Biology subjects

Choudhury, C.

Publications and source records attributed to Choudhury, C..

12 recordsLinked to original sources

bioRxiv2025

Plasma membrane folate transport in fungi and plants is mediated by members of the oligopeptide transporter (OPT) family

Folates are essential for all organisms. They are acquired either through de novo biosynthesis or from the diet. Yeast, fungi and plants make their own folates and it has not been clear if plasma membrane folate transporters exist in these organisms. Using a synthetic lethal screen in Saccharomyces cerevisiae we observed that deletions in a gene encoding the previously identified glutathione transporter, OPT1, was synthetically sick with a disruption in folate biosynthesis. Uptake experiments confirmed that Opt1p/Hgt1p can transport folinic acid and the naturally abundant methyl tetrahydrofolate. As S. cerevisiae Opt1p was able to transport both folate and glutathione, we used alanine-scanning mutants of the residues in the transmembrane domains of the channel to identify the residues required specifically for the uptake of folates and distinct from those required for glutathione. We further examined the oligopeptide transporter family of other organisms for the presence of folate transporters. In C. albicans, CaOPT1, the orthologue of S. cerevisiae OPT1 efficiently transported folate but not glutathione, while the previously characterized glutathione transporter, CaOPT7 could not transport folate. Aspergillus fumigatus has eight homologues of the oligopeptide transporter family, of which OptB and OptH could transport folates. In the plant Arabidopsis thaliana, the Opt1 homologs AtOpt2, AtOpt4, and AtOpt6 could transport folates. This discovery of folate transporters across fungi and plants fills a critical gap in our understanding of folate metabolism, and can benefit the exploitation of these pathways in pathogenic fungi, and in plants.

genetics↗

bioRxiv2025

A Humanized Mouse Model of Oral Squamous Cell Carcinoma for Investigating Resistance to Anti-PD1 Immunotherapy

Immunotherapy has transformed cancer therapeutics; however, its impact on oral squamous cell carcinoma (OSCC) remains limited due to acquired resistance, including to Pembrolizumab. To better understand this challenge, we develop a humanized mouse model using immune-deficient NCG mice engrafted with human peripheral blood mononuclear cells (hPBMC), followed by orthotopic implantation of head and neck squamous cell carcinoma (HNSCC) stem cells. This model closely mimics the human tumor-immune microenvironment, showing aggressive tumor growth and metastasis. Pembrolizumab treatment significantly decreases CD8+ T cells, dendritic cells, and MHC-class-I expression at tumor sites, which are related to immunotherapy resistance. Further, pembrolizumab treatment confirms the elevated PD-L1 levels, immune evasion pathways and downregulation of MHC-class-I. qPCR further validates human-specific HLA expression. Our model offers a valuable tool for studying immunotherapy resistance and advancing treatment strategies in OSCC and could be useful for other cancers.

cancer biology↗

bioRxiv2025

Unique TMJ-specific transcriptomic signature and its medial layer: Implications in osteoarthritis

ObjectivesOsteoarthritis (OA) is a debilitating joint disease that affects millions of people worldwide, with the temporomandibular joint (TMJ) and knee joint being prominently affected. Despite its prevalence, TMJ-OA remains understudied. This study aimed to investigate the transcriptional signature of the TMJ compared to that of the knee joint and to explore transcriptional differences in the medial and superficial layers of the TMJ-OA. DesignSix-month-old C57BL/6J mice TMJ and knee samples were collected. Goat TMJ superficial and medial layer cartilage was separated and treated with IL-1{beta}. All samples were subjected to bulk RNA sequencing followed by differential expression and gene set enrichment analysis. ResultsWe identified 4,031 protein-coding genes differentially expressed in the TMJ compared to the knee, with significant enrichment of neuronal system genes and lower enrichment of innate immune system genes. Key osteoarthritis biomarkers such as Mmp13, Postn, and Col1a1 were more highly expressed in the TMJ, indicating a higher vulnerability to OA development. IL-1{beta} treatment in goat TMJ chondrocytes mimicked the natural TMJ-OA-like transcriptional changes and immune responses, which are also observed in the rabbit TMJ-OA model. This validated the in vitro goat TMJ-OA model. The IL-1{beta}-treated goat TMJ medial cartilage layer was enriched in OA-associated transcription factors (TFs), senescence genes, and epigenetic regulators. ConclusionOur study demonstrated the unique transcriptomic signature of the TMJ compared with the knee joint, highlighting its vulnerability to OA and pain. These findings provide valuable insights into the molecular mechanisms of TMJ and offer a resource for potential therapeutic target selection for TMJ-OA treatment. HighlightsO_LISignificant enrichment of neuronal system genes and lower enrichment of innate immune system genes in temporomandibular joint. C_LIO_LIKey osteoarthritis biomarkers such as Mmp13, Postn, and Col1a1 have higher expression in temporomandibular joint, indicating a higher vulnerability to osteoarthritis development. C_LIO_LIInterleukin-1beta treatment in goat temporomandibular joint medial layer cartilage mimics natural temporomandibular joint osteoarthritis-like transcriptional changes and immune responses observed in rabbit temporomandibular joint osteoarthritis model. C_LI

molecular biology↗

bioRxiv2025

LOXL2 Deletion Triggers TMJ Osteoarthritis While Overexpression Protects Against NF-κB-Induced Chondrocyte Apoptosis

Temporomandibular joint osteoarthritis (TMJ-OA) affects a significant proportion of the population worldwide. However, there has been no substantial progress in the development of FDA-approved drugs for treatment due to a lack of understanding of the specific factors regulating key TMJ-OA molecular mechanisms. Lysyl Oxidase Like-2 (LOXL2) promotes knee joint cartilage protection, and it is downregulated in TMJ-OA animal model. We evaluated the role of LOXL2 in TMJ cartilage, its molecular mechanism and gene networks using in vivo Loxl2 knockout mice (Acan-Cre; Loxl2flox/flox) and ex vivo goat TMJ cartilage. Our results show that Loxl2 knockout in mice cartilage upregulates Il1b, Mmp9, Mmp13, Adamts4, and Adamts5, whereas it reduces the levels of aggrecan and proteoglycan. Loxl2 deleted TMJ cartilage show a higher enrichment of inflammatory response, TNFA signaling via NF-kB, extracellular matrix (ECM), and collagen degradation pathway network. Conversely, LOXL2 treatment reduces interleukin-1 beta (IL-1{beta})-induced expression of Mmp13, protects mitochondrial function and ECM from degeneration. Importantly, LOXL2 attenuates IL-1{beta}-induced chondrocyte apoptosis via phosphorylation of NF-{kappa}B and expression of pain-related gene PTGS2 (encodes COX2). Taken together, Loxl2 knockout mice exacerbate TMJ-OA through cartilage/ECM degradation, mitochondrial dysfunction, chondrocyte apoptosis, and inflammatory gene expression, whereas LOXL2 treatment mitigates these effects. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/653519v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@cb7540org.highwire.dtl.DTLVardef@17ebe2eorg.highwire.dtl.DTLVardef@1f7fd59org.highwire.dtl.DTLVardef@19fdd7_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

LOXL2 alleviates post-traumatic knee osteoarthritis and pain

Cartilage has limited potential for self-regeneration, and damage can lead to structural, molecular, and functional aberrations, leading to osteoarthritis (OA). Traumatic knee injuries can also lead to cartilage degeneration and post-traumatic OA (PTOA). This study aimed to explore whether lysyl oxidase-like 2 (LOXL2) deletion promotes PTOA-induced transcriptional changes similar to those in human OA, as well as the upregulation of collagen degradation, inflammation, and pain-related gene networks. LOXL2 was found to be downregulated in mouse knee PTOA. Aggrecan promotes specific deletion of Loxl2 in knee cartilage, shows OA-like molecular changes, and aggravates mouse PTOA. Furthermore, transcriptional analysis revealed the upregulation of cartilage degeneration factors, signatures of inflammatory M1 macrophages, and pain. These Loxl2 deleted PTOA mice have a molecular resemblance to the human knee OA pathogenic gene signature, which could lead to OA and pain. Interestingly, intra-articular injection of adenovirus-delivered LOXL2 rescued knee joint function, alleviated cartilage degeneration, restored treadmill-running capabilities, and reduced mechanical allodynia by relieving knee joint disability and pain. Taken together, LOXL2 deletion in mice knee promotes the severity PTOA, similar to human OA, implying its potential as a therapeutic candidate for human PTOA.

molecular biology↗

bioRxiv2025

LSD1 inhibition corrects dysregulated MHC-I and dendritic cells activation through IFNγ-CXCL9-CXCR3 axis to promote antitumor immunity in HNSCC

Poor infiltration of CD8+ T cells and dysregulated MHC-I confer resistance to anticancer clinical therapies. This study aimed to elucidate the mechanisms of lysine-specific demethylase 1 (LSD1, encoded by KDM1A gene) in antitumor immunity in Head and Neck Squamous cell carcinoma (HNSCC). LSD1 inhibition in syngeneic and chronic tobacco carcinogen-induced HNSCC mice recruited activated dendritic cells (DCs), CD4+ and CD8+ T cells, enriched interferon-gamma (IFN{gamma}) in T cells, CXCL9 in DCs, and CXCR3 in T cells, as evaluated using flow cytometry and single-cell RNA-seq analysis. Humanized HNSCC mice and TCGA data validated the inverse correlation of KDM1A with DC markers, CD8+ T cells, and their activating chemokines. Kdm1a knockout in mouse HNSCC and LSD1 inhibitor treatment to co-culture of human HNSCC cells with human peripheral blood mononuclear cells (PBMCs) resulted in MHC-I upregulation in cancer cells for efficient antigen presentation in tumors. Overall, LSD1 inhibition in tumor cells upregulates MHC class I and induces DCs to produce CXCL9, which in turn activates CD8+ T cells through the CXCL9-CXCR3 axis to produce IFN{gamma}. Finally, we identified a novel mechanism by which LSD1 inhibition promotes the activation of H3K4me2 and its direct interaction with MHC-I to induce antitumor immunity. This may have implications in poorly immunogenic and immunotherapy-resistant cancers. Statement of SignificanceLSD1-mediated unique mechanisms have impact on epigenetic therapy, MHC-I resistant HNSCC therapies, and poor CD8+ and dendritic cell infilterated tumors.

cancer biology↗

bioRxiv2025

Shared molecular regulation of quiescence in neural and glioma stem cells reveals therapeutic vulnerabilities.

Quiescence, a reversible state of cell-cycle arrest, is an adaptive feature of many adult tissue stem cells, including those in the adult brain. In gliomas, brain tumour stem cells that reside in a quiescent state preferentially survive chemotherapy and radiotherapy, highlighting their critical role in therapy resistance and disease progression. To date, it remains unclear whether the molecular programs governing these states are functionally conserved between neural stem cells and brain tumour stem cells. Here, we establish novel in vitro models to study quiescence and find that glioma stem cells are markedly more resistant to entering quiescence than neural stem cells, suggesting that glioma stem cell quiescence more closely resembles a slow-cycling phenotype or shallow quiescence. Nonetheless, direct comparison of quiescent neural stem cells and quiescent/slow-cycling glioma stem cells, as they transition towards proliferation, reveals conserved gene expression trajectories, indicating shared molecular mechanisms. Furthermore, we find that pathways influencing quiescence in neural stem cells exert similar effects in glioma stem cells, underscoring the functional parallels between these populations. Finally, we identify that inhibition of TGF-{beta} signalling might provide an avenue to improve current standard-of-care treatments by targeting quiescent glioma stem cells. Note on version 2This version corrects two figure errors and expands the Methods. The conclusions of are unchanged. ImagesIn version 1, debris adjacent to the organoid had been digitally removed from the drug-treated image in Figure 6D. The unmodified original is restored here. Images within Figures 3A and 6D have each been processed identically to one another. Scattered cellular debris was present to a similar extent in control and drug-treated samples. O_FIG O_LINKSMALLFIG WIDTH=153 HEIGHT=200 SRC="FIGDIR/small/634421v2_fig6.gif" ALT="Figure 6"> View larger version (51K): org.highwire.dtl.DTLVardef@19aef83org.highwire.dtl.DTLVardef@e2c1e1org.highwire.dtl.DTLVardef@126bf22org.highwire.dtl.DTLVardef@ce7acd_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 6:C_FLOATNO TGF--R1 inhibition drives GSCs out of a quiescent state. (A) QBC395 cells were cultured in BMP4 quiescence media plus inhibitors for three days. Graphs show percentage of proliferating GSCs (SOX2+Ki67+) over total GSCs (SOX2+). Data were normalised to mean of quiescence media alone. (B) QBC395 cells were cultured in palbociclib quiescence media plus inhibitors for three days. Graphs show proportion of proliferating GSCs (SOX2+Ki67+) over total GSCs (SOX2+). Data were first normalised to vehicle control for each independent experiment and then quiescence media alone. (C) Schematic of glioblastoma organoid generation. Experiments began within 1-2 weeks of derivation to maximise cellular heterogeneity. Protocol adapted from Jacob and colleagues (60). (D) Image of glioblastoma organoid treated with 30 {micro}M LY-364397 or DMSO vehicle (control) for three days, stained for SOX2 (cyan) and Ki67 (magenta). Dashed lines demarcate organoid boundary. Scale bar: 100 {micro}m. Scattered cellular debris was observed in both control and drug-treated samples to a similar extent. (E) Proportion of proliferating GSCs (SOX2+Ki67+) over total GSCs (SOX2+) after glioblastoma organoids were cultured in the presence of inhibitors. Data were normalised to mean of respective vehicle control. Graphs in (A, B, E) show mean {+/-} SEM. Experiments in (A, B) represent independent experiments on separate passages (n=3-4). Dots in (E) represent individual organoids. Statistics: one-way repeated measures ANOVA with Holm-Sidaks multiple comparisons test in (A, B). Two-way ANOVA in (E) with multiple comparisons t-test. *P < 0.05, **P < 0.01, ***P < 0.001. Panel C created with BioRender (Agreement number: FY289ELRZ5). C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=52 SRC="FIGDIR/small/634421v2_fig3.gif" ALT="Figure 3"> View larger version (21K): org.highwire.dtl.DTLVardef@1e04d0corg.highwire.dtl.DTLVardef@e25e26org.highwire.dtl.DTLVardef@1934ab0org.highwire.dtl.DTLVardef@10c77fb_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 3:C_FLOATNO BMP4 and palbociclib treatment induces patient-derived GSCs into shallow versus deep states of quiescence, respectively. (A) Treatment with 16 ng/mL BMP4 markedly reduces the proportion of proliferating NSCs (SOX2+Ki67+) in primary adult mouse SVZ cultures. (B) Treatment with 16 ng/mL BMP4 modestly reduces the proportion of proliferating GSCs (SOX2+Ki67+) in primary patient-derived cultures (QBC395 cell line). (C) Concentration-response curve of mouse NSCs and patient-derived GSCs (QBC395 cell line) treated with BMP4, measuring proportion of proliferating stem cells (SOX2+Ki67+) relative to control media. Data were normalised to mean of control. (D) Palbociclib treatment markedly reduces the proportion of proliferating GSCs (SOX2+Ki67+) relative to control media. Data were normalised to mean of control. GSC graphs in (C, D) show mean {+/-} SEM of independent experiments conducted on separate passages (n=3-4). NSC graph in (C) shows mean {+/-} SEM of individual cell lines derived from SVZ of different mice (n=4). Scale bar: 15 {micro}m in (A, B). Cross in (D) represents concentration-response at 200 nM. Statistics in (C): two-way ANOVA reporting main effect of cell-line. C_FIG DataThe glioblastoma organoid data in Figure 6E have been updated following re-imaging and blinded re-analysis. Reported effect sizes have changed slightly; the conclusions are unchanged. Figure S2 has likewise been updated following re-imaging and re-analysis. MethodsThe Methods have been expanded to describe the analysis more fully, including data exclusion criteria, normalisation, and blinding. Minor changes to figure legends and the main text are not itemised.

cancer biology↗

bioRxiv2024

Sequential transcriptional programs underpin activation of quiescent hippocampal stem cells.

Postnatal neural stem cells are primarily quiescent, which is a cellular state that exists as a continuum from deep to shallow quiescence. The molecular changes that occur along this continuum are beginning to be understood but the transcription factor network governing these changes has not been defined. We show that these transitions are regulated by sequential transcription factor programs. Single-cell transcriptomic analyses of mice with loss- or gain-of-function of the essential activation factor Ascl1, reveal that Ascl1 promotes the activation of hippocampal neural stem cells by driving these cells out of deep quiescence, despite its low protein expression. Subsequently, during the transition from deep to shallow quiescence, Ascl1 induces the expression of Mycn, which drives progression through shallow states of quiescence towards an active state. Together, these results define the required sequence of transcription factors during hippocampal neural stem cell activation.

neuroscience↗

bioRxiv2024

Identification of Juglone, a "first-in-class" inhibitor of the human glutathione degrading enzyme, ChaC1, using yeast-based high throughput screens

The cytosolic glutathione-degrading enzyme, ChaC1, is highly upregulated in several cancers, with the upregulation correlating to poor prognosis. The ability to inhibit ChaC1 thus becomes important in pathophysiological situations where elevated glutathione levels would be beneficial. As no inhibitors of ChaC1 are known, in this study we have focussed on this goal. We have initially taken a computational approach where a systemic structure-based virtual screening was performed. However, none of the predicted hits proved to be effective inhibitors. We also evaluated synthetic substrate analogs, but these too were not inhibitory. As both these approaches targeted the active site, we shifted to developing two high-throughput, robust, yeast-based assays that were active site independent. A small molecule compound library was screened using an automated liquid handling system using these screens. The hits were further analyzed using in vitro assays. Among them, juglone, a naturally occurring naphthoquinone, completely inhibited ChaC1 activity with an IC50 of 8.7 {micro}M. It was also effective against the ChaC2 enzyme. Kinetic studies indicated that the inhibition was not competitive with the substrate. Juglone is known to form adducts with glutathione and is also known to selectively inhibit enzymes by covalently binding to their active site cysteine residues. However, juglone continued to inhibit a cysteine-free ChaC1 variant, indicating that it was acting through a novel mechanism. We evaluated different inhibitory mechanisms, and also analogues of juglone, and found plumbagin effective as an inhibitor. These compounds represent the first-in-class inhibitors of the ChaC enzymes discovered using a robust yeast screen.

biochemistry↗

bioRxiv2023

Arylamine N-acetyltransferase-1 reveals a subpopulation of ALS patients with altered metabolic capacity

Amyotrophic lateral sclerosis (ALS) is a heterogeneous disease characterised by metabolic changes at onset and throughout disease progression. Here, we investigate the role of arylamine N-acetyltransferase 1 (NAT1), a cytosolic protein associated with mitochondrial function, in ALS. We demonstrate that expression of the murine homolog (mNat2) increases in skeletal muscle of SODG93A mice, but not control animals, at onset of symptoms and remains elevated until end stage of the disease. Measurement of mitochondrial respiration in peripheral blood mononuclear cells of patients with ALS identified patient sub-populations with low and high metabolic potential, which was strongly associated with NAT1 activity. Those patients with high NAT1 activity had elevated basal respiration, ATP production, mitochondrial reserve, and aerobic glycolysis. NAT1 predicted increased whole body metabolic index, which may be clinically significant as these patients show increased functional decline and shorter survival. NAT1 may be a novel target in those patients with elevated activity.

biochemistry↗

bioRxiv2021

Synergistic collagen-condiment: Streptococcal collagen-like (Scl) protein in cell-adhesion and diabetic wound-closure matrix

Group A streptococcus (GAS), Streptococcus pyogenes manifests plethora of diseases through its explicit virulence factors. Among these, the recently deciphered MSCRAMMs, Streptococcal collagen-like (Scls) adhesins are most studied proteins in context of their biophysically stable collagenous-sequence (Gly-X-Y) despite the difference from analogous mammalian-collagen. Based on recent evidence on collagen-mimetic Scls, we elucidated biomaterial-potential of the unmodified, recombinant Scl1 (rScl1). Initially, rScl1 trimeric- assembly yielded its stability in silico than the monomeric-unit. Thereby, rScl1 matrix characterization was confirmed in vitro. rScl1 exhibited high A549 and HepG2 cell- viability--rScl1 dose incremented to 20.0 {micro}g/ml at time points up to 24 hr, and on 24 hr stored-dishes--deliberating it non-cytotoxic. Imploring cell-adhesion potential, we observed increased cell-counts tangential to rScl1-gradient. This affirmative prelude on rScl1 as a supporting-matrix cued its synergy to collagen; we discerned it through rScl1-augmented, full-thickness diabetic wound-closure in vivo and as a first, we studied > 18-month rabbit alloxan-models. We have ascertained re-epithelialization with higher type III collagen in absence of inflammation evidenced morphometrically and histologically. Finally, we correlated our observations through atomistic-evaluation of rScl1-2{beta}1-integrin interaction, surprisingly, with augmented binding-energy compared to collagen. Hence, connoting recombinant-streptococcal collagen as an alternate; with further characterization, rScl1 can potentiate important revelations conceding homogeneous and safe, bio-available, biomaterial.

microbiology↗

bioRxiv2020

Hybrid Pharmacophore Approach as a Novel Strategy to identify Anti-Mycobacterial Chemotypes: Investigation with DapB Enzyme

Antimicrobial resistance (AMR) is one of the most serious global public health threats as it compromises the successful treatment of deadly infectious diseases like tuberculosis. New therapeutics are constantly needed but it takes a long time and is expensive to explore new biochemical space. One way to address this issue is to repurpose the validated targets and identify novel chemotypes that can simultaneously bind to multiple binding pockets of these targets as a new lead generation strategy. This study reports such a strategy, dynamic hybrid pharmacophore model (DHPM), which represents the combined interaction features of different binding pockets contrary to the conventional approaches, where pharmacophore models are generated from single binding sites. We have considered Mtb-DapB, a validated mycobacterial drug target, as our model system to explore the effectiveness of DHPMs to screen novel unexplored compounds. Mtb-DapB has a cofactor binding site (CBS) and an adjacent substrate binding site (SBS). Four different model systems of Mtb-DapB were designed where, either NADPH/NADH occupies CBS in presence/absence of an inhibitor 2, 6-PDC in the adjacent SBS. Two more model systems were designed, where 2, 6-PDC was linked to NADPH and NADH to form hybrid molecules. The six model systems were subjected to 200ns molecular dynamics simulations and trajectories were analyzed to identify stable ligand-receptor interaction features. Based on these interactions, conventional pharmacophore models (CPM) were generated from the individual binding sites while DHPMs were created from hybrid-molecules occupying both binding sites. A huge library of 15, 63,764 publicly available molecules were screened by CPMs and DHPMs. The screened hits obtained from both types of models were compared based on their Hashed binary molecular fingerprints and 4-point pharmacophore fingerprints using Tanimoto, Cosine, Dice and Tversky similarity matrices. Molecules screened by DHPM exhibited significant structural diversity, better binding strength and drug like properties as compared to the compounds screened by CPMs indicating the efficiency of DHPM to explore new chemical space for anti-TB drug discovery. The idea of DHPM can be applied for a wide range of mycobacterial or other pathogen targets to venture into unexplored chemical space.

bioinformatics↗