Biology subjects

Tiwari, A.

Publications and source records attributed to Tiwari, A..

At least 55 records · Page 3Linked to original sources

bioRxiv2021

How Our Cells Become Our Selves: The Cellular Phylodynamic Biology of Growth and Development

Our lives begin with 1 cell, then 2, then 4, then the trillion cell adult, comprised of cell lineages, tissues, organs. How does this occur? Examination in numbers of cells, N, Cellular Phylodynamics, revealed two previously unappreciated processes: UNI-GROWTH, the slowing of growth that occurs as we become larger, caused by fewer cells dividing, captured by the Universal Mitotic Fraction and Universal Growth Equations, with accuracy confirmed for 13 species, including nematodes, mollusks, and vertebrates; and ALLO-GROWTH, the creation of body parts from Founder Cells, captured by the Cellular Allometric Growth Equation, which describes mitotic expansion by Cell-Heritable change in the Cell Cycle Time. These equations can generate cell lineage approximations, bringing the power of coalescent theory to developmental biology.

developmental biology↗

bioRxiv2021

In vitro reconstitution demonstrates the amyloid-beta mediated myelin membrane deformation

Amyloid-beta (A{beta}) aggregation mediated neuronal membrane deformation, although poorly understood, is implicated in Alzheimers Disease (AD). Particularly, whether A{beta} aggregation can induce neuronal demyelination remains unknown. Here we show that A{beta}-40 binds and induces extensive tubulation in the myelin membrane in vitro. The binding of A{beta}-40 depends predominantly on the lipid packing defect densities and electrostatic interactions and results in rigidification of the myelin membrane in the early time scales. Furthermore, elongation of A{beta}-40 into higher oligomeric and fibrillar species leads to eventual fluidization of the myelin membrane followed by extensive membrane tubulation observed in the late phase. Taken together, our results capture mechanistic insights into snapshots of temporal dynamics of A{beta}-40 - myelin membrane interaction and demonstrate how short timescale, local phenomena of binding, and fibril mediated load generation manifests into long timescale, global phenomena of myelin tubulation and demonstrates the ability of A{beta}-40 to demyelinate.

biophysics↗

bioRxiv2021

Captivity-induced behaviour and spatial learning abilities in an enigmatic, aquifer-dwelling blind eel, Rakthamichthys digressus

We investigated the impact of captive life on behaviour and learning abilities in an enigmatic, aquifer-dwelling blind eel, Rakthamichthys digressus. Of eight major behavioural traits related to exploration and activity in a novel arena, four were significantly altered by life in captivity. While the startle response upon introduction into the arena and overall swimming away from the walls increased after captivity, inactivity exhibited immediately after the startle and the reaction to an external disturbance decreased. We also observed behavioural syndromes between startle responses and horizontal wall following, and between overall activity and vertical wall following; however, these behavioural syndromes were not altered by maintenance in captivity. Interestingly, this blind-eel failed to learn a simple spatial task in a Y-maze apparatus. Captive-associated behavioural changes in R. digressus may influence their survival after reintroduction into natural habitats, and such changes must be taken into account while developing protocols for ex-situ conservation and subsequent release.

animal behavior and cognition↗

bioRxiv2021

Modulation of endometrial E-Cadherin and N-Cadherin by ovarian steroids and embryonic stimuli

The endometrium is a dynamic tissue that undergoes extensive remodelling to attain a receptive state which is further modulated in presence of an embryo for successful initiation of pregnancy. Cadherins are the proteins of junctional complex of which E-cadherin (E-Cad) is crucial for maintaining epithelial cell state and integrity of the epithelial barrier; gain of N-cadherin (N-Cad) in epithelial cells leads to epithelial to mesenchymal transition (EMT). In the present study, we aimed to investigate the expression of E-Cad and N-Cad in the mouse endometrial luminal epithelium and its modulation by estrogen, progesterone and embryonic stimuli. We observed that E-Cad is diffusely expressed in the luminal epithelium of mouse endometrium during the estrus stage and upon estrogen treatment. It is apico-laterally and basolaterally sorted at the diestrus stage and in response to combined treatment of estrogen and progesterone. In 3D spheroids of human endometrial epithelial cells, combined treatment with estrogen and progesterone led to lateral sorting of E-Cad. In the mouse endometrium at the time of embryo implantation, there is loss of E-Cad which was associated with the gain of N-Cad suggestive of EMT in the luminal epithelium. This EMT is possibly driven by embryonic stimuli as treatment with estrogen and progesterone did not lead to gain of N-Cad expression. In conclusion, the present study demonstrates that steroid hormones directly affect E-Cad sorting in the endometrial epithelium.

developmental biology↗

bioRxiv2020

The C99 domain of the amyloid precursor protein is a disordered membrane phase-preferring protein

Processing of the amyloid precursor protein (APP) via the amyloidogenic pathway is associated with the etiology of Alzheimers disease. The cleavage of APP by {beta}-secretase to generate the transmembrane 99-residue C-terminal fragment (C99) and subsequent processing of C99 by {gamma}-secretase to yield amyloid-{beta} (A{beta}) peptides are essential steps in this pathway. Biochemical evidence suggests amyloidogenic processing of C99 occurs in cholesterol- and sphingolipid-enriched liquid ordered phase membrane raft domains. However, direct evidence that C99 preferentially associates with rafts has remained elusive. Here, we test this idea by quantifying the affinity of C99-GFP for raft domains in cell-derived giant plasma membrane vesicles. We find that C99 is essentially excluded from ordered domains in HeLa cells, SH-SY5Y cells and neurons, instead exhibiting a strong (roughly 90%) affinity for disordered domains. The strong association of C99 with disordered domains occurs independently of its cholesterol binding activity, homodimerization, or the familial Alzheimer disease Arctic mutation. Finally, we confirm previous studies suggesting that C99 is processed in the plasma membrane by -secretase, in addition to the well-known {gamma}-secretase. These findings suggest that C99 itself lacks an intrinsic affinity for raft domains, implying either that amyloidogenic processing of the protein occurs in disordered regions of the membrane, that processing involves a marginal sub-population of C99 found in rafts, or that as-yet-unidentified protein-protein interactions involving C99 in living cells drive it into rafts to promote its cleavage therein.

neuroscience↗

bioRxiv2020

Ratio of hydrophobic-hydrophilic and positive-negative residues at lipid-water-interface influences surface expression and channel-gating of TRPV1

During evolution, TRPV1 has lost, retained or selected certain residues at Lipid-Water-Interface (LWI) and formed specific patterns there. The ratio of "hydrophobic-hydrophilic" and "positive-negative charged" residues at the inner LWI remains conserved throughout vertebrate evolution and play important role in regulating TRPV1 trafficking, localization and functions. Arg575 is an important residue as Arg575Asp mutant has reduced Capsaicin-sensitivity, surface expression, colocalization with lipid-raft markers, cell area, and increased cell lethality. This lethality is due to the disruption of the ratio between positive-negative charges there. Such lethality can be rescued by either using TRPV1-specfic inhibitor 5-IRTX or by restoring the positive-negative charge ratio at that position, i.e. by introducing Asp576Arg mutation in Arg575Asp backbone. We propose that Arg575Asp mutant confers TRPV1 in a "constitutive-open-like" condition. These findings have broader implication in understanding the molecular basis of thermo-gating and channel-gating and the microenvironments involved in such process that goes erratic in different diseases.

cell biology↗

bioRxiv2020

Designing of Epitope-Based Vaccine from the Conserved Region of the Spike Glycoprotein of SARS-CoV-2

The emergence of COVID-19 as a pandemic with a high morbidity rate is posing serious global concern. There is an urgent need to design a suitable therapy or vaccine that could fight against SARS-CoV-2 infection. As spike glycoprotein of SARS-CoV-2 plays a crucial role in receptor binding and membrane fusion inside the host, it could be a suitable target for designing of an epitope-based vaccine. SARS-CoV-2 is an RNA virus and thus has a property to mutate. So, a conserved peptide region of spike glycoprotein was used for predicting suitable B cell and T cell epitopes. 4 T cell epitopes were selected based on stability, antigenicity, allergenicity and toxicity. Further, MHC-I were found from the immune database that could best interact with the selected epitopes. Population coverage analysis was also done to check the presence of identified MHC-I, in the human population of the affected countries. The T cell epitope that binds with the respective MHC-I with highest affinity was chosen. Molecular dynamic simulation results show that the epitope is well selected. This is an in-silico based study that predicts a novel T cell epitope from the conserved spike glycoprotein that could act as a target for designing of the epitope-based vaccine. Further, B cell epitopes have also been found but the main work focuses on T cell epitope as the immunity generated by it is long lasting as compared to B cell epitope.

bioinformatics↗

bioRxiv2020

PT150 is a modulator of glucocorticoid and androgen receptors with antiviral activity against SARS-CoV-2.

PT150 is a clinical stage molecule, taken orally, with a strong safety profile having completed Phase 1 and Phase 2 clinical trials for its original use as an anti-depressant. It has an active IND for COVID-19. Antiviral activities have been found for PT150 and other members of its class in a variety of virus families; thus, it was now tested against SARS-CoV-2 in human bronchial epithelial lining cells and showed effective 90% inhibitory antiviral concentration (EC90) of 5.55 M. PT150 is a member of an extended platform of novel glucocorticoid receptor (GR) and androgen receptor (AR) binding molecules. In vivo, their predominant net effect is one of systemic glucocorticoid antagonism, but they also show direct downregulation of AR and minor GR agonism at the cellular level. We hypothesize that anti-SARS-CoV-2 activity depends in part on this AR downregulation through diminished TMPRSS2 expression and modulation of ACE2 activity. Given that hypercortisolemia is now suggested to be a significant co-factor for COVID-19 progression, we also postulate an additive role for its potent immunomodulatory effects through systemic antagonism of cortisol.

microbiology↗

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

Network-based Machine Learning Approach for Structural Domain Identification in Proteins

In the era of structural genomics, with a large number of protein structures becoming available, identification of domains is an important problem in protein function analysis as it forms the first step in protein classification. Domain identification has been an active area of research for over four decades and a wide range of automated methods have been proposed. In the proposed network-based machine learning approach, NML-DIP, a combination of supervised (SVM) and unsupervised (k-means) machine learning techniques are used for domain identification in proteins. The algorithm proceeds by first representing protein structure as a protein contact network and using topological properties, viz., length, density, and interaction strength (that assesses inter- and intra-domain interactions) as feature vectors in the first SVM to distinguish between single and multi-domain proteins. A second SVM is used to identify the number of domains in multi-domain proteins. Thus, it does not require a prior information of the number of domains. The k-means algorithm is then used to identify the domain boundaries that are assessed using CATH annotation. The performance of the proposed algorithm is evaluated on four benchmark datasets and compared with four state-of-the-art domain identification methods. The performance of the approach is comparable to other domain identification tools and works well even when the domains are formed with non-contiguous segments. The performance of the program is significantly improved for prior information about the number of domains. The algorithm is available at: https://bit.ly/NML-DIP.

bioinformatics↗

bioRxiv2020

KLF4 coordinates corneal epithelial apicobasal polarity and plane of cell division, and is downregulated in ocular surface squamous neoplasia

Apicobasal polarity (ABP) is an important feature of many epithelial cells, including those in the stratified squamous corneal epithelium (CE). Previously, we demonstrated that KLF4 promotes CE homeostasis by suppressing epithelial-mesenchymal transition (EMT) and TGF-{beta} signaling. As dysregulation of TGF-{beta} signaling affects ABP, we investigated the role of KLF4 in regulating cell polarity and plane of division by spatiotemporally regulated ablation of Klf4 in adult ternary transgenic Klf4{Delta}/{Delta}CE (Klf4LoxP/LoxP/Krt12rtTA/rtTA/Tet-O-Cre) mouse CE. Klf4{Delta}/{Delta}CE cells displayed decreased expression and mis-localization of apical polarity markers Pals1 and Crumbs1, apicolateral Par3, and basolateral Scribble. Cdc42 was upregulated, while Rac and Rho were mis-localized in the Klf4{Delta}/{Delta}CE cytoplasm unlike their cortical expression in the control. Phalloidin staining revealed disrupted actin cytoskeleton in the Klf4{Delta}/{Delta}CE cells. Survivin and phospho-histone-H3 immunostaining revealed a tilt in the plane of cell division favoring symmetrical divisions with vertical axis in the Klf4{Delta}/{Delta}CE compared with predominantly asymmetrical divisions with horizontal axis in the control. Human ocular surface squamous neoplasia (OSSN) tissues displayed signs of EMT and loss of ABP markers PAR3, PALS1 and SCRIB, coupled with downregulation of KLF4. By demonstrating that Klf4 ablation affects CE expression of ABP markers and Rho family GTPases, cytoskeletal actin organization and the plane of cell division, and that KLF4 is downregulated in OSSN tissues that display EMT and lack ABP, these results elucidate the key integrative role of KLF4 in coordinating CE cell polarity and plane of division, loss of which results in OSSN.

cell biology↗

bioRxiv2019

Role of HP1β during spermatogenesis and DNA replication

Maintaining genomic stability in a continually dividing cell population requires accurate DNA repair, especially in male germ cells. Repair and replication protein access to DNA, however, is complicated by chromatin compaction. The HP1{beta} chromatin protein, encoded by Cbx1, is associated with chromatin condensation but its role in meiosis is not clear. To investigate the role of Cbx1 in male germ cells, we generated testis specific Cbx1 deficient transgenic mice by crossing Cbx1flox/flox (Cbx1f/f) mice with Stra8 Cre+/- mice. Loss of Cbx1 in testes adversely affected sperm maturation and Cbx1 deletion increased seminiferous tubule degeneration and basal level DNA damage., We observed that Cbx1-/- MEF cells displayed reduced resolution of stalled DNA replication forks as well as decreased fork restart, indicating defective DNA synthesis. Taken together, these results suggest that loss of Cbx1 in growing cells leads to DNA replication defects and associated DNA damage that impact cell survival.

cell biology↗

bioRxiv2019

A commonly used photosynthetic inhibitor fails to block electron flow to photosystem I in intact systems

In plant science, 2,4-dinitrophenylether of iodonitrothymol (DNP-INT) is frequently used as an alternative to 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone (DBMIB) to examine the capacity of plastoquinol and semiquinone to reduce O2. DNP-INT is considered an effective inhibitor of the photosynthetic electron transfer chain (PETC) through its binding at the Q0 site of Cyt-b6f. The binding and action of DNP-INT has been previously characterized spectroscopically in purified Cyt-b6f complex reconstituted with Plastocyanin, PSII membranes and plastoquinone, as well as in isolated thylakoids based on its property to block MV-mediated O2 consumption. Contrary to the conclusions made from these experiments, we observed clear reduction of P700+ in samples incubated with DNP-INT during our recent investigation into the sites of oxygen consumption in isolated thylakoids. Therefore, we carried out an extensive investigation of DNP-INTs chemical efficacy in isolated thylakoids and intact leaves. This included examination of its capacity to block the PETC before PSI, and therefore its inhibition of CO2 fixation. P700 redox kinetics were measured using Dual-PAM whilst Membrane Inlet Mass Spectrometry (MIMS) was used for simultaneous determination of the rates of O2 evolution and O2 consumption in isolated thylakoids and CO2 fixation in intact leaves, using two stable isotopes of oxygen (16O2,18O2) and CO2 (12C,13C), respectively. Based on these investigations we confirmed that DNP-INT is unable to completely block the PETC and CO2 fixation, therefore its use may produce artefacts if applied to isolated thylakoids or intact cells, especially when determining the locations of reactive oxygen species formation in the photosynthetic apparatus.

plant biology↗

bioRxiv2019

Interplay of bacterial membrane vesicle and host lipid physicochemical parameters during host-bacterial membrane vesicle interaction

Bacterial membrane vesicles (MVs) facilitate long-distance delivery of virulence factors crucial for pathogenicity. The entry and trafficking mechanisms of virulence factors inside host cells are recently emerging, however, if bacterial MVs can fuse and modulate the physicochemical properties of the host lipid membrane and membrane lipid parameter for fusion remains unknown. Here we reconstitute the interaction of bacterial MV with host cell lipid membranes and quantitatively show that bacterial MV interaction increases the fluidity, dipole potential, and Compressibility of a biologically relevant multi-component host membrane upon fusion. The presence of cylindrical lipids such as phosphatidylcholine and a moderate acyl chain length of C16 helps the MV interaction. While significant binding of bacterial MVs to the raft-like lipid membranes with phase-separated regions of the membrane was observed, however, MVs prefer binding to the liquid-disordered regions of the membrane. Further, the elevated levels of cholesterol tend to hinder the interaction of bacterial MVs as evident from the favorable excess Gibbs free energy of mixing of bacterial MVs with host lipid membranes. The findings provide new insights that might have implications for the regulation of host machinery by bacterial pathogens through the manipulation of host membrane properties.

biochemistry↗

bioRxiv2019

In silico investigation of Y220C mutant p53 for lead design

p53 protein coded by the Tp53 gene is considered as one of the most intensively researched protein and mainly due to its role as a tumor suppressor, it acts as a tumor suppressor by carrying out two biologically complex processes namely Cell cycle arrest and apoptosis, In the oncogenic Y220C mutant p53, tyrosine is replaced by cysteine at 220th residue of the DNA binding Domain which causes the formation of a surface crevice, this specific mutation is responsible for approx. 100,000 cancer cases per year due to the destabilization and denaturation of the protein, as a result, the protein degrades at room temperature. In this work we carry out intensive Molecular Dynamic Simulations and Molecular Docking Studies to understand the structural dynamics of wild type p53 and changes the occurs in the mutant protein and also try to design lead against the druggable crevice and at the end of our study we used fragment-based optimization to come up with lead molecules which can act as scaffold for further drug development process

bioinformatics↗

bioRxiv2019

Genomics to aid species delimitation and effective conservation of the Sharpnose Guitarfish (Glaucostegus granulatus)

The Sharpnose Guitarfish (Glaucostegus granulatus) is one of fifteen critically endangered Rhino Rays which has been exploited as incidental catch, leading to severe population depletions and localized disappearances. Like many chondrichthyan species, there are no species-specific time-series data available for the Sharpnose Guitarfish that can be used to calculate population reduction, partly due to a lack of species-specific reporting as well as limitations in accurate taxonomic identification. We here present the first complete mitochondrial genome and partial nuclear genome of the species and the first detail phylogenetic assessment of the species. We expect that data presented in the current manuscript will aid in accurate species-specific landing and population assessments of the species in the future and will enable conservation efforts to protect and recover remaining populations.

genomics↗

bioRxiv2019

Increased expression of mitochondrial dysfunction stimulon genes affects chloroplast redox status and photosynthetic electron transfer in Arabidopsis

Mitochondrial retrograde signals control expression of nuclear mitochondrial dysfunction stimulon (MDS) genes. Although MDS gene products mostly affect mitochondrial functions, they also influence production of reactive oxygen species (ROS) and redox status of chloroplasts. To study this inter-organellar interaction, we analysed the response of the Arabidopsis MDS-overexpressor mutant rcd1 to methyl viologen (MV), which catalyses electron transfer from Photosystem I (PSI) to molecular oxygen, generating ROS in Mehlers reaction. The response of plants to MV was investigated by imaging chlorophyll fluorescence in aerobic and hypoxic environments, and by membrane inlet mass spectrometry. Hypoxic treatment abolished the effect of MV on photosynthetic electron transfer in rcd1, but not in wild type. A similar reaction to hypoxia was observed in other MDS-activating lines and treatments. This suggests that MDS gene products contribute to oxygen depletion at the PSI electron-acceptor side. In unstressed growth conditions this MDS-related effect is likely masked by endogenous oxygen evolution and gas exchange with the atmosphere. In rcd1, altered Mehlers reaction coincided with more reduced state of the chloroplast NADPH-thioredoxin oxidoreductase C (NTRC) and its targets, suggesting that NTRC performs feedback control of photosynthesis. This regulation may represent a novel mechanism whereby mitochondrial retrograde signalling affects chloroplast functions.

plant biology↗

bioRxiv2019

Discovery of compounds inhibiting the ADP-ribosyltransferase activity of pertussis toxin

Targeted pathogen-selective approach to antibiotic development holds promise to minimize collateral damage to the beneficial microbiome. The AB5-topology pertussis toxin (PtxS1-S5, 1:1:1:2:1) is a major virulence factor of Bordetella pertussis, the causative agent of the highly contagious respiratory disease whooping cough. Once internalized into the host cell, PtxS1 ADP-ribosylates -subunits of the heterotrimeric Gi-superfamily, thereby disrupting G-protein-coupled receptor signaling. Here, we report the discovery of the first small molecules inhibiting the ADP-ribosyltransferase activity of pertussis toxin. We developed protocols to purify mg-levels of truncated but highly active recombinant B. pertussis PtxS1 from Escherichia coli and an in vitro high throughput-compatible assay to quantify NAD+ consumption during PtxS1-catalyzed ADP-ribosylation of Gi. Two inhibitory compounds (NSC228155 and NSC29193) with low micromolar IC50-values (3.0 {micro}M and 6.8 {micro}M) were identified in the in vitro NAD+ consumption assay that also were potent in an independent in vitro assay monitoring conjugation of ADP-ribose to Gi. Docking and molecular dynamics simulations identified plausible binding poses of NSC228155 and in particular of NSC29193, most likely owing to the rigidity of the latter ligand, at the NAD+-binding pocket of PtxS1. NSC228155 inhibited the pertussis AB5 holotoxin-catalyzed ADP-ribosylation of Gi in living human cells with a low micromolar IC50-value (2.4 {micro}M). NSC228155 and NSC29193 might prove useful hit compounds in targeted B. pertussis-selective drug development.

microbiology↗