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<label for="email-include-supplemental" class="email-include-supplemental-label">MeSH and other data</label>
</div>
<div class="form-field recaptcha ">
<div class="g-recaptcha" id="id-recaptcha" data-sitekey="6LfsWHMdAAAAAClKbtOpjQ2pMjgsGxvv7NdZW9uI"></div>
</div>
<div id="captcha-error-message" class="usa-input-error-message captcha-validation-message" role="alert"></div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Sending..."
data-ga-category="save_share"
data-ga-action="email"
data-ga-label="send">
Send email
</button>
<button class="action-panel-cancel"
aria-label="Close 'Email citations' panel"
ref="linksrc=close_email_panel"
aria-controls="email-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="email"
data-ga-label="cancel">
Cancel
</button>
</div>
<input type="hidden" name="email-search-details" value="" />
<input type="hidden" name="email-search-details-hash" value="0e42663a6c3bd85498fcb88798998fed7bfdc45d457db35281e41afe13cc0524" />
</form>
</div>
</div>
<div id="collections-action-panel"
class="collections-action-panel action-panel in-progress-dots-panel"
aria-hidden="true"
data-collections-open-panel-enabled="false"
data-collections-open-panel-url-hash="#open-collections-panel">
<div class="inner-wrap">
<h3 class="action-panel-heading">
Add to Collections
</h3>
<form id="collections-action-panel-form"
class="collections-action-panel-form action-panel-content action-form action-panel-smaller-selectors"
data-existing-collections-url="/list-existing-collections/"
data-add-to-existing-collection-url="/add-to-existing-collection/"
data-create-and-add-to-new-collection-url="/create-and-add-to-new-collection/"
data-get-article-ids-by-search-url="/get-article-ids-by-search/"
data-myncbi-max-collection-name-length="100"
data-add-to-collection-max-amount="1000"
data-collections-root-url="https://www.ncbi.nlm.nih.gov/myncbi/collections/">
<input type="hidden" name="csrfmiddlewaretoken" value="nBBwy534rhTflUxzvwnH6Wzt1kO0qD4UEolBbVwq1SGJflDp8P0JA457zowcjeCe">
<div class="choice-group" role="radiogroup">
<ul class="radio-group-items">
<li>
<input type="radio"
id="collections-action-panel-new"
class="collections-new"
name="collections"
value="new"
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="collections_radio_new">
<label for="collections-action-panel-new">Create a new collection</label>
</li>
<li>
<input type="radio"
id="collections-action-panel-existing"
class="collections-existing"
name="collections"
value="existing"
checked="true"
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="collections_radio_existing">
<label for="collections-action-panel-existing">Add to an existing collection</label>
</li>
</ul>
</div>
<div class="controls-wrapper">
<div class="action-panel-control-wrap new-collections-controls">
<label for="collections-action-panel-add-to-new" class="action-panel-label required-field-asterisk">
Name your collection:
</label>
<input
type="text"
name="add-to-new-collection"
id="collections-action-panel-add-to-new"
class="collections-action-add-to-new"
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/"
maxlength="100"
data-ga-category="save_share"
data-ga-action="create_collection"
data-ga-label="non_favorties_collection">
<div class="collections-new-name-too-long usa-input-error-message selection-validation-message">
Name must be less than 100 characters
</div>
</div>
<div class="action-panel-control-wrap existing-collections-controls">
<label for="collections-action-panel-add-to-existing" class="action-panel-label">
Choose a collection:
</label>
<select id="collections-action-panel-add-to-existing"
class="action-panel-selector collections-action-add-to-existing"
data-ga-category="save_share"
data-ga-action="select_collection"
data-ga-label="($('#collections-action-add-to-existing').val() === 'Favorites') ? 'Favorites' : 'non_favorites_collection'">
</select>
<div class="collections-retry-load-on-error usa-input-error-message selection-validation-message">
Unable to load your collection due to an error<br>
<a href="#">Please try again</a>
</div>
</div>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Adding..."
data-pinger-ignore
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="add">
Add
</button>
<button class="action-panel-cancel"
aria-label="Close 'Add to Collections' panel"
ref="linksrc=close_collections_panel"
aria-controls="collections-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="bibliography-action-panel"
class="bibliography-action-panel action-panel in-progress-dots-panel"
aria-hidden="true"
data-bibliography-open-panel-enabled="false"
data-bibliography-open-panel-url-hash="#open-bibliography-panel">
<div class="inner-wrap">
<h3 class="action-panel-heading">
Add to My Bibliography
</h3>
<form id="bibliography-action-panel-form"
class="bibliography-action-panel-form action-panel-content action-form action-panel-smaller-selectors"
data-add-to-bibliography-max-amount="100"
data-add-to-bibliography-batch-size="10"
data-bibliography-delegates-url="/list-bibliography-delegates/"
data-add-to-bibliography-url="/add-to-bibliography/"
data-get-article-ids-by-search-url="/get-article-ids-by-search/"
data-mybib-root-url="https://www.ncbi.nlm.nih.gov/myncbi/collections/mybibliography/">
<input type="hidden" name="csrfmiddlewaretoken" value="nBBwy534rhTflUxzvwnH6Wzt1kO0qD4UEolBbVwq1SGJflDp8P0JA457zowcjeCe">
<div class="action-panel-control-wrap bibliographies-controls">
<div class="choice-group">
<ul class="bibliographies-action-add radio-group-items">
<li>
<input name="bibliography" id="my-bibliography" class="my-bibliography" type="radio" checked/>
<label for="my-bibliography">My Bibliography</label>
</li>
</ul>
</div>
</div>
<div class="bibliographies-retry-load-on-error usa-input-error-message selection-validation-message">
Unable to load your delegates due to an error<br>
<a href="#">Please try again</a>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Adding..."
data-pinger-ignore>
Add
</button>
<button class="action-panel-cancel"
aria-label="Close 'Add to bibliography' panel"
ref="linksrc=close_bibliography_panel"
aria-controls="bibliography-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="mybib"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="saved-search-action-panel" class="saved-search-action-panel action-panel " aria-hidden="true"
data-saved-search-open-panel-enabled="false"
data-saved-search-open-panel-url-hash="#open-saved-search-panel">
<div class="inner-wrap">
<h2 class="action-panel-heading">
Your saved search
</h2>
<form id="saved-search-action-panel-form"
class="saved-search-action-panel-form action-panel-content action-form"
data-create-saved-search-url="/create-saved-search/"
data-try-search-terms-url="/try-search-term/"
data-saved-search-root-url="https://www.ncbi.nlm.nih.gov/myncbi/searches/">
<input type="hidden" name="csrfmiddlewaretoken" value="nBBwy534rhTflUxzvwnH6Wzt1kO0qD4UEolBbVwq1SGJflDp8P0JA457zowcjeCe">
<div class="action-panel-control-wrap">
<label for="saved-search-name" class="action-panel-label saved-search-name-label required-field-asterisk">
Name of saved search:
</label>
<input maxlength="200"
type="text"
name="saved-search-name"
id="saved-search-name"
class="saved-search-name"
value=""
required
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/">
</div>
<div class="action-panel-control-wrap">
<label for="saved-search-term" class="action-panel-label required-field-asterisk">
Search terms:
</label>
<textarea name="saved-search-term" id="saved-search-term" class="saved-search-term" required></textarea>
</div>
<div class="test-search-term-wrap">
<a href="#" class="try-search-term">Test search terms</a>
</div>
<div class="choice-group action-panel-extra-margin-top">
<span class="action-panel-label" id="fieldset-label">
Would you like email updates of new search results?
</span>
<fieldset id="saved-search-alert" aria-describedby="fieldset-label">
<legend class="usa-sr-only">Saved Search Alert Radio Buttons</legend>
<ul class="radio-group-items">
<li>
<input type="radio" id="saved-search-alert-yes" class="saved-search-alert-yes" name="saved-search-alert" value="yes" checked>
<label for="saved-search-alert-yes" class="action-panel-label">Yes</label>
</li>
<li>
<input aria-label="No radio input" type="radio" id="saved-search-alert-no" class="saved-search-alert-no" name="saved-search-alert" value="no">
<label for="saved-search-alert-no" class="action-panel-label">No</label>
</li>
</ul>
</fieldset>
</div>
<div class="alert-schedule-wrap">
<div class="action-panel-control-wrap">
<label class="action-panel-label">
Email:
</label>
<span aria-label="Email address" id="saved-search-email" class="action-panel-label"><span class="action-panel-label-bold"></span> (<a class="myncbi-account-settings" href="https://www.ncbi.nlm.nih.gov/account/settings/">change</a>)</span>
</div>
<div class="action-panel-control-wrap action-panel-extra-margin-top">
<label for="saved-search-frequency" class="action-panel-label">
Frequency:
</label>
<select id="saved-search-frequency" class="no-border-panel-selector saved-search-frequency">
<option value="monthly">Monthly</option>
<option value="weekly">Weekly</option>
<option value="daily">Daily</option>
</select>
</div>
<div class="action-panel-control-wrap saved-search-monthly-additional">
<label for="saved-search-monthly-on-day" class="action-panel-label">
Which day?
</label>
<select id="saved-search-monthly-on-day" class="no-border-panel-selector">
<option value="Sunday">The first Sunday</option>
<option value="Monday">The first Monday</option>
<option value="Tuesday">The first Tuesday</option>
<option value="Wednesday">The first Wednesday</option>
<option value="Thursday">The first Thursday</option>
<option value="Friday">The first Friday</option>
<option value="Saturday">The first Saturday</option>
<option value="day">The first day</option>
<option value="weekday">The first weekday</option>
</select>
</div>
<div class="action-panel-control-wrap saved-search-weekly-additional">
<label for="saved-search-weekly-on-day" class="action-panel-label">
Which day?
</label>
<select id="saved-search-weekly-on-day" class="no-border-panel-selector saved-search-weekly-on-day">
<option value="Sunday">Sunday</option>
<option value="Monday">Monday</option>
<option value="Tuesday">Tuesday</option>
<option value="Wednesday">Wednesday</option>
<option value="Thursday">Thursday</option>
<option value="Friday">Friday</option>
<option value="Saturday">Saturday</option>
</select>
</div>
<div class="action-panel-control-wrap">
<label for="saved-search-report" class="action-panel-label">
Report format:
</label>
<select id="saved-search-report" class="no-border-panel-selector saved-search-report">
<option value="DocSum">Summary</option>
<option value="DocSumText">Summary (text)</option>
<option value="Abstract">Abstract</option>
<option value="AbstractText">Abstract (text)</option>
<option value="MEDLINE">PubMed</option>
</select>
</div>
<div class="action-panel-control-wrap">
<label for="saved-search-amount" class="action-panel-label">
Send at most:
</label>
<select id="saved-search-amount" class="no-border-panel-selector saved-search-amount">
<option value="1">1 item</option>
<option value="5" selected>5 items</option>
<option value="10">10 items</option>
<option value="20">20 items</option>
<option value="50">50 items</option>
<option value="100">100 items</option>
<option value="200">200 items</option>
</select>
</div>
<div>
<input type="checkbox" id="saved-search-send-if-no-result" class="saved-search-send-if-no-result" name="saved-search-send-if-no-result">
<label for="saved-search-send-if-no-result" class="action-panel-label smaller-checkbox">
Send even when there aren't any new results
</label>
</div>
<div class="action-panel-control-wrap option-text-in-email-wrap">
<label for="saved-search-email-text" class="action-panel-label">
Optional text in email:
</label>
<textarea name="saved-search-email-text"
id="saved-search-email-text"
class="saved-search-email-text"></textarea>
</div>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Saving..."
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="save">
Save
</button>
<button class="action-panel-cancel"
aria-label="Close 'Your saved search' panel"
ref="linksrc=close_saved_search_panel"
aria-controls="saved-search-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="citation-manager-action-panel" class="citation-manager-action-panel action-panel" aria-hidden="true">
<div class="inner-wrap">
<h2 class="action-panel-heading">
Create a file for external citation management software
</h2>
<form id="citation-manager-action-panel-form"
class="action-panel-content action-form"
action="/results-export-ids/"
data-by-search-action="/results-export-search-data/"
data-by-ids-action="/results-export-ids/"
method="post"
data-by-search-method="post"
data-by-ids-method="post">
<input type="hidden" name="csrfmiddlewaretoken" value="nBBwy534rhTflUxzvwnH6Wzt1kO0qD4UEolBbVwq1SGJflDp8P0JA457zowcjeCe">
<input name="results-format" type="hidden" value="pubmed"/>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Sending..."
data-ga-category="save_share"
data-ga-action="citation_manager"
data-ga-label="save">
Create file
</button>
<button class="action-panel-cancel"
aria-label="Close 'Send citations to citation manager' panel"
ref="linksrc=close_citation_manager_panel"
aria-controls="citation-manager-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="citation_manager"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="rss-action-panel" class="rss-action-panel action-panel " aria-hidden="true">
<div class="inner-wrap">
<h2 class="action-panel-heading">
Your RSS Feed
</h2>
<form id="rss-action-panel-form"
class="rss-action-panel-form action-panel-content action-form"
data-create-rss-feed-url="/create-rss-feed-url/"
data-search-form-term-value="">
<input type="hidden" name="csrfmiddlewaretoken" value="nBBwy534rhTflUxzvwnH6Wzt1kO0qD4UEolBbVwq1SGJflDp8P0JA457zowcjeCe">
<div class="action-panel-control-wrap">
<label for="rss-name" class="action-panel-label required-field-asterisk">
Name of RSS Feed:
</label>
<input maxlength="200"
placeholder="Name"
type="text"
name="rss-name"
id="rss-name"
class="rss-name"
value=''
required
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/">
</div>
<div class="rss-limit-wrap">
<div class="action-panel-control-wrap action-panel-extra-margin-top">
<label for="rss-limit" class="action-panel-label">
Number of items displayed:
</label>
<select id="rss-limit" class="no-border-panel-selector rss-limit">
<option value="5">5</option>
<option value="10">10</option>
<option value="15" selected="selected">15</option>
<option value="20">20</option>
<option value="50">50</option>
<option value="100">100</option>
</select>
</div>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Creating..."
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="save">
Create RSS
</button>
<button class="action-panel-cancel"
aria-label="Close 'Your RSS' panel"
ref="linksrc=close_rss_panel"
aria-controls="rss-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="cancel">
Cancel
</button>
</div>
<div class="action-panel-control-wrap rss-link-copy-wrap">
<label for="rss-link" class="usa-sr-only">RSS Link</label>
<input placeholder="Your RSS Feed Link" type="text" name="rss-link" id="rss-link" class="rss-link" title="RSS Link">
<button
type="button"
disabled
class="rss-link-copy-button disabled"
data-ga-category="save_share"
data-ga-action="rss"
data-ga-label="copy">
Copy
</button>
</div>
</form>
</div>
</div>
</div>
</div>
<div class="article-page" id="article-page" data-article-pmid="38111640">
<aside class="page-sidebar">
<div class="inner-wrap">
<div class="full-text-links">
<div class="full-view">
<h3 class="title">
Full text links
</h3>
<div class="full-text-links-list">
<a class="link-item
dialog-focus"
href="https://doi.org/10.3389/fmicb.2023.1276951"
target="_blank"
rel="noopener"
ref="linksrc=fulltextorjournal_fulltext&amp;is_pmc=False&amp;PrId=6956&amp;itool=Abstract-def&amp;log$=linkouticon&amp;uid=38111640&amp;db=pubmed&amp;nlmid=101548977"
title="See full text options at Frontiers Media SA"
data-ga-category="full_text"
data-ga-action="Frontiers Media SA"
data-ga-label="38111640"
><img src="https://cdn.ncbi.nlm.nih.gov/corehtml/query/egifs/https:--www.frontiersin.org-alerts-logo-PubMed.png" alt="Frontiers Media SA full text link"><span class="text">
Frontiers Media SA
</span></a><a class="link-item
pmc
"
href="https://pmc.ncbi.nlm.nih.gov/articles/pmid/38111640/"
target="_blank"
rel="noopener"
ref="linksrc=fulltextorjournal_fulltext&amp;is_pmc=True&amp;PrId=3494&amp;itool=Abstract-def&amp;log$=linkouticon&amp;uid=38111640&amp;db=pubmed&amp;nlmid=101548977"
title="Free full text at PubMed Central"
data-ga-category="full_text"
data-ga-action="PMC"
data-ga-label="38111640"
><span class="text">
Free PMC article
</span></a>
</div>
</div>
<div class="short-view">
<a href="#" class="full-text-links-button full-text-links-dialog-trigger">
Full text links
</a>
</div>
</div>
<div class="actions-buttons sidebar"><h3 class="title">Actions</h3><div class="inner-wrap"><button class="citation-button citation-dialog-trigger"
aria-label="Open dialog with citation text in different styles"
data-ga-category="save_share"
data-ga-action="cite"
data-ga-label="open"
data-all-citations-url="/38111640/citations/"
data-citation-style="nlm"
data-pubmed-format-link="/38111640/export/"><span class="button-label">Cite</span></button><link type="text/css" href="ncbi-overlay-block/src/overlay-block.css"><div class="collections-button-container" data-article-id="38111640" data-article-db="pubmed"><button class="collections-button collections-dialog-trigger"
aria-label="Save article in MyNCBI collections."
data-ga-category="collections_button"
data-ga-action="click"
data-ga-label="collections_button"
data-collections-open-dialog-enabled="false"
data-collections-open-dialog-url="https://account.ncbi.nlm.nih.gov/?back_url=https%3A%2F%2Fpubmed.ncbi.nlm.nih.gov%2F38111640%2F%23open-collections-dialog"
data-in-collections="false"><span class="button-label">Collections</span></button><div class="overlay" role="dialog"><div id="collections-action-dialog"
class="dialog collections-dialog"
aria-hidden="true"><div class="title">Add to Collections</div><div class="collections-action-panel action-panel"><form id="collections-action-dialog-form"
class="collections-action-panel-form action-panel-content action-form action-panel-smaller-selectors"
data-existing-collections-url="/list-existing-collections/"
data-add-to-existing-collection-url="/add-to-existing-collection/"
data-create-and-add-to-new-collection-url="/create-and-add-to-new-collection/"
data-myncbi-max-collection-name-length="100"
data-collections-root-url="https://www.ncbi.nlm.nih.gov/myncbi/collections/"><input type="hidden" name="csrfmiddlewaretoken" value="nBBwy534rhTflUxzvwnH6Wzt1kO0qD4UEolBbVwq1SGJflDp8P0JA457zowcjeCe"><div class="choice-group" role="radiogroup"><ul class="radio-group-items"><li><input type="radio"
id="collections-action-dialog-new"
class="collections-new"
name="collections"
value="new"
data-ga-category="collections_button"
data-ga-action="click"
data-ga-label="collections_radio_new"><label for="collections-action-dialog-new">Create a new collection</label></li><li><input type="radio"
id="collections-action-dialog-existing"
class="collections-existing"
name="collections"
value="existing"
checked="true"
data-ga-category="collections_button"
data-ga-action="click"
data-ga-label="collections_radio_existing"><label for="collections-action-dialog-existing">Add to an existing collection</label></li></ul></div><div class="controls-wrapper"><div class="action-panel-control-wrap new-collections-controls"><label for="collections-action-dialog-add-to-new" class="action-panel-label required-field-asterisk">
Name your collection:
</label><input
type="text"
name="add-to-new-collection"
id="collections-action-dialog-add-to-new"
class="collections-action-add-to-new"
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/"
maxlength="100"
data-ga-category="collections_button"
data-ga-action="create_collection"
data-ga-label="non_favorties_collection"><div class="collections-new-name-too-long usa-input-error-message selection-validation-message">
Name must be less than 100 characters
</div></div><div class="action-panel-control-wrap existing-collections-controls"><label for="collections-action-dialog-add-to-existing" class="action-panel-label">
Choose a collection:
</label><select id="collections-action-dialog-add-to-existing"
class="action-panel-selector collections-action-add-to-existing"
data-ga-category="collections_button"
data-ga-action="select_collection"
data-ga-label="($('.collections-action-add-to-existing').val() === 'Favorites') ? 'Favorites' : 'non_favorites_collection'"></select><div class="collections-retry-load-on-error usa-input-error-message selection-validation-message">
Unable to load your collection due to an error<br><a href="#">Please try again</a></div></div></div><div class="action-panel-actions"><button class="action-panel-submit"
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Insight into the mechanism of gallstone disease by proteomic and metaproteomic characterization of human bile
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data-ga-label="Ling Du">Ling Du</a><sup class="affiliation-links"><span class="author-sup-separator">&nbsp;</span><a class="affiliation-link" title="Key Laboratory of Digestive Cancer Full Cycle Monitoring and Precise Intervention of Shanghai Municipal Health Commission, Minhang Hospital, Fudan University, Shanghai, China." href="#short-view-affiliation-2" ref="linksrc=author_aff">
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data-ga-label="Jun Li">Jun Li</a><sup class="affiliation-links"><span class="author-sup-separator">&nbsp;</span><a class="affiliation-link" title="Department of Surgery, Tongren Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China." href="#short-view-affiliation-3" ref="linksrc=author_aff">
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data-ga-label="Feng Liu">Feng Liu</a><sup class="affiliation-links"><span class="author-sup-separator">&nbsp;</span><a class="affiliation-link" title="Minhang Hospital, Fudan University, and Shanghai Key Laboratory of Medical Epigenetics, The International Co-laboratory of Medical Epigenetics and Metabolism, Ministry of Science and Technology, Institutes of Biomedical of Sciences, Fudan University, Shanghai, China." href="#short-view-affiliation-1" ref="linksrc=author_aff">
1
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Affiliations
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<li data-affiliation-id="short-view-affiliation-1"
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><sup class="key">1</sup> Minhang Hospital, Fudan University, and Shanghai Key Laboratory of Medical Epigenetics, The International Co-laboratory of Medical Epigenetics and Metabolism, Ministry of Science and Technology, Institutes of Biomedical of Sciences, Fudan University, Shanghai, China.</li>
<li data-affiliation-id="short-view-affiliation-2"
id="short-view-affiliation-2"
><sup class="key">2</sup> Key Laboratory of Digestive Cancer Full Cycle Monitoring and Precise Intervention of Shanghai Municipal Health Commission, Minhang Hospital, Fudan University, Shanghai, China.</li>
<li data-affiliation-id="short-view-affiliation-3"
id="short-view-affiliation-3"
><sup class="key">3</sup> Department of Surgery, Tongren Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China.</li>
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<div class="equal-contrib-explanation" id="short-view-equal-contrib-explanation"><sup class="key">#</sup> Contributed equally.</div>
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Abstract
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<p>
<strong class="sub-title">
Introduction:
</strong>
Cholesterol gallstone disease is a prevalent condition that has a significant economic impact. However, the role of the bile microbiome in its development and the host's responses to it remain poorly understood.
</p>
<p>
<strong class="sub-title">
Methods:
</strong>
In this study, we conducted a comprehensive analysis of microbial and human bile proteins in 40 individuals with either gallstone disease or gallbladder polyps. We employed a combined proteomic and metaproteomic approach, as well as meta-taxonomic analysis, functional pathway enrichment, and Western blot analyses.
</p>
<p>
<strong class="sub-title">
Results:
</strong>
Our metaproteomic analysis, utilizing the lowest common ancestor algorithm, identified 158 microbial taxa in the bile samples. We discovered microbial taxa that may contribute to gallstone formation, including β-glucuronidase-producing bacteria such as Streptococcus, Staphylococcus, and Clostridium, as well as those involved in biofilm formation like Helicobacter, Cyanobacteria, Pseudomonas, <i>Escherichia coli</i>, and Clostridium. Furthermore, we identified 2,749 human proteins and 87 microbial proteins with a protein false discovery rate (FDR) of 1% and at least 2 distinct peptides. Among these proteins, we found microbial proteins crucial to biofilm formation, such as QDR3, ompA, ndk, pstS, nanA, pfIB, and dnaK. Notably, QDR3 showed a gradual upregulation from chronic to acute cholesterol gallstone disease when compared to polyp samples. Additionally, we discovered other microbial proteins that enhance bacterial virulence and gallstone formation by counteracting host oxidative stress, including sodB, katG, rbr, htrA, and ahpC. We also identified microbial proteins like lepA, rtxA, pckA, tuf, and tpiA that are linked to bacterial virulence and potential gallstone formation, with lepA being upregulated in gallstone bile compared to polyp bile. Furthermore, our analysis of the host proteome in gallstone bile revealed enhanced inflammatory molecular profiles, including innate immune molecules against microbial infections. Gallstone bile exhibited overrepresented pathways related to blood coagulation, folate metabolism, and the IL-17 pathway. However, we observed suppressed metabolic activities, particularly catabolic metabolism and transport activities, in gallstone bile compared to polyp bile. Notably, acute cholelithiasis bile demonstrated significantly impaired metabolic activities compared to chronic cholelithiasis bile.
</p>
<p>
<strong class="sub-title">
Conclusion:
</strong>
Our study provides a comprehensive metaproteomic analysis of bile samples related to gallstone disease, offering new insights into the microbiome-host interaction and gallstone formation mechanism.
</p>
</div>
<p>
<strong class="sub-title">
Keywords:
</strong>
bile; cholecystitis; gallstone; metaproteomics; microbiome; proteomics.
</p>
</div>
<p class="copyright" id="copyright">
Copyright © 2023 Yang, Wang, Jiang, Zhang, Du, Li and Liu.
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<p xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:p1="http://pubmed.gov/pub-one">The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>
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Figures
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<div class="figure-caption-contents"><p> Proteomic and metaproteomic analysis of… </p></div>
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<div class="figure-caption-contents"><p> Proteomic and metaproteomic analysis of human bile with or without gallstone. <b> (A) </b> The… </p></div>
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Figure 1
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<div class="figure-caption-contents">Proteomic and metaproteomic analysis of human bile with or without gallstone. <b>(A)</b> The cohort enrolled in this study contains 14 acute CHLs, 17 chronic CHLs and 9 gallstone-free patients with gallbladder polyps. The bile fluids were collected during gallbladder resection of gallstone patients or polyp remove of patients. The proteins were extracted and separated using SDS-PAGE, followed by in-gel tryptic digestion. LCMS analysis was performed using TIMS-TOF pro mass spectrometer. <b>(B)</b> The peptides were identified by two-step database searching using Metalab against a comprehensive protein database (UniProtDB) and the peptides were further subjected to taxonomic lowest common ancestor (LCA) analysis. Metataxonomic analysis using public 16S rRNA datasets of human bile by Kraken2 program was performed independently to verify the metaproteomic results. <b>(C)</b> The correlation plot of the bile samples based on protein identification. <b>(D)</b> PLS-DA of bile samples based on identified microbial taxa. The ellipses illustrated the confidence interval.</div>
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<div class="figure-caption-contents"><p> Identification of microbes from gallstone… </p></div>
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</strong>
<div class="figure-caption-contents"><p> Identification of microbes from gallstone and polyp bile samples. <b> (A) </b> The proportion of… </p></div>
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<figcaption id="figure-caption-1" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description">
<strong class="figure-label">
Figure 2
</strong>
<div class="figure-caption-contents">Identification of microbes from gallstone and polyp bile samples. <b>(A)</b> The proportion of unique peptides identified in human, fungi, bacteria, archaea, and virus. <b>(B)</b> The structure and abundance of the top 10 phyla in both polyp and gallstone bile samples were analyzed using the intensity of identified microbial peptides. CHL, cholecystitis. <b>(C)</b> The structure and abundance of the top 10 microbial species in polyp and gallstone bile samples. <b>(D)</b> The taxa we identified were found in other Human body sites through data mining of public references. The details were deposited in Supplementary Table S2. <b>(E)</b> The tandem MS spectrum of peptide LYCEEVGWVICGIK identified from lepA protein (LEPA_BUCCC) of <i>Buchnera aphidicola</i>. <b>(F)</b> The Sankey plot shows the metataxonomic analysis of human bile samples. The dataset was retrieved from GenBank with the BioProject accession number PRJNA580086 (<ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA580086">https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA580086</ext-link>). In this project, 45 biliary bile samples were analyzed using 16S rRNA sequencing. We re-analyzed the dataset against kraken2-microbial database (<ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="https://lomanlab.github.io/mockcommunity/mc_databases.html">https://lomanlab.github.io/mockcommunity/mc_databases.html</ext-link>) using kraken2 program (Lu et al., 2022). The red arrowhead represents the clade of <i>Buchnera aphidicola</i>, which has been detected in 44 out of 45 bile samples, comprising a total of 7,313 reads.</div>
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<p> Figure 3 </p>
</strong>
<div class="figure-caption-contents"><p> Taxon changes associated with gallstone… </p></div>
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<strong class="figure-label">
<p> Figure 3 </p>
</strong>
<div class="figure-caption-contents"><p> Taxon changes associated with gallstone formation determined by the microbial peptides. <b> (A) </b> Hierarchical… </p></div>
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<figcaption id="figure-caption-2" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description">
<strong class="figure-label">
Figure 3
</strong>
<div class="figure-caption-contents">Taxon changes associated with gallstone formation determined by the microbial peptides. <b>(A)</b> Hierarchical cluster analysis of the microbial taxa significantly changed in gallstone bile samples (<i>n</i>=31) comparing to polyp bile samples (<i>n</i>=9). <b>(B,C)</b> Linear discriminant analysis Effect Size (LEfSe) analysis of microbial biomarkers of CHL (<i>n</i>=31) and polyp (<i>n</i>=9) using an LDA threshold of 2. The numbers indicate the major marker clades in CHL. <b>(D)</b> The abundance of Helicobacteraceae between CHL (<i>n</i>=31) and polyp (<i>n</i> =9) patients based on the proteomic data. The <i>p</i> values were calculated using Welchs <i>t</i> test. MS, mass spectrometry. <b>(E)</b> The abundance of Helicobacteraceae between CHL (<i>n</i>=14) and healthy (<i>n</i>=13) patients based on the 16S rRNA data (dataset PRJNA439241). 16S, 16S rRNA dataset analysis. Chole, cholecystitis. Healt, healthy.</div>
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</strong>
<div class="figure-caption-contents"><p> The microbial proteins identified from… </p></div>
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<p> Figure 4 </p>
</strong>
<div class="figure-caption-contents"><p> The microbial proteins identified from gallstone and polyp bile samples. <b> (A) </b> Abundance rank… </p></div>
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Figure 4
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<div class="figure-caption-contents">The microbial proteins identified from gallstone and polyp bile samples. <b>(A)</b> Abundance rank of identified proteins. The intensity of each protein was determined by summing up the LFQ values across all samples. Microbial proteins were highlighted. <b>(B)</b> The abundance of differential microbial proteins between CHL (<i>n</i>=31) and polyp (<i>n</i>=9) patients. <b>(C)</b> The differential microbial proteins between acute CHL (<i>n</i>=14) and polyp (<i>n</i>=9) patients. <b>(D)</b> The differential microbial proteins between chronic CHL (<i>n</i>=17) and polyp (<i>n</i>=9) patients. <b>(E)</b> Three microbial proteins upregulated in CHL compared to polyp. <i>p</i> values were calculated using Welchs <i>t</i> test. <b>(F)</b> The differential microbial proteins between acute CHL (<i>n</i>=14) and chronic CHL (<i>n</i>=17) patients.</div>
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<p> Figure 5 </p>
</strong>
<div class="figure-caption-contents"><p> The host proteins identified from… </p></div>
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<p> Figure 5 </p>
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<div class="figure-caption-contents"><p> The host proteins identified from gallstone and polyp bile samples. <b> (A) </b> Gene ontology… </p></div>
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Figure 5
</strong>
<div class="figure-caption-contents">The host proteins identified from gallstone and polyp bile samples. <b>(A)</b> Gene ontology (GO) enrichment was performed on the upregulated human proteins in CHL compared to polyp using the g:Profiler tool (<ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="https://biit.cs.ut.ee/gprofiler/gost">https://biit.cs.ut.ee/gprofiler/gost</ext-link>) (Raudvere et al., 2019). <b>(B)</b> Differential expression of complement proteins between CHL and polyp samples. <i>p</i> values were calculated using unpaired Welchs <i>t</i> test. <b>(C)</b> Two mucin proteins were upregulated in CHL compared to polyp. <b>(D)</b> GO enrichment of the downregulated human proteins in CHL compared to polyp. GO_MF, Gene ontology of molecular function; GO_CC, cellular component; GO_BP biological process; REAC, Reactome; WP, WikiPathways. <b>(E)</b> Proteins associated with bile secretion were downregulated in CHL compared to polyp.</div>
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