[\citeproctext]{ref-palmerActivityReconstruction2016} Palmer, J. L. A., Hoogland, M. H. L., \& Waters-Rist, A. L. (2016). Activity {Reconstruction} of {Post}-{Medieval Dutch Rural Villagers} from {Upper Limb Osteoarthritis} and {Entheseal Changes}. \emph{International Journal of Osteoarchaeology}, \emph{26}(1), 78--92. \url{https://doi.org/10.1002/oa.2397} -\leavevmode\vadjust pre{\hypertarget{ref-Rbase}{}}% +\bibitem[\citeproctext]{ref-Rbase} R Core Team. (2020). \emph{R: {A} language and environment for statistical computing} {[}Manual{]}. {R Foundation for Statistical -Computing}; {R Foundation for Statistical Computing}. +Computing}. -\leavevmode\vadjust pre{\hypertarget{ref-raffertyCurrentResearch2012}{}}% +\bibitem[\citeproctext]{ref-raffertyCurrentResearch2012} Rafferty, S. M., Lednev, I., Virkler, K., \& Chovanec, Z. (2012). Current research on smoking pipe residues. \emph{Journal of Archaeological Science}, \emph{39}(7), 1951--1959. \url{https://doi.org/10.1016/j.jas.2012.02.001} -\leavevmode\vadjust pre{\hypertarget{ref-rehImpactTobacco2012}{}}% +\bibitem[\citeproctext]{ref-rehImpactTobacco2012} Reh, D. D., Higgins, T. S., \& Smith, T. L. (2012). Impact of {Tobacco -Smoke} on {Chronic Rhinosinusitis} {\textendash} {A Review} of the +Smoke} on {Chronic Rhinosinusitis} \textendash{} {A Review} of the {Literature}. \emph{International Forum of Allergy \& Rhinology}, \emph{2}(5), 362. \url{https://doi.org/10.1002/alr.21054} -\leavevmode\vadjust pre{\hypertarget{ref-Rpsych}{}}% +\bibitem[\citeproctext]{ref-Rpsych} Revelle, W. (2022). \emph{Psych: {Procedures} for psychological, psychometric, and personality research} {[}Manual{]}. {Northwestern University}. -\leavevmode\vadjust pre{\hypertarget{ref-rogersPalaeopathologyJoint2000}{}}% +\bibitem[\citeproctext]{ref-rogersPalaeopathologyJoint2000} Rogers, J. (2000). The palaeopathology of joint disease. In M. Cox \& S. Mays (Eds.), \emph{Human osteology : {In} archaeology and forensic science.} (1st ed, pp. 163--182). {Cambridge University Press}. -\leavevmode\vadjust pre{\hypertarget{ref-scannapiecoRoleOral1999}{}}% +\bibitem[\citeproctext]{ref-scannapiecoRoleOral1999} Scannapieco, F. A. (1999). Role of {Oral Bacteria} in {Respiratory Infection}. \emph{Journal of Periodontology}, \emph{70}(7), 793--802. \url{https://doi.org/10.1902/jop.1999.70.7.793} -\leavevmode\vadjust pre{\hypertarget{ref-scannapiecoPotentialAssociations2001}{}}% +\bibitem[\citeproctext]{ref-scannapiecoPotentialAssociations2001} Scannapieco, F. A., \& Ho, A. W. (2001). Potential {Associations Between Chronic Respiratory Disease} and {Periodontal Disease}: {Analysis} of {National Health} and {Nutrition Examination Survey III}. \emph{Journal of Periodontology}, \emph{72}(1), 50--56. \url{https://doi.org/10.1902/jop.2001.72.1.50} -\leavevmode\vadjust pre{\hypertarget{ref-scheltemaOpiumTrade1907}{}}% +\bibitem[\citeproctext]{ref-scheltemaOpiumTrade1907} Scheltema, J. F. (1907). The {Opium Trade} in the {Dutch East Indies}. {I}. \emph{American Journal of Sociology}, \emph{13}(1), 79--112. -\leavevmode\vadjust pre{\hypertarget{ref-schuijtemakerTeTheegasten2011}{}}% +\bibitem[\citeproctext]{ref-schuijtemakerTeTheegasten2011} Schuijtemaker, D. (2011). {Te Theegasten}. \emph{De Nieuwe Schouwschuit}, \emph{9}, 16--17. -\leavevmode\vadjust pre{\hypertarget{ref-slavinDiagnosisManagement2005}{}}% +\bibitem[\citeproctext]{ref-slavinDiagnosisManagement2005} Slavin, R. G., Spector, S. L., Bernstein, I. L., Slavin, R. G., Kaliner, M. A., Kennedy, D. W., Virant, F. S., Wald, E. R., Khan, D. A., Blessing-Moore, J., Lang, D. M., Nicklas, R. A., Oppenheimer, J. J., @@ -7331,66 +7136,73 @@ \section*{References cited}\label{references-cited-4}} Allergy and Clinical Immunology}, \emph{116}(6, Supplement), S13--S47. \url{https://doi.org/10.1016/j.jaci.2005.09.048} -\leavevmode\vadjust pre{\hypertarget{ref-smithDetectionOpium2018}{}}% +\bibitem[\citeproctext]{ref-smithDetectionOpium2018} Smith, R. K., Stacey, R. J., Bergström, E., \& Thomas-Oates, J. (2018). Detection of opium alkaloids in a {Cypriot} base-ring juglet. \emph{Analyst}, \emph{143}(21), 5127--5136. \url{https://doi.org/10.1039/C8AN01040D} -\leavevmode\vadjust pre{\hypertarget{ref-sorensenEffectAntioxidants2018}{}}% +\bibitem[\citeproctext]{ref-sorensenEffectAntioxidants2018} Sørensen, L. K., \& Hasselstrøm, J. B. (2018). The effect of antioxidants on the long-term stability of {THC} and related cannabinoids in sampled whole blood. \emph{Drug Testing and Analysis}, \emph{10}(2), 301--309. \url{https://doi.org/10.1002/dta.2221} -\leavevmode\vadjust pre{\hypertarget{ref-sorensenDrugsCalculus2021}{}}% +\bibitem[\citeproctext]{ref-sorensenDrugsCalculus2021} Sørensen, L. K., Hasselstrøm, J. B., Larsen, L. S., \& Bindslev, D. A. -(2021). Entrapment of drugs in dental calculus {\textendash} {Detection} +(2021). Entrapment of drugs in dental calculus \textendash{} {Detection} validation based on test results from post-mortem investigations. \emph{Forensic Science International}, \emph{319}, 110647. \url{https://doi.org/10.1016/j.forsciint.2020.110647} -\leavevmode\vadjust pre{\hypertarget{ref-srdjenovicSimultaneousHPLC2008}{}}% +\bibitem[\citeproctext]{ref-srdjenovicSimultaneousHPLC2008} Srdjenovic, B., Djordjevic-Milic, V., Grujic, N., Injac, R., \& Lepojevic, Z. (2008). Simultaneous {HPLC Determination} of {Caffeine}, {Theobromine}, and {Theophylline} in {Food}, {Drinks}, and {Herbal Products}. \emph{Journal of Chromatographic Science}, \emph{46}(2), 144--149. \url{https://doi.org/10.1093/chromsci/46.2.144} -\leavevmode\vadjust pre{\hypertarget{ref-stavricVariabilityCaffeine1988}{}}% +\bibitem[\citeproctext]{ref-stavricVariabilityCaffeine1988} Stavric, B., Klassen, R., Watkinson, B., Karpinski, K., Stapley, R., \& Fried, P. (1988). Variability in caffeine consumption from coffee and tea: {Possible} significance for epidemiological studies. \emph{Food and Chemical Toxicology}, \emph{26}(2), 111--118. \url{https://doi.org/10.1016/0278-6915(88)90107-X} -\leavevmode\vadjust pre{\hypertarget{ref-sunMetabolomicsEvaluation2016}{}}% +\bibitem[\citeproctext]{ref-sunMetabolomicsEvaluation2016} Sun, J., Jin, J., Beger, R. D., Cerniglia, C. E., Yang, M., \& Chen, H. (2016). Metabolomics evaluation of the impact of smokeless tobacco exposure on the oral bacterium {Capnocytophaga} sputigena. \emph{Toxicology in Vitro}, \emph{36}, 133--141. \url{https://doi.org/10.1016/j.tiv.2016.07.020} -\leavevmode\vadjust pre{\hypertarget{ref-takahashiOralMicrobiome2015}{}}% +\bibitem[\citeproctext]{ref-takahashiOralMicrobiome2015} Takahashi, N. (2015). Oral {Microbiome Metabolism}: {From} {``{Who Are They}?''} To {``{What Are They Doing}?''} \emph{Journal of Dental Research}, \emph{94}(12), 1628--1637. \url{https://doi.org/10.1177/0022034515606045} -\leavevmode\vadjust pre{\hypertarget{ref-tushinghamHuntergathererTobacco2013}{}}% +\bibitem[\citeproctext]{ref-tushinghamHuntergathererTobacco2013} Tushingham, S., Ardura, D., Eerkens, J. W., Palazoglu, M., Shahbaz, S., \& Fiehn, O. (2013). Hunter-gatherer tobacco smoking: Earliest evidence from the {Pacific Northwest Coast} of {North America}. \emph{Journal of Archaeological Science}, \emph{40}(2), 1397--1407. \url{https://doi.org/10.1016/j.jas.2012.09.019} -\leavevmode\vadjust pre{\hypertarget{ref-valenDetermination212017}{}}% +\bibitem[\citeproctext]{ref-unoSexAgedependent2017} +Uno, Y., Takata, R., Kito, G., Yamazaki, H., Nakagawa, K., Nakamura, Y., +Kamataki, T., \& Katagiri, T. (2017). Sex- and age-dependent gene +expression in human liver: {An} implication for drug-metabolizing +enzymes. \emph{Drug Metabolism and Pharmacokinetics}, \emph{32}(1), +100--107. \url{https://doi.org/10.1016/j.dmpk.2016.10.409} + +\bibitem[\citeproctext]{ref-valenDetermination212017} Valen, A., Leere Øiestad, Å. M., Strand, D. H., Skari, R., \& Berg, T. (2017). Determination of 21 drugs in oral fluid using fully automated supported liquid extraction and {UHPLC-MS}/{MS}. \emph{Drug Testing and Analysis}, \emph{9}(5), 808--823. \url{https://doi.org/10.1002/dta.2045} -\leavevmode\vadjust pre{\hypertarget{ref-velskoMicrobialDifferences2019}{}}% +\bibitem[\citeproctext]{ref-velskoMicrobialDifferences2019} Velsko, I. M., Fellows Yates, J. A., Aron, F., Hagan, R. W., Frantz, L. A. F., Loe, L., Martinez, J. B. R., Chaves, E., Gosden, C., Larson, G., \& Warinner, C. (2019). Microbial differences between dental plaque and @@ -7398,7 +7210,7 @@ \section*{References cited}\label{references-cited-4}} \emph{Microbiome}, \emph{7}(1), 102. \url{https://doi.org/10.1186/s40168-019-0717-3} -\leavevmode\vadjust pre{\hypertarget{ref-velskoDentalCalculus2017}{}}% +\bibitem[\citeproctext]{ref-velskoDentalCalculus2017} Velsko, I. M., Overmyer, K. A., Speller, C., Klaus, L., Collins, M. J., Loe, L., Frantz, L. A. F., Sankaranarayanan, K., Lewis, C. M., Martinez, J. B. R., Chaves, E., Coon, J. J., Larson, G., \& Warinner, C. (2017). @@ -7406,7 +7218,7 @@ \section*{References cited}\label{references-cited-4}} \emph{Metabolomics}, \emph{13}(11), 134. \url{https://doi.org/10.1007/s11306-017-1270-3} -\leavevmode\vadjust pre{\hypertarget{ref-warinnerEvidenceMilk2014}{}}% +\bibitem[\citeproctext]{ref-warinnerEvidenceMilk2014} Warinner, C., Hendy, J., Speller, C., Cappellini, E., Fischer, R., Trachsel, C., Arneborg, J., Lynnerup, N., Craig, O. E., Swallow, D. M., Fotakis, A., Christensen, R. J., Olsen, J. V., Liebert, A., Montalva, @@ -7415,18 +7227,18 @@ \section*{References cited}\label{references-cited-4}} human dental calculus. \emph{Scientific Reports}, \emph{4}, 7104. \url{https://doi.org/10.1038/srep07104} -\leavevmode\vadjust pre{\hypertarget{ref-whiteDentalCalculus1997}{}}% +\bibitem[\citeproctext]{ref-whiteDentalCalculus1997} White, D. J. (1997). Dental calculus: Recent insights into occurrence, formation, prevention, removal and oral health effects of supragingival and subgingival deposits. \emph{European Journal of Oral Sciences}, \emph{105}(5), 508--522. \url{https://doi.org/10.1111/j.1600-0722.1997.tb00238.x} -\leavevmode\vadjust pre{\hypertarget{ref-ggplot2}{}}% +\bibitem[\citeproctext]{ref-ggplot2} Wickham, H. (2016). \emph{Ggplot2: {Elegant Graphics} for {Data Analysis}}. {Springer-Verlag}. -\leavevmode\vadjust pre{\hypertarget{ref-tidyverse2019}{}}% +\bibitem[\citeproctext]{ref-tidyverse2019} Wickham, H., Averick, M., Bryan, J., Chang, W., McGowan, L. D., François, R., Grolemund, G., Hayes, A., Henry, L., Hester, J., Kuhn, M., Pedersen, T. L., Miller, E., Bache, S. M., Müller, K., Ooms, J., @@ -7434,7 +7246,7 @@ \section*{References cited}\label{references-cited-4}} Welcome to the {tidyverse}. \emph{Journal of Open Source Software}, \emph{4}(43), 1686. \url{https://doi.org/10.21105/joss.01686} -\leavevmode\vadjust pre{\hypertarget{ref-willeRelationshipOral2009}{}}% +\bibitem[\citeproctext]{ref-willeRelationshipOral2009} Wille, S. M. R., Raes, E., Lillsunde, P., Gunnar, T., Laloup, M., Samyn, N., Christophersen, A. S., Moeller, M. R., Hammer, K. P., \& Verstraete, A. G. (2009). Relationship {Between Oral Fluid} and {Blood @@ -7442,14 +7254,14 @@ \section*{References cited}\label{references-cited-4}} Under} the {Influence} of {Drugs}. \emph{Therapeutic Drug Monitoring}, \emph{31}(4), 511. \url{https://doi.org/10.1097/FTD.0b013e3181ae46ea} -\leavevmode\vadjust pre{\hypertarget{ref-yaussyCalculusSurvivorship2019}{}}% +\bibitem[\citeproctext]{ref-yaussyCalculusSurvivorship2019} Yaussy, S. L., \& DeWitte, S. N. (2019). Calculus and survivorship in medieval {London}: {The} association between dental disease and a demographic measure of general health. \emph{American Journal of Physical Anthropology}, \emph{168}(3), 552--565. \url{https://doi.org/10.1002/ajpa.23772} -\leavevmode\vadjust pre{\hypertarget{ref-ziesemer16SChallenges2015}{}}% +\bibitem[\citeproctext]{ref-ziesemer16SChallenges2015} Ziesemer, K. A., Mann, A. E., Sankaranarayanan, K., Schroeder, H., Ozga, A. T., Brandt, B. W., Zaura, E., Waters-Rist, A., Hoogland, M., Salazar-Garcia, D. C., Aldenderfer, M., Speller, C., Hendy, J., Weston, @@ -7458,7 +7270,7 @@ \section*{References cited}\label{references-cited-4}} microbiome reconstruction using {16S rRNA} gene amplification. \emph{Sci Rep}, \emph{5}, 16498. \url{https://doi.org/10.1038/srep16498} -\leavevmode\vadjust pre{\hypertarget{ref-ziesemerGenomeCalculus2018}{}}% +\bibitem[\citeproctext]{ref-ziesemerGenomeCalculus2018} Ziesemer, K. A., Ramos-Madrigal, J., Mann, A. E., Brandt, B. W., Sankaranarayanan, K., Ozga, A. T., Hoogland, M., Hofman, C. A., Salazar-García, D. C., Frohlich, B., Milner, G. R., Stone, A. C., @@ -7467,7 +7279,7 @@ \section*{References cited}\label{references-cited-4}} ancient dental calculus and dentin. \emph{American Journal of Physical Anthropology}. \url{https://doi.org/10.1002/ajpa.23763} -\leavevmode\vadjust pre{\hypertarget{ref-zijngeBiofilmArchitecture2010}{}}% +\bibitem[\citeproctext]{ref-zijngeBiofilmArchitecture2010} Zijnge, V., van Leeuwen, M. B. M., Degener, J. E., Abbas, F., Thurnheer, T., Gmür, R., \& M. Harmsen, H. J. (2010). Oral {Biofilm Architecture} on {Natural Teeth}. \emph{PLoS ONE}, \emph{5}(2), e9321. @@ -7477,8 +7289,7 @@ \section*{References cited}\label{references-cited-4}} \bookmarksetup{startatroot} -\hypertarget{chap-discussion}{% -\chapter{Discussion}\label{chap-discussion}} +\chapter{Discussion}\label{chap-discussion} Archaeological researchers are presented with a unique challenge. Because time eventually degrades everything, the archaeological record @@ -7507,46 +7318,42 @@ \chapter{Discussion}\label{chap-discussion}} they there? If the thing in question was consumed, but not entrapped in the dental calculus; why is this the case? -As shown in \protect\hyperlink{fig-plot-and-wordclouds}{Chapter 1}, -dental calculus has become a very popular substance within -archaeological research. One of its primary uses is to reconstruct the -diet of past populations. It's not surprising why this is the case. It -forms and grows inside our mouth over time, and it is in direct contact -with everything we put in our mouth. However, there is limited -systematic and fundamental research and experimentation being conducted -within the fields that make use of archaeological dental calculus. There -are of course exceptions -(\protect\hyperlink{ref-fagernasMicrobialBiogeography2021}{Fagernäs et -al., 2021}; \protect\hyperlink{ref-leonardPlantMicroremains2015}{Leonard -et al., 2015}; \protect\hyperlink{ref-powerChimpCalculus2015}{R. C. -Power et al., 2015}; -\protect\hyperlink{ref-powerRepresentativenessDental2021}{Robert C. -Power et al., 2021}; -\protect\hyperlink{ref-sotoCharacterizationDecontamination2019}{Soto et -al., 2019}; \protect\hyperlink{ref-trompEDTACalculus2017}{Tromp et al., -2017}; \protect\hyperlink{ref-velskoMicrobialDifferences2019}{Velsko et -al., 2019}, \protect\hyperlink{ref-velskoHighConservation2023}{2023}), -but they have not addressed the full extent of dental calculus -limitations (nor should they). This type of research should aim to -validate aspects of our current analytical methods on synthetic -materials or through detailed observation and documentation of dietary -habits in living humans (or non-human primates), and critically evaluate -the patterns of information we extract. Methods-validation has also been -conducted on archaeological material -(\protect\hyperlink{ref-fagernasMicrobialBiogeography2021}{Fagernäs et -al., 2021}; \protect\hyperlink{ref-modiCalculusMethodologies2020}{Modi -et al., 2020}; \protect\hyperlink{ref-trompEDTACalculus2017}{Tromp et -al., 2017}), but these studies are limited by the fact that we have no -way of knowing what the original diet looked like. At least not at the -resolution necessary to really scrutinize the results of a method. All -we have are pieces of information from the, likely incomplete, dietary -remains that ended up in the calculus, and from contextual remains, such -as animal bones, food residues, and plant remains, both macro- and -microscopic. And even then we have no way of saying for certain whether -the materials were included in the diet, or just there because our -somewhat crucial requirement for oxygen means the oral cavity is not a -closed system (\protect\hyperlink{ref-radiniFoodPathways2017}{Radini et -al., 2017}). +As shown in \hyperref[fig-plot-and-wordclouds]{Chapter 1}, dental +calculus has become a very popular substance within archaeological +research. One of its primary uses is to reconstruct the diet of past +populations. It's not surprising why this is the case. It forms and +grows inside our mouth over time, and it is in direct contact with +everything we put in our mouth. However, there is limited systematic and +fundamental research and experimentation being conducted within the +fields that make use of archaeological dental calculus. There are of +course exceptions +(\citeproc{ref-fagernasMicrobialBiogeography2021}{Fagernäs et al., +2021}; \citeproc{ref-leonardPlantMicroremains2015}{Leonard et al., +2015}; \citeproc{ref-powerChimpCalculus2015}{R. C. Power et al., 2015}; +\citeproc{ref-powerRepresentativenessDental2021}{Robert C. Power et al., +2021}; \citeproc{ref-sotoCharacterizationDecontamination2019}{Soto et +al., 2019}; \citeproc{ref-trompEDTACalculus2017}{Tromp et al., 2017}; +\citeproc{ref-velskoMicrobialDifferences2019}{Velsko et al., 2019}, +\citeproc{ref-velskoHighConservation2023}{2023}), but they have not +addressed the full extent of dental calculus limitations (nor should +they). This type of research should aim to validate aspects of our +current analytical methods on synthetic materials or through detailed +observation and documentation of dietary habits in living humans (or +non-human primates), and critically evaluate the patterns of information +we extract. Methods-validation has also been conducted on archaeological +material (\citeproc{ref-fagernasMicrobialBiogeography2021}{Fagernäs et +al., 2021}; \citeproc{ref-modiCalculusMethodologies2020}{Modi et al., +2020}; \citeproc{ref-trompEDTACalculus2017}{Tromp et al., 2017}), but +these studies are limited by the fact that we have no way of knowing +what the original diet looked like. At least not at the resolution +necessary to really scrutinize the results of a method. All we have are +pieces of information from the, likely incomplete, dietary remains that +ended up in the calculus, and from contextual remains, such as animal +bones, food residues, and plant remains, both macro- and microscopic. +And even then we have no way of saying for certain whether the materials +were included in the diet, or just there because our somewhat crucial +requirement for oxygen means the oral cavity is not a closed system +(\citeproc{ref-radiniFoodPathways2017}{Radini et al., 2017}). In this dissertation, I have mainly focused on the development, validation, and application of an oral biofilm model and its potential @@ -7554,28 +7361,27 @@ \chapter{Discussion}\label{chap-discussion}} to develop a protocol for an oral biofilm model with a relatively simple setup, and use it to grow artificial dental calculus, and that it can serve as a reasonable proxy to natural dental calculus -{[}\protect\hyperlink{byoc-valid}{Chapter 3}; Bartholdy, Velsko, et al. -(\protect\hyperlink{ref-bartholdyAssessingValidity2023}{2023})). I -demonstrated how the oral biofilm model can answer questions and -identify hidden biases related to using dental calculus for paleodietary +{[}\hyperref[byoc-valid]{Chapter 3}; Bartholdy, Velsko, et al. +(\citeproc{ref-bartholdyAssessingValidity2023}{2023})). I demonstrated +how the oral biofilm model can answer questions and identify hidden +biases related to using dental calculus for paleodietary reconstructions, specifically addressing the identification and quantification of starch granules. The results from this study showed that what goes in, doesn't necessarily come out. And the loss of information is not evenly distributed across the different types of starches, depending on size and morphology -{[}\protect\hyperlink{byoc-starch}{Chapter 4}; Bartholdy \& Henry -(\protect\hyperlink{ref-bartholdyInvestigatingBiases2022}{2022}){]}. In -\protect\hyperlink{mb11CalculusPilot}{Chapter 5} I present a study that -goes beyond the model and looks at archaeological dental calculus. This -is, after all, a dissertation in archaeology. We analysed dental -calculus samples from a rural Dutch archaeological site in -Middenbeemster, using ultra high performance liquid chromatography -tandem mass spectrometry (UHPLC-ESI-MS/MS). This allowed us to identify -a number of residues from plants that may have been consumed for -nutrition, medicine, recreation, or all of the above. - -\hypertarget{the-dental-calculus-model}{% -\section{The dental calculus model}\label{the-dental-calculus-model}} +{[}\hyperref[byoc-starch]{Chapter 4}; Bartholdy \& Henry +(\citeproc{ref-bartholdyInvestigatingBiases2022}{2022}){]}. In +\hyperref[mb11CalculusPilot]{Chapter 5} I present a study that goes +beyond the model and looks at archaeological dental calculus. This is, +after all, a dissertation in archaeology. We analysed dental calculus +samples from a rural Dutch archaeological site in Middenbeemster, using +ultra high performance liquid chromatography tandem mass spectrometry +(UHPLC-ESI-MS/MS). This allowed us to identify a number of residues from +plants that may have been consumed for nutrition, medicine, recreation, +or all of the above. + +\section{The dental calculus model}\label{the-dental-calculus-model} While the use of oral biofilm models in dental research is well-established, even long-term calcifying models to produce dental @@ -7601,11 +7407,10 @@ \section{The dental calculus model}\label{the-dental-calculus-model}} The model we chose was a simple model using a shaking incubator and a 24 deepwell plate with the plastic lids as a substratum. The artificial saliva we used was based on the basal modified medium used by Sissons -and colleagues -(\protect\hyperlink{ref-sissonsMultistationPlaque1991}{1991}, -\protect\hyperlink{ref-sissonsPHResponse1994}{1994}; -\protect\hyperlink{ref-sissonsArtificialPlaque1997}{1997}) to grow -dental calculus. We also made use of their calcifying solution, calcium +and colleagues (\citeproc{ref-sissonsMultistationPlaque1991}{1991}, +\citeproc{ref-sissonsPHResponse1994}{1994}; +\citeproc{ref-sissonsArtificialPlaque1997}{1997}) to grow dental +calculus. We also made use of their calcifying solution, calcium phosphate monofluorophosphate urea (CPMU) to speed up the mineralisation process (natural dental calculus can take weeks, even months, to form). To make sure the calculus we were growing in the lab was a good model @@ -7624,28 +7429,26 @@ \section{The dental calculus model}\label{the-dental-calculus-model}} anaerobes. The natural samples also had a more diverse representation of bacteria from all stages of biofilm development, including early- middle-, and late-colonisers, while model calculus samples were -predominantly late-colonisers -(\protect\hyperlink{byoc-valid}{\textbf{Chapter 3}}, Bartholdy, Velsko, -et al. (\protect\hyperlink{ref-bartholdyAssessingValidity2023}{2023})). -Results from our metagenomic analysis were similar to a comparable -\emph{in vitro} biofilm model. In their study, the authors also used a -24-well plate with pooled saliva as inoculate. The growth medium was -similar but also contained a sheep's-blood serum, and the samples were -only grown for 24 hours -(\protect\hyperlink{ref-edlundUncoveringComplex2018}{Edlund et al., -2018}). As with our model, the comparison with natural oral samples -showed a lower overall richness and diversity, and a distinct microbial -profile (\protect\hyperlink{byoc-valid}{\textbf{Chapter 3}}, Bartholdy, -Velsko, et al. -(\protect\hyperlink{ref-bartholdyAssessingValidity2023}{2023})). Given -that our results are similar to a short-term biofilm model, we may be -replacing the medium too often (every three days), and not allowing -communities to establish more complex metabolic pathways that are -normally present in mature biofilms. To resolve this and other issues, -our protocol will benefit from further refinement. Using serum in the -medium may help to establish thicker and more stable biofilms, and allow -slow-growing organisms to become more established -(\protect\hyperlink{ref-ammannZurichBiofilm2012}{Ammann et al., 2012}). +predominantly late-colonisers (\hyperref[byoc-valid]{\textbf{Chapter +3}}, Bartholdy, Velsko, et al. +(\citeproc{ref-bartholdyAssessingValidity2023}{2023})). Results from our +metagenomic analysis were similar to a comparable \emph{in vitro} +biofilm model. In their study, the authors also used a 24-well plate +with pooled saliva as inoculate. The growth medium was similar but also +contained a sheep's-blood serum, and the samples were only grown for 24 +hours (\citeproc{ref-edlundUncoveringComplex2018}{Edlund et al., 2018}). +As with our model, the comparison with natural oral samples showed a +lower overall richness and diversity, and a distinct microbial profile +(\hyperref[byoc-valid]{\textbf{Chapter 3}}, Bartholdy, Velsko, et al. +(\citeproc{ref-bartholdyAssessingValidity2023}{2023})). Given that our +results are similar to a short-term biofilm model, we may be replacing +the medium too often (every three days), and not allowing communities to +establish more complex metabolic pathways that are normally present in +mature biofilms. To resolve this and other issues, our protocol will +benefit from further refinement. Using serum in the medium may help to +establish thicker and more stable biofilms, and allow slow-growing +organisms to become more established +(\citeproc{ref-ammannZurichBiofilm2012}{Ammann et al., 2012}). Filter-sterilising the heat-sensitive solutions that are not autoclaved, such as CPMU and starch solutions, may prevent environmental contamination from entering the biofilm during the setup, such as @@ -7655,7 +7458,7 @@ \section{The dental calculus model}\label{the-dental-calculus-model}} changes to the model setup, the model will have to be re-validated, as the concentrations of nutrients, let alone the type of nutrients, will impact the community composition of the biofilms -(\protect\hyperlink{ref-edlundBiofilmModel2013}{Edlund et al., 2013}). +(\citeproc{ref-edlundBiofilmModel2013}{Edlund et al., 2013}). We also used Fourier Transform Infrared (FTIR) spectroscopy to assess the mineral content of our model and compare it to natural dental @@ -7673,48 +7476,47 @@ \section{The dental calculus model}\label{the-dental-calculus-model}} compared to archaeological calculus. Not only did the archaeological calculus spend a few hundred years maturing in the ground, allowing crystals to expand into the gaps created by degraded organic matter -(\protect\hyperlink{ref-weinerBiologicalMaterials2010}{Weiner, 2010}), -but given the known lack of oral hygiene practices in the past, the -calculus was surely older than 25 days before being buried. We also only -analysed a single archaeological sample, so we don't know how -representative this sample is of archaeological samples in general. -Perhaps this was a particularly under- or over-mineralised sample. It -would be more appropriate to compare to the modern reference samples, -since we are actually trying to recreate something that mimics natural -modern calculus, not something that has been buried for hundreds of -years or more. Unfortunately we didn't have access to new modern samples -and couldn't produce modern calculus grind curves for this analysis. - -\hypertarget{model-application}{% -\subsection{Model application}\label{model-application}} +(\citeproc{ref-weinerBiologicalMaterials2010}{Weiner, 2010}), but given +the known lack of oral hygiene practices in the past, the calculus was +surely older than 25 days before being buried. We also only analysed a +single archaeological sample, so we don't know how representative this +sample is of archaeological samples in general. Perhaps this was a +particularly under- or over-mineralised sample. It would be more +appropriate to compare to the modern reference samples, since we are +actually trying to recreate something that mimics natural modern +calculus, not something that has been buried for hundreds of years or +more. Unfortunately we didn't have access to new modern samples and +couldn't produce modern calculus grind curves for this analysis. + +\subsection{Model application}\label{model-application} After establishing that our model dental calculus mimics, at least to some extent, the real deal, we assessed what biases may occur in starch incorporation. It is a mistake to think you can solve any major problems -just with potatoes (\protect\hyperlink{ref-adamsLifeUniverse2002}{Adams, -2002a}), so we also included wheat starch in the model to cover a wider -range of granule shapes and sizes. Put simply, we added a known amount -of starch granules---well, to the extent we could estimate the large -quantities in our starch solutions without counting every single -granule---to our biofilm over the course of the 25-day experiment. -Starch solutions were added on day nine of the experiment. This was a -somewhat arbitrary decision; we only needed to ensure that there was -enough separation between the last saliva donation and the introduction -of starch treatments. We did this to prevent our starch counts from -being affected by \(\alpha\)-amylase activity from the donated saliva, -thereby getting somewhat `pure' counts from the added starches. However, -we found no evidence of the model retaining \(\alpha\)-amylase from the -donated saliva, there is no reason the starch treatments couldn't start -sooner in the experiment. For future experiments looking at the effect -of amylase activity, it's important to still keep this under -consideration, as amylase activity from natural saliva can fluctuate in -individuals throughout the day based on both physical and psychological -influences (\protect\hyperlink{ref-naterHumanAmylase2005}{Nater et al., -2005}). Controlling the level of amylase activity in the experiment is -more easily done with amylase artificiallsupplier of scientificy added -to the model. Amylase can be purchased from your local supplier of -scientific equipment along with some overpriced sugar and baking soda. -If it's not `analytical grade' it's not +just with potatoes (\citeproc{ref-adamsLifeUniverse2002}{Adams, 2002a}), +so we also included wheat starch in the model to cover a wider range of +granule shapes and sizes. Put simply, we added a known amount of starch +granules---well, to the extent we could estimate the large quantities in +our starch solutions without counting every single granule---to our +biofilm over the course of the 25-day experiment. Starch solutions were +added on day nine of the experiment. This was a somewhat arbitrary +decision; we only needed to ensure that there was enough separation +between the last saliva donation and the introduction of starch +treatments. We did this to prevent our starch counts from being affected +by \(\alpha\)-amylase activity from the donated saliva, thereby getting +somewhat `pure' counts from the added starches. However, we found no +evidence of the model retaining \(\alpha\)-amylase from the donated +saliva, there is no reason the starch treatments couldn't start sooner +in the experiment. For future experiments looking at the effect of +amylase activity, it's important to still keep this under consideration, +as amylase activity from natural saliva can fluctuate in individuals +throughout the day based on both physical and psychological influences +(\citeproc{ref-naterHumanAmylase2005}{Nater et al., 2005}). Controlling +the level of amylase activity in the experiment is more easily done with +amylase artificiallsupplier of scientificy added to the model. Amylase +can be purchased from your local supplier of scientific equipment along +with some overpriced sugar and baking soda. If it's not `analytical +grade' it's not At the end of the experiment, we dissolved the calculus and counted the number of starches that were inside. Those who are familiar with @@ -7729,45 +7531,39 @@ \subsection{Model application}\label{model-application}} that a very, VERY, low proportion of the starch granules that we `fed' our samples actually made it into the dental calculus; only 0.06\% to 0.16\% of granules from the treatment solutions were extracted from the -dental calculus (\protect\hyperlink{byoc-starch}{\textbf{Chapter 4}}, -Bartholdy \& Henry -(\protect\hyperlink{ref-bartholdyInvestigatingBiases2022}{2022})). Given +dental calculus (\hyperref[byoc-starch]{\textbf{Chapter 4}}, Bartholdy +\& Henry (\citeproc{ref-bartholdyInvestigatingBiases2022}{2022})). Given how few actually make it in, this may suggest that evidence for dietary starches are the result of repeated exposure to a large quantity of granule-containing foods. -\hypertarget{disc-model-limitations}{% -\subsection{Model limitations}\label{disc-model-limitations}} +\subsection{Model limitations}\label{disc-model-limitations} So far I have covered what our biofilm model can do. It is equally important to talk about what our model can't do. After all, we demand rigidly defined areas of doubt and uncertainty -(\protect\hyperlink{ref-adamsHitchhikersGuide2002}{Adams, 2002c}). While -we have a high degree of control and reproducibility, especially when -compared to \emph{in vivo} models, there are certain conditions we -cannot regulate with our current setup. This includes environmental -conditions such as CO\textsubscript{2} and oxygen availability, which -rely on the conditions in the lab where the experiments take place. To -some extent, the bacterial communities within a biofilm can generate -favorable conditions in a local environment through metabolic -processes---one of the adaptive benefits from being part of a -biofilm---but these are still somewhat dependent on the extrinsic -environment in which they are situated. Biofilms on hard tissues will -differ in composition from those found on soft tissues. And biofilms -found closer to the front of the mouth will differ from those found -towards the back -(\protect\hyperlink{ref-kolenbranderOralMultispecies2010}{Kolenbrander -et al., 2010}; \protect\hyperlink{ref-marshDentalPlaque2005}{Marsh, -2005}; -\protect\hyperlink{ref-palmerCoaggregationInteractions2003}{Palmer et -al., 2003}; \protect\hyperlink{ref-proctorSpatialGradient2018}{Proctor -et al., 2018}). This difference is also something that is difficult to -mimic in a single experimental setup; as is the ability to control -salivary flow rates and circadian rhythms, both of which can influence -the growth of plaque -(\protect\hyperlink{ref-dawesCircadianRhythms1972}{Dawes, 1972}; -\protect\hyperlink{ref-proctorSpatialGradient2018}{Proctor et al., -2018}). +(\citeproc{ref-adamsHitchhikersGuide2002}{Adams, 2002c}). While we have +a high degree of control and reproducibility, especially when compared +to \emph{in vivo} models, there are certain conditions we cannot +regulate with our current setup. This includes environmental conditions +such as CO\textsubscript{2} and oxygen availability, which rely on the +conditions in the lab where the experiments take place. To some extent, +the bacterial communities within a biofilm can generate favorable +conditions in a local environment through metabolic processes---one of +the adaptive benefits from being part of a biofilm---but these are still +somewhat dependent on the extrinsic environment in which they are +situated. Biofilms on hard tissues will differ in composition from those +found on soft tissues. And biofilms found closer to the front of the +mouth will differ from those found towards the back +(\citeproc{ref-kolenbranderOralMultispecies2010}{Kolenbrander et al., +2010}; \citeproc{ref-marshDentalPlaque2005}{Marsh, 2005}; +\citeproc{ref-palmerCoaggregationInteractions2003}{Palmer et al., 2003}; +\citeproc{ref-proctorSpatialGradient2018}{Proctor et al., 2018}). This +difference is also something that is difficult to mimic in a single +experimental setup; as is the ability to control salivary flow rates and +circadian rhythms, both of which can influence the growth of plaque +(\citeproc{ref-dawesCircadianRhythms1972}{Dawes, 1972}; +\citeproc{ref-proctorSpatialGradient2018}{Proctor et al., 2018}). The effect of circadian differences in microbiome between individuals can influence replication of the microbial composition of our model, @@ -7786,20 +7582,19 @@ \subsection{Model limitations}\label{disc-model-limitations}} for \(\alpha\)-amylase once the inoculations have been completed. There are streptococcal species present in the model that are known for their ability to bind amylase -(\protect\hyperlink{ref-haaseComparativeGenomics2017}{Haase et al., -2017}; \protect\hyperlink{ref-nikitkovaStarchBiofilms2013}{Nikitkova et -al., 2013}); however, we did not investigate whether the strains present -in our model contain these genes. Starch solutions were only introduced -on day 9 of the experiment. Prior to this, all samples were treated with -the sucrose solution. The absence of starch during inoculation could -have suppressed bacterial production of amylase-binding proteins -(\protect\hyperlink{ref-nikitkovaEffectStarch2012}{Nikitkova et al., -2012}). Frequent medium replacements may also be clearing out all of the -unbound host salivary amylase. We don't know exactly why -\(\alpha\)-amylase is absent, and need to look into this. In the -meantime, this absence opens up opportunities to examine its role in the -incorporation process of dietary materials (see -\protect\hyperlink{bfmodels-in-arch}{below}). +(\citeproc{ref-haaseComparativeGenomics2017}{Haase et al., 2017}; +\citeproc{ref-nikitkovaStarchBiofilms2013}{Nikitkova et al., 2013}); +however, we did not investigate whether the strains present in our model +contain these genes. Starch solutions were only introduced on day 9 of +the experiment. Prior to this, all samples were treated with the sucrose +solution. The absence of starch during inoculation could have suppressed +bacterial production of amylase-binding proteins +(\citeproc{ref-nikitkovaEffectStarch2012}{Nikitkova et al., 2012}). +Frequent medium replacements may also be clearing out all of the unbound +host salivary amylase. We don't know exactly why \(\alpha\)-amylase is +absent, and need to look into this. In the meantime, this absence opens +up opportunities to examine its role in the incorporation process of +dietary materials (see \hyperref[bfmodels-in-arch]{below}). A well-known limitation of biofilm models in general is the difficulty in capturing the diversity and complexity of the natural oral biome. @@ -7808,16 +7603,15 @@ \subsection{Model limitations}\label{disc-model-limitations}} communities, or as an environmental complexity determined by nutrient availability, host immune-responses to biofilms, and fluctuating microenvironments across the biofilm in response to these factors -(\protect\hyperlink{ref-bjarnsholtVivoBiofilm2013}{Bjarnsholt et al., -2013}; \protect\hyperlink{ref-edlundUncoveringComplex2018}{Edlund et -al., 2018}). These limitations can be mitigated by complex experimental -setups, but at the cost of lower throughput and higher financial cost. -Increasing the number of species included in a model can approach the -diversity found in the natural microbiome, but still falls short of -capturing the complete diversity -(\protect\hyperlink{ref-edlundBiofilmModel2013}{Edlund et al., 2013}), -and the use of whole saliva introduces another set of limitations (as -discussed above). +(\citeproc{ref-bjarnsholtVivoBiofilm2013}{Bjarnsholt et al., 2013}; +\citeproc{ref-edlundUncoveringComplex2018}{Edlund et al., 2018}). These +limitations can be mitigated by complex experimental setups, but at the +cost of lower throughput and higher financial cost. Increasing the +number of species included in a model can approach the diversity found +in the natural microbiome, but still falls short of capturing the +complete diversity (\citeproc{ref-edlundBiofilmModel2013}{Edlund et al., +2013}), and the use of whole saliva introduces another set of +limitations (as discussed above). Then of course there's the inevitable limitation that we're dealing with a model. An attempt to recreate the real thing under controlled @@ -7831,10 +7625,9 @@ \subsection{Model limitations}\label{disc-model-limitations}} constantly at risk of removal by the tongue, salivary flow, oral hygiene practices, even the act of chewing---processes which help shape the biofilm (this is counterintuitive since they are processes of removal) -(\protect\hyperlink{ref-shawCommonalityElastic2004}{Shaw et al., 2004}). +(\citeproc{ref-shawCommonalityElastic2004}{Shaw et al., 2004}). -\hypertarget{further-model-validation}{% -\subsection{Further model validation}\label{further-model-validation}} +\subsection{Further model validation}\label{further-model-validation} Going forward, we aim to further assess the validity of our model, as well as optimise the protocol. While we have established that our model @@ -7856,13 +7649,13 @@ \subsection{Further model validation}\label{further-model-validation}} within the first few hours of consuming carbohydrates, after which the saliva will work to balance the pH back to pre-carbohydrate levels, also known as the `Stephan curve' -(\protect\hyperlink{ref-stephanStudiesChanges1947}{Stephan \& Hemmens, -1947}). By acting as a buffer and restoring the oral pH-level, saliva -can help prevent high levels of acid from demineralising the tooth -surface and causing caries. Since our model is fed both with sucrose and -starch, it is important to know that the pH levels don't permanently -drop to levels that are unfavourable to mineral supersaturation and -plaque mineralisation. +(\citeproc{ref-stephanStudiesChanges1947}{Stephan \& Hemmens, 1947}). By +acting as a buffer and restoring the oral pH-level, saliva can help +prevent high levels of acid from demineralising the tooth surface and +causing caries. Since our model is fed both with sucrose and starch, it +is important to know that the pH levels don't permanently drop to levels +that are unfavourable to mineral supersaturation and plaque +mineralisation. Since FTIR only addresses the overall mineral composition, we will need to further investigate whether there are any other structural/chemical @@ -7870,9 +7663,8 @@ \subsection{Further model validation}\label{further-model-validation}} microbial profiles, and microscopically examine the model to determine the micro-architecture. -\hypertarget{bfmodels-in-arch}{% \subsection{Potential biofilm model applications in -archaeology}\label{bfmodels-in-arch}} +archaeology}\label{bfmodels-in-arch} Biofilm models are an untapped resource in archaeological research, especially for dental calculus research. Coupled with existing @@ -7904,17 +7696,17 @@ \subsection{Potential biofilm model applications in hydrophobicity of molecules, etc) influence their ability to become incorporated or penetrate the mineralised surface? This question of incorporation also came up during the analysis of archaeological dental -calculus in \protect\hyperlink{mb11CalculusPilot}{\textbf{Chapter 5}} -(\protect\hyperlink{ref-bartholdyMultiproxyAnalysis2023}{Bartholdy, -Hasselstrøm, et al., 2023}). Based on the presence of many metabolites, -it seems that this may not have been during consumption, but rather -during excretion through saliva, or, put more simply, when the molecules -are on their way out of the body again. This makes some sense, since -food actually spends relatively little time in our mouth while we're -eating, and significantly longer travelling through our body. This may -also explain the very low retention of starch granules we found in -\protect\hyperlink{byoc-starch}{\textbf{Chapter 4}}. It seems that most -of the starch granules are swallowed, while few become lodged in our +calculus in \hyperref[mb11CalculusPilot]{\textbf{Chapter 5}} +(\citeproc{ref-bartholdyMultiproxyAnalysis2023}{Bartholdy, Hasselstrøm, +et al., 2023}). Based on the presence of many metabolites, it seems that +this may not have been during consumption, but rather during excretion +through saliva, or, put more simply, when the molecules are on their way +out of the body again. This makes some sense, since food actually spends +relatively little time in our mouth while we're eating, and +significantly longer travelling through our body. This may also explain +the very low retention of starch granules we found in +\hyperref[byoc-starch]{\textbf{Chapter 4}}. It seems that most of the +starch granules are swallowed, while few become lodged in our teeth/plaque and are eventually trapped in dental calculus. Without looking into the mechanism by which starches and other food molecules are incorporated into dental plaque, we are always going to be guessing @@ -7926,25 +7718,24 @@ \subsection{Potential biofilm model applications in retention of dietary molecules and microremains. It is likely that they will cause differential retention given that they make use of a lot of the food that passes through our mouths with the help of digestive -enzymes (\protect\hyperlink{ref-rogersRoleStreptococcus2001}{Rogers et -al., 2001}). The important question to answer is how, and, to what -extent, they influence this process. A systematic approach would be to -set up multiple experiments with different sets of defined consortia -grown under the same conditions. On a related note, the absence of host +enzymes (\citeproc{ref-rogersRoleStreptococcus2001}{Rogers et al., +2001}). The important question to answer is how, and, to what extent, +they influence this process. A systematic approach would be to set up +multiple experiments with different sets of defined consortia grown +under the same conditions. On a related note, the absence of host salivary \(\alpha\)-amylase activity in our model (as shown in -\protect\hyperlink{byoc-starch}{\textbf{Chapter 4}}, Bartholdy \& Henry -(\protect\hyperlink{ref-bartholdyInvestigatingBiases2022}{2022})) -provides an opportunity to explore the effect of various amylase levels -on the incorporation and retention of dietary compounds, especially -starches, in dental calculus. Alpha-amylase can be purchased from most -laboratory supply companies, and can therefore be added to the model and -explored as a controlled variable. Some bacteria have the ability to -bind \(\alpha\)-amylase in order to use the degradation products of -starches as nutrients -(\protect\hyperlink{ref-nikitkovaEffectStarch2012}{Nikitkova et al., -2012}; \protect\hyperlink{ref-rogersRoleStreptococcus2001}{Rogers et -al., 2001}), so the abundance of these bacteria coupled with -\(\alpha\)-amylase activity will likely influence starch retention. +\hyperref[byoc-starch]{\textbf{Chapter 4}}, Bartholdy \& Henry +(\citeproc{ref-bartholdyInvestigatingBiases2022}{2022})) provides an +opportunity to explore the effect of various amylase levels on the +incorporation and retention of dietary compounds, especially starches, +in dental calculus. Alpha-amylase can be purchased from most laboratory +supply companies, and can therefore be added to the model and explored +as a controlled variable. Some bacteria have the ability to bind +\(\alpha\)-amylase in order to use the degradation products of starches +as nutrients (\citeproc{ref-nikitkovaEffectStarch2012}{Nikitkova et al., +2012}; \citeproc{ref-rogersRoleStreptococcus2001}{Rogers et al., 2001}), +so the abundance of these bacteria coupled with \(\alpha\)-amylase +activity will likely influence starch retention. Finally, it's worth noting how important it is to be able to generate an unlimited number of samples for validating current methods and @@ -7954,9 +7745,8 @@ \subsection{Potential biofilm model applications in be a great substance to try out new things, and even for training researchers on the range of methods at our disposal. -\hypertarget{dental-calculus-in-archaeology-and-future-challenges}{% \section{Dental calculus in archaeology and future -challenges}\label{dental-calculus-in-archaeology-and-future-challenges}} +challenges}\label{dental-calculus-in-archaeology-and-future-challenges} Dental calculus has provided unique perspectives on multiple activities of humans in the past, from dietary practices to the evolution of the @@ -7976,8 +7766,7 @@ \section{Dental calculus in archaeology and future small deposits of minerals, bacteria, food debris, and whatever else made its way into the mouth during life. -\hypertarget{incorporation-pathways}{% -\subsection{Incorporation pathways}\label{incorporation-pathways}} +\subsection{Incorporation pathways}\label{incorporation-pathways} As discussed above, one of the main challenges of working with dental calculus is our lack of understanding of incorporation pathways. We need @@ -7991,7 +7780,7 @@ \subsection{Incorporation pathways}\label{incorporation-pathways}} physicochemical properties allows them to enter and become trapped is still unknown. The surfaces of starch granules mainly contain polar phospholipids -(\protect\hyperlink{ref-cornejo-ramirezStructuralCharacteristics2018}{Cornejo-Ramírez +(\citeproc{ref-cornejo-ramirezStructuralCharacteristics2018}{Cornejo-Ramírez et al., 2018}), making the phospholipid bilayer of a starch granule compatible with, or even attracted to, a biofilm consisting largely of water. Conversely, hydrophobic molecules might be less likely to @@ -8008,16 +7797,14 @@ \subsection{Incorporation pathways}\label{incorporation-pathways}} underrepresented. We know that this happens, but not why. Smaller molecules may be able to hitch a ride through diffusion channels that transport nutrients into the biofilm -(\protect\hyperlink{ref-flemmingBiofilmMatrix2010}{Flemming \& -Wingender, 2010}), although biofilms are known for their ability to -limit diffusion of specific molecules, such as antibiotics -(\protect\hyperlink{ref-stewartAntimicrobialTolerance2015}{Stewart, -2015}). Diffusion of molecules has been explored clinically, but mainly -focusing on antibacterial agents -(\protect\hyperlink{ref-maModelingDiffusion2010}{R. Ma et al., 2010}; -\protect\hyperlink{ref-stewartAntimicrobialTolerance2015}{Stewart, -2015}; -\protect\hyperlink{ref-takenakaDiffusionMacromolecules2009}{Takenaka et +(\citeproc{ref-flemmingBiofilmMatrix2010}{Flemming \& Wingender, 2010}), +although biofilms are known for their ability to limit diffusion of +specific molecules, such as antibiotics +(\citeproc{ref-stewartAntimicrobialTolerance2015}{Stewart, 2015}). +Diffusion of molecules has been explored clinically, but mainly focusing +on antibacterial agents (\citeproc{ref-maModelingDiffusion2010}{R. Ma et +al., 2010}; \citeproc{ref-stewartAntimicrobialTolerance2015}{Stewart, +2015}; \citeproc{ref-takenakaDiffusionMacromolecules2009}{Takenaka et al., 2009}). So far nothing has been done to explore the dietary perspective in which we're interested. @@ -8028,9 +7815,8 @@ \subsection{Incorporation pathways}\label{incorporation-pathways}} deposits on the molars. However, mucous-rich saliva, produced by the sublingual and submandibular glands (located in the front of the mouth), preferentially binds toxins -(\protect\hyperlink{ref-doddsHealthBenefits2005}{Dodds et al., 2005}), -making the anterior teeth a good hypothetical target for detecting these -compounds. +(\citeproc{ref-doddsHealthBenefits2005}{Dodds et al., 2005}), making the +anterior teeth a good hypothetical target for detecting these compounds. Another potential pathway is the presence of molecules in dental calculus as a result of excretion from the body through the saliva. If @@ -8040,77 +7826,72 @@ \subsection{Incorporation pathways}\label{incorporation-pathways}} bloodstream and are distributed throughout the body. The molecules can then re-enter the mouth through the saliva and spend significantly more time in the mouth the second time around, as excretion may take days -(\protect\hyperlink{ref-leeOralFluid2011}{Lee et al., 2011}). At this -point the original compounds may have been broken down by, for example, -the liver or kidneys, in which case mainly the metabolites will be -present. The plausibility of finding molecules via this pathway depends -on the size of the molecules and the ability to diffuse from -serum/plasma to saliva and enter the oral cavity. Given this -incorporation pathway, the molecules are, hypothetically, more likely to -be secreted in higher concentrations through the serum-rich saliva of -the parotid glands, located next to the molars -(\protect\hyperlink{ref-doddsHealthBenefits2005}{Dodds et al., 2005}). -Molecules originating from this pathway would mean that it, -unfortunately, wouldn't be possible to determine the mode of consumption -(e.g.~chewing vs.~smoking) based on the mass spectrometric results -alone, but would require further analysis of the dentition to identify. -For example, if nicotine is detected, it would be useful to identify -tooth staining and periodontal disease caused by tobacco smoking -(\protect\hyperlink{ref-nessEpidemiologicStudy1977}{Ness et al., 1977}). +(\citeproc{ref-leeOralFluid2011}{Lee et al., 2011}). At this point the +original compounds may have been broken down by, for example, the liver +or kidneys, in which case mainly the metabolites will be present. The +plausibility of finding molecules via this pathway depends on the size +of the molecules and the ability to diffuse from serum/plasma to saliva +and enter the oral cavity. Given this incorporation pathway, the +molecules are, hypothetically, more likely to be secreted in higher +concentrations through the serum-rich saliva of the parotid glands, +located next to the molars (\citeproc{ref-doddsHealthBenefits2005}{Dodds +et al., 2005}). Molecules originating from this pathway would mean that +it, unfortunately, wouldn't be possible to determine the mode of +consumption (e.g.~chewing vs.~smoking) based on the mass spectrometric +results alone, but would require further analysis of the dentition to +identify. For example, if nicotine is detected, it would be useful to +identify tooth staining and periodontal disease caused by tobacco +smoking (\citeproc{ref-nessEpidemiologicStudy1977}{Ness et al., 1977}). It would also require relying on contextual materials found at the site, but that's something which should be done anyway. To bridge this essential gap in our knowledge, further testing through systematic sampling of different parts of the dentition is needed. -\hypertarget{identification-of-fragmented-remains}{% \subsection{Identification of fragmented -remains}\label{identification-of-fragmented-remains}} +remains}\label{identification-of-fragmented-remains} Identifying and quantifying plant microremains has a particular set of challenges, even before the food has entered our mouth. Humans have become reliant on processing foods to aid digestion and to maximise the energy acquired from eating. Unfortunately, this also means that the microremains are put through various damaging processes during -preparation -(\protect\hyperlink{ref-graneroStarchTaphonomy2020}{García-Granero, +preparation (\citeproc{ref-graneroStarchTaphonomy2020}{García-Granero, 2020}). Pre-cooking processing may already render starch granules -unidentifiable (\protect\hyperlink{ref-liInfluenceGrinding2020}{Li et -al., 2020}). During cooking, starch granules are, at best, modified and, -at worst, completely destroyed depending on the cooking method -(\protect\hyperlink{ref-henryCookingStarch2009}{Henry et al., 2009}). -The granules that survive the cooking process are then submitted to -further harm in the oral cavity by the act of chewing and the presence -of digestive enzymes. After death, the starch granules that are trapped -in dental calculus will have to resist degradation from the burial +unidentifiable (\citeproc{ref-liInfluenceGrinding2020}{Li et al., +2020}). During cooking, starch granules are, at best, modified and, at +worst, completely destroyed depending on the cooking method +(\citeproc{ref-henryCookingStarch2009}{Henry et al., 2009}). The +granules that survive the cooking process are then submitted to further +harm in the oral cavity by the act of chewing and the presence of +digestive enzymes. After death, the starch granules that are trapped in +dental calculus will have to resist degradation from the burial environment, including bacteria, fungi, and water damage -(\protect\hyperlink{ref-graneroStarchTaphonomy2020}{García-Granero, -2020}). To add final insult to injury, further damage can occur during -excavation and processing of the dental calculus -(\protect\hyperlink{ref-trompEDTACalculus2017}{Tromp et al., 2017}), and -even during preparation for microscopic identification -(\protect\hyperlink{ref-graneroStarchTaphonomy2020}{García-Granero, -2020}). Through all this, there are still dietary molecules and -microremains that somehow survive hundreds-to-thousands of years inside -dental calculus, and remain identifiable. Our next challenge is to -determine how to interpret these remaining microremains. To date, most +(\citeproc{ref-graneroStarchTaphonomy2020}{García-Granero, 2020}). To +add final insult to injury, further damage can occur during excavation +and processing of the dental calculus +(\citeproc{ref-trompEDTACalculus2017}{Tromp et al., 2017}), and even +during preparation for microscopic identification +(\citeproc{ref-graneroStarchTaphonomy2020}{García-Granero, 2020}). +Through all this, there are still dietary molecules and microremains +that somehow survive hundreds-to-thousands of years inside dental +calculus, and remain identifiable. Our next challenge is to determine +how to interpret these remaining microremains. To date, most experimental methods have addressed the damage and modifications occurring to microremains present on tools and cooking utensils -(\protect\hyperlink{ref-langejansRemainsDay2010}{Langejans, 2010}; -\protect\hyperlink{ref-liInfluenceGrinding2020}{Li et al., 2020}; -\protect\hyperlink{ref-maMorphologicalChanges2019}{Z. Ma et al., 2019}), -and not in the context of dental calculus. Given the added processes -affecting the survival and morphology of microremains unique to the oral -cavity, this context is very important. Validation conducted on -archaeological remains will suffer from the same limitations as \emph{in -vivo} studies, namely the variability of dental calculus growth. The -variability can affect comparisons between two or more individuals, as -well as between dental calculus deposits within the oral cavity of a -single individual. The human oral cavity is home to many unique -environments causing differences in the chemical and bacterial makeup of -dental calculus -(\protect\hyperlink{ref-fagernasMicrobialBiogeography2022}{Fagernäs et -al., 2022}; -\protect\hyperlink{ref-hayashizakiSiteSpecific2008}{Hayashizaki et al., +(\citeproc{ref-langejansRemainsDay2010}{Langejans, 2010}; +\citeproc{ref-liInfluenceGrinding2020}{Li et al., 2020}; +\citeproc{ref-maMorphologicalChanges2019}{Z. Ma et al., 2019}), and not +in the context of dental calculus. Given the added processes affecting +the survival and morphology of microremains unique to the oral cavity, +this context is very important. Validation conducted on archaeological +remains will suffer from the same limitations as \emph{in vivo} studies, +namely the variability of dental calculus growth. The variability can +affect comparisons between two or more individuals, as well as between +dental calculus deposits within the oral cavity of a single individual. +The human oral cavity is home to many unique environments causing +differences in the chemical and bacterial makeup of dental calculus +(\citeproc{ref-fagernasMicrobialBiogeography2022}{Fagernäs et al., +2022}; \citeproc{ref-hayashizakiSiteSpecific2008}{Hayashizaki et al., 2008}). Our best option to control these many factors and explore the precise nature of their individual impact on the incorporation and retention of dietary materials in dental calculus, is to isolate these @@ -8126,9 +7907,9 @@ \subsection{Identification of fragmented the millions of microbes inhabiting the oral cavity. Further complicating the matter is the inability to assign damaged DNA sequences to a single precise species designation, and instead relying on low -resolution estimates (\protect\hyperlink{ref-mannHaveSomething2023}{Mann -et al., 2023}). Similar issues are encountered in protein identification -(\protect\hyperlink{ref-hendyAncientProtein2021}{Hendy, 2021}). +resolution estimates (\citeproc{ref-mannHaveSomething2023}{Mann et al., +2023}). Similar issues are encountered in protein identification +(\citeproc{ref-hendyAncientProtein2021}{Hendy, 2021}). Adding to the challenge is the fact that not all materials will degrade in a similar manner. Some materials/molecules are more robust than @@ -8141,78 +7922,72 @@ \subsection{Identification of fragmented tetrahydrocannabinol, are relatively slim since these molecules are unstable and have a hard enough time surviving decades, let alone (pre-)historic timescales -(\protect\hyperlink{ref-lindholstLongTerm2010}{Lindholst, 2010}). -Protein and bacterial abundances are also impacted by differential -degradation (\protect\hyperlink{ref-hendyAncientProtein2021}{Hendy, -2021}). This makes it hard to determine whether the quantities of -molecules are an accurate reflection of the quantities during life, -which in turn complicates interpretations we make on the health and diet -of individuals. - -\hypertarget{contamination-and-lab-processing}{% +(\citeproc{ref-lindholstLongTerm2010}{Lindholst, 2010}). Protein and +bacterial abundances are also impacted by differential degradation +(\citeproc{ref-hendyAncientProtein2021}{Hendy, 2021}). This makes it +hard to determine whether the quantities of molecules are an accurate +reflection of the quantities during life, which in turn complicates +interpretations we make on the health and diet of individuals. + \subsection{Contamination and lab -processing}\label{contamination-and-lab-processing}} +processing}\label{contamination-and-lab-processing} It has been shown that dental calculus preserves well, and that little external contamination enters the calculus after burial -(\protect\hyperlink{ref-warinnerPathogensHost2014}{Warinner, Rodrigues, -et al., 2014}). Dental calculus is a robust material. After all, it's -made from a lot of the same material as bone. It can clearly provide -good protection to the microremains and various molecules trapped -inside, and survive thousands of years -(\protect\hyperlink{ref-yatesOralMicrobiome2021}{Fellows Yates et al., -2021}; \protect\hyperlink{ref-henryNeanderthalCalculus2014}{Henry et -al., 2014}). It is, however, not impenetrable. In fact, it can be quite -porous (\protect\hyperlink{ref-friskoppComparativeScanning1980}{Friskopp -\& Hammarström, 1980}; -\protect\hyperlink{ref-powerSynchrotronRadiationbased2022}{Robert C. -Power et al., 2022}). This means it's important to consider what may -have been originally trapped within the calculus during life, and what -could have entered post-mortem. The proportions of original to exogenous -material may also change with time, depending on the physicochemical -properties of the molecules. It seems that small hydrophilic molecules -are more often lost from dental calculus than larger hydrophobic -molecules, suggesting postmortem movement of water through the substrate -(\protect\hyperlink{ref-velskoDentalCalculus2017}{Velsko et al., 2017}). -In addition, these molecules may also be present as contamination in -labs or in the burial environment. I cannot stress enough how important -it is to collect control samples from surrounding soil and to replicate +(\citeproc{ref-warinnerPathogensHost2014}{Warinner, Rodrigues, et al., +2014}). Dental calculus is a robust material. After all, it's made from +a lot of the same material as bone. It can clearly provide good +protection to the microremains and various molecules trapped inside, and +survive thousands of years +(\citeproc{ref-yatesOralMicrobiome2021}{Fellows Yates et al., 2021}; +\citeproc{ref-henryNeanderthalCalculus2014}{Henry et al., 2014}). It is, +however, not impenetrable. In fact, it can be quite porous +(\citeproc{ref-friskoppComparativeScanning1980}{Friskopp \& Hammarström, +1980}; \citeproc{ref-powerSynchrotronRadiationbased2022}{Robert C. Power +et al., 2022}). This means it's important to consider what may have been +originally trapped within the calculus during life, and what could have +entered post-mortem. The proportions of original to exogenous material +may also change with time, depending on the physicochemical properties +of the molecules. It seems that small hydrophilic molecules are more +often lost from dental calculus than larger hydrophobic molecules, +suggesting postmortem movement of water through the substrate +(\citeproc{ref-velskoDentalCalculus2017}{Velsko et al., 2017}). In +addition, these molecules may also be present as contamination in labs +or in the burial environment. I cannot stress enough how important it is +to collect control samples from surrounding soil and to replicate findings in separate labs, with clear identification of potential contaminants -(\protect\hyperlink{ref-crowtherDocumentingContamination2014}{Crowther -et al., 2014}). - -In the study from \protect\hyperlink{mb11CalculusPilot}{\textbf{Chapter -5}} (\protect\hyperlink{ref-bartholdyMultiproxyAnalysis2023}{Bartholdy, -Hasselstrøm, et al., 2023}), we detected various compounds in dental -calculus using UHPLC-MS/MS, including salicylic acid, a phytohormone -from willow trees (\emph{Salix alba}, for example) with medicinal -properties. Willow bark has long been known for its medicinal -properties, and is present in many common foods. It is therefore not -surprising that we found it in the dental calculus of people from the -19th century. We also know, however, that salicylic acid is abundant and -very mobile in soil. With this in mind, how do we interpret our -findings? There are currently no standards for authenticating results -from GC/LC-MS/MS analyses on archaeological samples. Research in aDNA -uses, among other things, damage patterns from the sequences to -determine whether a sequence is old or not, and there are many tools -available to accomplish this, such as decontam -(\protect\hyperlink{ref-Rdecontam}{Davis et al., 2018}), PMD tools -(\protect\hyperlink{ref-skoglundSeparatingEndogenous2014}{Skoglund et -al., 2014}), HOPS -(\protect\hyperlink{ref-hublerHOPSAutomated2019}{Hübler et al., 2019}), -and cuperdec (\protect\hyperlink{ref-yatesOralMicrobiome2021}{Fellows +(\citeproc{ref-crowtherDocumentingContamination2014}{Crowther et al., +2014}). + +In the study from \hyperref[mb11CalculusPilot]{\textbf{Chapter 5}} +(\citeproc{ref-bartholdyMultiproxyAnalysis2023}{Bartholdy, Hasselstrøm, +et al., 2023}), we detected various compounds in dental calculus using +UHPLC-MS/MS, including salicylic acid, a phytohormone from willow trees +(\emph{Salix alba}, for example) with medicinal properties. Willow bark +has long been known for its medicinal properties, and is present in many +common foods. It is therefore not surprising that we found it in the +dental calculus of people from the 19th century. We also know, however, +that salicylic acid is abundant and very mobile in soil. With this in +mind, how do we interpret our findings? There are currently no standards +for authenticating results from GC/LC-MS/MS analyses on archaeological +samples. Research in aDNA uses, among other things, damage patterns from +the sequences to determine whether a sequence is old or not, and there +are many tools available to accomplish this, such as decontam +(\citeproc{ref-Rdecontam}{Davis et al., 2018}), PMD tools +(\citeproc{ref-skoglundSeparatingEndogenous2014}{Skoglund et al., +2014}), HOPS (\citeproc{ref-hublerHOPSAutomated2019}{Hübler et al., +2019}), and cuperdec (\citeproc{ref-yatesOralMicrobiome2021}{Fellows Yates et al., 2021}). Similarly paleoproteomic research can look at markers of degradation, such as deamidation -(\protect\hyperlink{ref-ramsoeDeamiDATESitespecific2020}{Ramsøe et al., -2020}). We attempted to provide a method to authenticate our finds by -plotting the quantity of compounds in three washes and comparing these -quantities with the quantity extracted from the calculus itself. We -expect to see a decrease in quantities over the three washes as surface -contaminants are removed, and a subsequent increase in quantity as the -calculus is dissolved and the compounds that were embedded within the -calculus are extracted -(\protect\hyperlink{ref-bartholdyMultiproxyAnalysis2023}{Bartholdy, +(\citeproc{ref-ramsoeDeamiDATESitespecific2020}{Ramsøe et al., 2020}). +We attempted to provide a method to authenticate our finds by plotting +the quantity of compounds in three washes and comparing these quantities +with the quantity extracted from the calculus itself. We expect to see a +decrease in quantities over the three washes as surface contaminants are +removed, and a subsequent increase in quantity as the calculus is +dissolved and the compounds that were embedded within the calculus are +extracted (\citeproc{ref-bartholdyMultiproxyAnalysis2023}{Bartholdy, Hasselstrøm, et al., 2023}). This assumes that the embedded compounds were incorporated during life, and does not in any way verify that the molecules are actually old. So what does this mean for our @@ -8227,65 +8002,60 @@ \subsection{Contamination and lab been present in the past. These included MDMA, Fentanyl, Amphetamine, and others. We detected cocaine in nine individuals. Cocaine is not a modern compound, since it has been used for millennia in the Americas -(\protect\hyperlink{ref-abucaCocaTrade2019}{Abduca, 2019}; -\protect\hyperlink{ref-indriatiCocaPrehistoric2001}{Indriati \& -Buikstra, 2001}; -\protect\hyperlink{ref-springfieldCocaineMetabolites1993}{Springfield et -al., 1993}), however, it didn't become known to Europeans until -colonisation in the late 15th century, and was only widely adopted in -the late 19th century after cocaine was isolated by Albert Niemann -(\protect\hyperlink{ref-abucaCocaTrade2019}{Abduca, 2019}; -\protect\hyperlink{ref-marianiCoca1886}{Company, 1886}). This -complicated things. Cocaine is an alkaloid found naturally in the leaves -of various species of coca plants. While we wouldn't expect a rural -population from 19th century Netherlands to have access to coca leaves, -it wasn't impossible to imagine. It was commonly observed to prevent -fatigue and suppress appetite, potentially useful to farmers. There was -some Dutch presence in South America with the Dutch West Indies, and -they even established the \emph{Nederlandsche Cocainefabriek} in -Amsterdam in 1900 (\protect\hyperlink{ref-bosHistoryLicit2006}{Bos, -2006}). Given the possible impact of such a finding, we analysed new -samples from the same individuals in a separate lab on different -equipment. We were unable to detect cocaine in any of the replicated -individuals, and it was probably a case of some sort of lab -contamination that managed to slip past our blanks -(\protect\hyperlink{ref-bartholdyMultiproxyAnalysis2023}{Bartholdy, +(\citeproc{ref-abucaCocaTrade2019}{Abduca, 2019}; +\citeproc{ref-indriatiCocaPrehistoric2001}{Indriati \& Buikstra, 2001}; +\citeproc{ref-springfieldCocaineMetabolites1993}{Springfield et al., +1993}), however, it didn't become known to Europeans until colonisation +in the late 15th century, and was only widely adopted in the late 19th +century after cocaine was isolated by Albert Niemann +(\citeproc{ref-abucaCocaTrade2019}{Abduca, 2019}; +\citeproc{ref-marianiCoca1886}{Company, 1886}). This complicated things. +Cocaine is an alkaloid found naturally in the leaves of various species +of coca plants. While we wouldn't expect a rural population from 19th +century Netherlands to have access to coca leaves, it wasn't impossible +to imagine. It was commonly observed to prevent fatigue and suppress +appetite, potentially useful to farmers. There was some Dutch presence +in South America with the Dutch West Indies, and they even established +the \emph{Nederlandsche Cocainefabriek} in Amsterdam in 1900 +(\citeproc{ref-bosHistoryLicit2006}{Bos, 2006}). Given the possible +impact of such a finding, we analysed new samples from the same +individuals in a separate lab on different equipment. We were unable to +detect cocaine in any of the replicated individuals, and it was probably +a case of some sort of lab contamination that managed to slip past our +blanks (\citeproc{ref-bartholdyMultiproxyAnalysis2023}{Bartholdy, Hasselstrøm, et al., 2023}). Upon further research, we were unable to find historic evidence of coca leaf-use in Europe for anything other than study, and the only small-scale botanical imports were recorded prior to the late 19th century (the most recent individuals included in our study were buried in the 1860s). Coca leaves are also susceptible to decay during travel and may not have been viable for their intended use -once they arrived in Europe -(\protect\hyperlink{ref-abucaCocaTrade2019}{Abduca, 2019}). +once they arrived in Europe (\citeproc{ref-abucaCocaTrade2019}{Abduca, +2019}). Contamination is widely recognised as a risk in all aspects of archaeological research, including paleobotany -(\protect\hyperlink{ref-crowtherDocumentingContamination2014}{Crowther -et al., 2014}) and aDNA -(\protect\hyperlink{ref-cooperAncientDNA2000}{Cooper \& Poinar, 2000}; -\protect\hyperlink{ref-gilbertBiochemicalPhysical2005}{Gilbert, Rudbeck, -et al., 2005}; -\protect\hyperlink{ref-gilbertAssessingAncient2005}{Gilbert, Bandelt, et -al., 2005}; \protect\hyperlink{ref-knappSettingStage2012}{Knapp et al., -2012}; \protect\hyperlink{ref-llamasFieldLaboratory2017}{Llamas et al., +(\citeproc{ref-crowtherDocumentingContamination2014}{Crowther et al., +2014}) and aDNA (\citeproc{ref-cooperAncientDNA2000}{Cooper \& Poinar, +2000}; \citeproc{ref-gilbertBiochemicalPhysical2005}{Gilbert, Rudbeck, +et al., 2005}; \citeproc{ref-gilbertAssessingAncient2005}{Gilbert, +Bandelt, et al., 2005}; \citeproc{ref-knappSettingStage2012}{Knapp et +al., 2012}; \citeproc{ref-llamasFieldLaboratory2017}{Llamas et al., 2017}), often because of bold claims made in the past (no specifics will be mentioned here). Protocols for dental calculus sampling include various steps for decontaminating dental calculus, and range from brushing the surface to UV-radiation and sonication. However, the use of liquids for decontamination may be problematic when there are plans to -do biomolecular analyses -(\protect\hyperlink{ref-velskoDentalCalculus2017}{Velsko et al., 2017}). -Sodium hydroxide (NaOH) has been suggested as a better decontamination -solution based on testing on synthetic precipitates of calcium phosphate -(the principal component of dental calculus) -(\protect\hyperlink{ref-sotoCharacterizationDecontamination2019}{Soto et -al., 2019}). It's not clear how valid this approach is since the -synthetic dental calculus was grown without bacteria, and they're -generally responsible for the channels (supplying nutrients) in dental -calculus that would allow a decontaminating agent to seep into the -calculus and affect the microremains. Nevertheless, it is a step in the -right direction. +do biomolecular analyses (\citeproc{ref-velskoDentalCalculus2017}{Velsko +et al., 2017}). Sodium hydroxide (NaOH) has been suggested as a better +decontamination solution based on testing on synthetic precipitates of +calcium phosphate (the principal component of dental calculus) +(\citeproc{ref-sotoCharacterizationDecontamination2019}{Soto et al., +2019}). It's not clear how valid this approach is since the synthetic +dental calculus was grown without bacteria, and they're generally +responsible for the channels (supplying nutrients) in dental calculus +that would allow a decontaminating agent to seep into the calculus and +affect the microremains. Nevertheless, it is a step in the right +direction. After decontamination, the dental calculus is dissolved to extract the remains trapped inside. The exact method for dissolving dental calculus @@ -8294,28 +8064,26 @@ \subsection{Contamination and lab hydrochloric acid (HCl) and ethylenediaminetetratetraacetic acid (EDTA). HCl has long been the preferred method for decalcification of dental calculus for extraction of plant microremains -(\protect\hyperlink{ref-hardyDentalCalculus2016}{Hardy et al., 2016}, -\protect\hyperlink{ref-hardyRecoveringInformation2018}{2018}). However, -there was no apparent testing on the original use of HCl -(\protect\hyperlink{ref-middletonImprovedMethod1990}{Middleton, 1990}), -which was originally developed for extraction of phytoliths, which are -very resistant to chemical degradation -(\protect\hyperlink{ref-cabanesPhytolithAnalysis2020}{Cabanes, 2020}). -It has since become clear that dental calculus is also a rich source of -starch granules (\protect\hyperlink{ref-henryCalculusSyria2008}{Henry \& -Piperno, 2008}; \protect\hyperlink{ref-cummingsMayanCalculus1997}{Scott -Cummings \& Magennis, 1997}), though it's not entirely clear how -resistant starch granules are to degradation by acids. It was briefly -mentioned in Henry \& Piperno -(\protect\hyperlink{ref-henryCalculusSyria2008}{2008}) that weak -solutions of HCl would not affect starch granules, but more recent -research suggests that EDTA can recover more material from -archaeological dental calculus than HCl -(\protect\hyperlink{ref-trompEDTACalculus2017}{Tromp et al., 2017}) and -cause less damage to the starches -(\protect\hyperlink{ref-lemoyneCalculusPretreatments2021}{Le Moyne \& -Crowther, 2021}). Validation of methods on archaeological material is -difficult since we don't really know the starting point. +(\citeproc{ref-hardyDentalCalculus2016}{Hardy et al., 2016}, +\citeproc{ref-hardyRecoveringInformation2018}{2018}). However, there was +no apparent testing on the original use of HCl +(\citeproc{ref-middletonImprovedMethod1990}{Middleton, 1990}), which was +originally developed for extraction of phytoliths, which are very +resistant to chemical degradation +(\citeproc{ref-cabanesPhytolithAnalysis2020}{Cabanes, 2020}). It has +since become clear that dental calculus is also a rich source of starch +granules (\citeproc{ref-henryCalculusSyria2008}{Henry \& Piperno, 2008}; +\citeproc{ref-cummingsMayanCalculus1997}{Scott Cummings \& Magennis, +1997}), though it's not entirely clear how resistant starch granules are +to degradation by acids. It was briefly mentioned in Henry \& Piperno +(\citeproc{ref-henryCalculusSyria2008}{2008}) that weak solutions of HCl +would not affect starch granules, but more recent research suggests that +EDTA can recover more material from archaeological dental calculus than +HCl (\citeproc{ref-trompEDTACalculus2017}{Tromp et al., 2017}) and cause +less damage to the starches +(\citeproc{ref-lemoyneCalculusPretreatments2021}{Le Moyne \& Crowther, +2021}). Validation of methods on archaeological material is difficult +since we don't really know the starting point. One way to explore the external contamination of calculus and how it may affect already present compounds and microremains, is to set up an @@ -8330,10 +8098,9 @@ \subsection{Contamination and lab but no further results were written up because of the aforementioned issue with the protocol, and intrusion by a pandemic. This particular failure motivated me to revise the protocol and properly validate the -grown model dental calculus (see -\protect\hyperlink{byoc-valid}{\textbf{Chapter 3}} and Bartholdy, -Velsko, et al. -(\protect\hyperlink{ref-bartholdyAssessingValidity2023}{2023})). +grown model dental calculus (see \hyperref[byoc-valid]{\textbf{Chapter +3}} and Bartholdy, Velsko, et al. +(\citeproc{ref-bartholdyAssessingValidity2023}{2023})). There is an art, or rather, a knack to decontamination and dissolution of dental calculus. The knack lies in learning how to make sure all @@ -8347,9 +8114,8 @@ \subsection{Contamination and lab and processing methods, and more extensive research on the effects of processing methods needs to be done. -\hypertarget{deliberate-and-efficient-sampling-and-analysis}{% \subsection{Deliberate and efficient sampling and -analysis}\label{deliberate-and-efficient-sampling-and-analysis}} +analysis}\label{deliberate-and-efficient-sampling-and-analysis} Dental calculus has many advantages over other elements from skeletal remains, especially when it comes to dietary reconstructions. With @@ -8362,8 +8128,8 @@ \subsection{Deliberate and efficient sampling and the enamel of teeth, would likely have gotten there after death as the result of environmental contamination. This doesn't mean we can throw caution to the wind and interpret everything in dental calculus as food -(\protect\hyperlink{ref-radiniFoodPathways2017}{Radini et al., 2017}), -but it is one of the likelier scenarios. +(\citeproc{ref-radiniFoodPathways2017}{Radini et al., 2017}), but it is +one of the likelier scenarios. Because the formation of dental calculus is continuous throughout life, the information we extract about diet more likely reflects a broader @@ -8379,13 +8145,13 @@ \subsection{Deliberate and efficient sampling and unlikely to have formed during childhood. Here, enamel represents the most appropriate choice. Enamel is formed during childhood and remains largely unchanged during life -(\protect\hyperlink{ref-hillsonDentalAnthropology1996}{Hillson, 1996}), -so any dietary influences from childhood during the time of enamel -formation, which spans around 28 weeks \emph{in utero} to around 16 -years (\emph{ex utero}, of course) -(\protect\hyperlink{ref-hillsonDentalAnthropology1996}{Hillson, 1996}), -will be present in the enamel of the adult dentition. Similarly, bone -and dentine (depending on where you sample the dentine) have a slower +(\citeproc{ref-hillsonDentalAnthropology1996}{Hillson, 1996}), so any +dietary influences from childhood during the time of enamel formation, +which spans around 28 weeks \emph{in utero} to around 16 years (\emph{ex +utero}, of course) +(\citeproc{ref-hillsonDentalAnthropology1996}{Hillson, 1996}), will be +present in the enamel of the adult dentition. Similarly, bone and +dentine (depending on where you sample the dentine) have a slower turnover, and represent a more stable source of dietary patterns. And since they are generally not exposed to environmental contamination during life (otherwise you're in trouble), they may, in some cases, be @@ -8393,22 +8159,21 @@ \subsection{Deliberate and efficient sampling and from a low resolution, since they can generally ``only'' (highly exaggerated air quotes since it's still incredibly useful) offer insights into very broad dietary trends -(\protect\hyperlink{ref-katzenbergStableIsotope2008}{Katzenberg, 2008}), -whereas methods used on dental calculus can be much more specific, -sometimes even incredibly so -(\protect\hyperlink{ref-hendyProteomicCalculus2018}{Hendy et al., 2018}; -\protect\hyperlink{ref-scottExoticFoods2021}{Scott et al., 2021}). +(\citeproc{ref-katzenbergStableIsotope2008}{Katzenberg, 2008}), whereas +methods used on dental calculus can be much more specific, sometimes +even incredibly so (\citeproc{ref-hendyProteomicCalculus2018}{Hendy et +al., 2018}; \citeproc{ref-scottExoticFoods2021}{Scott et al., 2021}). Others have also noted that the source of collagen protein in dental calculus, the primary target for stable isotope analyses, can be difficult to determine given all the microorganisms residing in plaque and dental calculus. This leaves questions about what the isotopes are actually saying about diet, if anything -(\protect\hyperlink{ref-priceTestingValidity2018}{Price et al., 2018}; -\protect\hyperlink{ref-salazar-garciaDentalCalculus2014}{Salazar-García -et al., 2014}), and may be more related to dental disease or -contamination from other archaeological materials -(\protect\hyperlink{ref-mackiePreservationMetaproteome2017}{Mackie et -al., 2017}). +(\citeproc{ref-priceTestingValidity2018}{Price et al., 2018}; +\citeproc{ref-salazar-garciaDentalCalculus2014}{Salazar-García et al., +2014}), and may be more related to dental disease or contamination from +other archaeological materials +(\citeproc{ref-mackiePreservationMetaproteome2017}{Mackie et al., +2017}). If sheer quantity of DNA is what you're after then there really is no better substance than dental calculus. It is estimated to contain up to @@ -8418,29 +8183,28 @@ \subsection{Deliberate and efficient sampling and though more variable. Dental calculus contains limited host DNA, which may be difficult to capture given the lower relative abundance compared than bacterial DNA, and it can be more fragmented -(\protect\hyperlink{ref-mannDifferentialPreservation2018}{Mann et al., -2018}; \protect\hyperlink{ref-ziesemerGenomeCalculus2018}{Ziesemer et -al., 2018}). This difference is due to the nature of the two substances. -During life, plaque is primarily made up of bacteria, while dentine does -not contain any bacteria. The exception is in some cases of oral -disease, such as periodontitis, where the presence of bacteria is a -byproduct of the disease process. Since dental calculus is also a trap -for food debris, dental calculus can contain plant DNA and food proteins -(\protect\hyperlink{ref-fagernasMicrobialBiogeography2022}{Fagernäs et -al., 2022}; \protect\hyperlink{ref-hendyProteomicCalculus2018}{Hendy et -al., 2018}; \protect\hyperlink{ref-scottExoticFoods2021}{Scott et al., -2021}; \protect\hyperlink{ref-warinnerEvidenceMilk2014}{Warinner, Hendy, -et al., 2014}). The problem with detecting dietary DNA in dental -calculus is the same as for human host DNA; there is very little of it, -and it may be highly damaged. This causes problems when trying to -identify the source of the DNA. If the DNA sequences are not long enough -to distinguish between multiple related sources (e.g.~mammals), then -interpretations can be made difficult -(\protect\hyperlink{ref-mannHaveSomething2023}{Mann et al., 2023}). That -being said, as our techniques develop and we accumulate more complete -reference databases that allow us to make more robust identifications on -smaller DNA fragments, dental calculus can become even more of a -treasure trove of information than it is already. +(\citeproc{ref-mannDifferentialPreservation2018}{Mann et al., 2018}; +\citeproc{ref-ziesemerGenomeCalculus2018}{Ziesemer et al., 2018}). This +difference is due to the nature of the two substances. During life, +plaque is primarily made up of bacteria, while dentine does not contain +any bacteria. The exception is in some cases of oral disease, such as +periodontitis, where the presence of bacteria is a byproduct of the +disease process. Since dental calculus is also a trap for food debris, +dental calculus can contain plant DNA and food proteins +(\citeproc{ref-fagernasMicrobialBiogeography2022}{Fagernäs et al., +2022}; \citeproc{ref-hendyProteomicCalculus2018}{Hendy et al., 2018}; +\citeproc{ref-scottExoticFoods2021}{Scott et al., 2021}; +\citeproc{ref-warinnerEvidenceMilk2014}{Warinner, Hendy, et al., 2014}). +The problem with detecting dietary DNA in dental calculus is the same as +for human host DNA; there is very little of it, and it may be highly +damaged. This causes problems when trying to identify the source of the +DNA. If the DNA sequences are not long enough to distinguish between +multiple related sources (e.g.~mammals), then interpretations can be +made difficult (\citeproc{ref-mannHaveSomething2023}{Mann et al., +2023}). That being said, as our techniques develop and we accumulate +more complete reference databases that allow us to make more robust +identifications on smaller DNA fragments, dental calculus can become +even more of a treasure trove of information than it is already. Detecting metabolites in dental calculus has its own set of considerations. Until now, the most common separation method for @@ -8453,18 +8217,17 @@ \subsection{Deliberate and efficient sampling and degradation of the compounds. Some metabolites, particularly alkaloids, are less volatile, and are therefore not easily vaporised and detected following derivatization -(\protect\hyperlink{ref-zimmermanBiomolecularArchaeology2023}{Zimmerman -\& Tushingham, 2023}). This is not a great feature when looking for +(\citeproc{ref-zimmermanBiomolecularArchaeology2023}{Zimmerman \& +Tushingham, 2023}). This is not a great feature when looking for potentially interesting dietary and non-dietary alkaloids. Methods using liquid chromatography coupled with mass-spectrometry (LC-MS) use lower temperatures and are able to detect these compounds directly, without the step of derivatization -(\protect\hyperlink{ref-rustichelliSimultaneousSeparation1996}{Rustichelli -et al., 1996}; -\protect\hyperlink{ref-sorensenSensitiveDetermination2017}{Sørensen \& -Hasselstrøm, 2017}). This reduces sample preparation time, but comes at -a higher cost, financially for instrumentation and operators (a serious -consideration for archaeological budgets). +(\citeproc{ref-rustichelliSimultaneousSeparation1996}{Rustichelli et +al., 1996}; \citeproc{ref-sorensenSensitiveDetermination2017}{Sørensen +\& Hasselstrøm, 2017}). This reduces sample preparation time, but comes +at a higher cost, financially for instrumentation and operators (a +serious consideration for archaeological budgets). If dental calculus is the best substance for the particular research goal, then it's important to maximise the information extracted from the @@ -8473,16 +8236,16 @@ \subsection{Deliberate and efficient sampling and there have been attempts to unify extraction protocols for different analyses to save on time and minimise destructive sampling, such as a combined extraction protocol for aDNA and proteomics -(\protect\hyperlink{ref-fagernasUnifiedProtocol2020}{Fagernäs et al., -2020}) and aDNA and plant microremains -(\protect\hyperlink{ref-modiCalculusMethodologies2020}{Modi et al., -2020}). The sequence of analyses should also be considered, as some +(\citeproc{ref-fagernasUnifiedProtocol2020}{Fagernäs et al., 2020}) and +aDNA and plant microremains +(\citeproc{ref-modiCalculusMethodologies2020}{Modi et al., 2020}). The +sequence of analyses should also be considered, as some `non-destructive' techniques may cause invisible damage to the samples. For example, high-powered imaging techniques involving radiation may affect the quantity and quality of extracted DNA -(\protect\hyperlink{ref-immelEffectXray2016}{Immel et al., 2016}). We -should continue to explore ways to minimise the amount of material -required to conduct our studies. +(\citeproc{ref-immelEffectXray2016}{Immel et al., 2016}). We should +continue to explore ways to minimise the amount of material required to +conduct our studies. While they are abundant in the past, dental calculus deposits are quite small, ranging from less than one to around a hundred milligrams. It is @@ -8499,8 +8262,7 @@ \subsection{Deliberate and efficient sampling and material, maybe it should. After all, it does contain human DNA, and our microbiomes are unique. -\hypertarget{thoughts-on-the-future}{% -\section{Thoughts on the future}\label{thoughts-on-the-future}} +\section{Thoughts on the future}\label{thoughts-on-the-future} It's hard to imagine the future of dental calculus to be anywhere else than in the hands of biomolecular methods. Further refinement of our @@ -8529,8 +8291,7 @@ \section{Thoughts on the future}\label{thoughts-on-the-future}} mechanical degradation. They were created as an alternative to \emph{Lycopodium} spore tablets in places where you might expect to find indigenous \emph{Lycopodium} spores -(\protect\hyperlink{ref-kitabaBlackCeramic2017}{Kitaba \& Nakagawa, -2017}). +(\citeproc{ref-kitabaBlackCeramic2017}{Kitaba \& Nakagawa, 2017}). The wide range of analytical methods that can provide important insights on dental calculus require a similarly wide range of expertise. @@ -8542,9 +8303,8 @@ \section{Thoughts on the future}\label{thoughts-on-the-future}} are growing; as they should. Paleoproteomics has already shown that it's possible to detect very specific information about dietary molecules present in dental calculus, down to the type of food, its source, and -method of processing -(\protect\hyperlink{ref-hendyProteomicCalculus2018}{Hendy et al., -2018}). It also has the advantage over DNA in that proteins seem to +method of processing (\citeproc{ref-hendyProteomicCalculus2018}{Hendy et +al., 2018}). It also has the advantage over DNA in that proteins seem to preserve for longer. Further development of reference databases and analytical methods is continuously improving the fields of paleoproteomics and (oral) metagenomics by increasing quantity of, and @@ -8555,18 +8315,18 @@ \section{Thoughts on the future}\label{thoughts-on-the-future}} Another area which may lead to exciting discoveries is accessing the layered structure of dental calculus through high-powered imaging techniques (e.g. -\protect\hyperlink{ref-powerSynchrotronRadiationbased2022}{Robert C. -Power et al., 2022}). We know that the formation of a biofilm is -sequential, with new layers of biofilm continuously forming on the -already established layers. Sequential analysis of dental calculus -layers might therefore be able to determine a sequence of incorporation -events for dietary material in dental calculus. However, since we can't -yet access information about the age of occurrence of the seemingly -haphazard mineralisation events in dental plaque, it is difficult to -envision a scenario where we can talk about dietary activities and the -age of individuals. Until then, though, it will still be beneficial to -be able to generate a sequence of deposition events and talk about the -dietary material found in each layer. +\citeproc{ref-powerSynchrotronRadiationbased2022}{Robert C. Power et +al., 2022}). We know that the formation of a biofilm is sequential, with +new layers of biofilm continuously forming on the already established +layers. Sequential analysis of dental calculus layers might therefore be +able to determine a sequence of incorporation events for dietary +material in dental calculus. However, since we can't yet access +information about the age of occurrence of the seemingly haphazard +mineralisation events in dental plaque, it is difficult to envision a +scenario where we can talk about dietary activities and the age of +individuals. Until then, though, it will still be beneficial to be able +to generate a sequence of deposition events and talk about the dietary +material found in each layer. Amidst a scientific revolution, it's important to remember that there are things that can be said about dental calculus without using @@ -8574,25 +8334,24 @@ \section{Thoughts on the future}\label{thoughts-on-the-future}} calculus deposits is cheaper and requires no specialised equipment. The presence of dental calculus and the size of the deposit can be meaningful. For example, Yaussy \& DeWitte -(\protect\hyperlink{ref-yaussyCalculusSurvivorship2019}{2019}) found a -decreased survivorship in individuals with dental calculus formations. -Past populations are also a well-suited target to explore the -relationship between dental diseases, such as dental calculus and -periodontitis; and between dental diseases and diet, since oral hygiene -interventions were less widespread in the past. Therefore, it's crucial -to record the deposit \emph{in situ} before proceeding with destructive -sampling. This means taking photos and scoring the deposit using -existing methods, such as -(\protect\hyperlink{ref-brothwellDiggingBones1981}{Brothwell, 1981}), -and recording detailed information allowing researchers to filter out +(\citeproc{ref-yaussyCalculusSurvivorship2019}{2019}) found a decreased +survivorship in individuals with dental calculus formations. Past +populations are also a well-suited target to explore the relationship +between dental diseases, such as dental calculus and periodontitis; and +between dental diseases and diet, since oral hygiene interventions were +less widespread in the past. Therefore, it's crucial to record the +deposit \emph{in situ} before proceeding with destructive sampling. This +means taking photos and scoring the deposit using existing methods, such +as (\citeproc{ref-brothwellDiggingBones1981}{Brothwell, 1981}), and +recording detailed information allowing researchers to filter out unnecessary information in downstream analyses rather than missing out on something that was never recorded. Ideally, each surface of the tooth should be scored separately to retain the most information for future analyses, and allows calculating a dental calculus index -(\protect\hyperlink{ref-greeneQuantifyingCalculus2005}{Greene et al., -2005}). Calculating an index with calculus scored on multiple surfaces -of the teeth allows us to reveal more patterns related to the presence -and absence of dental calculus, such as uneven distribution within the +(\citeproc{ref-greeneQuantifyingCalculus2005}{Greene et al., 2005}). +Calculating an index with calculus scored on multiple surfaces of the +teeth allows us to reveal more patterns related to the presence and +absence of dental calculus, such as uneven distribution within the dental arcade, allowing more fine-grained comparisons between populations and within different groups in the same population. No analytical method should be considered the be-all and end-all of our @@ -8612,14 +8371,14 @@ \section{Thoughts on the future}\label{thoughts-on-the-future}} not because the results were deemed ``positive'' or ``novel'', but because their methodology was sound and their results contribute to a robust, scientific foundation of knowledge -(\protect\hyperlink{ref-chambersInsteadPlaying2014}{Chambers et al., -2014}; \protect\hyperlink{ref-nosekRegisteredReports2014}{Nosek \& -Lakens, 2014}). Opening our methods will facilitate faster improvements -to existing protocols, as well as open up opportunities for researchers -in smaller labs. Here I'm not talking about vague, cryptic methods -sections in papers, but detailed protocols accessible to anyone with the -necessary materials and equipment. Platforms like protocols.io are a -great solution (e.g.~ +(\citeproc{ref-chambersInsteadPlaying2014}{Chambers et al., 2014}; +\citeproc{ref-nosekRegisteredReports2014}{Nosek \& Lakens, 2014}). +Opening our methods will facilitate faster improvements to existing +protocols, as well as open up opportunities for researchers in smaller +labs. Here I'm not talking about vague, cryptic methods sections in +papers, but detailed protocols accessible to anyone with the necessary +materials and equipment. Platforms like protocols.io are a great +solution (e.g.~ \href{dx.doi.org/10.17504/protocols.io.bvt9n6r6}{10.17504/protocols.io.bvt9n6r6} and \href{dx.doi.org/10.17504/protocols.io.dm6gpj9rdgzp/v1}{10.17504/protocols.io.dm6gpj9rdgzp/v1}). @@ -8627,16 +8386,16 @@ \section{Thoughts on the future}\label{thoughts-on-the-future}} incorporate multiple proxies in research studies, as this will no longer be limited to those with access to enough material and range of materials to conduct large-scale analyses (such as -\protect\hyperlink{ref-yatesOralMicrobiome2021}{Fellows Yates et al., -2021}). Ensuring that we publish our data in a manner that is Findable, +\citeproc{ref-yatesOralMicrobiome2021}{Fellows Yates et al., 2021}). +Ensuring that we publish our data in a manner that is Findable, Accessible, Interoperable, and Reusable (FAIR) will promote reproducibility and replication, two crucial aspects of scientific -research (\protect\hyperlink{ref-wilkinsonFAIRGuiding2016}{Wilkinson et -al., 2016}). Creating communities that can promote these practices -within specific fields and subfields can be effective in creating -relevant standards and fostering an environment that promotes equitable -research practices. This has been realised by the SPAAM community and -Open Phytoliths with +research (\citeproc{ref-wilkinsonFAIRGuiding2016}{Wilkinson et al., +2016}). Creating communities that can promote these practices within +specific fields and subfields can be effective in creating relevant +standards and fostering an environment that promotes equitable research +practices. This has been realised by the SPAAM community and Open +Phytoliths with \href{https://zenodo.org/record/7789069}{AncientMetagenomeDir} and the \href{https://zenodo.org/record/6435441}{FAIR Phytoliths Project}, respectively. Unfortunately, many of these initiatives fall on @@ -8648,8 +8407,7 @@ \section{Thoughts on the future}\label{thoughts-on-the-future}} widespread adoption of open practices and disproportionately impacting young scholars and early career researchers. -\hypertarget{concluding-remarks}{% -\section{Concluding remarks}\label{concluding-remarks}} +\section{Concluding remarks}\label{concluding-remarks} In my dissertation I set out to put dental calculus under the microscope, scrutinising what we know about dental calculus, what we @@ -8718,138 +8476,131 @@ \section{Concluding remarks}\label{concluding-remarks}} fundamental assumptions that we have been making without actually going through the trouble of testing them. After all, ``You can't possibly be a scientist if you mind people thinking that you're a fool'' -(\protect\hyperlink{ref-adamsLongThanks2002}{Adams, 2002b}). +(\citeproc{ref-adamsLongThanks2002}{Adams, 2002b}). -\hypertarget{references-cited-5}{% -\section*{References cited}\label{references-cited-5}} +\section*{References cited}\label{references-cited-5} \addcontentsline{toc}{section}{References cited} \markright{References cited} -\leavevmode\vadjust pre{\hypertarget{appendices}{}}% -\cleardoublepage -\phantomsection -\addcontentsline{toc}{part}{Appendices} -\appendix - -\hypertarget{refs-6}{} +\phantomsection\label{refs-6} \begin{CSLReferences}{1}{0} -\leavevmode\vadjust pre{\hypertarget{ref-abucaCocaTrade2019}{}}% +\bibitem[\citeproctext]{ref-abucaCocaTrade2019} Abduca, R. (2019). Coca leaf transfers to {Europe}. {Effects} on the consumption of coca in {North-western Argentina}. In M. Kaller \& F. Jacob (Eds.), \emph{Transatlantic {Trade} and {Global Cultural Transfers Since} 1492: {More} than {Commodities}}. {Routledge}. -\leavevmode\vadjust pre{\hypertarget{ref-adamsLifeUniverse2002}{}}% +\bibitem[\citeproctext]{ref-adamsLifeUniverse2002} Adams, D. (2002a). \emph{Life, the {Universe} and {Everything}}. {Picador}. -\leavevmode\vadjust pre{\hypertarget{ref-adamsLongThanks2002}{}}% +\bibitem[\citeproctext]{ref-adamsLongThanks2002} Adams, D. (2002b). \emph{So {Long}, and {Thanks} for {All} the {Fish}}. {Picador}. -\leavevmode\vadjust pre{\hypertarget{ref-adamsHitchhikersGuide2002}{}}% +\bibitem[\citeproctext]{ref-adamsHitchhikersGuide2002} Adams, D. (2002c). \emph{The {Hitchhiker}'s {Guide} to the {Galaxy}}. {Picador}. -\leavevmode\vadjust pre{\hypertarget{ref-ammannZurichBiofilm2012}{}}% +\bibitem[\citeproctext]{ref-ammannZurichBiofilm2012} Ammann, T. W., Gmür, R., \& Thurnheer, T. (2012). Advancement of the 10-species subgingival {Zurich} biofilm model by examining different nutritional conditions and defining the structure of the in vitro biofilms. \emph{BMC Microbiology}, \emph{12}, 227. \url{https://doi.org/10.1186/1471-2180-12-227} -\leavevmode\vadjust pre{\hypertarget{ref-bartholdyMultiproxyAnalysis2023}{}}% +\bibitem[\citeproctext]{ref-bartholdyMultiproxyAnalysis2023} Bartholdy, B. P., Hasselstrøm, J. B., Sørensen, L. K., Casna, M., Hoogland, M., Beemster, H. G., \& Henry, A. G. (2023). \emph{Multiproxy analysis exploring patterns of diet and disease in dental calculus and skeletal remains from a 19th century {Dutch} population}. {Zenodo}. \url{https://doi.org/10.5281/zenodo.7649151} -\leavevmode\vadjust pre{\hypertarget{ref-bartholdyInvestigatingBiases2022}{}}% +\bibitem[\citeproctext]{ref-bartholdyInvestigatingBiases2022} Bartholdy, B. P., \& Henry, A. G. (2022). Investigating {Biases Associated With Dietary Starch Incorporation} and {Retention With} an {Oral Biofilm Model}. \emph{Frontiers in Earth Science}, \emph{10}. \url{https://doi.org/10.3389/feart.2022.886512} -\leavevmode\vadjust pre{\hypertarget{ref-bartholdyAssessingValidity2023}{}}% +\bibitem[\citeproctext]{ref-bartholdyAssessingValidity2023} Bartholdy, B. P., Velsko, I. M., Gur-Arieh, S., Fagernäs, Z., Warinner, C., \& Henry, A. G. (2023). \emph{Assessing the validity of a calcifying oral biofilm model as a suitable proxy for dental calculus} (p. 2023.05.23.541904). {bioRxiv}. \url{https://doi.org/10.1101/2023.05.23.541904} -\leavevmode\vadjust pre{\hypertarget{ref-bjarnsholtVivoBiofilm2013}{}}% +\bibitem[\citeproctext]{ref-bjarnsholtVivoBiofilm2013} Bjarnsholt, T., Alhede, M., Alhede, M., Eickhardt-Sørensen, S. R., Moser, C., Kühl, M., Jensen, P. Ø., \& Høiby, N. (2013). The in vivo biofilm. \emph{Trends in Microbiology}, \emph{21}(9), 466--474. \url{https://doi.org/10.1016/j.tim.2013.06.002} -\leavevmode\vadjust pre{\hypertarget{ref-bosHistoryLicit2006}{}}% +\bibitem[\citeproctext]{ref-bosHistoryLicit2006} Bos, A. (2006). The {History} of {Licit Cocaine} in the {Netherlands}. \emph{De Economist}, \emph{154}(4), 581--586. \url{https://doi.org/10.1007/s10645-006-9031-0} -\leavevmode\vadjust pre{\hypertarget{ref-brothwellDiggingBones1981}{}}% +\bibitem[\citeproctext]{ref-brothwellDiggingBones1981} Brothwell, D. (1981). \emph{Digging up {Bones}: {The} excavation, treatment and study of human skeletal remains} (3rd ed.). {British Museum (Natural History)}. -\leavevmode\vadjust pre{\hypertarget{ref-cabanesPhytolithAnalysis2020}{}}% +\bibitem[\citeproctext]{ref-cabanesPhytolithAnalysis2020} Cabanes, D. (2020). Phytolith {Analysis} in {Paleoecology} and {Archaeology}. In A. G. Henry (Ed.), \emph{Handbook for the {Analysis} of {Micro-Particles} in {Archaeological Samples}} (pp. 255--288). {Springer International Publishing}. \url{https://doi.org/10.1007/978-3-030-42622-4_11} -\leavevmode\vadjust pre{\hypertarget{ref-chambersInsteadPlaying2014}{}}% +\bibitem[\citeproctext]{ref-chambersInsteadPlaying2014} Chambers, C. D., Feredoes, E., Muthukumaraswamy, S. D., \& Etchells, P. (2014). Instead of "playing the game" it is time to change the rules: {Registered Reports} at {AIMS Neuroscience} and beyond. \emph{AIMS Neuroscience}, \emph{1}(1), 4--17. \url{https://doi.org/10.3934/Neuroscience.2014.1.4} -\leavevmode\vadjust pre{\hypertarget{ref-marianiCoca1886}{}}% +\bibitem[\citeproctext]{ref-marianiCoca1886} Company, M. (1886). \emph{Coca {Erythroxylon}: {Its Uses} in the -{Treatment} of {Disease}} (4th ed.). {Mariani \& Co.} +{Treatment} of {Disease}} (Fourth). {Mariani \& Co.} -\leavevmode\vadjust pre{\hypertarget{ref-cooperAncientDNA2000}{}}% +\bibitem[\citeproctext]{ref-cooperAncientDNA2000} Cooper, A., \& Poinar, H. N. (2000). Ancient {DNA}: {Do} it right or {Not} at all. \emph{Science}, \emph{289}. \url{https://doi.org/10.1126/science.289.5482.1139b} -\leavevmode\vadjust pre{\hypertarget{ref-cornejo-ramirezStructuralCharacteristics2018}{}}% +\bibitem[\citeproctext]{ref-cornejo-ramirezStructuralCharacteristics2018} Cornejo-Ramírez, Y. I., Martínez-Cruz, O., Del Toro-Sánchez, C. L., Wong-Corral, F. J., Borboa-Flores, J., \& Cinco-Moroyoqui, F. J. (2018). The structural characteristics of starches and their functional properties. \emph{CyTA - Journal of Food}, \emph{16}(1), 1003--1017. \url{https://doi.org/10.1080/19476337.2018.1518343} -\leavevmode\vadjust pre{\hypertarget{ref-crowtherDocumentingContamination2014}{}}% +\bibitem[\citeproctext]{ref-crowtherDocumentingContamination2014} Crowther, A., Haslam, M., Oakden, N., Walde, D., \& Mercader, J. (2014). Documenting contamination in ancient starch laboratories. \emph{Journal of Archaeological Science}, \emph{49}, 90--104. \url{https://doi.org/10.1016/j.jas.2014.04.023} -\leavevmode\vadjust pre{\hypertarget{ref-Rdecontam}{}}% +\bibitem[\citeproctext]{ref-Rdecontam} Davis, N. M., Proctor, D. M., Holmes, S. P., Relman, D. A., \& Callahan, B. J. (2018). Simple statistical identification and removal of contaminant sequences in marker-gene and metagenomics data. \emph{Microbiome}, \emph{6}(1), 226. \url{https://doi.org/10.1186/s40168-018-0605-2} -\leavevmode\vadjust pre{\hypertarget{ref-dawesCircadianRhythms1972}{}}% +\bibitem[\citeproctext]{ref-dawesCircadianRhythms1972} Dawes, C. (1972). \href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1331668}{Circadian rhythms in human salivary flow rate and composition}. \emph{The Journal of Physiology}, \emph{220}(3), 529--545. -\leavevmode\vadjust pre{\hypertarget{ref-doddsHealthBenefits2005}{}}% +\bibitem[\citeproctext]{ref-doddsHealthBenefits2005} Dodds, M. W. J., Johnson, D. A., \& Yeh, C.-K. (2005). Health benefits of saliva: A review. \emph{Journal of Dentistry}, \emph{33}(3), 223--233. \url{https://doi.org/10.1016/j.jdent.2004.10.009} -\leavevmode\vadjust pre{\hypertarget{ref-edlundBiofilmModel2013}{}}% +\bibitem[\citeproctext]{ref-edlundBiofilmModel2013} Edlund, A., Yang, Y., Hall, A. P., Guo, L., Lux, R., He, X., Nelson, K. E., Nealson, K. H., Yooseph, S., Shi, W., \& McLean, J. S. (2013). An in vitrobiofilm model system maintaining a highly reproducible species and @@ -8857,28 +8608,28 @@ \section*{References cited}\label{references-cited-5}} \emph{Microbiome}, \emph{1}(1), 25. \url{https://doi.org/10.1186/2049-2618-1-25} -\leavevmode\vadjust pre{\hypertarget{ref-edlundUncoveringComplex2018}{}}% +\bibitem[\citeproctext]{ref-edlundUncoveringComplex2018} Edlund, A., Yang, Y., Yooseph, S., He, X., Shi, W., \& McLean, J. S. (2018). Uncovering complex microbiome activities via metatranscriptomics during 24 hours of oral biofilm assembly and maturation. \emph{Microbiome}, \emph{6}(1), 217. \url{https://doi.org/10.1186/s40168-018-0591-4} -\leavevmode\vadjust pre{\hypertarget{ref-fagernasUnifiedProtocol2020}{}}% +\bibitem[\citeproctext]{ref-fagernasUnifiedProtocol2020} Fagernäs, Z., García-Collado, M. I., Hendy, J., Hofman, C. A., Speller, C., Velsko, I. M., \& Warinner, C. (2020). A unified protocol for simultaneous extraction of {DNA} and proteins from archaeological dental calculus. \emph{Journal of Archaeological Science}, \emph{118}, 105135. \url{https://doi.org/10.1016/j.jas.2020.105135} -\leavevmode\vadjust pre{\hypertarget{ref-fagernasMicrobialBiogeography2021}{}}% +\bibitem[\citeproctext]{ref-fagernasMicrobialBiogeography2021} Fagernäs, Z., Salazar-García, D. C., Avilés, A., Haber, M., Henry, A., Maurandi, J. L., Ozga, A., Velsko, I. M., \& Warinner, C. (2021). Understanding the microbial biogeography of ancient human dentitions to guide study design and interpretation. \emph{bioRxiv}, 2021.08.16.456492. \url{https://doi.org/10.1101/2021.08.16.456492} -\leavevmode\vadjust pre{\hypertarget{ref-fagernasMicrobialBiogeography2022}{}}% +\bibitem[\citeproctext]{ref-fagernasMicrobialBiogeography2022} Fagernäs, Z., Salazar-García, D. C., Haber Uriarte, M., Avilés Fernández, A., Henry, A. G., Lomba Maurandi, J., Ozga, A. T., Velsko, I. M., \& Warinner, C. (2022). Understanding the microbial biogeography of @@ -8886,7 +8637,7 @@ \section*{References cited}\label{references-cited-5}} \emph{FEMS Microbes}, \emph{3}, xtac006. \url{https://doi.org/10.1093/femsmc/xtac006} -\leavevmode\vadjust pre{\hypertarget{ref-yatesOralMicrobiome2021}{}}% +\bibitem[\citeproctext]{ref-yatesOralMicrobiome2021} Fellows Yates, J. A., Velsko, I. M., Aron, F., Posth, C., Hofman, C. A., Austin, R. M., Parker, C. E., Mann, A. E., Nägele, K., Arthur, K. W., Arthur, J. W., Bauer, C. C., Crevecoeur, I., Cupillard, C., Curtis, M. @@ -8896,31 +8647,31 @@ \section*{References cited}\label{references-cited-5}} National Academy of Sciences}, \emph{118}(20). \url{https://doi.org/10.1073/pnas.2021655118} -\leavevmode\vadjust pre{\hypertarget{ref-flemmingBiofilmMatrix2010}{}}% +\bibitem[\citeproctext]{ref-flemmingBiofilmMatrix2010} Flemming, H.-C., \& Wingender, J. (2010). The biofilm matrix. \emph{Nature Reviews Microbiology}, \emph{8}(9), 623--633. \url{https://doi.org/10.1038/nrmicro2415} -\leavevmode\vadjust pre{\hypertarget{ref-friskoppComparativeScanning1980}{}}% +\bibitem[\citeproctext]{ref-friskoppComparativeScanning1980} Friskopp, J., \& Hammarström, L. (1980). A {Comparative}, {Scanning Electron Microscopic Study} of {Supragingival} and {Subgingival Calculus}. \emph{Journal of Periodontology}, \emph{51}(10), 553--562. \url{https://doi.org/10.1902/jop.1980.51.10.553} -\leavevmode\vadjust pre{\hypertarget{ref-graneroStarchTaphonomy2020}{}}% +\bibitem[\citeproctext]{ref-graneroStarchTaphonomy2020} García-Granero, J. J. (2020). Starch taphonomy, equifinality and the importance of context: {Some} notes on the identification of food processing through starch grain analysis. \emph{Journal of Archaeological Science}, \emph{124}, 105267. \url{https://doi.org/10.1016/j.jas.2020.105267} -\leavevmode\vadjust pre{\hypertarget{ref-gilbertAssessingAncient2005}{}}% +\bibitem[\citeproctext]{ref-gilbertAssessingAncient2005} Gilbert, M. T. P., Bandelt, H.-J., Hofreiter, M., \& Barnes, I. (2005). Assessing ancient {DNA} studies. \emph{Trends in Ecology \& Evolution}, \emph{20}(10), 541--544. \url{https://doi.org/10.1016/j.tree.2005.07.005} -\leavevmode\vadjust pre{\hypertarget{ref-gilbertBiochemicalPhysical2005}{}}% +\bibitem[\citeproctext]{ref-gilbertBiochemicalPhysical2005} Gilbert, M. T. P., Rudbeck, L., Willerslev, E., Hansen, A. J., Smith, C., Penkman, K. E. H., Prangenberg, K., Nielsen-Marsh, C. M., Jans, M. E., Arthur, P., Lynnerup, N., Turner-Walker, G., Biddle, M., @@ -8930,47 +8681,47 @@ \section*{References cited}\label{references-cited-5}} Science}, \emph{32}(5), 785--793. \url{https://doi.org/10.1016/j.jas.2004.12.008} -\leavevmode\vadjust pre{\hypertarget{ref-greeneQuantifyingCalculus2005}{}}% +\bibitem[\citeproctext]{ref-greeneQuantifyingCalculus2005} Greene, T. R., Kuba, C. L., \& Irish, J. D. (2005). Quantifying calculus: {A} suggested new approach for recording an important indicator of diet and dental health. \emph{HOMO - Journal of Comparative Human Biology}, \emph{56}(2), 119--132. \url{https://doi.org/10.1016/j.jchb.2005.02.002} -\leavevmode\vadjust pre{\hypertarget{ref-haaseComparativeGenomics2017}{}}% +\bibitem[\citeproctext]{ref-haaseComparativeGenomics2017} Haase, E. M., Kou, Y., Sabharwal, A., Liao, Y.-C., Lan, T., Lindqvist, C., \& Scannapieco, F. A. (2017). Comparative genomics and evolution of the amylase-binding proteins of oral streptococci. \emph{BMC Microbiology}, \emph{17}(1), 94. \url{https://doi.org/10.1186/s12866-017-1005-7} -\leavevmode\vadjust pre{\hypertarget{ref-hardyRecoveringInformation2018}{}}% +\bibitem[\citeproctext]{ref-hardyRecoveringInformation2018} Hardy, K., Buckley, S., \& Copeland, L. (2018). Pleistocene dental calculus: {Recovering} information on {Paleolithic} food items, medicines, paleoenvironment and microbes. \emph{Evolutionary Anthropology: Issues, News, and Reviews}, \emph{27}(5), 234--246. \url{https://doi.org/10.1002/evan.21718} -\leavevmode\vadjust pre{\hypertarget{ref-hardyDentalCalculus2016}{}}% +\bibitem[\citeproctext]{ref-hardyDentalCalculus2016} Hardy, K., Radini, A., Buckley, S., Sarig, R., Copeland, L., Gopher, A., \& Barkai, R. (2016). Dental calculus reveals potential respiratory irritants and ingestion of essential plant-based nutrients at {Lower Palaeolithic Qesem Cave Israel}. \emph{Quaternary International}, \emph{398}, 129--135. \url{https://doi.org/10.1016/j.quaint.2015.04.033} -\leavevmode\vadjust pre{\hypertarget{ref-hayashizakiSiteSpecific2008}{}}% +\bibitem[\citeproctext]{ref-hayashizakiSiteSpecific2008} Hayashizaki, J., Ban, S., Nakagaki, H., Okumura, A., Yoshii, S., \& Robinson, C. (2008). Site specific mineral composition and microstructure of human supra-gingival dental calculus. \emph{Archives of Oral Biology}, \emph{53}(2), 168--174. \url{https://doi.org/10.1016/j.archoralbio.2007.09.003} -\leavevmode\vadjust pre{\hypertarget{ref-hendyAncientProtein2021}{}}% +\bibitem[\citeproctext]{ref-hendyAncientProtein2021} Hendy, J. (2021). Ancient protein analysis in archaeology. \emph{Science Advances}, \emph{7}(3), eabb9314. \url{https://doi.org/10.1126/sciadv.abb9314} -\leavevmode\vadjust pre{\hypertarget{ref-hendyProteomicCalculus2018}{}}% +\bibitem[\citeproctext]{ref-hendyProteomicCalculus2018} Hendy, J., Warinner, C., Bouwman, A., Collins, M. J., Fiddyment, S., Fischer, R., Hagan, R., Hofman, C. A., Holst, M., Chaves, E., Klaus, L., Larson, G., Mackie, M., McGrath, K., Mundorff, A. Z., Radini, A., Rao, @@ -8979,114 +8730,114 @@ \section*{References cited}\label{references-cited-5}} \emph{Proceedings. Biological Sciences}, \emph{285}(1883), 20180977. \url{https://doi.org/10.1098/rspb.2018.0977} -\leavevmode\vadjust pre{\hypertarget{ref-henryNeanderthalCalculus2014}{}}% +\bibitem[\citeproctext]{ref-henryNeanderthalCalculus2014} Henry, A. G., Brooks, A. S., \& Piperno, D. R. (2014). Plant foods and the dietary ecology of {Neanderthals} and early modern humans. \emph{Journal of Human Evolution}, \emph{69}, 44--54. \url{https://doi.org/10.1016/j.jhevol.2013.12.014} -\leavevmode\vadjust pre{\hypertarget{ref-henryCookingStarch2009}{}}% +\bibitem[\citeproctext]{ref-henryCookingStarch2009} Henry, A. G., Hudson, H. F., \& Piperno, D. R. (2009). Changes in starch grain morphologies from cooking. \emph{Journal of Archaeological Science}, \emph{36}(3), 915--922. \url{https://doi.org/10.1016/j.jas.2008.11.008} -\leavevmode\vadjust pre{\hypertarget{ref-henryCalculusSyria2008}{}}% +\bibitem[\citeproctext]{ref-henryCalculusSyria2008} Henry, A. G., \& Piperno, D. R. (2008). Using plant microfossils from dental calculus to recover human diet: A case study from {Tell} -al-{Raq{ā}}'i, {Syria}. \emph{Journal of Archaeological Science}, +al-{Raqā}'i, {Syria}. \emph{Journal of Archaeological Science}, \emph{35}(7), 1943--1950. \url{https://doi.org/10.1016/j.jas.2007.12.005} -\leavevmode\vadjust pre{\hypertarget{ref-hillsonDentalAnthropology1996}{}}% +\bibitem[\citeproctext]{ref-hillsonDentalAnthropology1996} Hillson, S. (1996). \emph{Dental {Anthropology}}. {Cambridge University Press}. -\leavevmode\vadjust pre{\hypertarget{ref-hublerHOPSAutomated2019}{}}% +\bibitem[\citeproctext]{ref-hublerHOPSAutomated2019} Hübler, R., Key, F. M., Warinner, C., Bos, K. I., Krause, J., \& Herbig, A. (2019). {HOPS}: Automated detection and authentication of pathogen {DNA} in archaeological remains. \emph{Genome Biology}, \emph{20}(1), 280. \url{https://doi.org/10.1186/s13059-019-1903-0} -\leavevmode\vadjust pre{\hypertarget{ref-immelEffectXray2016}{}}% +\bibitem[\citeproctext]{ref-immelEffectXray2016} Immel, A., Le Cabec, A., Bonazzi, M., Herbig, A., Temming, H., Schuenemann, V. J., Bos, K. I., Langbein, F., Harvati, K., Bridault, A., Pion, G., Julien, M.-A., Krotova, O., Conard, N. J., Münzel, S. C., Drucker, D. G., Viola, B., Hublin, J.-J., Tafforeau, P., \& Krause, J. (2016). Effect of {X-ray} irradiation on ancient {DNA} in sub-fossil -bones {\textendash} {Guidelines} for safe {X-ray} imaging. +bones \textendash{} {Guidelines} for safe {X-ray} imaging. \emph{Scientific Reports}, \emph{6}(1), 32969. \url{https://doi.org/10.1038/srep32969} -\leavevmode\vadjust pre{\hypertarget{ref-indriatiCocaPrehistoric2001}{}}% +\bibitem[\citeproctext]{ref-indriatiCocaPrehistoric2001} Indriati, E., \& Buikstra, J. E. (2001). Coca chewing in prehistoric coastal {Peru}: {Dental} evidence. \emph{American Journal of Physical Anthropology}, \emph{114}(3), 242--257. \url{https://doi.org/10.1002/1096-8644(200103)114:3\%3C242::AID-AJPA1023\%3E3.0.CO;2-J} -\leavevmode\vadjust pre{\hypertarget{ref-katzenbergStableIsotope2008}{}}% +\bibitem[\citeproctext]{ref-katzenbergStableIsotope2008} Katzenberg, M. A. (2008). Stable {Isotope Analysis}: {A Tool} for {Studying Past Diet}, {Demography}, and {Life History}. In M. A. Katzenberg \& S. R. Saunders (Eds.), \emph{Biological {Anthropology} of the {Human Skeleton}} (pp. 301--340). {John Wiley and Sons}. -\leavevmode\vadjust pre{\hypertarget{ref-kitabaBlackCeramic2017}{}}% +\bibitem[\citeproctext]{ref-kitabaBlackCeramic2017} Kitaba, I., \& Nakagawa, T. (2017). Black ceramic spheres as marker grains for microfossil analyses, with improved chemical, physical, and optical properties. \emph{Quaternary International}, \emph{455}, 166--169. \url{https://doi.org/10.1016/j.quaint.2017.08.052} -\leavevmode\vadjust pre{\hypertarget{ref-knappSettingStage2012}{}}% +\bibitem[\citeproctext]{ref-knappSettingStage2012} Knapp, M., Clarke, A. C., Horsburgh, K. A., \& Matisoo-Smith, E. A. -(2012). Setting the stage {\textendash} {Building} and working in an +(2012). Setting the stage \textendash{} {Building} and working in an ancient {DNA} laboratory. \emph{Annals of Anatomy - Anatomischer Anzeiger}, \emph{194}(1), 3--6. \url{https://doi.org/10.1016/j.aanat.2011.03.008} -\leavevmode\vadjust pre{\hypertarget{ref-kolenbranderOralMultispecies2010}{}}% +\bibitem[\citeproctext]{ref-kolenbranderOralMultispecies2010} Kolenbrander, P. E., Palmer, R. J., Periasamy, S., \& Jakubovics, N. S. (2010). Oral multispecies biofilm development and the key role of -cell{\textendash}cell distance. \emph{Nature Reviews Microbiology}, +cell\textendash cell distance. \emph{Nature Reviews Microbiology}, \emph{8}(7), 471--480. \url{https://doi.org/10.1038/nrmicro2381} -\leavevmode\vadjust pre{\hypertarget{ref-langejansRemainsDay2010}{}}% +\bibitem[\citeproctext]{ref-langejansRemainsDay2010} Langejans, G. H. J. (2010). Remains of the day-preservation of organic micro-residues on stone tools. \emph{Journal of Archaeological Science}, \emph{37}(5), 971--985. \url{https://doi.org/10.1016/j.jas.2009.11.030} -\leavevmode\vadjust pre{\hypertarget{ref-lemoyneCalculusPretreatments2021}{}}% +\bibitem[\citeproctext]{ref-lemoyneCalculusPretreatments2021} Le Moyne, C., \& Crowther, A. (2021). Effects of chemical pre-treatments on modified starch granules: {Recommendations} for dental calculus decalcification for ancient starch research. \emph{Journal of Archaeological Science: Reports}, \emph{35}, 102762. \url{https://doi.org/10.1016/j.jasrep.2020.102762} -\leavevmode\vadjust pre{\hypertarget{ref-leeOralFluid2011}{}}% +\bibitem[\citeproctext]{ref-leeOralFluid2011} Lee, D., Milman, G., Barnes, A. J., Goodwin, R. S., Hirvonen, J., \& Huestis, M. A. (2011). Oral {Fluid Cannabinoids} in {Chronic}, {Daily Cannabis Smokers} during {Sustained}, {Monitored Abstinence}. \emph{Clinical Chemistry}, \emph{57}(8), 1127--1136. \url{https://doi.org/10.1373/clinchem.2011.164822} -\leavevmode\vadjust pre{\hypertarget{ref-leonardPlantMicroremains2015}{}}% +\bibitem[\citeproctext]{ref-leonardPlantMicroremains2015} Leonard, C., Vashro, L., O'Connell, J. F., \& Henry, A. G. (2015). Plant microremains in dental calculus as a record of plant consumption: {A} test with {Twe} forager-horticulturalists. \emph{Journal of Archaeological Science: Reports}, \emph{2}, 449--457. \url{https://doi.org/10.1016/j.jasrep.2015.03.009} -\leavevmode\vadjust pre{\hypertarget{ref-liInfluenceGrinding2020}{}}% +\bibitem[\citeproctext]{ref-liInfluenceGrinding2020} Li, W., Pagán-Jiménez, J. R., Tsoraki, C., Yao, L., \& Van Gijn, A. (2020). Influence of grinding on the preservation of starch grains from rice. \emph{Archaeometry}, \emph{62}(1), 157--171. \url{https://doi.org/10.1111/arcm.12510} -\leavevmode\vadjust pre{\hypertarget{ref-lindholstLongTerm2010}{}}% +\bibitem[\citeproctext]{ref-lindholstLongTerm2010} Lindholst, C. (2010). Long term stability of cannabis resin and cannabis extracts. \emph{Australian Journal of Forensic Sciences}, \emph{42}(3), 181--190. \url{https://doi.org/10.1080/00450610903258144} -\leavevmode\vadjust pre{\hypertarget{ref-llamasFieldLaboratory2017}{}}% +\bibitem[\citeproctext]{ref-llamasFieldLaboratory2017} Llamas, B., Valverde, G., Fehren-Schmitz, L., Weyrich, L. S., Cooper, A., \& Haak, W. (2017). From the field to the laboratory: {Controlling DNA} contamination in human ancient {DNA} research in the @@ -9094,34 +8845,34 @@ \section*{References cited}\label{references-cited-5}} Archaeological Research}, \emph{3}(1), 1--14. \url{https://doi.org/10.1080/20548923.2016.1258824} -\leavevmode\vadjust pre{\hypertarget{ref-maModelingDiffusion2010}{}}% +\bibitem[\citeproctext]{ref-maModelingDiffusion2010} Ma, R., Liu, J., Jiang, Y., Liu, Z., Tang, Z., Ye, D., Zeng, J., \& Huang, Z. (2010). Modeling of {Diffusion Transport} through {Oral Biofilms} with the {Inverse Problem Method}. \emph{International Journal of Oral Science}, \emph{2}(4), 190--197. \url{https://doi.org/10.4248/IJOS10075} -\leavevmode\vadjust pre{\hypertarget{ref-maMorphologicalChanges2019}{}}% +\bibitem[\citeproctext]{ref-maMorphologicalChanges2019} Ma, Z., Perry, L., Li, Q., \& Yang, X. (2019). Morphological changes in starch grains after dehusking and grinding with stone tools. \emph{Scientific Reports}, \emph{9}(1), 2355. \url{https://doi.org/10.1038/s41598-019-38758-6} -\leavevmode\vadjust pre{\hypertarget{ref-mackiePreservationMetaproteome2017}{}}% +\bibitem[\citeproctext]{ref-mackiePreservationMetaproteome2017} Mackie, M., Hendy, J., Lowe, A. D., Sperduti, A., Holst, M., Collins, M. J., \& Speller, C. F. (2017). Preservation of the metaproteome: Variability of protein preservation in ancient dental calculus. \emph{STAR: Science \& Technology of Archaeological Research}, \emph{3}(1), 58--70. \url{https://doi.org/10.1080/20548923.2017.1361629} -\leavevmode\vadjust pre{\hypertarget{ref-mannHaveSomething2023}{}}% +\bibitem[\citeproctext]{ref-mannHaveSomething2023} Mann, A. E., Fellows Yates, J. A., Fagernäs, Z., Austin, R. M., Nelson, E. A., \& Hofman, C. A. (2023). Do {I} have something in my teeth? {The} trouble with genetic analyses of diet from archaeological dental calculus. \emph{Quaternary International}, \emph{653--654}, 33--46. \url{https://doi.org/10.1016/j.quaint.2020.11.019} -\leavevmode\vadjust pre{\hypertarget{ref-mannDifferentialPreservation2018}{}}% +\bibitem[\citeproctext]{ref-mannDifferentialPreservation2018} Mann, A. E., Sabin, S., Ziesemer, K., Vågene, Å. J., Schroeder, H., Ozga, A. T., Sankaranarayanan, K., Hofman, C. A., Fellows Yates, J. A., Salazar-García, D. C., Frohlich, B., Aldenderfer, M., Hoogland, M., @@ -9131,18 +8882,18 @@ \section*{References cited}\label{references-cited-5}} \emph{Scientific Reports}, \emph{8}(1), 9822. \url{https://doi.org/10.1038/s41598-018-28091-9} -\leavevmode\vadjust pre{\hypertarget{ref-marshDentalPlaque2005}{}}% +\bibitem[\citeproctext]{ref-marshDentalPlaque2005} Marsh, P. D. (2005). Dental plaque: Biological significance of a biofilm and community life-style. \emph{Journal of Clinical Periodontology}, \emph{32}(s6), 7--15. \url{https://doi.org/10.1111/j.1600-051X.2005.00790.x} -\leavevmode\vadjust pre{\hypertarget{ref-middletonImprovedMethod1990}{}}% +\bibitem[\citeproctext]{ref-middletonImprovedMethod1990} Middleton, W. D. (1990). An {Improved Method} for {Extraction} of {Opal Phytoliths} from {Tartar Residues} on {Herbivore Teeth}. \emph{Phytolitharien Newsletter}, \emph{6}(3), 2--5. -\leavevmode\vadjust pre{\hypertarget{ref-modiCalculusMethodologies2020}{}}% +\bibitem[\citeproctext]{ref-modiCalculusMethodologies2020} Modi, A., Pisaneschi, L., Zaro, V., Vai, S., Vergata, C., Casalone, E., Caramelli, D., Moggi-Cecchi, J., Mariotti Lippi, M., \& Lari, M. (2020). Combined methodologies for gaining much information from ancient dental @@ -9151,47 +8902,47 @@ \section*{References cited}\label{references-cited-5}} Sciences}, \emph{12}(1), 10. \url{https://doi.org/10.1007/s12520-019-00983-5} -\leavevmode\vadjust pre{\hypertarget{ref-naterHumanAmylase2005}{}}% +\bibitem[\citeproctext]{ref-naterHumanAmylase2005} Nater, U. M., Rohleder, N., Gaab, J., Berger, S., Jud, A., Kirschbaum, C., \& Ehlert, U. (2005). Human salivary alpha-amylase reactivity in a psychosocial stress paradigm. \emph{International Journal of Psychophysiology}, \emph{55}(3), 333--342. \url{https://doi.org/10.1016/j.ijpsycho.2004.09.009} -\leavevmode\vadjust pre{\hypertarget{ref-nessEpidemiologicStudy1977}{}}% +\bibitem[\citeproctext]{ref-nessEpidemiologicStudy1977} Ness, L., Rosekrans, D. L., \& Welford, J. F. (1977). An epidemiologic study of factors affecting extrinsic staining of teeth in an {English} population. \emph{Community Dentistry and Oral Epidemiology}, \emph{5}(1), 55--60. \url{https://doi.org/10.1111/j.1600-0528.1977.tb01617.x} -\leavevmode\vadjust pre{\hypertarget{ref-nikitkovaEffectStarch2012}{}}% +\bibitem[\citeproctext]{ref-nikitkovaEffectStarch2012} Nikitkova, A. E., Haase, E. M., \& Scannapieco, F. A. (2012). Effect of starch and amylase on the expression of amylase-binding protein {A} in {Streptococcus} gordonii. \emph{Molecular Oral Microbiology}, \emph{27}(4), 284--294. \url{https://doi.org/10.1111/j.2041-1014.2012.00644.x} -\leavevmode\vadjust pre{\hypertarget{ref-nikitkovaStarchBiofilms2013}{}}% +\bibitem[\citeproctext]{ref-nikitkovaStarchBiofilms2013} Nikitkova, A. E., Haase, E. M., \& Scannapieco, F. A. (2013). Taking the {Starch} out of {Oral Biofilm Formation}: {Molecular Basis} and {Functional Significance} of {Salivary} {\(\alpha\)}-{Amylase Binding} to {Oral Streptococci}. \emph{Applied and Environmental Microbiology}, \emph{79}(2), 416--423. \url{https://doi.org/10.1128/AEM.02581-12} -\leavevmode\vadjust pre{\hypertarget{ref-nosekRegisteredReports2014}{}}% +\bibitem[\citeproctext]{ref-nosekRegisteredReports2014} Nosek, B. A., \& Lakens, D. (2014). Registered {Reports}. \emph{Social Psychology}, \emph{45}(3), 137--141. \url{https://doi.org/10.1027/1864-9335/a000192} -\leavevmode\vadjust pre{\hypertarget{ref-palmerCoaggregationInteractions2003}{}}% +\bibitem[\citeproctext]{ref-palmerCoaggregationInteractions2003} Palmer, R. J., Gordon, S. M., Cisar, J. O., \& Kolenbrander, P. E. (2003). Coaggregation-{Mediated Interactions} of {Streptococci} and {Actinomyces Detected} in {Initial Human Dental Plaque}. \emph{Journal of Bacteriology}, \emph{185}(11), 3400--3409. \url{https://doi.org/10.1128/JB.185.11.3400-3409.2003} -\leavevmode\vadjust pre{\hypertarget{ref-powerSynchrotronRadiationbased2022}{}}% +\bibitem[\citeproctext]{ref-powerSynchrotronRadiationbased2022} Power, Robert C., Henry, A. G., Moosmann, J., Beckmann, F., Temming, H., Roberts, A., \& Cabec, A. L. (2022). Synchrotron radiation-based phase-contrast microtomography of human dental calculus allows @@ -9199,61 +8950,61 @@ \section*{References cited}\label{references-cited-5}} samples. \emph{Journal of Medical Imaging}, \emph{9}(3), 031505. \url{https://doi.org/10.1117/1.JMI.9.3.031505} -\leavevmode\vadjust pre{\hypertarget{ref-powerChimpCalculus2015}{}}% +\bibitem[\citeproctext]{ref-powerChimpCalculus2015} Power, R. C., Salazar-Garcia, D. C., Wittig, R. M., Freiberg, M., \& Henry, A. G. (2015). Dental calculus evidence of {Tai Forest Chimpanzee} plant consumption and life history transitions. \emph{Scientific Reports}, \emph{5}, 15161. \url{https://doi.org/10.1038/srep15161} -\leavevmode\vadjust pre{\hypertarget{ref-powerRepresentativenessDental2021}{}}% +\bibitem[\citeproctext]{ref-powerRepresentativenessDental2021} Power, Robert C., Wittig, R. M., Stone, J. R., Kupczik, K., \& Schulz-Kornas, E. (2021). The representativeness of the dental calculus -dietary record: Insights from {Ta{ï}} chimpanzee faecal phytoliths. +dietary record: Insights from {Taï} chimpanzee faecal phytoliths. \emph{Archaeological and Anthropological Sciences}, \emph{13}(6), 104. \url{https://doi.org/10.1007/s12520-021-01342-z} -\leavevmode\vadjust pre{\hypertarget{ref-priceTestingValidity2018}{}}% +\bibitem[\citeproctext]{ref-priceTestingValidity2018} Price, S. D. R., Keenleyside, A., \& Schwarcz, H. P. (2018). Testing the validity of stable isotope analyses of dental calculus as a proxy in paleodietary studies. \emph{Journal of Archaeological Science}, \emph{91}, 92--103. \url{https://doi.org/10.1016/j.jas.2018.01.008} -\leavevmode\vadjust pre{\hypertarget{ref-proctorSpatialGradient2018}{}}% +\bibitem[\citeproctext]{ref-proctorSpatialGradient2018} Proctor, D. M., Fukuyama, J. A., Loomer, P. M., Armitage, G. C., Lee, S. A., Davis, N. M., Ryder, M. I., Holmes, S. P., \& Relman, D. A. (2018). A spatial gradient of bacterial diversity in the human oral cavity shaped by salivary flow. \emph{Nature Communications}, \emph{9}(1), 681. \url{https://doi.org/10.1038/s41467-018-02900-1} -\leavevmode\vadjust pre{\hypertarget{ref-radiniFoodPathways2017}{}}% +\bibitem[\citeproctext]{ref-radiniFoodPathways2017} Radini, A., Nikita, E., Buckley, S., Copeland, L., \& Hardy, K. (2017). Beyond food: {The} multiple pathways for inclusion of materials into ancient dental calculus. \emph{American Journal of Physical Anthropology}, \emph{162}, 71--83. \url{https://doi.org/10.1002/ajpa.23147} -\leavevmode\vadjust pre{\hypertarget{ref-ramsoeDeamiDATESitespecific2020}{}}% +\bibitem[\citeproctext]{ref-ramsoeDeamiDATESitespecific2020} Ramsøe, A., van Heekeren, V., Ponce, P., Fischer, R., Barnes, I., Speller, C., \& Collins, M. J. (2020). {DeamiDATE} 1.0: {Site-specific} deamidation as a tool to assess authenticity of members of ancient proteomes. \emph{Journal of Archaeological Science}, \emph{115}, 105080. \url{https://doi.org/10.1016/j.jas.2020.105080} -\leavevmode\vadjust pre{\hypertarget{ref-rogersRoleStreptococcus2001}{}}% +\bibitem[\citeproctext]{ref-rogersRoleStreptococcus2001} Rogers, J. D., Palmer, R. J., Kolenbrander, P. E., \& Scannapieco, F. A. (2001). Role of {Streptococcus} gordonii {Amylase-Binding Protein A} in {Adhesion} to {Hydroxyapatite}, {Starch Metabolism}, and {Biofilm Formation}. \emph{Infection and Immunity}, \emph{69}(11), 7046--7056. \url{https://doi.org/10.1128/IAI.69.11.7046-7056.2001} -\leavevmode\vadjust pre{\hypertarget{ref-rustichelliSimultaneousSeparation1996}{}}% +\bibitem[\citeproctext]{ref-rustichelliSimultaneousSeparation1996} Rustichelli, C., Ferioli, V., Vezzalini, F., Rossi, M. C., \& Gamberini, G. (1996). Simultaneous separation and identification of hashish constituents by coupled liquid chromatography-mass spectrometry ({HPLC-MS}). \emph{Chromatographia}, \emph{43}(3), 129--134. \url{https://doi.org/10.1007/BF02292940} -\leavevmode\vadjust pre{\hypertarget{ref-salazar-garciaDentalCalculus2014}{}}% +\bibitem[\citeproctext]{ref-salazar-garciaDentalCalculus2014} Salazar-García, D. C., Richards, M. P., Nehlich, O., \& Henry, A. G. (2014). Dental calculus is not equivalent to bone collagen for isotope analysis: A comparison between carbon and nitrogen stable isotope @@ -9262,7 +9013,7 @@ \section*{References cited}\label{references-cited-5}} {Spain}). \emph{Journal of Archaeological Science}, \emph{47}, 70--77. \url{https://doi.org/10.1016/j.jas.2014.03.026} -\leavevmode\vadjust pre{\hypertarget{ref-scottExoticFoods2021}{}}% +\bibitem[\citeproctext]{ref-scottExoticFoods2021} Scott, A., Power, R. C., Altmann-Wendling, V., Artzy, M., Martin, M. A. S., Eisenmann, S., Hagan, R., Salazar-García, D. C., Salmon, Y., Yegorov, D., Milevski, I., Finkelstein, I., Stockhammer, P. W., \& @@ -9271,52 +9022,52 @@ \section*{References cited}\label{references-cited-5}} \emph{Proceedings of the National Academy of Sciences}, \emph{118}(2), e2014956117. \url{https://doi.org/10.1073/pnas.2014956117} -\leavevmode\vadjust pre{\hypertarget{ref-cummingsMayanCalculus1997}{}}% +\bibitem[\citeproctext]{ref-cummingsMayanCalculus1997} Scott Cummings, L., \& Magennis, A. (1997). A phytolith and starch record of food and grit in {Mayan} human tooth tartar. In A. Pinilla, J. Juan-Tresserras, \& M. J. Machado (Eds.), \emph{The {State-of-the-Art} of {Phytoliths} in {Soils} and {Plants}}. {CSIC Press}. -\leavevmode\vadjust pre{\hypertarget{ref-shawCommonalityElastic2004}{}}% +\bibitem[\citeproctext]{ref-shawCommonalityElastic2004} Shaw, T., Winston, M., Rupp, C. J., Klapper, I., \& Stoodley, P. (2004). Commonality of {Elastic Relaxation Times} in {Biofilms}. \emph{Physical Review Letters}, \emph{93}(9), 098102. \url{https://doi.org/10.1103/PhysRevLett.93.098102} -\leavevmode\vadjust pre{\hypertarget{ref-sissonsArtificialPlaque1997}{}}% +\bibitem[\citeproctext]{ref-sissonsArtificialPlaque1997} Sissons, C. H. (1997). Artificial {Dental Plaque Biofilm Model Systems}. \emph{Advances in Dental Research}, \emph{11}(1), 110--126. \url{https://doi.org/10.1177/08959374970110010201} -\leavevmode\vadjust pre{\hypertarget{ref-sissonsMultistationPlaque1991}{}}% +\bibitem[\citeproctext]{ref-sissonsMultistationPlaque1991} Sissons, C. H., Cutress, T. W., Hoffman, M. P., \& Wakefield, J. S. J. (1991). A {Multi-station Dental Plaque Microcosm} ({Artificial Mouth}) for the {Study} of {Plaque Growth}, {Metabolism}, {pH}, and {Mineralization}: \emph{Journal of Dental Research}. \url{https://doi.org/10.1177/00220345910700110301} -\leavevmode\vadjust pre{\hypertarget{ref-sissonsPHResponse1994}{}}% +\bibitem[\citeproctext]{ref-sissonsPHResponse1994} Sissons, C. H., Wong, L., Hancock, E. M., \& Cutress, T. W. (1994). The {pH} response to urea and the effect of liquid flow in {``artificial mouth''} microcosm plaques. \emph{Archives of Oral Biology}, \emph{39}(6), 497--505. \url{https://doi.org/10.1016/0003-9969(94)90146-5} -\leavevmode\vadjust pre{\hypertarget{ref-skoglundSeparatingEndogenous2014}{}}% +\bibitem[\citeproctext]{ref-skoglundSeparatingEndogenous2014} Skoglund, P., Northoff, B. H., Shunkov, M. V., Derevianko, A. P., Pääbo, S., Krause, J., \& Jakobsson, M. (2014). Separating endogenous ancient {DNA} from modern day contamination in a {Siberian Neandertal}. \emph{Proceedings of the National Academy of Sciences}, \emph{111}(6), 2229--2234. \url{https://doi.org/10.1073/pnas.1318934111} -\leavevmode\vadjust pre{\hypertarget{ref-sorensenSensitiveDetermination2017}{}}% +\bibitem[\citeproctext]{ref-sorensenSensitiveDetermination2017} Sørensen, L. K., \& Hasselstrøm, J. B. (2017). Sensitive {Determination} -of {Cannabinoids} in {Whole Blood} by {LC}{\textendash}{MS-MS After -Rapid Removal} of {Phospholipids} by {Filtration}. \emph{Journal of -Analytical Toxicology}, \emph{41}(5), 382--391. -\url{https://doi.org/10.1093/jat/bkx030} +of {Cannabinoids} in {Whole Blood} by +{LC}\textendash{{MS-MS After Rapid Removal}} of {Phospholipids} by +{Filtration}. \emph{Journal of Analytical Toxicology}, \emph{41}(5), +382--391. \url{https://doi.org/10.1093/jat/bkx030} -\leavevmode\vadjust pre{\hypertarget{ref-sotoCharacterizationDecontamination2019}{}}% +\bibitem[\citeproctext]{ref-sotoCharacterizationDecontamination2019} Soto, M., Inwood, J., Clarke, S., Crowther, A., Covelli, D., Favreau, J., Itambu, M., Larter, S., Lee, P., Lozano, M., Maley, J., Mwambwiga, A., Patalano, R., Sammynaiken, R., Vergès, J. M., Zhu, J., \& Mercader, @@ -9325,14 +9076,14 @@ \section*{References cited}\label{references-cited-5}} Anthropological Sciences}, \emph{11}(9), 4847--4872. \url{https://doi.org/10.1007/s12520-019-00830-7} -\leavevmode\vadjust pre{\hypertarget{ref-springfieldCocaineMetabolites1993}{}}% +\bibitem[\citeproctext]{ref-springfieldCocaineMetabolites1993} Springfield, A. C., Cartmell, L. W., Aufderheide, A. C., Buikstra, J., \& Ho, J. (1993). Cocaine and metabolites in the hair of ancient {Peruvian} coca leaf chewers. \emph{Forensic Science International}, \emph{63}(1-3), 269--275. \url{https://doi.org/10.1016/0379-0738(93)90280-N} -\leavevmode\vadjust pre{\hypertarget{ref-stephanStudiesChanges1947}{}}% +\bibitem[\citeproctext]{ref-stephanStudiesChanges1947} Stephan, R. M., \& Hemmens, E. S. (1947). Studies of changes in {pH} produced by pure cultures of oral micro-organisms; effects of varying the microbic cell concentration; comparison of different micro-organisms @@ -9340,26 +9091,26 @@ \section*{References cited}\label{references-cited-5}} micro-organisms. \emph{Journal of Dental Research}, \emph{26}(1), 15--41. \url{https://doi.org/10.1177/00220345470260010201} -\leavevmode\vadjust pre{\hypertarget{ref-stewartAntimicrobialTolerance2015}{}}% +\bibitem[\citeproctext]{ref-stewartAntimicrobialTolerance2015} Stewart, P. S. (2015). Antimicrobial {Tolerance} in {Biofilms}. \emph{Microbiology Spectrum}, \emph{3}(3), 10.1128/microbiolspec.mb-0010-2014. \url{https://doi.org/10.1128/microbiolspec.mb-0010-2014} -\leavevmode\vadjust pre{\hypertarget{ref-takenakaDiffusionMacromolecules2009}{}}% +\bibitem[\citeproctext]{ref-takenakaDiffusionMacromolecules2009} Takenaka, S., Pitts, B., Trivedi, H. M., \& Stewart, P. S. (2009). Diffusion of {Macromolecules} in {Model Oral Biofilms}. \emph{Applied and Environmental Microbiology}, \emph{75}(6), 1750--1753. \url{https://doi.org/10.1128/AEM.02279-08} -\leavevmode\vadjust pre{\hypertarget{ref-trompEDTACalculus2017}{}}% +\bibitem[\citeproctext]{ref-trompEDTACalculus2017} Tromp, M., Buckley, H., Geber, J., \& Matisoo-Smith, E. (2017). {EDTA} decalcification of dental calculus as an alternate means of microparticle extraction from archaeological samples. \emph{Journal of Archaeological Science: Reports}, \emph{14}, 461--466. \url{https://doi.org/10.1016/j.jasrep.2017.06.035} -\leavevmode\vadjust pre{\hypertarget{ref-velskoMicrobialDifferences2019}{}}% +\bibitem[\citeproctext]{ref-velskoMicrobialDifferences2019} Velsko, I. M., Fellows Yates, J. A., Aron, F., Hagan, R. W., Frantz, L. A. F., Loe, L., Martinez, J. B. R., Chaves, E., Gosden, C., Larson, G., \& Warinner, C. (2019). Microbial differences between dental plaque and @@ -9367,14 +9118,14 @@ \section*{References cited}\label{references-cited-5}} \emph{Microbiome}, \emph{7}(1), 102. \url{https://doi.org/10.1186/s40168-019-0717-3} -\leavevmode\vadjust pre{\hypertarget{ref-velskoHighConservation2023}{}}% +\bibitem[\citeproctext]{ref-velskoHighConservation2023} Velsko, I. M., Gallois, S., Stahl, R., Henry, A. G., \& Warinner, C. (2023). High conservation of the dental plaque microbiome across populations with differing subsistence strategies and levels of market integration. \emph{Molecular Ecology}. \url{https://doi.org/10.1111/mec.16988} -\leavevmode\vadjust pre{\hypertarget{ref-velskoDentalCalculus2017}{}}% +\bibitem[\citeproctext]{ref-velskoDentalCalculus2017} Velsko, I. M., Overmyer, K. A., Speller, C., Klaus, L., Collins, M. J., Loe, L., Frantz, L. A. F., Sankaranarayanan, K., Lewis, C. M., Martinez, J. B. R., Chaves, E., Coon, J. J., Larson, G., \& Warinner, C. (2017). @@ -9382,7 +9133,7 @@ \section*{References cited}\label{references-cited-5}} \emph{Metabolomics}, \emph{13}(11), 134. \url{https://doi.org/10.1007/s11306-017-1270-3} -\leavevmode\vadjust pre{\hypertarget{ref-warinnerEvidenceMilk2014}{}}% +\bibitem[\citeproctext]{ref-warinnerEvidenceMilk2014} Warinner, C., Hendy, J., Speller, C., Cappellini, E., Fischer, R., Trachsel, C., Arneborg, J., Lynnerup, N., Craig, O. E., Swallow, D. M., Fotakis, A., Christensen, R. J., Olsen, J. V., Liebert, A., Montalva, @@ -9391,7 +9142,7 @@ \section*{References cited}\label{references-cited-5}} human dental calculus. \emph{Scientific Reports}, \emph{4}, 7104. \url{https://doi.org/10.1038/srep07104} -\leavevmode\vadjust pre{\hypertarget{ref-warinnerPathogensHost2014}{}}% +\bibitem[\citeproctext]{ref-warinnerPathogensHost2014} Warinner, C., Rodrigues, J. F., Vyas, R., Trachsel, C., Shved, N., Grossmann, J., Radini, A., Hancock, Y., Tito, R. Y., Fiddyment, S., Speller, C., Hendy, J., Charlton, S., Luder, H. U., Salazar-Garcia, D. @@ -9400,12 +9151,12 @@ \section*{References cited}\label{references-cited-5}} oral cavity. \emph{Nature Genetics}, \emph{46}(4), 336--344. \url{https://doi.org/10.1038/ng.2906} -\leavevmode\vadjust pre{\hypertarget{ref-weinerBiologicalMaterials2010}{}}% +\bibitem[\citeproctext]{ref-weinerBiologicalMaterials2010} Weiner, S. (2010). Biological {Materials}: {Bones} and {Teeth}. In \emph{Microarchaeology: {Beyond} the {Visible Archaeological Record}} (pp. 99--134). {Cambridge University Press}. -\leavevmode\vadjust pre{\hypertarget{ref-wilkinsonFAIRGuiding2016}{}}% +\bibitem[\citeproctext]{ref-wilkinsonFAIRGuiding2016} Wilkinson, M. D., Dumontier, M., Aalbersberg, Ij. J., Appleton, G., Axton, M., Baak, A., Blomberg, N., Boiten, J.-W., da Silva Santos, L. B., Bourne, P. E., Bouwman, J., Brookes, A. J., Clark, T., Crosas, M., @@ -9414,14 +9165,14 @@ \section*{References cited}\label{references-cited-5}} management and stewardship. \emph{Scientific Data}, \emph{3}(1), 160018. \url{https://doi.org/10.1038/sdata.2016.18} -\leavevmode\vadjust pre{\hypertarget{ref-yaussyCalculusSurvivorship2019}{}}% +\bibitem[\citeproctext]{ref-yaussyCalculusSurvivorship2019} Yaussy, S. L., \& DeWitte, S. N. (2019). Calculus and survivorship in medieval {London}: {The} association between dental disease and a demographic measure of general health. \emph{American Journal of Physical Anthropology}, \emph{168}(3), 552--565. \url{https://doi.org/10.1002/ajpa.23772} -\leavevmode\vadjust pre{\hypertarget{ref-ziesemerGenomeCalculus2018}{}}% +\bibitem[\citeproctext]{ref-ziesemerGenomeCalculus2018} Ziesemer, K. A., Ramos-Madrigal, J., Mann, A. E., Brandt, B. W., Sankaranarayanan, K., Ozga, A. T., Hoogland, M., Hofman, C. A., Salazar-García, D. C., Frohlich, B., Milner, G. R., Stone, A. C., @@ -9430,7 +9181,7 @@ \section*{References cited}\label{references-cited-5}} ancient dental calculus and dentin. \emph{American Journal of Physical Anthropology}. \url{https://doi.org/10.1002/ajpa.23763} -\leavevmode\vadjust pre{\hypertarget{ref-zimmermanBiomolecularArchaeology2023}{}}% +\bibitem[\citeproctext]{ref-zimmermanBiomolecularArchaeology2023} Zimmerman, M., \& Tushingham, S. (2023). The {Biomolecular Archaeology} of {Psychoactive Substances}. In A. M. Pollard, R. A. Armitage, \& C. Makarevicz (Eds.), \emph{Handbook of {Archaeological Sciences}} (Second @@ -9438,8 +9189,10 @@ \section*{References cited}\label{references-cited-5}} \end{CSLReferences} -\hypertarget{supplementary-information}{% -\chapter*{Supplementary information}\label{supplementary-information}} +\phantomsection\label{supplementary-information} +\bookmarksetup{startatroot} + +\chapter*{Supplementary information} \addcontentsline{toc}{chapter}{Supplementary information} \markboth{Supplementary information}{Supplementary information} @@ -9503,13 +9256,13 @@ \chapter*{Supplementary information}\label{supplementary-information}} \tightlist \item \textbf{DOI:} - \href{https://doi.org/10.5281/zenodo.10069669}{10.5281/zenodo.10069669} + \href{https://doi.org/10.24072/pcjournal.414}{10.24072/pcjournal.414} \item \textbf{Supplementary material:} \href{https://doi.org/10.5281/zenodo.10069669}{10.5281/zenodo.10069669} \item \textbf{Code:} - \href{https://doi.org/10.5281/zenodo.7649824}{10.5281/zenodo.7649824} + \href{https://doi.org/10.5281/zenodo.11040640}{10.5281/zenodo.11040640} \item \textbf{Data:} \href{https://doi.org/10.5281/zenodo.7648756}{10.5281/zenodo.7648756} @@ -9518,8 +9271,10 @@ \chapter*{Supplementary information}\label{supplementary-information}} \href{https://doi.org/10.24072/pci.archaeo.100389}{10.24072/pci.archaeo.100389} \end{itemize} -\hypertarget{summary-6}{% -\chapter*{Summary}\label{summary-6}} +\phantomsection\label{summary-6} +\bookmarksetup{startatroot} + +\chapter*{Summary} \addcontentsline{toc}{chapter}{Summary} \markboth{Summary}{Summary} @@ -9632,8 +9387,10 @@ \chapter*{Summary}\label{summary-6}} mechanisms causing various dietary (and non-dietary) markers to become entrapped in dental calculus. -\hypertarget{samenvatting}{% -\chapter*{Samenvatting}\label{samenvatting}} +\phantomsection\label{samenvatting} +\bookmarksetup{startatroot} + +\chapter*{Samenvatting} \addcontentsline{toc}{chapter}{Samenvatting} \markboth{Samenvatting}{Samenvatting} @@ -9759,8 +9516,10 @@ \chapter*{Samenvatting}\label{samenvatting}} onderliggende mechanismen beter te begrijpen waardoor verschillende voedingsmarkers (en niet-dieetmarkers) ingebed worden in tandsteen. -\hypertarget{curriculum-vitae}{% -\chapter*{Curriculum Vitae}\label{curriculum-vitae}} +\phantomsection\label{curriculum-vitae} +\bookmarksetup{startatroot} + +\chapter*{Curriculum Vitae} \addcontentsline{toc}{chapter}{Curriculum Vitae} \markboth{Curriculum Vitae}{Curriculum Vitae} @@ -9803,5 +9562,7 @@ \chapter*{Curriculum Vitae}\label{curriculum-vitae}} started as a Data Steward at TU Delft. I've done lots of other stuff, too, which you can find in the CV I can actually keep updated. +\phantomsection\label{3ade8a4a-fb1d-4a6c-8409-ac45482d5fc9} + \backmatter \end{document} diff --git a/latex/preamble.tex b/latex/preamble.tex index f6a6dfd..1644c60 100644 --- a/latex/preamble.tex +++ b/latex/preamble.tex @@ -19,7 +19,19 @@ % chapter and section names in header \renewcommand{\chaptermark}[1]{\markboth{\MakeLowercase{\chaptername\ \thechapter\ -\ #1}}{}} -\renewcommand{\sectionmark}[1]{ \markright{\MakeLowercase{#1}}{} } +\renewcommand{\chaptermark}[1]{\markboth{\MakeLowercase{\chaptername\ \thechapter\ -\ #1}}{}} +\renewcommand{\sectionmark}[1]{\markright{\MakeLowercase{#1}}{} } + +% fix header names for toc, lof, and lot to match chapter and section names +\usepackage{etoolbox} +\patchcmd{\tableofcontents}{\MakeUppercase\contentsname}{\MakeLowercase\contentsname}{}{} +\patchcmd{\tableofcontents}{\MakeUppercase\contentsname}{\MakeLowercase\contentsname}{}{} % twice to remove on both sides of header +\patchcmd{\listoffigures}{\MakeUppercase\listfigurename}{\MakeLowercase\listfigurename}{}{} +\patchcmd{\listoffigures}{\MakeUppercase\listfigurename}{\MakeLowercase\listfigurename}{}{} % twice to remove on both sides of header +\patchcmd{\listoftables}{\MakeUppercase\listtablename}{\MakeLowercase\listtablename}{}{} +\patchcmd{\listoftables}{\MakeUppercase\listtablename}{\MakeLowercase\listtablename}{}{} % twice to remove on both sides of header + + % modify captions for figures and tables \usepackage[labelfont=bf,