Table1_Alcohol consumption and colorectal cancer risk: A mendelian randomization study.DOCX

Background: Previous observational studies have provided inconsistent evidence for the association between alcohol consumption and the risk of colorectal cancer (CRC). To assess this potential causal effect, we performed bidirectional Mendelian randomization (MR) analysis.Methods: We selected six single nucleotide polymorphisms (SNPs) as instrumental variables (IVs) associated with alcohol consumption (ever versus never drinker) and two SNPs representing the number of drinks per week from a genome-wide association study (GWAS) of the Japanese population. Summary data for CRC were obtained from a GWAS meta-analysis in the Japanese population of 6,692 CRC cases and 27,178 controls. MR analysis was performed by the inverse-variance weighted (IVW) method primarily, supplemented with several sensitivity methods including the weighted median method, maximum likelihood method, MR pleiotropy residual sum and outlier (MR-PRESSO) test, MR-Egger regression, Causal Analysis Using Summary Effect estimates (CAUSE) method, as well as constrained maximum likelihood and model averaging and Bayesian information criterion (cML-MA-BIC) method. Multivariable Mendelian randomization (MMR) analyses were used to adjust for potential confounders. Reverse MR analyses were also performed to assess the potential causal effect of CRC on alcohol consumption.Results: Genetically predicted alcohol consumption (ever versus never drinker) was positively associated with the risk of CRC (odds ratio (OR) = 1.08, 95% confidence interval (CI): 1.05–1.12, p = 1.51 × 10–5 by IVW). The number of alcoholic drinks per week was also associated with an increased risk of CRC (OR = 1.39, 95%CI: 1.27–1.52, p = 5.29 × 10–13 by IVW). Sensitivity analysis yielded similar results. Reverse MR analyses found no evidence that CRC contributes to either ever drinkers (OR = 1.00, 95%CI: 0.99–1.00, p = 0.339 by IVW) or added number of drinks per week (OR = 1.01, 95%CI: 0.98–1.05, p = 0.545 by IVW).Conclusion: Our study suggested a potential causal association between alcohol consumption and the risk of CRC among Asians. Reducing drinking may be beneficial to the prevention and management of CRC.</p

Syntaxin 16 Regulates Lumen Formation during Epithelial Morphogenesis

<div><p>The formation and maintenance of cell-cell junctions, both under physiological and pathological conditions, requires the targeting and trafficking of junctional proteins. Proteins of the syntaxin (Stx)-family localize to a variety of subcellular membranes and contribute to intracellular transport of cargo by regulating vesicle fusion events at these sites. Unlike plasma membrane localized Stxs, the roles of endosome- and Golgi-localized stx proteins in epithelial morphogenesis are less understood. Here we show that Stx16– an endosome- and Golgi-localized target-membrane soluble N-ethylmaleimide attachment protein receptor (t-SNARE) that plays a role in membrane trafficking between these compartments – is essential for lumen development. In cultured Madin Darby Canine Kidney (MDCK) cells, Stx16 was selectively upregulated as sparsely plated cells attained confluency. Stx16-depleted confluent monolayers consistently showed lower transepithelial resistance than control monolayers, and failed to maintain endogenous and ectopically expressed E-cadherin at the adherens junctions due to decreased recycling. We further found that whereas cysts formed by MDCK cells cultured in Matrigel have a single hollow lumen, those formed by stx16-depleted counterparts had multiple lumens, due to abnormal orientiation of the mitotic spindle. Finally, a similar role for stx16 function <i>in vivo</i> is indicated by our analysis of pronephric-duct development in zebrafish expressing the <i>claudinB:lynGFP</i> transgene; lack of stx16 function in this structure (in <i>stx16-</i>morphant embryos) led to the development of enlarged, torturous pronephric ducts with more than one lumen. Taken together, our <i>in vitro</i> and <i>in vivo</i> studies establish a role for Stx16 in maintaining the integrity of cell-cell junctions, and thereby in morphogenesis of the kidney epithelial lumen.</p></div

Expression of Stx proteins in MDCK cells over the development and maintenance of a confluent monolayer.

<p>(<b>A–C</b>) MDCK cells (3.5×10⁵ cells/35 mm dish) were seeded and analyzed after achieving varying degree of confluency. (<b>A</b>) Confluency of MDCK monolayers at indicated times after initial cell seeding. Representative images are shown. (<b>B</b>) Western blotting for Stx protein and E-cadherin expression. Total cell lysates were prepared from cultures at varying degrees of confluency. (<b>C</b>) Quantitation of band densities in <b><i>B</i></b>. Values represent relative protein levels at different cell densities, after normalization to the 25%-confluency value, set arbitrarily at 100%. The percentage represents the mean (±SD) for n = 3.</p

Stx16 depletion leads to decrease in E-cadherin levels and formation of multiple lumens in epithelial cysts.

<p>(<b>A, B</b>) Immunoflourescence-based analysis of morphology and localization of basolateral and apical proteins in cysts formed from MDCK cells stably expressing scr-shRNA or sh<i>Stx16</i> 4d after plating in Matrigel. Cysts were fixed and immunostained for E-cadherin, β-catenin, ZO-1, and podocalyxin (gp135), and DNA was visualized using DAPI. Scale bar, 5 µm. Representative cysts; confocal sections through the top to bottom of a cyst are shown. (<b>C</b>) Quantification of cysts with single or multiple lumens as in <b><i>A</i></b>, <b><i>B</i></b>. Values are mean ± SD from three independent experiments; n >100 cysts/experiment. p<0.01.</p

Stx16 depletion leads to spindle misorientation and cystogenesis defects.

<p>(<b>A</b>) Confocal images of metaphase cells in the middle region of the cysts grown for 1–4 d, following fixation and staining with anti-α-tubulin antibody (red) and DAPI (blue). Note that in control cysts: orientation of the spindle with respect to the apical-basal axis is perpendicular (arrow);cell division occurs in the plane of the monolayer (2-cell stage); abscission is asymmetrical; and deposition of nascent apical surface occurs around a single central lumen (asterisk, multi-cell stage). (<b>B</b>) Schematic illustration of spindle-angle measurement. A line was drawn to connect the two spindle poles (thick black line). Another was drawn from the centroid of the apical domain to the midpoint of the spindle axis (thin black lines), and the acute angle (red) between the two lines was assessed. (<b>C</b>) Quantification of metaphase spindle angles in MDCK cells stably expressing scr-shRNA or sh<i>Stx16</i>. Data show means ± SEM, n  = 70 cysts from three independent experiments. Statistical significance was evaluated using a Mann-Whitney test. *, <i>p</i>≤0.002; **, <i>p</i>≤0.001.</p

DataSheet_1_Circulating levels of cytokines and risk of inflammatory bowel disease: evidence from genetic data.docx

BackgroundPrior epidemiological studies have established a correlation between inflammatory cytokines and inflammatory bowel disease (IBD). However, the nature of this relationship remains uncertain. Mendelian randomization (MR) study has the advantages of avoiding confounding and reverse causality compared with traditional observational research.ObjectiveWe aimed to evaluate whether genetically determined circulating levels of cytokines are associated with the risk of IBD by using the MR approach.Materials and methodsWe selected genetic variants associated with circulating levels of 28 cytokines at the genome-wide significance level from a genome-wide association study (GWAS) including 8,293 individuals. Summary-level data for IBD (including Crohn’s disease and ulcerative colitis) were obtained from the International Inflammatory Bowel Disease Genetics Consortium and UK Biobank. We performed the primary analysis using the inverse-variance weighted method, as well as sensitivity analyses to test the stability of our results. We subsequently replicated the results of IBD in the UK Biobank dataset. A reverse MR analysis was also conducted to evaluate the possibility of reverse causation.ResultsGenetically predicted elevated levels of interleukin-17 (IL-17) and monokine induced by interferon-gamma (MIG) were associated with an increased risk of IBD[odds ratio (OR): 1.52, 95% confidence interval (CI):1.10-2.08, P =0.010 for IL-17 and OR: 1.58, 95% CI: 1.24-2.00, P = 1.60×10-4 for MIG]. Moreover, we observed suggestive associations between β-NGF and MIP-1β with the risk of Crohn’s disease (OR: 0.71, 95% CI: 0.52-0.98, P = 0.039) and ulcerative colitis (OR: 1.08, 95% CI: 1.01-1.15, P= 0.019). In the reverse MR study, there was no evidence of causal effects of IBD and these cytokines.ConclusionOur study suggests the potential causal associations of IL-17 and MIG with IBD. Further studies are needed to determine whether IL-17 and MIG or their downstream effectors could be useful in the management of IBD.</p

Stx16 depleted MDCK cells show decreased E-cadherin at cell-cell junctions.

<p>(<b>A</b>) Immunofluorescence-based analysis of E-cadherin localization at MDCK cell-cell junctions during the development and maintenance of a monolayer. (<b>B</b>) Western blotting for E-cadherin following preparation of Triton-soluble (<i>S</i>) and -insoluble (<i>I</i>) fractions from scr-shRNA and shStx16 MDCK confluent monolayer 24 hrs after cell seeding. (<b>C</b>) Densitometric quantitation of E-cadherin bands as in <b><i>B</i></b>. Values represent the relative protein levels after normalization to an arbitrary value of 100% for src-shRNA expressing cells. The percentage represents the mean (±SD) for n = 3. (<b>D</b>) Immunofluorescence-based analysis of -catenin localization in scr-shRNA and shStx16 MDCK at various times post cell seeding.</p

Stx16 depletion causes abnormal pronephric-duct formation in zebrafish.

<p>(<b>A</b>) Diagram of the exon and intron organization of zebrafish <i>stx16</i>. Thick red lines indicate the binding regions of two antisense morpholinos (MOs), one of which targets the start codon (AUG) to block translation of <i>stx16</i>, and the other the mRNA splice acceptor site of exon 4 (splicing) to block splicing of the <i>stx16</i> mRNA. The two arrows indicate the locations of primers used in RT-PCR analysis in control and <i>stx16</i> morphants. (<b>B</b>) Western blot analysis showing the expression of Stx16 in control and <i>stx16</i>-MO^AUG-injected embryos at 48 hpf; expression of alpha-actin served as a loading control. (<b>C</b>) RT-PCR showing 319 bp and 867 bp amplicons, which result, respectively, from amplification from exon 4 and exon 4 plus unspliced intron 3 in the control uninjected and <i>stx16</i>-MO^sp-injected embryos. (<b>D</b>) Expression of <i>stx16</i> at 48 hrs, as detected by ISH. Left-hand panel: lateral view with anterior to the left. Right-hand panel: cross section of the region shown to left. Arrow: pronephric duct. (<b>E</b>) Normarski images (lateral view) of control and <i>stx16</i> MO^sp-injected embryos at 48 hpf. Bar graph at right shows the percentage of embryos showing abnormalities (edema and curvature). Arrow: pericardial edema; arrowhead: body curvature. (<b>F</b>) <i>pax2.1</i> expression at 48 hpf, as detected by ISH in the indicated embryos. Left-hand panels: Lateral view of the trunk region of indicated embryos, with anterior to the left. Right-hand panels: Transverse sections of embryos of the left-hand panel. Arrows: pronephric duct. (<b>G</b>) Top panel (i, ii): Representative epifluorescence images of 48-hpf <i>Tg(cldnb:lynEGFP)</i> uninjected control and <i>stx16</i>-MO^sp-injected embryos. Lateral view, anterior to the right, pronephric ducts. Middle panel (iii, iv): Confocal Z-stack images of pronephric ducts of <i>Tg(cldnb:lynEGFP)</i> taken at the regions shown in top panel. Bottom panel (v, vi): transverse sections of <i>Tg(cldnb:lynEGFP)</i> embryos showing the pronephric ducts (arrows). Nucleoli are stained with DAPI. Graph showing the percentages of embryos exhibiting pronephric-duct dilation. (<b>H</b>, <b>I</b>) Confocal (Z-stack) images of transverse sections of <i>Tg(cldnb:lynEGFP)</i> embryos at 48 hpf, showing the expression of PKCζ and pan-cadherin on one side of pronephric duct. Arrowheads: lumens.</p

Stx16 colocalizes with Rab11 and E-cadherin in MDCK cells.

<p>(<b>A</b>) Immunofluorescence based assessment of Rab11 localization in control (src-shRNA) and shStx16-expressing MDCK cells. Graph show quantification of overlap in Stx16 and Rab11 fluorescence signals, from images as in <b><i>A</i></b>. Value represents mean ± S.D. (n = 50 cells for each condition, from 5 separate experiments; p≤0.05). Intracellular localization of Rab11 is not altered in Stx16-depleted MDCK cells. (<b>B</b>) Immunofluorescence-based analysis of localization of E-cadherin-GFP in cells stably expressing Src-shRNA or sh<i>Stx16</i>. (<b>C</b>) Quantitation of relative levels of E-cadherin-GFP at the PM (staining with human E-cadherin antibody) along the cell edges vs. total throughout the cell, quantified using the Metamorph software. Values represent relative change in the levels of E-cadherin-GFP at the cell periphery (calculated as % of total cell-associated protein). Results are expressed as mean ± SEM (n = 70 cells for each condition, from three separate experiments and p≤0.05). (<b>D</b>) MDCK cells stably expressing scr-shRNA or sh<i>Stx16</i> were surface biotinylated. Samples were then incubated at 16°C for 30 min, to allow biotinylated E-cadherin to accumulate in endosomes. Biotin remaining at the surface was removed by treatment with MesNa and quenching of MesNa with iodoacetamide (0-min time point); samples were further incubated at 37°C for the indicated periods and, at each time point shown, subjected to a second MesNa treatment and then assessed for recycling of internalized E-cadherin. After each time point, the cells were lysed and the amount of biotinylated E-cadherin was determined by capture ELISA, using an Ab against E-cadherin. The fraction of internalized E-cadherin recycled back to the PM is expressed as a percentage of surface-labeled protein originally internalized (from the 0-min chase time point). Values are mean ± S.D. from 3 independent experiments; p≤0.005.</p

Stx16-depleted MDCK monolayer show decreased transepithelial resistance (TER).

<p>(<b>A</b>) Immunofluorescence staining for Stx16 expression in src-shRNA-MDCK and sh<i>Stx16</i>-MDCK cells. (<b>B</b>) Western blotting of Stx16 expression in src-shRNA-MDCK and sh<i>Stx16</i>-MDCK cells. (<b>C</b>) Quantitation of band densities from <b><i>B</i></b>. Values represent relative protein levels after normalization to an arbitrary value of 100% for src-shRNA expressing cells. The percentage represents the mean (±SD) for n = 3. (<b>D</b>) Transepithelial resistance (TER) measured in monolayers generated from src-shRNA-MDCK and shStx16-MDCK cells at indicated time intervals. (<b>E</b>) Recovery of TER in MDCK monolayers following a Ca²⁺-switch. Stx16 depletion caused a delayed recovery of TER. Graph represents the averages from 3 independent experiments (plus 3 replicates per experiment) and error bars show SEM.</p