Biology subjects

Kumar, N.

Publications and source records attributed to Kumar, N..

At least 163 records · Page 9Linked to original sources

bioRxiv2021

Novel RT-ddPCR assays for determining the transcriptional profile of SARS-CoV-2

The exact mechanism of coronavirus replication and transcription is not fully understood; however, a hallmark of coronavirus transcription is the generation of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and an array of subgenomic mRNAs (sgRNAs) encompassing sequences arising from discontinuous transcription. Existing PCR-based diagnostic assays for SAR-CoV-2 are qualitative or semi-quantitative and do not provide the resolution needed to assess the complex transcription dynamics of SARS-CoV-2 over the course of infection. We developed and validated a novel panel of specially designed SARS-CoV-2 ddPCR-based assays to map the viral transcription profile. Application of these assays to clinically relevant samples will enhance our understanding of SARS-CoV-2 replication and transcription and may also inform the development of improved diagnostic tools and therapeutics. HighlightsO_LIWe developed a novel panel of 7 quantitative RT-ddPCRs assays for SARS-Cov-2 C_LIO_LIOur panel targets nongenic and genic regions in genomic and subgenomic RNAs C_LIO_LIAll assays detect 1-10 copies and are linear over 3-4 orders of magnitude C_LIO_LIAll assays correlated with the clinical Abbott SARS-CoV-2 Viral Load Assay C_LIO_LIClinical samples showed higher copy numbers for targets at the 3 end of the genome C_LI

microbiology↗

bioRxiv2020

MAPPER: A new image analysis pipeline unmasks differential regulation of Drosophila wing features

Phenomics requires quantification of large volumes of image data, necessitating high throughput image processing approaches. Existing image processing pipelines for Drosophila wings, a powerful model for studying morphogenesis, are limited in speed, versatility, and precision. To overcome these limitations, we developed MAPPER, a fully-automated machine learning-based pipeline that quantifies high dimensional phenotypic signatures, with each dimension representing a unique morphological feature. MAPPER magnifies the power of Drosophila genetics by rapidly identifying subtle phenotypic differences in sample populations. To demonstrate its widespread utility, we used MAPPER to reveal new insights connecting patterning and growth across Drosophila genotypes and species. The morphological features extracted using MAPPER identified the presence of a uniform scaling of proximal-distal axis length across four different species of Drosophila. Observation of morphological features extracted by MAPPER from Drosophila wings by modulating insulin signaling pathway activity revealed the presence of a scaling gradient across the anterior-posterior axis. Additionally, batch processing of samples with MAPPER revealed a key function for the mechanosensitive calcium channel, Piezo, in regulating bilateral symmetry and robust organ growth. MAPPER is an open source tool for rapid analysis of large volumes of imaging data. Overall, MAPPER provides new capabilities to rigorously and systematically identify genotype-to-phenotype relationships in an automated, high throughput fashion. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/422888v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@bdbecdorg.highwire.dtl.DTLVardef@139a7fdorg.highwire.dtl.DTLVardef@43d5b9org.highwire.dtl.DTLVardef@1846af9_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2020

Emetine as an antiviral agent suppresses SARS-CoV-2 replication by inhibiting interaction of viral mRNA with eIF4E: An in vitro study

Emetine is a FDA-approved drug for the treatment of amebiasis. In the recent times we had also demonstrated the antiviral efficacy of emetine against some RNA and DNA viruses. Following emergence of the COVID-19, we further evaluated thein vitro antiviral activity of emetine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The therapeutic index of emetine was determined to be 10910.4, at a cytotoxic concentration 50 (CC50) of 1603.8 nM and effective concentration 50 (EC50) of 0.147 nM.Besides, we also demonstrated the protective efficacy of emetine against lethal challenge with infectious bronchitis virus (IBV; a chicken coronavirus) in the embryonated chicken egg infection model. Emetine treatment was shown to decrease viral RNA and protein synthesis without affecting other steps of viral life cycle such as attachment, entry and budding.In a chromatin immunoprecipitation (CHIP) assay, emetine was shown to disrupt the binding of SARS-CoV-2 RNA with eIF4E (eukaryotic translation initiation factor 4E, a cellular cap-binding protein required for initiation ofprotein translation). Further, SARS-CoV-2 was shown to exploit ERK/MNK1/eIF4E signalling pathwayfor its effective replication in the target cells. To conclude, emetine targets SARS-CoV-2 protein synthesis which is mediated via inhibiting the interaction of SARS-CoV-2 RNA with eIF4E. This is a novel mechanistic insight on the antiviral efficacy of emetine. In vitro antiviral efficacy against SARS-CoV-2 and its ability to protect chicken embryos against IBV suggests that emetine could be repurposed to treat COVID-19.

microbiology↗

bioRxiv2020

Human PRE-PIK3C2B exhibits long-range intra- and inter-chromosomal interactions with genomic regions enriched in repressive marks.

Human PRE-PIK3C2B is a dual nature polycomb response element that interacts with both polycomb as well as trithorax members. In the current study, using 4C-Seq (Capturing Circular Chromosomal Conformation-Sequencing), we identified long-range chromatin interactions associated with PRE-PIK3C2B and validated them with 3C-PCR. We identified both intra-as well as inter-chromosomal interactions, a large proportion of which were found to be closely distributed around transcriptional start sites (TSS). A significant number of interactions were also found to be associated with heterochromatic regions. Meta-analysis of ENCODE ChIP-Seq data identified an overall enrichment of YY1, CTCF as well as histone modification such as H3K4me3 and H3K27me marks in different cell lines. Almost 90% interactions were derived from either intronic or intergenic regions. among which large proportions of intronic interactors were either unique sequences or LINE/SINE derived. In case of intergenic interactions, majority of the interaction were associated with LINE/SINE repeats. We further found that genes proximal to the interactor sequences were co-expressed, they showed reduced expression. To the best of our knowledge this is one of the early demonstrations of long-range interaction of PRE sequences in human genome.

genetics↗

bioRxiv2020

Computational prediction of SARS-CoV-2 encoded miRNAs and their putative host targets

Over the past two decades, there has been a continued research on the role of small non-coding RNAs including microRNAs (miRNAs) in various diseases. Studies have shown that viruses modulate the host cellular machinery and hijack its metabolic and immune signalling pathways by miRNA mediated gene silencing. Given the immensity of coronavirus disease 19 (COVID-19) pandemic and the strong association of viral encoded miRNAs with their pathogenesis, it is important to study Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) miRNAs. To address this unexplored area, we identified 8 putative novel miRNAs from SARS-CoV-2 genome and explored their possible human gene targets. A significant proportion of these targets populated key immune and metabolic pathways such as MAPK signalling pathway, maturity-onset diabetes, Insulin signalling pathway, endocytosis, RNA transport, TGF-{beta} signalling pathway, to name a few. The data from this work is backed up by recently reported high-throughput transcriptomics datasets obtains from SARS-CoV-2 infected samples. Analysis of these datasets reveal that a significant proportion of the target human genes were down-regulated upon SARS-CoV-2 infection. The current study brings to light probable host metabolic and immune pathways susceptible to viral miRNA mediated silencing in a SARS-CoV-2 infection, and discusses its effects on the host pathophysiology.

bioinformatics↗

bioRxiv2020

Resource reallocation in engineered Escherichia coli strains with reduced genomes

A major challenge in synthetic biology is properly balancing evolved and engineered functions without compromising microbial fitness. Many microbial proteins are not required for growth in regular laboratory conditions, but it is unclear what fraction of the proteome can be eliminated to increase bioproduction and maintain fitness. Here, we investigated the effects of massive genome reduction in E. coli on the expression level and evolutionary stability of a model biosynthetic pathway to produce the pigment protodeoxyviolacein (PDV). We identified an amino acid metabolism imbalance and compromised growth that were correlated with elimination of genes associated with significant proteome fraction. Proteomic profiling suggested that increased amino acid pools are responsible for an alleviation of fitness defects associated with PDV expression. In addition, all strains with genome reductions that significantly affected the proteome exhibited decreased stability of PDV production compared to the wild-type strain under persistent PDV expression conditions despite the alleviation of fitness defects. These findings exhibit the importance of balancing evolved functions with engineered ones to achieve an optimal balance of fitness and bioproduction.

bioengineering↗

bioRxiv2020

Hemispheric asymmetry of entrained auditory steady state responses arise from the structural connectome

Structure-function relationships are fundamental to studies of neural systems, yet the mechanistic underpinnings of how structural networks shape hemispheric lateralization remain elusive. For instance, the asymmetric neuroanatomic embedding of primary auditory cortices was shown when connectivity with all other brain areas were considered. Concomitantly, functional lateralization e.g., left hemispheric dominance of speech processing and right hemispheric dominance of music perception, is widely acknowledged. The present article provides a parsimonious mechanistic explanation based on computational modelling and empirical recordings to demonstrate emergence of hemispheric lateralization of brain function. For validation of the model, empirical EEG recordings of auditory steady state responses (ASSR) were undertaken, and empirical findings suggest right hemispheric dominance at the level of cortical sources in binaural and monaural hearing conditions. Subsequently, we demonstrate the entrainment and phase of oscillations in connected brain regions based on a neurodynamic model constrained by empirically derived structural connectivity matrix from diffusion data. For relevance, we have taken into consideration time-delays in neural communication stemming from fibre distances and neuronal coupling arising from fibre densities. Based on relevant network parameters, we could demonstrate the emergence of right hemispheric dominance of ASSR in binaural and monaural hearing conditions when auditory cortical areas were chosen as triggers of entrained phase oscillators. Furthermore, we discovered emergence of left-lateralized functional response when bilateral Brocas area replaced auditory areas as triggers. Hence, a single unified mechanism based on entrainment of phase oscillators in a large-scale brain network could explain both emergence of right and left hemispheric laterality. Significance statementThe origin of hemispheric specialization of sensory processing is a fundamental question in neuroscience. For instance, speech and language are predominantly processed in the left hemispheric regions, while the right hemisphere is specialized for processing rhythmic, tonal, and melodic stimuli. Identification of the network mechanisms that give rise to such functional lateralization from structural constraints remains elusive. In the present study, we simulate neural activity observed during human EEG recordings of auditory steady-state responses from a biophysically realistic large-scale model constrained by underlying structural connectivity. Subsequently, we demonstrate how hemispheric lateralization of brain responses to sensory stimuli emerge from the time-delayed interactions among whole-brain neuronal ensembles.

neuroscience↗

bioRxiv2020

Physiological synaptic activity and recognition memory are fueled by astroglial glutamine

Presynaptic glutamate replenishment is fundamental to brain function. In high activity regimes, such as epileptic episodes, this process is thought to rely on the glutamate-glutamine cycle between neurons and astrocytes. However the presence of an astroglial glutamine supply, as well as its functional relevance in vivo in the healthy brain remain controversial, partly due to a lack of tools that can directly examine glutamine transfer. Here, we generated a novel fluorescent probe that tracks glutamine in live cells, which provided direct visual evidence of an activity-dependent glutamine supply from astroglial networks to presynaptic structures under physiological conditions. This mobilization is mediated by connexin43, an astroglial protein with both gap-junction and hemichannel functions, and is essential for synaptic transmission and object recognition memory. Our findings uncover an indispensable recruitment of astroglial glutamine in physiological synaptic activity and memory via an unconventional pathway, thus providing an astrocyte basis for cognitive processes.

neuroscience↗

bioRxiv2020

Isolation and characterization of lumpy skin disease virus from cattle in India

Lumpy skin disease (LSD) has devastating economic impact. During the last decade, LSD had spread to climatically new and previously disease-free countries, which also includes its recent emergence in the Indian subcontinent (2019). This study deals with the LSD outbreak(s) from cattle in Ranchi (India). Virus was isolated from the scabs (skin lesions) in the primary goat kidney cells. Phylogenetic analysis based on nucleotide sequencing of LSD virus (LSDV) ORF011, ORF012 and ORF036 suggested that the isolated virus (LSDV/Bos taurus-tc/India/2019/Ranchi) is closely related to Kenyan LSDV strains. Further, we adapted the isolated virus in Vero cells, an alternative cell line that was found suitable for LSDV propagation. In addition, kinetics of viral DNA synthesis and one-step growth curve analysis suggested that LSDV initiates synthesizing its genome at [~]24 hours post-infection (hpi) with a peak level at [~]96 hpi whereas evidence of progeny virus particles was observed at 36-48 hours (h) with a peak titre at [~]120 h. This study reports the first successful isolation of LSDV in India, besides describing alternative cells for virus isolation (primary goat kidney cells) and in vitro propagation (Vero cells) as well as providing some insights on LSDV life cycle. Author SummaryThis study describes the first successful isolation of LSDV in India. Nucleotide sequencing of LSDV ORF011, ORF012 and ORF036 suggested that the isolated virus is closely related to Kenyan LSDV strains. This is in accordance with the previous (only) study, thereby suggesting that a single LSDV strain is circulating in India. For the first time, we employed primary goat kidney (PGK) cells for the isolation of LSDV, where PGK cells were found more sensitive for LSDV isolation as compared to the primary lamb testicle (PLT) and primary goat testicle (PGT) cells. We also adapted the isolated virus in Vero cells and demonstrated that Vero cells are also suitable for in vitro propagation of LSDV. Besides, we also provide some insights on the LSDV life cycle.

microbiology↗

bioRxiv2020

Higher Order AMMI (HO-AMMI) analysis: A novel stability model to study genotype-location interactions

Additive main effects and multiplicative interaction (AMMI) model is most widely used to analyse genotype*environment interactions (GEI) wherein interaction effects of location is masked by year effect. Hence, presently available models are not able to estimate interaction effects of genotype*location (GLI) and genotype*year (GYI) separately. Moreover, genotype ranking differs as number of years of evaluation vary making selection of genotype for target location difficult. In the present study we propose a novel stability model i.e Higher-order-AMMI (HO-AMMI) analysis which is capable of calculating GLI without the confounding effect of GYI and GLYI. GEI of AMMI model and all 2-way interactions of HO-AMMI model follow {chi}2 distribution, whereas 3-way interaction (GLYI) of HO-AMMI follow noncentral {chi}2 distribution. With increase in number of years of evaluation contribution of GLI towards total variation increased whereas in AMMI model contribution of GEI towards total variation decreased. Variation explained by multiplicative components is higher in HO-AMMI compared to AMMI model. Genotypes were ranked using GL, GY and GL+GY+GLY interactions of HO-AMMI and GEI of AMMI for stability and yield and compared their ranks with field ranking. Correlation and linear regression analysis have indicated high association of GLI (HO-AMMI) with field ranking with high R2 values. Further, HO-AMMI model was able to remove the confounding effect of GYI and GLYI on GLI for accurate identification of genotype for target location irrespective of number of years of evaluation. Hence, HO-AMMI model can be used under multi-environment trials (MET) for selecting genotypes efficiently. Availability and InformationSource code implemented in R is available from corresponding author.

plant biology↗

bioRxiv2020

A cell-autonomous PD-1/PD-L1 circuit promotes tumorigenicity of thyroid cancer cells by activating a SHP2/Ras/MAPK signalling cascade

The programmed cell death-1 (PD-1) and its ligands PD-L1 and PD-L2 are immune checkpoints. Typically, cancer cells express the PD-Ls that bind PD-1 on immune cells, inhibiting their anti-cancer activity. Recently, PD-1 expression has been found in cancer cells. We analysed expression and functions of PD-1 in thyroid cancer (TC). Human TC specimens (47%), but not normal thyroids, displayed PD-1 expression in epithelial cells, which significantly correlated with tumour stage and lymph-node metastasis. PD-1 overexpression/stimulation promoted TC cell proliferation and migration in culture. PD-1 recruited the SHP2 phosphatase, potentiated its phosphatase activity thus enhancing Ras activation by dephosphorylation of inhibitory tyrosine 32 and triggering the MAPK cascade. PD-1 inhibition decreased, while PD-1 overexpression facilitated, TC cell xenograft growth by affecting cell proliferation. PD-1 circuit blockade in TC, besides restoring anti-cancer immunity, could also directly impair TC cell growth by inhibiting the Ras/MAPK pathway.

cancer biology↗

bioRxiv2020

Elicitation of potent serum neutralizing antibody responses in rabbits by immunization with an HIV-1 clade C trimeric Env derived from an Indian elite neutralizer

Evaluating the structure-function relationship of viral envelope (Env) evolution and the development of broadly cross-neutralizing antibodies (bnAbs) in natural infection can inform rational immunogen design. In the present study, we examined the magnitude and specificity of autologous neutralizing antibodies induced in rabbits by a novel HIV-1 clade C Env protein (1PGE-THIVC) vis-a-vis those developed in an elite neutralizer from whom the env sequence was obtained that was used to prepare the soluble Env protein. The thermostable 1PGE-THIVC Env displayed a native like pre-fusion closed conformation in solution as determined by small angle X-ray scattering (SAXS) and negative stain electron microscopy (EM). This closed spike conformation of 1PGE-THIVC Env trimers was correlated with weak or undetectable binding of non-neutralizing monoclonal antibodies (mAbs) compared to neutralizing mAbs. Furthermore, 1PGE-THIVC SOSIP induced potent neutralizing antibodies in rabbits to autologous virus variants. The autologous neutralizing antibody specificity induced in rabbits by 1PGE-THIVC was mapped to the C3/V4 region (T362/P401) of viral Env. This observation agreed with electron microscopy polyclonal epitope mapping (EMPEM) of the Env trimer complexed with IgG Fab prepared from the immunized rabbit sera. While the specificity of antibodies elicited in rabbits associated with neutralizing autologous viruses were distinct to those developed in the elite neutralizer, EMPEM analysis demonstrated significant changes to Env conformations when incubated with polyclonal antibody sera from the elite neutralizer, suggesting these antibodies lead to the destabilization of Env trimers. Our study not only shows distinct mechanisms associated with potent neutralization of sequence matched and unmatched autologous viruses by antibodies induced in rabbits and in the elite neutralizer, but also highlights how neutralizing antibodies developed during the course of natural infection can impact viral Env conformations. Author SummaryThe interplay between circulating virus variants and broadly cross neutralizing polyclonal antibodies developed in a subset of elite neutralizers is widely believed to provide strategies for rational immunogen design. In the present study, we studied the structural, antigenic and immunogenic properties of a thermostable soluble trimeric protein with near native pre-fusion conformation prepared using the primary sequence of an HIV-1 clade C env isolated from the broadly cross neutralizing plasma of an elite neutralizer. This novel SOSIP Env trimer demonstrated comparable antigenic, structural and immunogenic properties that favoured several ongoing subunit vaccine design efforts. The novel clade C SOSIP induced polyclonal neutralizing antibody response developed in rabbits not only differed in its epitope specificity compared to that elicited in natural infection in presence of pool of viral quasispecies but also showed how they differ in their ability to influence Env structure and conformation. A better understanding of how vaccine-induced polyclonal neutralizing antibody responses compares to responses that developed in natural infection will improve our knowledge in designing better vaccine design strategies.

immunology↗

bioRxiv2020

Structure of the Human Secretory Immunoglobulin M Core

Immunoglobulins (Ig) A and M are the only human antibodies that form oligomers and undergo transcytosis to mucosal secretions via the polymeric Ig receptor (pIgR). When complexed with the J-chain (JC) and the secretory component (SC) of pIgR, secretory IgA and IgM (sIgA and sIgM) play critical roles in host-pathogen defense. Recently, we determined the structure of sIgA-Fc which elucidated the mechanism of polymeric IgA assembly and revealed an extensive binding interface between IgA-Fc, JC, and SC. Despite low sequence identity shared with IgA-Fc, IgM-Fc also undergoes JC-mediated assembly and binds pIgR. Here, we report the structure of sIgM-Fc and carryout a systematic comparison to sIgA-Fc. Our structural analysis reveals a remarkably conserved mechanism of JC-templated oligomerization and SC recognition of both IgM and IgA through highly a conserved network of interactions. These studies reveal the structurally conserved features of sIgM and sIgA required for function in mucosal immunity.

immunology↗

bioRxiv2020

Host adaptation in gut Firmicutes is associated with sporulation loss and altered colonisation patterns

Human-to-human transmission of symbiotic, anaerobic bacteria is a fundamental evolutionary adaptation essential for membership of the human gut microbiota. However, despite its importance, the genomic and biological adaptations underpinning symbiont transmission remain poorly understood. Here, we show that sporulation ability, which promotes transmission of anaerobic bacteria, has been independently lost in many distinct evolutionary lineages of gut bacteria belonging to the Firmicutes phyla. Analysis of 1358 genome-sequenced Firmicutes reveals loss of sporulation is associated with features of host-adaptation such as genome reduction and specialized metabolic capabilities. Consistent with these data, analysis of 28,000 gut metagenomes from people around the world demonstrates that bacteria now incapable of sporulation are more abundant but less prevalent in the human population compared to spore-forming bacteria. Our results suggest host adaptation in gut Firmicutes is an evolutionary trade-off between transmission range and colonisation abundance, leading to distinct transmission cycles. We reveal host transmission as an underappreciated process that shapes the evolution, assembly and functions of gut Firmicutes.

microbiology↗

bioRxiv2020

BAP1 and YY1 regulate expression of death receptors in malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is a rare, aggressive, and incurable cancer arising from the mesothelial lining of the lungs with few treatment options. We recently reported loss of function of the nuclear deubiquitinase BRCA associated protein-1 (BAP1), a frequent event in MPM, is associated with sensitivity to tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). As a potential underlying mechanism, here we report that BAP1 negatively regulates the expression of TRAIL receptors: death receptors 4 (DR4) and 5 (DR5). Using tissue microarray (TMAs) of tumour samples from MPM patients, we found a strong inverse correlation between BAP1 and TRAIL receptors. BAP1 knockdown increased DR4 and DR5 expression, whereas overexpression of BAP1 had the opposite effect. Reporter assays confirmed wild-type BAP1, but not catalytically-inactive mutant BAP1, reduced promoter activities of DR4 and DR5, suggesting deubiquinase activity plays an important role in the regulation of gene expression. Co-IP studies demonstrated direct binding of BAP1 and the transcription factor Ying Yang 1 (YY1) and ChIP assays revealed BAP1 and YY1 to be enriched in the promoter regions of DR4 and DR5. Notably, shRNA knockdown of YY1 also increased DR4 and DR5 expression, and sensitivity to TRAIL. These results demonstrate that BAP1 and YY1 together negatively regulate transcriptional activity of TRAIL receptors. BAP1 and YY1 may both therefore be strong therapeutic targets to enhance the efficacy of TRAIL-induced apoptosis. Statement of significanceWe describe how the most-frequently mutated tumour suppressor gene in mesothelioma regulates the response to TNF-related apoptosis-inducing ligand (TRAIL). These findings will accelerate a biomarker-driven cancer therapy.

cancer biology↗

bioRxiv2020

Rapid Single Cell Evaluation of Human Disease and Disorder Targets Using REVEAL: SingleCell™

Single-cell (sc) sequencing performs unbiased profiling of individual cells and enables evaluation of less prevalent cellular populations, often missed using bulk sequencing. However, the scale and the complexity of the sc datasets poses a great challenge in its utility and this problem is further exacerbated when working with larger datasets typically generated by consortium efforts. As the scale of single cell datasets continues to increase exponentially, there is an unmet technological need to develop database platforms that can evaluate key biological hypothesis by querying extensive single-cell datasets. Large single-cell datasets like human cell atlas and COVID-19 cell atlas (collection of annotated sc datasets from various human organs) are excellent resources for profiling target genes involved in human diseases and disorders ranging from oncology, auto-immunity, as well as infectious diseases like COVID-19 caused by SARS-CoV-2 virus. SARS-CoV-2 infections have led to a worldwide pandemic with massive loss of lives, infections exceeding 7 million cases. The virus uses ACE2 and TMPRSS2 as key viral entry associated proteins expressed in human cells for infections. Evaluating the expression profile of key genes in large single-cell datasets can facilitate testing for diagnostics, therapeutics and vaccine targets; as the world struggles to cope with the on-going spread of COVID-19 infections. In this manuscript we describe, REVEAL: SingleCell which enables storage, retrieval and rapid query of single-cell datasets inclusive of millions of cells. The analytical database described here enables selecting and analyzing cells across multiple studies. Cells can be selected using individual metadata tags, more complex hierarchical ontology filtering, and gene expression threshold ranges, including co-expression of multiple genes. The tags on selected cells can be further evaluated for testing biological hypothesis. One such example includes identifying the most prevalent cell type annotation tag on returned cells. We used REVEAL: SingleCell to evaluate expression of key SARS-CoV-2 entry associated genes, and queried the current database (2.2 Million cells, 32 projects) to obtain the results in <60 seconds. We highlighted cells expressing COVID-19 associated genes are expressed on multiple tissue types, thus in part explains the multi-organ involvement in infected patients observed worldwide during the on-going COVID-19 pandemic.

molecular biology↗

bioRxiv2020

Canagliflozin Extends Lifespan in Genetically Heterogeneous Male But Not Female Mice

Canagliflozin (Cana) is an inhibitor of the sodium glucose transporter 2 (SGLT2), and is thought to act by blocking renal reuptake and intestinal absorption of glucose. Cana is FDA-approved for treatment of diabetes, and affords protection from cardiovascular and kidney diseases. In the context of the mouse Interventions Testing Program, genetically heterogeneous mice were given chow containing 180 ppm Cana at 7 months of age until their death. Cana extended median survival of male mice by 14%, with p < 0.001 by log-rank test. Cana also increased by 9% the age for 90th percentile survival (p < 0.001 by Wang/Allison test), with parallel effects seen at each of three test sites. Cana did not alter the distribution of inferred cause of death, nor of incidental pathology findings at end-of-life necropsies. No benefits were seen in female mice. The lifespan benefit of Cana is likely to reflect blunting of peak glucose levels, because similar longevity effects are seen in mice given acarbose, a diabetes drug that blocks glucose surges through a distinct mechanism, i.e. slowing breakdown of carbohydrate in the intestine. Interventions that control daily peak glucose levels deserve attention as possible preventive medicines to protect from a wide range of late-life neoplastic and degenerative diseases.Competing Interest StatementThe authors have declared no competing interest.View Full Text

pathology↗

bioRxiv2020

Detection of a multi-disease biomarker in Salivawith Graphene Field Effect Transistors

Human carbonic anhydrase 1 (CA1) has been suggested as a biomarker for identification of several diseases including cancers, pancreatitis, diabetes, and Sjogrens syndrome. However, the lack of a rapid, cheap, accurate, and easy-to-use quantification technique has prevented widespread utilization of CA1 for practical clinical applications. To this end, we present a label-free electronic biosensor for detection of CA1 utilizing highly sensitive graphene field effect transistors (G-FETs) as a transducer and specific RNA aptamers as a probe. The binding of CA1 with aptamers resulted in a positive shift in Dirac voltage VD of the G-FETs, the magnitude of which depended on target concentration. These aptameric G-FET biosensors showed the binding affinity (KD) of [~] 2.3 ng/ml (70 pM), which is four orders lower than that reported using a gel shift assay. This lower value of KD enabled us to achieve a detection range (10 pg/ml - 100 ng/ml) which is well in line with the clinically relevant range. These highly sensitive devices allowed us to further prove their clinical relevance by successfully detecting the presence of CA1 in human saliva samples. Utilization of this label-free biosensor could facilitate the early stage identification of various diseases associated with changes in concentration of CAs.

biophysics↗