Homocysteine alters cerebral microvascular integrity and causes remodeling by antagonizing GABA-A receptor

High levels of homocysteine (Hcy), known as hyperhomocysteinemia (HHcy), are associated with cerebrovascular diseases, such as vascular dementia, stroke, and Alzheimer’s disease. The γ-amino butyric acid (GABA) is an inhibitory neurotransmitter and a ligand of GABA-A receptor. By inhibiting excitatory response, it may decrease complications associated with vascular dementia and stroke. Hcy specifically competes with the GABA-A receptors and acts as an excitotoxic neurotransmitter. Previously, we have shown that Hcy increases levels of NADPH oxidase and reactive oxygen species (ROS), and decreases levels of thioredoxin and peroxiredoxin by antagonizing the GABA-A receptor. Hcy treatment leads to activation of matrix metalloproteinases (MMPs) in cerebral circulation by inducing redox stress and ROS. The hypothesis is that Hcy induces MMPs and suppresses tissue inhibitors of metalloproteinase (TIMPs), in part, by inhibiting the GABA-A receptor. This leads to degradation of the matrix and disruption of the blood brain barrier. The brain cortex of transgenic mouse model of HHcy (cystathionine β-synthase, CBS?/+) and GABA-A receptor null mice treated with and without muscimol (GABA-A receptor agonist) was analysed. The mRNA levels were measured by Q-RT-PCR. Levels of MMP-2, -9, -13, and TIMP-1, -2, -3, and -4 were evaluated by in situ labeling and PCR-gene arrays. Pial venular permeability to fluorescence-labeled albumin was assessed with intravital fluorescence microscopy. We found that Hcy increases metalloproteinase activity and decreases TIMP-4 by antagonizing the GABA-A receptor. The results demonstrate a novel mechanism in which brain microvascular permeability changes during HHcy and vascular dementias, and have therapeutic ramifications for microvascular disease in Alzheimer’s patients.     

Massive gene acquisitions in Mycobacterium indicus pranii provide a perspective on mycobacterial evolution

Understanding the evolutionary and genomic mechanisms responsible for turning the soil-derived saprophytic mycobacteria into lethal intracellular pathogens is a critical step towards the development of strategies for the control of mycobacterial diseases. In this context, Mycobacterium indicus pranii (MIP) is of specific interest because of its unique immunological and evolutionary significance. Evolutionarily, it is the progenitor of opportunistic pathogens belonging to M. avium complex and is endowed with features that place it between saprophytic and pathogenic species. Herein, we have sequenced the complete MIP genome to understand its unique life style, basis of immunomodulation and habitat diversification in mycobacteria. As a case of massive gene acquisitions, 50.5% of MIP open reading frames (ORFs) are laterally acquired. We show, for the first time for Mycobacterium, that MIP genome has mosaic architecture. These gene acquisitions have led to the enrichment of selected gene families critical to MIP physiology. Comparative genomic analysis indicates a higher antigenic potential of MIP imparting it a unique ability for immunomodulation. Besides, it also suggests an important role of genomic fluidity in habitat diversification within mycobacteria and provides a unique view of evolutionary divergence and putative bottlenecks that might have eventually led to intracellular survival and pathogenic attributes in mycobacteria.     

Spectrum of large copy number variations in 26 diverse Indian populations: potential involvement in phenotypic diversity

Copy number variations (CNVs) have provided a dynamic aspect to the apparently static human genome. We have analyzed CNVs larger than 100 kb in 477 healthy individuals from 26 diverse Indian populations of different linguistic, ethnic and geographic backgrounds. These CNVRs were identified using the Affymetrix 50K Xba 240 Array. We observed 1,425 and 1,337 CNVRs in the deletion and amplification sets, respectively, after pooling data from all the populations. More than 50% of the genes encompassed entirely in CNVs had both deletions and amplifications. There was wide variability across populations not only with respect to CNV extent (ranging from 0.04–1.14% of genome under deletion and 0.11–0.86% under amplification) but also in terms of functional enrichments of processes like keratinization, serine proteases and their inhibitors, cadherins, homeobox, olfactory receptors etc. These did not correlate with linguistic, ethnic, geographic backgrounds and size of populations. Certain processes were near exclusive to deletion (serine proteases, keratinization, olfactory receptors, GPCRs) or duplication (homeobox, serine protease inhibitors, embryonic limb morphogenesis) datasets. Populations having same enriched processes were observed to contain genes from different genomic loci. Comparison of polymorphic CNVRs (5% or more) with those cataloged in Database of Genomic Variants revealed that 78% (2473) of the genes in CNVRs in Indian populations are novel. Validation of CNVs using Sequenom MassARRAY revealed extensive heterogeneity in CNV boundaries. Exploration of CNV profiles in such diverse populations would provide a widely valuable resource for understanding diversity in phenotypes and disease.     

RAmiRNA: Software suite for generation of SVMbased prediction models of mature miRNAs

MicroRNAs (miRNAs) are short endogenous non-coding RNA molecules that regulate protein coding gene expression in animals, plants, fungi, algae and viruses through the RNA interference pathway. By virtue of their base complementarity, mature miRNAs stop the process of translation, thus acting as one of the important molecules in vivo. Attempts to predict precursor-miRNAs and mature miRNAs have been achieved in a significant number of model organisms but development of prediction models aiming at relatively less studied organisms are rare. In this work, we provide a suite of standalone softwares called RAmiRNA (RAdicalmiRNA detector), to solve the problem of custom development of prediction models for mature miRNAs using support vector machine (SVM) learning. RAmiRNA could be used to develop SVM based model for prediction of mature miRNAs in an organism or a group of organisms in a UNIX based local machine. Additionally RAmiRNA generates training accuracy for a quick estimation of prediction ability of generated model. AVAILABILITY: The database is available for free at http://ircb.iiita.ac.in.     

Ligand-mediated conformational changes and positioning of tryptophans in reconstituted human sodium/D-glucose cotransporter1 (hSGLT1) probed by tryptophan fluorescence

Recombinant purified human sodium/D-glucose cotransporter1 (hSGLT1) was reconstituted in a functional form into phospholipid vesicles and its conformational states in the absence and presence of ligands and inhibitors were probed by intrinsic tryptophan fluorescence. In the presence of sodium, sugars increase intrinsic fluorescence (maximum 17%) in a saturable manner in the following order alpha-MDG >D-Glu approximately D-Gal > D-Man >D-All, with no effect of L-Glu. Apparent affinities ranging from 0.65 to 10.4 mM were observed. In addition, D-Glu increased the accessibility of the Trps to hydrophilic collisional quenchers. On the contrary, the transport inhibitor phlorizin decreased Trps fluorescence in a sodium-dependent manner by 50% with a red shift of 4-6 nm and decreased quencher accessibility, these effects were saturable with a high affinity of 5 microM. Furthermore, the positioning of the tryptophans in the reconstituted transporter was investigated. hSGLT1 Trps fluorescence was reduced by N-bromosuccinimide treatment maximally 25% in membranes and 65% in solution. The fluorescence was also significantly but differently quenched by the lipid-soluble spin labeled probes 5-Doxyl-phosphatidylcholine (40%) and 12-Doxyl-phosphatidylcholine (26%). Depth-calculation using the parallax method suggested a location of Trps at an average depth of 10 angstrom from the center of the bilayer. These studies demonstrate the existence of different conformational states of the membrane-embedded transporter in its glucose-free form, as sodium-glucose-carrier complex and as sodium-phlorizin-carrier complex. They further indicate that most of the Trp residues in hSGLT1 are located in hydrophobic regions of the protein or in contact with the lipid bilayer of the membrane. There, they are located close to the membrane-water interface contributing to the vectorial nature of the transporter.     

Isolation and Characterization of Bacteriophages from Inland Saline Aquaculture Environments to Control Vibrio parahaemolyticus Contamination in Shrimp

Among the various bacterial pathogens associated with the aquaculture environment, Vibrio parahaemolyticus the important one from shrimp and human health aspects. Though having been around for several decades, phage-based control of bacterial pathogens (phage therapy) has recently re-emerged as an attractive alternative due to the availability of modern phage characterization tools and the global emergence of antibiotic-resistant bacteria. In the present study, a total of 12 V. parahaemolyticus specific phages were isolated from 264 water samples collected from inland saline shrimp culture farms. During the host range analysis against standard/field isolates of V. parahaemolyticus and other bacterial species, lytic activity was observed against 2.3–45.5% of tested V. parahaemolyticus isolates. No lytic activity was observed against other bacterial species. For genomic characterization, high-quality phage nucleic acid with concentrations ranging from 7.66 to 220 ng/µl was isolated from 9 phages. After digestion treatments with DNase, RNase and S1 nuclease, the nature of phage nucleic acid was determined as ssDNA and dsDNA for 7 and 2 phages respectively. During transmission electron microscopy analysis of phage V5, it was found to have a filamentous shape making it a member of the family Inoviridae. During efficacy study of phage against V. parahaemolyticus in shrimp, 78.1% reduction in bacterial counts was observed within 1 h of phage application. These results indicate the potential of phage therapy for the control of V. parahaemoyticus in shrimp.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12088-021-00934-6.     

An Epichlorohydrin-Crosslinked Semi-Interpenetrating GG-PEO Network as a Xerogel Matrix for Sustained Release of Sulpiride

The current study involved the development of a novel sustained release crosslinked semi-IPN xerogel matrix tablet prepared by chemical crosslinking of poly(ethylene) oxide (PEO) and gellan gum (GG) employing epichlorohydrin (EPI) as crosslinker. A Box–Behnken design was employed for the statistical optimization of the matrix system to ascertain the ideal combination of native polymeric and crosslinking agents. Characterization studies were performed by employing standard polymer characterization techniques such as Fourier transform infrared spectrometry, differential scanning calorimetry, and scanning electron microscopy. Formulated matrix tablets displayed zero-order release kinetics, extending over 24 h. The mechanism of drug release was primarily by swelling and surface erosion. Crosslinked semi-IPN xerogel matrix tablets were compared to non-crosslinked polymer blends; results from the study conducted showed that the physiochemical properties of the PEO and GG were sufficiently modified to allow for sustained release of sulpiride with a 100% drug release at 24 h in a controlled manner as compared to non-crosslinked formulations which displayed further release beyond the test period. Crosslinked formulations displayed water uptake between 450 and 500% indicating a controlled rate of swelling and erosion allowing for sustained release. Surface morphology of the crosslinked system depicted a porous structure formed by interpenetrating networks of polymers, allowing for a greater degree of controlled penetration into the system affording it the ability to sustain drug release. Therefore, conclusively, based on the study performed, crosslinked PEO-GG allows for the sustained release of sulpiride from a hydrophilic semi-IPN xerogel matrix system.KEY WORDS: epichlorohydrin, matrix tablet, semi-interpenetrating polymer network, sustained release, sulpiride     

Simultaneous single-cell protein production and COD removal with characterization of residual protein and intermediate metabolites during whey fermentation by K. marxianus

Cheese whey fermentation with Kluyveromyces marxianus was carried out at 40 °C and pH 3.5 to examine simultaneous single-cell protein production and chemical oxygen demand (COD) removal, determine the fate of soluble whey protein and characterize intermediate metabolites. After 36 h of batch fermentation, the biomass concentration increased from 2.0 to 6.0 g/L with 55 % COD reduction (including protein), whereas soluble whey protein concentration decreased from 5.6 to 4.1 g/L. It was confirmed through electrophoresis (SDS-PAGE) that the fermented whey protein was different from native whey protein. HPLC and GC–MS analysis revealed a change in composition of organic compounds post-fermentation. High inoculum concentration in batch fermentation resulted in an increase in biomass concentration from 10.3 to 15.9 g/L with 80 % COD reduction (including protein) within 36 h with residual protein concentration of 4.5 g/L. In third batch fermentation, the biomass concentration increased from 7.3 to 12.4 g/L with 71 % of COD removal and residual protein concentration of 4.3 g/L after 22 h. After 22 h, the batch process was shifted to a continuous process with cell recycle, and the steady state was achieved after another 60 h with biomass yield of 0.19 g biomass/g lactose and productivity of 0.26 g/L h. COD removal efficiency was 78–79 % with residual protein concentration of 3.8–4.2 g/L. The aerobic continuous fermentation process with cell recycle could be applied to single-cell protein production with substantial COD removal at low pH and high temperature from cheese whey.     

New insights into the evolutionary history of Plasmodium falciparum from mitochondrial genome sequence analyses of Indian isolates

Estimating genetic diversity and inferring the evolutionary history of Plasmodium falciparum could be helpful in understanding origin and spread of virulent and drug‐resistant forms of the malaria pathogen and therefore contribute to malaria control programme. Genetic diversity of the whole mitochondrial (mt) genome of P. falciparum sampled across the major distribution ranges had been reported, but no Indian P. falciparum isolate had been analysed so far, even though India is highly endemic to P. falciparum malaria. We have sequenced the whole mt genome of 44 Indian field isolates and utilized published data set of 96 genome sequences to present global genetic diversity and to revisit the evolutionary history of P. falciparum. Indian P. falciparum presents high genetic diversity with several characteristics of ancestral populations and shares many of the genetic features with African and to some extent Papua New Guinean (PNG) isolates. Similar to African isolates, Indian P. falciparum populations have maintained high effective population size and undergone rapid expansion in the past with oldest time to the most recent common ancestor (TMRCA). Interestingly, one of the four single nucleotide polymorphisms (SNPs) that differentiates P. falciparum from P. falciparum‐like isolates (infecting non‐human primates in Africa) was found to be segregating in five Indian P. falciparum isolates. This SNP was in tight linkage with other two novel SNPs that were found exclusively in these five Indian isolates. The results on the mt genome sequence analyses of Indian isolates on the whole add to the current understanding on the evolutionary history of P. falciparum.