Biology subjects

Sharma, V.

Publications and source records attributed to Sharma, V..

At least 109 records · Page 6Linked to original sources

bioRxiv2021

A novel approach to the detection of unusual mitochondrial protein change suggests hypometabolism of ancestral simians

The mitochondrial genome encodes core subunits involved in the process of oxidative phosphorylation. The sequence and structure of these mitochondria-encoded polypeptides are expected to be shaped by bioenergetic requirements linked to diet and environment. Here, we have developed a robust and effective method for highlighting phylogenetic tree edges with unexpectedly rapid, and likely consequential, substitutions within mitochondrial proteins. Further, our approach allows detection of discrete protein substitutions likely to alter enzyme performance. A survey of mammalian taxonomic groups performed using our method indicates that widely conserved residues in mitochondria-encoded proteins are more likely to rapidly mutate toward variants providing lower OXPHOS activity within specific clades. Intriguingly, our data suggest reduced cellular metabolism of ancestral anthropoids, and our findings have potential implications regarding primate encephalization. Significance StatementMitochondria harbor DNA (mtDNA) that encodes proteins important for converting food into energy. The environment and lifestyle of an organism shapes, and is shaped by, the sequences of these mitochondrial genomes. We developed a new approach for the detection of rapid functional change to proteins, and we applied our method to the mitochondria-encoded polypeptides of mammals. We found that primates displayed a general signature of relative hypometabolism that is shared with other mammals characterized by a low metabolic rate. Indications of reduced cellular metabolism extend even to the earliest anthropoids. Our findings have potential implications regarding the evolution of an enlarged primate brain.

evolutionary biology↗

bioRxiv2021

Stress induced Differential Expression of THAP9 & THAP9-AS1 in the S-phase of cell cycle.

Transposable elements function as one of the major effectors in response to biological or environmental stress. Under normal conditions, host organisms deploy epigenetic and post-transcriptional machinery (histone modifications, chromatin remodelers, long non-coding RNAs (lncRNAs)) at the TE sites to contain their mobility. But many a times, the chromatin architecture undergoes TE induced changes under the effect of stress that in turn might lead to unprecedented gene expression. LncRNAs are emerging as a crucial tool in the regulation of TEs. TEs possess remarkable abilities to respond in the face of stress, ranging from undetected mutations to changing the regulatory landscape of the host. Although the relationship between stress response and TE activation/deactivation is well acknowledged but our understanding of the mechanism of regulation remains poor. This study focuses on the gene expression of THAP9, a domesticated transposon and lncRNA THAP9-AS1 (THAP9-anti sense1), which form a sense and anti-sense gene pair with a promoter overlap of approximately 350bp. The two genes exhibit different patterns of gene expression under different types of stresses in the S-phase of the cell cycle. THAP9-AS1 is always upregulated under stress whereas THAP9 exhibits both downregulation and upregulation in different stresses. Both THAP9 and THAP9-AS1 exhibit a periodic gene expression throughout the S-phase which is a characteristic of cell cycle regulated genes.

molecular biology↗

bioRxiv2021

Genome sequencing of the bacteriophage CL31 and interaction with the host strain Corynebacterium glutamicum ATCC 13032

In this study, we provide a comprehensive analysis of the genomic features of the phage CL31 and the infection dynamics with the biotechnologically relevant host strain Corynebacterium glutamicum ATCC 13032. Genome sequencing and annotation of CL31 revealed a 45-kbp genome composed of 72 open reading frames, mimicking the GC content of its host strain (54.4 %). An ANI-based distance matrix showed the highest similarity of CL31 to the temperate corynephage {Phi}16. While the C. glutamicum ATCC 13032 wild type strain showed only mild propagation of CL31, a strain lacking the cglIR-cglIIR-cglIM restriction-modification system was efficiently infected by this phage. Interestingly, the prophage-free strain C. glutamicum MB001 featured an even accelerated amplification of CL31 compared to the {Delta}resmod strain suggesting a role of cryptic prophage elements in phage defense. Proteome analysis of purified phage particles and transcriptome analysis provide important insights into structural components of the phage and the response of C. glutamicum to CL31 infection. Isolation and sequencing of CL31-resistant strains revealed SNPs in genes involved in mycolic acid biosynthesis suggesting a role of this cell envelope component in phage adsorption. Altogether, these results provide an important basis for further investigation of phage-host interactions in this important biotechnological model organism.

microbiology↗

bioRxiv2021

Primary tumor associated macrophages activate programs of invasion and dormancy in disseminating tumor cells.

Metastases are initiated by disseminated tumor cells (DTCs) that depart from the primary tumor and colonize target organs. Growing evidence suggests that the microenvironment of the primary tumor lesion primes DTCs to display dormant or proliferative fates in target organs. However, the manner in which events taking place in the primary tumor influence DTC fate, sometimes long after dissemination, remains poorly understood. With the advent of a novel intravital imaging technique called the Window for High-Resolution Intravital Imaging of the Lung (WHRIL), we have, for the first time, been able to study the live lung longitudinally and follow the fate of individual DTCs that spontaneously disseminate from orthotopic breast tumors. We find, across several models, a high rate of success for tumor cells to complete the initial steps of the metastatic cascade in the secondary site, including retention of DTCs in the lung vasculature, speed of extravasation, and survival after extravasation. Importantly, initiation of metastatic growth was controlled primarily by a rate-limiting step that occurred post-extravasation and at the stage of the conversion of single DTCs from a dormant to a proliferative state. Detailed analysis of these events revealed that, even before dissemination, a subset of macrophages within the primary tumor induces, in tumor cells that are about to disseminate, the expression of proteins that regulate a pro- dissemination (MenaINV) and pro-dormancy (NR2F1) phenotype. Surprisingly, if cancer cells are intravenously injected, the rate limiting stages of MenaINV-associated extravasation, dormancy, and other parameters, are lost or altered in a way that impacts how DTCs progress through the metastatic cascade. Our work provides novel insight into how specific primary tumor microenvironments prime a subpopulation of cells for dissemination and dormancy. We also propose that dissecting mechanisms of metastasis, or testing anti-metastatic therapies, may yield results of limited application if derived from models that do not follow spontaneous dissemination. SIGNIFICANCEThis study provides important insight into the contribution of primary tumor microenvironmental niches to cancer metastasis by identifying the manner in which these niches spawn subpopulations of DTCs that are primed for dissemination and dormancy in the secondary site. This study may provide novel targets that could be inhibited to prevent successful colonization of the secondary site and, hence, metastasis.

cancer biology↗

bioRxiv2021

Immunogenic Potential of DNA Vaccine candidate, ZyCoV-D against SARS-CoV-2 in Animal Models

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), initially originated in China in year 2019 and spread rapidly across the globe within 5 months, causing over 96 million cases of infection and over 2 million deaths. Huge efforts were undertaken to bring the COVID-19 vaccines in clinical development, so that it can be made available at the earliest, if found to be efficacious in the trials. We developed a candidate vaccine ZyCoV-D comprising of a DNA plasmid vector carrying the gene encoding the spike protein (S) of the SARS-CoV-2 virus. The S protein of the virus includes the receptor binding domain (RBD), responsible for binding to the human angiotensin converting enzyme (ACE-2) receptor. The DNA plasmid construct was transformed into E. coli cells for large scale production. The immunogenicity potential of the plasmid DNA has been evaluated in mice, guinea pig, and rabbit models by intradermal route at 25, 100 and 500g dose. Based on the animal studies proof-of-concept has been established and preclinical toxicology (PCT) studies were conducted in rat and rabbit model. Preliminary animal study demonstrates that the candidate DNA vaccine induces antibody response including neutralizing antibodies against SARS-CoV-2 and also provided Th-1 response as evidenced by elevated IFN-{gamma} levels.

immunology↗

bioRxiv2020

THAP9 transposase cleaves DNA via conserved acidic residues in an RNaseH-like domain

The catalytic domain of most cut and paste DNA transposases have the canonical RNase-H fold which is also shared by other polynucleotidyl transferases like retroviral integrases and the RAG1 subunit of V(D)J recombinase. The RNase-H fold is a mixture of beta sheets and alpha helices with three acidic residues (Asp, Asp, Glu/Asp - DDE/D) that are involved in metal-mediated cleavage and subsequent integration of DNA. Human THAP9 (hTHAP9), homologous to the well-studied Drosophila P-element transposase (DmTNP), is an active DNA transposase that, although domesticated, still retains the catalytic activity to mobilize transposons. In this study we have modelled the structure of hTHAP9 using the recently available cryo-EM structure of DmTNP as a template to identify an RNase-H like fold along with important acidic residues in its catalytic domain. Site-directed mutagenesis of the predicted catalytic residues followed by screening for DNA excision and integration activity, led to the identification of candidate Ds and Es in the RNaseH fold that appear to constitute the catalytic triad of hTHAP9. Significance statementMany DNA transposases execute DNA excision via a catalytic domain, which has a canonical RNase-H fold. Despite the similar nature of the catalytic domain, these transposases exhibit mechanistically different strategies of transposition. We identify a potential RNase-H fold in hTHAP9 with a conserved DDE motif required for cutting DNA. Additionally, we have found a residue, which when mutated, leads to an increase in hTHAP9s transposition activity. Such hyperactive transposase mutants can be exploited as tools in genome engineering and gene therapy. This study has helped widen our knowledge about the catalytic activity of a functionally uncharacterized transposon-derived gene in the human genome.

molecular biology↗

bioRxiv2020

Single Cell Transcriptomics of Human Native Kidney and Kidney Allografts and New Insights into the Mechanism of Fibrosis

We tested the hypothesis that single-cell RNA-sequencing (scRNA-seq) analysis of human kidney allograft biopsies will reveal distinct cell types and states and yield insights to decipher the complex heterogeneity of alloimmune injury. We selected 3 biopsies of kidney cortex from 3 individuals for scRNA-seq and processed them fresh using an identical protocol on the 10x Chromium platform; (i) HK: native kidney biopsy from a living donor, (ii) AK1: allograft kidney with transplant glomerulopathy, tubulointerstitial fibrosis, and worsening graft function, and (iii) AK2: allograft kidney after successful treatment of active antibody-mediated rejection. We did not study T-cell-mediated rejections. We generated 7217 high-quality single cell transcriptomes. Taking advantage of the recipient-donor sex mismatches revealed by X and Y chromosome autosomal gene expression, we determined that in AK1 with fibrosis, 42 months after transplantation, more than half of the kidney allograft fibroblasts were recipient-derived and therefore likely migratory and graft infiltrative, whereas in AK2 without fibrosis, 84 months after transplantation, most fibroblasts were donor-organ-derived. Furthermore, AK1 was enriched for tubular progenitor cells overexpressing profibrotic extracellular matrix genes. AK2, eight months after successful treatment of rejection, contained endothelial cells that expressed T-cell chemoattractant cytokines. In addition to these key findings, our analysis revealed unique cell types and states in the kidney. Altogether, single-cell transcriptomics yielded novel mechanistic insights, which could pave the way for individualizing the care of transplant recipients.

molecular biology↗

bioRxiv2020

Genome sequence and characterization of five bacteriophages infecting Streptomyces coelicolor and Streptomyces venezuelae: Alderaan, Coruscant, Dagobah, Endor1 and Endor2

Streptomyces are well-known antibiotic producers, and are also characterized by a complex morphological differentiation. Streptomyces, like all bacteria, are confronted with the constant threat of phage predation, which in turn shapes bacterial evolution. However, despite significant sequencing efforts recently, relatively few phages infecting Streptomyces have been characterized compared to other genera. Here, we present the isolation and characterization of five novel Streptomyces phages. All five phages belong to the Siphoviridae family, based on their morphology as determined by transmission electron microscopy. Genome sequencing revealed that four of them were temperate phages, while one had a lytic lifestyle. Moreover, one of the newly sequenced phages shows very little homology to already described phages, highlighting the still largely untapped viral diversity. Altogether, this study expands the number of characterized phages of Streptomyces and sheds light on phage evolution and phage-host dynamics in Streptomyces.

microbiology↗

bioRxiv2020

Coxsackievirus B3 Infection Early in Pregnancy Induces Congenital Heart DefectsThrough Suppression of Fetal Cardiomyocyte Proliferation

AimsCoxsackievirus B (CVB), the most common cause of viral myocarditis, targets cardiomyocytes through Coxsackie and Adenovirus Receptor, which is highly expressed in the fetal heart. We hypothesized CVB3, a well-recognized culprit for viral myocarditis, can precipitate congenital heart defects (CHD), when fetal infection occurs during critical window of gestation. Methods & ResultsWe infected C57Bl/6 pregnant mice with CVB3 during serial time points in early gestation (E5, E7, E9 and E11). We used different viral titers to examine possible dose- response relationship and assessed viral loads in various fetal organs as well as kinetics of virus passage into the fetus during gestation. Provided viral exposure occurred between E7-E9, we observed characteristic features of ventricular septal defect (33.6%), abnormal myocardial architecture resembling non-compaction (23.5%), and double outlet right ventricle (4.4%) among 209 viable fetuses examined. We observed a direct relationship between viral titers, severity and incidence of CHD, with apparent predominance among female fetuses. Infected dams remained healthy; we did not observe any maternal heart or placental injury suggestive of direct viral effects on developing heart as likely cause of CHD. We examined signaling pathways in CVB3-exposed hearts using RNAseq, KEGG enrichment analysis and immunohistochemistry. Signaling proteins of the Hippo, tight junction, transforming growth factor {beta}1 and extracellular matrix proteins were the most highly enriched in CVB3-infected fetuses with VSD (log fold change >1.9, P<0.02). Moreover, cardiomyocyte proliferation was 50% lower in fetuses with VSD compared with uninfected controls. ConclusionPrenatal CVB3 infection can induce CHD, provided the infection occurs during a critical window. Alterations in myocardial proliferate capacity and consequent changes in cardiac architecture and trabeculation appear to account for the majority of observed phenotypes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=179 HEIGHT=200 SRC="FIGDIR/small/104844v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@79d320org.highwire.dtl.DTLVardef@1419b8org.highwire.dtl.DTLVardef@1a49082org.highwire.dtl.DTLVardef@10b72bb_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2020

Investigating the role of N-terminal domain in phosphodiesterase4B-inhibition by molecular dynamics simulation

Phosphodiesterase 4B (PDE4B) is a potential therapeutic target for the inflammatory respiratory diseases such as congestive obstructive pulmonary disease (COPD) and asthma. The sequence identity of [~]88% with its isoform PDE4D is the key barrier in developing selective PDE4B inhibitors which may help to overcome associated side effects. Despite high sequence identity, both isoforms differ in few residues present in N-terminal (UCR2) and C-terminal (CR3) involved in catalytic site formation. Previously, we designed and tested specific PDE4B inhibitors considering N-terminal residues as a part of the catalytic cavity. In continuation, current work thoroughly presents an MD simulation-based analysis of N-terminal residues and their role in ligand binding. The various parameters viz. root mean square deviation (RMSD), radius of gyration (Rg), root mean square fluctuation (RMSF), principal component analysis (PCA), dynamical cross-correlation matrix (DCCM) analysis, secondary structure analysis, and residue interaction mapping were investigated to establish rational. Results showed that UCR2 reduced RMSF values for the metal binding pocket (31.5{+/-}11 to 13.12{+/-}6 [A]2) and the substrate-binding pocket (38.8{+/-}32 to 17.3{+/-}11 [A]2). UCR2 enhanced anti-correlated motion at the active site region that led to the improved ligand-binding affinity of PDE4B from -24.57{+/-}3 to -35.54{+/-}2 kcal/mol. Further, the atomic-level analysis indicated that T-pi and {pi}-{pi} interactions between inhibitors and residues are vital forces that regulate inhibitor association to PDE4B with high affinity. In conclusion, UCR2, the N-terminal domain, embraces the dynamics of PDE4B active site and stabilizes PDE4B inhibitor interactions. Therefore the N-terminal domain needs to be included by designing next-generation, selective PDE4B-inhibitors as potential anti-inflammatory drugs.

bioinformatics↗

bioRxiv2020

Presence of polyadenylated 3' tail in RNA of Pepper mild mottle virus allows the Oligo(dT)18 in Priming cDNA Synthesis of its genomic RNA template

The PCR amplification of majority of the ssRNA of both genomic and non-genomic mRNA is accomplished by RT-PCR. The mRNA is subjected to cDNA synthesis using reverse transcriptase and either Oligo(dT)18, or random or gene specific reverse primers based priming strategies. The choice of primer largely depends on the nature of 3 prime terminus of mRNA and length of cDNA synthesized. In general, oligo(dT)18 is the preferred choice for mRNAs having poly(A) tail at 3 prime terminus. In general, tobamoviruses lack any poly(A) tail at their 3 prime untranslated region (UTR) which forms a tRNA like structure and upstream pseudoknot domain except tow viruses viz., Hibiscus latent Fort Pierce virus (HLFPV) and Hibiscus latent Singapore virus (HLSV) which accommodate internal poly(A) sequences of 46 and 87 nucleotides long, respectively in their 3 prime UTR. However, determination of full nucleotide sequence of Pepper mild mottle virus (PMMoV) using an oligo(dT)18 primed cDNA as template indicated the libertinism of oligo(dT)18 in priming cDNA synthesis of RNA template which are known to lack poly(A) tail. at the end or internally at its 3 prime end. Moreover, coat protein (CP) gene of PMMoV and bean common mosaic virus (BCMV) (Potyvirus with a poly(A) tract at its 3 prime end) was amplified using cDNA primed with random primer as well as oligo(dT)18 was successfully amplified but with significant variation in the intensity of the amplification band in case of PMMoV but not in BCMV. This clearly indicated the presence of PMMoV mRNA with polyadenylated 3 prime tail in total RNA isolated from PMMoV infected capsicum leaves with abundance of non-polyadenylated PMMoV genomic RNA (gRNA). Hence, we hypothesize that the generation of polyadenylated RNA population during the infection cycle of PMMoV in pepper may be possible reason for libertinism of oligo(dT)18 in priming cDNA synthesis of RNA template isolated from PMMoV infected leaves followed by amplification of entire PMMoV genome through RT-PCR. This is first study indicating the presence of polyadenylated or polyadenylated rich regions in PMMoV gRNA acquired during the infection cycle in pepper.

pathology↗

bioRxiv2020

Deletion of Autophagy gene ATG1 and F-box motif encoding gene YDR131C together leads to Synthetic Growth Defects and Flocculation behaviour in Saccharomyces cerevisiae

F-box motif encoding YDR131C is functionally uncharacterized gene which forms the complex with the SCF-E3 ligase. The F-box motif containing proteins are involved in substrate recruitment for the ubiquitination and subsequent degradation through 26S proteasome. Autophagy gene, ATG1 (ULK1in human) is a well conserved serine-threonine kinase, required for vesicle formation and cytoplasm to vacuole targeting pathway. Atg1p forms the complex with Atg13p and Atg17p during autophagy. The understanding of crosstalk between ubiquitin and autophagy pathways is crucial for synthetic lethality screen and drug targeting. Here we have conducted the study for genetic interaction between uncharacterized YDR131C and ATG1 gene representing both specific and non-specific protein degradation pathways. The single and double gene knockout strains of YDR131Cand ATG1 genes were constructed in the BY4741 genetic background and analysed for growth fitness. The strains were also evaluated for cellular growth response in presence of hydroxyurea (HU), methyl methane sulfonate (MMS), and hydrogen peroxide (H2O2) stress causing agents by spot assay. The ydr131c{Delta}atg1{Delta} showed the synthetic growth defect phenotype with floc formation in rich medium which showed floc disruption in presence of EDTA. The ydr131c{Delta}atg1{Delta} cells showed the sensitivity to stress agents HU, MMS, and H2O2 when compared with ydr131c{Delta}, atg1{Delta}, and WT cells.. Based on the observations, we report that YDR131C and ATG1 functions in parallel pathways for growth fitness and cellular growth response to stress agents. Interestingly this study also revealed the crosstalk between ubiquitination and autophagy pathways. The defects in both the pathways could lead to synthetic growth defects which may have implication for the precision medicine initiatives.

genetics↗

bioRxiv2020

Deletion of Autophagy gene ATG1 and Glyoxylate cycle Regulator encoding gene UCC1 together leads to Synthetic Growth Defects and Sensitivity to Genotoxic agents in Saccharomyces cerevisiae

Atg1 of S. cerevisiae is a key component of autophagy encoded by ATG1 gene, involved in the process of degradation of cytosolic components through autophagy. UCC1, an F-box encoding gene is involved in the negative regulation of glyoxylate pathway via degradation of Cit2 enzyme by ubiquitin proteasome system. We investigated the genetic interaction between ATG1 and UCC1 using the gene deletion approach. The atg1{Delta}ucc1{Delta} cells showed the synthetic growth defects with abnormal budding and sensitivity to genotoxic and oxidative stress agents. Based on the observations, we report that ATG1 and UCC1 interact genetically to regulate the cell growth fitness and function in parallel pathway in cellular response to the genotoxic stress agents. The present investigation also revealed the cross talks among autophagy, ubiquitin proteasome system, and glyoxylate pathways.

genetics↗

bioRxiv2020

Convergent losses of TLR5 suggest altered extracellular flagellin detection in four mammalian lineages

Toll-like receptors (TLRs) play an important role for the innate immune system by detecting pathogen-associated molecular patterns. TLR5 encodes the major extracellular receptor for bacterial flagellin and frequently evolves under positive selection, consistent with coevolutionary arms races between the host and pathogens. Furthermore, TLR5 is inactivated in several vertebrates and a TLR5 stop codon polymorphism is widespread in human populations. Here, we analyzed the genomes of 120 mammals and discovered that TLR5 is convergently lost in four independent lineages, comprising guinea pigs, Yangtze river dolphin, pinnipeds, and pangolins. Validated inactivating mutations, absence of protein-coding transcript expression, and relaxed selection on the TLR5 remnants confirm these losses. PCR analysis further confirmed the loss of TLR5 in the pinniped stem lineage. Finally, we show that TLR11, encoding a second extracellular flagellin receptor, is also absent in these four lineages. Independent losses of TLR5 and TLR11 suggests that a major pathway for detecting flagellated bacteria is not essential for different mammals and predicts an impaired capacity to sense extracellular flagellin

genomics↗

bioRxiv2020

LC/MS-QToF Profiling, Anti-Diabetic and Anti-Adipogenic potential of Divya MadhuKalp: A Novel Herbo-mineral Formulation

The incidence rate of diabetes mellitus is increasing worldwide. Herbal formulations have recently gained importance as an alternative therapeutic option in controlling diabetes without causing any side effects. In the present study, we have demonstrated maintenance of glycemic homeostasis and anti-adipogenic potential of a herbo-mineral formulation Divya MadhuKalp (DMK). Initially, we evaluated the presence of bioactive compounds in DMK using LC/MS-QToF analysis. In-vitro analysis of DMK in L6 (skeletal muscle) cells showed a significant increase in cellular glucose uptake. Similarly, a human equivalent dose of DMK significantly reduced blood glucose level in normoglycemic and oral glucose tolerance rat model. DMK extract also inhibited formation of advanced glycation end product and showed anti--glucosidase activity. Further analysis of DMK in 3T3 L1 pre-adipocytes demonstrated anti-adipogenic activity through reduction in intracellular lipid accumulation and triglyceride contents along with downregulation of major adipogenic transcriptional factors (PPAR-{gamma} and C/EBP) and, adipocytes marker genes (LPL, AP2 and adiponectin). In conclusion, DMK exhibited anti-diabetic and anti-adipogenic activities by synergistic effect of its bioactive compounds and can be considered as a potent herbo-mineral formulation for treating metabolic diseases.

cell biology↗

bioRxiv2020

Cellular internalization of Godanti Bhasma (anhydrous CaSO4) induces massive cytoplasmic reversible vacuolation and survival response of mammalian cells.

Bhasmas are Ayurvedic herbo-mineral formulations that have been used since ancient times for therapeutic benefits. Godanti Bhasma (GB) is an anhydrous calcium sulfate preparation processed by heating of gypsum powder with herbal extracts. Thermo-transformation of gypsum into the anhydrous GB was confirmed by Raman and FT-IR spectroscopy. GB particle showed size range of 0.5-5 {micro}m and neutral surface charge. Exposure to mammalian cells with GB particles showed massive vacuolation in their cytoplasm. Interestingly, no vacuolation was observed with parent gypsum particle. The result indicated that the cytoplasmic vacuolation by GB was due to its unique physicochemical property obtained during the thermo-transformation of gypsum. Using lysosomal inhibitors Bafilomycin A1 (BFA1) and Chloroquine (CQ), the process of vacuole formation was suppressed indicating GB induced vacuolation require acidic environment. The GB induced vacuolation was also found to follow dose and time dependent manner. Vacuolation often accompany with the sign of cell death whereas, in our study, massive vacuolation by GB did not induce any cell death. Moreover, GB treated cells survive with massive vacuolar process, which was reversed following post-treatment with vacuole inhibitors in GB treated cells, suggesting normal vacuolar function is essential for cell survival. Treatment of cells with GB was also found to induce translocation of LC3 protein from the nucleus to vacuolar membrane, indicating LC3 associated phagocytosis (LAP) is involved in the vacuolar process. Interestingly, the LAP function was found to be reversed in the cells treated with vacuole inhibitors. Our results provide a mechanistic correlation with GB induced vacuolation and associated LAP function, essential for cell survival. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=164 SRC="FIGDIR/small/930594v3_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@169ed89org.highwire.dtl.DTLVardef@169c339org.highwire.dtl.DTLVardef@900004org.highwire.dtl.DTLVardef@1f13823_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2020

Semecarpus anacardium Linn. leaf extract exhibits activities against breast cancer and prolongs the survival of tumor bearing mice

Semecarpus anacardium Linn. is commonly used in various traditional medicines from ancient times. The nuts have been described in Ayurveda medication systems to treat numerous clinical ailments. However, isolating phytochemical constituents from nuts remains challenging and exhibits cytotoxic effects on other cells. In this study, we have standardized procedures for isolating phytochemicals from the leaf extract. The ethyl acetate leaf extract selectively affects cancer cells in a dose-dependent manner (IC50: 0.57 g/ml in MCF-7 cells) in various cancer cell lines. Next, we examined if the extract incubation could induce cell cycle arrest and suppress cell migration in the cell culture model. Consistent with this idea, the leaf extract could potentially affect the aggressive migration nature of cancer cells. Moreover, oral administration of extract significantly restored tumor growth in mice. Together, these observations suggest the anti-cancer activities of S. anacardium leaf potential for both in vitro and in vivo models. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/898940v3_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@3fefborg.highwire.dtl.DTLVardef@594510org.highwire.dtl.DTLVardef@c525d2org.highwire.dtl.DTLVardef@1362978_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2019

DNA replication protein Cdc45 directly interacts with PCNA via its PIP box in Leishmania donovani and the Cdc45 PIP box is essential for cell survival

DNA replication protein Cdc45 is an integral part of the eukaryotic replicative helicase whose other components are the Mcm2-7 core, and GINS. We identified a PIP box motif in Leishmania donovani Cdc45. This motif is typically linked to interaction with the eukaryotic clamp proliferating cell nuclear antigen (PCNA). The homotrimeric PCNA can potentially bind upto three different proteins simultaneously via a loop region present in each monomer. Multiple binding partners have been identified from among the replication machinery in other eukaryotes, and the concerted /sequential binding of these partners are central to the fidelity of the replication process. Though conserved in Cdc45 across Leishmania species and Trypanosoma cruzi, the PIP box is absent in Trypanosoma brucei Cdc45. Here we investigate the possibility of Cdc45-PCNA interaction and the role of such an interaction in the in vivo context. Having confirmed the importance of Cdc45 in Leishmania DNA replication we establish that Cdc45 and PCNA interact stably in whole cell extracts, interacting with each other directly in vitro also. The interaction is mediated via the Cdc45 PIP box. This PIP box is essential for Leishmania survival. The importance of the Cdc45 PIP box is also examined in Schizosaccharomyces pombe, and it is found to be essential for cell survival in this organism also. Our results implicate a role for the Leishmania Cdc45 PIP box in recruiting or stabilizing PCNA on chromatin. The Cdc45-PCNA interaction might help tether PCNA and associated replicative DNA polymerase to the DNA template, thus facilitating replication fork elongation. Though multiple replication proteins have been identified to associate with PCNA in other eukaryotes, this is the first report demonstrating a direct interaction between Cdc45 and PCNA, and while our analysis suggests the interaction may not occur in human cells, it indicates that it is not confined to trypanosomatids.\n\nAuthor SummaryLeishmaniases are manifested in three forms: cutaneous, sub-cutaneous and visceral. The prevalent form in the Indian subcontinent is visceral Leishmaniasis (VL), which is fatal if not treated on time. While there are drugs for treatment, the hunt for additional drugs continues due to emerging drug resistance patterns. The parasite is transmitted by the bite of the sandfly, whereupon it establishes itself within cells of the host immune system (macrophages) and reproduces by binary fission. The replication of its genome is essential for parasite survival. Eukaryotic DNA replication is generally conserved across species. This study targets Cdc45, a protein that helps unwind the DNA double helix to enable copying of the two strands into two daughter strands. The new chains of DNA are synthesized by DNA polymerases, and a trimeric protein, proliferating cell nuclear antigen (PCNA), helps clamp the polymerases onto the template. In this study we find Cdc45 to interact with PCNA, and have identified the motif in Cdc45 via which it does so. Our results suggest this interaction is seen in some other eukaryotes as well. Based on the results of our experiments we propose that Cdc45 may help moor PCNA-polymerase complexes to template DNA.

molecular biology↗