Biology subjects

Mitra, D.

Publications and source records attributed to Mitra, D..

29 records · Page 2Linked to original sources

bioRxiv2022

Assessing combinatorial diversity of aureochrome bZIPs through genome-wide screening

Aureochromes are unique blue light-responsive LOV (Light Oxygen Voltage) photoreceptors cum basic leucine zipper (bZIP) transcription factors (TFs), present exclusively in photosynthetic marine stramenopiles. Considering the availability of the complete genome sequence, this study focuses particularly on aureochromes from Ectocaupus siliculosus. Aureochromes mediate light-regulated developmental responses in this brown photosynthetic algae. Both the LOV sensor and the bZIP effector shows sequence-structure conservation. The LOV+bZIP modules of aureochrome homologs/ paralogs are not only structurally similar but also show an identical oligomeric state -- preferably dimeric. Aureochromes execute diverse cellular responses in different photosynthetic stramenopiles-- though their activities can vary even within a given algal species. Besides a heterogeneous linker connecting the sensor-effector and a flexible N-terminal region, the sequence composition of both the domains is vital. Therefore, it is important to understand whether aureochromes select dimerization partners from the same family or interact with other bZIPs as well. To regulate multifarious bio-logical activities, it is possible that aureochromes activate the global TF interaction network. Following homo/heterodimer modeling, we address the compatibility of dimerization partners by screening through heptad repeats. We evaluate the dimer interface area in terms of gain in solvation energy as well as the number of hydrogen bonds/salt bridge interactions. We further explore the relative stability of these structures from a graph-theoretic perspective through well-studied measures such as the energy of the graph and average participation coefficient. Furthermore, we also conduct an information-theoretic analysis using network information centrality and Kullback-Leibler divergence. We find that all our investigations into the relative stability of these dimers using diverse methods from bioinformatics, network science, and, information theory are in harmonious agreement. Our approach and findings should facilitate the design of experiments.

bioinformatics↗

bioRxiv2022

Basic Leucine Zippers: Aureochromes Versus the Rest

The blue light photoreceptor cum transcription factors, Aureochromes (Aureos), are present exclusively in photosynthetic stramenopiles. Co-existence of Light-Oxygen-Voltage (LOV) and basic leucine zipper (bZIP) is unique to Aureos - therefore ideal to study light-dependent DNA binding/transcriptional regulation. Further, Aureos inverse effector-sensor topology, resembling several sensory eukaryotic transcription factors, makes them prototypical optogenetic scaffolds. In absence of 3D data, this study aims for a thorough investigation of the bZIP domains from Aureos and others, and their interaction with substrate DNA using tools from sequence/structural bioinformatics, network theory, molecular dynamics simulation and in vitro experiments. An in-depth comparison of 173 Aureo/plant/opisthokont bZIPs reveals Aureos uniqueness and evolutionary significance in DNA binding specificity as well as dimer stability. An all-atom network analysis on representative bZIP-DNA co-crystal structures, especially the measurement of eigenvector centrality, further adds importance to hydrophobic interactions in the zipper region to stabilize bZIP dimer and facilitate DNA binding in Aureos and other bZIPs. Perhaps the most notable finding is the unique histidine substitution at the basic region of Aureos unlike any other bZIPs. Not only is this residue important for DNA binding, this can serve as a potential switch point in Aureo/bZIP evolution. HighlightsO_LIAureochrome is perhaps the only light-responsive transcription factor in bZIP superfamily. C_LIO_LIWe draw a comparative between aureochromes and other bZIPs via in-silico/in-vitro methods. C_LIO_LIAureochromes form a distinctly separate lineage, midway in bZIP evolution. C_LIO_LIThe unique histidine facilitates aureochromes interaction with cognate DNA substrates. C_LI

plant biology↗

bioRxiv2021

A Robust, Low-Cost Instrument for Real-time Colorimetric Isothermal Nucleic Acid Amplification

The COVID-19 pandemic has highlighted the need for broader access to molecular diagnostics. Colorimetric isothermal nucleic acid amplification assays enable simplified instrumentation over more conventional PCR diagnostic assays and, as such, represent a promising approach for addressing this need. In particular, colorimetric LAMP (loop-mediated isothermal amplification) has received a great deal of interest recently. However, there do not currently exist robust instruments for performing these kinds of assays in high throughput with real-time readout of amplification signals. To address this need, we developed LARI, the LAMP Assay Reader Instrument. We have deployed over 50 LARIs for routine use in R&D and production environments, with over 12,000 assays run to date. In this paper, we present the design and construction of LARI along with thermal, optical, and assay performance characteristics. LARI can be produced for under $1500 and has broad applications in R&D, point-of-care diagnostics, and global health.

bioengineering↗

bioRxiv2020

PAN-INDIA 1000 SARS-CoV-2 RNA Genome Sequencing Reveals Important Insights into the Outbreak

The PAN-INDIA 1000 SARS-CoV-2 RNA Genome Sequencing Consortium has achieved its initial goal of completing the sequencing of 1000 SARS-CoV-2 genomes from nasopharyngeal and oropharyngeal swabs collected from individuals testing positive for COVID-19 by Real Time PCR. The samples were collected across 10 states covering different zones within India. Given the importance of this information for public health response initiatives investigating transmission of COVID-19, the sequence data is being released in GISAID database. This information will improve our understanding on how the virus is spreading, ultimately helping to interrupt the transmission chains, prevent new cases of infection, and provide impetus to research on intervention measures. This will also provide us with information on evolution of the virus, genetic predisposition (if any) and adaptation to human hosts. One thousand and fifty two sequences were used for phylodynamic, temporal and geographic mutation patterns and haplotype network analyses. Initial results indicate that multiple lineages of SARS-CoV-2 are circulating in India, probably introduced by travel from Europe, USA and East Asia. A2a (20A/B/C) was found to be predominant, along with few parental haplotypes 19A/B. In particular, there is a predominance of the D614G mutation, which is found to be emerging in almost all regions of the country. Additionally, mutations in important regions of the viral genome with significant geographical clustering have also been observed. The temporal haplotype diversities landscape in each region appears to be similar pan India, with haplotype diversities peaking between March-May, while by June A2a (20A/B/C) emerged as the predominant one. Within haplotypes, different states appear to have different proportions. Temporal and geographic patterns in the sequences obtained reveal interesting clustering of mutations. Some mutations are present at particularly high frequencies in one state as compared to others. The negative estimate Tajimas D (D = -2.26817) is consistent with the rapid expansion of SARS-CoV-2 population in India. Detailed mutational analysis across India to understand the gradual emergence of mutants at different regions of the country and its possible implication will help in better disease management.

genomics↗

bioRxiv2020

Effective Inhibition of SARS-CoV-2 Entry by Heparin and Enoxaparin Derivatives

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has caused a pandemic of historic proportions and continues to spread globally, with enormous consequences to human health. Currently there is no vaccine, effective therapeutic or prophylactic. Like other betacoronaviruses, attachment and entry of SARS-CoV-2 is mediated by the spike glycoprotein (SGP). In addition to its well-documented interaction with its receptor, human angiotensin converting enzyme 2 (hACE2), SGP has been found to bind to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we pseudotyped SARS-CoV-2 SGP on a third generation lentiviral (pLV) vector and tested the impact of various sulfated polysaccharides on transduction efficiency in mammalian cells. The pLV vector pseudotyped SGP efficiently and produced high titers on HEK293T cells. Various sulfated polysaccharides potently neutralized pLV-S pseudotyped virus with clear structure-based differences in anti-viral activity and affinity to SGP. Concentration-response curves showed that pLV-S particles were efficiently neutralized by a range of concentrations of unfractionated heparin (UFH), enoxaparin, 6-O-desulfated UFH and 6-O-desulfated enoxaparin with an IC50 of 5.99 {micro}g/L, 1.08 mg/L, 1.77 {micro}g/L, and 5.86 mg/L respectively. The low serum bioavailability of intranasally administered UFH, along with data suggesting that the nasal epithelium is a portal for initial infection and transmission, suggest that intranasal administration of UFH may be an effective and safe prophylactic treatment.

microbiology↗

bioRxiv2020

Multi-epitope based peptide vaccine design against SARS-CoV-2 using its spike protein

SARS-CoV-2 has been efficient in ensuring that many countries are brought to a standstill. With repercussions ranging from rampant mortality, fear, paranoia, and economic recession, the virus has brought together countries to look at possible therapeutic countermeasures. With prophylactic interventions possibly months away from being particularly effective, a slew of measures and possibilities concerning the design of vaccines are being worked upon. We attempted a structure-based approach utilizing a combination of epitope prediction servers and Molecular dynamic (MD) simulations to develop a multi-epitope-based subunit vaccine that involves the two subunits of the spike glycoprotein of SARS-CoV-2 (S1 and S2) coupled with a substantially effective chimeric adjuvant to create stable vaccine constructs. The designed constructs were evaluated based on their docking with Toll-Like Receptor (TLR) 4. Our findings provide an epitope-based peptide fragment that can be a potential candidate for the development of a vaccine against SARS-CoV-2. Recent experimental studies based on determining immunodominant regions across the spike glycoprotein of SARS-CoV-2 indicate the presence of the predicted epitopes included in this study.

bioinformatics↗

bioRxiv2019

Polarity protein distribution on the metaphase furrow regulates hexagon dominated plasma membrane organization in syncytial Drosophila embryos

Epithelial cells have a polarised distribution of protein complexes on the lateral membrane and are present as a polygonal array dominated by hexagons. Metazoan embryogenesis enables the study of temporal formation of the polygonal array and mechanisms that regulate its distribution. The plasma membrane of the syncytial Drosophila blastoderm embryo is organized as a polygonal array during cortical division cycles with an apical membrane and lateral furrow in between adjacent nuclei. We find that polygonal plasma membrane organization arises in syncytial division cycle 11 and hexagon dominance occurs with increase in furrow length in cycle 12. This is coincident with DE-cadherin and Bazooka enrichment at edges and the septin, Peanut enrichment at vertices of the base of the furrow. DE-cadherin depletion leads to loss of hexagon dominance. Bazooka and Peanut depletion leads to a delay in occurrence of hexagon dominance from nuclear cycle 12 to 13. Hexagon dominance in Bazooka and Peanut mutants occurs with furrow extension and correlates with increase in DE-cadherin in syncytial cycle 13. We conclude that a change in polarity complex distribution leads to loss of furrow stability thereby changing the polygonal organization of the blastoderm embryo.\n\nHighlight Summary for TOCMetazoan embryogenesis starts with the formation of polygonal epithelial-like cells. We show that hexagon dominance in polygonal epithelial-like plasma membrane organization occurs in nuclear cycle 12 in the syncytial blastoderm Drosophila embryo. DE-cadherin and Bazooka distribution along the lateral furrow regulates this hexagon dominance.

developmental biology↗

bioRxiv2019

A fast microfluidic device to measure the deformability of red blood cells

We report a microfluidic device to determine the shear elastic modulus and the Youngs modulus of red blood cells (RBCs). Our device consists of a single channel opening into a funnel, with a semi-circular obstacle placed at the mouth of the funnel. As a RBC passes the obstacle, it deflects from its original path. Using populations of artificially-stiffened RBCs, we show that the stiffer RBCs deflect more compared to the normal RBCs. We use calibration curves obtained from numerical simulations to map a trajectory of each RBC to its elastic constants. Our estimates of the shear elastic modulus and the Youngs modulus of normal RBCs are within the same range of values reported in the literature using AFM, optical tweezers and micropipette measurements. We also estimate indirectly the elongation index of normal and artificially hardened RBCs from their tracks, without any direct observation of their shapes. Finally, we sort a mixed population of RBCs based on their deformability alone. Our device could potentially be further miniaturized to sort and obtain the elastic constants of nanoscale objects, whose shape change is difficult to monitor by optical microscopy.

biophysics↗

bioRxiv2019

VHL Synthetic Lethality Signatures Uncovered by Genotype-specific CRISPR-Cas9 Screens

BackgroundGenome-wide CRISPR-Cas9 essentiality screening represents a powerful approach to identify genetic vulnerabilities in cancer cells. Here, we applied this technology and designed a strategy to identify target genes that are synthetic lethal (SL) with von Hippel-Lindau (VHL) tumor suppressor gene. Inactivation of VHL has been frequently found in clear cell renal cell carcinoma (ccRCC). Its SL partners serve as potential drug targets for the development of targeted cancer therapies.\n\nResultsWe performed parallel genome-wide CRISPR screens in two pairs of isogenic ccRCC cell lines that differ only in the VHL status. Comparative analyses of screening results not only confirmed a well-known role for mTOR signaling in renal carcinoma, but also identified DNA damage response and selenocysteine biosynthesis pathways as major SL targets in VHL-inactivated cancer cells. Follow-up studies provided cellular and mechanistic insights into SL interactions of these pathway genes with the VHL gene.\n\nConclusionsUsing isogenic CRISPR screening approach, we uncovered novel biological processes that are SL with VHL, which can be exploited for drug development for ccRCC. Our CRISPR and RNA-seq datasets provide a rich resource for future investigation of the function of the VHL tumor suppressor protein. Our work demonstrates the efficiency of CRISPR-based synthetic lethality screening in human isogenic cell pairs. Similar strategies could be employed to unveil SL partners with other oncogenic drivers.

cancer biology↗

bioRxiv2018

Dynamin is required for efficient cytomegalovirus maturation and envelopment

Cytomegalovirus secondary envelopment occurs in a virus-induced cytoplasmic assembly compartment (vAC) generated via a drastic reorganization of the membranes of the secretory and endocytic systems. Dynamin is a eukaryotic GTPase that is implicated in membrane remodeling and endocytic membrane fission events; however, the role of dynamin in cellular trafficking of viruses beyond virus entry is only partially understood. Mouse embryonic fibroblasts (MEF) engineered to excise all three isoforms of dynamin were infected with mouse cytomegalovirus (MCMV-K181). Immediate early (IE1; m123) viral protein was detected in these triple dynamin knockout (TKO) cells as well as in mock-induced parental MEF at early times post infection although levels were reduced in TKO cells, indicating that virus entry was affected but not eliminated. Levels of IE1 protein and another viral early protein (m04) were normalized by 48 hours post infection; however, late protein (m55; gB) expression was significantly reduced in infected TKO cells compared to parental MEF. Ultrastructural analysis revealed intact stages of nuclear virus maturation in both cases with equivalent numbers of nucleocapsids containing packaged viral DNA (C-capsids) indicating successful viral DNA replication, capsid assembly and genome packaging. Most importantly, severe defects in virus envelopment were visualized in TKO cells but not in parental cells. Dynamin inhibitor (dynasore) treated MEF showed a phenotype similar to TKO cells upon MCMV infection confirming the role of dynamin in late maturation processes. In summary, dynamin-mediated endocytic pathways are critical for the completion of cytoplasmic stages of cytomegalovirus maturation.\n\nImportanceViruses are known to exploit specific cellular functions at different stages of their life cycle in order to replicate, avoid immune recognition by the host and to establish a successful infection. Cytomegalovirus (CMV) infected cells are characterized by a prominent cytoplasmic inclusion (virus assembly compartment; vAC) that is the site of virus maturation and envelopment. While endocytic membranes are known to be the functional components of vAC, knowledge of specific endocytic pathways implicated in CMV maturation and envelopment is lacking. Here we show that dynamin, which is an integral part of host endocytic machinery, is largely dispensable for early stages of CMV infection but is required at a late stage of CMV maturation. Studies on dynamin function in CMV infection will help us understand the host-virus interaction pathways amenable to targeting by conventional small molecules as well as by newer generation nucleotide-based therapeutics (e.g. siRNA, CRISPR/CAS gRNA, etc.).

microbiology↗

bioRxiv2018

Microtubule-associated protein IQ67 DOMAIN5 regulates interdigitation of leaf pavement cells in Arabidopsis thaliana

Plant microtubules form a highly dynamic intracellular network with important roles for regulating cell division, cell proliferation and cell morphology. Its organization and dynamics are coordinated by various microtubule-associated proteins (MAPs) that integrate environmental and developmental stimuli to fine-tune and adjust cytoskeletal arrays. IQ67 DOMAIN (IQD) proteins recently emerged as a class of plant-specific MAPs with largely unknown functions. Here, using a reverse genetics approach, we characterize Arabidopsis IQD5 in terms of its expression domains, subcellular localization and biological functions. We show that IQD5 is expressed mostly in vegetative tissues, where it localizes to cortical microtubule arrays. Our phenotypic analysis of iqd5 loss-of-function lines reveals functions of IQD5 in pavement cell (PC) shape morphogenesis, as indicated by reduced interdigitation of neighboring cells in the leaf epidermis of iqd5 mutants. Histochemical analysis of cell wall composition further suggests reduced rates of cellulose deposition in anticlinal cell walls, which correlate with reduced asymmetric expansion. Lastly, we provide evidence for IQD5-dependent recruitment of calmodulin calcium sensors to cortical microtubule arrays. Our work thus identifies IQD5 as a novel player in PC shape regulation, and, for the first time, links calcium signaling to developmental processes that regulate multi-polar growth in PCs.\n\nHighlightMicrotubule-localized Arabidopsis IQ67 DOMAIN5 regulates pavement cell morphogenesis in the leaf epidermis and links calcium-calmodulin signaling to lobe initiation and asymmetric expansion during early phases of interdigitated cell growth.

plant biology↗