Biology subjects

Kaur, K.

Publications and source records attributed to Kaur, K..

27 records · Page 2Linked to original sources

bioRxiv2022

Distinct airway progenitor cells drive epithelial heterogeneity in the developing human lung

Recent advances using single cell genomic approaches have identified new epithelial cell types and uncovered cellular heterogeneity in the murine and human lung (1). Here, using scRNA-seq and microscopy we identify and describe a secretory-like cell that is enriched in the small airways of the developing human lung and identified by the unique co-expression of SCGB3A2/SFTPB/CFTR. To place these cells in the hierarchy of airway development, we apply a single cell barcode-based lineage tracing method track the fate of SCGB3A2/SFTPB/CFTR cells during airway organoid differentiation in vitro (2). Lineage tracing revealed that these cells have distinct developmental potential from basal cells, giving rise predominantly to pulmonary neuroendocrine cells (PNECs) and a subset of multiciliated cells distinguished by high C6 and low MUC16 expression. We conclude that SCGB3A2/SFTPB/CFTR cells act as a progenitor cell contributing to the cellular diversity and heterogeneity in the developing human airway. SIGNIFICANCE STATEMENTThe current study identifies a novel secretory cell type that is present predominantly in the small airway of the developing human lung. These secretory cells are defined by co-expression of SCGB3A2/SFTPB/CFTR, and functional studies show that this cell gives rise to pulmonary neuroendocrine cells and a sub-population of multiciliated cells, thereby leading to cellular heterogeneity.

developmental biology↗

bioRxiv2022

Metabolic switching and cell wall remodelling of Mycobacterium tuberculosis during bone tuberculosis

Bone tuberculosis is widely characterized by irreversible bone destruction caused by Mycobacterium tuberculosis. Mycobacterium has the ability to adapt to various environmental stresses by altering its transcriptome in order to establish infection in the host. Thus, it is of critical importance to understand the transcriptional profile of M. tuberculosis during infection in the bone environment compared to axenic cultures of exponentially growing M.tb. In the current study, we characterized the in vivo transcriptome of M. tuberculosis within abscesses or necrotic specimens obtained from patients with bone TB using whole genome microarrays in order to gain insight into the M. tuberculosis adaptive response within this host microenvironment. A total of 914 mycobacterial genes were found to be significantly over-expressed and 1688 were repressed (fold change>2; p-value [≤]0.05) in human bone TB specimens. Overall, the mycobacteria displayed a hypo-metabolic state with significant (p[≤]0.05) downregulation of major pathways involved in translational machinery, cellular and protein metabolism and response to hypoxia. However, significant enrichment (p [≤]0.05) of amino-sugar metabolic processes, membrane glycolipid biosynthesis, amino acid biosynthesis (serine, glycine, arginine and cysteine) and accumulation of mycolyl-arabinogalactan-peptidoglycan complex suggests possible mycobacterial survival strategies within the bone lesions by strengthening its cell wall and cellular integrity. Data were also screened for M.tb virulence proteins using Virulent Pred and VICM Pred tools, which revealed five genes (Rv1046c, Rv1230c, DppD, PE_PGRS26 and PE_PGRS43) with a possible role in the pathogenesis of bone TB. Next, an osteoblast cell line model for bone TB was developed allowing for significant intracellular multiplication of M.tb. Interestingly, three virulence genes (Rv1046c, DppD and PE_PGRS26) identified from human bone TB microarray data were also found to be overexpressed by intracellular M. tuberculosis in osteoblast cell lines. Overall, these data demonstrate that M. tuberculosis alters its transcriptome as an adaptive strategy to survive in the host and establish infection in bone. Additionally, the in vitro osteoblast model we describe may facilitate our understanding of the pathogenesis of bone TB. Author SummaryMusculoskeletal tuberculosis is the third most common manifestation of extra-pulmonary tuberculosis and massive bone destruction along with vertebral discs are one of the hallmarks of this disease. Mycobacterium tuberculosis, the causative agent, has the tremendous potential to adapt itself to different host environments due to its ability to alter the expression of genes/proteins belonging to different pathways. This study shows that the mycobacterial infection in bone is driven by the increased expression of genes belonging to cell wall remodelling and DNA damage repair pathways important for its survival. Further data analysis showed that some of these genes are coding for proteins possessing virulence potential that may be essential for survival of M. tuberculosis under such hostile environment of bone. We also developed an in vitro model of bone tuberculosis using an osteoblast cell line and validated the expression of these virulence factors. Identification of such virulence factors in the bone environment by M. tuberculosis may aid to identify new therapeutic targets for bone TB. Further, development of cell line model for bone TB is important to understand some unknown facets of this disease.

microbiology↗

bioRxiv2021

A modular protein subunit vaccine candidate produced in yeast confers protection against SARS-CoV-2 in non-human primates

Vaccines against SARS-CoV-2 have been distributed at massive scale in developed countries, and have been effective at preventing COVID-19. Access to vaccines is limited, however, in low- and middle-income countries (LMICs) due to insufficient supply, high costs, and cold storage requirements. New vaccines that can be produced in existing manufacturing facilities in LMICs, can be manufactured at low cost, and use widely available, proven, safe adjuvants like alum, would improve global immunity against SARS-CoV-2. One such protein subunit vaccine is produced by the Serum Institute of India Pvt. Ltd. and is currently in clinical testing. Two protein components, the SARS-CoV-2 receptor binding domain (RBD) and hepatitis B surface antigen virus-like particles (VLPs), are each produced in yeast, which would enable a low-cost, high-volume manufacturing process. Here, we describe the design and preclinical testing of the RBD-VLP vaccine in cynomolgus macaques. We observed titers of neutralizing antibodies (>104) above the range of protection for other licensed vaccines in non-human primates. Interestingly, addition of a second adjuvant (CpG1018) appeared to improve the cellular response while reducing the humoral response. We challenged animals with SARS-CoV-2, and observed a ~3.4 and ~2.9 log10 reduction in median viral loads in bronchoalveolar lavage and nasal mucosa, respectively, compared to sham controls. These results inform the design and formulation of current clinical COVID-19 vaccine candidates like the one described here, and future designs of RBD-based vaccines against variants of SARS-CoV-2 or other betacoronaviruses.

immunology↗

bioRxiv2021

Engineered SARS-CoV-2 receptor binding domain improves immunogenicity in mice and elicits protective immunity in hamsters

Global containment of COVID-19 still requires accessible and affordable vaccines for low- and middle-income countries (LMICs).1 Recently approved vaccines provide needed interventions, albeit at prices that may limit their global access.2 Subunit vaccines based on recombinant proteins are suited for large-volume microbial manufacturing to yield billions of doses annually, minimizing their manufacturing costs.3 These types of vaccines are well-established, proven interventions with multiple safe and efficacious commercial examples.4-6 Many vaccine candidates of this type for SARS-CoV-2 rely on sequences containing the receptor-binding domain (RBD), which mediates viral entry to cells via ACE2.7,8 Here we report an engineered sequence variant of RBD that exhibits high-yield manufacturability, high-affinity binding to ACE2, and enhanced immunogenicity after a single dose in mice compared to the Wuhan-Hu-1 variant used in current vaccines. Antibodies raised against the engineered protein exhibited heterotypic binding to the RBD from two recently reported SARS-CoV-2 variants of concern (501Y.V1/V2). Presentation of the engineered RBD on a designed virus-like particle (VLP) also reduced weight loss in hamsters upon viral challenge.

molecular biology↗

bioRxiv2021

Synthesis, Physiochemical and Biological evaluation of Inclusion Complex of Benzyl Isothiocyanate encapsulated in cyclodextrins for triple negative breast cancer

Benzyl isothiocyanate (BITC), an organic dietary compound, is allied with a major role in the potential chemopreventive effects. BITC has acknowledged rising attention as a therapeutic compound to be used in medicine because of its high potency and characteristic biopharmaceutical properties, like high permeability with marginal aqueous solubility. The highly volatile and hydrophobic nature brought a need to provide a suitable delivery-matrix to BITC to exploit its pharmacological potential to the fullest. It has been successfully incorporated in {beta}-CD and HP-{beta}-CD using acoustic forces and thoroughly characterized using UV-vis spectroscopy, FTIR, DSC, TEM, and SAXS. The complexation helped in masking the acute odour, achieving a controlled release of BITC, and made its use viable by prolonging the retention time and thereby sustaining the biological effects. Different models like Higuchi, first-order kinetic decay, Korsmeyer-Peppas model were applied, suggesting a diffusion-controlled mechanism of release. Also, the bioaccessibility and stability of BITC in an in vitro digestion model was evaluated. The main objective of the present work was to systemically study the credibility of BITC-CD complexes in well-established tumor mimicking 2D cell culture models and produce a conclusive report on its chemotherapeutic activity. The in vitro anti-cancer activity of BITC and the formed sonochemical complexes was confirmed by MTT assay and further evaluated using apoptosis assay and production of ROS like moieties. Cell cycle analysis was done to evaluate the growth inhibitory mechanism of BITC. Strikingly, BITC and its complexes showcased ROS generation and lysosome-mediated cell death. Effect on cell migration was assessed using wound healing assay. The results promptly suggest the functional efficacy of the CDs in releasing BITC and attest the ability of the complexes to provide alternate to otherwise remedially sparse triple-negative breast cancer.

cancer biology↗

bioRxiv2020

Molecular engineering improves antigen quality and enables integrated manufacturing of a trivalent subunit vaccine candidate for rotavirus

BackgroundVaccines comprising recombinant subunit proteins are well-suited to low-cost and high-volume production for global use. The design of manufacturing processes to produce subunit vaccines depends, however, on the inherent biophysical traits presented by an individual antigen of interest. New candidate antigens typically require developing custom processes for each one and may require unique steps to ensure sufficient yields without product-related variants. ResultsWe describe a holistic approach for the molecular design of recombinant protein antigens--considering both their manufacturability and antigenicity--informed by bioinformatic analyses such as RNA-seq, ribosome profiling, and sequence-based prediction tools. We demonstrate this approach by engineering the product sequences of a trivalent non-replicating rotavirus vaccine (NRRV) candidate to improve titers and mitigate product variants caused by N-terminal truncation, hypermannosylation, and aggregation. The three engineered NRRV antigens retained their original antigenicity and immunogenicity, while their improved manufacturability enabled concomitant production and purification of all three serotypes in a single, end-to-end perfusion-based process using the biotechnical yeast Komagataella phaffii. ConclusionsThis study demonstrates that molecular engineering of subunit antigens using advanced genomic methods can facilitate their manufacturing in continuous production. Such capabilities have potential to lower the cost and volumetric requirements in manufacturing vaccines based on recombinant protein subunits.

bioengineering↗

bioRxiv2020

In silico analysis of SNPs in human phosphofructokinase, Muscle (PFKM) gene: An apparent therapeutic target of aerobic glycolysis and cancer

Phosphofructokinase, muscle (PFKM), a key glycolytic regulatory enzyme is a potential target for cancer therapeutic studies accredited to the employed inefficient phenomenon known as Warburg effect. PFKM is encoded by PFKM gene located at chromosome 12q13.11. Single nucleotide polymorphisms (SNPs) are known to profoundly affect gene expression and protein function. Therefore, the first attempt was made to computationally identify putative functional PFKM variants. These SNPs were further explored to find their probable association with different cancer types. A total of 9694 SNPs were retrieved from dbSNP database. Of which, only 85 validated SNPs with [≥]10% minor allele frequency (MAF) were subjected to analysis by softwares including Ensembl Genome browser, FuncPred (SNPinfo), regulomeDB (v 2.0), SIFT and PolyPhen-2. The relative analysis of output obtained classified the selected-SNPs into 11 highly prioritized (HP), 20 moderately prioritized and 54 not/poorly prioritized SNPs. The 11 HP-SNPs were found to have the highest likelihood of being functionally important, evidenced by previous association of rs2269935, rs11168417, rs11609399 and rs2228500 HP-SNPs with cachexia, lung and breast cancer. The study warrants further experiments to confirm the predictive role of prioritized SNPs in cancer etiology and also provides directions to fellow researchers.

cancer biology↗

bioRxiv2019

Competency assessment of the medical interns and nurses and prevailing practices to provide family planning services in teaching hospitals in three states of India.

ObjectivesThe objectives of the study was to assess the knowledge and skills of medical interns and nurses regarding family planning (FP) services, and document the prevailing FP practices in the teaching hospitals in India.\n\nStudy DesignA cross-sectional study was conducted in three states (Delhi, Rajasthan, and Maharashtra) of India, among randomly selected 163 participants, including medical interns (n=81) and in-service nurses (n=82), during 2017. Semi-structured, pre-tested interview schedule, was used to assess the knowledge and status of training received; and objective structured clinical examination (OSCE) based checklist was used to assess the skills.\n\nResultsAbout 60% of the interns and 48% of the nurses knew more than five contraceptives that could be offered to the clients. About 22% (11.1% interns and 33.3% nurses) respondents believed that contraceptives should not be given to a married woman coming alone, and 31.9% (17.3% interns and 46.3% nurses) respondents reported that it was illegal to provide contraceptives to unmarried people. Nearly 43.3% interns and 69.5% nurses refused to demonstrate intrauterine contraceptive device (IUCD) insertion in the dummy uterus as per OSCE, and among those who did, 12.3% interns and 18.3% nurses had failed. About 63% interns and 63.4% of nurses had observed IUCD insertion, and 12.3% interns and 17.1% had performed IUCD insertion, during their training.\n\nConclusionsKnowledge and skills of interns and nurses regarding FP services were only partial. The medical training during graduation or internship, and during the job, was found to be inadequate to provide quality FP services.\n\nImplicationsThe partial knowledge and skills of medical interns and nurses regarding family planning services indicated inadequate training received, and substandard quality of services rendered by them, which may put the universal access to sexual and reproductive health care services and rights in the developing countries at risk.

epidemiology↗

bioRxiv2017

Increased glutaminolytic flux and activation of mitochondrial metabolism by BCL2 hyperactivity in lymphoma

B-cell lymphoma 2 (BCL2) is an important apoptosis regulator during developmental and pathological states, and its overexpression is a key feature of several malignancies. Genomic data from The Cancer Genome Atlas (TCGA) reveals significant somatic copy number amplification, overexpression, and/or elevated protein activity of BCL2 in 50 % of diffuse large B-cell lymphoma (DLBC) patients. While its canonical role in mitochondria-directed apoptosis is well established, the effect of BCL2 on transcriptional and metabolic networks remains elusive. Using an established lymphocytic pro-B-cell line overexpressing BCL2, we identified dysregulated transcriptional and metabolic networks by transcriptomic profiling arrays. Elevated BCL2 levels affect transcription factor complexes and mitogenic programs of NF-{kappa}B/REL, HIF1A/ARNT, AP1, E2F, and STAT factors. Using stable isotope-assisted metabolic flux measurements we quantify that elevated BCL2 expression increases carbon utilization boosting cellular proliferation. Tumorigenic overexpression of BCL2 significantly increases glycolytic flux, glutaminolysis, and anaplerotic flux into the TCA cycle. At the same time, the mitochondrial acetyl-CoA pool is separated from the glycolytic one by inactivating the pyruvate dehydrogenase complex via transcriptional regulation of pyruvate dehydrogenase kinase (PDK3). As compensatory fuel, mitochondrial TCA cycle metabolism is supported by asparagine synthase (ASNS) and oxidative glutaminolysis creating targets for small molecule inhibition of glutaminase. Lymphoma cells overexpressing BCL2 contained more mitochondrial mass and were more sensitive to L-glutamine deprivation and glutaminase inhibition. Cells overexpressing a mutant BCL2 G145E, which is incapable of binding BH domain members, failed to increase proliferation, glycolysis, or glutaminolysis. Taken together, the oncogene BCL2 has the ability to ramp up a metabolic phenotype supporting proliferation independent of its anti-apoptotic role. The cellular model of BCL2 activation supports NF-KB-positive subtypes of DLBC and identifies metabolic bottlenecks with dependency on anaplerotic flux as an actionable BCL2 effector network in cancer.

systems biology↗