Biology subjects

Gupta, M. K.

Publications and source records attributed to Gupta, M. K..

25 records · Page 2Linked to original sources

bioRxiv2020

beta-blocker reverses inhibition of beta-2 adrenergic receptor resensitization by hypoxia

Ischemia/hypoxia is major underlying cause for heart failure and stroke. Although beta-adrenergic receptor ({beta}AR) is phosphorylated in response to hypoxia, less is known about the underlying mechanisms. Hypoxia results in robust GRK2-mediated {beta}2AR phosphorylation but does not cause receptor internalization. However, hypoxia leads to significant endosomal-{beta}2AR phosphorylation accompanied by inhibition of {beta}2AR-associated protein phosphatase 2A (PP2A) activity impairing resensitization. Phosphoinositide 3-kinase {gamma} (PI3K{gamma}) impedes resensitization by phosphorylating endogenous inhibitor of protein phosphatase 2A, I2PP2A that inhibits PP2A activity. Hypoxia increased PI3K{gamma} activity leading to significant phosphorylation of I2PP2A resulting in inhibition of PP2A and consequently resensitization. Surprisingly, {beta}-blocker abrogated hypoxia-mediated {beta}2AR phosphorylation instead of phosphorylation in normoxia. Subjecting mice to hypoxia leads to significant cardiac dysfunction and {beta}2AR phosphorylation showing conservation of non-canonical hypoxia-mediated pathway in vivo. These findings provide mechanistic insights on hypoxia-mediated {beta}AR dysfunction which is rescued by {beta}-blocker and will have significant implications in heart failure and stroke.

biochemistry↗

bioRxiv2020

Characterizing and controlling nanoscale self-assembly of suckerin-12

Structural proteins such as the "suckerins" present promising avenues for fabricating functional materials. Suckerins are a family of naturally occurring block copolymer-type proteins that comprise the sucker ring teeth of cephalopods and are known to self-assemble into supramolecular networks of nanoconfined {beta}-sheets. Here, we report characterization and controllable, nanoscale self-assembly of suckerin-12 (S12). We characterize impacts of salt, pH, and protein concentration on S12 solubility, secondary structure, and self-assembly. In doing so, we identify conditions for fabricating ~100 nm nanoassemblies (NAs) with narrow size distributions. Finally, by installing a non-canonical amino acid (ncAA) into S12, we demonstrate the assembly of NAs that are covalently conjugated with a hydrophobic fluorophore, and the ability to change self-assembly and {beta}-sheet content by PEGylation. This work presents new insights into the biochemistry of suckerin-12 and demonstrates how ncAAs can be used to expedite and fine-tune the design of protein materials.

bioengineering↗

bioRxiv2020

Lyophilized cell-free systems display tolerance to organic solvent exposure

Cell-free systems offer a powerful way to deliver biochemical activity to the field without cold chain storage. These systems are capable of sensing as well as biosynthesis of useful molecules at the point of need. So far, cell-free protein synthesis (CFPS) reactions have been studied as aqueous solutions in test tubes or absorbed into paper or cloth. Embedding biological functionality into broadly-used materials, such as plastic polymers, represents an attractive goal. Unfortunately, this goal has for the most part remained out of reach, presumably due to the fragility of biological systems outside of aqueous environments. Here, we describe a surprising and useful feature of lyophilized cell-free lysate systems: tolerance to a variety of organic solvents. Screens of individual CFPS reagents and different CFPS methods reveal that solvent tolerance varies by CFPS reagent composition. Tolerance to suspension in organic solvents may facilitate the use of polymers to deliver dry cell-free reactions in the form of coatings or fibers, or allow dosing of analytes or substrates dissolved in non-aqueous solvents, among other processing possibilities.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsCFPScell-free protein synthesisGFPgreen fluorescent proteinDCMdichloromethaneDMFdimethylformamideDMSOdimethylsulfoxideTHFtetrahydrofuranView Full Text

synthetic biology↗

bioRxiv2020

Silk Fibroin as an Additive for Cell-Free Protein Synthesis

Cell-free systems contain many proteins and metabolites required for complex functions such as transcription and translation or multi-step metabolic conversions. Research into expanding the delivery of these systems by drying or by embedding into other materials is enabling new applications in sensing, point-of-need manufacturing, and responsive materials. Meanwhile, silk fibroin from the silk worm, Bombyx mori, has received attention as a protective additive for dried enzyme formulations and as a material to build biocompatible hydrogels for controlled localization or delivery of biomolecular cargoes. In this work, we explore the effects of silk fibroin as an additive in cell-free protein synthesis (CFPS) reactions. Impacts of silk fibroin on CFPS activity and stability after drying, as well as the potential for incorporation of CFPS into hydrogels of crosslinked silk fibroin are assessed. We find that simple addition of silk fibroin increased productivity of the CFPS reactions by up to 42%, which we attribute to macromolecular crowding effects. However, we did not find evidence that silk fibroin provides a protective effects after drying as previously described for purified enzymes. Further, the enzymatic crosslinking transformations of silk fibroin typically used to form hydrogels are inhibited in the presence of the CFPS reaction mixture. Crosslinking attempts did not impact CFPS activity, but did yield localized protein aggregates rather than a hydrogel. We discuss the mechanisms at play in these results and how the silk fibroin-CFPS system might be improved for the design of cell-free devices.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsCFPSCell-free Protein SynthesisSFSilk FibroinHRPhorse radish peroxidasesfGFPsuperfolder green fluorescent proteinSEMScanning Electron MicroscopyView Full Text

synthetic biology↗

bioRxiv2019

De novo assembly and characterization of transcriptome towards understanding molecular mechanism associated with MYMIV-resistance in Vigna mungo - A computational study

The fast climate change affects yield in Vigna mungo via enhancing both biotic and abiotic stresses. Out of all factors, the yellow mosaic disease has the most damaging effect. However, due to lack of reference genome of Vigna mungo, the complete mechanism associated with MYMIV (Mungbean Yellow Mosaic Indian Virus) resistance in Vigna mungo remain elusive to date. Considering this, the authors made an attempt to release new transcriptome and its annotation by employing computational approaches. Quality assessment of the generated transcriptomes reveals that it successfully aligned with 99.03% of the raw reads and hence can be employed for future research. Functional annotation of the transcriptome reveals that 31% and [~]14% of the total transcripts encode lncRNAs and protein-coding sequences, respectively. Further, analysis reveals that, out of total transcripts, only 4536 and 78808 are significantly down and up-regulated during MYMIV infection in Vigna mungo, respectively. These significant transcripts are mainly associated with ribosome, spliceosome, glycolysis /gluconeogenesis, RNA transport, oxidative phosphorylation, protein processing in the endoplasmic reticulum, MAPK signaling pathway - plant, methionine and cysteine metabolism, purine metabolism and RNA degradation. Unlike the previous study, this is for the first time, the present study identified these pathways may play key role in MYMIV resistance in Vigna mungo. Thus, information and transcriptomes data available in the present study make a significant contribution to understanding the genomic structure of Vigna mungo, enabling future analyses as well as downstream applications of gene expression, sequence evolution, and genome annotation.

bioinformatics↗

bioRxiv2019

Assessment of knowledge, attitudes and practices about antibiotic resistance among medical students in India

BackgroundTo reduce the magnitude of antimicrobial resistance, there is a need to change the knowledge and behavior of future prescribers regarding use and prescription of antibiotics. This can be ensured through the appropriate training of next generation doctors and medical students. But, before planning or strengthening any teaching or training program for any group, it is required to have a conclusive evidence about knowledge, attitude and practices of that group. With this background this study was conducted to assess the knowledge, attitudes and the practices of medical students in India with respect to antibiotic resistance and usage MethodsIt was a cross-sectional study which was done online through google forms. A semi-structured questionnaire containing a five point Likert scale was used for the data collection. The questionnaire was sent to medical students across India by sharing link through contacts of Medical Students Association of India. Respondent-driven sampling technique was also adopted for the study. Data was analyzed using SPSS v.25 and Microsoft Excel 2016. ResultsThe overall mean score of awareness for the students was 4.36 + 0.39. As compared to first year students, mean score of awareness was significantly higher among students of all the years. A significantly better awareness was also observed among pre final year students as compared to other years. Variable practices have been observed regarding use of antibiotics among medical students. ConclusionThe awareness level of medical students regarding antibiotics and its resistance was quite satisfactory. As far as attitude and practices are concerned, there is a significant need for improvements.

scientific communication and education↗

bioRxiv2018

Modulation of antibiotic sensitivity and biofilm formation in Pseudomonas aeruginosa by interspecies diffusible signal factor analogues

The opportunistic pathogen Pseudomonas aeruginosa can participate in inter-species communication through signaling by cis-2-unsaturated fatty acids of the diffusible signal factor (DSF) family. Sensing these signals involves the histidine kinase PA1396 and leads to altered biofilm formation and increased tolerance to various antibiotics. Here, we show that the membrane-associated sensory input domain of PA1396 has five trans-membrane helices, two of which are required for DSF sensing. DSF binding is associated with enhanced auto-phosphorylation of PA1396 incorporated into liposomes. Further, we examined the ability of synthetic DSF analogues to modulate or inhibit PA1396 activity. Several of these analogues block the ability of DSF to trigger auto-phosphorylation and gene expression, whereas others act as inverse agonists reducing biofilm formation and antibiotic tolerance, both in vitro and in murine infection models. These analogues may thus represent lead compounds for novel adjuvants to improve the efficacy of existing antibiotics.

microbiology↗