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Objective To investigate the diversity and composition of gut microbiota in patient with cholestatic liver disease, and explore their correlation with clinical parameters. Methods Fresh stool samples from 16 patients with cholestatic liver disease and 19 healthy controls were collected, and 16S rRNA sequencing was used to analyze the diversity of gut microbiota and composition characteristics. Pearson analysis was employed to analyze the correlation between the different bacteria and clinical parameters. Results There were no statistical differences in the baseline data, such as sex, age and body mass index (BMI) in study subjects (P>0.05), but significant differences were observed in serum indexes among them (P < 0.01). The results of α diversity showed that Chao1 and Shannon indexes were lower in the study group, but there was no statistical difference (P>0.05). β diversity results indicated that the species diversity had significant difference between 2 group (P < 0.01). Further analysis of microbiota composition showed largely relative abundance of Bacteroides, Lachnoclostridium, Veillonella, Roseburia and Escherichia shigella (P < 0.05), while the abundance of Blautia were dramatically decreased (P < 0.05). Pearson analysis displayed that Lachnoclostridium was strongly correlated with serum level of alkaline phosphatase (ALP) (P < 0.01) and gamma-glutamyl transferase (GGT) (P < 0.05), and Veillonella was also strongly correlated with GGT (P < 0.05). Conclusion Compared with healthy controls, the patients with cholestatic liver disease have obvious characteristic changes in the gut microbiota. The relatively high abundance of Veillonella is closely assocatied with cholestatic liver disease.

Additional file 1: Supplemental Figure 1. Confirmation of mouse and human Hpa encoding genes expressions across all experimental conditions. (a) Genotype identification of Hpa- Tg mice. Semiquantitative PCR amplification of DNA extracted from WT (n=1) and Hpa-Tg mice (n=10). Human Hpa specific primer was used to detect transgene vector sequences in the mouse genome. Total DNA was assessed by specific primer against a genomic sequence of the ribosomal protein L-19. The expression levels of mouse Hpa (b) and human Hpa (c) in WT+SW/WT+Cn/Hpa-Tg+SW/Hpa-Tg+Cn group were confirmed. The expression levels of mouse vs human Hpa in in Hpa-Tg SW/HpaTg+Cn groups were confirmed (d). (e) Before 16S rRNA sequencing of WT and HpaTg mice, the expression levels of mouse Hpa and human Hpa were confirmed. The expression levels of mouse or human Hpa, the expression levels of mouse vs human Hpa were also confirmed in ABX (f), FMT (g-i), cohousing (j), Parabacteroides administration (k-m) and acetate supplementation (n-p) experiments. Data were expressed as mean ± SEM. Significance between two groups was determined by unpaired, two-tailed t test or Mann-Whitney test depending on the sample distribution. Significance between multiple groups was determined through ordinary one-way ANOVA or Kruskal-Wallis test depending on the sample distribution. Exact p levels were all provided. ABX, antibiotic cocktail; Cn, caerulein; CoHo, cohoused; FMT, fecal microbiota transplantation; Hpa-Tg, heparanase-transgenic; P. distasonis, Parabacteroides distasonis; SiHo, housed singly; SW, sterile water; rRNA, ribosomal RNA; WT, wild-type. Supplemental Figure 2. Alpha-diversity, beta-diversity and bar plots of the phylum/class/order/family/genus taxonomic levels in WT and Hpa-Tg mice. (a) Alpha-diversity (Based on ACE, Observed features, Fisher alpha, Shannon and Faith pd). (b) PCoA of beta-diversity using Jaccard, Generalized Unifrac and Weighted Unifrac metric distance. (c) Quantification of dissimilarity values based on (b), presented as dissimilarity values (first (box bottom), third (box top) quartiles, the median (line inside box) and 2.5 interquartile range (line ends)). (d) Bar plots of the phylum taxonomic levels in WT and Hpa-Tg mice. Relative abundance is plotted for each group. The relative abundances of Firmicutes and Bacteroidetes were shown. (e) Bar plots of the class taxonomic levels in WT and Hpa-Tg mice. Relative abundance is plotted for each group. (f) Bar plots of the order taxonomic levels in WT and Hpa-Tg mice. Relative abundance is plotted for each group. Bacterial genera with relative abundance greater than 0.01% were analyzed. (g) Bar plots of the family taxonomic levels in WT and Hpa-Tg mice. Relative abundance is plotted for each group. Bacterial genera with relative abundance greater than 0.1% were analyzed. (h) Bar plots of the genus taxonomic levels in WT and Hpa-Tg mice. Relative abundance is plotted for each group. Bacterial genera with relative abundance greater than 1% were analyzed. Data were expressed as mean ± SEM. n=15 individuals/group. Significance between two groups was determined by unpaired, two-tailed t test or Mann-Whitney test depending on the sample distribution. For (b-c), differences of data were assessed by ANOSIM test. Exact p levels were all provided. ANOSIM, analysis of similarities Hpa-Tg, heparanase-transgenic; WT, wild-type. Supplemental Figure 3. Exacerbated acute pancreatitis in Hpa-Tg mice depended on gut microbiota. (a) ABX experimental design, WT and Hpa-Tg littermates were put on a course of intragastrically antibiotic cocktail administration for 5 days for gut microbiota depletion After that, 16S rRNA sequencing analysis and AP induction were performed. (b) Pancreas (g)/body (g) weight x 1‰. (c) Serum amylase. (d) Serum lipase. (e) Representative images of pancreatic H&E staining. (f) Histologic score. (g) Serum TNF-α measured by ELISA. (h) Serum IL-6 measured by ELISA. (a-h) n=5 individuals/group. Data were expressed as mean ± SEM. Differences of data were assessed by assessed by unpaired, two-tailed t test or Mann-Whitney test depending on the sample distribution. Exact p levels were all provided. Scale bars, 500 and 200 μm, respectively. ABX, antibiotic cocktail; AP, acute pancreatitis; Cn, caerulein; Hpa-Tg, heparanase-transgenic; rRNA, ribosomal RNA; WT, wild-type. Supplemental Figure 4. Beta-diversity among ABX experimental groups. 16S rRNA sequencing analysis in fecal bacterial DNA from ABX WT mice (n=5) and Hpa Tg mice (n=3) was performed. PCoA of beta-diversity using Bray-Curtis (a), Jaccard (b), Generalized Unifrac (c) and Weighted Unifrac (d) metric distance. Differences of data were assessed by ANOSIM test. Exact p levels were all provided. ABX, antibiotic cocktail; ANOSIM, analysis of similarities; Hpa-Tg, heparanase-transgenic; PCoA, principal coordinate analysis; rRNA, ribosomal RNA; WT, wild-type. Supplemental Figure 5. Beta-diversity among FMT experimental groups. 16S rRNA sequencing analysis in fecal bacterial DNA from FMT groups was performed. n=7 individuals/group. PCoA of beta-diversity using Bray-Curtis (a), Jaccard (b), Generalized Unifrac (c) and Weighted Unifrac (d) metric distance. Differences of data were assessed by ANOSIM test. Exact p levels were all provided. ANOSIM, analysis of similarities; Hpa-Tg, heparanase-transgenic; PCoA, principal coordinate analysis FMT, fecal microbiota transplantation; rRNA, ribosomal RNA; WT, wild-type. Supplemental Figure 6. Beta-diversity among CoHo WT and CoHo Hpa-Tg groups. 16S rRNA sequencing analysis in fecal bacterial DNA from CoHo WT and CoHo Hpa-Tg groups was performed. n=7 individuals/group. PCoA of beta-diversity using Bray-Curtis (a), Jaccard (b), Generalized Unifrac (c) and Weighted Unifrac (d) metric distance. Differences of data were assessed by ANOSIM test. Exact p levels were all provided. ANOSIM, analysis of similarities; CoHo, cohoused; Hpa-Tg, heparanase-transgenic; PCoA, principal coordinate analysis; rRNA, ribosomal RNA; WT, wild-type. Supplemental Figure 7. Parabacteroides was the most important biomarker to distinguish WT and Hpa-Tg mice. (a) VIP score of OPLS-DA. VIP score (calculated based on 16S rRNA sequencing data of Fig. 2) was used to rank the ability of different taxa to discriminate between WT and Hpa-Tg mice. A taxon with VIP score >1.5 was considered important in the discrimination. n=15 individuals/group. (b) In ABX experiment, Parabacteroides abundance was confirmed by 16S rRNA sequencing (n=5/3 in ABX WT/ABX Hpa-Tg group, respectively) and qRT-PCR (using specific primers of P. distasonis, n=5 individuals/group). (c) In FMT experiment, Parabacteroides abundance was confirmed by 16S rRNA sequencing and qRT-PCR (using specific primers of P. distasonis). n=7 individuals/group. (d) In Cohousing experiment, Parabacteroides abundance was confirmed by 16S rRNA sequencing and qRT-PCR (using specific primers of P. distasonis). n=7 individuals/group. (e-j) The correlations between severity indicators of AP and Parabacteroides abundance were analyzed using Spearman’s correlations. 6 mice each in WT and Hpa-Tg mice were included in the statistics. (k) In Parabacteroides administration experiment, Parabacteroides abundance was confirmed by 16S rRNA sequencing. n=5 individuals/group. Data were expressed as mean ± SEM. Differences of data in two groups were assessed by assessed by unpaired, two-tailed t test or Mann-Whitney test depending on the sample distribution. Differences of data in more than two groups were assessed by ordinary one-way ANOVA or Kruskal-Wallis test depending on the sample distribution. Exact p levels were all provided. ABX, antibiotic cocktail; AP, acute pancreatitis; FMT, fecal microbiota transplantation; Hpa-Tg, heparanase-transgenic; OPLS-DA, orthogonal partial least squares discrimination analysis; P. distasonis, Parabacteroides. distasonis; qRT-PCR, Quantitative RT-PCR; rRNA, ribosomal RNA; VIP, valuable influence on projection; WT, wild-type. Supplemental Figure 8. Acetate concentration in ABX, FMT and cohousing experiments. (a) The whole annotation of microbial gene function of WT and Hpa-Tg mice on KEGG pathway analysis. Analysis of 16S rRNA sequencing data from Figure 2. n = 15 individuals/group. (b) Relative fold change of acetate concentration from cecal content in ABX experiment. n=5 individuals/group. (c) Relative fold change of acetate concentration from cecal content in FMT experiment. n=7 individuals/group. (d) Relative fold change of acetate concentration from cecal content in CoHo WT and CoHo Hpa-Tg mice. n=7 individuals/group. Data were expressed as mean ± SEM. Differences of data in two groups were assessed by assessed by Mann-Whitney test. Differences of data in FMT experiment was assessed by ordinary one-way ANOVA. Exact p levels were all provided. ABX, antibiotic cocktail; CoHo, cohoused; FMT, fecal microbiota transplantation; Hpa-Tg, heparanase-transgenic; rRNA, ribosomal RNA; KEGG, kyoto encyclopedia of genes and genomes; WT, wild-type. Supplemental Figure 9. Confirmation of acetate enrichment in acetate supplementation experiment. Relative fold change of acetate concentration from cecal content in acetate supplementation experiment. n=9 individuals/group. Data were expressed as mean ± SEM. Differences of data were assessed by ordinary one-way ANOVA. Exact p levels were all provided. WT, wild-type; Hpa-Tg, heparanase transgenic. Supplemental Figure 10. Neutrophil infiltration depended on gut microbiota in heparanase-exacerbated acute pancreatitis. WT and Hpa-Tg mice were treated with/without Cn. Representative plots of immunohistochemistry and bar plots of neutrophils in pancreas were shown in (a) and (b), respectively. Representative plots of immunohistochemistry and bar plots of neutrophils in pancreas of ABX experiment were shown in (c) and (d), respectively. Representative plots of immunohistochemistry and bar plots of neutrophils in pancreas of FMT experiment were shown in (e) and (f), respectively. Representative plots of immunohistochemistry and bar plots of neutrophils in pancreas of cohousing experiment were shown in (g) and (h), respectively. Differences of data in two groups were assessed by unpaired, two-tailed t test. Differences of data in more than two groups were assessed by ordinary one-way ANOVA. Exact p levels were all provided. Scale bars, 200 and 50 μm, respectively. ABX, antibiotic cocktail; FMT, fecal microbiota transplantation; Hpa-Tg, heparanase- X transgenic; Cn, caerulein; CoHo, cohoused. SiHo, housed singly; SW, sterile water; WT, wild-type. Supplemental Figure 11. Alpha rarefaction curves of all 16S rRNA sequencing analyses. Alpha rarefaction curves (Faith pd) in WT and Hpa-Tg groups (a), ABX experiment (b), CoHo WT and CoHo Hpa-Tg groups (c), FMT experiment (d) and the administration of Parabacteroides experiment (e). ABX, antibiotic cocktail; CoHo, cohoused; FMT, fecal microbiota transplantation; Hpa-Tg, heparanase-transgenic; P. distasonis, Parabacteroides distasonis; WT, wild-type. Supplemental Table 1. Schmidt's score system of pancreatic histopathology. Supplemental Table 2. Primers for qRT-PCR detection (SYBR Green). Supplemental Table 3. Primers for qRT-PCR detection (Taqman).

Along with the globally rapid development of e-commerce, multichannel retail including both brick-and-mortar and online stores is in 2016 the standard for the sellers. However, such justified strategy not always guarantee retailers’ success in commercial campaigns. Most of the time the difficulties arised from the lack of efficient coordination between the online store and its offline counterpart based upon their respective attributes. On the basis of the percieved congruence concept, this dissertation investigates subjects’ evaluation on store attributes between the different channels of the Chinese multichannel retail market. Through the Depth Interview (Study 1), it was found that Chinese multichannel consumers prefer the multichannel shopping behavior. They like the between-channel congruence but also expect to enjoy the incongruent situation which indicates their propensity to compare the between-channel prices and assortment. The two quantitative studies identified the perceived congruence as a determinant to the subjects’ evaluation towards the retailer. More precisely, the more congruence subjects perceive from the cross-channel price policy, the more price fairness subjects judge, turns in more favorable attitude toward the retailer (Study 2). In contrast, when the subjects perceive more schema incongruity, from the cross-channel assortment, they evaluate more variety (Study 3). But this consequence only occurs when the cognitive process is not constrained. In addition, both price fairness perception and variety perception are identified to mediate the effect of congruence perception on subjects’ attitude toward retailers. Besides, subjects’ experimental involvement, does not statistically support the moderation effect interacting with congruence perception on subjects’ evaluation regarding channel store attributes, but the marginal effect in visualization indicates that researchers must, in future study, keep investigating the relevant influences between involvement and congruence perception.; Avec le développement rapide du commerce électronique, la vente multi-canaux, du type « brick-and-mortar » et vente en ligne est le standard pour les vendeurs en 2016. Cependant, cette stratégie ne garantit pas toujours le succès des détaillants dans les campagnes commerciales. La plupart du temps les difficultés proviennent d’un manque de coordination efficace entre la boutique en ligne et son homologue hors ligne selon leurs attributs respectifs. Sur la base du concept de congruence perçu, cette thèse étudie l'évaluation par les sujets des attributs des magasins entre les différents canaux du marché multicanaux chinois. Les résultats d’études du type « Depth Interview » (étude 1), montrent que les consommateurs chinois préfèrent le comportement commercial multicanaux. Ils aiment la congruence entre les canaux, mais espèrent aussi profiter des situations non-congruentes, ce qui indique leur propension à comparer les prix et l'assortiment entre canaux. Les deux études quantitatives ont identifié la congruence perçue comme un facteur déterminant pour l'évaluation des détaillants par les sujets. Plus précisément, plus les sujets perçoivent de la congruence dans la politique de prix entre les canaux, plus les sujets jugent les prix justes. des prix équitables, se traduisent par une attitude plus favorable envers le vendeur (étude 2). En revanche, lorsque les sujets perçoivent plus d’incongruité dans l'assortiment cross-canaux, ils considèrent que le choix est plus important (étude 3). Mais cette conséquence ne se produit que lorsque le processus cognitif n’est pas contraint. En outre, quand un prix équitable est perçu et qu’une variété est perçue les deux sont identifiés comme médiateurs de l'effet de la perception de congruence sur l'attitude des sujets envers les détaillants. En outre, l’implication des sujets dans l’expérimentation ne supporte pas statistiquement l'effet de modération en interaction avec la congruence perception sur des sujets de l'évaluation en ce qui concerne les attributs du canal de distribution, mais l'effet marginal dans la visualisation indique que les chercheurs doivent, dans les études futures, continuer l'étude des influences pertinentes entre l’implioation et la congruence perçue.

Along with the globally rapid development of e-commerce, multichannel retail including both brick-and-mortar and online stores is in 2016 the standard for the sellers. However, such justified strategy not always guarantee retailers’ success in commercial campaigns. Most of the time the difficulties arised from the lack of efficient coordination between the online store and its offline counterpart based upon their respective attributes. On the basis of the percieved congruence concept, this dissertation investigates subjects’ evaluation on store attributes between the different channels of the Chinese multichannel retail market. Through the Depth Interview (Study 1), it was found that Chinese multichannel consumers prefer the multichannel shopping behavior. They like the between-channel congruence but also expect to enjoy the incongruent situation which indicates their propensity to compare the between-channel prices and assortment. The two quantitative studies identified the perceived congruence as a determinant to the subjects’ evaluation towards the retailer. More precisely, the more congruence subjects perceive from the cross-channel price policy, the more price fairness subjects judge, turns in more favorable attitude toward the retailer (Study 2). In contrast, when the subjects perceive more schema incongruity, from the cross-channel assortment, they evaluate more variety (Study 3). But this consequence only occurs when the cognitive process is not constrained. In addition, both price fairness perception and variety perception are identified to mediate the effect of congruence perception on subjects’ attitude toward retailers. Besides, subjects’ experimental involvement, does not statistically support the moderation effect interacting with congruence perception on subjects’ evaluation regarding channel store attributes, but the marginal effect in visualization indicates that researchers must, in future study, keep investigating the relevant influences between involvement and congruence perception.; Avec le développement rapide du commerce électronique, la vente multi-canaux, du type « brick-and-mortar » et vente en ligne est le standard pour les vendeurs en 2016. Cependant, cette stratégie ne garantit pas toujours le succès des détaillants dans les campagnes commerciales. La plupart du temps les difficultés proviennent d’un manque de coordination efficace entre la boutique en ligne et son homologue hors ligne selon leurs attributs respectifs. Sur la base du concept de congruence perçu, cette thèse étudie l'évaluation par les sujets des attributs des magasins entre les différents canaux du marché multicanaux chinois. Les résultats d’études du type « Depth Interview » (étude 1), montrent que les consommateurs chinois préfèrent le comportement commercial multicanaux. Ils aiment la congruence entre les canaux, mais espèrent aussi profiter des situations non-congruentes, ce qui indique leur propension à comparer les prix et l'assortiment entre canaux. Les deux études quantitatives ont identifié la congruence perçue comme un facteur déterminant pour l'évaluation des détaillants par les sujets. Plus précisément, plus les sujets perçoivent de la congruence dans la politique de prix entre les canaux, plus les sujets jugent les prix justes. des prix équitables, se traduisent par une attitude plus favorable envers le vendeur (étude 2). En revanche, lorsque les sujets perçoivent plus d’incongruité dans l'assortiment cross-canaux, ils considèrent que le choix est plus important (étude 3). Mais cette conséquence ne se produit que lorsque le processus cognitif n’est pas contraint. En outre, quand un prix équitable est perçu et qu’une variété est perçue les deux sont identifiés comme médiateurs de l'effet de la perception de congruence sur l'attitude des sujets envers les détaillants. En outre, l’implication des sujets dans l’expérimentation ne supporte pas statistiquement l'effet de modération en interaction avec la congruence perception sur des sujets de l'évaluation en ce qui concerne les attributs du canal de distribution, mais l'effet marginal dans la visualisation indique que les chercheurs doivent, dans les études futures, continuer l'étude des influences pertinentes entre l’implioation et la congruence perçue.