Score plot and loading plot obtained by PCA or O-PLS.

<p>(A) A PCA score plot of young controls (shadowed), aged controls (opened), and SSCs (closed) (R2X[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142645#pone.0142645.ref001" target="_blank">1</a>] = 0.329635; R2X[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142645#pone.0142645.ref002" target="_blank">2</a>] = 0.201184; Ellipse: Hotelling T2 (95%)) using the peak area ratios of each <i>N</i>-glycan to the total peak area of all identified <i>N</i>-glycans. (B) An O-PLS score plot between young controls (shadowed), aged controls (opened), and SSCs (closed). R2X [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142645#pone.0142645.ref001" target="_blank">1</a>] = 0.295051, R2X [XSide Comp. 1] = 0.106066, Ellipse: Hotelling T2 (95%) (C) A loading plot with jack-knifed confidence intervals by O-PLS. The pq[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142645#pone.0142645.ref001" target="_blank">1</a>] value is the weight that combines the X and Y variables. The error bar indicates the standard error (SE) of pq[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142645#pone.0142645.ref001" target="_blank">1</a>] values obtained from 16 samples independently. Glycan compositions were deduced by the accurate mass. Numbers in parentheses indicate isomers. “N” or “P” in parentheses indicates the data obtained from the negative or positive ion mode, respectively. Closed and shadowed columns represent [pq1] / SE > 1.5 and < 1.5, respectively. Row data were summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142645#pone.0142645.s009" target="_blank">S2 Table</a>. Hex, hexose; HexNAc, <i>N</i>-acetylhexosamine; NeuNAc, <i>N</i>-acetylneuraminic acid; dHex, deoxyhexose; NH₄, ammonium.</p

Deduced structures of characteristic <i>N</i>-glycans in SSCs.

<p>The number of the <i>N</i>-glycan corresponded to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142645#pone.0142645.t001" target="_blank">Table 1</a>. No. 1~14 were increased and No. 15~18 were decreased in SSCs, respectively. blue square, <i>N</i>-acetylglucosamine; yellow circle, galactose; green circle, mannose; purple diamond, <i>N</i>-acetylneuraminic acid; red triangle, fucose.</p

Typical <i>N</i>-glycan profile from plasma proteins in SSC.

<p>Deduced <i>N</i>-glycan structures were added to the base peak chromatogram of the SSC sample. Top and bottom charts represent the positive and negative ion modes, respectively. Vertical axis, relative abundance; horizontal axis, retention time; blue square, <i>N</i>-acetylglucosamine; yellow circle, galactose; green circle, mannose; purple diamond, <i>N</i>-acetylneuraminic acid; red triangle, fucose.</p

Summary of characteristic <i>N</i>-glycan profiles in SSCs.

<p>Increased and decreased <i>N-</i>glycans in SSCs were sorted by pq[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142645#pone.0142645.ref001" target="_blank">1</a>] values in the loading plot of O-PLS shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142645#pone.0142645.g002" target="_blank">Fig 2C</a>.</p><p>^a The number of the <i>N</i>-glycan corresponded to Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142645#pone.0142645.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142645#pone.0142645.s007" target="_blank">S7</a>.</p><p>^b The number in parentheses indicates isomers.</p><p>^c Hex, hexose; HexNAc, <i>N</i>-acetylhexosamine; NeuNAc, <i>N</i>-acetylneuraminic acid; dHex, deoxyhexose.</p><p>Summary of characteristic <i>N</i>-glycan profiles in SSCs.</p

Comparison of physiological and inflammatory parameters between SSCs and young/aged controls.

<p>^aData represent the mean± standard deviation.</p><p>^<b>b</b>Data represent the median (interquartile range).</p><p>^<b>c</b>Numbers in parentheses indicate the numbers of subjects.</p><p>^dDifferences from SSCs were calculated by ANOVA (*<i>p</i> < 0.05, **<i>p</i> < 0.01).</p><p>^eDifferences from SSCs were calculated by ANOVA using logarithmically transformed data (*<i>p</i> < 0.05, **<i>p</i> < 0.01).</p><p>^fnot determined</p><p>Comparison of physiological and inflammatory parameters between SSCs and young/aged controls.</p

DataSheet1_Distinguishing two distinct types of salivary extracellular vesicles: a potential tool for understanding their pathophysiological roles.docx

Extracellular vesicles (EVs), which are found in almost all cells and human body fluids, are currently being studied as a source of pathophysiological information. Previously, we demonstrated that at least two types of EVs can be isolated from human whole saliva (WS) using enzymatic activity of dipeptidyl peptidase IV (DPP IV) as a marker for differentiating the EV subsets. In the present study, EV fractions, termed EV-I 20 k-ppt and EV-II 100 k-ppt, were prepared by a combination of size-exclusion chromatography of improved condition and sequential centrifugation. The EV-I 20 k-ppt fraction contained medium/large EVs with a diameter of 100–1,000 nm, including aminopeptidase N (APN), mucin 1, ezrin, and Annexin A1. EV-II 100 k-ppt contained small EVs with a diameter of 20–70 nm, with DPP IV and CD9, programmed cell death 6-interacting protein, and tumor susceptibility gene 101 as characteristic proteins. Proteomic analyses also revealed distinctive repertoires of constituent proteins. Immunoprecipitation of several membrane proteins of the EVs with respective antibodies suggested their differential local membrane environment between the two types of salivary vesicles. Thus, we identified two distinctive types of EVs, one is APN/MUC1- rich EVs (EV-I, large/medium EVs) and the other is DPP IV/CD9-rich EVs (EV-II, small EVs). Furthermore, analysis of the binding of the EVs to coronavirus spike proteins showed that EV-II 100 k-ppt, but not EV-I 20 k-ppt, significantly bound to the spike protein of Middle East respiratory syndrome coronavirus (MERS-CoV). Finally, we developed a simple method to prepare two distinctive EVs from only 1 mL of human WS using sequential immunoprecipitation. Elucidating the features and functions of these two types of salivary EVs may help us understand their pathophysiological roles in the oral cavity and gastrointestinal tract.</p

Table1_Distinguishing two distinct types of salivary extracellular vesicles: a potential tool for understanding their pathophysiological roles.xlsx

Extracellular vesicles (EVs), which are found in almost all cells and human body fluids, are currently being studied as a source of pathophysiological information. Previously, we demonstrated that at least two types of EVs can be isolated from human whole saliva (WS) using enzymatic activity of dipeptidyl peptidase IV (DPP IV) as a marker for differentiating the EV subsets. In the present study, EV fractions, termed EV-I 20 k-ppt and EV-II 100 k-ppt, were prepared by a combination of size-exclusion chromatography of improved condition and sequential centrifugation. The EV-I 20 k-ppt fraction contained medium/large EVs with a diameter of 100–1,000 nm, including aminopeptidase N (APN), mucin 1, ezrin, and Annexin A1. EV-II 100 k-ppt contained small EVs with a diameter of 20–70 nm, with DPP IV and CD9, programmed cell death 6-interacting protein, and tumor susceptibility gene 101 as characteristic proteins. Proteomic analyses also revealed distinctive repertoires of constituent proteins. Immunoprecipitation of several membrane proteins of the EVs with respective antibodies suggested their differential local membrane environment between the two types of salivary vesicles. Thus, we identified two distinctive types of EVs, one is APN/MUC1- rich EVs (EV-I, large/medium EVs) and the other is DPP IV/CD9-rich EVs (EV-II, small EVs). Furthermore, analysis of the binding of the EVs to coronavirus spike proteins showed that EV-II 100 k-ppt, but not EV-I 20 k-ppt, significantly bound to the spike protein of Middle East respiratory syndrome coronavirus (MERS-CoV). Finally, we developed a simple method to prepare two distinctive EVs from only 1 mL of human WS using sequential immunoprecipitation. Elucidating the features and functions of these two types of salivary EVs may help us understand their pathophysiological roles in the oral cavity and gastrointestinal tract.</p