The alpha2 isoform of Na,K-ATPase mediates ouabain-induced cardiac inotropy in mice

Inhibition of Na,K-ATPase activity by cardiac glycosides is believed to be the major mechanism by which this class of drugs increases heart contractility. However, direct evidence demonstrating this is lacking. Furthermore it is unknown which specific alpha isoform of Na,K-ATPase is responsible for the effect of cardiac glycosides. Several studies also suggest that cardiac glycosides, such as ouabain, function by mechanisms other than inhibition of the Na,K-ATPase. To determine whether Na,K-ATPase, specifically the alpha2 Na,K-ATPase isozyme, mediates ouabain-induced cardiac inotropy, we developed animals expressing a ouabain-insensitive alpha2 isoform of the Na,K-ATPase using Cre-Lox technology and analyzed cardiac contractility after administration of ouabain. The homozygous knock-in animals were born in normal Mendelian ratio and developed normally to adulthood. Analysis of their cardiovascular function demonstrated normal heart function. Cardiac contractility analysis in isolated hearts and in intact animals demonstrated that ouabain-induced cardiac inotropy occurred in hearts from wild type but not from the targeted animals. These results clearly demonstrate that the Na,K-ATPase and specifically the alpha2 Na,K-ATPase isozyme mediates ouabain-induced cardiac contractility in mice.     

Modulating EGFR-MTORC1-autophagy as a potential therapy for persistent fetal vasculature (PFV) disease

ABSTRACT

Persistent fetal vasculature (PFV) is a human disease that results from failure of the fetal vasculature to regress normally. The regulatory mechanisms responsible for fetal vascular regression remain obscure, as does the underlying cause of regression failure. However, there are a few animal models that mimic the clinical manifestations of human PFV, which can be used to study different aspects of the disease. One such model is the Nuc1 rat model that arose from a spontaneous mutation in the Cryba1 (crystallin, beta 1) gene and exhibits complete failure of the hyaloid vasculature to regress. Our studies with the Nuc1 rat indicate that macroautophagy/autophagy, a process in eukaryotic cells for degrading dysfunctional components to ensure cellular homeostasis, is severely impaired in Nuc1 ocular astrocytes. Further, we show that CRYBA1 interacts with EGFR (epidermal growth factor receptor) and that loss of this interaction in Nuc1 astrocytes increases EGFR levels. Moreover, our data also show a reduction in EGFR degradation in Nuc1 astrocytes compared to control cells that leads to over-activation of the mechanistic target of rapamycin kinase complex 1 (MTORC1) pathway. The impaired EGFR-MTORC1-autophagy signaling in Nuc1 astrocytes triggers abnormal proliferation and migration. The abnormally migrating astrocytes ensheath the hyaloid artery, contributing to the pathogenesis of PFV in Nuc1, by adversely affecting the vascular remodeling processes essential to regression of the fetal vasculature. Herein, we demonstrate in vivo that gefitinib (EGFR inhibitor) can rescue the PFV phenotype in Nuc1 and may serve as a novel therapy for PFV disease by modulating the EGFR-MTORC1-autophagy pathway.     

The nature of the carbohydrate binding module determines the catalytic efficiency of xylanase Z of Clostridium thermocellum

Xylanase Z of Clostridium thermocellum exists as a complex in the cellulosome with N-terminus feruloyl esterase, a carbohydrate binding module (CBM6) and a dockerin domain. To study the role of the binding modules on the activity of XynZ, different variants with the CBM6 attached to the catalytic domain at its C-terminal (XynZ-CB) and N-terminal (XynZ-BC), and the CBM22 attached at N-terminus (XynZ-B′C) were expressed in Escherichia coli at levels around 30% of the total cell proteins. The activities of XynZ-BC, XynZ-CB and XynZ-B′C were 4200, 4180 and 20,700 U μM⁻¹ against birchwood xylan, respectively. Substrate binding studies showed that in case of XynZ-BC and XynZ-CB the substrate birchwood xylan remaining unbound were 51 and 52%, respectively, whereas in the case of XynZ-B′C the substrate remaining unbound was 39% under the assay conditions used. The molecular docking studies showed that the binding site of CBM22 in XynZ-B′C is more exposed and thus available for substrate binding as compared to the tunnel shape binding pocket produced in XynZ-BC and thus hindering the substrate binding. The substrate binding data for the two constructs are in agreement with this explanation.     

Nematicidal and allelopathic potential of Argemone mexicana,a tropical weed

Argemone mexicana L. (Papaveraceae), a tropical annual weed, is phytotoxic to many crop species. This study was designed to examine the allelochemical and nematicidal potential of A. mexicana and to better understand the role of this weed in the ecosystem. A methanol-soluble extract of the leaf material caused greater juvenile mortality of Meloidogyne javanica than did ethyl acetate or hexane extracts indicating the polar nature of the toxins. Decomposing tissues of A. mexicana in soil at 50 g kg^–1 were highly deleterious causing 80% mortality of tomato plants. At 10 g kg^–1 plant growth was enhanced, while at 30 g kg^–1 plant growth was substantially retarded. M. javanica population densities in the rhizosphere and in roots, and gall formation were significantly suppressed when 10, 30 or 50 g kg^–1 A. mexicana was allowed to decompose in the soil. To establish whether decomposition was necessary to produce phytotoxic symptoms, or whether the shoot extract alone could interfere with plant growth, an aqueous shoot extract was applied to soil. Whereas a 50% extract promoted plant growth, a 100% (100 g/500 mL distilled water) concentration significantly reduced plant height, and fresh weights of shoot and root. In general, decomposing plant material caused greater phytotoxicity compared to the aqueous extract. Addition of N as NH₄NO₃ partially alleviated the phytotoxic action of A. mexicana,and also reduced severity of root-knot disease. Adding Pseudomonas aeruginosa to soil amended with A. mexicana resulted in decreased density of M. javanicain the rhizosphere and in tomato roots, suppressed galling rates and enhanced plant growth.     

Facile synthesis,spectroscopic evaluation and antimicrobial screening of metal endowed triazole compounds

The scientific interest in developing new complexes as inhibitors of bacterial biofilm related infections is constantly rising. The present work describes the chemical synthesis, structural and biological scrutiny of a triazole Schiff base ligand and its corresponding complexes. Triazole Schiff base, (2-methoxy-4-[(1H-1,2,4-triazol-3-ylimino)methyl]phenol) was synthesized from the condensation reaction of 3-amino-1,2,4-triazole and 4-hydroxy-3-methoxybenzaldehyde in an equimolar ratio. The triazole ligand (H₂L) was characterized by physical (solubility, color, melting point), spectroscopic [UV–visible (UV–Vis), Fourier transform infrared spectroscopy (FT-IR), proton nuclear magnetic resonance (¹H-NMR) and mass spectra (MS)] and micro analysis to evaluate their elemental composition. The bidentate ligand was complexed with transition metal [VO(IV), Fe(II), Co(II), Ni(II), Cu(II) and Zn(II)] in 1:2 molar ratio. The complexes were characterized by physical (color, solubility, decomposition temperature, conductance and magnetic moment), FT-IR, UV–Vis and elemental analysis. Thermal stability and fluorescence properties of the compounds were also determined. Density functional theory based theoretical calculations were accomplished to gain more insight into spectroscopic properties. The frontier molecular orbital analysis revealed that the ligand was less reactive with reduced electron donating capability and more kinetic stability than complexes. The as-synthesized compounds were scrutinized for anti-bacterial and anti-fungal activity against selected strains. Cobalt complex exhibited highest antibacterial activity against Escherichia coli and nickel complex has shown highest antifungal activity against Aspergillus niger. All the compounds also showed good antioxidant activity. The theoretical results reflect consistency with the experimental findings signifying that such compounds could be the promising chemical scaffolds in the near future against microbial infectious.

Graphic abstract

     

Screening of inhibitors against SARS-CoV-2 spike protein and their capability to block the viral entry mechanism: A viroinformatics study

SARS-CoV-2, previously named 2019 novel coronavirus (2019-nCoV), has been associated with the global pandemic of acute respiratory distress syndrome. First reported in December 2019 in the Wuhan province of China, this new RNA virus has several folds higher transmission among humans than its other family member (SARS-CoV and MERS-CoV). The SARS-CoV-2 spike receptor-binding domain (RBD) is the region mediating the binding of the virus to host cells via Angiotensin-converting enzyme 2 (ACE2), a critical step of viral. Here in this study, we have utilized in silico approach for the virtual screening of antiviral library extracted from the Asinex database against the Receptor binding domain (RBD) of the S1 subunit of the SARS-CoV-2 spike glycoprotein. Further, the molecules were ranked based on their binding affinity against RBD, and the top 15 molecules were selected. The affinity of these selected molecules to interrupt the ACE2-Spike interaction was also studied. It was found that the chosen molecules were demonstrating excellent binding affinity against spike protein, and these molecules were also very effectively interrupting the ACE2-RBD interaction.Furthermore, molecular dynamics (MD) simulation studies were utilized to investigate the top 3 selected molecules' stability in the ACE2-RBD complexes. To the best of our knowledge, this is the first study where molecules' inhibitory potential against the Receptor binding domain (RBD) of the S1 subunit of the SARS-CoV-2 spike glycoprotein and their inhibitory potential against the ACE2-Spike has been studied. We believe that these compounds can be further tested as a potential therapeutic option against COVID-19.