Laser microdissection and pressure-catapulting technique to study gene expression in the reoxygenated myocardium

For focal events such as myocardial infarction, it is important to dissect infarction-induced biological responses as a function of space with respect to the infarct core. Laser microdissection pressure catapulting (LMPC) represents a recent variant of laser capture microdissection that enables robot-assisted rapid capture of catapulted tissue without direct user contact. This work represents the maiden effort to apply laser capture microdissection to study spatially resolved biological responses in myocardial infarction. Infarcted areas of the surviving ischemic-reperfused murine heart were identified using a standardized hematoxylin QS staining procedure. Standard staining techniques fail to preserve tissue RNA. Exposure of the tissue to an aqueous medium (typically used during standard immunohistochemical staining), with or without RNase inhibitors, resulted in a rapid degradation of genes, with approximately 80% loss in the 1st h. Tissue elements (1 x 10(4)-4 x 10(6) microm(2)) captured from infarcted and noninfarcted sites with micrometer-level surgical precision were collected in a chaotropic RNA lysis solution. Isolated RNA was analyzed for quality by microfluidics technology and reverse transcribed to generate high-quality cDNA. Real-time PCR analysis of the cDNA showed marked (200- and 400-fold, respectively) induction of collagen Ia and IIIa at the infarcted site compared with the noninfarcted site. This work reports a sophisticated yet rapid approach to measurement of relative gene expressions from tissue elements captured from spatially resolved microscopic regions in the heart with micrometer-level precision.     

Synthesis, structural characterization and electrochemical activity of oxidovanadium(IV/V) complexes of a diprotic ONS chelating ligand

The present work reports the chemistry of a few oxidovanadium(IV) and (V) complexes of the ONS chelating ligand S-benzyl-β-N-(2-hydroxyphenylethylidine) dithiocarbazate (H₂L). Major objective of this work is to arrive at some general conclusions about the influence of binding environment generated by the replacement of an O-donor center by a S-donor point in a ligand (of a similar arrangement of the other O- and N-donor points) on the redox behavior and on the structural features of comparable [VO(OEt)(ONS)] and [VO(OEt)(ONO)] complexes. Synthesis, characterization by various physicochemical techniques (UV-Vis, IR, EPR and elemental analysis), exploration of electrochemical activity of the oxidovanadium(V) complex [V^VO(OEt)L] (1), the mixed ligand complex [V^VO(N-O)L] (3) (where N-O is the mono anion of 8-hydroxyquinoline) and a binuclear complex [V^VO(OEt)L]₂(μ-4,4′-bipy) (2) are reported. Similar studies on of mixed ligand oxidovanadium(IV) complexes of the formula [V^VO(N-N)L] (4,5) (where N-N = 2,2′-bipy and o-phen) are also presented here. The [V^VO(OEt)L] complex is pentacoordinated and distorted square pyramidal, while the [V^IV(N-N)L] complexes are hexacoordinated and octahedral. Structural features of the complex 1 were compared with the corresponding aspects of the previously reported analogous complex [V^VO(OEt)(ONO)] (1′).     

Alpha-lipoic acid does not alter stress protein response to acute exercise in diabetic brain

Heat shock proteins (HSPs) are molecular chaperones which may act protective in cerebrovascular insults and peripheral diabetic neuropathy. We hypothesized that alpha-lipoic acid (LA), a natural thiol antioxidant, may enhance brain HSP response in diabetes. Rats with or without streptozotocin-induced diabetes were treated with LA or saline for 8 weeks. Half of the rats were subjected to exhaustive exercise to investigate HSP induction, and the brain tissue was analyzed. Diabetes increased constitutive HSC70 mRNA, and decreased HSP90 and glucose-regulated protein 75 (GRP75) mRNA without affecting protein levels. Exercise increased HSP90 protein and mRNA, and also GRP75 and heme oxygenase-1 (HO-1) mRNA only in non-diabetic animals. LA had no significant effect on brain HSPs, although LA increased HSC70 and HO-1 mRNA in diabetic animals and decreased HSC70 mRNA in non-diabetic animals. Eukaryotic translation elongation factor-2, essential for protein synthesis, was decreased by diabetes and suggesting a mechanism for the impaired HSP response related to translocation of the nascent chain during protein synthesis. LA supplementation does not offset the adverse effects of diabetes on brain HSP mRNA expression. Diabetes may impair HSP translation through elongation factors related to nascent chain translocation and subsequent responses to acute stress.     

Pseudomonas aeruginosa WS-1 for biological control of leaf blight disease of Withania somnifera

Withania somnifera (L.) Dunal, also known Indian ginseng is one of the most widespread tranquillizers tranquillisers used for the treatment of nervous disorders, intestinal infection, leprosy, and cancer; it also suffers a leaf blight disease caused by the fungus Alternaria dianthicola in various districts of South Bengal, India: Pseudomonas aeruginosa strain WS-1 isolated from the rhizosphere, showed both in vitro and in vivo antagonistic activity against the pathogen. The antifungal activity of the isolate has been found to be linked to theproduction of a siderophore, volatile substances (hydrocyanic acid), proteases and chitinases. Foliar application of a talc talc-based formulation of P. aeruginosa strain WS-1 to field grown W. somnifera reduced disease severity by 80% compared to non-treated control.     

Acetylcholine's effect on vascular resistance and compliance in the pulmonary circulation

Acetylcholine's effect on the distribution of vascular resistance and compliance in the canine pulmonary circulation was determined under control and elevated vascular tone by the arterial, venous, and double occlusion techniques in isolated blood-perfused dog lungs at both constant flow and constant pressure. Large and small blood vessel resistances and compliances were studied in lungs given concentrations of acetylcholine ranging from 2.0 ng/ml to 200 micrograms/ml. The results of this study indicate that acetylcholine dilates large arteries at low concentrations (less than or equal to 20 ng/ml) and constricts small and large veins at concentrations of at least 2 micrograms/ml. Characterization of acetylcholine's effects at constant pulmonary blood flow indicates that 1) large artery vasodilation may be endothelial-derived relaxing factor-mediated because the dilation is blocked with methylene blue; 2) a vasodilator of the arachidonic acid cascade (blocked by ibuprofen), probably prostacyclin, lessens acetylcholine's pressor effects; 3) when vascular tone was increased, acetylcholine's hemodynamic effects were attenuated; and 4) acetylcholine decreased middle compartment and large vessle compliance under control but not elevated vascular tone. Under constant pressure at control vascular tone acetylcholine increases resistance in all segments except the large artery, and at elevated vascular tone the pressor effects were enhanced, and large artery resistance was increased.     

In silico evaluation of flavonoids as effective antiviral agents on the spike glycoprotein of SARS-CoV-2

The novel coronavirus pandemic has spread over in 213 countries as of July 2020. Approximately 12 million people have been infected so far according to the reports from World Health Organization (WHO). Preventive measures are being taken globally to avoid the rapid spread of virus. In the current study, an in silico approach is carried out as a means of inhibiting the spike protein of the novel coronavirus by flavonoids from natural sources that possess both antiviral and anti-inflammatory properties. The methodology is focused on molecular docking of 10 flavonoid compounds that are docked with the spike protein of SARS-CoV-2, to determine the highest binding affinity at the binding site. Molecular dynamics simulation was carried out with the flavonoid-protein complex showing the highest binding affinity and highest interactions. The flavonoid naringin showed the least binding energy of −9.8 Kcal/mol with the spike protein which was compared with the standard drug, dexamethasone which is being repurposed to treat critically ill patients. MD simulation was carried out on naringin-spike protein complex for their conformational stability in the active site of the novel coronavirus spike protein. The RMSD of the complex appeared to be more stable when compared to that of the protein from 0.2 nm to 0.4 nm. With the aid of this in silico approach further in vitro studies can be carried out on these flavonoids against the novel coronavirus as a means of viral protein inhibitors.