Biology subjects

Das, S.

Publications and source records attributed to Das, S..

At least 325 records · Page 18Linked to original sources

bioRxiv2021

Convergent evolution of SARS-CoV-2 spike mutations, L452R, E484Q and P681R, in the second wave of COVID-19 in Maharashtra, India

As the global severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic expands, genomic epidemiology and whole genome sequencing are being constantly used to investigate its transmissions and evolution. In the backdrop of the global emergence of "variants of concern" (VOCs) during December 2020 and an upsurge in a state in the western part of India since January 2021, whole genome sequencing and analysis of spike protein mutations using sequence and structural approaches was undertaken to identify possible new variants and gauge the fitness of current circulating strains. Phylogenetic analysis revealed that the predominant clade in circulation was a distinct newly identified lineage B.1.617 possessing common signature mutations D111D, G142D, L452R, E484Q, D614G and P681R, in the spike protein including within the receptor binding domain (RBD). Of these, the mutations at residue positions 452, 484 and 681 have been reported in other globally circulating lineages. The structural analysis of RBD mutations L452R and E484Q along with P681R in the furin cleavage site, revealed that these may possibly result in increased ACE2 binding and rate of S1-S2 cleavage resulting in better transmissibility. The same two RBD mutations indicated decreased binding to select monoclonal antibodies (mAbs) and may affect their neutralization potential. Experimental validation against a wider panel of mAbs, sera from vaccinees and those that recovered from natural infection needs to be studied. The emergence of such local variants through the accumulation of convergent mutations during the COVID-19 second wave needs to be further investigated for their public health impact in the rest of the country and its possibility of becoming a VOC.

molecular biology↗

bioRxiv2021

Replication timing analysis in polyploid cells reveals Rif1 uses multiple mechanisms to promote underreplication in Drosophila.

Regulation of DNA replication and copy number are necessary to promote genome stability and maintain cell and tissue function. DNA replication is regulated temporally in a process known as replication timing (RT). Rif1 is key regulator of RT and has a critical function in copy number control in polyploid cells. In a previous study (Munden et al., 2018), we demonstrated that Rif1 functions with SUUR to inhibit replication fork progression and promote underreplication (UR) of specific genomic regions. How Rif1-dependent control of RT factors into its ability to promote UR is unknown. By applying a computational approach to measure RT in Drosophila polyploid cells, we show that SUUR and Rif1 have differential roles in controlling UR and RT. Our findings reveal that Rif1 functions both upstream and downstream of SUUR to promote UR. Our work provides new mechanistic insight into the process of UR and its links to RT.

molecular biology↗

bioRxiv2021

Rapalogs downmodulate intrinsic immunity and promote cell entry of SARS-CoV-2

SARS-CoV-2 infection in immunocompromised individuals is associated with prolonged virus shedding and evolution of viral variants. Rapamycin and its analogs (rapalogs, including everolimus, temsirolimus, and ridaforolimus) are FDA-approved as mTOR inhibitors for the treatment of human diseases, including cancer and autoimmunity. Rapalog use is commonly associated with increased susceptibility to infection, which has been traditionally explained by impaired adaptive immunity. Here, we show that exposure to rapalogs increases susceptibility to SARS-CoV-2 infection in tissue culture and in immunologically naive rodents by antagonizing the cell-intrinsic immune response. By identifying one rapalog (ridaforolimus) that is less potent in this regard, we demonstrate that rapalogs promote Spike-mediated entry into cells by triggering the degradation of antiviral proteins IFITM2 and IFITM3 via an endolysosomal remodeling program called microautophagy. Rapalogs that increase virus entry inhibit the mTOR-mediated phosphorylation of the transcription factor TFEB, which facilitates its nuclear translocation and triggers microautophagy. In rodent models of infection, injection of rapamycin prior to and after virus exposure resulted in elevated SARS-CoV-2 replication and exacerbated viral disease, while ridaforolimus had milder effects. Overall, our findings indicate that preexisting use of certain rapalogs may elevate host susceptibility to SARS-CoV-2 infection and disease by activating lysosome-mediated suppression of intrinsic immunity. SignificanceRapamycin is an immunosuppressant used in humans to treat cancer, autoimmunity, and other disease states. Here, we show that rapamycin and related compounds promote the first step of the SARS-CoV-2 infection cycle--entry into cells--by disarming cell-intrinsic immune defenses. We outline the molecular basis for this effect by identifying a rapamycin derivative that is inactive, laying the foundation for improved mTOR inhibitors that do not suppress intrinsic immunity. We find that rapamycin analogs that promote SARS-CoV-2 entry are those that activate TFEB, a transcription factor that triggers the degradation of antiviral membrane proteins inside of cells. Finally, rapamycin administration to rodents prior to SARS-CoV-2 challenge results in enhanced viral disease, revealing that its use in humans may increase susceptibility to infection.

microbiology↗

bioRxiv2021

An AI-guided invariant signature places MIS-C with Kawasaki disease in a continuum of host immune responses

A significant surge in cases of multisystem inflammatory syndrome in children (MIS-C, also called Pediatric Inflammatory Multisystem Syndrome - PIMS) has been observed amidst the COVID-19 pandemic. MIS-C shares many clinical features with Kawasaki disease (KD), although clinical course and outcomes are divergent. We analyzed whole blood RNA sequences, serum cytokines, and formalin fixed heart tissues from these patients using a computational toolbox of two gene signatures, i.e., the 166-gene viral pandemic (ViP) signature, and its 20-gene severe (s)ViP subset that were developed in the context of SARS-CoV-2 infection and a 13-transcript signature previously demonstrated to be diagnostic for KD. Our analyses revealed that KD and MIS-C are on the same continuum of the host immune response as COVID-19. While both the pediatric syndromes converge upon an IL15/IL15RA-centric cytokine storm, suggestive of shared proximal pathways of immunopathogenesis, they diverge in other laboratory parameters and cardiac phenotypes. The ViP signatures also revealed unique targetable cytokine pathways in MIS-C, place MIS-C farther along in the spectrum in severity compared to KD and pinpoint key clinical (reduced cardiac function) and laboratory (thrombocytopenia and eosinopenia) parameters that can be useful to monitor severity.

immunology↗

bioRxiv2021

The CTEXT complex in Saccharomyces cerevisiae plays a crucial role in degrading distinct sets of aberrant mRNAs by the nuclear exosome

In Saccharomyces cerevisiae, DRN (Decay of RNA in the Nucleus) requiring Cbc1/2p, Tif4631p, and Upf3p promotes the exosomal degradation of aberrantly long 3'-extended-, export-defective transcripts and a small group of normal (special) mRNAs. In this study, using a systematic proteomic analysis we show that each of the known components interacts with one another and they exist as a separate complex, which was dubbed CTEXT (CBC-Tif4631p-dependent EXosome Targeting). We also identified a DEAD-box RNA helicase Dbp2p as an additional novel component of CTEXT during this analysis which was further bolstered by the finding that genomic deletions of Dbp2p led to the stabilization of all the signature nuclear messages. Interestingly, the RRM domain of Tif4631p located at the extreme N-termini of this polypeptide was found to play a vital role in in mediating the interaction of the CTEXT with the core exosome complex. These inferences were substantiated by the finding that deletion of this domain led to the functional impairment of the CTEXT complex. Thus, the CTEXT constitutes an independent complex that assists the nuclear exosome in degrading the select classes of nuclear transcripts in Saccharomyces cerevisiae.

molecular biology↗

bioRxiv2021

A potential clue for the application of prime editing in tomato, a dicot plant

Precision genome editing is highly desired for crop improvement. The recently emerged CRISPR/Cas technology offers great potential applications in precision plant genome engineering. A prime editing (PE) approach combining a reverse transcriptase (RT) with a Cas9 nickase and a "priming" extended guide RNA has shown a high frequency for precise genome modification in mammalian cells and several plant species. Nevertheless, the applications of the PE approach in dicot plants are still limited and inefficient. We designed and tested prime editors for precision editing of a synthetic sequence in a transient assay and for desirable alleles of 10 loci in tomato by stable transformation. Our data obtained by targeted deep sequencing also revealed only low PE efficiencies in both the tobacco and tomato systems. Further assessment of the activities of the PE components uncovered potential reasons for the inefficiency of the PE complexes. Modifying the pegRNA sequences by shortening or introducing mismatches to the primer binding sequences (PBS) in order to reduce their melting temperatures (Tm) did not enhance the PE efficiency at the SlBMP21, SlALC and SlALS1 loci. Our data show challenges of PE approach in tomato, indicating a further improvement of the PE system for the successful applications such as use of improved expression systems. Our work provides an important clue for the successful application of the PE approach in crop improvement.

molecular biology↗

bioRxiv2021

Deep learning from multiple experts improves identification of amyloid neuropathologies

Pathologists can label pathologies differently, making it challenging to yield consistent assessments in the absence of one ground truth. To address this problem, we present a DL approach that draws on a cohort of experts, weighs each contribution, and is robust to noisy labels. We collected 100,495 annotations on 20,099 candidate amyloid beta neuropathologies (cerebral amyloid angiopathy (CAA), and cored and diffuse plaques) from three institutions, independently annotated by five experts. DL methods trained on a consensus-of-two strategy yielded 12.6-26% improvements by area under the precision recall curve (AUPRC) when compared to those that learned individualized annotations. This strategy surpassed individual-expert models, even when unfairly assessed on benchmarks favoring them. Moreover, ensembling over individual models was robust to hidden random annotators. In blind prospective tests of 52,555 subsequent expert-annotated images, the models labeled pathologies like their human counterparts (consensus model AUPRC=0.74 cored; 0.69 CAA). This study demonstrates a means to combine multiple ground truths into a common-ground DL model that yields consistent diagnoses informed by multiple and potentially variable expert opinion.

pathology↗

bioRxiv2021

Effect of chronic JUUL aerosol inhalation on inflammatory states of the brain, lung, heart and colon in mice

While health effects of conventional tobacco are well defined, data on vaping devices, including the most popular e-cigarette JUUL, are less established. Prior acute e-cigarette studies demonstrated inflammatory and cardiopulmonary physiology changes while chronic studies demonstrated extra-pulmonary effects, including neurotransmitter alterations in reward pathways. In this study we investigated effects of chronic flavored JUUL aerosol inhalation on inflammatory markers in brain, lung, heart, and colon. JUUL induced upregulation of cytokine and chemokine gene expression and increased HMGB1 and RAGE in the nucleus accumbens. Inflammatory gene expression increased in colon, and cardiopulmonary inflammatory responses to acute lung injury with lipopolysaccharide were exacerbated in the heart. Flavor-dependent changes in several responses were also observed. Our findings raise concerns regarding long-term risks of e-cigarette use as neuroinflammation may contribute to behavioral changes and mood disorders, while gut inflammation has been tied to poor systemic health and cardiac inflammation to development of heart disease. One Sentence SummaryChronic, daily inhalation of pod-based e-cigarette aerosols alters the inflammatory state across multiple organ systems in mice.

immunology↗

bioRxiv2021

Domain dependent orchestrated regulation of bacterial growth, persistence and chemotaxis by an essential GTPase, CgtA, in Vibrio cholerae

CgtA, an evolutionarily conserved GTPase, associated with the 50S ribosome controls a broad spectrum of physiological processes in bacteria. It has three structural domains, viz., N-terminal domain (NTD), GTPase domain and C-terminal domain (CTD). CgtA regulates expression of several of genes during nutritional stress in Vibrio cholerae. The mechanism of transcriptional regulation by CgtA is unknown, though the NTD concomitantly with the GTPase domain participates in the process. Here, we show that the in vivo deletion of the 57 amino acids long CTD of CgtA GTPase of V. cholerae is dispensable for viability, contrary to the complete knockdown of cgtA gene. Slower growth was observed in cgtA knockdown strain with intermittent diauxic lags in minimal media than the CTD deleted strain. Irreversible defect in colony morphology was observed in the cells with CTD deletion. Resuscitation of persister cells occurred when nutritionally deprived complete cgtA knockdown cells after growing for longer periods were transferred to nutritionally enriched media. The motility of the cgtA knockdown strain was significantly reduced than the wild type cells. Furthermore, CTD deleted cells were also found to be defective in motility, but comparatively lower than cgtA knockdown cells. Elongated and slender V. cholerae cells were observed by SEM imaging upon cgtA depletion, whereas, upon CTD deletion cellular elongation did not occur. Based on our study here, we propose that the CTD of CgtA perceives the nutritional stress response, to which the NTD and GTPase responds.

molecular 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

Germline activity of the heat shock factor HSF-1 programs the insulin-receptor daf-2 in C. elegans

The mechanisms by which maternal stress alters offspring phenotypes remain poorly understood. Here we report that the heat shock transcription factor HSF-1, activated in the C. elegans maternal germline upon stress, epigenetically programs the insulin-like receptor daf-2 by increasing repressive H3K9me2 levels throughout the daf-2 gene. This increase occurs by the recruitment of the C. elegans SETDB1 homolog MET-2 by HSF-1. Increased H3K9me2 levels at daf-2 persist in offspring to downregulate daf-2, activate the C. elegans FOXO ortholog DAF-16 and enhance offspring stress resilience. Thus, HSF-1 activity in the mother promotes the early life programming of the insulin/IGF-1 signaling (IIS) pathway and determines the strategy of stress resilience in progeny. One Sentence SummaryHSF-1 recruits MET-2 to silence daf-2 and mediate early life programming of C. elegans upon stress

cell biology↗

bioRxiv2021

Cell lineage as a predictor of immune response in neuroblastoma

Immunotherapy for patients with neuroblastoma has met with limited success, partly due to an incomplete understanding of the mechanisms underlying immune responsiveness in this clinically and genetically heterogenic tumor. Here, we undertook an unbiased analysis using dimension reduction and UMAP visualization of transcriptional signatures derived from 498 primary neuroblastoma tumors. Four distinct clusters based on differentially expressed genes emerged, of which one, representing about 30% and comprising mainly of MYCN-nonamplified tumors, was notable for the high expression of genes associated with both immune response activation and suppression. This capacity to elicit a productive immune response resided exclusively in tumors with dominant populations of undifferentiated, neural crest-like or mesenchymal cells; by contrast, tumors comprising primarily of committed, adrenergic neuron-like cells were less immunogenic. Mesenchymal neuroblastoma cells were enriched for innate and adaptive immune gene signatures, demonstrated engagement with cytotoxic T and natural killer cells, and induced immune cell infiltration in an immunocompetent mouse model. Transcriptional or targeted therapy-induced reprogramming of adrenergic cells to the mesenchymal state led to reactivation of tumor cell-intrinsic immune genes. Key immune response genes in adrenergic tumor cells were found to be epigenetically silenced by the PRC2 complex, and such repression could be relieved by either mesenchymal cell state reprogramming or EZH2 inhibition, leading to increased activation of natural killer cells by the tumor cells. These data identify cell lineage as a major determinant of the immunogenic potential in neuroblastoma that could be used to stratify patients who are most likely to benefit from immunotherapy.

cancer biology↗

bioRxiv2021

Actin fence therapy with exogenous V12Rac1 protects against Acute Lung Injury

High mortality in Acute Lung Injury (ALI) results from sustained proinflammatory signaling by alveolar receptors, such as TNF receptor type 1 (TNFR1). Factors that determine the sustained signaling are not known. Unexpectedly, optical imaging of live alveoli revealed a major TNF-induced surge of alveolar TNFR1 due to a Ca2+-dependent mechanism that decreased the cortical actin fence. Mouse mortality due to inhaled LPS was associated with cofilin activation, actin loss and the TNFR1 surge. The constitutively active form of the GTPase, Rac1 (V12Rac1), given intranasally as a non-covalent construct with a cell-permeable peptide, enhanced alveolar F-actin and blocked the TNFR1 surge. V12Rac1 also protected against ALI-induced mortality resulting from intranasal (i.n.) instillation of LPS, or of Pseudomonas aeruginosa. We propose a new therapeutic paradigm in which actin enhancement by exogenous Rac1 strengthens the alveolar actin fence, protecting against proinflammatory receptor hyperexpression, hence blocking ALI.

physiology↗

bioRxiv2020

Liver Electrical Impedance Tomography for Early Identification of Fatty Infiltrate in Obesity

Non-alcoholic fatty liver disease (NAFLD) is endemic in developed countries and is one of the most common causes of cardiometabolic diseases in overweight/obese individuals. While liver biopsy or magnetic resonance imaging (MRI) is the current gold standard to diagnose NAFLD, the former is prone to bleeding and the latter is costly. We hereby demonstrated liver electrical impedance tomography (EIT) as a non-invasive and portable detection method for fatty infiltrate. We enrolled 19 subjects (15 females and 4 males; 27 to 74 years old) to undergo liver MRI scans, followed by EIT measurements via a multi-electrode array. The liver MRI scans provided subject-specific a priori knowledge of the liver boundary conditions for segmentation and EIT reconstruction, and the 3-D multi-echo MRI data quantified liver proton-density fat fraction (PDFF%) as a recognized reference standard for validating liver fat infiltrate. Using acquired voltage data and the reconstruction algorithm for the EIT imaging, we computed the absolute conductivity distribution of abdomen in 2-D. Correlation analyses were performed to compare the individual EIT conductivity vs. MRI PDFF with their demographics in terms of gender, BMI (kg{middle dot}m-2), age (years), waist circumference (cm), height (cm), and weight (kg). Our results indicate that EIT conductivity (S{middle dot}m-1) and liver MRI for PDFF were not correlated with the demographics, whereas the decrease in EIT conductivity was correlated with the increase in MRI PDFF (R = - 0.69, p= 0.003). Thus, EIT conductivity holds promise for developing a non-invasive, portable, and quantitative method to detect fatty liver disease.

bioengineering↗

bioRxiv2020

Niclosamide inhibits SARS-CoV2 entry by blocking internalization through pH-dependent CLIC/GEEC endocytic pathway

Many viruses utilize the host endo-lysosomal network to infect cells. Tracing the endocytic itinerary of SARS-CoV2 can provide insights into viral trafficking and aid in designing new therapeutic targets. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV2 is internalized via the clathrin and dynamin-independent, pH-dependent CLIC/GEEC (CG) endocytic pathway. Endosomal acidification inhibitors like BafilomycinA1 and NH4Cl, which inhibit the CG pathway, strongly block the uptake of RBD. Using transduction assays with SARS-CoV2 Spike-pseudovirus, we confirmed that these acidification inhibitors also impede viral infection. By contrast, Chloroquine neither affects RBD uptake nor extensively alters the endosomal pH, yet attenuates Spike-pseudovirus entry, indicating a pH-independent mechanism of intervention. We screened a subset of FDA-approved acidification inhibitors and found Niclosamide to be a potential SARS-CoV2 entry inhibitor. Niclosamide, thus, could provide broader applicability in subverting infection of similar category viruses entering host cells via this pH-dependent endocytic pathway.

cell biology↗

bioRxiv2020

Rrp6p/Rrp47p constitutes an independent nuclear turnover system of mature small non-coding RNAs in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the nuclear exosome/Rrp6p/TRAMP participates in the 3-end processing of several precursor non-coding RNAs. Here we demonstrate that the depletion of nucleus-specific 3[->]5 exoribonuclease Rrp6p and its cofactor, Rrp47p led to the specific and selective enhancement of steady-state levels of mature small non-coding RNAs (sncRNAs) that include 5S and 5.8S rRNAs, snRNAs and snoRNAs, but not 18S and 25S rRNAs. Most importantly, their steady-state enhancement does not require the exosome, TRAMP, CTEXT, or Rrp6p-associated Mpp6p. Rrp6p/47p-dependent enhancement of the steady-state levels of sncRNAs is associated with the diminution of their nuclear decay-rate and requires their polyadenylation before targeting by Rrp6p, which is catalyzed by both the canonical and non-canonical poly(A) polymerases, Pap1p and Trf4p. Consistent with this finding, we also demonstrated that Rrp6p and Rrp47p exist as an exosome-independent complex. Thus, Rrp6p-Rrp47p defines a core nuclear exosome-independent novel turnover system that targets the small non-coding RNAs.

molecular biology↗

bioRxiv2020

TiMEG: an integrative approach for partially missing multi-omics data with an application to tuberous sclerosis

1Multi-omics data integration is widely used to understand the genetic architecture of disease. In multi-omics association analysis, data collected on multiple omics for the same set of individuals are immensely important for biomarker identification. But when the sample size of such data is limited, the presence of partially missing individual-level observations poses a major challenge in data integration. More often, genotype data are available for all individuals under study but gene expression and/or methylation information are missing for different subsets of those individuals. Here, we develop a statistical model TiMEG, for the identification of disease-associated biomarkers in a case-control paradigm by integrating the above-mentioned data types, especially, in presence of missing omics data. Based on a likelihood approach, TiMEG exploits the inter-relationship among multiple omics data to capture weaker signals, that remain unidentified in single-omics analyses. Its application on a real tuberous sclerosis dataset identified functionally relevant genes in the disease pathway.

bioinformatics↗

bioRxiv2020

Communicating Regulatory High Throughput Sequencing Data Using BioCompute Objects

For regulatory submissions of next generation sequencing (NGS) data it is vital for the analysis workflow to be robust, reproducible, and understandable. This project demonstrates that the use of the IEEE 2791-2020 Standard, (BioCompute objects [BCO]) enables complete and concise communication of NGS data analysis results. One arm of a clinical trial was replicated using synthetically generated data made to resemble real biological data. Two separate, independent analyses were then carried out using BCOs as the tool for communication of analysis: one to simulate a pharmaceutical regulatory submission to the FDA, and another to simulate the FDA review. The two results were compared and tabulated for concordance analysis: of the 118 simulated patient samples generated, the final results of 117 (99.15%) were in agreement. This high concordance rate demonstrates the ability of a BCO, when a verification kit is included, to effectively capture and clearly communicate NGS analyses within regulatory submissions. BCO promotes transparency and induces reproducibility, thereby reinforcing trust in the regulatory submission process.

scientific communication and education↗