Schlafen 12 Interaction with SerpinB12 and Deubiquitylases Drives Human Enterocyte Differentiation

Background/Aims: Human enterocytic differentiation is altered during development, fasting, adaptation, and bariatric surgery, but its intracellular control remains unclear. We hypothesized that Schlafen 12 (SLFN12) regulates enterocyte differentiation. Methods: We used laser capture dissection of epithelium, qRT-PCR, and immunohistochemistry to evaluate SLFN12 expression in biopsies of control and fasting human duodenal mucosa, and viral overexpression and siRNA to trace the SLFN12 pathway in human Caco-2 and HIEC6 intestinal epithelial cells. Results: Fasting human duodenal mucosa expressed less SLFN12 mRNA and protein, accompanied by decreases in enterocytic markers like sucrase-isomaltase. SLFN12 overexpression increased Caco-2 sucrase-isomaltase promoter activity, mRNA, and protein independently of proliferation, and activated the SLFN12 putative promoter. SLFN12 coprecipitated Serpin B12 (SERPB12). An inactivating SLFN12 point mutation prevented both SERPB12 binding and sucrase-isomaltase induction. SERPB12 overexpression also induced sucrase-isomaltase, while reducing SERPB12 prevented the SLFN12 effect on sucrase-isomaltase. Sucrase-isomaltase induction by both SLFN12 and SERPB12 was attenuated by reducing UCHL5 or USP14, and blocked by reducing both. SERPB12 stimulated USP14 but not UCHL5 activity. SERPB12 coprecipitated USP14 but not UCHL5. Moreover, SLFN12 increased protein levels of the sucrase-isomaltase-promoter-binding transcription factor cdx2 without altering Cdx2 mRNA. This was prevented by reducing UCHL5 and USP14. We further validated this pathway in vitro and in vivo. SLFN12 or SERPB12 overexpression induced sucrase-isomaltase in human non-malignant HIEC-6 enterocytes. Conclusions: SLFN12 regulates human enterocytic differentiation by a pathway involving SERPB12, the deubiquitylases, and Cdx2. This pathway may be targeted to manipulate human enterocytic differentiation in mucosal atrophy, short gut or obesity

Deposition of silicon nitride films using chemical vapor deposition for photovoltaic applications

AbstractSilicon nitride is a versatile material since many decades due to its compatibility with conventional fabrication technology. Other than its potential applications in microelectronics, this material has been regarded as an antireflection coating in solar cells. In this paper, we present the fabrication and characterization of silicon nitride (Si3N4) films deposited at 750, 800 and 850°C using atmospheric chemical vapor deposition. We have investigated the effect of deposition temperature on the physical and optical properties of the prepared films. The refractive index and film thickness were found to be increased in accordance with an increase in the deposition temperature. Fourier transform infrared (FTIR) spectroscopy analysis showed the Si–N–Si stretching peak at 917cm−1 further, the full width at half maxima and Si–N–Si peak position showed their dependency on the deposition temperature. Finally, the optimal reflectance was observed through Si3N4 film deposited at temperature 800°C which confirms its suitability as an antireflection coating

Association Study between T2DM and CAPN10 SNP-19 (rs3842570) Polymorphism in Navi Mumbai Population

Genetic research has brought a lot of new knowledge in the area of genetic predisposition of type 2 diabetes mellitus (T2DM). It has been proposed that excessive insulin resistance and obesity are also responsible for the higher incidence of type 2 diabetes. Calpain-10 (CAPN10) is a member of a large family of intracellular proteases. The polymorphism at deletion/insertion SNP19 of this gene influences susceptibility to T2DM. The aim of the study was to determine whether calpain-10 (ins/del SNP19) polymorphism contributes significantly to susceptibility to T2DM in population of Navi Mumbai. The study included randomly selected 75 patients of which 33 had T2DM and 42 served as control subjects. Mean waist-to-hip ratio, HDL, LDL, VLDL, cholesterol, and triglyceride showed no difference whereas mean of age, FBS, and body mass index showed significant differences between the control and diabetes subjects. Genotyping of calpain-10 (ins/del SNP19) polymorphism was performed by polymerase chain reaction method. Among 75 participants, for allele-specific SNP19, genotype frequencies of allele1 (2R-32 bp), heterozygous allele (2R-3R 32 bp), and allele 2 (3R-32 bp) were 20 (26.6%), 36 (48%), and 19 (25.3%) observed, respectively. The results from the present study have indicated that CAPN10 (SNP19) shows no significant association with T2DM and more extensive studies on T2DM using candidate gene approach may provide better preventive measures and potential disease diagnostic tools

Biomarker Potential of Vimentin in Oral Cancers

Oral carcinogenesis is a multistep process. As much as 5% to 85% of oral tumors can develop from potentially malignant disorders (PMD). Although the oral cavity is accessible for visual examination, the ability of current clinical or histological methods to predict the lesions that can progress to malignancy is limited. Thus, developing biological markers that will serve as an adjunct to histodiagnosis has become essential. Our previous studies comprehensively demonstrated that aberrant vimentin expression in oral premalignant lesions correlates to the degree of malignancy. Likewise, overwhelming research from various groups show a substantial contribution of vimentin in oral cancer progression. In this review, we have described studies on vimentin in oral cancers, to make a compelling case for vimentin as a prognostic biomarker

ZINC40099027 activates human focal adhesion kinase by accelerating the enzymatic activity of the FAK kinase domain

Abstract Focal adhesion kinase (FAK) regulates gastrointestinal epithelial restitution and healing. ZINC40099027 (Zn27) activates cellular FAK and promotes intestinal epithelial wound closure in vitro and in mice. However, whether Zn27 activates FAK directly or indirectly remains unknown. We evaluated Zn27 potential modulation of the key phosphatases, PTP‐PEST, PTP1B, and SHP2, that inactivate FAK, and performed in vitro kinase assays with purified FAK to assess direct Zn27‐FAK interaction. In human Caco‐2 cells, Zn27‐stimulated FAK‐Tyr‐397 phosphorylation despite PTP‐PEST inhibition and did not affect PTP1B‐FAK interaction or SHP2 activity. Conversely, in vitro kinase assays demonstrated that Zn27 directly activates both full‐length 125 kDa FAK and its 35 kDa kinase domain. The ATP‐competitive FAK inhibitor PF573228 reduced basal and ZN27‐stimulated FAK phosphorylation in Caco‐2 cells, but Zn27 increased FAK phosphorylation even in cells treated with PF573228. Increasing PF573228 concentrations completely prevented activation of 35 kDa FAK in vitro by a normally effective Zn27 concentration. Conversely, increasing Zn27 concentrations dose‐dependently activated kinase activity and overcame PF573228 inhibition of FAK, suggesting the direct interactions of Zn27 with FAK may be competitive. Zn27 increased the maximal activity (Vmax) of FAK. The apparent Km of the substrate also increased under laboratory conditions less relevant to intracellular ATP concentrations. These results suggest that Zn27 is highly potent and enhances FAK activity via allosteric interaction with the FAK kinase domain to increase the Vmax of FAK for ATP. Understanding Zn27 enhancement of FAK activity will be important to redesign and develop a clinical drug that can promote mucosal wound healing

Role of tumor cell surface lysosome-associated membrane protein-1 (LAMP1) and its associated carbohydrates in lung metastasis

Purpose: Expression of lysosome-associated membrane protein-1 (LAMP1) on the surface correlates with metastatic potential of B16 melanoma cells. Downregulation of their expression in high metastatic (B16F10) cells reduced their surface expression and metastatic potential. Present investigations explore if overexpression of LAMP1 on the surface of low metastatic (B16F1) cells augment their metastatic ability, and if so, how? Methods: B16F1 cells were transduced with lentiviral vector carrying mutant-LAMP1 (Y386A) (mutLAMP1). Surface expression of LAMP1 and carbohydrates was analyzed by flow cytometry, immunofluorescence and/or immunoprecipitation and Western blotting. Cell spreading and motility were assessed on components of extracellular matrix (ECM) (fibronectin) and basement membrane (BM) (matrigel), and galectin-3-coated coverslips/plates. Metastatic potential was assessed using experimental metastasis assay. Results: Pre-incubation with anti-LAMP1 antibodies significantly reduced lung metastasis of B16F10 cells. Overexpression of mutLAMP1 significantly increased its surface expression on B16F1 cells, resulting in increased cellular spreading and motility on fibronectin and matrigel. LAMP1 is the major carrier of poly-N-acetyllactosamine (polyLacNAc) on B16F10 cells. However, significantly higher expression of mutLAMP1 had no effect on galectin-3 binding on cell surface or on spreading or motility of cells on galectin-3-coated coverslips/plates. These cells also failed to show any gain in metastatic ability. This could be because LAMP1 from these cells carried significantly lower levels of polyLacNAc in comparison with B16F10 cells. Conclusions: PolyLacNAc on B16F10 cells and galectin-3 on lungs are the major participants in melanoma metastasis. Although surface LAMP1 promotes interactions with organ ECM and BM, carbohydrates on LAMP1 play a decisive role in dictating lung metastasis

N-glycans and metastasis in galectin-3 transgenic mice

Poly-N-acetyl-lactosamine (polyLacNAc) on N-glycans facilitate lung specific metastasis of melanoma cells by serving as high affinity ligands for galectin-3, expressed in highest amounts in the lungs, on almost all its tissue compartments including on the surface of vascular endothelium. PolyLacNAc not only aids in initial arrest on the organ endothelium but in all the events of extravasation. Inhibition of polyLacNAc synthesis, or competitive inhibition of its interaction with galectin-3 all inhibited these processes and experimental metastasis. Transgenic galectin-3 mice, viz., gal-3 (wild type), gal-3 (hemizygous) and gal-3 (null) have been used to prove that galectin-3/polyLacNAc interactions are indeed critical for lung specific metastasis. Gal-3 mice which showe

Failure of pentavalent antimony in visceral leishmaniasis in India: report from the center of the Indian epidemic

In India, 320 patients with visceral leishmaniasis (209 in the state of Bihar and 11 in the neighboring state of Uttar Pradesh) received identical pentavalent antimony (Sb) treatment. Sb induced long-term cure in 35% (95% confidence interval [CI], 28%-42%) of those in Bihar versus 86% (95% CI, 79%-93%) of those in Uttar Pradesh. In Bihar, the center of the Indian epidemic, traditional Sb treatment should be abandoned