Biology subjects

Kumar, S.

Publications and source records attributed to Kumar, S..

At least 505 records · Page 28Linked to original sources

bioRxiv2021

Recombinant binder of sperm protein1 (rec-BSP1) as a potent fertility factor mediates its effect on embryo production

ObjectiveTo understand the effect of recombinant BSP1 (rec-BSP1) on in vitro capacitation of sperm and fertilization study Method(s)Articles were screened for reports including rec-BSP1, Capacitation, in vitro fertilization InterventionNone Main Outcome Measure(s)Reproductive outcomes, effect on gametes and embryos Result(s)Here we report an optimization of condition for rec-BSP1 production which was used for in vitro capacitation and enhancement of buffalo embryo production. The sequence of the protein was used for multiple sequence alignment which has 99% similarity with PDC 109 protein. The expression of rec-BSP1 was carried out successfully with 1 mM IPTG at 160 C for 22 hrs and purified it in soluble form. The structure of rec-BSP1 was generated using 3D modelling and analysed its mode of binding with heparin and PC by molecular docking and the structural stability of rec-BSP1-PC and rec-BSP1-heparin complexes by using molecular dynamic (MD) simulation. The effect of rec-BSP1 was observed on in vitro capacitation of spermatozoa and buffalo blastocyst production. It was found that the rec-BSP1 enhanced the sperm motility at a concentration of 50 g/ml for 1 h of incubation without having any detrimental effect on the sperm morphology and a significant increase in blastocyst production at concentration of 50 g/ml rec-BSP1. Hence this finding represents a new insight and advance the prospective approach to develop a potential fertility factor in reproduction. Conclusion(s)The purified rec-BSP1 may enhance on male fertility and mediated its effect on in vitro blastocyst production in buffalo.

bioengineering↗

bioRxiv2021

eIF2α integrates proteotoxic signals both from ER and cytoplasm: Hsp70-Bag3 module regulates HRI-dependent phosphorylation of eIF2α

The major heat shock protein Hsp70 has been implicated in many stages of cancer development. These effects are mediated by a scaffold protein Bag3 that binds to Hsp70 and links it to components of multiple cancer-related signaling pathways. Accordingly, the Hsp70-Bag3 complex has been targeted by small molecules, which showed strong anti-cancer effects. Here, our initial question was how JG-98, an allosteric inhibitor of Hsp70 that blocks its interaction with Bag3, causes cell death. Breast epithelial cells MCF10A transformed with a single oncogene Her2 showed higher sensitivity to JG-98 then parental MCF10A cells. RNA expression analysis showed that this enhanced sensitivity correlated with higher induction of the UPR genes. Indeed, depletion of the pro-apoptotic UPR responsive transcription factor CHOP significantly protected cells from JG-98. Surprisingly, only the eIF2-associated branch of the UPR was activated by JG-98, suggesting that the response was not related to the ER proteotoxicity. Indeed, it was dependent on activation of a distinct cytoplasmic eIF2 kinase HRI. HRI-dependent phosphorylation of eIF2 was also activated by the cytoplasmic proteotoxicity via Hsp70-Bag3 complex, which directly associates with HRI. Dissociation of Hsp70-Bag3 complex led to Bag3-dependent degradation of HRI via autophagy. Therefore, eIF2 integrates proteotoxicity signals from both ER and cytoplasm, and the cytoplasmic response mediates cytotoxicity of the Hsp70-Bag3 inhibitors.

cell biology↗

bioRxiv2021

Immunophenotyping and transcriptional profiling of human plasmablasts in dengue.

Previous studies have shown that plasmablasts expand massively in dengue patients as compared to many other situations such as influenza infection or vaccination. However, a detailed understanding of the phenotypes and transcriptional features of these cells is lacking. Moreover, despite India having nearly a third of global dengue disease burden, there is virtually no information on plasmablasts responses in dengue patients from India. Here, we provide a detailed characterization of plasmablast responses from dengue confirmed febrile children in India. Immunophenotyping and RNA seq analysis showed that in addition to secreting dengue specific antibodies, these massively expanding cells expressed several adhesion molecules, chemokines and chemokine receptors that are involved in endothelial interactions, homing to skin or mucosal tissues including intestine. Surprisingly, we found that these cells also upregulated expression of several cytokine genes that are involved in angiogenesis, leukocyte extravasation and vascular permeability. These transcriptional features were qualitatively similar to plasmablasts from influenza vaccinees. Interestingly, the expansion of the plasmablasts in dengue patients was significantly lower in patients with primary dengue infection compared to those with secondary dengue. Moreover, within the primary dengue patients, their expansion was significantly lower in patients with mild dengue infection (DI) compared to patients with dengue with warning signs (DW) or severe dengue (SD). These results significantly improve our understanding of human plasmablast responses in dengue. Importance Dengue is a globally spreading with over 100 million clinical cases annually with symptoms ranging from mild self-limiting febrile illness to more severe and sometimes life-threatening dengue hemorrhagic fever or shock, especially among children. India contributes nearly a third of global dengue disease burden. The pathophysiology of dengue is complex and remains poorly understood despite many advances indicating a key role for antibody dependent enhancement of infection. While serum antibodies have been extensively studied, the characteristics of the cellular factories responsible for antibody production, i.e., plasmablasts, are only beginning to emerge. This study provides a comprehensive understanding of the magnitude, phenotype, functional and transcriptional profiles of human plasmablasts from dengue patients in India.

immunology↗

bioRxiv2021

Transfer RNA-derived non-coding RNAs (tncRNAs): Uncovering hidden regulators of transcriptional regulatory circuits in plants

The lateral emergence of distinct classes of non-coding RNAs has led to better insights into the eukaryotic regulatory networks. Among them, the existence of transfer RNA (tRNA)-derived non-coding RNAs (tncRNAs) demands their exploration in the plant kingdom. Here, we have designed a methodology to uncover the bigger picture of tncRNAome in plants. Using this, we have identified diverse tncRNAs of length 14-50 nt in ~2500 small RNA sequencing (sRNA-seq) samples of six major angiosperms, and further studied their various features including length, codon-usage, cleavage pattern, and modified tRNA nucleosides. Codon-dependent generation of tncRNAs indicates that the process is highly specific rather than being mere random tRNA degradation. Analysis for nucleotide composition of tncRNA cleavage positions indicates that they are generated through precise endoribonucleolytic machinery. Certain tRNA nucleoside modifications on tncRNAs were found to be conserved across the plants, and hence may influence tRNA cleavage, as well as tncRNA functions. Pathway enrichment analysis revealed that common tncRNA targets were majorly involved in the metabolic and developmental processes of plants. Also, many tRFs were found to be associated with transposable elements. Distinct tissue-specific tncRNA clusters indicate their role in plant development under normal physiological conditions. Furthermore, the identification of a significant number of differentially expressed tncRNAs under several abiotic and biotic stresses highlights their probable role as gene expression modulators during various stress conditions. Thus, this study will be beneficial to investigate the emerging role of tncRNAs as prospective biomarkers in plant development and stress. HighlightsO_LIComputational pipeline for accurate identification of genuine transfer RNA-derived non-coding RNAs (tncRNAs) using small RNA sequencing (sRNA-seq) datasets. C_LIO_LISix major tncRNA classes of length ranging from 14 to 50 nt were identified in ~2,500 sRNA-seq datasets in six different angiosperms. C_LIO_LItRNA nucleoside modifications may affect tncRNA cleavage in plants. C_LIO_LIConserved tncRNAs target transcripts are involved in plant growth, development, and metabolism. C_LIO_LItncRNAs are expressed in a tissue-dependent, and stress-specific manner in plants. C_LI

bioinformatics↗

bioRxiv2021

Phylomedicine of mutational processes in somatic cancer cell populations

Mutational processes in somatic cancer cell populations are constantly changing, leaving their signatures in the accumulated genomic variation in tumors. The inference of mutational signatures from the observed genetic variation enables spatiotemporal tracking of tumor mutational processes that evolve due to cellular environmental changes, mutations, and treatment regimes. Ultimately, mutational patterns illuminate the mechanistic understanding of their evolution in cancer progression. We show that the integration of cancer cell phylogeny with mutational signature deconvolution enables higher-resolution detection of gain and loss of mutational processes within the phylogeny. This approach to analyzing somatic genomic variations in 61 lung cancer patients revealed a high turn-over of mutational processes over time and closely related clonal lineages. Some mutational signatures (e.g., smoking-related) showed a higher propensity to be lost, whereas others (e.g., AID/APOBEC) were gained during lung tumors evolution. These observations shed light on the evolution of mutational processes in somatic cell evolution.

evolutionary biology↗

bioRxiv2021

An immunoinformatics approach to study the epitopes contributed by Nsp13 of SARS-CoV-2

The on-going coronavirus disease-19 (COVID-19) pandemic caused by SARS-CoV-2 has infected hundreds of millions of people and killed more than two million people worldwide. Currently, there are no effective drugs available for treating SARS-CoV-2 infections; however, vaccines are now being administered worldwide to control this virus. In this study, we have studied SARS-CoV-2 helicase, Nsp13, which is critical for viral replication. We compared the Nsp13 sequences reported from India with the first reported sequence from Wuhan province, China to identify and characterize the mutations occurring in this protein. To correlate the functional impact of these mutations, we characterised the most prominent B cell and T cell epitopes contributed by Nsp13. Our data revealed twenty-one epitopes, which exhibited high antigenicity, stability and interactions with MHC class-I and class-II molecules. Subsequently, the physiochemical properties of these epitopes were also analysed. Furthermore, several of these Nsp13 epitopes harbour mutations, which were further characterised by secondary structure and per-residue disorderness, stability and dynamicity predictions. Altogether, we report the candidate epitopes of Nsp13 that may help the scientific community to understand the evolution of SARS-CoV-2 variants and their probable implications.

molecular biology↗

bioRxiv2021

Data-driven speciation tree prior for better species divergence times in calibration-poor molecular phylogenies

MotivationPrecise time calibrations needed to estimate ages of species divergence are not always available due to fossil records incompleteness. Consequently, clock calibrations available for Bayesian dating analyses can be few and diffused, i.e., phylogenies are calibration-poor, impeding reliable inference of the timetree of life. We examined the role of speciation birth-death tree prior on Bayesian node age estimates in calibration-poor phylogenies and tested the usefulness of an informative, data-driven tree prior to enhancing the accuracy and precision of estimated times. ResultsWe present a simple method to estimate parameters of the birth-death tree prior from the molecular phylogeny for use in Bayesian dating analyses. The use of a data-driven birth-death (ddBD) tree prior leads to improvement in Bayesian node age estimates for calibration-poor phylogenies. We show that the ddBD tree prior, along with only a few well-constrained calibrations, can produce excellent node ages and credibility intervals, whereas the use of an uninformative, uniform (flat) tree prior may require more calibrations. Relaxed clock dating with ddBD tree prior also produced better results than a flat tree prior when using diffused node calibrations. We also suggest using ddBD tree priors to improve the detection of outliers and influential calibrations in cross-validation analyses. ConclusionEmpirical Bayesian dating analyses with ddBD tree priors enable more accurate and precise node age estimates for calibration-poor phylogenies. Our results have practical applications because the ddBD tree prior reduces the number of well-constrained calibrations necessary to obtain reliable node age estimates. This would help address key impediments in building the grand timetree of life, revealing the process of speciation, and elucidating the dynamics of biological diversification. AvailabilityAn R module for computing the ddBD tree prior, simulated datasets, and empirical datasets are available at https://github.com/cathyqqtao/ddBD-tree-prior.

evolutionary biology↗

bioRxiv2021

Comparison of SARS-CoV-2 VOC 202012/01 (UK variant) and D614G variant transmission by different routes in Syrian hamsters

Many SARS-CoV-2 variants of concern has been reported recently which were linked to increased transmission. In our earlier study on virus shedding using VOC 202012/01(UK variant) and D614G variant in hamster model, we observed significantly higher viral RNA shedding through nasal wash in case of UK variant. Hence, we compared the transmission of both the UK and D614G variant by various routes in Syrian hamsters to understand whether the high viral RNA shedding could enhance the transmission efficiency of the variant. The current study demonstrated comparable transmission efficiency of both UK and D614G variants of SARS-CoV-2 in Syrian hamsters.

microbiology↗

bioRxiv2021

Robust optical autofocus system utilizing neural networks trained for extended range and time-course and automated multiwell plate imaging including single molecule localization microscopy

We present a robust, long-range optical autofocus system for microscopy utilizing machine learning. This can be useful for experiments with long image data acquisition times that may be impacted by defocusing resulting from drift of components, e.g. due to changes in temperature or mechanical drift. It is also useful for automated slide scanning or multiwell plate imaging where the sample(s) to be imaged may not be in the same horizontal plane throughout the image data acquisition. To address the impact of (thermal or mechanical) fluctuations over time in the optical autofocus system itself, we utilise a convolutional neural network (CNN) that is trained over multiple days to account for such fluctuations. To address the trade-off between axial precision and range of the autofocus, we implement orthogonal optical readouts with separate CNN training data, thereby achieving an accuracy well within the 600 nm depth of field of our 1.3 numerical aperture objective lens over a defocus range of up to approximately +/- 100 m. We characterise the performance of this autofocus system and demonstrate its application to automated multiwell plate single molecule localisation microscopy.

bioengineering↗

bioRxiv2021

Significance of the Wnt canonical pathway in radiotoxicity via oxidative stress of electron beam radiation and its molecular control in mice

Radiation triggers the cell death events through signaling proteins, but the combined mechanism of events is unexplored. We intend to investigate how the combined cascade works throughout the radiation process and its significance over radiotoxicity. Thirty adult mice were irradiated with electron beam radiation, and five were served as a control (Non treated). Mice were sacrificed after post 24 hours and 30 days of irradiation. We assessed the oxidative stress parameter and mRNA profile (Liver, Kidney, spleen, and germ cells), sperm viability, and motility. The mRNA profile study established the network of combined cascade work during radiation to combat oxidative response and cell survival. The quantitative examination of mRNA uncovers unique critical changes in all mRNA levels in all the cases, particularly in germ cells. Recuperation was likewise seen in post 30 days radiation in the liver, spleen, and kidney followed by oxidative stress parameters, however not in germ cells. It proposes reproductive physiology is exceptionally sensitive towards radiation, even at the molecular level. It also suggests the suppression of Lef1/axin2 could be the main reason for permanent failure in the sperm function process. Post irradiation likewise influences the morphology of sperms. The decrease in mRNA level of Lef1, Axin2, survivin, Ku70 suggests radiation inhibits the Wnt canonical pathway and failure in DNA repair mechanism in a coupled manner. Likewise, the increase in BAX and BCL2 suggests apoptosis activation followed by the decreased expression of enzymatic antioxidants. In summary, controlled several interlinked cascades execute when body exposure to radiation may further be used in a study to counterpart and better comprehend medication focus on radiation treatment.

molecular biology↗

bioRxiv2021

Identifying Dihydropyrimidine Dehydrogenase as a Novel Regulator of Hepatic Steatosis

Pyrimidine catabolism is implicated in hepatic steatosis. Dihydropyrimidine Dehydrogenase (DPYD) is an enzyme responsible for uracil and thymine catabolism, and DPYD human genetic variability affects clinically observed toxicity following 5-Fluorouracil (5-FU) administration. In an in vitro model of diet-induced steatosis, the pharmacologic inhibition of DPYD resulted in protection from lipid accumulation. Additionally, a gain-of-function mutation of DPYD, created through clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR-Cas9) engineering, led to an increased lipid burden, which was associated with altered mitochondrial functionality in a hepatocarcionma cell line. The studies presented herein describe a novel role for DPYD in hepatocyte metabolic regulation as a modulator of hepatic steatosis.

cell biology↗

bioRxiv2021

Structural basis for the hyperthermostability of an archaeal glutaminase induced by post-translational succinimide formation

Stability of proteins from hyperthermophiles enabled by reduction of conformational flexibility is realized through various mechanisms. Presence of a stable, hydrolysis-resistant succinimide arising from cyclization of the side chains of aspartyl/asparaginyl residues with backbone amide -NH of the succeeding residue would restrain the torsion angle {Psi}. Here, we describe the crystal structure of Methanocaldococcus jannaschii glutamine amidotransferase (MjGATase) and address the mechanism of a succinimide-induced increased thermostability using molecular dynamics simulations. This study reveals the interplay of negatively charged electrostatic shield and n[->]{pi}* interactions in preventing succinimide hydrolysis. The stable succinimidyl residue induces formation of a conformational-lock, reducing protein flexibility. Protein destabilization upon replacement with the {Phi}-restricted prolyl residue highlights the specificity of the conformationally restrained succinimidyl residue in imparting hyperthermostability. The conservation of succinimide-forming tripeptide sequence (E(N/D)(E/D)) in a group of archaeal GATases suggests an adaptation of this otherwise detrimental post-translational modification as an inducer of thermostability.

biochemistry↗

bioRxiv2021

Negative DNA supercoiling tunes protein-mediated looping

Protein-mediated DNA looping is fundamental to gene regulation and such loops occur stochastically in purified systems. Additional proteins increase the probability of looping, but these probabilities maintain a broad distribution. For example, the probability of lac repressor-mediated looping in individual molecules ranged 0-100%, and individual molecules exhibited representative behavior only in observations lasting an hour or more. Titrating with HU protein progressively compacted the DNA without narrowing the 0-100% distribution. Increased negative supercoiling produced an ensemble of molecules in which all individual molecules more closely resembled the average. Furthermore, in only twelve minutes of observation, well within the doubling time of the bacterium, most molecules exhibited the looping probability of the ensemble. DNA supercoiling, an inherent feature of all genomes, appears to impose time-constrained, emergent behavior on otherwise random molecular activity.

biophysics↗

bioRxiv2021

Comparison of the pathogenicity and virus shedding of SARS CoV-2 VOC 202012/01 and D614G variant in hamster model

The emergence of SARS-CoV-2 variants has posed a serious challenge to public health system and vaccination programs across the globe. We have studied the pathogenicity and virus shedding pattern of the SARS-CoV-2 VOC 202012/01 and compared with D614G variant in Syrian hamsters. VOC 202012/01 could produce disease in hamsters characterized by body weight loss and respiratory tract tropism but mild lung pathology. Further, we also documented that neutralizing antibodies developed against VOC 202012/01 could equally neutralize D614G variant. Higher load of VOC 202012/01 in the nasal wash specimens was observed during the first week of infection outcompeting the D614G variant. The findings suggest increased fitness of VOC 202012/01 to the upper respiratory tract which could lead to higher transmission. Further investigations are needed to understand the transmissibility of new variants. One-Sentence SummarySARS-CoV-2 VOC 202012/01 infected hamsters demonstrated high viral RNA shedding through the nasal secretions and significant body weight loss with mild lung pathology compared to the D614G variant.

microbiology↗

bioRxiv2021

Similar neural and perceptual masking effects of low-power optogenetic stimulation in primate V1

Can direct stimulation of primate V1 substitute for a visual stimulus and mimic its perceptual effect? To address this question, we developed an optical-genetic toolkit to "read" neural population responses using widefield calcium imaging, while simultaneously using optogenetics to "write" neural responses into V1 of behaving macaques. We focused on the phenomenon of visual masking, where detection of a dim target is significantly reduced by a co-localized medium-brightness pedestal. Using our toolkit, we tested whether V1 optogenetic stimulation can recapitulate the perceptual masking effect of a visual pedestal. We find that, similar to a visual pedestal, low-power optostimulation can significantly reduce visual detection sensitivity, that a sublinear interaction between visual and optogenetic evoked V1 responses could account for this perceptual effect, and that these neural and behavioral effects are spatially selective. Our toolkit and results open the door for further exploration of perceptual substitutions by direct stimulation of sensory cortex.

neuroscience↗

bioRxiv2021

Identification of common key genes and pathways between Covid-19 and lung cancer by using protein-protein interaction network analysis

COVID-19 is indeed an infection that is caused by a recently found coronavirus group, a type of virus proven to cause human respiratory diseases. The high mortality rate was observed in patients who had pre-existing health conditions like cancer. However, the molecular mechanism of SARS-CoV-2 infection in lung cancer patients was not discovered yet at the pathway level. This study was about determining the common key genes of COVID-19 and lung cancer through network analysis. The hub genes associated with COVID-19 and lung cancer were identified through Protein-Protein interaction analysis. The hub genes are ALB, CXCL8, FGF2, IL6, INS, MMP2, MMP9, PTGS2, STAT3 and VEGFA. Through gene enrichment, it is identified both COVID-19 and lung cancer have a common pathway in EGFR tyrosine kinase inhibitor resistance, IL-17 signalling pathway, AGE-RAGE signalling pathway in diabetic complications, HIF-1 signalling pathway and pathways in cancer.

bioinformatics↗

bioRxiv2021

The neural G protein Gαo tagged with GFP at an internal loop is functional in C. elegans

Go is the alpha subunit of the major heterotrimeric G protein in neurons and mediates signaling by every known neurotransmitter, yet the signaling mechanisms activated by Go remain to be fully elucidated. Genetic analysis in Caenorhabditis elegans has shown that Go signaling inhibits neuronal activity and neurotransmitter release, but studies of the molecular mechanisms underlying these effects have been limited by lack of tools to complement genetic studies with other experimental approaches. Here we demonstrate that inserting the green fluorescent protein (GFP) into an internal loop of the Go protein results in a tagged protein that is functional in vivo and that facilitates cell biological and biochemical studies of Go. Transgenic expression of Go-GFP rescues the defects caused by loss of endogenous Go in assays of egg laying and locomotion behaviors. Defects in body morphology caused by loss of Go are also rescued by Go-GFP. The Go-GFP protein is localized to the plasma membrane of neurons, mimicking localization of endogenous Go. Using GFP as an epitope tag, Go-GFP can be immunoprecipitated from C. elegans lysates to purify Go protein complexes. The Go-GFP transgene reported in this study enables studies involving in vivo localization and biochemical purification of Go to complement the already well-developed genetic analysis of Go signaling.

neuroscience↗

bioRxiv2021

AlnC: An extensive database of long non-coding RNAs in Angiosperms

Long non-coding RNAs (lncRNAs) play a major role in diverse biological processes that are contemplated to have diverse regulatory roles in plants. While lncRNAs have been described and annotated in major plant species, many of plant species lack annotated lncRNAs despite the availability of transcriptome data in public repositories. In this study, we have identified and annotated the lncRNAs in the transcriptomic data from the 1000 plant genome project (1KP), and developed a user-friendly, open-access database called AlnC. This can be used for the exploration of lncRNAs in 682 Angiosperm plants. The current version of AlnC offers 10,855,598 annotated lncRNA transcripts across 809 sample tissues of diverse flowering plants. To enhance the AlnC interface, we have provided the features for browsing, searching and downloading of lncRNA data, interactive graphics, and online BLAST service. In addition to these information, each lncRNA record is annotated with possible Short open reading frames (e.g. sORFs) to facilitate the study of peptides encoded within lncRNAs. With this user-friendly interface, we assume that AlnC will serve as a rich source of lncRNAs that will contribute to small-and large-scale studies in a wide range of flowering plants.

bioinformatics↗