Biology subjects

Pradhan, S.

Publications and source records attributed to Pradhan, S..

53 records · Page 3Linked to original sources

bioRxiv2022

Comparative Radiopathology of Male Reproductive Organs in Sprague Dawley Rats Administered with Curcuma longa and Ocimum sanctum Against Acute Gamma Irradiation

ObjectiveThe study was designed to evaluate the deleterious e{square}ects of acute lethal gamma irradiation on male reproductive organs of Sprague Dawley rats and the radioprotective potential by Curcuma longa and Ocimum sanctum in experimental set up. Methods25 Male Spraque Dawley rats were taken and randomized into 5 groups (T1, T2, T3, T4, T5). Each group containing 5 animals. The groups were named as Group T1 (Negative Control), Group T2 (irradiation positive control), Group T3 (irradiation and Curcuma longa), Group T4 (irradiation and Ocimum sanctum), Group T5 (irradiation, Curcuma longa and Ocimum sanctum). The rats in groups T1 and T2 were given a daily dosage of normal saline for 14 days. Group T3 and T4 rats received aqueous extracts of Curcuma longa (40 mg/kg) and Ocimum sanctum (40 mg/kg), respectively, whereas group T5 rats received a combination of aqueous extracts of Curcuma longa (40 mg/kg) and Ocimum sanctum (40 mg/kg) for 14 days. prior to gamma irradiation. Animals of group T2, T3, T4, T5 were irradiated with 10 Gy of {gamma} - radiation on 15th day. ResultsExposure of animals to 10 Gy gamma radiation resulted in significant decrease in body weight and testicular weight. Histopathological observations revealed testicular degenerative changes in the seminiferous tubules with primary spermatocytes being most affected and epididymis showed decreased spermatids density with cell debris in the lumen. Pretreatment with Curcuma longa at 40mg/kg for 14 days alleviated the radiation induced pathological changes in testis and epididymis. Curcuma longa demonstrated much better protective effect in combination with Ocimum sanctum in alleviating the radio pathological changes showing the possibility of synergistic effect. However, administration of Ocimum sanctum alone at 40mg/kg for 14 days did not reveal any significant radioprotective property after histomorphological evaluation. ConclusionGamma irradiation of 10 Gy induces severe radio pathological damage to organs of male reproductive system of Spraque Dawley rats. Pre-treatment with Curcuma longa prior to radiotherapy may be effective in preventing radiation-induced damage in the male reproductive organs. Better radioprotection can be achieved with the combination of Curcuma longa and Ocimum sanctum than using alone.

pathology↗

bioRxiv2022

Differential scanning fluorimetry as a measure of functionality of refolded anti-malarial antibody fragments

Transmission blocking monoclonal antibodies, 4B7 and 1245, targeting Pfs25, have been demonstrated to block the developmental stages of the malarial parasite inside the mosquitoes. In this work, anti-Plasmodium antibody fragments of 4B7 and 1245, designated as diabodies, were expressed in bacteria and refolded using a standardized method, with a yield of 0.1 and 0.4 mg per litre of culture, respectively. Dimeric species was detected for 4B7-Db, but not for 1245-Db, using glutaraldehyde cross-linking experiment. The quality of the purified refolded fraction was assessed with differential scanning fluorimetry (DSF). Refolded 4B7-Db, with a melting temperature of 59{degrees}C, recognized Pfs25 expressed on the surface of ookinete and zygote stages of Plasmodium in an immunofluorescence-based assay, whereas, 1245-Db, exhibiting a skewed melt profile, showed weak recognition. Further, refolded 4B7-Db recognized the linear epitope, on purified Pfs25 protein, both in the denatured and native state. Differential scanning fluorimetry can be potentially employed as a qualitative measure of functionality, to evaluate refolded proteins, with applicability for antibody engineering in passive immunization.

biochemistry↗

bioRxiv2022

Newcastle disease burden in Nepal and efficacy of Tablet I-2 vaccine in commercial and backyard poultry production

Poultry (Gallus domesticus) farming plays an important role as an income generating enterprise in a developing country like Nepal, contributing more than 4% to the national GDP. It is also one of the major sources of protein for growing population. Newcastle Disease (ND) is a major poultry disease affecting both commercial and backyard poultry production worldwide. There were more than 90 reported cases of ND outbreaks in Nepal in 2018, with over 74,986 birds being affected. ND might be responsible for over 7% of total poultry mortality in the country. Recent outbreak of ND in 2021 affected many farms throughout Nepal, and caused massive poultry production loss. ND is caused by a single stranded RNA virus which presents very similar clinical symptoms as Influenza A (commonly known as Bird flu), adding much complexity to clinical disease identification and intervention. We conducted a nationwide ND and Influenza A prevalence study, collecting samples from commercial and backyard poultry farms from across the major poultry production hubs of Nepal, and conducted both serological and molecular assessments-giving us disease exposure history and identification of floating strains of ND Virus (NDV). Of 600 commercial chickens tested from various farms, both NDV (n=381, 64%) and IA (n=125, 21%) antibodies were detected in the majority of the samples. In backyard chicken (n=108, 39 farms), sero-prevalence was also relatively high for both NDV (n=38, 35%) and IA (n=17, 16%). Out of the 40 commercial farms, majority had detectable NDV (n=31, 78%) and IA (n=15, 38%) virus present. In backyard farms (n=36), we also detected NDV (n=6, 16%) and IA (n=1, 3%) virus. We Genotyped (strain) detected NDV, and found Genotype II to be present in most of the commercial farms (which might be coming from live vaccine usage) and Genotype I in some backyard poultry samples. The identified Genotype I strain is reported for the first time, and hence could be an endemic NDV strain found in Nepal. Our 2021 ND outbreak investigation identified Genotype VII c as the causative strain. Additionally, we have developed a thermostable I-2 NDV vaccine (Ranigoldunga) in tablet formulation and tested on various (mixed) breeds of chicken (G. domesticus). This vaccine seems to be highly effective against NDV, including a virulent 2021 outbreak strain (Genotype VII c). The I-2 Tablet ND vaccine showed more than 85% efficacy when administered either ocularly or in water, and has a stability of 30 days in room temperature.

microbiology↗

bioRxiv2022

A general, highly efficient and facile synthesis of biocompatible rhodamine dyes and probes for live-cell multicolor nanoscopy

The development of live-cell fluorescence nanoscopy is powered by the availability of suitable fluorescent probes. Rhodamines are among the best fluorophores for labeling intracellular structures. Isomeric tuning is a powerful method for optimizing biocompatibility of the rhodamine-containing probes without affecting their spectral properties. However, the efficient synthesis pathway for rhodamine 4-isomers is still lacking. Herein, we present a facile protecting-group-free 4-carboxyrhodamines synthesis based on nucleophilic addition of lithium dicarboxybenzenide to the corresponding xanthone. This approach drastically reduces the number of synthesis steps and expands the achievable structural diversity, increases overall yields and permits a cheap gram-scale synthesis of the dyes. We prepared a wide range of symmetric and asymmetric 4-carboxyrhodamines covering the whole visible spectrum and targeted them to multiple structures in living cells - microtubules, DNA, actin, mitochondria, lysosomes, Halo-tagged and SNAP-tagged proteins. The enhanced permeability fluorescent probes operate at submicromolar concentrations allowing high contrast STED and confocal microscopy of living cells and tissues.

biochemistry↗

bioRxiv2021

Development of Web Application for the Comparison of Segment Variability with Sequence Evolution and Immunogenic Properties for Highly Variable Proteins: An Application to Viruses.

High rate of mutation and structural flexibilities in viral proteins quickly make them resistant to the host immune system and existing antiviral strategies. For most of the pathogenic viruses, the key survival strategies lie in their ability to evolve rapidly through mutations that affects the protein structure and function. Along with the experimental research related to antiviral development, computational data mining also plays an important role in deciphering the molecular and genomic signatures of the viral adaptability. Uncovering conserved regions in viral proteins with diverse chemical and biological properties is an important area of research for developing antiviral therapeutics, though assigning those regions is not a trivial work. Advancement in protein structural information databases and repositories, made by experimental research accelerated the in-silico mining of the data to generate more integrative information. Despite of the huge effort on correlating the protein structural information with its sequence, it is still a challenge to defeat the high mutability and adaptability of the viral genomics structure. In this current study, the authors have developed a user-friendly web application interface that will allow users to study and visualize protein segment variabilities in viral proteins and may help to find antiviral strategies. The present work of web application development allows thorough mining of the surface properties and variabilities of viral proteins which in combination with immunogenicity and evolutionary properties make the visualization robust. In combination with previous research on 20-Dimensional Euclidian Geometry based sequence variability characterization algorithm, four other parameters has been considered for this platform: [1] predicted solvent accessibility information, [2] B-Cell epitopic potential, [3] T-Cell epitopic potential and [4] coevolving region of the viral protein. Uniqueness of this study lies in the fact that a protein sequence stretch is being characterized rather than single residue-based information, which helps to compare properties of protein segments with variability. In current work, as an example, beside presenting the web application platform, five proteins of SARS-CoV2 was presented with keeping focus on protein-S. Current web-application database contains 29 proteins from 7 viruses including a GitHub repository of the raw data used in this study. The web application is up and running in the following address: http://www.protsegvar.com.

bioinformatics↗

bioRxiv2021

Poly ADP-ribosylation of SET8 leads to aberrant H4K20me1 domains in mammalian cells

In mammalian cells, SET8 mediated Histone H4 Lys 20 monomethylation (H4K20me1) has been implicated in regulating mitotic condensation, DNA replication, DNA damage response, and gene expression. Here we show SET8, the only known enzyme for H4K20me1 is post-translationally poly ADP-ribosylated by PARP1 on lysine residues. PARP1 interacts with SET8 in a cell cycle-dependent manner. Poly ADP-ribosylation on SET8 renders it catalytically compromised, and degradation via ubiquitylation pathway. Knockdown of PARP1 led to an increase of SET8 protein levels, leading to aberrant H4K20me1 and H4K20me3 domains genome-wide. H4K20me1 is associated with higher gene transcription levels while the increase of H4K20me3 levels was predominant in DNA repeat elements correlating with modulation of chromatin states. Hence, SET8 mediated chromatin remodeling in mammalian cells are modulated by poly ADP-ribosylation by PARP1.

molecular 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

Heterochromatin diversity modulates genome compartmentalization and loop extrusion barriers

Two dominant processes organizing chromosomes are loop extrusion and the compartmental segregation of active and inactive chromatin. The molecular players involved in loop extrusion during interphase, cohesin and CTCF, have been extensively studied and experimentally validated. However, neither the molecular determinants nor the functional roles of compartmentalization are well understood. Here, we distinguish three inactive chromatin states using contact frequency profiling, comprising two types of heterochromatin and a previously uncharacterized inactive state exhibiting a neutral interaction preference. We find that heterochromatin marked by long continuous stretches of H3K9me3, HP1 and HP1{beta} correlates with a conserved signature of strong compartmentalization and is abundant in HCT116 colon cancer cells. We demonstrate that disruption of DNA methyltransferase activity dramatically remodels genome compartmentalization as a consequence of the loss of H3K9me3 and HP1 binding. Interestingly, H3K9me3-HP1/{beta} is replaced by the neutral inactive state and retains late replication timing. Furthermore, we show that H3K9me3-HP1/{beta} heterochromatin is permissive to loop extrusion by cohesin but refractory to CTCF, explaining a paucity of visible loop extrusion-associated patterns in Hi-C. Accordingly, CTCF loop extrusion barriers are reactivated upon loss of H3K9me3-HP1/{beta}, not as a result of canonical demethylation of the CTCF binding motif but due to an intrinsic resistance of H3K9me3-HP1/{beta} heterochromatin to CTCF binding. Together, our work reveals a dynamic structural and organizational diversity of the inactive portion of the genome and establishes new connections between the regulation of chromatin state and chromosome organization, including an interplay between DNA methylation, compartmentalization and loop extrusion. HighlightsO_LIThree inactive chromatin states are distinguishable by long-range contact frequencies in HCT116, respectively associated with H3K9me3, H3K27me3 and a H3K9me2 state with neutral contact preferences. C_LIO_LIH3K9me3-HP1/{beta} heterochromatin has a high degree of homotypic affinity and is permissive to loop extrusion but depleted in extrusion barriers. C_LIO_LIDisrupting DNA methylation causes widespread loss of H3K9me3-HP1/{beta} and dramatic remodeling of genome compartmentalization. C_LIO_LIH3K9me3-HP1/{beta} is replaced by the neutral inactive state, which gains CTCF loop extrusion barriers and associated contact frequency patterns. C_LIO_LIDNA methylation suppresses CTCF binding via two distinct mechanisms. C_LI

genomics↗

bioRxiv2021

A high content lipidomics method using scheduled MRM with variable retention time window and relative dwell time weightage

Lipid compositions of cells, tissues and bio-fluids are complex, with varying concentrations and structural diversity, which makes their identification challenging. Newer methods for comprehensive analysis of lipids are thus necessary. Herein, we propose a targeted-mass spectrometry based method for large-scale lipidomics using a combination of variable retention time window and relative dwell time weightage. Using this, we detected more than 1000 lipid species, including structural isomers. The limit of detection varied from femtomolar to nanomolar range and the coefficient of variance <30% for 849 lipid species. We used this method to identify lipids altered due to Vitamin B12 deficiency and found that the levels of lipids with {omega}-3 fatty acid chains decreased while those with {omega}-6 increased. This method enables identification of by far the largest number of lipid species with structural isomers in a single experiment and would significantly advance our understanding of the role of lipids in biological processes.

biochemistry↗

bioRxiv2020

Universal NicE-seq for high resolution accessible chromatin profiling for formaldehyde fixed and FFPE tissues

Accessible chromatin plays a central role in gene expression and chromatin architecture. Current accessible chromatin approaches depend on limited digestion/cutting and pasting adaptors at the accessible DNA, thus requiring additional materials and time for optimization. Universal NicE-seq (UniNicE-seq) is an improved accessible chromatin profiling method that negate the optimization step and is suited to a variety of mammalian cells and tissues. Addition of 5-methyldeoxycytidine triphosphate during accessible chromatin labeling and an on-bead library making step substantially improved the signal to noise ratio while protecting the accessible regions from repeated nicking in cell lines, mouse T cells, mouse kidney, and human frozen tissue sections. We also demonstrate one tube UniNicE-seq for FFPE tissue section for direct NGS library preparation without sonication and DNA purification steps. These refinements allowed reliable mapping of accessible chromatin for high resolution genomic feature studies.

genetics↗

bioRxiv2020

Visualization and sequencing of accessible chromatin reveals cell cycle and post romidepsin treatment dynamics

Chromatin accessibility is a predictor of gene expression, cell division and cell type specificity. NicE-viewSeq (Nicking Enzyme assisted viewing and Sequencing) allows accessible chromatin visualization and sequencing with overall lower mitochondrial DNA and duplicated sequences interference relative to ATAC-see. Using NicE-viewSeq, we interrogated the accessibility of chromatin in a cell cycle (G1, S and G2/M) - specific manner using mammalian cells. Despite DNA replication and subsequent condensation of chromatin to chromosomes, chromatin accessibility remained generally preserved with minimal subtle alterations. Genome-wide alteration of chromatin accessibility within TSS and enhancer elements gradually decreased as cells progressed from G1 to G2M, with distinct differential accessibility near consensus transcription factors sites. Inhibition of histone deacetylases promoted accessible chromatin within gene bodies, correlating with apoptotic gene expression. In addition, reduced chromatin accessibility for the MYC oncogene pathway correlated with down regulation of pertinent genes. Surprisingly, repetitive RNA loci expression remained unaltered following histone acetylation-mediated increased accessibility. Therefore, we suggest that subtle changes in chromatin accessibility is a prerequisite during cell cycle and histone deacetylase inhibitor mediated therapeutics.

genetics↗

bioRxiv2019

EM-seq: Detection of DNA Methylation at Single Base Resolution from Picograms of DNA

Bisulfite sequencing is widely used to detect 5mC and 5hmC at single base resolution. However, bisulfite treatment damages DNA resulting in fragmentation, loss of DNA and biased sequencing data. To overcome this, we developed Enzymatic Methyl-seq (EM-seq), an enzymatic based approach that uses as little as 100 pg of DNA. EM-seq outperformed bisulfite converted libraries in all metrics examined including coverage, duplication, sensitivity and nucleotide composition. EM-seq libraries displayed even GC distribution, improved correlation across input amounts as well as increased representation of genomic features. These data indicate that EM-seq is more accurate and reliable than whole genome bisulfite sequencing (WGBS).

genomics↗

bioRxiv2019

Non-destructive enzymatic deamination enables single molecule long read sequencing for the determination of 5-methylcytosine and 5-hydroxymethylcytosine at single base resolution.

The predominant methodology for DNA methylation analysis relies on the chemical deamination by sodium bisulfite of unmodified cytosine to uracil to permit the differential readout of methylated cytosines. Bisulfite treatment damages the DNA leading to fragmentation and loss of long-range methylation information. To overcome this limitation of bisulfite treated DNA we applied a new enzymatic deamination approach, termed EM-seq (Enzymatic Methyl-seq) to long-range sequencing technologies. Our methodology, named LR-EM-seq (Long Range Enzymatic Methyl-seq) preserves the integrity of DNA allowing long-range methylation profiling of 5-mC and 5-hmC over several kilobases of genomic DNA. When applied to known differentially methylated regions (DMR), LR-EM-seq achieves phasing of over 5 kb resulting in broader and better defined DMRs compared to previously reported. This result demonstrated the importance of phasing methylation for biologically relevant questions and the applicability of LR-EM-seq for long range epigenetic analysis at single molecule and single nucleotide resolution.

genomics↗

bioRxiv2019

Transcription factor LSF facilitiates lysine methylation of α-tubulin by microtubule-associated SET8

Microtubules are critical for mitosis, cell motility, and protein and organelle transport, and are a validated target for anticancer drugs. However, tubulin regulation and recruitment in these cellular processes is less understood. Post-translational modifications of tubulin are proposed to regulate microtubule functions and dynamics. Although many such modifications have been investigated, tubulin methylations and enzymes responsible for methylation have only recently begun to be described. Here we report that N-lysine methyl transferase KMT5A (SET8/PR-Set7), which methylates histone H4K20, also methylates -tubulin. Furthermore, the transcription factor LSF binds both tubulin and SET8, and enhances -tubulin methylation in vitro, countered by FQI1, a specific small molecule inhibitor of LSF. Thus, the three proteins SET8, LSF, and tubulin, all essential for mitotic progression, interact with each other. Overall, these results point to dual functions for both SET8 and LSF not only in chromatin regulation, but also for cytoskeletal modification.

biochemistry↗

bioRxiv2018

Environmental programming of adult foraging behavior in C. elegans

Foraging strategies must be tuned to the availability and distribution of resources in the environment. This can occur over generations and lead to genetic differences in foraging behavior, or it can occur on shorter time scales within an individuals life span. Both genetic and experience-based strategies must be implemented by neural circuits that respond to environmental cues and track internal states, and the analysis of such circuits provides insight into the neural basis of complex decision making. In C. elegans, between-strain genetic differences and within-strain plasticity in foraging has been observed. Most individual changes in foraging are short-term, based on experience over several hours. Here, we tested if developmental experience could permanently alter foraging. We found that in wild strains that are normally highly exploratory, early-life starvation leads to \"cautious\" foraging behavior in which exploration is reduced. We characterize the behavioral bases for these strategies and identify changes in the dynamics of a locomotory circuit involved in navigation. Overall, we show that some C. elegans strains exhibit adaptive tuning of their foraging behavior based on early-life experience, and this is associated with changes in a core navigation circuit.

neuroscience↗

bioRxiv2017

Hypermethylation of human DNA: Fine-tuning transcription associated with development

Tissue-specific gene transcription can be affected by DNA methylation in ways that are difficult to discern from studies focused on genome-wide analyses of differentially methylated regions (DMRs). We studied 95 genes in detail using available epigenetic and transcription databases to detect and elucidate less obvious associations between development-linked hypermethylated DMRs in myoblasts (Mb) and cell-and tissue-specific expression. Many of these genes encode developmental transcription factors and display DNA hypermethylation also in skeletal muscle (SkM) and a few heterologous samples (e.g., aorta, mammary epithelial cells, or brain) among the 38 types of human cell cultures or tissues examined. Most of the DMRs overlapped transcription regulatory elements, including canonical, alternative, or cryptic promoters; enhancers; CTCF binding sites; and long-noncoding RNA (lncRNA) gene regions. Among the prominent relationships between DMRs and expression was promoter-region hypermethylation accompanying repression in Mb but not in many other repressed samples (26 genes). Another surprising relationship was down-modulated (but not silenced) expression in Mb associated with DNA hypermethylation at cryptic enhancers in Mb although such methylation was absent in both non-expressing samples and highly expressing samples (24 genes). The tissue-specificity of DNA hypermethylation can be explained for many of the genes by their roles in prenatal development or by the tissue-specific expression of neighboring genes. Besides elucidating developmental epigenetics, our study provides insights into the roles of abnormal DNA methylation in disease, e.g., cancer, Duchenne muscular dystrophy, and congenital heart malformations.

genomics↗

bioRxiv2017

Mechanochemical feedback and control of endocytosis and membrane tension

Plasma membrane tension is an important factor that regulates many key cellular processes. Membrane trafficking is tightly coupled to membrane tension and can modulate the latter by addition or removal of the membrane. However, the cellular pathway(s) involved in these processes are poorly understood. Here we find that, among a number of endocytic processes operating simultaneously at the cell surface, a dynamin and clathrin-independent pathway, the CLIC/GEEC (CG) pathway, is rapidly and specifically upregulated upon reduction of tension. On the other hand, inhibition of the CG pathway results in lower membrane tension, while up regulation significantly enhances membrane tension. We find that vinculin, a well-studied mechanotransducer, mediates the tension-dependent regulation of the CG pathway. Vinculin negatively regulates a key CG pathway regulator, GBF1, at the plasma membrane in a tension dependent manner. Thus, the CG pathway operates in a negative feedback loop with membrane tension which leads to a homeostatic regulation of membrane tension.

cell biology↗