Biology subjects

Dixit, M.

Publications and source records attributed to Dixit, M..

14 recordsLinked to original sources

bioRxiv2026

SOX9-mediated G1 elongation confers reserve stem cell-associated injury resistance in human intestinal stem cells

Background & Aims Dynamic cell cycle control is critical for intestinal crypt maintenance and injury responses, yet genetic regulators driving these changes remain poorly defined. As reserve intestinal stem cells (rISCs) are often considered to be slowly-cycling and can resist replication-dependent injury, factors that restrain proliferation may confer cytoprotection. Here, we define SOX9 as a regulator of intestinal stem cell (ISC) cycling and injury resistance. Methods Primary human ISCs were engineered to tune SOX9 levels, visualize cell cycle state, and manipulate cell cycle regulators. Using this system, we tested how SOX9 dosage impacts stemness, differentiation, proliferative recovery after SOX9 washout, and survival after 5-FU-mediated injury. Transcriptional analyses identified candidate links between SOX9 levels and cell cycle control, which were functionally tested using inducible INK4A (CDKN2A) and Cyclin D2 (CCND2) ISC lines. Results SOX9 induction lengthens the cell cycle in a dose-dependent manner largely by elongating G1 phase through the INK4A-Rb pathway. The effects of high SOX9 levels repressing proliferation and stem cell activity are reversible. SOX9 induction protects against 5-FU toxicity. This protection is mimicked by INK4A overexpression or pharmacological G1 phase arrest and repressed by CCND2 induction. Conclusions These findings identify SOX9-mediated G1 elongation as a reversible cytoprotective program that confers key functional properties associated with rISCs: proliferative restraint, retained stem cell potential, and resistance to replication-dependent injury. This positions G1 length as a potential determinant of which crypt cells survive injury to act as reserve stem cells.

cell biology↗

bioRxiv2026

Insulin regulates lymphocyte traction on fibronectin-coated compliant substrates in a calcium-dependent manner.

Lymphocyte-extracellular matrix (ECM) interactions occur intermittently throughout the lymphocytes life cycle. Alterations in blood insulin levels following feeding modulates naive lymphocyte trafficking and adhesion to fibronectin via a pathway involving insulin-like growth factor-1 receptor (IGF-1R), phospholipase C gamma 1 (PLC-{gamma}1) and {beta}2 integrin activation. Lymphocytes exert traction forces, on the ECM during the process of extravasation. While these forces are essential for several homeostatic processes, the role of insulin in modulating lymphocyte-derived traction forces upon ECM adhesion is unknown. The aim of the current study was to investigate the effect of insulin on the traction generated by lymphocytes when adhered onto a fibronectin-coated substrate. Jurkat T-cells were placed on a fibronectin layer (50{micro}g/ml, 100{micro}m thickness) coated on polyacrylamide gels of stiffness 400Pa with red fluorescence beads as fiduciary markers. The cellular force generated by Jurkat T-cells was mapped using traction force microscopy. To elucidate the role of PLC-{gamma}1 in cellular force generation, the traction of Jurkat T-cells lacking PLC-{gamma}1, as well as those of a knockout cell where PLC-{gamma}1 was restored were quantified and compared with wild-type Jurkat T-cells. Lack of PLC-{gamma}1 attenuated adhesion when compared to wild-type Jurkat T-cells. Additionally, the traction force generated by each cell type decreased with increasing concentration of extracellular calcium. Treatment of adherent Jurkat T-cells with insulin increased traction in lower extracellular calcium condition while a dip was observed when a high extracellular calcium was present, in comparison to the untreated cells. However, the effect of insulin treatment was lost in the case of Jurkat T-cells lacking PLC-{gamma}1. Together these results indicate that insulin regulates traction force generated by adherent Jurkat T-cells via a process involving PLC-{gamma}1, in a calcium dependent manner.

immunology↗

bioRxiv2025

FRG1 Regulates Nonsense-Mediated mRNA Decay by Modulating UPF1 Levels

FRG1 acts as a molecular double-edged sword: while its overexpression is linked to facioscapulohumeral muscular dystrophy (FSHD), its under expression activates tumorigenic signalling pathways--highlighting the importance of tightly regulated FRG1 levels. Although prior studies have hinted at FRG1s involvement in RNA biogenesis, its core molecular functions have remained largely elusive. Our previous work identified FRG1 as a transcriptional regulator of genes in the nonsense-mediated decay (NMD) pathway; however, the underlying mechanisms were not fully delineated. In this study, using a range of cellular models with altered FRG1 expression, we demonstrate that reduced FRG1 levels enhance NMD activity. We reveal that FRG1 is a structural component of both the spliceosome and the exon junction complex (EJC), and that it directly modulates NMD by interacting with UPF1--regulating its ubiquitination and degradation. Furthermore, we show that DUX4 inversely regulates the NMD machinery through FRG1, establishing a critical molecular axis. Despite its structural association with the EJC and spliceosome, the absence of FRG1 does not compromise the integrity of these complexes. Polysome profiling showed that FRG1 co-sediments with eIF4A3, a core EJC component, across translating ribosomal fractions. This interaction was further supported by proximity ligation assays confirming the close spatial proximity of FRG1 and eIF4A3. Importantly, we validated the impact of FRG1 perturbation on NMD efficiency and UPF1 levels in vivo using a transgenic FRG1 knockout zebrafish model. Together, these findings establish a direct, DUX4-independent role for FRG1 in regulating the NMD pathway and uncover a previously unrecognized function for FRG1 in post-transcriptional gene regulation. Our work provides mechanistic insight into FRG1s molecular roles and lays the groundwork for therapeutic strategies targeting diseases associated with its dysregulation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/689056v2_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1ef00e1org.highwire.dtl.DTLVardef@22cd63org.highwire.dtl.DTLVardef@da4080org.highwire.dtl.DTLVardef@c0d283_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

molecular biology↗

bioRxiv2025

Nutritional Triggers of PCOS and NAFLD: A Comparative Analysis of Diets in Female C57BL/6J Mice

ObjectiveObesity is a key factor in the development of Polycystic Ovary Syndrome (PCOS) and Non- alcoholic fatty liver disease (NAFLD). Its relationship with reproductive health and metabolic disorders remains an area of interest. Here we aimed to study the effects of two different obesogenic diets in female C57BL/6J mice on their metabolic and reproductive function. MethodsFemale C57BL/6J mice were fed high-fat high-sugar (HFHS) (45% fat and 10% sugar) diet and High fat diet (60% fat) for 21 weeks to investigate the effects on metabolic health and reproductive health. Mice were assessed for body weight, abdominal circumference, glucose tolerance (OGTT) and estrous cyclicity. At the end of the study biochemical analysis was done on isolated serum and, histological assessment of organs including liver and ovarian tissue was done to score for cystic follicle development and steatosis respectively. ResultsMice on the HFHS diet did not gain weight but developed dyslipidaemia, abnormal liver enzyme levels and severe hepatic steatosis. They did not show impaired glucose tolerance and did not develop any PCOS-like ovarian morphology indicating hepatic metabolic disturbances without significant impact on reproductive health. In contrast, mice on the 60% high-fat diet (HFD) showed marginal increase in body weight and developed altered estrous cyclicity and PCOS-like ovarian morphology. They also exhibited impaired glucose tolerance and insulin resistance, confirming a metabolic disturbance associated with PCOS. These mice did not develop significant liver fibrosis, suggesting a more specific impact on the ovaries and glucose metabolism. Our findings suggest that diet composition plays a crucial role in determining the specific metabolic and reproductive outcomes in obesity-related disorders in females.

physiology↗

bioRxiv2024

Deletion of absent in melanoma (AIM) 2 gene alters bone morphology

Absent in Melanoma (AIM) 2 is a gene that is induced by interferon and acts as a cytosolic sensor for double-stranded (ds) DNA. It forms the AIM2 inflammasome, leading to the production of interleukin (IL)-1{beta} and IL-18. Our previous research demonstrated that mice lacking AIM2 exhibit spontaneous obesity, insulin resistance, and inflammation in adipose tissue. In this study, we aimed to explore the impact of AIM2 gene deletion on bone structure in adult and aged mice. Utilizing micro-computed tomography (micro-CT), we discovered that female mice lacking AIM2 showed an increase in the total cross-sectional area at 5 months of age, accompanied by an increase in cortical thickness in the mid-diaphysis of the femur at both 5 and 15 months of age. At 15 months of age, the cortical bone mineral density (BMD) significantly decreased in AIM2 null females compared to wild-type (WT) mice. In AIM2 null mice, both trabecular bone volume and BMD at the distal metaphysis of the femur significantly decreased at 5 and 15 months of age. Similarly, micro-CT analysis of the L4 vertebra revealed significant decreases in trabecular bone volume and BMD in aged AIM2 null females compared to WT mice. Histological examination of femurs from aged mice demonstrated increased bone marrow adiposity in AIM2 null mice, accompanied by a significant increase in CD45-/CD31-/Sca1+/Pdgfa+ adipose progenitor cells, and a decrease in the ratio of CD31-/CD31+ osteogenic progenitor cells, as determined by flow cytometry of bone marrow cells. Our findings suggest that AIM2 deficiency affects bone health by promoting adipogenesis in bone marrow cells and inducing a pro-inflammatory environment, potentially contributing to the decreased bone mineral density.

pathology↗

bioRxiv2023

Role of Tyrosine Phosphorylation in PTP-PEST

We study the influence of tyrosine phosphorylation on PTP-PEST, a cytosolic protein tyrosine phosphatase. Utilizing a combination of experimental data and computational modeling, specific tyrosine sites, notably Y64 and Y88, are identified for potential phosphorylation. Phosphorylation at these sites affects loop dynamics near the catalytic site, altering interactions among key residues and modifying the binding pockets size. This, in turn, impacts substrate binding, as indicated by changes in binding energy. Our findings provide insights into the structural and functional consequences of tyrosine phosphorylation on PTP-PEST, enhancing our understanding of its effects on substrate binding and catalytic conformation.

biophysics↗

bioRxiv2023

An in vitro platform for quantifying cell cycle phase lengths in primary human intestinal stem cells

Background and AimsThe intestinal epithelium exhibits dynamic control of cell cycle phase lengths, yet no experimental platform exists for directly analyzing cell cycle phases in living human intestinal stem cells (ISCs). Here, we develop primary human ISC lines with two different reporter constructs to provide fluorescent readouts to analyze cell cycle phases in cycling ISCs. Methods3D printing was used to construct a collagen press for making chamber slides that support primary human ISC growth and maintenance within the working distance of a confocal microscope objective. The PIP-FUCCI fluorescent cell cycle reporter and a variant with H2A-mScarlet that allows for automated tracking of cell cycle phases (PIP-H2A) were used in human ISCs along with live imaging and EdU pulsing. An analysis pipeline combining free-to-use programs and publicly available code was compiled to analyze live imaging results. ResultsChamber slides with soft collagen pressed to a thickness of 0.3 mm concurrently support ISC cycling and confocal imaging. PIP-FUCCI ISCs were found to be optimal for snapshot analysis wherein all nuclei are assigned to a cell cycle phase from a single image. PIP-H2A ISCs were better suited for live imaging since constant nuclear signal allowed for more automated analysis. CellPose2 and TrackMate were used together to track cycling cells. ConclusionsWe present two complete platforms for analyzing cell cycle phases in living primary human ISCs. The PIP-FUCCI construct allows for cell cycle phase assignment from one image of living cells, the PIP-H2A construct allows for semi-automated direct quantification of cell cycle phase lengths in human ISCs using our computational pipeline. These platforms hold great promise for future studies on how pharmaceutical agents affect the intestinal epithelium, how cell cycle is regulated in human ISCs, and more.

cell biology↗

bioRxiv2023

Targeting mitochondrial dysfunction using methylene blue or mitoquinone to improve skeletal aging

Declines in mitochondrial content and impaired cytochrome c oxidase activity (complex IV) can result in reduced energy metabolism and increased levels of oxidants. Methylene blue (MB) is a well-established antioxidant that has been shown to improve mitochondrial function in both in vitro and in vivo settings. Mitoquinone (MitoQ) is a selective antioxidant that specifically targets mitochondria and effectively reduces the accumulation of reactive oxygen species (ROS) within cells. To investigate the effect of acute and long-term administration of MB on skeletal morphology, we administered MB to aged (18 months old) female C57BL/J6 mice, as well as to adult male and female mice with a genetically diverse background (UM-HET3). Additionally, we used MitoQ as an alternative approach to target mitochondrial oxidative stress during aging in adult female and male UM-HET3 mice. Although we observed some beneficial effects of MB and MitoQ in vitro, the administration of these compounds in vivo did not alter the progression of age-induced bone loss. Specifically, treating 18-month-old female mice with MB for 6 or 12 months did not have an effect on age-related bone loss. Similarly, long-term treatment with MB from 7 to 22 months or with MitoQ from 4 to 22 months of age did not affect the morphology of cortical bone at the mid-diaphysis of the femur, trabecular bone at the distal-metaphysis of the femur, or trabecular bone at the lumbar vertebra-5 (L5) in UM-HET3 mice. Based on our findings, it appears that long-term treatment with MB or MitoQ alone, as a means to reduce skeletal oxidative stress, is insufficient to inhibit age-associated bone loss. This supports the notion that interventions solely with antioxidants may not provide adequate protection against skeletal aging.

pharmacology and toxicology↗

bioRxiv2022

In-silico identification of Tyr232 in AMPKα2 as a dephosphorylation site for the protein tyrosine phosphatase PTP-PEST

The AMP-activated protein kinase (AMPK) is known to be activated by the protein tyrosine phosphatase non-receptor type 12 (PTP-PEST) under hypoxic conditions. This activation is mediated by tyrosine dephosphorylation of the AMPK subunit. However, the identity of the phosphotyrosine residues remains unknown. In this study we first predicted the structure of the complex of the AMPK2 subunit and PTP-PEST catalytic domain using bioinformatics tools and further confirm the stability of the complex using molecular dynamics simulations. Evaluation of the protein-protein interfaces indicates that residue Tyr232 is the most likely site of dephosphorylation on AMPK2. In addition, we explored the effect of phosphorylation of PTP-PEST residue Tyr64 on the stability of the complex. The phosphorylation of Tyr64, an interface residue, enhances the stability of the complex via the rearrangement of a network of electrostatic interactions in conjunction with conformational changes in the catalytic WPD loop. Our findings present a plausible structural basis of AMPK regulation mediated by PTP-PEST and shows how phosphorylation of PTP-PEST could be involved in its activation.

biochemistry↗

bioRxiv2021

The SPICA Coarse-Grained Force Field for Proteins and Peptides

A coarse-grained (CG) model for peptides and proteins was developed as an extension of the SPICA (Surface Property fItting Coarse grAined) force field (FF). The model was designed to examine membrane proteins that are fully compatible with the lipid membranes of the SPICA FF. A preliminary version of this protein model was created using thermodynamic properties, including the surface tension and density in the SPICA (formerly called SDK) FF. In this study, we improved the CG protein model to facilitate molecular dynamics (MD) simulation with a reproduction of multiple properties from both experiments and all-atom (AA) simulations. The side chain analogs reproduced the transfer free energy profiles across the lipid membrane and demonstrated reasonable dimerization free energies in water compared to those from AA-MD. A series of peptides/proteins adsorbed or penetrated into the membrane simulated by the CG-MD correctly predicted the penetration depths and tilt angles of peripheral and transmembrane peptides/proteins comparable to those in the orientation of protein in membrane (OPM) database. In addition, the dimerization free energies of several transmembrane helices within a lipid bilayer were comparable to those from experimental estimation. Application studies on a series of membrane protein assemblies, scramblases, and poliovirus capsids demonstrated a good performance of the SPICA FF.

biophysics↗

bioRxiv2021

Oral glucose feeding enhances adherence of quiescent lymphocytes to fibronectin via non-canonical insulin signalling.

Impaired glucose metabolism is associated with chronic inflammation, aberrant immunity and anomalous leukocyte trafficking. Conversely, infusion of functional immune cells restores glucose metabolism. Despite being exposed to periodic alterations in blood insulin levels upon fasting and feeding, studies exploring the physiological effects of these hormonal changes on quiescent circulating lymphocytes are missing. Here we find that oral glucose load in healthy men and mice enhance adherence of circulating peripheral blood mononuclear cells (PBMCs) to fibronectin. This led to increased homing of post-load PBMCs to injured blood vessels. Cell culture based experiments on Jurkat-T cells and PBMCs demonstrated that insulin elicits these adhesive effects through a non-canonical signalling involving insulin growth factor-1 receptor (IGF-1R) and phospholipase C gamma-1 (PLC{gamma}-1) mediated activation of integrin {beta}1. Our findings point to the relevance of post-prandial insulin spikes in regulating homing of circulating T-cells to various organs for tissue repair and immunity. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/464163v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@11edce6org.highwire.dtl.DTLVardef@1389835org.highwire.dtl.DTLVardef@90c96eorg.highwire.dtl.DTLVardef@1988a63_HPS_FORMAT_FIGEXP M_FIG Insulin mediates fibronectin adherence of lymphocytes through non-canonical signalling. Insulin mediates auto-phosphorylation of IGF-1 receptor at Tyr1135 leading to activation of PLC-{gamma}1 through Tyr783 phosphorylation, which in turn leads to the activation of integrin 1 through intracellular calcium to ultimately enhance adhesion of quiescent lymphocytes to fibronectin. C_FIG

physiology↗

bioRxiv2020

Protein tyrosine phosphatase-PEST (PTP-PEST) mediates hypoxia-induced endothelial autophagy and angiogenesis through AMPK activation

Global and endothelial loss of PTP-PEST is associated with impaired cardiovascular development and embryonic lethality. Although hypoxia is implicated in vascular morphogenesis and remodelling, its effect on PTP-PEST remains unexplored. Here we report that hypoxia (1% oxygen)increases protein levels and catalytic activity of PTP-PEST in primary endothelial cells. Immunoprecipitation followed by mass spectrometry (LC/MS/MS) revealed that AMP-activated protein kinase alpha subunits (AMPK 1 and 2) interact with PTP-PEST under normoxia but not in hypoxia. Co-immunoprecipitation experiments confirmed this observation and determined that AMPK subunits interact with the catalytic domain of PTP-PEST. Knock-down of PTP-PEST abrogated hypoxia mediated tyrosine dephosphorylation and activation of AMPK (Thr172 phosphorylation). Absence of PTP-PEST also blocked hypoxia-induced autophagy (measured as LC3 degradation and puncta formation) which was rescued by AMPK activator, metformin (500{micro}M). Since endothelial autophagy is a pre-requisite for angiogenesis, knock-down of PTP-PEST also attenuated endothelial cell migration and capillary tube formation with autophagy inducer rapamycin (200nM) rescuing these effects. In conclusion, this work identifies for the first time PTP-PEST as a regulator of hypoxia-induced AMPK activation and endothelial autophagy to promote angiogenesis.

cell biology↗

bioRxiv2020

Altered kinetics of circulating progenitor cells in cardiopulmonary bypass (CPB) associated vasoplegic patients: An observational study

Vasoplegia observed post cardiopulmonary bypass (CPB) is associated with substantial morbidity, multiple organ failure and mortality. Circulating counts of hematopoietic stem cells (HSCs) and endothelial progenitor cells (EPC) are potential markers of neo-vascularization and vascular repair. However, the significance of changes in the circulating levels of these progenitors in perioperative CPB, and their association with post-CPB vasoplegia, are currently unexplored. We enumerated HSC and EPC counts, via flow cytometry, at different time-points during CPB in 19 individuals who underwent elective cardiac surgery. These 19 individuals were categorized into two groups based on severity of post-operative vasoplegia, a clinically insignificant vasoplegic Group 1 (G1) and a clinically significant vasoplegic Group 2 (G2). Differential changes in progenitor cell counts during different stages of surgery were compared across these two groups. Machine-learning classifiers (logistic regression and gradient boosting) were employed to determine if differential changes in progenitor counts could aid the classification of individuals into these groups. Enumerating progenitor cells revealed an early and significant increase in the circulating counts of CD34+ and CD34+CD133+ hematopoietic stem cells (HSC) in G1 individuals, while these counts were attenuated in G2 individuals. Additionally, EPCs (CD34+VEGFR2+) were lower in G2 individuals compared to G1. Gradient boosting outperformed logistic regression in assessing the vasoplegia grouping based on the fold change in circulating CD 34+ levels. Our findings indicate that a lack of early response of CD34+ cells and CD34+CD133+ HSCs might serve as an early marker for development of clinically significant vasoplegia after CPB.

physiology↗

bioRxiv2019

IQGAP2 acts as a tumor suppressor in breast cancer and its reduced expression promotes cancer growth and metastasis by MEK/ERK signalling pathways

IQGAP2 is a member of IQGAPs scaffolding protein family. It has been reported as a tumor suppressor in various cancers, as well as, an oncogene in some cancers, suggesting organ specific role. Need to identify therapeutic targets which function in ER/PR independent way, prompted us to explore role of IQGAP2 in molecular mechanism in breast cancer, which was completely unknown. In vitro studies in estrogen receptor positive breast cancer cell line (MCF7) showed that low IQGAP2 expression results in increased cell proliferation, migration and invasion of cells whereas an opposite effect was observed with ectopic expression of IQGAP2. Triple negative breast cancer cell line (MDA-MB-468), with IQGAP2 depletion showed similar effect, supporting its role in ER/PR independent manner. Furthermore, we found that reduced IQGAP2 expression induces the expression of EMT markers; twist and N-cadherin and decreases the expression of MET marker, E-cadherin via the MEK/ERK pathway but not via AKT pathway. Validation of findings in patients showed a reduced IQGAP2 expression in breast cancer tissues compared to normal tissue. Patients with low levels of IQGAP2 showed correlation with higher tumor stage. Our results suggest that IQGAP2 acts as a tumor suppressor and its down regulation results in cell growth, cell invasion and EMT through the MEK/ERK signalling pathways and it hence may be a potential therapeutic target in breast cancer.

cancer biology↗