Biology subjects

Mishra, R. K.

Publications and source records attributed to Mishra, R. K..

At least 37 records · Page 2Linked to original sources

bioRxiv2021

B.1.617.3 SARS CoV-2 spike E156G/Δ157-158 mutations contribute to reduced neutralization sensitivity and increased infectivity

SARS CoV-2 variants raise significant concerns due to their ability to cause vaccine breakthrough infections. Here, we sequence-characterized the spike gene, isolated from a breakthrough infection, that corresponded to B.1.617.3 lineage. Delineating the functional impact of spike mutations using reporter pseudoviruses (PV) revealed that N-terminal domain (NTD)-specific E156G/{Delta}157-158 contributed to increased infectivity and reduced sensitivity to ChAdOx1 nCoV-19 vaccine (Covishield)-elicited neutralizing antibodies. A six-nucleotide deletion (467-472) in the spike coding region introduced this change in the NTD. We confirmed the presence of E156G/{Delta}157-158 in the RT-PCR-positive cases concurrently screened, in addition to other circulating spike (S1) mutations like T19R, T95I, L452R, E484Q, and D614G. Notably, E156G/{Delta}157-158 was present in more than 85% of the sequences reported from the USA, UK, and India in August 2021. The spike PV bearing combination of E156G/{Delta}157-158 and L452R further promoted infectivity and conferred immune evasion. Additionally, increased cell-to-cell fusion was observed when spike harbored E156G/{Delta}157-158, L452R, and E484Q, suggesting a combinatorial effect of these mutations. Notwithstanding, the plasma from a recovered individual robustly inhibited mutant spike PV, indicating the increased breadth of neutralization post-recovery. Our data highlights the importance of spike NTD-specific changes in determining infectivity and immune escape of variants.

microbiology↗

bioRxiv2021

In situ Nuclear Matrix preparation in Drosophila melanogaster and its use in studying the components of nuclear architecture

The study of Nuclear Matrix (NuMat) over the last 40 years has been limited to either isolated nuclei from tissues or cells grown in culture. Here, we provide a protocol for NuMat preparation in intact Drosophila melanogaster embryos and its use in dissecting the components of nuclear architecture. The protocol does not require isolation of nuclei and therefore maintains the three-dimensional milieu of an intact embryo, which is biologically more relevant compared to cells in culture. One of the advantages of this protocol is that only a small number of embryos are required. The protocol can be extended to larval tissues like salivary glands and imaginal discs with little modification. Taken together, it becomes possible to carry out such studies in parallel to genetic experiments using mutant and transgenic flies. This protocol, therefore, opens the powerful field of fly genetics to cell biology in the study of nuclear architecture. SummaryNuclear Matrix is a biochemically defined entity and a basic component of the nuclear architecture. Here we present a protocol to isolate and visualize Nuclear Matrix in situ in the intact embryos and tissues of Drosophila melanogaster and its potential applications.

cell biology↗

bioRxiv2021

Free spermidine evokes superoxide radicals that manifest toxicity

Spermidine and other polyamines alleviate oxidative stress, yet excess spermidine seems toxic to Escherichia coli unless it is neutralized by SpeG, an enzyme for the spermidine N-acetyl transferase function. Besides, a specific mechanism of SpeG function conferring pathogenic fitness to Staphylococcus aureus USA300 strain is unknown. Here, we provide evidence that although spermidine mitigates oxidative stress by lowering hydroxyl radical and hydrogen peroxide levels, excess of it simultaneously triggers the production of superoxide radicals, thereby causing toxicity in the E. coli {Delta}speG strain as well as naturally SpeG-deficient S. aureus RN4220 strain. However, wild-type E. coli and S. aureus USA300 with a horizontally-acquired speG gene tolerate applied exogenous spermidine stress. Furthermore, we demonstrate that while RNA-bound spermidine inhibits iron oxidation, free spermidine interacts and oxidizes the iron to evoke superoxide radicals directly. Superoxide radicals thus generated, further affects redox balance and iron homeostasis. Since iron acquisition and metabolism in the host tissues is a challenging task for S. aureus, the current findings helped us explain that the evolutionary gain of SpeG function by S. aureus USA300 strain allows it to neutralize exogenous spermidine- and spermine-mediated toxicity towards iron metabolism inside the host tissues.

biochemistry↗

bioRxiv2021

Implications of critical nodes-dependent unidirectional cross-talk between Plasmodium and Human SUMO

The endoparasitic pathogen, Plasmodium falciparum (Pf), modulates protein-protein interactions to employ post-translational modifications like SUMOylation in order to establish successful infections. The interaction between E1 and E2 (Ubc9) enzymes governs species specificity in the Plasmodium SUMOylation pathway. Here, we demonstrate that a unidirectional cross-species interaction exists between Pf-SUMO and Human-E2, whereas Hs-SUMO1 failed to interact with Pf-E2. Biochemical and biophysical analysis revealed that surface-accessible Aspartates of Pf-SUMO determine the efficacy and specificity of SUMO-Ubc9 interactions. Furthermore, we demonstrate that critical residues of the Pf-Ubc9 N-terminal are responsible for the lack of interaction between Hs-SUMO1 and Pf-Ubc9. Mutating these residues to corresponding Hs-Ubc9 residues restore electrostatic, {pi}-{pi}, and hydrophobic interactions and allows efficient cross-species interactions. We suggest that the critical changes acquired on the surfaces of Plasmodium SUMO and Ubc9 proteins as nodes can help Plasmodium exploit the host SUMOylation machinery. Thus, Pf-SUMO interactions can be targeted for developing antimalarials.

biochemistry↗

bioRxiv2021

How does literacy affect speech processing? Not by enhancing cortical responses to speech, but by promoting connectivity of acoustic-phonetic and graphomotor cortices.

Previous research suggest that literacy, specifically learning alphabetic letter-to-phoneme mappings, modifies online speech processing, and enhances brain responses to speech in auditory areas associated with phonological processing (Dehaene et al., 2010). However, alphabets are not the only orthographic systems in use in the world, and hundreds of millions of individuals speak languages that are not written using alphabets. In order to make claims that literacy per se has broad and general consequences for brain responses to speech, one must seek confirmatory evidence from non-alphabetic literacy. To this end, we conducted a longitudinal fMRI study in India probing the effect of literacy in Devanagari, an abubgida, on functional connectivity and cerebral responses to speech in 91 variously literate Hindi-speaking individuals. Twenty-two completely illiterate participants underwent six months of reading and writing training. Devanagari literacy increases functional connectivity between acoustic-phonetic and graphomotor brain areas, but we find no evidence that literacy changes the way speech is processed, either in cross-sectional or longitudinal analyses. These findings shows that a radical reconfiguration of the neurofunctional substrates of online speech processing is not a universal result of learning to read, and raise the possibility that writing, not only reading, may be instrumental in moulding literate speech perception. Significance StatementIt has come to be accepted that a consequence of being able to read is enhanced auditory processing of speech, reflected by increased cortical responses in areas associated with phonological processing. Here we find no relationship between literacy and the magnitude of brain response to speech stimuli in individuals who speak Hindi, which is written using a non-alphabetic script, Devanagari - an abugida. We propose that the exact nature of the script under examination must be considered before making sweeping claims about the consequences of literacy for the brain. Further, we find evidence that literacy enhances functional connectivity between auditory processing areas and graphomotor areas, suggesting a mechanism whereby learning to write, not only to read, might influence speech perception.

neuroscience↗

bioRxiv2021

Host transcriptional response to SARS-CoV-2 infection in COVID-19 patients

BackgroundOne of the most perplexing aspects of infection with the SARS-CoV-2 virus has been the variable response elicited in its human hosts. Investigating the transcriptional changes in individuals affected by COVID-19 can help understand and predict the degree of illness and guide clinical outcomes in diverse backgrounds. MethodsAnalysis of host transcriptome variations via RNA sequencing from naso/oropharyngeal swabs of COVID-19 patients. ResultsWe report strong upregulation of the innate immune response, especially type I interferon pathway, upon SARS-CoV-2 infection. Upregulated genes were subjected to a comparative meta-analysis using global datasets to identify a common network of interferon stimulated and viral response genes that mediate the host response and resolution of infection. A large proportion of mis-regulated genes showed a reduction in expression level, suggesting an overall decrease in host mRNA production. Significantly downregulated genes included those encoding olfactory, taste and neuro-sensory receptors. Many pro-inflammatory markers and cytokines were also downregulated or remained unchanged in the COVID-19 patients. Finally, a large number of non-coding RNAs were identified as down-regulated, with a few of the lncRNAs associated with functional roles in directing the response to viral infection. ConclusionsSARS-CoV-2 infection results in the robust activation of the bodys innate immunity. Reduction of gene expression is well correlated with the clinical manifestations and symptoms of COVID-19 such as the loss of smell and taste, and myocardial and neurological complications. This study provides a critical dataset of genes that will enhance our understanding of the nature and prognosis of COVID-19.

bioinformatics↗

bioRxiv2021

Genomic organization of the autonomous regulatory domain of eyeless locus in Drosophila melanogaster

In Drosophila, expression of eyeless (ey) gene is restricted to the developing eyes and central nervous system. However, the flanking genes, myoglianin (myo), and bent (bt) have different temporal and spatial expression patterns as compared to the ey. How distinct regulation of ey is maintained is mostly unknown. Earlier, we have identified a boundary element intervening myo and ey genes (ME boundary) that prevents the crosstalk between the cis-regulatory elements of myo and ey genes. In the present study, we further searched for the cis-elements that define the domain of ey and maintain its expression pattern. We identify another boundary element between ey and bt, the EB boundary. The EB boundary separates the regulatory landscapes of ey and bt genes. The two boundaries, ME and EB, show a long-range interaction as well as interact with the nuclear architecture. This suggests functional autonomy of the ey locus and its insulation from differentially regulated flanking regions. We also identify a new Polycomb Response Element, the ey-PRE, within the ey domain. The expression state of the ey gene, once established during early development is likely to be maintained with the help of ey- PRE. Our study proposes a general regulatory mechanism by which a gene can be maintained in a functionally independent chromatin domain in gene-rich euchromatin.

genetics↗

bioRxiv2021

Dry Swab Method of sample collection for SARS-CoV2 testing can be used for culturing virus

Back groundEarlier studies suggested the use of dry swab method for SARS-CoV-2 detection as it does not need VTM and subsequent RNA extraction step making the process cheaper, safer and faster. In this study we explore whether the virus in the dry swab is viable and can be cultured and propagated. MethodSwabs were spiked with SARS-CoV-2 and stored in three different conditions: a) as dry swab (SD, eluted in 1 mL DMEM), b) in 1 mL of Viral Transport Medium (SVTM), and c) in 1 mL of Tris-EDTA buffer (STE). The sample groups were stored either at room temperature (RT, 25{degrees}C{+/-}1{degrees}C) or at 4{degrees}C for 1, 4, 8, 12, 24, 48 and 72 hours before being used as viral inoculums for the propagation studies in Vero cells. ResultsThe RT-qPCR data suggests that SD incubated both at RT and 4{degrees}C harbors viral particles that are viable and culturable at par with SVTM and STE. ConclusionThe dry swab method, in addition to its advantages in detection of the virus, also renders viable viral particles that can be cultured and propagated.

microbiology↗

bioRxiv2020

Satb2 acts as a gatekeeper for major developmental transitions during early vertebrate embryogenesis

Zygotic genome activation (ZGA) initiates regionalized transcription responsible for the acquisition of distinct cellular identities. ZGA is dependent upon dynamic chromatin architecture sculpted by conserved DNA-binding proteins. However, whether the tissue-specific transcription is mechanistically linked with the onset of ZGA is unknown. Here, we have addressed the involvement of chromatin organizer SATB2 in orchestrating these processes during vertebrate embryogenesis. Integrative analysis of transcriptome, genome-wide occupancy and chromatin accessibility revealed contrasting molecular functions of maternal and zygotic pools of Satb2. Maternal Satb2 represses zygotic genes by influencing the interplay between the pluripotency factors. By contrast, zygotic Satb2 activates transcription of the same group of genes during neural crest development and organogenesis. Comparative analysis of maternal versus zygotic function of Satb2 underscores how these antithetical activities are temporally coordinated and functionally implemented. We discuss the evolutionary implications of the biphasic and bimodal regulation of landmark developmental transitions by a single determinant.

developmental biology↗

bioRxiv2020

An Inexpensive RT-PCR Endpoint Diagnostic Assay for SARS-CoV-2 Using Nested PCR: Direct Assessment of Detection Efficiency of RT-qPCR Tests and Suitability for Surveillance

With a view to extending testing capabilities for the ongoing SARS-CoV-2 pandemic we have developed a test that lowers cost and does not require real time quantitative reverse transcription polymerase chain reaction (RT-qPCR). We developed a reverse transcription nested PCR endpoint assay (RT-nPCR) and showed that RT-nPCR has comparable performance to the standard RT-qPCR test. In the course of comparing the results of both tests, we found that the standard RT-qPCR test can have low detection efficiency (less than 50%) in a real testing scenario which may be only partly explained by low viral representation in many samples. This finding points to the importance of directly monitoring detection efficiency in test environments. We also suggest measures that would improve detection efficiency.

molecular biology↗

bioRxiv2020

Improved and Simplified Diagnosis of Covid-19 using TE Extraction from Dry Swabs

Rigorous testing is the way forward to fight the Covid-19 pandemic. Here we show that the currently used and most reliable RT-PCR based SARS-CoV-2 procedure can be further simplified to make it faster, safer and economical by bypassing the RNA isolation step. The modified method is not only fast and convenient but also at par with the traditional method in terms of accuracy, and therefore, can be used for mass screening. Our method takes about half the time and is cheaper by about 40% compared to current most widely used method. We also provide a variant of the new method that increases the efficiency of detection by about 20% compared to the currently used method. Taken together, we demonstrate a more effective and reliable method of SARS-CoV-2 detection.

molecular biology↗

bioRxiv2020

A distinct phylogenetic cluster of Indian SARS-CoV-2 isolates

From an isolated epidemic, COVID-19 has now emerged as a global pandemic. The availability of genomes in the public domain following the epidemic provides a unique opportunity to understand the evolution and spread of the SARS-CoV-2 virus across the globe. The availability of whole genomes from multiple states in India prompted us to analyse the phylogenetic clusters of genomes in India. We performed whole-genome sequencing for 64 genomes making a total of 361 genomes from India, followed by phylogenetic clustering, substitution analysis, and dating of the different phylogenetic clusters of viral genomes. We describe a distinct phylogenetic cluster (Clade I / A3i) of SARS-CoV-2 genomes from India, which encompasses 41% of all genomes sequenced and deposited in the public domain from multiple states in India. Globally 3.5% of genomes, which till date could not be mapped to any distinct known cluster fall in this newly defined clade. The cluster is characterized by a core set of shared genetic variants - C6312A (T2016K), C13730T (A88V/A97V), C23929T, and C28311T (P13L). Further, the cluster is also characterized by a nucleotide substitution rate of 1.4 x 10-3 variants per site per year, lower than the prevalent A2a cluster, and predominantly driven by variants in the E and N genes and relative sparing of the S gene. Epidemiological assessments suggest that the common ancestor emerged in the month of February 2020 and possibly resulted in an outbreak followed by countrywide spread, as evidenced by the low divergence of the genomes from across the country. To the best of our knowledge, this is the first comprehensive study characterizing the distinct and predominant cluster of SARS-CoV-2 in India.

genomics↗

bioRxiv2020

Overexpressed Nup88 stabilized through interaction with Nup62 promotes NFκB dependent pathways in cancer

Nuclear pores control nucleo-cytoplasmic trafficking and directly or indirectly regulate vital cellular processes. Nup88, important for Crm1 mediated nuclear export process, is overexpressed in many cancers. A positive correlation exists between progressive stages of cancer and Nup88 expression. However, links between Nup88 overexpression and head and neck cancer are insignificant, and mechanistic details are non-existent. Here, we report that Nup88 exhibits positive correlation in head and neck cancer in addition to elevated Nup62 levels. We demonstrate that Nup88 interacts with Nup62 in a cell-cycle and glycosylation independent manner. The overexpression of Nup88 or Nup62 imparts proliferation and migration advantages to cells. We further report that the interaction with Nup62 stabilizes Nup88 by inhibiting proteasome-mediated degradation of overexpressed Nup88. Overexpressed Nup88 is stabile and partly inside the nucleus and can interact with NF{kappa}B (p65). Nup88 overexpression induces proliferative and inflammatory responses downstream of p65. Altogether, we suggest that simultaneous overexpression of Nup62 and Nup88 in head and neck cancer stabilizes overexpressed Nup88. Stable Nup88 interacts with p65 and induces inflammatory, proliferative, and migratory advantages to cells, which perhaps is the underlying mechanism driving tumorigenic transformations.

cancer biology↗

bioRxiv2020

Identification of potential vaccine candidates against SARS-CoV-2, A step forward to fight novel coronavirus 2019-nCoV: A Reverse Vaccinology Approach

The recent Coronavirus Disease 2019 (COVID-19) causes an immense health crisis to global public health. The COVID-19 is the etiologic agent of a recently arose disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Presently, there is no vaccine available against this emerged viral disease. Therefore, it is indeed a need of the hour to develop an effectual and safe vaccine against this decidedly pandemic disease. In the current study, we collected SARS-CoV-2 genome which is prominent in India against human host, further more using reverse vaccinology here we claim effective vaccine candidates that can be mile stone in battle against COVID19. This novel study divulged one promising antigenic peptide GVYFASTEK from surface glycoprotein (protein accession no. - QIA98583.1) of SARS-CoV-2, which was predicated to be interacted with MHC alleles and showed up to 90% conservancy and high value of antigenicity. Subsequently, the molecular docking and simulation studies were verified molecular interaction of this prime antigenic peptide with the residues of HLA-A*11-01 allele for MHC Class I. After vigorous analysis, this peptide was predicted to be suitable epitope which is capable to induce the strong cell-mediated immune response against the SARS-CoV-2. Consequences from the current study could facilitate selecting SARS-CoV-2 epitopes for vaccine production pipelines in the immediate future. This novel research will certainly pave the way for a fast, reliable and virtuous platform to provide timely countermeasure of this dangerous pandemic disease, COVID-19.

bioinformatics↗

bioRxiv2020

Long-read genome sequencing and assembly of Leptopilina boulardi: a specialist Drosophila parasitoid

Leptopilina boulardi (Hymenoptera: Figitidae) is a specialist parasitoid of Drosophila. The Drosophila-Leptopilina system has emerged as a suitable model for understanding several aspects of host-parasitoid biology. However, a good quality genome of the wasp counterpart was lacking. Here, we report a whole-genome assembly of L. boulardi to bring it in the scope of the applied and fundamental research on Drosophila parasitoids with access to epigenomics and genome editing tools. The 375Mb draft genome has an N50 of 275Kb with 6315 scaffolds >500bp and encompasses >95% complete BUSCOs. Using a combination of ab-initio and RNA-Seq based methods, 25259 protein-coding genes were predicted and 90% (22729) of them could be annotated with at least one function. We demonstrate the quality of the assembled genome by recapitulating the phylogenetic relationship of L. boulardi with other Hymenopterans. The key developmental regulators like Hox genes and sex determination genes are well conserved in L. boulardi, and so is the basic toolkit for epigenetic regulation. The search for epigenetic regulators has also revealed that L. boulardi genome possesses DNMT1 (maintenance DNA methyltransferase), DNMT2 (tRNA methyltransferase) but lacks the de novo DNA methyltransferase (DNMT3). Also, the heterochromatin protein 1 family appears to have expanded as compared to other hymenopterans. The draft genome of L. boulardi (Lb17) will expedite the research on Drosophila parasitoids. This genome resource and early indication of epigenetic aspects in its specialization make it an interesting system to address a variety of questions on host-parasitoid biology.

genomics↗

bioRxiv2019

Identification of evolutionarily conserved nuclear matrix proteins and their prokaryotic origins

Compared to prokaryotic cells, a typical eukaryotic cell is much more complex along with its endomembrane system and membrane-bound organelles. Although the endosymbiosis theories convincingly explain the evolution of membrane-bound organelles such as mitochondria and chloroplasts, very little is understood about the evolutionary origins of the nucleus, the defining feature of eukaryotes. Most studies on nuclear evolution have not been able to take into consideration the underlying structural framework of the nucleus, attributed to the nuclear matrix (NuMat), a ribonucleoproteinaceous structure. This can largely be attributed to the lack of annotation of its core components. Since, NuMat has been shown to provide a structural platform for facilitating a variety of nuclear functions such as replication, transcription, and splicing, it is important to identify its protein components to better understand these processes. In this study, we address this issue using the developing embryos of D. melanogaster and D. rerio and identify 362 core NuMat proteins that are conserved between the two organisms. We find that of them, 132 protein groups have originated from pre-existing proteins in prokaryotes. While 51 were conserved across all eukaryotic supergroups, 17 new proteins evolved before the evolution of the last eukaryotic common ancestor and together these 68 proteins out of the 362 core conserved NuMat proteins are conserved across all eukaryotes indicating their indispensable nature for nuclear function for over 1.5 billion years of eukaryotic history. Our analysis paves the way to understand the evolution of the complex internal nuclear architecture and its functions.

molecular biology↗

bioRxiv2018

ELYS coordinates NF-κB pathway dynamics during development in Drosophila

Summary StatementELYS, a nucleoporin spatiotemporally regulates NF-{kappa}B pathway dynamics during development in Drosophila and its misregulation in post-embryonic stages leads to apoptosis mediated abnormalities.\n\nAbstractNuclear pores are the exclusive conduit to facilitate the nucleocytoplasmic transport in a precisely regulated manner. ELYS, a constituent protein of nuclear pores, initiates assembly of nuclear pore complexes (NPCs) into functional nuclear pores towards the end of mitosis. Using cellular, molecular and genetic tools, here, we report that ELYS orthologue (dElys) plays critical roles during Drosophila development. Through in silico analyses, we find all conserved structural features in dElys except for the presence of non-canonical AT-hook motif strongly binding with DNA. dElys localized to nuclear rim in interphase cells, but during mitosis, it was present on chromatin. RNAi mediated depletion of dElys leads to aberrant development and defects in the nuclear lamina and NPCs assembly at the cellular level. Furthermore, we demonstrate that in dElys depletion NF-{kappa}B is activated and accumulates inside the nucleus which results in illimed expression of critical molecules. dElys depletion sustains NF-{kappa}B into the nucleus in post-embryonic stages. Prolonged NF-{kappa}B inside nucleus induces apoptosis in response to hitherto unknown quality check mechanism and highlights on the under-appreciated apoptotic paradigm of NF-{kappa}B pathway.

cell biology↗

bioRxiv2018

Choline Transporter in α/β core neurons of Drosophila mushroom body non-canonically regulates pupal eclosion and maintains neuromuscular junction integrity

Insect mushroom bodies (MB) have an ensemble of synaptic connections well-studied for their role in experience-dependent learning and several higher cognitive functions. MB requires neurotransmission for an efficient flow of information across synapses with the different flexibility to meet the demand of the dynamically changing environment of an insect. Neurotransmitter transporters coordinate appropriate changes for an efficient neurotransmission at the synapse. Till date, there is no transporter reported for any of the previously known neurotransmitters in the intrinsic neurons of MB. In this study, we report a highly enriched expression of Choline Transporter (ChT) in Drosophila MB. We demonstrate that knockdown of ChT in a sub-type of MB neurons called /{beta} core (/{beta}c) neurons leads to eclosion failure, peristaltic defect in larvae, and altered NMJ phenotype. These defects were neither observed on knockdown of proteins of the cholinergic locus in /{beta}c neurons nor by knockdown of ChT in cholinergic neurons. Thus, our study provides insights into non-canonical roles of ChT in MB.

developmental biology↗