Biology subjects

Gupta, S.

Publications and source records attributed to Gupta, S..

At least 307 records · Page 17Linked to original sources

bioRxiv2020

Investigation of biochemical changes in barley inoculated with Trichoderma harzianum T-22 under salt stress

Increases in soil salinity impacts growth and yield of agricultural plants by inhibiting plant functions. Soil salinization is increasing because of the pressure of a growing population on food supply. Genetically modified crops and plant breeding techniques are being used to produce plants tolerant to salt stress. However, interactions of fungal endophytes with crop plants can also improve tolerance and is a less expensive approach. Here, the role of Trichoderma harzianum T-22 in alleviating NaCl-induced stress in two barley genotypes (cv. Vlamingh and cv. Gairdner) has been investigated. Metabolomics using GC-MS for polar metabolites and LC-MS for lipids was employed to provide insights into the biochemical changes in barley roots inoculated with fungus during the early stages of interaction. T. harzianum increased the root length of both genotypes under controlled and saline conditions. The fungus reduced the relative concentration of sugars in both genotypes and caused no change in organic acids under saline conditions. Amino acids decreased only in cv. Gairdner in fungus-inoculated roots under saline conditions. Lipid analyses suggest that salt stress causes large changes in the lipid profile of roots but that inoculation with fungus greatly reduces the extent of these changes. By studying a tolerant and a sensitive genotype and their responses to salt and inoculation we have been able to develop hypotheses about what lipid species and metabolites may be involved in the tolerant genotype for its tolerance to salt and how fungal inoculation changes the response of the sensitive genotype to improve its tolerance.

biochemistry↗

bioRxiv2020

A critical residue in the α1M2-M3 linker regulating GABAA receptor pore gating by diazepam

Benzodiazepines (BZDs) are a class of widely prescribed psychotropic drugs. Their anxiolytic and sedative effects are conferred by modulating the activity of GABAA receptors (GABAARs), which are the primary inhibitory neurotransmitter receptors throughout the central nervous system. However, the physical mechanism by which BZDs exert their effects on the receptor is poorly understood. In particular, BZDs require coapplication with an agonist to effectively open the channel pore, making it difficult to dissect whether the drug has altered either agonist binding or channel gating as these two processes are intimately coupled. To isolate effects on gating we used a spontaneously active gain of function mutant (1L9T{beta}2{gamma}2L) that is directly gated by BZDs alone in the absence of agonist. In the 1L9T background we explored effects of alanine substitutions throughout the 1M2-M3 linker on modulation of the channel pore by the BZD positive modulator diazepam (DZ). The M2-M3 linker is known to be an important element for channel activation. Linker mutations generally impaired unliganded pore opening, indicating that side chain interactions are important for channel gating in the absence of bound agonist. All but one mutation had no effect on the transduction of chemical energy from DZ binding to pore gating. Strikingly, 1V279A doubles DZs energetic contribution to gating, whereas larger side chains at this site do not. In a wild-type background 1V279A enhances DZ-potentiation of currents evoked by saturating GABA, consistent with a direct effect on the pore closed/open equilibrium. Our observations identify an important residue regulating coupling between the BZD site and the pore gate, thereby shedding new light on the molecular mechanism of a frequently prescribed class of psychotropic drugs.

molecular biology↗

bioRxiv2020

Vimentin intermediate filaments mediate cell shape on visco-elastic substrates

The ability of cells to take and change shape is a fundamental feature underlying development, wound repair, and tissue maintenance. Central to this process is physical and signaling interactions between the three cytoskeletal polymeric networks: F-actin, microtubules, and intermediate filaments (IFs). Vimentin is an IF protein that is essential to the mechanical resilience of cells and regulates cross-talk amongst the cytoskeleton, but its role in how cells sense and respond to the surrounding extracellular matrix is largely unclear. To investigate vimentins role in substrate sensing, we designed polyacrylamide hydrogels that mimic the elastic and viscoelastic nature of in vivo tissues. Using wild-type and vimentin-null mouse embryonic fibroblasts, we show that vimentin enhances cell spreading on viscoelastic substrates, even though it has little effect in the limit of purely elastic substrates. Our results provide compelling evidence that the vimentin cytoskeletal network is a physical modulator of how cells sense and respond to mechanical properties of their extracellular environment.

biophysics↗

bioRxiv2020

Learning distance-dependent motif interactions: an interpretable CNN model of genomic events

In many biological studies, prediction is used primarily to validate the model; the real quest is to understand the underlying phenomenon. Therefore, interpretable deep models for biological studies are required. Here, we propose the Hyper-parameter eXplainable Motif Pair framework (HyperXPair) to model biological motifs and their distance-dependent context through explicitly interpretable parameters. This makes HyperXPair more than a decision-support tool; it is also a hypothesis-generating tool designed to advance knowledge in the field. We demonstrate the utility of our model by learning distance-dependent motif interactions for two biological problems: transcription initiation and RNA splicing.

bioinformatics↗

bioRxiv2020

Replication dynamics and cytotoxicity of SARS-CoV-2 Swedish isolate in commonly used laboratory cell lines

The present pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is driving intense research activities to understand the basic biology of the virus and determine effective therapeutic strategies. The commonly used laboratory cell lines of human origin are the first line of experimental models to study the pathogenicity and performing antiviral assays. Thus, to find suitable cell models to study SARS-CoV-2, we assessed the tropism and cytopathogenicity of the first Swedish isolate of SARS-CoV-2 in six different cell lines of human origin and compared their growth characteristics to other globally isolated strains. Overall, Calu-3, Caco2, Huh7, and 293FT cell lines showed a high to moderate level of susceptibility to the majority of virus isolates. In Caco2 cells the virus can achieve high titers in the absence of any prominent cytopathic effect. The protein expression profile during SARS-CoV-2 infection revealed cell-type-specific regulation of cellular pathways. Type-I interferon signaling was identified as the common dysregulated cellular response in Caco2, Calu-3 and Huh7 cells. Overall, cell-type specific variability was noted for cytopathogenicity, susceptibility and cellular response to SARS-CoV-2. This study provides important clues regarding SARS-CoV-2 pathogenesis and can represent as a guide for future studies to design therapeutics.

microbiology↗

bioRxiv2020

Primary carbohydrate metabolism genes participate in heat stress memory at the shoot apical meristem of Arabidopsis thaliana

Although we have a good understanding of the development of shoot apical meristems (SAM) in higher plants, and the function of the stem cells (SCs) embedded in the SAM, there is surprisingly little known of its molecular responses to abiotic stresses. Here, we show that the SAM of Arabidopsis thaliana senses heat stress (HS) and retains an autonomous molecular memory of a previous non-lethal HS, allowing the SAM to regain growth after exposure to an otherwise lethal HS several days later. Using RNA-seq, we identified genes participating in establishing a SAM-specific HS memory. The genes include HEAT SHOCK TRANSCRIPTION FACTORs (HSFs), of which HSFA2 is essential, but not sufficient, for full HS memory in the SAM, the SC regulators CLAVATA1 (CLV1) and CLV3, and several primary carbohydrate metabolism genes, including FRUCTOSE-BISPHOSPHATE ALDOLASE 6 (FBA6). We found that expression of FBA6 during HS at the SAM complements that of FBA8 in the same organ. Furthermore, we show that sugar availability at the SAM is essential for survival at high-temperature HS. Collectively, plants have evolved a sophisticated protection mechanism to maintain SCs and, hence, their capacity to re-initiate shoot growth after stress release.

plant biology↗

bioRxiv2020

An ultra-high affinity synthetic nanobody blocks SARS-CoV-2 infection by locking Spike into an inactive conformation

Without an effective prophylactic solution, infections from SARS-CoV-2 continue to rise worldwide with devastating health and economic costs. SARS-CoV-2 gains entry into host cells via an interaction between its Spike protein and the host cell receptor angiotensin converting enzyme 2 (ACE2). Disruption of this interaction confers potent neutralization of viral entry, providing an avenue for vaccine design and for therapeutic antibodies. Here, we develop single-domain antibodies (nanobodies) that potently disrupt the interaction between the SARS-CoV-2 Spike and ACE2. By screening a yeast surface-displayed library of synthetic nanobody sequences, we identified a panel of nanobodies that bind to multiple epitopes on Spike and block ACE2 interaction via two distinct mechanisms. Cryogenic electron microscopy (cryo-EM) revealed that one exceptionally stable nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for SARS-CoV-2 Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains stability and function after aerosolization, lyophilization, and heat treatment. These properties may enable aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia, promising to yield a widely deployable, patient-friendly prophylactic and/or early infection therapeutic agent to stem the worst pandemic in a century.

biochemistry↗

bioRxiv2020

Analysis of single nucleotide polymorphisms between 2019-nCoV genomes and its impact on codon usage

The spread of COVID-19 is a global concern that has taken a toll on entire human health. Researchers across the globe has been working in devising the strategies to combat this dreadful disease. Studies focused on genetic variability helps design effective drugs and vaccines. Considering this, the present study entails the information regarding the genome-wide mutations detected in the 108 SARS CoV-2 genomes worldwide. We identified a few hypervariable regions localized in orf1ab, spike, and nucleocapsid gene. These nucleotide polymorphisms demonstrated their effect on both codon usage as well as amino acid usage pattern. Altogether the present study provides valuable information that would be helpful to ongoing research on 2019-nCoV vaccine development.

genomics↗

bioRxiv2020

A transitory signaling center controls timing of primordial germ cell differentiation

Organogenesis requires exquisite spatio-temporal coordination of cell morphogenesis, migration, proliferation and differentiation of multiple cell types. For gonads, this involves complex interactions between somatic and germline tissues. During Drosophila ovary morphogenesis primordial germ cells (PGCs) are either sequestered in stem cell niches and maintained in an undifferentiated, germline stem cell state, or transition directly towards differentiation. Here, we identify a mechanism that links hormonal triggers of somatic tissue morphogenesis with PGC differentiation. An early ecdysone pulse initiates somatic swarm cell (SwC) migration, positioning them close to PGCs. A second hormone peak activates Torso-like signal in SwCs, which stimulates the Torso RTK signaling pathway in PGCs promoting their differentiation by de-repression of the differentiation gene bag of marbles. Thus, systemic temporal cues generate a transitory signaling center that coordinate ovarian morphogenesis with stem cell self-renewal and differentiation programs, a concept applicable broadly to the integration of stem cells and their niches. HIGHLIGHTSO_LISteroid pulses coordinate gonadogenesis, stem cell self-renewal and differentiation C_LIO_LIAn early steroid pulse initiates migration of Swarm Cells, a transitory support cell type C_LIO_LIA late steroid pulse induces Torso-like expression, a Torso receptor tyrosine kinase (RTK) activator, in Swarm Cells C_LIO_LITorso RTK signaling in primordial germ cells activates the key differentiation gene bam by relieving Kruppel-mediated repression C_LI

developmental biology↗

bioRxiv2020

Sensitivity to perturbations of a cell migration under temporal regulation

Few studies have measured the robustness to perturbations of the final position of a long-range migrating cell. In the nematode Caenorhabditis elegans, the QR neuroblast migrates anteriorly in the young larva, while undergoing three rounds of division. The daughters of QR.pa stop their migration at an anterior body position and acquire a neuronal fate. Previous studies showed that the migration stops upon expression of the Wnt receptor MIG-1, which surprisingly is not induced by positional cues but by a timing mechanism (Mentink et al. 2014). Given this temporal regulation, we wondered 1) how precise QR.pax positioning is when confronted with various challenges, such as stochastic noise, environment or body size variation and 2) whether QR.pax position varies among C. elegans wild isolates. We find that the variance of QR.pax final position is similar to that of other long-range migrating neurons. Its mean position undergoes a slight posterior shift at higher temperature, while its variance is greatly increased following sustained starvation at hatching. We manipulated body size using mutants and tetraploid animals. As expected from the temporal mechanism, smaller mutants display anteriorly shifted QR.pax cells, while longer mutants and tetraploids display posteriorly shifted QR.pax cells. Using a mathematical model, we show however that body size variation is partially compensated. We find that cell speed is indeed altered in body size mutants. Finally, we could detect highly significant variation among C. elegans wild isolates. Overall, this study reveals that the final cell position of QR.pax shows some degree of sensitivity to external perturbations and natural genetic variation.

developmental biology↗

bioRxiv2020

DNA methylation mediated downregulation of histone H3 variant H3.3 affects cell proliferation contributing to the development of HCC

H3.3 variant is a versatile histone important for development and disease. We report a DNA methylation dependent decrease of histone H3 variant H3.3 in hepatocellular carcinoma (HCC) development and an increase in the level of the H3.2 variant. The loss of H3.3 correlates with a decrease in the histone PTMs associated with active transcription. The overexpression of H3.3 and H3.2 did not affect global PTMs and cell physiology, probably owing to the deregulation of specific histone chaperones CAF-1 (for H3.2) and HIRA (for H3.3) that we observed in HCC. Notably, upon P150 (CAF-1 subunit) knockdown in HCC cell lines, a cell cycle arrest in S-phase was observed, possibly due to the decrease in the histone levels necessary for DNA packaging. Furthermore, H3.3 knockdown in a preneoplastic liver cell line led to an increase in cell proliferation and a decreased transcription of tumor suppressor genes, recapitulating the tumor cell phenotype. Importantly, our data suggest that the use of DNA Methyl Transferase (DNMT) and Histone Deacetylase (HDAC) inhibitors to restore the expression of H3.3 and the altered chromatin state for the better clinical management of the disease.

cancer biology↗

bioRxiv2020

Unique roles of ATAC and SAGA KAT2A complexes in normal and malignant hematopoiesis

Epigenetic histone modifiers are key players in cell fate decisions. Significant research has focused on their enzymatic activity, but less is known about the contextual role of the complexes they integrate. We focus on KAT2A, a histone acetyltransferase we recently associated with leukemia stem cell maintenance, and which participates in ATAC and SAGA complexes. We show that ATAC is uniquely required for maintenance of normal and leukemia stem and progenitor cells, while SAGA more specifically contributes to cell identity. This dichotomy sets a paradigm for investigating epigenetic activities in their macromolecular context and informs epigenetic regulator targeting for translational purposes.

cell biology↗

bioRxiv2020

Dysregulation in mTOR/HIF-1 signaling identified by proteo-transcriptomics of SARS-CoV-2 infected cells

How Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infections engage cellular host pathways and innate immunity in infected cells remain largely elusive. We performed an integrative proteo-transcriptomics analysis in SARS-CoV-2 infected HuH7 cells to map the cellular response to the invading virus over time. We identified four pathways, ErbB, HIF-1, mTOR and TNF signaling, among others that were markedly modulated during the course of the SARS-CoV-2 infection in vitro. Western blot validation of the downstream effector molecules of these pathways revealed a significant reduction in activated S6K1 and 4E-BP1 at 72 hours post infection. Unlike other human respiratory viruses, we found a significant inhibition of HIF-1 through the entire time course of the infection, suggesting a crosstalk between the SARS-CoV-2 and the mTOR/HIF-1 signaling. Further investigations are required to better understand the molecular sequelae in order to guide potential therapy in the management of severe COVID-19 patients.

microbiology↗

bioRxiv2020

Convergent evolution of p38/MAPK activation in hormone resistant prostate cancer mediates pro-survival, immune evasive, and metastatic phenotypes

Adaptation of cancer cells to targeted therapy follows ecological paradigms observed in natural populations that encounter resource depletion and changing environments, including activation of pro-survival mechanisms, migration to new locations, and escape of predation. We identified the p38 MAPK pathway as a common molecular driver of these three responses during the adaptation to hormone therapy resistance in prostate cancer. The p38 pathway is activated in therapy-resistant cells and mechanistically drives these three convergent responses through sustained AR activity, enhanced invasion and metastasis, and immune evasion. Targeting p38 signaling may represent a new therapeutic strategy to treat men with metastatic, hormone therapy-resistant prostate cancer.

cancer biology↗

bioRxiv2020

Obtaining leaner deep neural networks for decoding brain functional connectome in a single shot

Neuroscientific knowledge points to the presence of redundancy in the correlations of brains functional activity. These redundancies can be removed to mitigate the problem of overfitting when deep neural network (DNN) models are used to classify neuroimaging datasets. We propose an algorithm that removes insignificant nodes of DNNs in a layerwise manner and then adds a subset of correlated features in a single shot. When performing experiments with functional MRI datasets for classifying patients from healthy controls, we were able to obtain simpler and more generalizable DNNs. The obtained DNNs maintained a similar performance as the full network with only around 2% of the initial trainable parameters. Further, we used the trained network to identify salient brain regions and connections from functional connectome for multiple brain disorders. The identified biomarkers were found to closely correspond to previously known disease biomarkers. The proposed methods have cross-modal applications in obtaining leaner DNNs that seem to fit the data better. The corresponding code is available at https://github.com/SCSE-Biomedical-Computing-Group/LEAN_CLIP.

bioinformatics↗

bioRxiv2020

HSPs, Ubiquitins and Antioxidants aid in Heat Tolerance in Tharparkar Indicine cattle

Transcriptome profiling of Vrindavani and Tharparkar cattle revealed that more numbers of genes dysregulated in Vrindavani than in Tharparkar. A contrast in gene expression was observed with 18.5 % of upregulated genes in Vrindavani were downregulated in Tharparkar and 17.5% upregulated genes in Tharparkar were downregulated in Vrindavani. Functional annotation of genes differentially expressed in Tharparkar and Vrindavani revealed that the systems biology in Tharparkar is moving towards counteracting the effects due to heat stress. Unlike Vrindavani, Tharparkar is not only endowed with higher expression of the scavengers (UBE2G1, UBE2S, and UBE2H) of misfolded proteins but also with protectors (VCP, Serp1, and CALR) of naive unfolded proteins. Further, higher expression of the antioxidants in Tharparkar enables it to cope up with higher levels of free radicals generated as a result of heat stress. In this study we found relevant genes dysregulated in Tharparkar in the direction that can counter heat stress.

physiology↗

bioRxiv2020

Mechanism of monoallelic expression and allelic rheostat role of DNA methylation

In diploid cells, maternal and paternal copies of genes usually have similar transcriptional activity. Mammalian allele-specific epigenetic mechanisms such as X-chromosome inactivation (XCI) and imprinting were historically viewed as rare exceptions to this rule. The discovery of mitotically stable monoallelic autosomal expression (MAE) a decade ago revealed an additional allele-specific mode regulating thousands of mammalian genes. However, despite its prevalence, the mechanistic basis of MAE remains unknown. To uncover the mechanism of MAE maintenance, we devised a small-molecule screen for reactivation of silenced alleles across multiple loci using targeted RNA sequencing. Contrary to previous reports, we identified DNA methylation as a key mechanism of MAE mitotic maintenance. In contrast with the binary choice of the active allele in XCI, stringent transcriptome-wide analysis revealed MAE as a regulatory mode with tunable control of allele-specific expression, dependent on the extent of DNA methylation. In a subset of MAE genes, allelic imbalance was insensitive to changes in DNA methylation, implicating additional mechanisms in MAE maintenance in these loci. Our findings identify a key mechanism of MAE maintenance, reveal tunability of this mode of gene regulation, and provide the essential platform for probing the biological role of MAE in development and disease.

genomics↗

bioRxiv2020

Unexpected variability of allelic imbalance estimates from RNA sequencing

RNA sequencing and other experimental methods that produce large amounts of data are increasingly dominant in molecular biology. However, the noise properties of these techniques have not been fully understood. We assessed the reproducibility of allele-specific expression measurements by conducting replicate sequencing experiments from the same RNA sample. Surprisingly, variation in the estimates of allelic imbalance (AI) between technical replicates was up to 7-fold higher than expected from commonly applied noise models. We show that AI overdispersion varies substantially between replicates and between experimental series, appears to arise during the construction of sequencing libraries, and can be measured by comparing technical replicates. We demonstrate that compensation for AI overdispersion greatly reduces technical variation and enables reliable differential analysis of allele-specific expression across samples and across experiments. Conversely, not taking AI overdispersion into account can lead to a substantial number of false positives in analysis of allele-specific gene expression

bioinformatics↗