Biology subjects

Mishra, A.

Publications and source records attributed to Mishra, A..

At least 145 records · Page 8Linked to original sources

bioRxiv2020

Uncanny similarity of unique inserts in the 2019-nCoV spike protein to HIV-1 gp120 and Gag

We are currently witnessing a major epidemic caused by the 2019 novel coronavirus (2019-nCoV). The evolution of 2019-nCoV remains elusive. We found 4 insertions in the spike glycoprotein (S) which are unique to the 2019-nCoV and are not present in other coronaviruses. Importantly, amino acid residues in all the 4 inserts have identity or similarity to those in the HIV-1 gp120 or HIV-1 Gag. Interestingly, despite the inserts being discontinuous on the primary amino acid sequence, 3D-modelling of the 2019-nCoV suggests that they converge to constitute the receptor binding site. The finding of 4 unique inserts in the 2019-nCoV, all of which have identity /similarity to amino acid residues in key structural proteins of HIV-1 is unlikely to be fortuitous in nature. This work provides yet unknown insights on 2019-nCoV and sheds light on the evolution and pathogenicity of this virus with important implications for diagnosis of this virus.

evolutionary biology↗

bioRxiv2019

Amyloid-like aggregates cause lysosomal defects in neurons via gain-of-function toxicity

The autophagy-lysosomal pathway is impaired in many neurodegenerative diseases characterized by protein aggregation, but the link between aggregation and lysosomal dysfunction remains poorly understood. Here, we combine cryo-electron tomography, proteomics and cell biology studies to investigate the effects of protein aggregates in primary neurons. We use artificial amyloid-like {beta}-sheet proteins ({beta} proteins) to focus on the gain-of-function aspect of aggregation. These proteins form fibrillar aggregates and cause neurotoxicity. We show that late stages of autophagy are impaired by the aggregates, resulting in lysosomal alterations reminiscent of lysosomal storage disorders. Mechanistically, {beta} proteins interact with and sequester AP-31, a subunit of the AP-3 adaptor complex involved in protein trafficking to lysosomal organelles. This leads to destabilization of the AP-3 complex, missorting of AP-3 cargo, and lysosomal defects. Restoring AP-31 expression ameliorates neurotoxicity caused by {beta} proteins. Altogether, our results highlight the link between protein aggregation and neurotoxicity, pointing to lysosomes as particularly vulnerable organelles.

cell biology↗

bioRxiv2019

Proteomics Analysis of Differential Expression of Lung Proteins in Response to Highly Pathogenic Avian Influenza Virus Infection in Chicken

Elucidation of molecular pathogenesis underlying virus-host interaction is important for the development of new diagnostic and therapeutic strategies against highly pathogenic avian influenza (HPAI) infection in chicken. However, chicken HPAI viral pathogenesis is not completely understood. To elucidate the intracellular signaling pathways and critical host proteins associated with influenza pathogenesis, we characterized the lung proteome of chicken infected with HPAI H5N1 virus (A/duck/India/02CA10/2011/Agartala). The chicken mass spectra data sets comprised1, 47, 451 MS scans and 19, 917 MS/MS scans. At local FDR 5% level, we identified total 3313 chicken proteins with presence of at least one unique peptide. At 12 hrs, 247 proteins are downregulated while 1754 proteins are downregulated at 48 hrs indicating that the host has succumbed to infection. There is expression of proteins of the predominant signaling pathways, such as TLR, RLR, NLR and JAK-STAT signaling. Activation of these pathways is associated with cytokine storm effect and thus may be the cause of severity of HPAI H5N1 infection in chicken. Further we identified proteins like MyD88, IKBKB, IRAK4, RELA, and MAVS involved in the critical signaling pathways and some other novel proteins(HNF4A, ELAVL1, FN1, COPS5, CUL1, BRCA1 and FYN) as main hub proteins that might play important roles in influenza pathogenesis in chicken. Taken together, we characterized the signaling pathways and the proteomic determinants responsible for disease pathogenesis in chicken infected with HPAI H5N1 virus.

immunology↗

bioRxiv2019

Three complete genome sequences of Penguin megrivirus from ornithogenic soil, Adelie penguin and Weddell seal of Antarctica

We report the complete genome sequences of Penguin megriviruses from three different sources from Antarctica namely feces of Adelie penguin, feces of Weddell seal and ornithogenic soil. Phylogenetic analysis indicates the prevalence of very similar viruses in different sources of Antarctic environment. These genome sequences aid to understand the evolution of megriviruses in Antarctic ecology and reveal their place in global megrivirus phylogeny.

microbiology↗

bioRxiv2019

CXCR4 intracellular protein regulates drug resistance and tumorigenic potential via modulating the expression of Death Receptor 5

CXCR4 overexpression in solid tumors has been strongly associated with poor prognosis and adverse clinical outcome. However, CXCR4 signaling inhibitor drug Plerixafor has shown limited clinical success in cancer treatment. Therefore, CXCR4 signaling may not be the exclusive contributor to its pro-tumorigenic functions. In our continuous effort to understand the chemokine receptor signaling inhibition as cancer therapy, here we unexpectedly discovered that instead of its signaling, intracellular CXCR4 protein augments therapy resistance and pro-tumorigenic functions. Unbiased proteome profiler apoptosis array followed by immunoblot, FACS, real-time PCR and ChIP analyses demonstrate that CXCR4 promotes DR5 downregulation via modulating differential recruitment of transcription factors p53 and YY1 to its promoter. Surprisingly, inhibiting CXCR4 mediated signals failed to block the above phenotype. Irrespective of CXCR4 surface expression, its loss compromised colon tumor growth in vivo. Finally, TCGA data mining and human patient sample data analysis showed CXCR4 and DR5 are inversely regulated in human cancers. Together, we showed evidence for the first time that targeting CXCR4 intracellular protein may be critical to dampen the pro-tumorigenic functions of CXCR4.

cancer biology↗

bioRxiv2019

Effects of low temperature and cold-acclimation on photoinhibition and singlet oxygen production in four natural accessions of Arabidopsis

To understand the effects of low temperature and cold-acclimation on reactive oxygen species and photoinhibition of photosystem II (PSII), light-induced inactivation of PSII was measured at 22 and 4 {degrees}C from four Arabidopsis thaliana accessions (Rschew, Tenela, Columbia-0 and Coimbra) grown under optimal conditions. Photoinhibition was also measured at 4 {degrees}C from plants cold-acclimated at 4 {degrees}C for two weeks. Measurements were done in the absence and presence of lincomycin that blocks PSII repair, and PSII activity was assayed with the ratio of variable to maximum chlorophyll a fluorescence (FV/FM) and with light-saturated rate of oxygen evolution using a quinone acceptor. Of the non-acclimated accessions, Rschew was the most tolerant to photoinhibition and Coimbra the least; the rate constants of photoinhibition of the most sensitive accession were 1.3-1.9 times as high as those of the tolerant ones. The damaging reaction of photoinhibition in non-acclimated plants was slower or equal at 4 {degrees}C than at 22 {degrees}C. The rate constants of photoinhibition of cold-acclimated plants, at 4 {degrees}C, were 0.55 to 1.25 times as high as those of non-acclimated plants; the protective effect of cold-acclimation on photoinhibition was consistent in Columbia-0 and Coimbra whereas Rschew and Tenela were either slightly more tolerant or susceptible, depending on the method used to assay photoinhibition. Production of singlet oxygen, measured from thylakoid membranes isolated from non-acclimated and cold-acclimated plants, did not decrease due to cold-acclimation, nor did singlet oxygen production correlate with the rate of photoinhibition or with flavonol contents of the leaves.

plant biology↗

bioRxiv2019

Mate-finding dispersal reduces local mate limitation and sex bias in dispersal

O_LISex-biased dispersal (SBD) often skews the local sex ratio in a population. This can result in a shortage of mates for individuals of the less-dispersive sex. Such mate limitation can lead to Allee effects in populations that are small or undergoing range expansion, consequently affecting their survival, growth, stability and invasion speed. C_LIO_LITheory predicts that mate shortage can lead to either an increase or a decrease in the dispersal of the less-dispersive sex. However, neither of these predictions have been empirically validated. C_LIO_LITo investigate how SBD-induced mate limitation affects dispersal of the less-dispersive sex, we used Drosophila melanogaster populations with varying dispersal propensities. To rule out any mate-independent density effects, we examined the behavioral plasticity of dispersal in presence of mates as well as same-sex individuals with differential dispersal capabilities. C_LIO_LIIn the presence of high-dispersive mates, the dispersal of both male and female individuals was significantly increased. However, the magnitude of this increase was much larger in males than in females, indicating that the former show greater mate-finding dispersal. Moreover, the dispersal of either sex did not change when dispersing alongside high- or low-dispersive individuals of the same sex. This suggested that the observed plasticity in dispersal was indeed due to mate-finding dispersal, and not mate-independent density effects. C_LIO_LIStrong mate-finding dispersal can diminish the magnitude of sex bias in dispersal. This can modulate the evolutionary processes that shape range expansions and invasions, depending on the population size. In small populations, mate-finding dispersal can ameliorate Allee effects. However, in large populations, it can dilute the effects of spatial sorting. C_LI

ecology↗

bioRxiv2019

Francisella novicida Cas9 interrogates genomic DNA with very high specificity and can be used for mammalian genome editing

Genome editing using the CRISPR Cas9 system has been used to manipulate eukaryotic DNA and make precise heritable changes. Although the widely used Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants have been efficiently harnessed for numerous gene-editing applications across different platforms, concerns remain, regarding their putative off targeting at multiple loci across the genome. Here we report that Francisella novicida Cas9 (FnCas9) shows a very high specificity of binding to its intended targets and negligible binding to off-target loci. The specificity is determined by its minimal binding affinity with DNA when mismatches to the target sgRNA are present in the sgRNA:DNA heteroduplex. FnCas9 produces staggered cleavage, higher HDR rates and very low non-specific genome editing compared to SpCas9. We demonstrate FnCas9 mediated correction of the sickle cell mutation in patient derived iPSCs and propose that it can be used for precise therapeutic genome editing for a wide variety of genetic disorders.\n\nSIGNIFICANCE STATEMENTTherapeutic genome editing has been significantly accentuated by the advent of CRISPR based gene correction. However, genomic off-targeting has been a major setback for clinical translation. Although high fidelity versions of Cas9 have been rationally designed, they recognize and bind to off-targets. In this study, we characterize a naturally occurring Cas9 from Francisella novicida (FnCas9) that shows negligible binding affinity to off targets differing by one or more mismatches, rendering it highly specific in target recognition and editing. We show that FnCas9 can direct both HDR and NHEJ mediated DNA repair, generates higher rate of HDR and negligible off-target editing. Finally we show its potential in therapeutic genome editing by correcting the sickle cell anemia mutation in patient derived iPSCs.

molecular biology↗

bioRxiv2019

Ontogeny and expression profiles of steroid hormone receptors in a mouse model of endometriosis

Endometriosis is a chronic incurable disorder of unknown etiology affecting a large proportion of women in reproductive age. In order to understand the pathogenesis and preclinical testing of drugs,animal models that recapitulate the key features of the disorder are highly desirous. Herein, we describe the ontogeny of the ectopic endometrial lesion in a mouse model where uterine tissue was ligated to the intestinal mesentery and the animals were followed up from day 5 to day 60 post-surgery. Out of 60 animals that underwent surgery, 58 developed endometriosis using this strategy. Most lesions were pale, fluid filled while red lesions were seen in ~10% of animals. Histologically, in most animals there was one large cystic gland with well differentiated epithelium, in 13% of animals there was mixed phenotype (well and poorly differentiated). There was extensive stromal compaction and increased number of macrophages in ectopic lesions. During the course of endometriosis, there was an increase in number of PCNA positive epithelial and stromal cells. The epithelial cells at all the time point were cytokeratin positive and the stroma was vimentin positive. However, at day 30 and 60, the stromal cells were also cytokeratin positive. The mRNA levels of estrogen receptors Esr1 and Gper1 were reduced while those of Esr2 were elevated as compared to normal endometrium, the levels of progesterone receptor (Pgr) were found to be downregulated in ectopic lesions as compared to control. However, these differences were not statistically significant due to high biological variability. Low abundance of Cyp19a1 transcripts (aromatase gene) were only detected in the ectopic endometrium. Immunohistochemically, the expression of ER and ER{beta} was significantly reduced only in stromal cells; the epithelial cell staining was maintained. GPER1 and PR immunoreactivity was significantly low in both epithelial and stromal cells. The immunostaining of all the steroid receptors was highly heterogeneous in the ectopic tissues with some areas of sections had stained intensely while others had negligible staining. We propose that temporal and spatial difference in the expression of steroid hormone receptors during the course of endometriosis development coupled with micro-heterogeneity may alter the effectiveness of steroid hormone analogues resulting in variable outcomes and often failure of therapy.

pathology↗

bioRxiv2019

Intra-mitochondrial protein degradationalleviates alpha-synuclein seeding and Abeta 42 aggregation

Mitochondria have long been implicated in Parkinsons disease (PD), however, it is not clear how mitochondrial impairment and alpha-synuclein pathology are coupled. We report here that intra-mitochondrial protein homeostasis plays a major role in alpha-synuclein fibril elongation, as interference with intra-mitochondrial proteases and mitochondrial protein import significantly aggravate alpha-synuclein aggregation. In contrast, direct inhibition of mitochondrial complex I, increase in intracellular calcium concentration or formation of reactive oxygen species (ROS), all of which have been associated with mitochondrial stress, did not affect alpha-synuclein pathology. We further demonstrate that similar mechanisms are involved in Amyloid {beta} 1-42 (A{beta}42) aggregation, suggesting that mitochondria are directly capable of influencing cytosolic protein homeostasis of aggregation-prone proteins.

neuroscience↗

bioRxiv2019

Thyroid hormone regulates distinct paths to maturation in pigment cell lineages

Circulating endocrine factors are critical for orchestrating complex developmental processes during the generation of adult form. One such factor, thyroid hormone, regulates diverse cellular processes during post-embryonic development and can drive disparate morphological outcomes through mechanisms that remain essentially unknown. We sought to define how thyroid hormone elicits opposite responses in the abundance of two pigment cell classes during development of the zebrafish adult pigment pattern. By profiling individual transcriptomes from thousands of neural crest derived cells, including pigment cells, we reconstructed developmental trajectories and identified lineage-specific changes in response to thyroid hormone. Our findings show that thyroid hormone and its receptors regulate distinct events of cellular maturation across lineages, and illustrate how a single, global factor integrates seemingly divergent morphogenetic outcomes across developmental time.

developmental biology↗

bioRxiv2018

Lipid Lowering Oxopropanylindole Hydrazone Derivatives with Antioxidant and Antihyperglycemic Activity

A series of substituted oxopropanylindole hydrazone derivatives was synthesized and evaluated for anti-oxidant and anti-dyslipidemic activity. Among these 12 compounds the three compounds 6c, 7b and 7d showed good anti-oxidant activity and the compound 6c attenuataed LDL oxidation by 32%. The compounds 6c and 7d also showed good anti-dyslipidemic activity by reducing serum levels of total cholesterol (TC), phospholipids (PL) and triglycerides (TG). These two compounds were further evaluated for anti-adipogenic and anti-hyperglycemic activity, where 6c was found most active compound with 44% reduction in lipid accumulation and 20.5% and 24.3% reduction in blood glucose at 5h and 24h respectively, as compared to standard drug metformin.

biochemistry↗

bioRxiv2018

Age-dependent deterioration of nuclear pore assembly in mitotic cells decreases transport dynamics

Nuclear transport is facilitated by the Nuclear Pore Complex (NPC) and is essential for life in eukaryotes. The NPC is a long-lived and exceptionally large structure. We asked whether NPC function is compromised in ageing mitotic cells. By imaging of single yeast cells during ageing, we show that the abundance of several NPC components and NPC assembly factors decreases while signs of misassembled NPCs appear. Consequently, nuclear permeability decreases, resulting in decreased dynamics of transcription factor shuttling and increased nuclear compartmentalisation. In support that declining NPC quality control is important in mitotic ageing, we find that the transport kinetics observed in ageing is mimicked in an NPC assembly mutant. Additionally, the single cell life histories reveal that cells that better maintain NPC function are longer lived. We conclude that assembly and quality control of NPCs are major challenges for ageing mitotic cells.

cell biology↗

bioRxiv2018

Synthesis, SAR and Docking Studies of Substituted Aryl phenylthiazolyl phenylcarboxamide as potential Protein Tyrosine Phosphatase 1B Inhibitors

Inspired by the potent PTP1B inhibitory activity reported in a novel series of substituted aryl thiazolyl phenylsulphonamides (I), sulfonyl moiety in the most active compound (I, R= OCH3, R1= CF3, 73.6% PTP1B inhibition) was replaced by benzoyl group (region B) to afford compound II which showed lesser activity (50.5% PTP1B inhibition). To optimize the activity, further structural modifications were done on compound II at region A, B and C to design and synthesize a series of 24 aryl phenylthiazolyl phenylcarboxamides for evaluation against PTP1B enzyme. Among these compounds six compounds showed good PTP1B inhibitory activity in the order of compound 38 > 30 > 29 > 37 > 22 > 19. The lowest energy conformer of compound 38 at PTP1B active site shows favorable binding similar to known PTP1B binders and explains its selectivity towards PTP1B. Compound 38 also showed promising antihyperglycemic, antidyslipidemic and insulin resistant reversal activities in vivo in STZ model and db/db mice model. Altogether, the compound 38 present an excellent candidate for future PTP1B targeted drug discovery.

pharmacology and toxicology↗

bioRxiv2018

Functional Interrogation of Lynch Syndrome Associated MSH2 Missense Variants Using CRISPR-Cas9 Gene Editing in Human Embryonic Stem Cells

Lynch syndrome (LS) is a hereditary cancer predisposition condition caused by inactivating germline mutations in one of the DNA mismatch repair (MMR) genes. Identifying a deleterious germline mutation by DNA sequencing is important for confirming an LS diagnosis. Frameshift and nonsense mutations significantly alter the protein product and likely impair MMR function. However, the implication of a missense mutation is often difficult to interpret. Referred to as variants of uncertain significance (VUS), their discovery hampers the definitive LS diagnosis. To determine the pathogenic significance of a VUS it is helpful to know its impact on protein function. Functional studies in the test tube and in cellular models have been performed for some VUS, however, these studies have been limited by the artificial nature of the assays. We report here an improved functional assay in which we engineered site-specific MSH2 VUS using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 gene editing in human embryonic stem cells. This approach introduces the variant into the endogenous MSH2 loci, while simultaneously eliminating the wild-type gene. We then characterized the impact of the variants on cellular MMR functions including DNA damage response signaling upon challenge with a DNA alkylating agent and the repair of DNA microsatellites. We classified the MMR functional capability of 8 of 10 VUS under study providing valuable information for determining their likelihood of being bona fide LS mutations. This improved human cell-based assay system for functionally testing MMR gene VUS will facilitate the identification of high risk LS patients.\n\nSignificance StatementUnderstanding how cancer-associated missense variants in MMR genes affect function helps determine whether they truly contribute to disease. Laboratory assays previously utilized are limited by their artificial nature. To improve this, we introduced variants directly into the endogenous MMR loci in hESCs using CRISPR-Cas9 gene editing. This approach allows us to assess each variant while being expressed by its normal regulatory elements in a cellular environment. Our results will help guide the management of patients world-wide who carry these variants. At the same time, this study provides a technical road map for assessing the functional effects of all LS-associated variants, as well as variants linked to other genetic diseases where a cell-based functional assay is available.

genomics↗

bioRxiv2018

Two-component systems regulate swarming in Pseudomonas aeruginosa PA14

Swarming in Pseudomonas aeruginosa is a quorum-dependant motility over semi-solid surfaces. On soft agar, P. aeruginosa exhibits a dendritic swarm pattern, with multiple levels of branching. Swarm patterns vary considerably depending upon the experimental design. In the present study, we show that the swarm pattern is plastic and media dependent. We define several quantifiable, macroscale features of the swarm to study the plasticity observed across media. Further, through a targeted screen of 113 genes encoding two-component system (TCS) components, we show that 44 TCS genes regulate PA14 swarming in a contextual fashion. However, only four TCS genes are essential for swarming. Many swarming-defective TCS mutants are highly efficient in biofilm formation indicating an antagonistic relationship between swarming and biofilm states in P. aeruginosa.

microbiology↗

bioRxiv2018

Genetic Determinants of Cortical Structure (Thickness, Surface Area and Volumes) among Disease Free Adults in the CHARGE Consortium

Cortical thickness, surface area and volumes (MRI cortical measures) vary with age and cognitive function, and in neurological and psychiatric diseases. We examined heritability, genetic correlations and genome-wide associations of cortical measures across the whole cortex, and in 34 anatomically predefined regions. Our discovery sample comprised 22,822 individuals from 20 cohorts within the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium and the United Kingdom Biobank. Significant associations were replicated in the Enhancing Neuroimaging Genetics through Meta-analysis (ENIGMA) consortium, and their biological implications explored using bioinformatic annotation and pathway analyses. We identified genetic heterogeneity between cortical measures and brain regions, and 161 genome-wide significant associations pointing to wnt/{beta}-catenin, TGF-{beta} and sonic hedgehog pathways. There was enrichment for genes involved in anthropometric traits, hindbrain development, vascular and neurodegenerative disease and psychiatric conditions. These data are a rich resource for studies of the biological mechanisms behind cortical development and aging.

genetics↗

bioRxiv2018

Amyloid β oligomers constrict human capillaries in Alzheimer’s disease via signalling to pericytes

Vascular compromise occurs early in Alzheimers disease (AD) and other dementias1-3. Amyloid {beta} (A{beta}) reduces cerebral blood flow4-6 and, as most of the cerebral vasculature resistance is in capillaries7, A{beta} might mainly act on contractile pericytes on capillary walls8-10. Employing human tissue to establish disease-relevance, and rodent experiments to define mechanism, we now show that A{beta} constricts brain capillaries at pericyte locations in human subjects with cognitive decline. Applying soluble A{beta}1-42 oligomers to live human cortical tissue constricted capillaries. Using rat cortical slices, this was shown to reflect A{beta} evoking capillary pericyte contraction, with an EC50 of 4.7 nM, via the generation of reactive oxygen species and activation of endothelin ET-A receptors. In freshly-fixed diagnostic biopsies from human patients investigated for cognitive decline, mean capillary diameters were less in subjects showing A{beta} deposition than in subjects without A{beta} deposition. For patients with A{beta} deposition, the capillary diameter was 31% less at pericyte somata than away from somata, predicting a halving of blood flow. Constriction of capillaries by A{beta} will contribute to the energy lack1-3 occurring in AD, which promotes further A{beta} generation11,12. This mechanism reconciles the amyloid hypothesis13-15 with the earliest events in AD being vascular1.

neuroscience↗