Biology subjects

Krishnan, P.

Publications and source records attributed to Krishnan, P..

15 recordsLinked to original sources

bioRxiv2025

G Protein Coupled Estrogen Receptor Signaling Maintains β Cell Identity in Female Mice

Type 2 diabetes (T2D) arises in the context of obesity and overnutrition; however, additional demographic features including age and biological sex contribute to T2D risk. Estradiol (E2) is thought to play a protective metabolic role that may govern sex differences in the development of T2D. The mechanisms by which E2 exerts these effects and the impact of reduced E2 signaling in {beta} cells during menopause remain incompletely understood. We analyzed publicly available whole islet transcriptome datasets from female and male cadaveric donors and showed significant age-related modulation of gene expression, including changes in pathways related to {beta} cell function, in islets from female donors. Importantly, these patterns were not observed in islets from male donors. To test the in vivo relationship between E2 signaling and {beta} cell function, 10-week- old female C57BL6/J mice underwent an ovariectomy (OVX) or sham (CTR) surgery, followed by 4 weeks of high-fat diet (HFD) treatment. HFD-OVX mice exhibited obesity-induced glucose intolerance, increased cell mass, and reduced expression of {beta} cell identity markers. Furthermore, ex vivo treatment of islets with the G protein coupled estrogen receptor (GPER)- specific agonist G-1 restored {beta} cell identity gene expression. Together, these data identify a novel connection between GPER signaling and {beta} cell identity and suggest that menopausal loss of E2 signaling through GPER may be linked with loss of {beta} cell identity.

physiology↗

bioRxiv2024

Zymoseptoria tritici stealth infection is facilitated by stage-specific down-regulation of a β-glucanase

Plant cell walls constitute a major defence barrier against pathogens, although it is unclear how specific cell wall components impact pathogen colonisation. Pathogens secrete cell wall degrading enzymes (CWDEs) to facilitate plant colonisation, but damaged, infected cells are often a source of cell wall-derived oligosaccharides that trigger host immunity. The mechanisms by which pathogens minimize the release of cell wall-derived oligosaccharides while colonizing the host remain to be elucidated. We combined biochemical, molecular genetics and transcriptomic analyses to functionally characterize a glycoside hydrolase (ZtGH45) from the wheat pathogen Zymoseptoria tritici. ZtGH45 gene is expressed during the necrotrophic phase of the fungus, coinciding with an accumulation of wheat {beta}-1,3/1,4-mixed-linked glucan (MLG)-derived oligosaccharides. We show that overexpression of ZtGH45 enhances {beta}-1,3/1,4-glucan hydrolysis and the derived oligosaccharides trigger an immune response in wheat, which hinders Z. tritici virulence. The results demonstrate that tight regulation of ZtGH45 is critical for the infection process to prevent early accumulation of MLG oligosaccharides that would prematurely induce host immunity counterbalancing fungal virulence. We suggest that the balance between plant cell wall degradation by fungal CWDE and the release of immunogenic wall-derived oligosaccharides governs the outcome of host invasion by pathogens.

plant biology↗

bioRxiv2024

Pharmacological inhibition of tyrosine protein-kinase 2 reduces islet inflammation and delays type 1 diabetes onset in mice

Tyrosine protein-kinase 2 (TYK2), a member of the Janus kinase family, mediates inflammatory signaling through multiple cytokines, including interferon- (IFN), interleukin (IL)-12, and IL-23. Missense mutations in TYK2 are associated with protection against type 1 diabetes (T1D), and inhibition of TYK2 shows promise in the management of other autoimmune conditions. Here, we evaluated the effects of specific TYK2 inhibitors (TYK2is) in pre-clinical models of T1D. First, human {beta} cells, cadaveric donor islets, and iPSC-derived islets were treated in vitro with IFN in combination with a small molecule TYK2i (BMS-986165 or a related molecule BMS-986202). TYK2 inhibition prevented IFN-induced {beta} cell HLA class I up-regulation, endoplasmic reticulum stress, and chemokine production. In co-culture studies, pre-treatment of {beta} cells with a TYK2i prevented IFN-induced activation of T cells targeting an epitope of insulin. In vivo administration of BMS-986202 in two mouse models of T1D (RIP-LCMV-GP mice and NOD mice) reduced systemic and tissue-localized inflammation, prevented {beta} cell death, and delayed T1D onset. Transcriptional phenotyping of pancreatic islets, pancreatic lymph nodes (PLN), and spleen during early disease pathogenesis highlighted a role for TYK2 inhibition in modulating signaling pathways associated with inflammation, translational control, stress signaling, secretory function, immunity, and diabetes. Additionally, TYK2i treatment changed the composition of innate and adaptive immune cell populations in the blood and disease target tissues, resulting in an immune phenotype with a diminished capacity for {beta} cell destruction. Overall, these findings indicate that TYK2i has beneficial effects in both the immune and endocrine compartments in models of T1D, thus supporting a path forward for testing TYK2 inhibitors in human T1D.

physiology↗

bioRxiv2024

miR-146a-5p mediates inflammation-induced β cell mitochondrial dysfunction and apoptosis

We previously showed that miR-146a-5p is upregulated in pancreatic islets treated with pro-inflammatory cytokines. Others have reported that miR-146a-5p overexpression is associated with {beta} cell apoptosis and impaired insulin secretion. However, the molecular mechanisms mediating these effects remain elusive. To investigate the role of miR-146a-5p in {beta} cell function, we developed stable MIN6 cell lines to either overexpress or inhibit the expression of miR-146a-5p. Monoclonal cell populations were treated with pro-inflammatory cytokines (IL-1{beta}, IFN{gamma}, and TNF) to model type 1 diabetes (T1D) in vitro. We found that overexpression of miR-146a-5p increased cell death under conditions of inflammatory stress and led to mitochondrial membrane depolarization, whereas inhibition of miR-146a-5p reversed these effects. Additionally, inhibition of miR-146a-5p increased insulin secretion, mitochondrial DNA copy number, respiration rate, and ATP production Further, RNA sequencing data showed enrichment of pathways related to insulin secretion, apoptosis, and mitochondrial function when the expression levels of miR-146a-5p were altered. Finally, a temporal increase in miR-146a-5p expression levels and a decrease in mitochondria function markers was observed in islets derived from NOD mice. Collectively, these data suggest that miR-146a-5p may promote {beta} cell dysfunction and death during inflammatory stress by suppressing mitochondrial function.

molecular biology↗

bioRxiv2023

β Cell microRNAs Function as Molecular Hubs of Type 1 Diabetes Pathogenesis and as Biomarkers of Diabetes Risk

MicroRNAs (miRNAs) are small non-coding RNAs that play a crucial role in modulating gene expression and are enriched in cell-derived extracellular vesicles (EVs). We investigated whether miRNAs from human islets and islet-derived EVs could provide insight into {beta} cell stress pathways activated during type 1 diabetes (T1D) evolution, therefore serving as potential disease biomarkers. We treated human islets from 10 cadaveric donors with IL-1{beta} and IFN-{gamma} to model T1D ex vivo. MicroRNAs were isolated from islets and islet-derived EVs, and small RNA sequencing was performed. We found 20 and 14 differentially expressed (DE) miRNAs in cytokine-versus control-treated islets and EVs, respectively. Interestingly, the miRNAs found in EVs were mostly different from those found in islets. Only two miRNAs, miR-155-5p and miR-146a-5p, were upregulated in both islets and EVs, suggesting selective sorting of miRNAs into EVs. We used machine learning algorithms to rank DE EV-associated miRNAs, and developed custom label-free Localized Surface Plasmon Resonance-based biosensors to measure top ranked EVs in human plasma. Results from this analysis revealed that miR-155, miR-146, miR-30c, and miR-802 were upregulated and miR-124-3p was downregulated in plasma-derived EVs from children with recent-onset T1D. In addition, miR-146 and miR-30c were upregulated in plasma-derived EVs of autoantibody positive (AAb+) children compared to matched non-diabetic controls, while miR-124 was downregulated in both T1D and AAb+ groups. Furthermore, single-molecule fluorescence in situ hybridization confirmed increased expression of the most highly upregulated islet miRNA, miR-155, in pancreatic sections from organ donors with AAb+ and T1D. One Sentence SummarymiRNA expression patterns in human pancreatic islets and EVs change under inflammatory conditions and can be leveraged to inform biomarkers strategies for T1D.

physiology↗

bioRxiv2023

Polygenic pathogen networks control of host and pathogen transcriptional plasticity in the Arabidopsis-Botrytis pathosystem

Bidirectional flow of information shapes the outcome of the host-pathogen interactions and depends on the genetics of each organism. Recent work has begun to use co-transcriptomic studies to shed light on this bidirectional flow, but it is unclear how plastic the co-transcriptome is in response to genetic variation in both the host and pathogen. To study co-transcriptome plasticity, we conducted transcriptomics using natural genetic variation in the pathogen, Botrytis cinerea, and large effect genetic variation abolishing defense signaling pathways within the host, Arabidopsis thaliana. We show that genetic variation in the pathogen has a greater influence on the co-transcriptome than mutations that abolish defense signaling pathways in the host. Genome wide association mapping using the pathogens genetic variation and both organisms transcriptomes allowed an assessment of how the pathogen modulates plasticity in response to the host. This showed that the differences in both organisms responses were linked to trans-eQTL hotspots within the pathogens genome. These hotspots control gene sets in either the host or pathogen and show differential allele sensitivity to the hosts genetic variation rather than qualitative host specificity. Interestingly, nearly all the trans-eQTL hotspots were unique to the host or pathogen transcriptomes. In this system of differential plasticity, the pathogen mediates the shift in the co-transcriptome more than the host.

plant biology↗

bioRxiv2023

Zinc nano-fertilization enhances wheat productivity and biofortification

Zinc (Zn) malnutrition has emerged as one of the major health challenges in developing nations across the globe. Development of Zn management protocols in staple food crops using modern scientific tools to enhance Zn concentration in grains along with augmented crop yields became utmost necessary. In this context a 2-year experiment was carried out to assess the effects of zinc oxide nanoparticles (ZnO-NPs) vis-a-vis bulk zinc sulfate (ZnSO4) on wheat growth, yield and Zn concentration in plant parts. Four levels of application of ZnO-NPs (0, 20, 25 and 30 mg kg-1) were compared with ZnSO4 (equal to Zinc concentration in ZnO-NPs). Results revealed that seed vigor was significantly (p <0.05) higher under 25 and 30 mg kg-1 soil ZnO-NPs treatments over ZnSO4. Among the crop yield parameters such as tillers (plant-1), grain weight (plant-1), biomass (plant-1) and grain yield were significantly (p <0.05) higher under ZnO-NPs 25 mg kg-1 treated soil as compared to any other treatment. Zinc concentration in grains increased with dose of ZnO-NPs and it was significantly more than ZnSO4 treated soil at each treatment level. ZnO-NPs and ZnSO4 treatments did not affect photosynthetic rate and chlorophyll (SPAD) content significantly. In conclusion, 25 mg kg-1 ZnO-NPs application could be recommended in wheat cultivation to improve growth, yield and grain Zn biofortification.

plant biology↗

bioRxiv2022

Stromal Interaction Molecule 1 Maintains β Cell Identity and Function in Female Mice through Preservation of G Protein-Coupled Estrogen Receptor 1 Signaling

Loss of pancreatic {beta} cell mass, identity, and function contribute to the development of diabetes. Here, we show that the endoplasmic reticulum (ER) calcium sensor, stromal interaction molecule 1 (STIM1), is critical for the maintenance of {beta} cell function in female mice. When mice with {beta} cell-specific deletion of STIM1 (STIM1{Delta}{beta}) were challenged with high-fat diet, {beta} cell dysfunction was observed in female, but not male, mice. Impaired glucose tolerance was accompanied by reductions in {beta} cell mass, a concomitant increase in cell mass, and significant reductions in the expression of markers of {beta} cell maturity, including MafA and UCN3. Mechanistic assays demonstrated that the sexually dimorphic phenotype observed in STIM1{Delta}{beta} mice was due in part to loss of signaling through the noncanonical 17-{beta} estradiol receptor, GPER1. Together, these data suggest that STIM1 orchestrates pancreatic {beta} cell function and identity through GPER1-mediated estradiol signaling.

cell biology↗

bioRxiv2022

SERCA2 regulates proinsulin processing and processing enzyme maturation in the pancreatic β cell

Increased circulating levels of incompletely processed insulin (i.e. proinsulin) are observed clinically in both type 1 and type 2 diabetes; however, the mechanisms underlying impaired proinsulin processing remain incompletely understood. Here, we identify the sarcoendoplasmic reticulum Ca2+ ATPase-2 (SERCA2) pump and {beta} cell ER Ca2+ as key regulators of systemic glucose tolerance and proinsulin processing. We generated mice with a {beta} cell-specific SERCA2 deletion ({beta}S2KO) and SERCA2 deficient INS-1 cells to show that SERCA2 loss increases systemic and pancreatic levels of proinsulin protein and leads to aberrant localization of proinsulin within the proximal {beta} cell secretory pathway. These defects in proinsulin processing were linked to reduced maturation of the proinsulin processing enzymes PC1/3 and PC2, suggesting a model whereby chronic ER Ca2+ depletion in the {beta} cell, which is observed in many pathological conditions, impairs the spatial regulation of prohormone trafficking, processing, and maturation within the {beta} cell secretory pathway.

physiology↗

bioRxiv2021

A discovery-based proteomics approach identifies protein disulfide isomerase (PDIA1) as a biomarker of β cell stress in type 1 diabetes

BackgroundActivation of stress pathways intrinsic to the {beta} cell are thought to both accelerate {beta} cell death and increase {beta} cell immunogenicity in type 1 diabetes (T1D). However, information on the timing and scope of these responses is lacking. MethodsTo identify temporal and disease-related changes in islet {beta} cell protein expression, data independent acquisition-mass spectrometry was performed on islets collected longitudinally from NOD mice and NOD-SCID mice rendered diabetic through T cell adoptive transfer. FindingsIn islets collected from female NOD mice at 10, 12, and 14 weeks of age, we found a time-restricted upregulation of proteins involved in the maintenance of {beta} cell function and stress mitigation, followed by loss of expression of protective proteins that heralded diabetes onset. Pathway analysis identified EIF2 signaling and the unfolded protein response, mTOR signaling, mitochondrial function, and oxidative phosphorylation as commonly modulated pathways in both diabetic NOD mice and NOD-SCID mice rendered acutely diabetic by adoptive transfer, highlighting this core set of pathways in T1D pathogenesis. In immunofluorescence validation studies, {beta} cell expression of protein disulfide isomerase A1 (PDIA1) and 14-3-3b were found to be increased during disease progression in NOD islets, while PDIA1 plasma levels were increased in pre-diabetic NOD mice and in the serum of children with recent-onset T1D compared to age and sex-matched non-diabetic controls. InterpretationWe identified a common and core set of modulated pathways across distinct mouse models of T1D and identified PDIA1 as a potential human biomarker of {beta} cell stress in T1D.

physiology↗

bioRxiv2021

Loss of Secretory Pathway Ca2+ ATPase (SPCA1) Impairs Insulin Secretion and Reduces Autophagy in the Pancreatic Islet

The {beta} cell Golgi apparatus serves as a significant store of intracellular Ca2+ and an important site of proinsulin maturation. However, the contribution of Golgi Ca2+ to diabetes pathophysiology is unknown. The Golgi primarily utilizes the Secretory Pathway Ca2+ ATPase (SPCA1) to maintain intraluminal Ca2+ stores, and loss of SPCA1 has been linked to impaired Golgi function in other cell types. Here, we demonstrated that SPCA1 expression is decreased in islets from diabetic mice and human organ donors with type 2 diabetes, suggesting SPCA1 may impact diabetes development. INS-1 {beta} cells lacking SPCA1 (SPCA1KO) showed reduced intraluminal Golgi Ca2+ levels, reduced glucose-stimulated insulin secretion (GSIS), and increased insulin content. Islets from SPCA1 haploinsufficient mice (SPCA1+/-) exhibited reduced GSIS, altered glucose-induced Ca2+ oscillations, and altered insulin granule maturation. Autophagy can regulate granule homeostasis, therefore we induced autophagy with Torin1 and found that SPCA1KO cells and SPCA1+/- islets had reduced levels of the autophagosome marker LC3-II. Furthermore, SPCA1KO LC3-II were unchanged after blocking autophagy initiation or autophagolysosome fusion and acidification. Thus, we concluded that {beta} cell SPCA1 plays an important role in the maintenance of Golgi Ca2+ homeostasis and reduced Golgi Ca2+ impairs autophagy initiation and may impact insulin granule homeostasis.

cell biology↗

bioRxiv2020

Virtual experiments provide plausible explanations for IVIVE underpredictions of hepatic clearance

We explore a virtual experiment strategy to discover plausible mechanism-based explanations for frequently inaccurate predictions of hepatic drug clearance made using established in vitro-in vivo extrapolation methods. We describe a three-protocol plan that uses validated software analogs of real in vitro and in vivo systems constructed using a common set of objects, components, spaces, and methods. Both systems utilize identical quasi-autonomous hepatocyte analogs containing enzyme-like objects. We parameterize concrete mobile objects (virtual compounds) to simulate the referent drugs disposition and removal characteristics in vitro and in vivo. The goal of Protocol one (Protocol two) is that measures of virtual compound removal using the in vitro analog (in vivo analog) map directly to the measurements used to compute intrinsic clearance (in vivo hepatic clearance). Protocol three, the focus of this work, requires achieving an essential cross-system validation target. For a subset of virtual compounds, measurements of unbound compounds entering hepatocytes (and their subsequent removal) during virtual in vitro experiments will directly predict corresponding measures made during virtual in vivo experiments. We study four highly permeable virtual compounds when their unbound fraction is fixed at one of seven values (0.05-1.0). Results span the range of hepatic extraction ratios. We achieve the cross-system validation target in 15 cases. In the other 13 cases, explanations of the in vitro-in vivo differences in disposition and removal trace to differences in compound-hepatocyte access within the two analogs during execution. The hepato-mimetic structural organization of hepatocytes within the in vivo analog, which is absent within the in vitro analog, is the determining factor. The results taken together support the feasibility of using the three-protocol plan to help explain observed in vitro-in vivo extrapolation discrepancies. We conjecture that, for some cases, the model mechanism-based explanations of discrepancies described herein will have wet-lab counterparts.

bioengineering↗

bioRxiv2019

Contrasting model mechanisms of alanine aminotransferase (ALT) release from damaged and necrotic hepatocytes as an example of general biomarker mechanisms

Interpretations of elevated blood levels of alanine aminotransferase (ALT) for drug-induced liver injury often assume that the biomarker is released passively from dying cells. However, the mechanisms driving that release have not been explored experimentally. The usefulness of ALT and related biomarkers will improve by developing mechanism-based explanations of elevated levels that can be expanded and elaborated incrementally. We provide the means to challenge the ability of closely related concretized model mechanisms to generate patterns of simulated hepatic injury and ALT release that scale (or not) to be quantitatively similar to the wet-lab validation targets. The validation targets for this work are elevated measures of plasma ALT following acetaminophen (APAP) exposure in mice. We build on a published model mechanism that helps explain the generation of characteristic spatiotemporal features of APAP hepatotoxicity within hepatic lobules. Discrete event and agent-oriented software methods are most prominent. We instantiate and leverage a small constellation of concrete model mechanisms. Their details during execution help bring into focus ways in which particular sources of uncertainty become entangled within and across several levels with cause-effect details. Monte Carlo sampling and simulations comprise a virtual experiment. Falsification of one (or more) of the model mechanisms provides new knowledge and shrinks the model mechanism constellation incrementally. We challenge the sufficiency of four potentially explanatory theories for ALT release. The first model mechanism tested failed to achieve the initial validation target, but each of the three others succeeded. We scale ALT amounts in virtual mice directly to target plasma ALT measures in individual mice. Results for one of the three model mechanisms matched all target ALT measures quantitatively. We assert that the actual mechanisms responsible for ALT measures in individual mice and the virtual causal processes occurring during model execution are strongly analogous within and among real hepatic lobular levels. Author summaryInterpretations of elevated biomarkers for drug-induced liver injury assume passive release during hepatocyte death, yet indirect evidence indicates that plasma levels can increase absent injury. Limitations on measurements make it infeasible to resolve causal linkages between drug disposition and plasma levels of biomarkers. To improve explanatory knowledge, we instantiate within virtual mice, plausible mechanism-based causal linkages between acetaminophen disposition and alanine aminotransferase (ALT) behavior that enables simulation results to meet stringent quantitative validation prerequisites. We challenge the sufficiency of four model mechanisms by scaling ALT measurements in virtual mice to corresponding plasma values. Virtual experiment results in which ALT release is a combined consequence of lobular-location-dependent hepatocyte death and drug-induced cellular damage, matches all validation targets. We assert that the actual mechanisms responsible for plasma ALT measures in individual mice and the virtual causal processes occurring during model execution are strongly analogous within and among real hepatic lobular levels.

pharmacology and toxicology↗

bioRxiv2019

Targeting the PI5P4K lipid kinase family in cancer using novel covalent inhibitors

The PI5P4Ks have been demonstrated to be important for cancer cell proliferation and other diseases. However, the therapeutic potential of targeting these kinases is understudied due to a lack of potent, specific small molecules available. Here we present the discovery and characterization of a novel pan-PI5P4K inhibitor, THZ-P1-2, that covalently targets cysteines on a disordered loop in PI5P4K/{beta}/{gamma}. THZ-P1-2 demonstrates cellular on-target engagement with limited off-targets across the kinome. AML/ALL cell lines were sensitive to THZ-P1-2, consistent with PI5P4Ks reported role in leukemogenesis. THZ-P1-2 causes autophagosome clearance defects and upregulation in TFEB nuclear localization and target genes, disrupting autophagy in a covalent-dependent manner and phenocopying the effects of PI5P4K genetic deletion. Our studies demonstrate that PI5P4Ks are tractable targets, with THZ-P1-2 as a useful tool to further interrogate the therapeutic potential of PI5P4K inhibition and inform drug discovery campaigns for these lipid kinases in cancer metabolism and other autophagy-dependent disorders.

cancer biology↗

bioRxiv2018

Transposable element insertions shape gene regulation and melanin production in a fungal pathogen

Background\n\nVariation in gene expression contributes to phenotypic diversity within species and adaptation. However, very few cases of adaptive regulatory changes have been reported and the mechanisms underlying variation in gene expression remain largely unexplored. Fungal pathogen genomes are highly plastic and harbour numerous insertions of transposable elements, which can potentially contribute to gene expression regulation. In this work we elucidated how transposable elements contribute to variation of melanin accumulation, a quantitative adaptive trait of fungal pathogens that is involved in survival under stress conditions.\n\nResults\n\nWe demonstrated that differential transcriptional regulation of the gene encoding the transcription factor Zmr1, which controls expression of the genes in the melanin biosynthetic gene cluster, is responsible for variation in melanin accumulation in the fungal plant pathogen Zymoseptoria tritici. We show that differences in melanin levels between two strains of Z. tritici are due to two levels of transcriptional regulation: 1) variation in the promoter sequence of Zmr1, and 2) an insertion of transposable elements upstream of the Zmr1 promoter. Remarkably, independent insertions of transposable elements upstream of Zmr1 occurred in 9% of Z. tritici strains from around the world and negatively regulated Zmr1 expression, contributing to melanin accumulation variation.\n\nConclusions\n\nOur studies demonstrate that different layers of transcriptional control fine-tune the synthesis of melanin. These regulatory mechanisms potentially evolved to balance the fitness costs associated with melanin production against its positive contribution to survival in stressful environments.

molecular biology↗