Regulation of ß-cell death by ADP-ribosylhydrolase ARH3 via lipid signaling in insulitis.

BACKGROUND: Lipids are regulators of insulitis and ß-cell death in type 1 diabetes development, but the underlying mechanisms are poorly understood. Here, we investigated how the islet lipid composition and downstream signaling regulate ß-cell death. METHODS: We performed lipidomics using three models of insulitis: human islets and EndoC-ßH1 ß cells treated with the pro-inflammatory cytokines interlukine-1ß and interferon-γ, and islets from pre-diabetic non-obese mice. We also performed mass spectrometry and fluorescence imaging to determine the localization of lipids and enzyme in islets. RNAi, apoptotic assay, and qPCR were performed to determine the role of a specific factor in lipid-mediated cytokine signaling. RESULTS: Across all three models, lipidomic analyses showed a consistent increase of lysophosphatidylcholine species and phosphatidylcholines with polyunsaturated fatty acids and a reduction of triacylglycerol species. Imaging assays showed that phosphatidylcholines with polyunsaturated fatty acids and their hydrolyzing enzyme phospholipase PLA2G6 are enriched in islets. In downstream signaling, omega-3 fatty acids reduce cytokine-induced ß-cell death by improving the expression of ADP-ribosylhydrolase ARH3. The mechanism involves omega-3 fatty acid-mediated reduction of the histone methylation polycomb complex PRC2 component Suz12, upregulating the expression of Arh3, which in turn decreases cell apoptosis. CONCLUSIONS: Our data provide insights into the change of lipidomics landscape in ß cells during insulitis and identify a protective mechanism by omega-3 fatty acids. Video Abstract.

Secondary metabolite profile of Streptomyces spp. changes when grown with the sub-lethal concentration of silver nanoparticles: possible implication in novel compound discovery.

The decline of new antibiotics and the emergence of multidrug resistance in pathogens necessitates a revisit of strategies used for lead compound discovery. This study proposes to induce the production of bioactive compounds with sub-lethal concentrations of silver nanoparticles (Ag-NPs). A total of Forty-two Actinobacteria isolates from four Saudi soil samples were grown with and without sub-lethal concentration of Ag-NPs (50 µg ml-1). The spent broth grown with Ag-NPs, or without Ag-NPs were screened for antimicrobial activity against four bacteria. Interestingly, out of 42 strains, broths of three strains grown with sub-lethal concentration of Ag-NPs exhibit antimicrobial activity against Staphylococcus aureus and Micrococcus luteus. Among these, two strains S4-4 and S4-21 identified as Streptomyces labedae and Streptomyces tirandamycinicus based on 16S rRNA gene sequence were selected for detailed study. The change in the secondary metabolites profile in the presence of Ag-NPs was evaluated using GC-MS and LC-MS analyses. Butanol extracts of spent broth grown with Ag-NPs exhibit strong antimicrobial activity against M. luteus and S. aureus. While the extracts of the controls with the same concentration of Ag-NPs do not show any activity. GC-analysis revealed a clear change in the secondary metabolite profile when grown with Ag-NPs. Similarly, the LC-MS patterns also differ significantly. Results of this study, strongly suggest that sub-lethal concentrations of Ag-NPs influence the production of secondary metabolites by Streptomyces. Besides, LC-MS results identified possible secondary metabolites, associated with oxidative stress and antimicrobial activities. This strategy can be used to possibly induce cryptic biosynthetic gene clusters for the discovery of new lead compounds.

Translation Rescue by Targeting Ppp1r15a through Its Upstream Open Reading Frame in Sepsis-Induced Acute Kidney Injury in a Murine Model.

BACKGROUND: Translation shutdown is a hallmark of late-phase, sepsis-induced kidney injury. Methods for controlling protein synthesis in the kidney are limited. Reversing translation shutdown requires dephosphorylation of the eukaryotic initiation factor 2 (eIF2) subunit eIF2 α ; this is mediated by a key regulatory molecule, protein phosphatase 1 regulatory subunit 15A (Ppp1r15a), also known as GADD34. METHODS: To study protein synthesis in the kidney in a murine endotoxemia model and investigate the feasibility of translation control in vivo by boosting the protein expression of Ppp1r15a, we combined multiple tools, including ribosome profiling (Ribo-seq), proteomics, polyribosome profiling, and antisense oligonucleotides, and a newly generated Ppp1r15a knock-in mouse model and multiple mutant cell lines. RESULTS: We report that translation shutdown in established sepsis-induced kidney injury is brought about by excessive eIF2 α phosphorylation and sustained by blunted expression of the counter-regulatory phosphatase Ppp1r15a. We determined the blunted Ppp1r15a expression persists because of the presence of an upstream open reading frame (uORF). Overcoming this barrier with genetic and antisense oligonucleotide approaches enabled the overexpression of Ppp1r15a, which salvaged translation and improved kidney function in an endotoxemia model. Loss of this uORF also had broad effects on the composition and phosphorylation status of the immunopeptidome-peptides associated with the MHC-that extended beyond the eIF2 α axis. CONCLUSIONS: We found Ppp1r15a is translationally repressed during late-phase sepsis because of the existence of an uORF, which is a prime therapeutic candidate for this strategic rescue of translation in late-phase sepsis. The ability to accurately control translation dynamics during sepsis may offer new paths for the development of therapies at codon-level precision. PODCAST: This article contains a podcast at.

IL-15 blockade and rapamycin rescue multifactorial loss of factor VIII from AAV-transduced hepatocytes in hemophilia A mice.

Hepatic adeno-associated viral (AAV) gene transfer has the potential to cure the X-linked bleeding disorder hemophilia A. However, declining therapeutic coagulation factor VIII (FVIII) expression has plagued clinical trials. To assess the mechanistic underpinnings of this loss of FVIII expression, we developed a hemophilia A mouse model that shares key features observed in clinical trials. Following liver-directed AAV8 gene transfer in the presence of rapamycin, initial FVIII protein expression declines over time in the absence of antibody formation. Surprisingly, loss of FVIII protein production occurs despite persistence of transgene and mRNA, suggesting a translational shutdown rather than a loss of transduced hepatocytes. Some of the animals develop ER stress, which may be linked to hepatic inflammatory cytokine expression. FVIII protein expression is preserved by interleukin-15/interleukin-15 receptor blockade, which suppresses CD8+ T and natural killer cell responses. Interestingly, mice with initial FVIII levels >100% of normal had diminishing expression while still under immune suppression. Taken together, our findings of interanimal variability of the response, and the ability of the immune system to shut down transgene expression without utilizing cytolytic or antibody-mediated mechanisms, illustrate the challenges associated with FVIII gene transfer. Our protocols based upon cytokine blockade should help to maintain efficient FVIII expression.

Effectiveness of Nonfunctionalized Graphene Oxide Nanolayers as Nanomedicine against Colon, Cervical, and Breast Cancer Cells.

Recent studies in nanomedicine have intensively explored the prospective applications of surface-tailored graphene oxide (GO) as anticancer entity. However, the efficacy of nonfunctionalized graphene oxide nanolayers (GRO-NLs) as an anticancer agent is less explored. In this study, we report the synthesis of GRO-NLs and their in vitro anticancer potential in breast (MCF-7), colon (HT-29), and cervical (HeLa) cancer cells. GRO-NLs-treated HT-29, HeLa, and MCF-7 cells showed cytotoxicity in the MTT and NRU assays via defects in mitochondrial functions and lysosomal activity. HT-29, HeLa, and MCF-7 cells treated with GRO-NLs exhibited substantial elevations in ROS, disturbances of the mitochondrial membrane potential, an influx of Ca2+, and apoptosis. The qPCR quantification showed the upregulation of caspase 3, caspase 9, bax, and SOD1 genes in GRO-NLs-treated cells. Western blotting showed the depletion of P21, P53, and CDC25C proteins in the above cancer cell lines after GRO-NLs treatment, indicating its function as a mutagen to induce mutation in the P53 gene, thereby affecting P53 protein and downstream effectors P21 and CDC25C. In addition, there may be a mechanism other than P53 mutation that controls P53 dysfunction. We conclude that nonfunctionalized GRO-NLs exhibit prospective biomedical application as a putative anticancer entity against colon, cervical, and breast cancers.

Sb-Doped Cerium Molybdate: An Emerging Material as Dielectric and Photocatalyst for the Removal of Diclofenac Potassium from Aqueous Media.

This work reports the influence of antimony substitution in a cerium molybdate lattice for improved dielectric and photocatalytic properties. For this purpose, a series of Ce2-xSbx(MoO4)3 (x = 0.00, 0.01, 0.03, 0.05, 0.07, and 0.09) were synthesized through a co-precipitation route. The as-synthesized materials were characterized for their optical properties, functional groups, chemical oxidation states, structural phases, surface properties, and dielectric characteristics using UV-Vis spectroscopy (UV-Vis), Fourier transform infrared (FTIR) and Raman spectroscopies, X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), Brunauer-Emmett-Teller (BET) analysis, and impedance spectroscopy, respectively. UV-Vis study showed a prominent red shift of absorption maxima and a continuous decrease in band gap (3.35 eV to 2.79 eV) by increasing the dopant concentration. The presence of Ce-O and Mo-O-Mo bonds, detected via FTIR and Raman spectroscopies, are confirmed, indicating the successful synthesis of the desired material. The monoclinic phase was dominant in all materials, and the crystallite size was decreased from 40.29 nm to 29.09 nm by increasing the Sb content. A significant increase in the dielectric constant (ε' = 2.856 × 108, 20 Hz) and a decrease in the loss tan (tanδ = 1.647, 20 Hz) were exhibited as functions of the increasing Sb concentration. Furthermore, the photocatalytic efficiency of pristine cerium molybdate was also increased by 1.24 times against diclofenac potassium by incorporating Sb (x = 0.09) in the cerium molybdate. The photocatalytic efficiency of 85.8% was achieved within 180 min of UV light exposure at optimized conditions. The photocatalytic reaction followed pseudo-first-order kinetics with an apparent rate constant of 0.0105 min-1, and the photocatalyst was recyclable with good photocatalytic activity even after five successive runs. Overall, the as-synthesized Sb-doped cerium molybdate material has proven to be a promising candidate for charge storage devices and a sustainable photocatalyst for wastewater treatment.

Advances in Graphene/Inorganic Nanoparticle Composites for Catalytic Applications.

Graphene-based nanocomposites with inorganic (metal and metal oxide) nanoparticles leads to materials with high catalytic activity for a variety of chemical transformations. Graphene and its derivatives such as graphene oxide, highly reduced graphene oxide, or nitrogen-doped graphene are excellent support materials due to their high surface area, their extended π-system, and variable functionalities for effective chemical interactions to fabricate nanocomposites. The ability to fine-tune the surface composition for desired functionalities enhances the versatility of graphene-based nanocomposites in catalysis. This review summarizes the preparation of graphene/inorganic NPs based nanocomposites and their use in catalytic applications. We discuss the large-scale synthesis of graphene-based nanomaterials. We have also highlighted the interfacial electronic communication between graphene/inorganic nanoparticles and other factors resulting in increased catalytic efficiencies.

Green Synthesized ZnO Nanoparticles as Biodiesel Blends and Their Effect on the Performance and Emission of Greenhouse Gases.

Pollution and global warming are a few of the many reasons for environmental problems, due to industrial wastes and greenhouse gases, hence there are efforts to bring down such emissions to reduce pollution and combat global warming. In the present study, zinc oxide nanoparticles are green synthesized using cow dung as fuel, through combustion. Synthesized material was characterized by FTIR, XRD, UV, and FESEM. The as-prepared ZnO-GS NPs were employed as a transesterification catalyst for the preparation of biodiesel from discarded cooking oil. The biodiesel obtained is termed D-COME (discarded cooking oil methyl ester), which is blended with 20% commercial diesel (B20). Additionally, this blend, i.e., B20, is further blended with varying amounts of as-prepared ZnO-GS NPs, in order to ascertain its effects on the quality of emissions of various greenhouse gases such as hydrocarbons, COx, NOx. Moreover, the brake thermal efficiency (BTHE) and brake specific fuel consumption (BSFC) were studied for their blends. The blend (B20) with 30 mg of ZnO-GS, i.e., B20-30, displays the best performance and reduced emissions. Comparative studies revealed that the ZnO-GS NPs are as efficient as the ZnO-C NPs, indicating that the green synthetic approach employed does not affect the efficiency of the ZnO NPs.

Development and Validation for Quantification of 7-Nitroso Impurity in Sitagliptin by Ultraperformance Liquid Chromatography with Triple Quadrupole Mass Spectrometry.

The purpose of this research study was to develop an analytical method for the quantification of 7-nitroso-3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4] triazolo [4,3-a] pyrazine (7-nitroso impurity), which is a potential genotoxic impurity. Since sitagliptin is an anti-diabetic medication used to treat type 2 diabetes and the duration of the treatment is long-term, the content of nitroso impurity must be controlled by using suitable techniques. To quantify this impurity, a highly sensitive and reproducible ultraperformance liquid chromatography with triple quadrupole mass spectrometry (UHPLC-MS/MS) method was developed. The analysis was performed on a Kromasil-100, with a C18 column (100 mm × 4.6 mm with a particle size of 3.5 µm) at an oven temperature of approximately 40 °C. The mobile phase was composed of 0.12% formic acid in water, with methanol as mobile phases A and B, and the flow rate was set to 0.6 mL/min. The method was validated according to the current International Council for Harmonisation (ICH) guidelines with respect to acceptable limits, specificity, reproducibility, accuracy, linearity, precision, ruggedness and robustness. This method is useful for the detection of the impurity at the lowest limit of detection (LOD), which was 0.002 ppm, and the lowest limit of quantification (LOQ), which was 0.005 ppm. This method was linear in the range of 0.005 to 0.06 ppm and the square of the correlation coefficient (R2) was determined to be > 0.99. This method could help to determine the impurity in the regular analysis of sitagliptin drug substances and drug products.

Green Synthesis of Silver Nanoparticles Using Aerial Part Extract of the Anthemis pseudocotula Boiss. Plant and Their Biological Activity.

Green syntheses of metallic nanoparticles using plant extracts as effective sources of reductants and stabilizers have attracted decent popularity due to their non-toxicity, environmental friendliness and rapid nature. The current study demonstrates the ecofriendly, facile and inexpensive synthesis of silver nanoparticles (AP-AgNPs) using the extract of aerial parts of the Anthemis pseudocotula Boiss. plant (AP). Herein, the aerial parts extract of AP performed a twin role of a reducing as well as a stabilizing agent. The green synthesized AP-AgNPs were characterized by several techniques such as XRD, UV-Vis, FT-IR, TEM, SEM and EDX. Furthermore, the antimicrobial and antibiofilm activity of as-prepared AP-AgNPs were examined by a standard two-fold microbroth dilution method and tissue culture plate methods, respectively, against several Gram-negative and Gram-positive bacterial strains and fungal species such as Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), multidrug-resistant Pseudomonas aeruginosa (MDR-PA) and Acinetobacter baumannii (MDR-AB), methicillin-resistant S. aureus (MRSA) and Candida albicans (C. albicans) strains. The antimicrobial activity results clearly indicated that the Gram-negative bacteria MDR-PA was most affected by AgNPs as compared to other Gram-negative and Gram-positive bacteria and fungi C. albicans. Whereas, in the case of antibiofilm activity, it has been found that AgNPs at 0.039 mg/mL, inhibit biofilms formation of Gram-negative bacteria i.e., MDR-PA, E. coli, and MDR-AB by 78.98 ± 1.12, 65.77 ± 1.05 and 66.94 ± 1.35%, respectively. On the other hand, at the same dose (i.e., 0.039 mg/mL), AP-AgNPs inhibits biofilm formation of Gram-positive bacteria i.e., MRSA, S. aureus and fungi C. albicans by 67.81 ± 0.99, 54.61 ± 1.11 and 56.22 ± 1.06%, respectively. The present work indicates the efficiency of green synthesized AP-AgNPs as good antimicrobial and antibiofilm agents against selected bacterial and fungal species.

A High-Performance Asymmetric Supercapacitor Based on Tungsten Oxide Nanoplates and Highly Reduced Graphene Oxide Electrodes.

Tungsten oxide/graphene hybrid materials are attractive semiconductors for energy-related applications. Herein, we report an asymmetric supercapacitor (ASC, HRG//m-WO3 ASC), fabricated from monoclinic tungsten oxide (m-WO3 ) nanoplates as a negative electrode and highly reduced graphene oxide (HRG) as a positive electrode material. The supercapacitor performance of the prepared electrodes was evaluated in an aqueous electrolyte (1 m H2 SO4 ) using three- and two-electrode systems. The HRG//m-WO3 ASC exhibits a maximum specific capacitance of 389 F g-1 at a current density of 0.5 A g-1 , with an associated high energy density of 93 Wh kg-1 at a power density of 500 W kg-1 in a wide 1.6 V operating potential window. In addition, the HRG//m-WO3 ASC displays long-term cycling stability, maintaining 92 % of the original specific capacitance after 5000 galvanostatic charge-discharge cycles. The m-WO3 nanoplates were prepared hydrothermally while HRG was synthesized by a modified Hummers method.

ResNet Based Deep Features and Random Forest Classifier for Diabetic Retinopathy Detection.

Diabetic retinopathy, an eye disease commonly afflicting diabetic patients, can result in loss of vision if prompt detection and treatment are not done in the early stages. Once the symptoms are identified, the severity level of the disease needs to be classified for prescribing the right medicine. This study proposes a deep learning-based approach, for the classification and grading of diabetic retinopathy images. The proposed approach uses the feature map of ResNet-50 and passes it to Random Forest for classification. The proposed approach is compared with five state-of-the-art approaches using two category Messidor-2 and five category EyePACS datasets. These two categories on the Messidor-2 dataset include 'No Referable Diabetic Macular Edema Grade (DME)' and 'Referable DME' while five categories consist of 'Proliferative diabetic retinopathy', 'Severe', 'Moderate', 'Mild', and 'No diabetic retinopathy'. The results show that the proposed approach outperforms compared approaches and achieves an accuracy of 96% and 75.09% for these datasets, respectively. The proposed approach outperforms six existing state-of-the-art architectures, namely ResNet-50, VGG-19, Inception-v3, MobileNet, Xception, and VGG16.

BF3-OEt2 Catalyzed C3-Alkylation of Indole: Synthesis of Indolylsuccinimidesand Their Cytotoxicity Studies.

A simple and efficient BF3-OEt2 promoted C3-alkylation of indole has been developed to obtain3-indolylsuccinimidesfrom commercially available indoles and maleimides, with excellent yields under mild reaction conditions. Furthermore, anti-proliferative activity of these conjugates was evaluated against HT-29 (Colorectal), Hepg2 (Liver) and A549 (Lung) human cancer cell lines. One of the compounds, 3w, having N,N-Dimethylatedindolylsuccinimide is a potent congener amongst the series with IC50 value 0.02 µM and 0.8 µM against HT-29 and Hepg2 cell lines, respectively, and compound 3i was most active amongst the series with IC50 value 1.5 µM against A549 cells. Molecular docking study and mechanism of reaction have briefly beendiscussed. This method is better than previous reports in view of yield and substrate scope including electron deficient indoles.

Straightforward Synthesis of Mn3O4/ZnO/Eu2O3-Based Ternary Heterostructure Nano-Photocatalyst and Its Application for the Photodegradation of Methyl Orange and Methylene Blue Dyes.

Zinc oxide-ternary heterostructure Mn3O4/ZnO/Eu2O3 nanocomposites were successfully prepared via waste curd as fuel by a facile one-pot combustion procedure. The fabricated heterostructures were characterized utilizing XRD, UV-Visible, FT-IR, FE-SEM, HRTEM and EDX analysis. The photocatalytic degradation efficacy of the synthesized ternary nanocomposite was evaluated utilizing model organic pollutants of methylene blue (MB) and methyl orange (MO) in water as examples of cationic dyes and anionic dyes, respectively, under natural solar irradiation. The effect of various experimental factors, viz. the effect of a light source, catalyst dosage, irradiation time, pH of dye solution and dye concentration on the photodegradation activity, was systematically studied. The ternary Mn3O4/ZnO/Eu2O3 photocatalyst exhibited excellent MB and MO degradation activity of 98% and 96%, respectively, at 150 min under natural sunlight irradiation. Experiments further conclude that the fabricated nanocomposite exhibits pH-dependent photocatalytic efficacy, and for best results, concentrations of dye and catalysts have to be maintained in a specific range. The prepared photocatalysts are exemplary and could be employed for wastewater handling and several ecological applications.

Antispasmodic activity and mechanism of action of polyherbal formulation DCD-684 on rabbit jejunum.

Digas colic drops (DCD-684) is a polyherbal formulation containing decoctions of five medicinal plants namely Carum carvi L., Foeniculum vulgare Mill, Mentha arvensis L., Mentha piperita L. and Zingiber officinale Roscoe. These plants have been extensively used in traditional medicine for the treatment of various gastrointestinal diseases including abdominal colic. This study was conducted to determine the spasmolytic effect of DCD-684 (100% v/v) and its individual plant components on isolated rabbit jejunum (in vitro) and their possible mechanism of action. The effects were evaluated on spontaneous and pre-contracted tissues using KCl (80mM) and other contractile agonists including acetylcholine (0.3µM), carbamylcholine (0.3µM), serotonin (10 µM) and histamine (100µM) in the presence and absence of DCD-684. The various concentrations of DCD-684 (0.1-3% v/v) demonstrated spasmolytic effects on both spontaneous (IC50=0.75%) and KCl-induced contractions (IC50=1.6%), respectively. It also inhibited the contractions induced by acetylcholine (IC50=0.45%), carbamylcholine (IC50=0.95%), serotonin (IC50=0.95%) and histamine (IC50=0.87%). The DCD-684 exhibited synergistic effect due to its five plant components suggesting that spasmolytic cascade is probably governed by muscarinic and/or nicotinic receptors, serotonergic histaminergic, as well as calcium channel blocking mechanisms. Thereby, providing the pharmacological basis of its therapeutic use in the gastrointestinal motility disorders and related inflammatory ailments.

Assessment of acute, sub-acute, chronic and genotoxicity of polyherbal formulation DCD-684 in mice.

Digas colic drops (DCD-684) a polyherbal formulation containing Carum carvi, Foeniculum vulgare, Mentha arvensis, Mentha piperita and Zingiber officinale is widely used in Pakistan against gastrointestinal ailments including infantile colic. The DCD-684 (0.03-3ml/kg.bw) administered orally in acute (7-days) and sub-acute toxicity (14-days) tests, displayed neither mortality nor toxicological changes in physical, behavioral, biochemical and histopathological parameters. In chronic study (90-days), DCD-684 (0.3-12ml/kg.bw) also revealed no changes. However, at 18 and 36 ml/kg.bw, liver demonstrated mild inflammation correlating with raised aspartate transaminase (AST), alkaline phosphatase (ALP) and alpha fetoprotein (AFP) levels. Increased levels of urea and inflamed renal parenchyma indicated mild nephro-toxicity with high alanine aminotransferase (ALT) at 36ml/kg.bw. The LD50 of DCD-684 in mice was 27.5 ml/kg.bw. In hepatocytes at 36ml/kg.bw, elevated mRNA expression of pro-inflammatory chemokines and cytokines were evident. DCD-684 neither damaged DNA nor induced cytotoxicity in micronucleus assay. In conclusion, polyherbal DCD-684 caused neither hepatic, renal, genotoxicity nor any undesirable effect in mice. Higher doses administered for 90 days showed mild toxic effects with no sign of necrosis, fibrosis or genotoxicity. Thus, in mice DCD-684 demonstrated a wide margin of safety to be used for the relief of infantile colic.

Endoplasmic reticulum stress alters ryanodine receptor function in the murine pancreatic ß cell.

Alterations in endoplasmic reticulum (ER) calcium (Ca2+) levels diminish insulin secretion and reduce ß-cell survival in both major forms of diabetes. The mechanisms responsible for ER Ca2+ loss in ß cells remain incompletely understood. Moreover, a specific role for either ryanodine receptor (RyR) or inositol 1,4,5-triphosphate receptor (IP3R) dysfunction in the pathophysiology of diabetes remains largely untested. To this end, here we applied intracellular and ER Ca2+ imaging techniques in INS-1 ß cells and isolated islets to determine whether diabetogenic stressors alter RyR or IP3R function. Our results revealed that the RyR is sensitive mainly to ER stress-induced dysfunction, whereas cytokine stress specifically alters IP3R activity. Consistent with this observation, pharmacological inhibition of the RyR with ryanodine and inhibition of the IP3R with xestospongin C prevented ER Ca2+ loss under ER and cytokine stress conditions, respectively. However, RyR blockade distinctly prevented ß-cell death, propagation of the unfolded protein response (UPR), and dysfunctional glucose-induced Ca2+ oscillations in tunicamycin-treated INS-1 ß cells and mouse islets and Akita islets. Monitoring at the single-cell level revealed that ER stress acutely increases the frequency of intracellular Ca2+ transients that depend on both ER Ca2+ leakage from the RyR and plasma membrane depolarization. Collectively, these findings indicate that RyR dysfunction shapes ER Ca2+ dynamics in ß cells and regulates both UPR activation and cell death, suggesting that RyR-mediated loss of ER Ca2+ may be an early pathogenic event in diabetes.

Copper-Promoted One-Pot Approach: Synthesis of Benzimidazoles.

A facile, one-pot, and proficient method was developed for the production of various 2-arylaminobenzimidazoles. This methodology is based for the first time on a copper catalyst promoted domino C-N cross-coupling reaction for the generation of 2-arylaminobenzimidazoles. Mechanistic investigations revealed that the synthetic pathway involves a copper-based desulphurization/nucleophilic substitution and a subsequent domino intra and intermolecular C-N cross-coupling reactions. Some of the issues typically encountered during the synthesis of 2-arylaminobezimidazoles, including the use of expensive catalytic systems and the low reactivity of bromo precursors, were addressed using this newly developed copper-catalyzed method. The reaction procedure is simple, generally with excellent substrate tolerance, and provides good to high yields of the desired products.

HIV-Nef Protein Persists in the Lungs of Aviremic Patients with HIV and Induces Endothelial Cell Death.

It remains a mystery why HIV-associated end-organ pathologies persist in the era of combined antiretroviral therapy (ART). One possible mechanism is the continued production of HIV-encoded proteins in latently HIV-infected T cells and macrophages. The proapoptotic protein HIV-Nef persists in the blood of ART-treated patients within extracellular vesicles (EVs) and peripheral blood mononuclear cells. Here we demonstrate that HIV-Nef is present in cells and EVs isolated from BAL of patients on ART. We hypothesize that HIV-Nef persistence in the lung induces endothelial apoptosis leading to endothelial dysfunction and further pulmonary vascular pathologies. The presence of HIV-Nef in patients with HIV correlates with the surface expression of the proapoptotic endothelial-monocyte-activating polypeptide II (EMAPII), which was implicated in progression of pulmonary emphysema via mechanisms involving endothelial cell death. HIV-Nef protein induces EMAPII surface expression in human embryonic kidney 293T cells, T cells, and human and mouse lung endothelial cells. HIV-Nef packages itself into EVs and increases the amount of EVs secreted from Nef-expressing T cells and Nef-transfected human embryonic kidney 293T cells. EVs from BAL of HIV+ patients and Nef-transfected cells induce apoptosis in lung microvascular endothelial cells by upregulating EMAPII surface expression in a PAK2-dependent fashion. Transgenic expression of HIV-Nef in vascular endothelial-cadherin+ endothelial cells leads to lung rarefaction, characterized by reduced alveoli and overall increase in lung inspiratory capacity. These changes occur concomitantly with lung endothelial cell apoptosis. Together, these data suggest that HIV-Nef induces endothelial cell apoptosis via an EMAPII-dependent mechanism that is sufficient to cause pulmonary vascular pathologies even in the absence of inflammation.

Episodic ß-cell death and dedifferentiation during diet-induced obesity and dysglycemia in male mice.

Loss of functional islet ß-cell mass through cellular death or dedifferentiation is thought to lead to dysglycemia during the progression from obesity to type 2 diabetes. To assess these processes in a mouse model of obesity, we performed measures of circulating cell-free differentially methylated insulin II ( Ins2) DNA as a biomarker of ß-cell death and aldehyde dehydrogenase 1 family member A3 (ALDH1A3) and forkhead box 01 (Foxo1) immunostaining as markers of ß-cell dedifferentiation. Eight-week-old, C57BL/6J mice were fed a low-fat diet (LFD; 10% kcal from fat) or a high-fat diet (HFD; 60% kcal from fat) and were followed longitudinally for up to 13 wk to measure glycemic control and ß-cell mass, death, and dedifferentiation. Compared with LFD controls, ß-cell mass increased during the feeding period in HFD animals, and statistically greater ß-cell death (unmethylated Ins2) was detectable at 2 and 6 wk after diet initiation. Those times correspond to periods when significant step increases in fasting glucose and glucose intolerance, respectively, were detected. ALDH1A3 and Foxo1 immunostaining of the pancreas revealed evidence of ß-cell dedifferentiation by 13 wk when fed an HFD, but not in LFD controls. In conclusion, early episodic ß-cell death may be a feature of cellular turnover correlated with changes in glycemia during ß-cell mass accrual in obesity, whereas ß-cell dedifferentiation may be a feature seen later in established disease.-Tersey, S. A., Levasseur, E. M., Syed, F., Farb, T. B., Orr, K. S., Nelson, J. B., Shaw, J. L., Bokvist, K., Mather, K. J., Mirmira, R. G. Episodic ß-cell death and dedifferentiation during diet-induced obesity and dysglycemia in male mice.