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The tight junction protein claudin‐7 is essential for tight junction function and intestinal homeostasis. Cldn7 deletion in mice leads to an inflammatory bowel disease‐like phenotype exhibiting severe intestinal epithelial damage, weight loss, inflammation, mucosal ulcerations, and epithelial hyperplasia. Claudin‐7 has also been shown to be involved in cancer metastasis and invasion. Here, we test our hypothesis that claudin‐7 plays an important role in regulating colonic intestinal stem cell function. Conditional knockout of Cldn7 in the colon led to impaired epithelial cell differentiation, hyperproliferative epithelium, a decrease in active stem cells, and dramatically altered gene expression profiles. In 3D colonoid culture, claudin‐7–deficient crypts were unable to survive and form spheroids, emphasizing the importance of claudin‐7 in stem cell survival. Inhibition of the Hippo pathway or activation of Notch signaling partially rescued the defective stem cell behavior. Concurrent Notch activation and Hippo inhibition resulted in restored colonoid survival, growth, and differentiation to the level comparable to those of wild‐type derived crypts. In this study, we highlight the essential role of claudin‐7 in regulating Notch and Hippo signaling–dependent colonic stem cell functions, including survival, self‐renewal, and differentiation. These new findings may shed light on potential avenues to explore for drug development in colorectal cancer. [ABSTRACT FROM AUTHOR]

Epithelial cells are polarized with defined apical tight junctions (TJs), lateral adherens junctions (AJs), and basal integrin–matrix interactions. However, it is increasingly recognized that resident cell junction proteins can be found in varying locations and with previously unrecognized functions. Our study here presents the nanoarchitecture and nanocolocalization of cell junction proteins in culture and tissue by stochastic optical reconstruction microscopy (STORM). The Z‐axial view of noncancerous MDCK‐II and PZ‐HPV‐7 cell–cell junctions resolved β‐catenin and p120ctn localizations to TJs and AJs, with p120ctn apical to β‐catenin and colocalizing with TJ protein claudin‐7. More basally, p120ctn and β‐catenin become colocalized. This topography was lost in isogenic Ras‐transformed MDCK cells and cancerous PC3 cells, where p120ctn becomes basally localized in relation to β‐catenin. Claudin‐7 gene conditional knockout (cKO) in mice also have altered polarity of p120ctn relative to β‐catenin, like that seen in normal‐to‐cancer cell phenotypic transformation. Additionally, claudin‐7 cKO resulted in redistribution and relocalization of other cell junction proteins, including claudin‐1, zonula occludens‐1, integrin α2, epithelial cell adhesion molecule, and focal adhesion kinase (FAK); specifically, integrin α2 and FAK were observed at the apical–lateral compartment. Our data show that STORM reveals regional cellular junction nanoarchitecture previously uncharacterized, providing new insight into potential trans‐compartmental modulation of protein functions. [ABSTRACT FROM AUTHOR]

Background: C9ORF72 plays important roles in many cellular processes, including autophagy, membrane trafficking and immune response. The C9ORF72 mutation is the most common cause of familial amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) which is attributed to both gain‐ and loss‐of‐function mechanisms. C9ORF72 forms a complex with SMCR8 and WDR41, which was reported to regulate signaling of small GTPases such as Rab and ARF proteins. Developing small molecules that modulate interfacial C9ORF72:SMCR8:WDR41 (CSW) and small GTPase interactions will aid in understanding their mode of actions and the potential roles of GTP‐dependent signaling in ALS/FTD. Since the C9ORF72 complex has been shown to elicit prominent GTPase activating protein (GAP) activity on ARF, a major regulator of membrane traffic, we aimed to develop novel small molecules that interfere with the binding between the C9ORF72 complex and ARF1. Method: To identify novel ligands to interfere with the binding between CSW complex and ARF1, we applied in silico virtual screening methods. To this end, the crystal structure of the CSW in complex with ARF1 (PDB: 7MGE) was used for screening of 40,183,714 chemicals available in Mcule database to find hit compound (s). Result: We defined the binding pocket in the interface of ARF1 and CSW complex including all essential amino acid residues. For filtering the compounds, stringent criteria such as the number of rotatable bonds and the number of heavy atoms were applied. The top 100 hits were identified using this structure‐based virtual screening which show various interactions with the defined binding site of target. Next, the prepared library (the 100 identified Hits) was used as input for virtual screening using Schrodinger software. Flexible docking was performed using Glide program running under LINUX operating system. Based on vina scoring function, 10% of most active compounds that showed the best docking scores were identified and kept for further analysis. Conclusion: Rational design, computer‐aided, and in silico screening help developing pharmacological modulators to regulate C9ORF72‐ARF signaling and investigate their potential roles in ALS/FTD. Acknowledgement: This study is supported in part by NIH GM146257 on ALS² Initiative and by The Wooten Foundation. [ABSTRACT FROM AUTHOR]

Background: The Maastricht V/Florence Consensus Report recommends amoxicillin‐fluoroquinolone triple or quadruple therapy as a second‐line treatment for Helicobacter pylori infection. An important caveat of amoxicillin‐fluoroquinolone rescue therapy is poor eradication efficacy in the presence of fluoroquinolone resistance. The study aimed to investigate the efficacies of tetracycline‐levofloxacin (TL) quadruple therapy and amoxicillin‐levofloxacin (AL) quadruple therapy in the second‐line treatment of H. pylori infection. Methods: Consecutive H. pylori‐infected subjects after the failure of first‐line therapies were randomly allocated to receive either TL quadruple therapy (tetracycline 500 mg QID, levofloxacin 500 mg QD, esomeprazole 40 mg BID, and tripotassium dicitrato bismuthate 300 mg QID) or AL quadruple therapy (amoxicillin 500 mg QID, levofloxacin 500 mg QD, esomeprazole 40 mg BID, and tripotassium dicitrato bismuthate 300 mg QID) for 10 days. Post‐treatment H. pylori status was assessed 6 weeks after the end of therapy. Results: The study was early terminated after an interim analysis. In the TL quadruple group, 50 out of 56 patients (89.3%) had successful eradication of H. pylori infection. Cure of H. pylori infection was achieved only in 39 of 52 patients (69.6%) receiving AL quadruple therapy. Intention‐to‐treat analysis showed that TL quadruple therapy achieved a markedly higher eradication rate than AL quadruple therapy (95% confidence interval: 4.8% to 34.6%; p = 0.010). Further analysis revealed that TL quadruple therapy had a high eradication rate for both levofloxacin‐susceptible and resistant strains (100% and 88.9%). In contrast, AL quadruple therapy yielded a high eradication for levofloxacin‐susceptible strains (90.9%) but a poor eradication efficacy for levofloxacin‐resistant strains (50.0%). The two therapies exhibited comparable frequencies of adverse events (37.5% vs 21.4%) and drug adherence (98.2% vs 94.6%). Conclusions: Ten‐day TL quadruple therapy is more effective than AL quadruple therapy in the second‐line treatment of H. pylori infection in a population with high levofloxacin resistance. [ABSTRACT FROM AUTHOR]

Background and Aim: Concomitant therapy is a recommended first‐line treatment for Helicobacter pylori infection in most national or international consensuses. Reverse hybrid therapy is a modified 14‐day concomitant therapy without clarithromycin and metronidazole in the final 7 days. This study aims to test whether 14‐day reverse hybrid therapy is non‐inferior to 14‐day concomitant therapy in the first‐line treatment of H. pylori infection. Methods: Helicobacter pylori‐infected adult patients were randomly assigned to receive either reverse hybrid therapy (dexlansoprazole 60 mg o.d. plus amoxicillin 1 g b.d. for 14 days, and clarithromycin 500 mg plus metronidazole 500 mg b.d. for initial 7 days) or concomitant therapy (dexlansoprazole 60 mg once o.d. plus amoxicillin 1 g, clarithromycin 500 mg, and metronidazole 500 mg b.d. for 14 days). H. pylori status was assessed 6 weeks after the end of treatment. Results: Helicobacter pylori‐infected participants (n = 248) were randomized to receive either 14‐day reverse hybrid therapy (n = 124) or 14‐day concomitant therapy (n = 124). Intention‐to‐treat analysis demonstrated that the two therapies had comparable eradication rate (95.2% vs 93.5%; 95% confidence interval, −4.0% to 7.4%; P = 0.582). However, reverse hybrid therapy had a much lower frequency of adverse events than concomitant therapy (20.2% vs 38.7%, P = 0.001). The two therapies exhibited comparable drug adherence (93.5% vs 87.9%, P = 0.125). Conclusions: Fourteen‐day reverse hybrid therapy and 14‐day concomitant therapy are equivalent in efficacy for the first‐line treatment of H. pylori infection. However, reverse hybrid therapy has fewer adverse events compared with concomitant therapy. [ABSTRACT FROM AUTHOR]

Background and Aims: Anti‐Helicobacter pylori therapy may lead to the growth of pathogenic or antibiotic‐resistant bacteria in the gut. The study aimed to investigate the short‐term and long‐term impacts of H. pylori eradication with reverse hybrid therapy on the components and macrolide resistance of the gut microbiota. Methods: Helicobacter pylori‐related gastritis patients were administered a 14‐day reverse hybrid therapy. Fecal samples were collected before treatment and at the end of week 2, week 8, and week 48. The V3–V4 region of the bacterial 16S rRNA gene in fecal specimens was amplified by polymerase chain reaction and sequenced on Illumina MiSeq platform. Additionally, amplification of erm(B) gene (encoding erythromycin resistance methylase) was performed. Results: Reverse hybrid therapy resulted in decreased relative abundances of Firmicutes (from 62.0% to 30.7%; P < 0.001) and Actinobacteria (from 3.4% to 0.6%; 0.032) at the end of therapy. In contrast, the relative abundance of Proteobacteria increased from 10.2% to 49.1% (0.002). These microbiota alterations did not persist but returned to the initial levels at week 8 and week 48. The amount of erm(B) gene in fecal specimens was comparable with the pretreatment level at week 2 but increased at week 8 (0.025) and then returned to the pretreatment level by week 48. Conclusions: Helicobacter pylori eradication with reverse hybrid therapy can lead to short‐term gut dysbiosis. The amount of erm(B) gene in the stool increased transiently after treatment and returned to the pretreatment level at 1‐year post‐treatment. [ABSTRACT FROM AUTHOR]

Widely recognized in channelopathy spectrum disorders, dominant mutations in TRPV4 (transient receptor potential vanilloid 4) has been found in both nervous system and skeletal diseases. Additionally, if TRPV4-RhoA activation is a prime suspect of TRPV4 neuropathy, why was RhoA not the most prominent component revealed in the unbiased proteomic screening? Thus, it was proposed that TRPV4, acting as a RhoGDI, and RhoA together constitute an environmentally sensitive signaling complex that contributes to pathological cytoskeletal alterations during neurological injury and disease. [Extracted from the article]

Claudins are a family of tight junction ( TJ) integral membrane proteins that play a crucial role in maintaining cell polarity, adhesion, and paracellular permeability. Changes in expression levels of claudin proteins have been associated with human lung cancer. Previously, we have reported that claudin-7 expression is significantly downregulated in human lung carcinomas. To investigate the role of claudin-7 in lung cancer cells after anti-cancer drug treatments, we transfected claudin-7 cDNA into human NCI-H522 lung cancer cells, which have no detectable expression of claudin-7 protein. Flow cytometry analysis demonstrated that cells transfected with claudin-7 had a significantly higher percentage of cell apoptosis when compared to that of vector transfected cell population. The cell viability assayed by MTT and Annexin V was significantly decreased and cell apoptosis was dramatically increased in claudin-7 transfected cells compared to that of vector transfected cells after cisplatin treatment. Cisplatin is an anti-cancer drug clinically used to treat tumors in several tissues including lung tumors. Most importantly, after cisplatin treatment, the expression levels of cleaved caspase-3, -8, and poly adenosine 5′-diphosphate ribose polymerase ( PARP) were much higher in claudin-7 transfected cells than in control cells. Furthermore, using the site-directed mutagenesis approach, we identified that claudin-7 was phosphorylated at serine 204 by protein kinase C. Non-phosphorylated claudin-7 mutant showed increased cell viability, suggesting that phosphorylation increases chemosensitivity to cisplatin treatment. We concluded that claudin-7 expression in H522 lung cancer cells increases chemosensitivity to cisplatin through the increased activation of caspase pathway. [ABSTRACT FROM AUTHOR]

10-Hydroxycamptothecin (HCPT) elicits strong anti-cancer effects and is less toxic than camptothecin (CPT), making it widely used in recent clinical trials. However, its low solubility limits its application as an effective anti-cancer therapy. In the present study we investigate the hypothesis that the unique water dispersible oleic acid-Triton X-100-coated FeO nanoparticles loaded with HCPT disrupt epithelial cell-cell junctions and induce human lung cancer cell apoptosis through the caspase-8 pathway. We characterized the HCPT-loaded nanoparticles and determined their effects on lung cancer cell viability and apoptosis by using immunofluorescence light microscopy and SDS-PAGE/immunoblots. We found that HCPT-loaded nanoparticles elicited an anti-proliferative effect in a dose-dependent manner. HCPT-loaded nanoparticles reduced the expression of cell-cell junction protein claudins, E-cadherin and ZO-1, and transmission electron microcopy demonstrated a disrupted tight junction ultrastructure. Transepithelial electric resistance was also reduced, indicating the reduction of tight junction functions. The HCPT-loaded nanoparticles increased phosphorylation of p38 and SAPK/JNK while it showed no effects on p42/44 MAP kinase. Compared with void FeO nanoparticles or HCPT drug alone, HCPT drug-loaded nanoparticles evoked synergistic effects by increasing cell apoptosis with enhanced activation of the caspase-8 pathway. Therefore, our current study highlights the potential of HCPT drug-loaded nanoparticles as a chemotherapeutic agent for increasing anti-cancer drug efficacy. ( Cancer Sci 2011; 102: 1216-1222) [ABSTRACT FROM AUTHOR]

Bartter's syndrome is a constellation of symptoms characterized by hyper-reninemic hypokalemia, metabolic alkalosis, elevated renin and aldosterone, low or normal blood pressure, and hyperplasia of the juxtaglomerular apparatus. So far, five gene mutations in proteins regulating the sodium chloride transport in the thick ascending limb of Henle's loop have been described. However, the molecular mechanisms underlying the presentation of hypomagnesemia in some of these patients remains unclear. Claudins are a family of transmembranous proteins within the tight junctions that have been shown to be important for the paracellular movement of ions. Mutations in claudin-16 have been identified in patients with familial hypomagnesemia with hypercalciuria and nephrocalcinosis. To test the hypothesis that mutations in claudin genes may be involved in the altered magnesium and calcium transport in Bartter's syndrome, we began to examine the genes of claudins known to be present in renal tubules in four pediatric patients with Bartter's syndrome. All four patients were African Americans with hypomagnesemia and hypercalciuria. In this study, we did not find any mutation in the coding regions of claudin-2, -3, -4, -7, -8, -10, -11, or -16 genes in these patients. However, all patients had a single nucleotide substitution of C for T at the position of 451 of claudin-8 gene sequence that changes amino acid residue from serine to proline at the position of 151 in the second extracellular domain of claudin-8 protein. The significance of this known single nucleotide polymorphism remains to be determined. [ABSTRACT FROM AUTHOR]

Abstract: Mutations in WNK4 have been linked to hypertension in PHAII. Paracellular ion transport has been reported to be involved in this disease process; however, the specific molecular target has not been identified. In this study, we found that TJ protein claudin-7 and WNK4 were partially co-localized in renal tubules of rat kidney and co-immunoprecipitated in kidney epithelial cells. The wild-type and PHAII-causing mutant, but not the kinase-dead mutant, phosphorylated claudin-7. We have identified ser206 in the COOH-terminus of claudin-7 as a putative phosphorylation site for WNK4. More importantly, disease-causing mutant enhanced claudin-7 phosphorylation and significantly increased paracellular permeability to Cl−. [Copyright &y& Elsevier]