Biology subjects

Gupta, P.

Publications and source records attributed to Gupta, P..

At least 109 records · Page 6Linked to original sources

bioRxiv2022

Whole-Genome Profiling of Ethyl Methanesulfonate Mutagenesis in Tomato

The induced mutations accelerate crop improvement by providing novel disease resistance and yield alleles. However, the alleles with no perceptible phenotype but having an altered function remain hidden in mutagenized plants. The whole-genome sequencing (WGS) of mutagenized individuals uncovers the complete spectrum of mutations in the genome. We sequenced 132 doubly ethyl methanesulfonate (EMS)-mutagenized lines of tomato and detected ca. 41 million SNPs and 5.5 million short-INDELs. We found a very high average density of mutations 1/3.05 Kb compared to other species. About 97% of the genome had mutations, including the genes, promoters, UTRs, and introns. More than 1/3rd of genes in the mutagenized population had one or more deleterious mutations predicted by SIFT. Nearly 1/4th of deleterious genes mapped on tomato metabolic pathways modulating multiple pathway steps. Contrary to the reported GC>AT transition bias for EMS, we found EMS also produced nearly equal AT>GC transitions. Comparing mutation frequency among synonymous codons revealed that the most preferred codon is least mutagenic towards EMS. The reduction in carotenoids in {zeta}-carotene isomerase mutant fruits and chloroplasts relocation loss in phototropin1 mutant validated the mutation discovery pipeline. Our study makes a large repertoire of mutations accessible to genetic studies and the breeding of tomato.

plant biology↗

bioRxiv2022

Genetic Impairment of Succinate Metabolism Disrupts Bioenergetic Sensing in Adrenal Neuroendocrine Tumor

Metabolic dysfunction mutations can impair energy sensing and cause cancer. Loss of function of mitochondrial TCA cycle enzyme, succinate dehydrogenase B (SDHB) results in various forms of cancer typified by pheochromocytoma (PC). Here we delineate a signaling cascade where the loss of SDHB induces the Warburg effect in PC tumors, triggers dysregulation of Ca2+ homeostasis, and aberrantly activates calpain and the protein kinase Cdk5, through conversion of its cofactor from p35 to p25. Consequently, aberrant Cdk5 initiates a cascade of phospho- signaling where GSK3 inhibition inactivates energy sensing by AMP-kinase through dephosphorylation of the AMP-kinase {gamma} subunit, PRKAG2. Overexpression of p25-GFP in mouse adrenal chromaffin cells also elicits this phosphorylation signaling and causes PC tumor formation. A novel Cdk5 inhibitor, MRT3-007, reversed this phospho-cascade, invoking an anti- Warburg effect, cell cycle arrest, and senescence-like phenotype. This therapeutic approach halted tumor progression in vivo. Thus, we reveal an important novel mechanistic feature of metabolic sensing and demonstrate that its dysregulation underlies tumor progression in PC and likely other cancers. HighlightsO_LILoss of SDHB function in pheochromocytoma causes Ca2+ dysregulation, calpain activation, and aberrant activation of the protein kinase Cdk5. C_LIO_LIHyperactive Cdk5 deregulates a GSK3/PRKAG2/AMPK signaling cascade. C_LIO_LIp25 overexpression and consequent aberrant Cdk5 activity in chromaffin cells causes pheochromocytoma. C_LIO_LIInhibition of Cdk5 activates the PRKAG2/AMPK/p53 signaling to rescue cell senescence and block PC tumor progression. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=175 SRC="FIGDIR/small/475410v2_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@16f0a48org.highwire.dtl.DTLVardef@10c1c7aorg.highwire.dtl.DTLVardef@21b100org.highwire.dtl.DTLVardef@1eb8674_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2021

Mechanistic Insights in Biofilm Eradication Potential of Tyrosol Functionalized Chitosan Gold Nanoparticles against Candida spp.

In the present study, tyrosol functionalized chitosan gold nanoparticles (Chi-TY-AuNPs) were prepared as an alternative treatment strategy to combat fungal infections. Various biophysical techniques were used to characterize the synthesized Chi-TY-AuNPs. The antifungal and antibiofilm activities of Chi-TY-AuNPs were evaluated against C. albicans and C. glabrata and efforts have been made to elucidate the possible mechanism of action. Chi-TY-AuNPs showed a high fungicidal effect against both sessile and planktonic cells of Candida spp. Additionally, Chi-TY-AuNPs completely eradicated (100%) the mature biofilms of both the Candida spp. FESEM analysis highlighted the morphological alterations in Chi-TY-AuNPs treated Candida biofilm cells. Effect of Chi-TY-AuNPs on the ECM components showed significant reduction in protein content in C. glabrata biofilm and substantial decrease in extracellular DNA (eDNA) content of both the Candida spp. ROS generation analysis using DCFDA-PI staining showed high ROS levels in both the Candida spp., whereas pronounced ROS production was observed in Chi-TY-AuNPs treated C. glabrata biofilm. Biochemical analysis revealed decreased ergosterol content in Chi-TY-AuNPs treated C. glabrata cells, while inconsequential changes were observed in C. albicans. Furthermore, the transcriptional expression of selected genes (ergosterol biosynthesis, efflux, sterol importer, and glucan biogenesis) was reduced in C. glabrata in response to Chi-TY-AuNPs except ERG11 and CDR1. Conclusively the result showed the biofilm inhibition and biofilm eradication efficacy of Chi-TY-AuNPs in both the Candida spp. Findings of the present study manifest Chi-TY-AuNPs as a potential therapeutic solution to Candida biofilm-related chronic infections and overcome biofilm antifungal resistance.

microbiology↗

bioRxiv2021

Potential Key Genes Associated with Stroke types and its subtypes: A Computational Approach

To investigate prospective key genes and pathways associated with the pathogenesis and prognosis of stroke types along with subtypes. Human genes using genome assembly build 38 patch release 13 with known gene symbols through NCBI gene database (https://www.ncbi.nlm.nih.gov/gene) were fetched. PubMed advanced queries were constructed using stroke-related keywords and associations were calculated using Normalized pointwise mutual information (nPMI) between each gene symbol and queries. Genes related with stroke risk within their types and subtypes were investigated in order to discover genetic markers to predict individuals who are at the risk of developing stroke with their subtypes. A total of 2,785 (9.4%) genes were found to be linked to the risk of stroke. Based on stroke types, 1,287 (46.2%) and 376 (13.5%) genes were found to be related with IS and HS respectively. Further stratification of IS based on TOAST classification, 86 (6.6%) genes were confined to Large artery atherosclerosis; 131 (10.1%) and 130 (10%) genes were related with the risk of small vessel disease and Cardioembolism subtypes of IS. Besides, a prognostic panel of 9 genes signature consisting of CYP4A11, ALOX5P, NOTCH, NINJ2, FGB, MTHFR, PDE4D, HDAC9, and ZHFX3 can be treated as a diagnostic marker to predict individuals who are at the risk of developing stroke with their subtypes.

bioinformatics↗

bioRxiv2021

Molecular characterization of carbapenem resistant Klebsiella pneumoniae clinical isolates: Preliminary experience from a tertiary care teaching hospital in the Himalayas

IntroductionKlebsiella pneumoniae is recognized as an urgent threat to human health because of the emergence of multidrug-resistant (MDR) and hypervirulent strains. Development of novel highly effective and safe treatment options is the need of the hour. One of the ways of achieving this goal is by conducting molecular characterization studies of antibiotic resistant bacteria. Hypothesis/Gap StatementThe government of India is committed to generate validated AMR data from different parts of the country. Efforts are being made to perform molecular characterization of MDR and potentially virulent bacteria by whole-genome sequencing (WGS). However, the data that we have at present is skewed as many parts of the country remain underrepresented. Uttarakhand is one such state located in the Himalay an belt of India, with relatively poor access to healthcare and a paucity of research. AimThis study was performed to generate WGS based preliminary data about the population structure, multi-locus sequence types (MLST), and virulence factors of CRKp isolates recovered from patients in a tertiary care teaching hospital and institute of national importance, located in Rishikesh, Uttarakhand, India. MethodologyA cross-sectional study was conducted at a tertiary care teaching hospital. It included twenty-nine randomly selected and archived carbapenem resistant Klebsiella pneumoniae (CR-Kp) isolates obtained from various clinical samples submitted in the Bacteriology laboratory for culture and sensitivity testing, from July 2018 to August 2019. After preliminary identification (ID) and antibiotic susceptibility testing (AST), as per standard guidelines, these isolates were sent to Central Research Laboratory (CRL), Bengaluru, India, for further characterization & WGS. ResultsTwenty-seven out of twenty-nine test isolates were CRKp. Among the 27 CRKp isolates, ST14 was the most common sequence type (8, 29.6%), followed by ST231 (5, 18.5%) and ST147(3, 11.1%) respectively. KL2 (9/27, 33.3%) and KL51 (8/27, 29.6%) were dominant K loci types in this study. Out of 5 O antigens identified, O1 and O2 together accounted for 88.9% (n=27) CRKp isolates. Yersiniabactin and Aerobactin were identified in 88.9% (24/27) & 29.6% (8/27) of the CRKp isolates of the isolate. Regulatory genes rmpA2 and rmpADC were found in 14.8% (4/27) and 3.7% (1/27) isolates respectively. The predominant plasmid replicons present were ColKP3 (55.5%), IncFII(K) (51.8%), IncFIB(pQil) (44.4%), IncFIB(K) (37%), IncR (33.3%) and Col44 0I (18.5%) respectively. A perfect agreement (100%) was observed between phenotypic and genotypic resistance profiles in the case of fluoroquinolones, penicillins, Beta Lactam/Beta Lactam Inhibitor combinations (BL/BLI), and cephalosporins respectively. As compared to phenotypic resistance, higher genotypic resistance for aminoglycosides (96.3%) and folate pathway inhibitors (92.6%) respectively, was observed. ConclusionThis study emphasizes the need for continued genomic surveillance of emerging CRKp and other MDR bacteria in Uttarakhand and neighbouring states of India. This in turn would help in generating critical information that can be used to assess the emergence, dissemination, and potential impact of important variants.

microbiology↗

bioRxiv2021

Involvement of translocon complex in hemoglobin import from infected erythrocyte cytoplasm into the Plasmodium parasite

Haemoglobin degradation is crucial for the growth and survival of Plasmodium falciparum in human erythrocytes. Although the process of Hb degradation has been studied in great detail, the mechanisms of Hb uptake remain ambiguous to date. Here, we characterized Heme Detoxification Protein (PfHDP), a protein localized in the parasitophorous vacuole, parasite food vacuole and infected erythrocyte cytosol for its role in Hb uptake. Immunoprecipitation of PfHDP-GFP fusion protein from a transgenic line using anti-GFP antibody and of Plasmodium parasite extract using anti-human Hb antibodies respectively, showed the association of PfHDP/Hb with each other as well as with the members of PTEX translocon complex. Some of these associations such as PfHDP/Hb and PfHDP/Pfexp-2 interactions were confirmed by in vitro protein-protein interaction tools. To know the roles of PfHDP and translocon complex in Hb import into the parasites, we next studied the Hb uptake by the parasite in PfHDP knock-down line using the GlmS ribozyme strategy. PfHDP knock-down significantly reduced the Hb uptake in these parasites in comparison to the wild type parasites. Further, the transient knock-down of one of the members of the translocon complex; PfHSP101 showed considerable reduction in Hb uptake. Morphological analysis of PfHDP-HA-GlmS transgenic parasites in the presence of GlcN showed food vacuole abnormalities and parasite stress, thereby causing a growth defect in the development of these parasites. Together, we implicate the translocon complex in the trafficking of PfHDP/Hb complex in the parasite and suggest a role for PfHDP in the uptake of Hb and parasite development. The study thus reveals new insights into the function of PfHDP, making it an extremely important target for developing new antimalarials.

molecular biology↗

bioRxiv2021

Development of an efficient single-cell cloning and expansion strategy for genome edited induced pluripotent stem cells

Disease-specific human induced pluripotent stem cells (hiPSCs) can be generated directly from individuals with known disease characteristics or alternatively be modified using genome editing approaches to introduce disease causing genetic mutations to study the biological response of those mutations. The genome editing procedure in hiPSCs is still inefficient, particularly when it comes to homology directed repair (HDR) of genetic mutations or targeted transgene insertion in the genome and single cell cloning of edited cells. In addition, genome editing processes also involve additional cellular stresses such as trouble with cell viability and genetic stability of hiPSCs. Therefore, efficient workflows are desired to increase genome editing application to hiPSC disease models and therapeutic applications. Apart from genome editing efficiency, hiPSC survival following single-cell cloning has proved to be challenging and has thus restricted the capability to easily isolate homogeneous clones from edited hiPSCs. To this end, we demonstrate an efficient workflow for feeder-free single cell clone generation and expansion in both CRISPR-mediated knock-out (KO) and knock-in (KI) hiPSC lines. Using StemFlex medium and CloneR supplement in conjunction with Matrigel cell culture matrix, we show that cell viability and expansion during single-cell cloning in edited and unedited cells is significantly enhanced. Our reliable single-cell cloning and expansion workflow did not affect the biology of the hiPSCs as the cells retained their growth and morphology, expression of various pluripotency markers and normal karyotype. This simplified and efficient workflow will allow for a new level of sophistication in generating hiPSC-based disease models to promote rapid advancement in basic research and also the development of novel cellular therapeutics.

cell biology↗

bioRxiv2021

Chemical inhibition of lycopene β-cyclases unmask operation of phytoene synthase 2 in ripening tomato fruits

In ripening tomato fruits, the leaf-specific carotenoids biosynthesis mediated by phytoene synthase 2 (PSY2) is replaced by a fruit-specific pathway by the expression of two chromoplast-specific genes: phytoene synthase 1 (PSY1) and lycopene-{beta}-cyclase (CYCB). Though both PSY1 and PSY2 genes express in tomato fruits, the functional role of PSY2 is not known. To decipher whether PSY2-mediated carotenogenesis operates in ripening fruits, we blocked the in vivo activity of lycopene-{beta}-cyclases in fruits of several carotenoids and ripening mutants by CPTA (2-(4-Chlorophenylthio)triethylamine hydrochloride), an inhibitor of lycopene-{beta}-cyclases. The CPTA-treatment induced accumulation of lycopene in leaves, immature-green and ripening fruits. Even, in psy1 mutants V7 and r that are deficient in fruit-specific carotenoid biosynthesis, CPTA triggered lycopene accumulation but lowered the abscisic acid level. Differing from fruit-specific carotenogenesis, CPTA-treated V7 and r mutant fruits accumulated lycopene but not phytoene and phytofluene. The lack of phytoene and phytofluene accumulation was reminiscent of PSY2-mediated leaf-like carotenogenesis, where phytoene and phytofluene accumulation is never seen. The lycopene accumulation was associated with the partial transformation of chloroplasts to chromoplasts bearing thread-like crystalline structures. Our study uncovers the operation of a parallel carotenogenesis pathway mediated by PSY2 that provides precursors for abscisic acid biosynthesis in ripening tomato fruits. Significance statementIt is believed that in ripening tomato fruits phytoene synthase 2 that drives carotenoid biosynthesis in leaves is redundant. Contrary to this, we show that in phytoene synthase 1 mutant fruit that is bereft of lycopene, the chemical inhibition of lycopene {beta}-cyclases triggers lycopene accumulation. Our results uncover that phytoene synthase 2 remains functional in ripening fruits and provides precursors for abscisic acid formation in fruits.

plant biology↗

bioRxiv2021

The Evolutionary History of Small RNAs in the Solanaceae

The Solanaceae or "nightshade" family is an economically important group that harbors a remarkable amount of diversity. To gain a better understanding of how the unique biology of the Solanaceae relates to the familys small RNA genomic landscape, we downloaded over 255 publicly available small RNA datasets that comprise over 2.6 billion reads of sequence data. We applied a suite of computational tools to predict and annotate two major small RNA classes: (1) microRNAs (miRNAs), typically 20-22 nt RNAs generated from a hairpin precursor and functioning in gene silencing, and (2) short interfering RNAs (siRNAs), including 24-nt heterochromatic siRNAs (hc-siRNAs) typically functioning to repress repetitive regions of the genome via RNA-directed DNA methylation, as well as secondary phased siRNAs (phasiRNAs) and trans-acting siRNAs (tasiRNAs) generated via miRNA-directed cleavage of a Pol II-derived RNA precursor. Our analyses described thousands of small RNA loci, including poorly-understood clusters of 22-nt siRNAs that accumulate during viral infection. The birth, death, expansion, and contraction of these small RNA loci are dynamic evolutionary processes that characterize the Solanaceae family. These analyses indicate that individuals within the same genus share similar small RNA landscapes, whereas comparisons between distinct genera within the Solanaceae reveal relatively few commonalities. ONE-SENTENCE SUMMARYWe use over 255 publicly-available small RNA datasets to characterize the small RNA landscape for the Solanaceae family.

plant biology↗

bioRxiv2021

miRador: a fast and precise tool for the prediction of plant miRNAs

Plant microRNAs (miRNAs) are short, non-coding RNA molecules that restrict gene expression via post-transcriptional regulation and function in several essential pathways including development, growth, and stress responses. Accurately identifying miRNAs in populations of small RNA (sRNA) sequencing libraries is a computationally intensive process which has resulted in the misidentification of inaccurately annotated miRNA sequences. In recent years, criteria for miRNA annotation have been refined to reduce these misannotations. Here, we describe miRador, a novel miRNA identification tool that utilizes the most up-to-date, community-established criteria for accurate identification of miRNAs in plants. We combine target prediction and Parallel Analysis of RNA Ends (PARE) data to assess the precision of the miRNAs identified by miRador. We compare miRador to other commonly used miRNA prediction tools and we find that miRador is at least as precise as other prediction tools while being significantly faster than other tools.

plant biology↗

bioRxiv2021

A Fragment-based approach to assess the ligandability of ArgB, ArgC, ArgD and ArgF in the L-arginine biosynthetic pathway of Mycobacterium tuberculosis.

The L-arginine biosynthesis pathway consists of eight enzymes that catalyse the conversion of L-glutamate to L-arginine, appears to be attractive target for anti-Tuberculosis (TB) drug discovery. Starvation of M. tuberculosis deleted for either argB or argF genes led to rapid sterilization of these strains in mice while Chemical inhibition of ArgJ with Pranlukast was also found to clear chronic M. tuberculosis infection in animal models. In this work, the ligandability of four enzymes of the pathway ArgB, ArgC, ArgD and ArgF is explored using a fragment-based approach. We reveal several hits for these enzymes validated with biochemical and biophysical assays, and X-ray crystallographic data, which in the case of ArgB were further confirmed to have on-target activity against M. tuberculosis. These results demonstrate the potential of more enzymes in this pathway to be targeted with dedicated drug discovery programmes.

biochemistry↗

bioRxiv2020

Chia (Salvia hispanica) gene expression atlas elucidates dynamic spatio-temporal changes associated with plant growth and development

Chia (Salvia hispanica L.), now a popular superfood, is one of the richest sources of dietary nutrients such as protein, fiber, and polyunsaturated fatty acids. At present, the genomic and genetic information available in the public domain for this crop is scanty, which hinders understanding its growth and developmental processes and impedes genetic improvement through genomics-assisted methods. We report RNA-seq based comprehensive transcriptome atlas of Chia across 13 different tissue types covering vegetative and reproductive growth stages. We generated [~]394 million raw reads from transcriptome sequencing, of which [~]355 million high-quality reads were used to generate de novo reference transcriptome assembly and the tissue-specific transcript assemblies. After quality assessment of merged assemblies and using redundancy reduction methods, 82,663 reference transcripts were identified. Of these, 53,200 transcripts show differential expression in at least one sample and provide information on spatio-temporal modulation of gene expression in Chia. We identified genes involved in the biosynthesis of omega-3 and omega-6 polyunsaturated fatty acids, and various terpenoid compounds. The study also led to the identification of 633 differentially expressed transcription factors from 53 gene families. The coexpression analysis suggested that members of the B3, bZIP, ERF, WOX, AP2, MYB, C3H, EIL, LBD, DBB, Nin-like, and HSF transcription factor gene families play key roles in the regulation of target gene expression across various developmental stages. This study also identified 2,411 simple sequence repeat (SSRs) as potential genetic markers residing in the transcribed regions. The transcriptome atlas provides essential genomic resources for basic research, applications in plant breeding, and annotation of the Chia genome.

genomics↗

bioRxiv2020

Cocculus hirsutus -derived phytopharmaceutical drug has potent anti-dengue activity

BackgroundDengue is a serious public health concern worldwide, with ~3 billion people at risk of contracting dengue virus (DENV) infections. Currently, no effective vaccine or drug is available for the prevention or treatment of dengue, which leaves only anti-mosquito strategies to combat this disease. The present study was initiated to determine the in-vitro and in vivo protective effects of a plant-derived phytopharmaceutical drug for the treatment of dengue. Methodology/Principal FindingsIn our previous report, we had identified methanolic extract of the aerial parts of Cissampelos pareira to exhibit in vitro and in vivo anti-dengue activity against all the four DENV serotypes. In the current study, we have identified another Indian medicinal plant, Cocculus hirsutus, which has a more potent anti-dengue activity than C. pareira. The activity has been evaluated through flow-cytometry-based virus inhibition assay. Interestingly, the stem of C. hirsutus was found to be more potent than the aerial part irrespective of the extraction solvent used viz., denatured spirit, hydro-alcohol (50:50) and water. Hence, the aqueous extract of stem of C. hirsutus (AQCH) was further advanced for investigations because of greater regulatory acceptance. The AQCH exhibited dose-dependent inhibition of release of DENV and its secretory antigen, NS1. Five chemical markers viz. Sinococuline, 20-Hydroxyecdysone, Makisterone-A, Magnoflorine and Coniferyl alcohol were identified as the major chemical ingredients of the AQCH extract. These chemicals were subsequently used for extract standardisation. Importantly, AQCH completely protected AG129 mice at 25 mg/kg/dose body weight when fed 4 times a day post-infection with a lethal dose of DENV-2 S221 strain. Because of its potential as an effective phytopharmaceutical drug against dengue, AQCH, has been formulated into tablets for further pre-clinical and clinical developments. Conclusions/SignificanceWe provide evidence of the pan anti-dengue potential of C. hirsutus-based phytopharmaceutical drug as determined through in vitro and in vivo experiments. We have also characterized five chemical entities in the drug substance, which provides means for standardization of drug substance and drug product. Based on these findings, a program to develop a safe and effective C. hirsutus-derived phytopharmaceutical drug for the treatment of dengue has been initiated. Author summaryThere is an urgent need to develop a safe and effective drug against dengue, which is a rapidly expanding mosquito-borne viral disease. Half of the worlds population has been estimated to be at risk of contracting this disease and the situation remains grim due to lack of an approved drug. We aimed to develop an ethnopharmacological drug against dengue by exploring traditional Indian medicinal science, Ayurveda. This led us to identify a creeper, Cocculus hirsutus, as a more potent anti-dengue plant than Cissampelos pareira, reported in our earlier published study. The stem part of C. hirsutus was found to be more efficacious in inhibiting the propagation of dengue viruses (DENVs) in cell culture than its aerial part. Hence, we chose to advance aqueous extract of stem of C. hirsutus (AQCH) for further studies. Importantly, AQCH also protected immune-compromised mice from lethal DENV infection, which is suggestive of its potential clinical relevance. We have identified five chemical marker compounds in AQCH to gauge the quality and consistency of extract preparation and its formulation into stable tablets. Based on the findings of this study, we have undertaken the development of a safe and effective C. hirsutus-derived phytopharmaceutical drug for the treatment of dengue.

microbiology↗

bioRxiv2020

RNA-binding protein FXR1 drives cMYC translation by mRNA circularization through eIF4F recruitment in ovarian cancer

BackgroundThe RNA-binding protein FXR1 (fragile X-related protein 1) has been implicated as an important regulator of post-transcriptional changes of mRNAs. However, its role in mRNA circularization and recruitment of eukaryotic translation initiation factors for protein translation remains obscure. Here, we aimed to investigate the molecular mechanisms and potential clinical applications of FXR1 in ovarian cancer growth and progression. MethodsFXR1 copy number variation, mRNA expression, protein levels, and their association with prognosis were determined in clinical datasets. An orthotopic ovarian cancer model and bioluminescence imaging were used for preclinical evaluation of FXR1 in vivo. Reverse phase protein arrays (RPPA) and qPCR arrays were performed to identify FXR1s key targets and downstream effects. SUnSET and polysome profiling were used to determine the translational effects of FXR1. Immunoprecipitation and immunofluorescence were performed to identify the interaction between FXR1 and cMYC mRNA and eIF4F complex. RNA-immunoprecipitation (RIP), RNA electrophoretic mobility shift assays (REMSA), proximity ligation assays (PLA), and biochemical assays were used to identify the specific site on cMYC mRNA to which FXR1 binds to promote mRNA circularization and translation. ResultsWe found that amplification and copy-gain of FXR1 increased the expression of FXR1 mRNA and FXR1 protein in ovarian cancer patients, and these events associated with poor prognosis. We demonstrated that FXR1 binds to AU-rich elements (ARE) within the 3 untranslated region (3UTR) of cMYC. As a consequence, FXR1 binding to cMYC 3UTR leads to the circularization of mRNA and facilitated the recruitment of eukaryotic translation initiation factors (eIFs) to translation start site for improving protein synthesis. ConclusionWe found that FXR1 upregulates a known oncogene, cMYC, by binding to AU-rich elements within the 3UTR, leading to the recruitment of the eIF4F complex for cMYC translation. Our findings uncover a novel mechanism of action of FXR1 in tumorigenesis and provides opportunities to use FXR1 and its downstream effectors as biomarkers or therapeutic targets in ovarian and other cancers.

cancer biology↗

bioRxiv2020

Repurposing the antidepressant sertraline as SHMT inhibitor to suppress serine/glycine synthesis addicted breast tumor growth.

Metabolic rewiring is a hallmark of cancer that supports tumor growth, survival and chemotherapy resistance. While normal cells often rely on extracellular serine and glycine supply, a significant subset of cancers becomes addicted to intracellular serine/glycine synthesis, offering an attractive drug target. Previously developed inhibitors of serine/glycine synthesis enzymes did not reach clinical trials due to unfavorable pharmacokinetic profiles, implying that further efforts to identify clinically applicable drugs targeting this pathway are required. In this study, we aimed to develop therapies that can rapidly enter the clinical practice by focusing on drug repurposing, as their safety and cost-effectiveness have been optimized before. Using a yeast model system, we repurposed two compounds, sertraline and thimerosal, for their selective toxicity against serine/glycine synthesis addicted breast cancer and T-cell acute lymphoblastic leukemia cell lines. Isotope tracer metabolomics, computational docking studies and an enzymatic activity assay revealed that sertraline and thimerosal inhibit serine/glycine synthesis enzymes serine hydroxymethyltransferase and phosphoglycerate dehydrogenase, respectively. In addition, we demonstrated that sertralines anti-proliferative activity was further aggravated by mitochondrial inhibitors, such as the antimalarial artemether, by causing G1-S cell cycle arrest. Most notably, this combination also resulted in serine-selective antitumor activity in breast cancer mouse xenografts. Collectively, this study provides molecular insights into the repurposed mode-of-action of the antidepressant sertraline and allows to delineate a hitherto unidentified group of cancers being particularly sensitive to treatment with sertraline. Furthermore, we highlight the simultaneous inhibition of serine/glycine synthesis and mitochondrial metabolism as a novel treatment strategy for serine/glycine synthesis addicted cancers.

cancer biology↗

bioRxiv2020

Integrated genomic view of SARS-CoV-2 in India

India first detected SARS-CoV-2, causal agent of COVID-19 in late January-2020, imported from Wuhan, China. March-2020 onwards; importation of cases from rest of the countries followed by seeding of local transmission triggered further outbreaks in India. We used ARTIC protocol based tiling amplicon sequencing of SARS-CoV-2 (n=104) from different states of India using a combination of MinION and MinIT from Oxford Nanopore Technology to understand introduction and local transmission. The analyses revealed multiple introductions of SARS-CoV-2 from Europe and Asia following local transmission. The most prevalent genomes with patterns of variance (confined in a cluster) remain unclassified, here, proposed as A4-clade based on its divergence within A-cluster. The viral haplotypes may link their persistence to geo-climatic conditions and host response. Despite the effectiveness of non-therapeutic interventions in India, multipronged strategies including molecular surveillance based on real-time viral genomic data is of paramount importance for a timely management of the pandemic.

microbiology↗

bioRxiv2020

Mutation landscape of SARS-CoV-2 reveals three mutually exclusive clusters of leading and trailing single nucleotide substitutions

The COVID-19 pandemic has spread across the globe at an alarming rate. However, unlike any of the previous global outbreaks the availability of a large number of SARS-CoV-2 sequences provides us with a unique opportunity to understand viral evolution in real time. We analysed 1448 full-length (>29000 nt) sequences available and identified 40 single-nucleotide substitutions occurring in >1% of the genomes. Majority of the substitutions were C to T or G to A. We identify C/Gs with an upstream TTT trinucleotide motif as hotspots for mutations in the SARS-CoV-2 genome. Interestingly, three of the 40 substitutions occur within highly conserved secondary structures in the 5 and 3 regions of the genomic RNA that are critical for the virus life cycle. Furthermore, clustering analysis revealed unique geographical distribution of SARS-CoV-2 variants defined by their mutation profile. Of note, we observed several co-occurring mutations that almost never occur individually. We define five mutually exclusive lineages (A1, B1, C1, D1 and E1) of SARS-CoV-2 which account for about three quarters of the genomes analysed. We identify lineage-defining leading mutations in the SARS-CoV-2 genome which precede the occurrence of sub-lineage defining trailing mutations. The identification of mutually exclusive lineage-defining mutations with geographically restricted patterns of distribution has potential implications for diagnosis, pathogenesis and vaccine design. Our work provides novel insights on the temporal evolution of SARS-CoV-2. ImportanceThe SARS-CoV-2 / COVID-19 pandemic has spread far and wide with high infectivity. However, the severeness of the infection as well as the mortality rates differ greatly across different geographic areas. Here we report high frequency mutations in the SARS-CoV-2 genomes which show the presence of linage-defining, leading and trailing mutations. Moreover, we propose for the first time, five mutually exclusive clusters of SARS-CoV-2 which account for 75% of the genomes analysed. This will have implications in diagnosis, pathogenesis and vaccine design

genomics↗

bioRxiv2020

Reanalysis of Genome Sequences of tomato accessions and its wild relatives: Development of tomato genomic variation (TGV) database integrating SNPs and INDELs polymorphisms

MotivationFacilitated by technological advances and expeditious decrease in the sequencing costs, whole-genome sequencing (WGS) is increasingly implemented to uncover variations in cultivars/accessions of many crop plants. In tomato (Solanum lycopersicum), the availability of the genome sequence, followed by the resequencing of tomato cultivars and its wild relatives, has provided a prodigious resource for the improvement of traits. A high-quality genome resequencing of 84 tomato accessions and wild relatives generated a dataset that can be used as a resource to identify agronomically important alleles across the genome. Converting this dataset into a searchable database, including information about the influence of SNPs on protein function, provides valuable information about the genetic variations. The database will assist in searching for functional variants of a gene for introgression into tomato cultivars. ResultsA recent release of better-quality tomato genome reference assembly SL3.0, and new annotation ITAG3.2 of SL3.0, dropped 3,857 genes, added 4,900 novel genes, and updated 20,766 genes. Using the above version, we remapped the data from the tomato lines resequenced under the "100 Tomato Genome ReSequencing Project" on new tomato genome assembly SL3.0 and made an online searchable Tomato Genomic Variations (TGV) database. The TGV contains information about SNPs and InDels and expands it by functional annotation of variants with new ITAG3.2 using SIFT4G software. This database with search function assists in inferring the influence of SNPs on the function of a target gene. This database can be used for selecting SNPs, which can be potentially deployed for improving tomato traits. Availability and ImplementationTGV is freely available at http://psd.uohyd.ac.in/tgv. Contactrameshwar.sharma@gmail.com

bioinformatics↗