Biology subjects

Kumar, B.

Publications and source records attributed to Kumar, B..

34 records · Page 2Linked to original sources

bioRxiv2021

BMP signaling is necessary and sufficient for osteoarthritis and a target for disease modifying therapy

ObjectiveTo investigate the role of BMP signaling in osteoarthritis' pathogenesis and propose a disease-modifying therapy for OA. MethodsC57BL6/J mouse line was used to perform ACLT surgery at P120 to study the expression pattern of the BMP signaling readout pSMAD1/5/9. To investigate whether activation of BMP signaling is sufficient and necessary to induce osteoarthritis, we have used conditional GOF and LOF mouse lines in which BMP signaling can be activated or depleted, respectively, upon intra-peritoneal injection of tamoxifen. Finally, we locally inhibited BMP signaling through intra-articular injection of LDN-193189 pre- and post-onset surgically induced OA. Most of the analysis has been done through immunohistochemistry, histopathological staining, and micro-CT to evaluate the status of the pathogenesis of the disease. ResultsWe observed concomitant activation of BMP signaling, as judged by pSMAD1/5/9 immunoreactivity in the articular cartilage, upon induction of osteoarthritis with simultaneous depletion of SMURF1, an intra-cellular BMP signaling inhibitor in articular cartilage. Even without surgical induction of osteoarthritis, only BMP gain-of-function mutation induces OA in mouse articular cartilage. Also, genetic, or pharmacological inhibition of BMP signaling offered significant protection against OA pathogenesis. Interestingly, post-onset of the disease, inhibition of BMP signaling by intra-articular injection of LDN-193189 retarded OA progression with a significant reduction in inflammatory markers. ConclusionOur study demonstrated that BMP signaling plays an essential role in the pathogenesis of OA and that local BMP inhibition can be an effective therapeutic strategy to mitigate osteoarthritis.

developmental biology↗

bioRxiv2020

PRAK-03202: A triple antigen VLP vaccine candidate against SARS CoV-2

The rapid development of safe and effective vaccines against SARS CoV-2 is the need of the hour for the coronavirus outbreak. Here, we have developed PRAK-03202, the worlds first triple antigen VLP vaccine candidate in a highly characterized S. cerevisiae-based D-CryptTM platform, which induced SARS CoV-2 specific neutralizing antibodies in BALB/c mice. Immunizations using three different doses of PRAK-03202 induces antigen specific (Spike, envelope and membrane proteins) humoral response and neutralizing potential. PBMCs from convalescent patients, when exposed to PRAK-03202, showed lymphocyte proliferation and elevated IFN-{gamma} levels suggestive of conservation of epitopes and induction of T helper 1 (Th1)-biased cellular immune responses. These data support the clinical development and testing of PRAK-03202 for use in humans.

immunology↗

bioRxiv2020

CBX2 and CBX7 antagonistically regulate metabolic reprogramming in breast cancer

Striking similarity exists between metabolic changes associated with embryogenesis and tumorigenesis. Chromobox proteins-CBX2/4/6/7/8, core components of canonical polycomb repressor complex 1 (cPRC1), play essential roles in embryonic development and aberrantly expressed in breast cancer. Understanding how altered CBX expression relates to metabolic reprogramming in breast cancer may reveal vulnerabilities of therapeutic pertinence. Using transcriptomic and metabolomic data from breast cancer patients (N>3000 combined), we performed pathway-based analysis and identified outstanding roles of CBX2 and CBX7 in positive and negative regulation of glucose metabolism, respectively. Genetic ablation experiments validated the contrasting roles of two isoforms in cancer metabolism and cell growth. Furthermore, we provide evidence for the role of mTORC1 signaling in mediating contrary effects of CBX2 and CBX7 on breast cancer metabolism. Underpinning the biological significance of metabolic roles, CBX2 and CBX7 were found to be the most up- and down-regulated isoforms, respectively, in breast tumors compared to normal tissues. Moreover, CBX2 and CBX7 expression (not other isoforms) correlated strongly, but oppositely, with breast tumor subtype aggressiveness and the proliferation markers. Consistently, genomic data also showed higher amplification frequency of CBX2, not CBX7, in breast tumors. Highlighting the clinical significance of findings, disease-specific survival and drug sensitivity analysis revealed that CBX2 and CBX7 predicted patient outcome and sensitivity to FDA-approved clinical drugs. In summary, this work identifies novel cross-talk between CBX2/7 and breast tumor metabolism, and the results presented may have implications in strategies targeting breast cancer.

cancer biology↗

bioRxiv2020

HAHmiR.DB : A Server Platform For High Altitude Human miRNA-Gene Coregulatory Networks And Associated Regulatory-Circuits.

Rapid ascent to High Altitude (HA) can cause severe damage to body organs and may lead to many fatal disorders. During induction to HA, human body undergoes various physiological, biochemical, hematological and molecular changes to adapt to the extreme environmental conditions. Many literature references hint that gene-expression-regulation and regulatory molecules like microRNAs (miRNAs) and Transcription Factors (TFs) control adaptive responses during HA-stress. These biomolecules are known to interact in a complex combinatorial manner to fine-tune the gene expression and help in controlling the molecular responses during this stress and ultimately help in acclimatization. HAHmiR.DB (High-Altitude Human miRNA Database) is a unique, comprehensive, curated collection of miRNAs that have been experimentally validated to be associated with HA-stress; their level of expression in different altitudes, fold change, experiment duration, biomarker association, disease and drug association, tissue-specific expression level, Gene Ontology (GO) and Kyoto Encyclopaedia of Gene and Genomes (KEGG) pathway associations. As a server platform it also uniquely constructs and analyses interactive miRNA-TF-Gene coregulatory networks and extracts regulatory-circuits/Feed Forward Loops (FFLs) using in-house scripts. These regulatory circuits help to offer mechanistic insights in complex regulatory mechanisms during HA stress. The server can also build these regulatory networks between two and more miRNAs of the database and also identify the regulatory-circuits from this network. Hence HAHmiR.DB is the first-of its-kind database in HA research which a reliable platform to explore, compare, analyse and retrieve miRNAs associated with HA stress, their coregulatory networks and FFL regulatory circuits. HAHmiR.DB is freely accessible at http://www.hahmirdb.in

bioinformatics↗

bioRxiv2020

Multi-Antigenic Virus-like Particle of SARS CoV-2 produced in Saccharomyces cerevisiae as a vaccine candidate

Spike, Envelope and Membrane proteins from the SARS CoV-2 virus surface coat are important vaccine targets. We hereby report recombinant co-expression of the three proteins (Spike, Envelope and Membrane) in a engineered Saccharomyces cerevisiae platform (D-Crypt) and their self-assembly as Virus-like particle (VLP). This design as a multi-antigenic VLP for SARS CoV-2 has the potential to be a scalable vaccine candidate. The VLP is confirmed by transmission electron microscopy (TEM) images of the SARS CoV-2, along with supportive HPLC, Dynamic Light Scattering (DLS) and allied analytical data. The images clearly outline the presence of a "Corona" like morphology, and uniform size distribution.

biochemistry↗

bioRxiv2020

miRFFLDB - An integrated resource for human miRNA-Transcription Factor-Target Gene coregulatory networks and associated Feed Forward Loops.

Gene regulation is viewed as a complex process where regulatory elements and their targets form highly complex network interactions thus affecting normal biological physiology and also disease-initiation and progress. Transcription factors (TF) and microRNA (miRNA) are fundamental transcriptional and post-transcriptional regulators of the gene expression controlling important biological processes. In recent years, many high throughput studies revealed that the complex regulatory interactions are mediated by the complex interplay between miRNA and TF regulating a Target Gene (TG) in conjunction. miRNAs and TFs are also known to regulate each other. This complex coregulatory mechanism may be represented in the form of miRNA:TF:TG coregulatory network. This network can be used to identify several small recurring subgraphs known as regulatory-circuits. One of these regulatory-circuits also called the Feed-Forward Loops (FFLs) is a three-node pattern which is composed of a miRNA and a TF, one of which regulates the other and both jointly regulate a TG. These regulatory-circuits have proven useful in elucidating the complex interplay of gene regulation during many physiological and pathological conditions. Human.miRFFL.DB is a comprehensive integrated resource for human miRNA:TF:TG coregulatory directed networks and their associated regulatory-circuits. In-house scripts based on the graph theory principle have been used to identify both types of FFL motifs i.e. miRNA-FFL and TF-FFL. The database additionally provides an interactive visualization of the coregulatory networks and associated FFLs. Human.miRFFL.DB can be used as a comprehensive ready reference for human miRNA:TF:TG coregulatory networks and associated FFLs for decrypting complex cellular interactions of these regulatory biomolecules. Human.miRFFL.DB is available online at http://mirffldb.in/human/

bioinformatics↗

bioRxiv2020

Pervasive promoter hypermethylation of silenced TERT alleles in human cancers

In cancers, maintenance of telomeres often occurs through activation of the catalytic subunit of telomerase, encoded by TERT. Yet, most cancers show only modest levels of telomerase gene expression, even in the context of activating hotspot promoter mutations (C228T and C250T). The role of epigenetic mechanisms, including DNA methylation, in regulating telomerase gene expression in cancer cells is not fully understood. Here, we have carried out the most comprehensive characterization to date of TERT promoter methylation using ultra-deep bisulfite sequencing spanning the CpG island surrounding the core TERT promoter in 96 different human cell lines. In general, we observed that immortalized and cancer cell lines were hypermethylated in a region upstream of the recurrent C228T and C250T TERT promoter mutations, while non-malignant primary cells were comparatively hypomethylated in this region. However, at the allele-level, we generally observe hypermethylation of promoter sequences in cancer cells is associated with repressed expression, and the remaining unmethylated alleles marked with open chromatin are largely responsible for the observed TERT expression in cancer cells. Our findings suggest that hypermethylation of the TERT promoter alleles signals transcriptional repression of those alleles, leading to the attenuation of TERT activation in cancer cells. SIGNIFICANCEHypermethylation of the TERT promoter alleles to attenuate TERT activation in cancer cells may account for the modest activation of TERT expression in most cancers.

cancer biology↗

bioRxiv2019

Pan-cancer single cell RNA-seq uncovers recurring programs of cellular heterogeneity

Cultured cell lines are the workhorse of cancer research, but it is unclear to what extent they recapitulate the cellular heterogeneity observed among malignant cells in tumors, given the absence of a native tumor microenvironment. Here, we used multiplexed single cell RNA-seq to profile ~200 cancer cell lines. We uncovered expression programs that are recurrently heterogeneous within many cancer cell lines and are largely independent of observed genetic diversity. These programs of heterogeneity are associated with diverse biological processes, including cell cycle, senescence, stress and interferon responses, epithelial-to-mesenchymal transition, and protein maturation and degradation. Notably, some of these recurrent programs recapitulate those seen in human tumors, suggesting a prominent role of intrinsic plasticity in generating intra-tumoral heterogeneity. Moreover, the data allowed us to prioritize specific cell lines as model systems of cellular plasticity. We used two such models to demonstrate the dynamics, regulation and drug sensitivities associated with a cancer senescence program also observed in human tumors. Our work describes the landscape of cellular heterogeneity in diverse cancer cell lines, and identifies recurrent patterns of expression heterogeneity that are shared between tumors and specific cell lines and can thus be further explored in follow up studies.

cancer biology↗

bioRxiv2019

Disaggregated Level Child Morbidity in Bangladesh: An Application of Small Area Estimation Method

Acute respiratory infection (ARI) and diarrhoea are two major causes of child morbidity and mortality in Bangladesh. National and regional level prevalence of ARI and diarrhoea are calculated from nationwide surveys; however, prevalence at micro-level administrative units (say, district and sub-district) is not possible due to lack of sufficient data. In such case, small area estimation (SAE) methods can be applied by combining a survey data with a census data. Using a SAE method for dichotomous response variable, this study aims to estimate the proportions of under-5 children experienced with ARI and diarrhoea separately as well as either ARI or diarrhoea within a period of two-week preceding the survey. The ARI and diarrhoea information extracted from Bangladesh Demographic and Health Survey 2011 are used to develop a random effect logistic model for each of the indicators, and then the prevalence is estimated adapting the World Bank SAE approach for the dichotomous response variable using the 5% data of the Census 2011. The estimated prevalence of each indicator significantly varied by district and sub-district (1.4-11.3% for diarrhoea, 2.2-11.8% for ARI and 4.3-16.5% for ARI/diarrhoea at sub-district level). In a number of districts and sub-district, the proportions are found double the national level. District and sub-district levels spatial distributions of the indicators might help the policy makers to identify the vulnerable disaggregated and remote hotspots. Particularly, aid industries can provide effective interventions at the highly vulnerable spots to overcome the gaps between micro and macro level administrative units.

scientific communication and education↗

bioRxiv2019

AML ImmunoPET: 64Cu-DOTA-anti-CD33 PET-CT imaging of AML in Xenograft mouse model

PurposeAcute myeloid leukemia (AML) is a highly aggressive form of leukemia that results in poor survival outcomes. Currently, diagnosis and prognosis are based on invasive single-point bone marrow biopsies (iliac crest). There is currently no AML-specific non-invasive imaging method to assess disease in the whole body, including in extramedullary organs, representing an unmet clinical need. About 85-90% of human myeloid leukemia cells express CD33 (an accepted biomarker for AML) cell surface receptors, highlighting CD33 as an ideal candidate for AML immunoPET.\n\nExperimental DesignIn this study, we evaluated if 64Cu-DOTA-anti-CD33 murine monoclonal antibody (mAb) can be used for ImmunoPET-CT imaging of AML in preclinical model. For translational purpose, a humanized anti-CD33 antibody was produced, and after confirming its anti-CD33 PET-CT imaging ability we further explored its therapeutic potential.\n\nResults64Cu-DOTA-anti-CD33 based PET-CT imaging detected CD33+ AML in mice with high sensitivity (95.65%) and specificity (100%). In the entire skeletal system, CD33+ PET activity was significantly high in the bones of AML bearing mice over non-leukemic control mice; femur (p<0.00001), tibia (p=0.0001), humerus (p=0.0014), and lumber spine (p<0.00001). Interestingly, the imaging showed CD33+ PET activity prevelant in epiphysis/diaphysis of the bones, indicating spatial heterogeneity. Anti-CD33 therapy using newly developed humanized anti-CD33 mAb as an ADC (p=0.02) and 225Ac-anti-CD33-RIT (p<0.00001), significantly reduced disease burden over that of respective controls.\n\nConclusionWe have successfully developed a novel anti-CD33 immunoPET-CT based non-invasive imaging tool for diagnosis of AML and its spatial distribution, which indicates a preferential skeletal niche.\n\nTranslational RelevanceAcute myeloid leukemia (AML) exhibits extensive heterogeneity in therapeutic response, even in patients sharing similar characteristics, indicating the inadequacy of currently used biopsy-based prognostic tools. There is a critical need for non-invasive quantitative imaging modality specific to AML, which is able to assess this disease in the whole body, including in extramedullary organs and to longitudinally monitor treatment response. CD33, an accepted AML biomarker is expressed on more than 85-90% of blast cells and Mylotarg (anti-CD33-ADC) is FDA approved immunotherapy for AML. In this preclinical study using anti-human CD33 mAb (64Cu-DOTA-anti-CD33), a CD33 PET-CT imaging modality was developed that detected AML with high sensitivity and specificity. The approach also indicated spatial heterogeneity in leukemic disease, dependent on disease burden, which warrants caution in analyzing results from single point bone marrow biopsies. Therefore, this imaging modality can be used to non-invasively detect AML in humans and also monitor treatment response, an unmet clinical need.

cancer biology↗

bioRxiv2019

Development of Kinetic Modeling to Assess Multi-functional Vascular Response to Low Dose Radiation in Leukemia

Vascular permeability, tissue transfer rate (Ktrans), fractional extracellular tissue space ({nu}ec) and blood perfusion are crucial parameters to assess bone marrow vasculature (BMV) function. However, quantitative measurements of these parameters in a mouse model are difficult because of limited resolution of standard macroscopic imaging modalities. Using intravital multiphoton microscopy (MPM), live imaging of dextran transfer from BMV to calvarium tissue of mice bearing acute lymphoblastic leukemia (ALL) was performed to obtain BMV parameters. Mice bearing ALL had increased BMV permeability, altered Ktrans, increased {nu}ec, decreased blood perfusion, and increased BMV permeability resulting in reduced drug uptake. Targeted 2 Gy radiation therapy (RT) to mice bearing ALL increased local BMV perfusion and ALL chemotherapy uptake (P<0.0001 and P=0.0036, respectively), suggesting RT prior to chemotherapy treatment may increase treatment efficacy. Developed MPM techniques allow for a quantitative assessment of BMV functional parameters not previously performed with microscopic or macroscopic imaging.

cancer biology↗

bioRxiv2019

Quantitative multiplexed ChIP reveals global alterations that shape promoter bivalency in ground state embryonic stem cells

To understand the epigenetic foundation of naive pluripotent cells, we implement a quantitative multiplexed ChIP-Seq method (MINUTE-ChIP) comparing mouse ESC grown in 2i versus Serum conditions. Combined with quantitative western blot and mass spectrometry, we find compelling evidence for a broad H3K27me3 hypermethylation of the genome, concomitant with the widespread loss of DNA CpG methylation. We show that opposing action of EZH2 and JMJD3/UTX shape the H3K27me3 landscape, with almost all bivalent promoters stably retaining high H3K27me3 levels in 2i. In parallel, we show a mechanistically uncoupled, global decrease of H3K4me3 and strong reduction at bivalent promoters, suggesting that low H3K4me3 and high H3K27me3 levels at bivalent promoters safeguard naive pluripotency.

molecular biology↗

bioRxiv2018

The Cuon Enigma: Genome survey and comparative genomics of the endangered Dhole (Cuon alpinus)

The Asiatic wild dog is an endangered monophyletic canid restricted to Asia; facing threats from habitat fragmentation and other anthropogenic factors. Dholes have unique adaptations as compared to other wolf-like canids for large litter size (larger number of mammae) and hypercarnivory making it evolutionarily notable. Over evolutionary time, dhole and the subsequent divergent wild canids have lost coat patterns found in African wild dog. Here we report the first high coverage genome survey of Asiatic wild dog and mapped it with African wild dog, dingo and domestic dog to assess the structural variants. We generated a total of 124.8 Gb data from 416140921 raw read pairs and retained 398659457 reads with 52X coverage and mapped 99.16% of the clean reads to the three reference genomes. We identified ~13553269 SNVs, ~2858184 InDels, ~41000 SVs, ~1854109 SSRs and about 1000 CNVs. We compared the annotated genome of dingo and domestic dog with dhole genome sequence to understand the role of genes responsible in pelage pattern, dentition and mammary glands. Positively selected genes for these phenotypes were looked for SNP variants and top ranked genes for coat pattern, dentition and mammary glands were found to play a role in signalling and developmental pathways. Mitochondrial genome assembly predicted 35 genes, 11 CDS and 24 tRNA. This genome information will help in understanding the divergence of two monophlyletic canids, Cuon and Lycaon, and the evolutionary adaptations of dholes with respect to other canids.

genomics↗

bioRxiv2018

Functional Balance between TCF21-Slug defines phenotypic plasticity and sub-classes in high-grade serous ovarian cancer

Cellular plasticity and transitional phenotypes add to complexities of cancer metastasis initiated by single cell epithelial to mesenchymal transition or cooperative cell migration (CCM). We identified novel regulatory cross-talks between Tcf21 and Slug in mediating phenotypic and migration plasticity in high-grade serous ovarian adenocarcinoma. Live imaging discerned CCM as being achieved either through rapid cell proliferation or sheet migration. Transitional states were enriched over the rigid epithelial or mesenchymal phenotypes under conditions of environmental stresses. The Tcf21-Slug interplay identified in HGSC tumors through effective stratification of subtypes also contributed to class-switching in response to disease progression or therapy. Our study effectively provides a framework for understanding the relevance of cellular plasticity in situ as a function of two transcription factors.

cancer biology↗

bioRxiv2018

Effects of different load on physiological, hematological, biochemical, cytokines indices of Zanskar ponies at high altitude

High altitude people required high endurance pack animals for load carrying and riding at prevalent mountainous terrains and rugged region. So far no studies have been taken to evaluate effect of loads on physiology of ponies in high altitude region. So, in this view we evaluated variation in physiological, hematological, biochemical, and cytokines indices of Zanskar ponies during load carrying at high altitude. Total twelve (12) numbers of Zanskar ponies, mare, age 4-6 years, were divided into three groups; group-A (without load), group-B (60 kg), and group-C (80 kg) of back pack loads. Track was very narrow and slippery with gravel, uneven with rocky surface and has a steep gradient of 4 km uphill at altitude 3291 to 3500 m. When we evaluate these parameters, it is understood that the heart rate, pulse rate and respiration rate was significantly (p<0.05) increased in 80 kg group among the three groups. The hematology parameters viz. hemoglobin, PCV, lymphocytes, monocytes%, ESR and eosinophil% significantly (p<0.05) changed in 80 kg group after load carrying among the three groups which was followed by control and 60 kg group. In biochemical parameters viz. LA, LDH, TP, HK, CORT, T3, CRT, AST, CK-MB, GPx, FRAP and IL-6 significantly (p<0.05) changed in 80 kg group after load carrying among the three groups which was followed by control and 60 kg group. The ALT, ALB, GLB, UR and UA significantly (p<0.05) changed in 80 kg group before and after load carrying among the three groups which was followed by control and 60 kg group. It has been concluded that, this result has revealed strong correlation of change in biomarkers level with performance in ponies during load carry. Hence, these parameters might be use for performance of endurance of Zanskar ponies in high mountain region.

physiology↗

bioRxiv2018

Cell-type specific expression of oncogenic and tumor suppressive microRNAs in the human prostate and prostate cancer

MiR-1 and miR-143 are frequently reduced in human prostate cancer (PCa), while miR-141 and miR-21 are frequently elevated. Consequently, these miRNAs have been studied as cell-autonomous tumor suppressors and oncogenes. However, the cell-type specificity of these miRNAs is not well defined in prostate tissue. Through two different microdissection techniques, and droplet digital RT-PCR, we quantified these miRNAs in the stroma and epithelium of radical prostatectomy specimens. In contrast to their purported roles as cell-autonomous tumor suppressors, we found miR-1 and miR-143 expression to be predominantly stromal. Conversely, miR-141 was predominantly epithelial. MiR-21 was detected in both stroma and epithelium. Strikingly, the levels of miR-1 and miR-143 were significantly reduced in tumor-associated stroma, but not tumor epithelium. Gene expression analyses in human cell lines, tissues, and prostate-derived stromal cultures support the cell-type selective expression of miR-1, miR-141, and miR-143. Analyses of the PCa Genome Atlas (TCGA-PRAD) showed a strong positive correlation between stromal markers and miR-1 and miR-143, and a strong negative correlation between stromal markers and miR-141. In these tumors, loss of miR-1 and gain of miR-21 was highly associated with biochemical recurrence. These data shed new light on stromal and epithelial miRNA expression in the PCa tumor microenvironment.

cancer biology↗