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Purpose. Traditional Chinese medicine (TCM) including Chinese patent medicine has been widely used to treat irritable bowel syndrome (IBS). Syndrome differentiation is the essence of TCM. However, the diagnostic ability of gastroenterologists to detect TCM syndromes in IBS in China remains unknown. The aim of this study was to investigate the ability of gastroenterologists to diagnose the TCM syndromes of IBS based on modified simple criteria compared with TCM practitioners. Methods. Patients meeting the Rome III criteria for IBS-D or IBS-C were recruited from six tertiary referral centers between January 2016 and December 2017. After learning the diagnosis criteria of the TCM syndromes in IBS, gastroenterologists first diagnosed the syndromes of the enrolled patients. Subsequently, the patients were diagnosed by TCM practitioners. The rate of agreement between the gastroenterologists and TCM practitioners was analyzed. In addition, demographic data and the distribution of TCM syndrome types in IBS were also analyzed. Results. A total of 178 patients (93 males and 85 females), including 131 patients with IBS-D and 47 patients with IBS-C, were enrolled in this study. The rate of agreement of the syndrome diagnosis between the gastroenterologists and TCM practitioners was 84.3%. The diagnosis consistency rates among IBS-D patients and IBS-C patients were 87.0% and 76.5%, respectively. The most common TCM syndrome type in IBS-D patients was liver depression and spleen deficiency syndrome (27.5%), followed by spleen-yang deficiency syndrome (19.8%). Dryness and heat in intestine syndrome was the most common TCM syndrome in IBS-C patients (57.4%). Conclusions. Gastroenterologists had good diagnostic agreement with TCM practitioners for diagnosing TCM syndrome types in IBS after learning the diagnostic criteria. This knowledge can aid gastroenterologists in selecting suitable Chinese patent medicine to treat IBS.

Facial micro-expression (FME) is a fast and subtle facial muscle movement that typically reflects person’s real mental state. It is a huge challenge in the FME recognition task due to the low intensity and short duration. FME can be decomposed into a combination of facial muscle action units (AU), and analyzing the correlation between AUs is a solution for FME recognition. In this paper, we propose a framework called spatio-temporal AU graph convolutional network (STA-GCN) for FME recognition. Firstly, pre-divided AU-related regions are input into the 3D CNN, and inter-frame relations are encoded by inserting a Non-Local module for focusing on apex information. Moreover, to obtain the inter-AU dependencies, we construct separate graphs of their spatial relationships and activation probabilities. The relationship feature we obtain from the graph convolution network (GCN) are used to activate on the full-face features. Our proposed algorithm achieves state-of-the-art accuracy of 76.08% accuracy and F1-score of 70.96% on the CASME II dataset, which outperformance all baselines.

As one of the most critical pathogens, Aeromonas hydrophila (AH) can cause motile aeromonad septicemia (MAS) in freshwater fish. In recent decades, a myriad of studies had been done for bacterial infect fish. However, the mechanism of bacterial infects fish especially freshwater fish was scanty. This study was conducted for investigating the invasion pathway of A. hydrophila in vivo of black carp. We have performed A. hydrophila 4332 transformed with a plasmid encoding the green fluorescent protein (pGFPuv) (AH4332GFPuv) in black carp. The AH4332GFPuv had similar growth properties and virulence as the wild-type strains under the simulated natural condition. In this study, black carp were divided into five groups: IM (challenged via immersion), IBD (increased stocking density), SAW (skin artificially wounded by scalpel), MR (mucus removed from the body surface), and C0 (control group). The number of AH4332GFPuv in gill, liver, spleen, intestine, mid kidney, head kidney, muscle, eye, brain, heart, and blood were examined after 72 h post-infection from all groups. Significantly high bacterial numbers were observed in the gills and intestine. The number of bacteria was significantly higher in IBD group than IM group. In conclusion, the gill, intestines, and injured skin are likely to be the primary infection routes. info:eu-repo/semantics/publishedVersion

Background: Functional dyspepsia (FD) is a common upper gastrointestinal disorder worldwide, but the current treatments for FD are still unsatisfactory. The aims of this study were to investigate the efficacy and safety of Qi-Zhi-Wei-Tong granules in patients with postprandial distress syndrome (PDS)-predominant FD. Methods: The study was conducted as a randomized, double-blinded, multicenter, placebo-controlled design in 197 patients with PDS. All participants received placebo treatment for 1 week. Patients whose total symptom score decreased by

The attachment of cell-surface carbohydrates to proteins mediated by the amino acids serine or threonine (O-glycan) is involved in tumor metastasis; the roles of O-glycans vary depending on their structure, but the detailed mechanisms by which O-glycans trigger signaling to control tumor metastasis are largely unknown. In this study, we found that the reduced expression of core 3 synthase correlated with metastasis to lymph nodes and distant organs, resulting in poor prognosis for colorectal cancer (CRC) patients. Mechanically, we revealed that mucin-type core 3 O-glycan was synthesized at the membrane-tethered MUC1 N terminus because of core 3 synthase expression in colon cancer cells. This further inhibited the translocation of MUC1-C to the nucleus, initiated p53 gene transcription that was dependent on the inhibition of MUC1-C nucleus translocation, activated p53-mediated miR-200c expression and resulted in mesenchymal-epithelial transition (MET). Inhibition of MUC1 via small interfering RNA (siRNA) in re-expressed core 3 synthase colon cancer cells further inhibited MUC1-C nucleus translocation, increased p53 and miR-200c expression, and enhanced MET. However, inhibition of p53 via siRNA or miR-200c via miR-200c inhibitor in re-expressed core 3 synthase colon cancer cells promoted the epithelial-mesenchymal transition (EMT) in a reversible manner. Core 3 synthase mRNA levels and the p53 mRNA levels or miR-200c levels in the colon cancerous samples were positively correlated. Our findings suggest a novel mechanism linking mucin-type core 3 O-glycan to the EMT-MET plasticity of CRC cells via MUC1/p53/miR-200c-dependent signaling cascade and shed light on therapeutic strategies to treat this malignancy.

Complement is traditionally recognized as part of the innate immune system, defending the host against the invasion of foreign pathogens. In complement system, C3 (complement component 3) is a central component. Therefore, research into C3 can help us better understand the functions of fish complement system. In this study, we detected the grass carp C3 (gcC3) mRNA expression in all sample tissues from healthy grass carp, which was highest in the liver, followed by the heart and the spleen, and lowest in the muscle, head kidney, trunk kidney, blood and intestine. After infection with Aeromonas hydrophila, gcC3 mRNA expression levels were significantly upregulated in the gill, liver, spleen, intestine, trunk kidney and head kidney. Interestingly, C3 protein levels were downregulated and subsequently upregulated in the liver and serum. Histologically, C3 protein at 24 h pi was over expressed in necrotic liver sites, and the liver index (LI) at this point was significantly higher than that of the control. These findings are indicated that C3 plays an important role in the immune response of grass carp after A. hydrophila infection, and C3 protein may play an assistant role in repairing liver tissues from A. hydrophila injury.

Background & Aims Multidrug resistance-associated protein 2 (MRP2) excretes conjugated organic anions including bilirubin and bile acids. Malfunction of MRP2 leads to jaundice in patients. Studies in rodents indicate that Radixin plays a critical role in determining Mrp2 canalicular membrane expression. However, it is not known how human hepatic MRP2 expression is regulated in cholestasis. Methods We assessed liver MRP2 expression in patients with obstructive cholestasis caused by gallstone blockage of bile ducts, and investigated the regulatory mechanism in HepG2 cells. Results Western blot detected that liver MRP2 protein expression in obstructive cholestatic patients (n=30) was significantly reduced to 25% of the non-cholestatic controls (n=23). Immunoprecipitation identified Ezrin but not Radixin associating with MRP2 in human livers, and the increased amount of phospho-Ezrin Thr567 was positively correlated with the amount of co-precipitated MRP2 in cholestatic livers, whereas Ezrin and Radixin total protein levels were unchanged in cholestasis. Further detailed studies indicate that Ezrin Thr567 phosphorylation plays an important role in MRP2 internalization in HepG2 cells. Since increased expression of PKCα, δ and e were detected in these cholestatic livers, we further confirmed that these PKCs stimulated Ezrin phosphorylation and reduced MRP2 membrane expression in HepG2 cells. Finally, we identified GP78 as the key ubiquitin ligase E3 involved in MRP2 proteasome degradation. Conclusions Activation of liver PKCs during cholestasis leads to Ezrin Thr567 phosphorylation resulting in MRP2 internalization and degradation where ubiquitin ligase E3 GP78 is involved. This process provides a mechanistic explanation for jaundice seen in patients with obstructive cholestasis.

// Yangyang Shang 1,* , Qiong Pan 1,* , Lei Chen 1,* , Jun Ye 1 , Xiaoli Zhong 1 , Xiaohuan Li 1 , Linkuan Meng 1 , Jin Guo 1 , Yin Tian 1 , Yonghong He 1 , Wensheng Chen 1 , Zhihong Peng 1 and Rongquan Wang 1 1 Department of Gastroenterology, Southwest Hospital, Third Military Medical University, Chongqing, P. R. China * These authors have contributed equally to this study Correspondence to: Zhihong Peng , email: // Rongquan Wang, email: // Keywords : Achaete scute-like 2, CDX2, transcriptional regulation, colorectal carcinoma, differentiation Received : March 26, 2015 Accepted : August 13, 2015 Published : August 17, 2015 Abstract The role of Achaete scute-like 2 (Ascl2) in colorectal cancer (CRC) cell differentiation is unknown. LS174T, HT-29 and Caco-2 cells have high Ascl2 expression, while Lovo and SW480 cells have low Ascl2 expression. LS174T and HT-29 cells with Ascl2 knockdown were transfected with caudal type homeobox 2 (CDX2) promoter constructs and used for luciferase assays and chromatin immunoprecipitation (ChIP) assays. Ascl2 knockdown promoted differentiation of CRC cells into a goblet cell phenotype, as determined by increased expression of MUC2, TFF3, and CDX2. Ascl2 knockdown activated CDX2 expression through a transcriptional mechanism via direct binding of Ascl2 to the proximal E-box of the CDX2 promoter. Ascl2 over-expression in Lovo and SW480 cells inhibited a goblet cell phenotype, as determined by reduced CDX2 and MUC2 expression. Inverse correlations between Ascl2 and CDX2, and Ascl2 and MUC2 mRNA levels, as well as Ascl2 and CDX2 protein levels were observed in CRC cancerous samples. This study demonstrates CDX2 repression by Ascl2 and highlights a role for Ascl2 in CRC cell differentiation. These findings suggest that the Ascl2/CDX2 axis may serve as a potential therapeutic target in colorectal cancer.

Ascl2, a basic helix-loop-helix transcription factor, is a downstream target of WNT signaling that controls the fate of intestinal cryptic stem cells and colon cancer progenitor cells. However, its involvement in colon cancer and downstream molecular events is largely undefined; in particular, the mechanism by which Ascl2 regulates the plasticity of epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) programs in colon cancer cells remains unknown. In this study, we systematically demonstrate that Ascl2 loss of function in colon cancer cells promotes MET by derepressing the expression of microRNA (miR)-200s (i.e. miR-200b, miR-200a, miR-429, miR-200c, and miR-141) and further activating their expression through a transcriptional mechanism that involves direct binding to the most proximal E-box (E-box2) in the miR-200b-a-429 promoter. Activation of miR-200s due to Ascl2 deficiency led to the inhibition of ZEB1/2 expression and the alteration of epithelial and mesenchymal features. Transfection of miR-200b, miR-200a, and miR-429 inhibitors into Ascl2-deficient colon cancer cells promoted the epithelial-mesenchymal transition in a reversible manner. Transfection of miR-200a or miR-429 inhibitors into Ascl2-deficient colon cancer cells increased cellular proliferation and migration. Ascl2 mRNA levels and the miR-200a, miR-200b, miR-200c, miR-141, or miR-429 levels in the colon cancerous samples were inversely correlated. These results provide the first evidence of a link between Ascl2 and miR-200s in the regulation of EMT-MET plasticity in colon cancer.

We have reported that Achaete scute-like 2 (Ascl2) transcriptionally repressed miR-200 family members and affected the epithelial-mesenchymal transition (EMT)-mesenchymal-epithelial transition (MET) plasticity in colorectal cancer (CRC) cells. However, little is known about the regulation of the Ascl2/miR-200 axis. Here, we found that hypoxia inducible factor-1α (HIF-1α) mRNA levels were positively correlated with Ascl2 mRNA levels and inversely correlated with miR-200b in CRC samples. Mechanistically, we showed that Ascl2 was a downstream target of HIF-1α and had a critical role in the EMT phenotype induced by hypoxia or HIF-1α over-expression. Hypoxia or HIF-1α over-expression activated Ascl2 expression in CRC cells in a direct transcriptional mechanism via binding with the hypoxia-response element (HRE) at the proximal Ascl2 promoter. HIF-1α-induced Ascl2 expression repressed miR-200b expression to induce EMT occurrence. Furthermore, we found HIF-1α was a direct target of miR-200b. MiR-200b bound with the 3′-UTR of HIF-1α in CRC cells. HIF-1α/Ascl2/miR-200b regulatory feedback circuit modulated the EMT-MET plasticity of CRC cells. Our results confirmed a novel HIF-1α/Ascl2/miR-200b regulatory feedback circuit in modulating EMT-MET plasticity of CRC cells, which could serve as a possible therapeutic target.

Cyclooxygenase-2 (COX-2) inhibitor, celecoxib, causes growth inhibition of human gastric carcinoma cells, but it remains unclear whether celecoxib inhibits Helicobacter pylori-induced invasion of gastric cancer cells. The adenine nucleotide translocator (ANT) is a mitochondrial bi-functional protein. We speculate that ANT-dependent pathways might contribute to H. pylori-induced invasion and metastasis of gastric cancer cells. Therefore, in the present study, we evaluate the effect of celecoxib on H. pylori-induced gastric cancer cell motility and invasion. We also explore the role of ANTs in H. pylori-induced gastric cancer cell motility and invasion of gastric cancer cell line AGS. Our results demonstrate that celecoxib induces anoikis-like apoptosis and suppresses the proliferation and invasion of gastric cancer cells induced by H. pylori in culture. RT-PCR and Western blot analysis indicates that celecoxib upregulates the expression of ANT1 and ANT3; however, celecoxib did not increase the expression of ANT2. Our results suggest that celecoxib could be an effective means for suppressing proliferation and invasion of gastric cancer cells induced by H. pylori through an adenine nucleotide translocator-dependent mechanism.

Chronic gastritis associated with Helicobacter pylori is a leading cause of gastric intestinal metaplasia (IM), which arises from abnormal cell differentiation of the epithelium in the gastric mucosa. However, the mechanisms involved in H. pylori-mediated IM remain elusive. The aim of our study was to explore the effects and the underlying mechanisms of H. pylori on the abnormal expression of Hath1 and Sox2 and to reveal its relationship to the development of gastric IM. We found that Hath1 and Sox2 were overexpressed in gastric IM tissue. Hath1 expression was up-regulated, whereas Sox2 expression, which was independent of the CagA virulence factor, was down-regulated in gastric epithelial cells and coincided with increased IL-6 and IL-8 levels in the culture media. Stimulation with H. pylori-related cytokine IL-8, but not IL-6 or IL-1β, was induced by Hath1 expression in the gastric epithelial cells. Although IL-8 and IL-6 levels correlated with STAT3 (signal transducer and activator of transcription) phosphorylation before and after H. pylori eradication in the gastric mucosa, only the blocking of IL-8-induced STAT3 activation using AG490 or STAT3-targeting RNA interference altered Hath1 expression. Additionally, we found that H. pylori down-regulated Hes1, which is a direct downstream target gene of Notch signaling and a repressor of Hath1 expression. These findings suggest that H. pylori induced inflammation up-regulate Hath1 expression via interleukin-8/STAT3 (IL-8) phosphorylation while suppressing Hes1, which provides a novel molecular connection between a H. pylori infection and intestinal metaplasia.

We characterized and identified the cDNA sequence of Toll-like receptor 20.2 in Ctenopharyngodon idella (gctlr20.2); it consisted of 3197 bp, with an open reading frame of 2835 bp that encoded a 944 amino acid polypeptide. Relatively, high expression levels of gctlr20.2 were observed in the spleen, head kidney, liver and brain tissues, with lower expression levels in the trunk kidney, intestine and heart tissues. In vivo and in vitro, after being challenged with Aeromonas hydrophila or grass carp reovirus (GCRV), gctlr20.2 expression was induced in C. idella kidney cells stimulated with lipopolysaccharide, flagellin or polyinosinic-polycytidylic acid. Overexpression of gctlr20.2 increased the expression of il1β, il8 and tnf-α, but not ifn, and also increased the activity of the nf-κB signal pathway. Silencing, via siRNA-tlr20.2, inhibited gctlr20.2 transcription by 65.7% and down-regulated the expression of inflammatory cytokine genes, but not tnf-α. This study increases understanding of the immune system in C. idella.

Multidrug resistance-associated protein 3 (MRP3, ABC subfamily C [ABCC]3) plays an important role in protecting hepatocytes and other tissues by excreting an array of toxic organic anion conjugates, including bile salts. MRP3/ABCC3 expression is increased in the liver of some cholestatic patients, but the molecular mechanism of this up-regulation remains elusive. In this report, we assessed liver MRP3/ABCC3 expression in patients (n = 22) with obstructive cholestasis caused by gallstone blockage of bile ducts and noncholestatic patient controls (n = 22). MRP3/ABCC3 messenger RNA (mRNA) and protein expression were significantly increased by 3.4- and 4.6-fold, respectively, in these cholestatic patients where elevated plasma tumor necrosis factor alpha (TNFα) (4.7-fold; P < 0.01) and hepatic specificity protein 1 transcription factor (SP1) and liver receptor homolog 1 expression (3.1- and 2.1-fold at mRNA level, 3.5- and 2.5-fold at protein level, respectively) were also observed. The induction of hepatic MRP3/ABCC3 mRNA expression is significantly positively correlated with the level of plasma TNFα in these patients. In HepG2 cells, TNFα treatment induced SP1 and MRP3/ABCC3 expression in a dose- and time-dependent manner, where increased phosphorylation of c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) was also detected. These inductions were significantly reduced in the presence of the JNK inhibitor, SP600125. TNFα treatment enhanced HepG2 cell nuclear extract-binding activity to the MRP3/ABCC3 promoter, but was abolished by SP600125, as demonstrated by electrophoretic mobility shift assay (EMSA). An increase in nuclear protein-binding activity to the MRP3/ABCC3 promoter, consisting primarily of SP1, was also observed in liver samples from cholestatic patients, as assessed by supershift EMSA assays. Conclusions: Our findings indicate that up-regulation of hepatic MRP3/ABCC3 expression in human obstructive cholestasis is likely triggered by TNFα, mediated by activation of JNK/SAPK and SP1. (HEPATOLOGY 2012)

The human cervical cancer oncogene HCCR-2 is overexpressed in various malignant tumors and cell lines, and might function as a negative regulator of the p53 tumor suppressor. Here, we used RNA interference strategies to evaluate the role of HCCR-2 in liver cancer, and to explore its potential therapeutic effect. Changes of HepG2 cells stably transfected by an HCCR-2 RNA interference vector were detected by real-time PCR, MTT staining, plate colony formation, flow cytometry, and cell migration experiments. Apoptosis-related protein Bcl-2 and Bax levels were measured by Western blot. Our results showed that of the three siRNA-expressing vectors, siRNA-H3 had a suppressive effect on the expression of HCCR-2 mRNA, interfering with proliferation and migration of HCCR-2. Moreover, the apoptotic rate also increased, and cells transfected by siRNA-H3 were blocked in the G0/G1 stage. Plate colony formation experiments demonstrated that the single cell clone formation capacity of HepG2-H3 cells was clearly lower than that of HepG2 and HepG2-N cells. Western blot results indicated that the expression of Bcl-2 was inhibited, and the expression of Bax was increased. In summary, RNAi targeting HCCR-2 could be an effective means for suppressing malignant features of hepatocellular carcinoma cells.

Tumor necrosis factor-related apoptosis induced ligand (TRAIL) is an important apoptosis inducer in a variety of tumor cells. In the present study, we determined the underlying molecular mechanisms by which certain gastric cancer cells are resistant to TRAIL. We first detected expression of programmed cell death 4 (PDCD4) in three gastric cancer cell lines and identified its association with the sensitivity of gastric cancer cells to TRAIL. We then stably transfected PDCD4 cDNA or shRNA into these gastric cell lines. Our data showed that restoration of PDCD4 expression induced TRAIL sensitivity, whereas knockdown of PDCD4 expression reduced the sensitivity of these tumor cells to TRAIL treatment. PDCD4 was able to suppress expression of FLICE-inhibiting protein (FLIP), a negative regulator of apoptosis. Knockdown of FLIP expression using FLIP shRNA had similar effects as those of restored PDCD4 expression. Furthermore, the proteasome inhibitor MG132 was able to inhibit expression of FLIP mRNA and protein and upregulate the sensitivity of these cells to TRAIL treatment. Taken together, the results from the current study demonstrated that PDCD4 plays an important role in mediating the sensitivity of gastric cancer cells to TRAIL-induced apoptosis through FLIP suppression. Therefore, the proteasome inhibitor MG132 should be further evaluated for combination therapy with TRAIL.

SOX2 and Hath1 are transcription factors that are critical for the control of terminal cell differentiation in the gastrointestinal mucosa. This study investigated the correlations between SOX2 and Hath1 expression in gastric carcinoma and patients’ clinicopathological features and prognosis. Hath1 and SOX2 were detected by immunohistochemistry in gastric carcinoma (n = 50). Probability of survival of patients after surgery was estimated by the Kaplan–Meier method and compared using Log-rank test. Hath1 and SOX2 were inversely expressed in gastric carcinoma. Patients with strong SOX2 expression (++ to +++) showed lower incidences of lymph node metastasis (p = 0.007), deeper invasion (p = 0.010), and III–IV clinical stages (p = 0.011) compared to patients with low SOX2 expression (− to +). There was no significant difference in SOX2 and Hath1 expression in the cancerous tissues of the patients with and without Helicobacter pylori infection (p > 0.05). The patients with strong expression of SOX2 in their cancerous tissues (++ to +++) had a better prognosis than those with low expression of SOX2 (− to +; p = 0.005). There was no correlation between Hath1 expression level and prognosis (p = 0.676). SOX2 and Hath1 are inversely expressed in gastric carcinoma. SOX2 provides a survival advantage to patients of gastric carcinoma and appears to be associated with metastasis and clinical stages.

Background/Aims: The transjugular intrahepatic portosystemic shunt (TIPS) is technically divided into TIPS through the left branch of the portal vein (TIPS-LBPV) and TIPS through the right branch of the portal vein (TIPS-RBPV). In order to compare their advantages and disadvantages, this randomized, controlled trial was designed to investigate their outcomes in advanced cirrhotic patients. Methods: Seventy-two patients were randomly placed into TIPS-LBPV (36 patients) and TIPS-RBPV (36 patients, with four failures) groups, and they were prospectively followed for 2 years after TIPS implantation. Results: Patients who underwent the two different kinds of TIPS were balanced during recruitment for this study. The incidences of overall encephalopathy and de novo encephalopathy in the TIPS-LBPV group were significantly lower than that of the TIPS-RBPV group during follow-up (P=0.036 and 0.012 respectively). The incidences of rebleeding or re-intervention and improvement of ascites were similar between groups (P>0.05). Patients undergoing TIPS-RBPV required more rehospitalization and incurred more costs than those who underwent TIPS-LBPV (P=0.030 and 0.039 respectively). There was no significant difference between the two groups in survival based on a survival curve constructed according to the Kaplan–Meier method (P>0.05). Conclusion: Patients undergoing TIPS-LBPV had a lower incidence of encephalopathy, less rehospitalization and lower costs after TIPS implantation compared with patients undergoing TIPS-RBPV.

Background & Aims Levels of bile acid metabolic enzymes and membrane transporters have been reported to change in cholestasis. These alterations (e.g. CYP7A1 repression and MRP4 induction) are thought to be adaptive responses that attenuate cholestatic liver injury. However, the molecular mechanisms of these adaptive responses in human obstructive cholestasis due to gallstone biliary obstruction remain unclear. Methods We collected liver samples from cholestatic patients with biliary obstruction due to gallstones and from control patients without liver disease (n = 22 per group). The expression levels of bile acid synthetic and detoxification enzymes, membrane transporters, and the related nuclear receptors and transcriptional factors were measured. Results The levels of bile acid synthetic enzymes, CYP7B1 and CYP8B1, and the detoxification enzyme CYP2B6 were increased in cholestatic livers by 2.4-fold, 2.8-fold, and 1.9-fold, respectively (p

The roles of host glycosylation in interactions with EPEC and EHEC O157:H7 are largely unclear; this study examined whether O-glycans are involved in EPEC and EHEC O157:H7 adherence to HT-29 cells. Bacterial adherence to the cultured cells was determined using the direct co-staining of adherent bacteria and host cells, the adherent bacteria plating, and/or the direct fluorescent observation of the adherent GFP-labeled bacteria. A comparison of the adherence of EPEC and EHEC O157:H7 to HT-29-Gal and HT-29 cells indicated that the differentiation of HT-29 cells led to a reduction in the adherence of EPEC and EHEC O157:H7. EPEC and EHEC O157:H7 adhesion decreased after the abrogation of O-glycan biosynthesis mediated by benzyl-α-GalNAc treatment. Core 2 O-glycan-deficient HT-29 cells induced by C2GnT2 knockdown had a significant reduction in EPEC and EHEC O157:H7 adhesion in C2GnT2-sh2/HT-29 cells compared with HT-29 and shRNA-Ctr/HT-29 cells. MUC2 expression in benzyl-α-GalNAc-treated HT-29 cells was significantly reduced but unchanged in C2GnT2-deficient HT-29 cells. EPEC or EHEC O157:H7 infection in C2GnT2-deficient HT-29 cells deteriorated the epithelial barrier function. The occludin expression in the shRNA-Ctr/HT-29 and C2GnT2-sh2/HT-29 cells after infection with EPEC or EHEC O157:H7 was pyknic and discontinuous at the cell surface compared with its continuous distribution of control cells. These data indicate that EPEC and EHEC O157:H7 adherence to HT-29 cells is related to mucin-type core 2 O-glycan. This study provides the concepts toward the design of carbohydrate-dependent inhibition of EPEC and EHEC O157:H7 adhesion to human intestinal epithelial cells.

In a previous study we showed, by transient expression studies in COS-1 cells, that the C-terminal domain of rat intestinal membrane mucin Muc3 was cleaved between glycine and serine within a GSIVV (one-letter) amino acid sequence during its residence in the endoplasmic reticulum. The extracellular domain fragment remained linked to the membrane-associated fragment by non-covalent interactions. The present study demonstrates that cleavage depends not only on the presence of the G/SIVV site (where G/S is the glycine↓serine cleavage site), but also on more distant C-terminal sequences in the SEA (sea-urchin sperm protein, enterokinase and agrin) module. Inhibition of N-glycosylation by tunicamycin treatment of transfected cells did not prevent re-association of fragments, although cleavage was partially impaired, as some of the non-glycosylated, non-cleaved products were seen to accumulate in cells. Membrane targeting of the Muc3 domain and its cleavage products occurred in transfected cells and was not impaired in mutants in which the cleavage site was mutated. Targeting was also not impaired for products devoid of N-linked oligosaccharides. Our studies thus indicate that (a) cleavage within the SEA module of rat Muc3 requires participation of peptide sequences located C-terminal of and distant from the cleavage site, (b) re-association of the fragments requires the SEA module, but is independent of N-linked oligosaccharides, and (c) membrane targeting of the mucin is independent of the SEA-module-cleavage reaction.

Since induction of hTERT expression and subsequent telomerase activation play a critical role in the multistep process of tumorigenesis, a better understanding of hTERT regulation may provide not only a rationale for the molecular basis of cancer progression but also a path to the development of cancer prevention. The c-Myc oncoprotein can function effectively in activating the transcriptional expression of hTERT through E-box elements on its promoter. E2F transcription factor 1 (E2F1) was found to be a repressor of hTERT transcription by directly binding to its promoter, thereby inhibiting hTERT protein expression. For the extensively crosstalk between c-Myc and E2F1 signals, which is now known to be vital to cell fate, we speculated that E2F1 may play a negative regulatory role in c-Myc-induced hTERT transcription. In the present study, we chose to use human embryonic fibroblast cells as an experimental model system, and present evidence that the E2F1 transcription factor constitutes a negative regulatory system to limit c-Myc transcriptional activation of hTERT in normal cells. Furthermore, we demonstrated that upregulation of the miR-17-92 cluster (miR-20a/miR-17-5p) is involved in the regulation of E2F1-mediated negative feedback of the c-Myc/hTERT pathway. Our results not only reveal novel insights into how normal cells control the transmission of c-Myc-mediated oncogenic signals, but also further establish E2F1 as an important molecular target for cancer therapy.

Aim To investigate the effects of the nitrous oxide (NO)-donor sodium nitroprusside (SNP) on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in human gastric cancer cells. Methods The MTT assay and flow cytometry were used to detect cellular proliferation and markers of apoptosis, respectively. Expression levels of caspases-8, and 9 were determined by Western blot. Changes in Nitric Oxide Synthase (NOS) activity, NO production, and caspase activation were also evaluated. Results We found that TRAIL induced apoptosis and cell cycle arrest in human gastric cancer cell lines, and that this effect was mediated by NO production, and activation of both the extrinsic and intrinsic signaling pathways of apoptosis. In addition, we found that the NO-donor SNP sensitizes gastric cancer cells to TRAIL-mediated apoptosis. Treatment of cells with both TRAIL and SNP resulted in increased activation of caspase-8 and caspase-9 and NO release. Inhibition of caspase-8 blocked cell TRAIL-induced apoptosis, while a selective caspase-9 inhibitor was unable to prevent apoptosis induced by either TRAIL or TRAIL plus SNP. Inhibition of NOS could block the activation of caspase-9, but had no obvious effect on cell apoptosis. Conclusions SNP-sensitized gastric cancer cells to TRAIL-induced cytotoxicity by stimulating the release of NO, in turn facilitating the mitochondria-mediated signal transduction pathway. The engagement of the mitochondria signaling pathways along with the TRAIL death receptor signaling pathway synergistically increase levels of apoptosis in these cells.

We investigated whether a synthetic tetrameric branched peptide based on the conserved TFLK motif from mammary-associated serum amyloid A3 (M-SAA3) is more efficient than the monomeric peptide at up-regulating MUC3 expression and examined the possible mechanism(s) and biological significance of this process. We used standard solid-phase methods to synthesize a tetrameric branched peptide (sequence GWLTFLKAAG) containing a trilysine core, termed the TFLK-containing 10-mer BP. The aberrant expression of transcription factors was analyzed using a transcription factor protein/DNA array. MUC3 and relevant transcription factors were detected using real-time PCR and/or Western blots. The luciferase assay, EMSA, and ChIP assays were used to analyze the activity of the human MUC3 promoter. The bacterial adherence assay was used to evaluate the in vitro inhibition of enteropathogenic Escherichia coli or enterohemorrhage E. coli serotype O157:H7 (EHEC O157:H7) adherence to HT-29-Gal cells after treatment with the TFLK-containing 10-mer BP. In HT-29-Gal cells, the TFLK-containing 10-mer BP induced higher levels of MUC3 expression than the M-SAA3-derived N-terminal 10-mer monomeric peptide, and MUC3 expression was activated through transcriptional mechanisms, including the induction of multiple transcription factors and further binding with their cis-elements between nucleotides −242 and −62 within MUC3 promoter. Interestingly, the TFLK-containing 10-mer BP dramatically inhibited enteropathogenic E. coli and EHEC O157:H7 adherence to the HT-29-Gal cells compared with the controls. This finding suggests a potential therapeutic use for this peptide to prevent gastrointestinal infection.

Resistance to anoikis, the subtype of apoptosis induced by lack of matrix adhesion, contributes to malignant transformation and development of metastasis. MicroRNAs play key regulatory roles in tumorigenesis and metastasis. In this study, we described that miR-26a, which is usually downregulated in tumor cells, is involved in the acquisition of anoikis-resistance of human esophageal adenocarcinoma (EA) cells. Results of qRT-PCR in clinical samples showed that downregulated miR-26a expression is related to tumorigenesis and metastasis of EA. In vitro experiments determined that miR-26a directly participates in the regulation of cell cycle and anoikis of human EA OE33 cells. Further, we identified that Rb1 is the direct functional target of miR-26a, and revealed that the reduction of miR-26a expression leads to increased Rb1 protein level and thus inhibits the function of E2F1, by which it influences the phenotypes of cell cycle and anoikis. The findings we reported here presented the evidence that miR-26a may be involved in regulation of anoikis-resistance of EA cells. Targeting miR-26a may provide a novel strategy to inhibit metastasis.

CpG oligodeoxynucleotides (CpG ODN) have the potential to enhance the antigen-presenting cells function of human naïve B cells. In this study, we aim to define the effect of CpG ODNs on the binding capacity of human naïve B cells for different Hepatitis B virus (HBV) epitopes. Three HLA-A2 restricted epitopes were selected to incubate with CpG ODN-primed human naïve B cells. Binding capacity for each epitope and expression of CD80, CD86, class I major histocompatibility complex (MHC), and class II MHC of naïve B cells was tested, respectively, by flow cytometry. CpG ODNs, especially ODN 2216, enhanced the binding capacity of human naïve B cells for HBV epitopes (p < 0.01), and induced markedly higher expression of CD80, CD86, class I MHC, and class II MHC. The binding capacity of CpG-treated naive B cells for each epitope was significantly different. In all the 3 subjects, CpG ODN 2216-primed naïve B cells showed the highest binding ability for Env172–180 compared with the other epitopes with a high expression of co-stimulatory and MHC molecules. CpG ODN showed the potential to selectively enhance the binding capacity of human naïve B cells for HBV epitopes. These results suggest new strategies for development of vaccine design.

This study was designed to determine whether a transjugular intrahepatic portosystemic shunt (TIPS) combined with embolotherapy was superior to TIPS alone.Seventy-nine patients were included in the study (43 in the TIPS and embolotherapy group and 36 in the TIPS alone group). Embolotherapy was performed after TIPS using coils and a tissue adhesive agent. The portosystemic pressure gradient (PPG) after TIPS was lower than 12 mm Hg in all patients. Multivariate analyses were performed using a Cox regression model, and the probabilities of survival and rebleeding were estimated with the Kaplan-Meier method.Baseline patient survey data showed similar distributions in both groups. The mean follow-up time was 45.6 months (range: 1 to 85.6 mo). There were no significant differences in the incidences of rebleeding (P=0.889), stent revision (P=0.728), encephalopathy (P=0.728), the cumulative survival rate (P=0.552), or the probability of being free of rebleeding (P=0.806) between the 2 groups. Of 9 patients with rebleeding after TIPS plus embolotherapy, 7 had a history of esophageal variceal bleeding and 2 had gastric variceal bleeding. Of 8 patients with rebleeding after TIPS alone, 4 had a history of esophageal variceal bleeding and 4 had gastric variceal bleeding (P=0.247). Multivariate analysis showed that PPG after TIPS was an independent predictor of rebleeding (P=0.036). Age and Model of End-stage Liver Disease score were independent predictors of survival (P=0.048 and 0.037).The results suggest that TIPS with embolotherapy cannot reduce the risk of rebleeding if PPG is less than 12 mm Hg after TIPS. PPG after TIPS is an independent predictor of rebleeding.

Numb was originally identified as an important cell fate determinant that is asymmetrically inherited during mitosis and controls the fate of sibling cells by inhibiting the Notch signaling pathway in neural tissue. The small intestinal epithelium originates from the division of stem cells that reside in the crypt, which further differentiate into goblet cells, absorptive cells, paneth cells, and enteroendocrine cells. However, Numb's involvement in the differentiation process of intestinal epithelium is largely unknown. In the present study, we confirm that both the Numb mRNA and protein isoforms are expressed in adult mouse intestinal mucosa. Numb protein is ubiquitously expressed throughout the crypt–villus axis of the small intestinal epithelium and is mainly localized to the cytoplasmic membrane. Down-regulation of endogenous Numb using RNA interference in cultured intestinal LS174T cells increased Notch signaling, leading to the up-regulation of Hes1 and the down-regulation of Hath1. Knockdown of Numb alleviated MUC2 protein expression and led to loss of the goblet cell phenotype in LS174Tl cells. Our results provide the first evidence that Numb, an important cell fate determinant, modulates intestinal epithelial cells towards the goblet cell phenotype by inhibiting the Notch signaling pathway.

Hepatic multidrug resistance-associated protein 4 (Mrp4) levels are low, but increase markedly in rodent cholestatic liver. Nuclear receptors (NRs) are essential for regulating Mrp4 expression in cholestasis models. However, information about MRP4 and related NRs, including constitutive androstane receptor (CAR), pregnane X receptor (PXR), and retinoic X receptor-α (RXRα), is relatively lacking in human obstructive cholestasis. We collected liver samples from patients with obstructive cholestasis or without liver disease and investigated the expression of MRP4 and NRs CAR, PXR, and RXRα by semi-quantitative RT-PCR, Western blot and immunostaining assays.MRP4 mRNA/protein levels were markedly increased in obstructive cholestasis. Concentration of serum total bile acids (TBA) was significantly correlated with MRP4 protein in cholestasis samples (P 0.01). PXR and RXRα mRNA/protein levels were significantly increased in obstructive cholestasis. CAR mRNA levels were unchanged while protein levels were markedly induced in obstructive cholestasis. There was a statistically positive correlation between MRP4 mRNA and CAR protein (P 0.05), suggesting that CAR may activate transcription of MRP4 genes by its nuclear translocation.Hepatic MRP4 levels were dramatically induced in human obstructive cholestasis, which may reduce liver injury by increasing efflux of toxic bile acids from hepatocytes into blood.

Recent investigations discovered that CIAPIN1 might be another drug resistance-associated molecule in cancer cells. However, the underlying mechanisms of CIAPIN1-related multidrug resistance (MDR) remain elusive. In the present study, we investigated the role and possible mechanisms of CIAPIN1 in MDR of human colon carcinoma LoVo/Adr cells which express the wild-type p53 gene. By using small interference RNA and gene transfection techniques, we found that knockdown of CIAPIN1 expression re-sensitized LoVo/Adr cells to anti-cancer drugs and up-regulation of CIAPIN1 in sensitive LoVo cells resulted in a distinct MDR phenotype. We further revealed that CIAPIN1 conferred the MDR phenotype in LoVo/Adr cells through up-regulating expression of MDR-1 (P-gp) and Bcl-xL. Finally, by analyzing the effect of inactivation of wild-type p53 on CIAPIN1-induced up-regulation of P-gp and Bcl-xL, we determined that CIAPIN1 could exhibit its MDR-related function independently of the p53 signaling pathway. Overall, the results presented here further suggest that over-expression of CIAPIN1 is an important mechanism of drug resistance in human cancers, even if not the sole one.

TRAIL has been reported to induce apoptosis in a variety of tumor cell types including hepato-cellular carcinoma (HCC) cell lines. However, considerable numbers of HCC cells, especially some highly malignant tumors, show resistance to TRAIL-induced apoptosis. The molecular mechanisms that regulate sensitivity versus resistance of tumor cells to TRAIL-induced apoptosis remain poorly defined. It has been shown that human telomerase catalytic subunit (hTERT) is overexpressed in human HCCs. In this study, we investigated the effects and the mechanisms of hTERT RNAi on the TRAIL-induced apoptosis of HCC cells that exhibit resistance to TRAIL. Our results indicate that hTERT RNAi sensitizes TRAIL-resistant HCC cells to TRAIL-induced apoptosis. hTERT RNAi-mediated sensitization to TRAIL-induced apoptosis is accompanied up-regulation of procaspases-8 and -9, inhibition of telomerase activity and loss of telomere length. Our results suggest that hTERT RNAi overcame the resistance of the HCC cells against TRAIL, at least in part, via the mitochondrial type II apoptosis pathway and telomerase-dependent pathway.

Background Achaete scute-like 2 (Ascl2), a basic helix-loop-helix (bHLH) transcription factor, controls the fate of intestinal stem cells. However, the role of Ascl2 in colon cancer progenitor cells remains unknown. The cell line HT-29 (47.5–95% of CD133+ population) and LS174T (0.45% of CD133+ population) were chosen for functional evaluation of Ascl2 in colon cancer progenitor cells after gene knockdown by RNA interference. Methodology/Principal Findings Immunohistochemistry demonstrated that Ascl2 was significantly increased in colorectal adenocarcinomas. Downregulation of Ascl2 using RNA interference in cultured colonic adenocarcinoma HT-29 and LS174T cells reduced cellular proliferation, colony-forming ability, invasion and migration in vitro, and resulted in the growth arrest of tumor xenografts in vivo. The Ascl2 protein level in CD133+ HT-29 cells was significantly higher than in CD133− HT-29 cells. Ascl2 blockade via shRNA interference in HT-29 cells (shRNA-Ascl2/HT-29 cells) resulted in 26.2% of cells staining CD133+ compared with 54.7% in control shRNA-Ctr/HT-29 cells. The levels of ‘stemness’ associated genes, such as CD133, Sox2, Oct4, Lgr5, Bmi1, and C-myc, were significantly decreased in shRNA-Ascl2/HT-29 and shRNA-Ascl2/LS174T cells in vitro as well as in the corresponding tumor xenograft (CD133 was not performed in shRNA-Ascl2/LS174T cells). The shRNA-Ascl2/HT-29 cells had inhibited abilities to form tumorspheres compared with control. The microRNA (miRNAs) microarrays, identified 26 up-regulated miRNAs and 58 down-regulated miRNAs in shRNA-Ascl2/HT-29 cells. Expression levels of let-7b, miRNA-124, miRNA-125b, miRNA-17, miRNA-20a and miRNA-302b, involved in the regulation of ‘stemness’, were quantified with qPCR, which confirmed their identities. Restoration of miRNA-302b, via its mimic, led to the restoration of shRNA-Ascl2/HT-29 ‘stemness’ characteristics, including tumorsphere formation and ‘stemness’ associated genes levels, and the recovery of cellular behaviors, including colony-forming ability, invasion and migration in vitro. Conclusions/Significance Ascl2 may be a potential target for the inhibition of colon cancer progenitor cells, and functions through a miR-302b-related mechanism.

Background The secreted proteins from goblet cells compose the intestinal mucus. The aims of this study were to determine how they exist in two intestinal mucus layers. Methodology/Principal Findings The intestinal mucosa was fixed with Carnoy solution and immunostained. Mucus from the loose layer, the firm layer was gently suctioned or scraped, respectively, lysed in SDS sample buffer with or without DTT, then subjected to the western blotting of rTFF3, rIgGFcγBP or rMuc2. The non-reduced or reduced soluble mucus samples in RIPA buffer were co-immunoprecipitated to investigate their possible interactions. Polyclonal antibodies for rTFF3, the rIgGFcγBP C-terminal domain and the rMuc2 C-terminal domain confirmed their localization in the mucus layer and in the mucus collected from the rat intestinal loose layer or firm layer in both western blot and immunoprecipitation experiments. A complex of rTFF3, which was approximately 250 kDa, and a monomer of 6 kDa were present in both layers of the intestinal mucus; rIgGFcγBP was present in the complex (250–280 kDa) under non-reducing conditions, but shifted to 164 kDa under reducing conditions in both of the layers. rMuc2 was found mainly in a complex of 214–270 kDa under non-reducing conditions, but it shifted to 140 kDa under reducing conditions. The co-immunoprecipitation experiments showed that binding occurs among rTFF3, rIgGFcγBP and rMuc2 in the RIPA buffer soluble intestinal mucus. Blocking the covalent interaction by 100 mM DTT in the RIPA buffer soluble intestinal mucus disassociated their binding. Conclusions/Significance Rat goblet cell-secreted TFF3, IgGFcγBP and Muc2, existing in the two intestinal mucus layers, are bound together by covalent interactions in the soluble fraction of intestinal mucus and form heteropolymers to be one of the biochemical mechanisms of composing the net-like structure of mucus.