Biology subjects

Gupta, V. K.

Publications and source records attributed to Gupta, V. K..

30 records · Page 2Linked to original sources

bioRxiv2021

The Effect of Minnelide against SARS-CoV-2 in a Murine Model

Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2, is the causative agent of coronavirus disease 2019, COVID-19, and the current COVID-19 pandemic. Even as more vaccine candidates are released, more treatment options are critically needed. Here, we investigated the use of Minnelide, a water soluble pro-drug with anti-inflammatory properties, for the treatment of COVID-19. To do this, k18-hACE2 mice were infected with SARS-CoV-2 or given PBS control intranasally. The next day mice were either treated daily with low dose (0.0025mg/day) or high dose Minnelide (0.005mg/day), or given vehicle control intraperitoneal. Mice were weighed daily, and sacrificed at day 6 and 10 post-infection to analyze viral burden, cytokine response, and pathology. We observed a reduction in viral load in the lungs of Minnelide-treated mice infected with SARS-CoV-2 at day 10 post-infection compared to day 6 post-infection. All SARS-CoV-2 infected non-treated mice were moribund six days post-infection while treatment with Minnelide extended survival for both low (60% survival) and high (100% survival) dose treated mice ten days post-infection. Interestingly, cytokine analysis demonstrated a significant reduction in IL-6 (lung and heart) and D-dimer (serum) in high dose treated SARS-CoV-2 infected mice compared to mice infected with SARS-CoV-2 alone at day 6 post-infection. Additionally, histology analysis revealed that Minnelide treatment significantly improved lung pathology ten days post-infection with SARS-CoV-2 with all the mice exhibiting normal lung tissue with thin alveolar septa and no inflammatory cells. Overall, our study exhibits potential for the use of Minnelide to improve survival in COVID-19 patients.

microbiology↗

bioRxiv2021

Host F-box protein 22 enhances the uptake of Brucella by macrophages and drives a sustained release of pro-inflammatory cytokines through degradation of the anti-inflammatory effector proteins of Brucella.

Brucella species are intracellular bacterial pathogens, causing the world-wide zoonotic disease, brucellosis. Brucella invade professional and non-professional phagocytic cells, followed by resisting intracellular killing and establishing a replication permissive niche. Brucella also modulate the innate and adaptive immune responses of the host for their chronic persistence. The complex intracellular cycle of Brucella majorly depends on multiple host factors but limited information is available on host and bacterial proteins that play essential role in the invasion, intracellular replication and modulation of host immune responses. By employing an siRNA screening, we identified a role for the host protein, FBXO22 in Brucella-macrophage interaction. FBXO22 is the key element in the SCF E3 ubiquitination complex where it determines the substrate specificity for ubiquitination and degradation of various host proteins. Downregulation of FBXO22 by siRNA or CRISPR-Cas9 system, resulted diminished uptake of Brucella into macrophages, which was dependent on NF-{kappa}B-mediated regulation of phagocytic receptors. FBXO22 expression was upregulated in Brucella-infected macrophages that resulted induction of phagocytic receptors and enhanced production of pro-inflammatory cytokines through NF-{kappa}B. Furthermore, we found that FBXO22 recruits the effector proteins of Brucella, including the anti-inflammatory proteins, TcpB and OMP25 for degradation through the SCF complex. We did not observe any role for another F-box containing protein of SCF complex, {beta}-TrCP in Brucella-macrophage interaction. Our findings unravel novel functions of FBXO22 in host-pathogen interaction and its contribution to pathogenesis of infectious diseases. Author SummaryBrucellosis is a major zoonotic disease world-wide that poses a serious veterinary and public health problem in various countries, impacting their economic development. Brucellosis is caused by the species of intracellular bacterial pathogen, Brucella that replicates in professional and non-professional phagocytic cells. Brucella is considered as a stealthy pathogen as it invades/suppresses host defense responses using various virulence strategies. Brucella hijacks many cellular processes for gaining entry into the target cells, followed by establishing a replication permissive niche. However, host proteins that are involved in Brucella-macrophage interaction remains obscure. Here, we identified the host protein, FBXO22 that recruits target proteins to SCF E3 ubiquitination complex for their ubiquitination and degradation. We found that down-regulation and upregulation of FBXO22 decreased and enhanced the uptake of Brucella by macrophages, respectively. Our subsequent studies revealed that Brucella induces the expression of FBXO22 that resulted activation of NF-{kappa}B and the concomitant upregulation of phagocytic receptors that might have contributed to the enhanced uptake of Brucella. The Brucella-induced expression of FBXO22 resulted enhanced production of pro-inflammatory cytokines. We have also found that FBXO22 targets Brucella effectors, including the anti-inflammatory effector proteins for degradation through the SCF complex. Our experimental data reveals that FBXO22 plays an important role in the uptake of microbial pathogens by macrophages and pathogenesis of infectious diseases that is resulting from overt inflammatory responses.

microbiology↗

bioRxiv2021

Diet induced hyperlipidemia confers resistance to standard therapy in pancreatic cancer by selecting for "tumor protective" microbial metabolites and treatment refractory cells.

Obesity causes a number of systemic alterations including chronic inflammation and changes in gut microbiome. However, whether these actively contribute to poor survival and therapy resistance in patients with pancreatic cancer remain undetermined. Our current study shows that high fat diet fed pancreatic tumor bearing mice do not respond to standard of care therapy with gemcitabine and paclitaxel when compared to corresponding control diet fed mice. Upon fecal matter transplant from control mice to high fat diet fed mice, the tumors became sensitive to standard of care therapy and showed extensive cell death. Analysis of gut microbiome showed an enrichment of queuosine (Q) producing bacteria in high fat diet fed mice and an enrichment of S-adenosyl methionine (SAM) producing bacteria in control diet fed mice. Further, treatment of high fat diet fed animals with SAM recapitulated the observation with lean to obese fecal matter transplant. Additionally, treatment of pancreatic and colon cancer cell lines in vitro with Q promoted resistance to the paclitaxel and oxaliplatin respectively, while treatment with SAM promoted sensitivity to these drugs. Treatment of pancreatic cancer cells with Q showed upregulation PRDX1, that is involved in oxidative stress protection. Analysis of tumor tissues in high fat diet fed mice showed high PRDX1, low apoptosis and increased proliferation, which were reversed upon treatment with SAM as well as by lean to obese fecal matter transplant. In parallel, high fat diet fed mice showed increase in CD133+ treatment refractory population compared to the control animals. Interestingly, treatment with Q in vitro did not enrich for CD133+ population, indicating that Q mediated protection from cell death was independent of enrichment of treatment refractory cells. These observations indicated that microbial metabolite Q accumulated in high fat diet fed mice protected tumors from chemotherapy induced oxidative stress by upregulating PRDX1. This protection could be reversed by treatment with SAM. We conclude that relative concentration of S-adenosyl methionine and queuosine in fecal samples of pancreatic cancer patients can be indicative of therapy response in this disease.

cancer biology↗

bioRxiv2020

The nature of mitotic forces in epithelial monolayers

Epithelial cells undergo striking morphological changes during mitosis to ensure proper segregation of genetic and cytoplasmic materials. These morphological changes occur despite dividing cells being mechanically restricted by neighboring cells, indicating the need for extracellular force generation. While forces generated during mitotic rounding are well understood, forces generated after rounding remain unknown. Here, we identify two distinct stages of mitotic force generation that follow rounding: (1) protrusive forces along the mitotic axis that drive mitotic elongation, and (2) outward forces that facilitate post-mitotic re-spreading. Cytokinetic ring contraction of the mitotic cell, but not activity of neighboring cells, generates extracellular forces that propel mitotic elongation and also contribute to chromosome separation. Forces from mitotic elongation are observed in epithelia across many model organisms. Thus, forces from mitotic elongation represent a universal mechanism that powers mitosis in confining epithelia.

cell biology↗

bioRxiv2020

Hypoxia driven oncometabolite L-2HG maintains "stemness"-differentiation balance and facilitates immune suppression in pancreatic cancer

2-hydroxyglutarate (2-HG) has gained considerable importance in glioma and blood cancers that have mutations in the IDH1/2 gene. In the current study we show for the first time that pancreatic tumors produce 2HG in the absence of IDH1/2 mutation. Our study shows that hypoxic pancreatic tumors that have activated lactate dehydrogenase (LDH) activity, produce the L-isoform of 2HG. Metabolic mass spectrometric analysis along with chiral derivatization showed that pancreatic cancer cells as well as stromal cells secrete the L-isomeric form of 2-hydroxyglutarate (L-2HG) when exposed to hypoxic environment. Serum analysis of human pancreatic cancer patients also showed similar accumulation of L-2HG. Our results showed that this abnormally accumulated L-2HG regulates self-renewal by increasing expression of genes associated with stemness (Sox-2, CD133) and by decreasing expression of differentiation genes (Pdx-1, HB9, NKX6.1). Further analysis showed that secreted L-2HG mediates cross talk with immune T-cells and hampers their proliferation and migration thereby suppressing the anti-tumor immunity. In vivo targeting of LDH enzyme with inhibitor (GSK2837808A) showed decrease in L-2HG as well as subsequent tumor regression and sensitization to immune-checkpoint therapy. Present study shows for the first time that hypoxia mediated accumulation of L-2HG drives self-renewal in pancreatic cancer by shifting critical balance of gene expression towards stemness and promotes immune suppression by impairing T cell activation in this disease. Additionally, it indicates that targeting LDH can sensitize pancreatic tumors to anti-PD1 therapy by decreasing L-2HG and reverting their immune evasive function.

cancer biology↗

bioRxiv2020

Stroma secreted IL6 selects for stem-like population and alters pancreatic tumor microenvironment by reprogramming metabolic pathways

Pancreatic adenocarcinoma is a devastating disease with an abysmal survival rate of 9%. A robust fibro-inflammatory and desmoplastic stroma, characteristic of pancreatic cancer, contributes to the challenges in developing viable therapeutic strategies in this disease. Apart from constricting blood vessels and preventing efficient drug delivery to the tumor, the stroma also contributes to aggressive biology of the cancer along with its immune-evasive microenvironment. In this study, we show that in pancreatic tumors, the developing stroma increases tumor initiation frequency in pancreatic cancer cells in vivo by enriching for CD133+ aggressive "stem-like" cells. Additionally, the stromal fibroblasts secrete IL6 as the major cytokine, increases glycolytic flux in the pancreatic tumor cells and increases lactate efflux in the microenvironment via activation of the STAT signaling pathway. We also show that the secreted lactate favors activation of M2 macrophages in the tumor microenvironment, which excludes CD8+ T-cells in the tumor. Our data additionally confirms that treatment of pancreatic tumors with anti-IL6 antibody results in tumor regression as well as decreased CD133+ population within the tumor. Furthermore, inhibiting the lactate efflux in the microenvironment reduces M2 macrophages, and makes pancreatic tumors more responsive to anti-PD1 therapy. This suggests that stromal IL6 driven metabolic reprogramming plays a significant role in the development of an immune evasive microenvironment. In conclusion, our study shows that targeting the metabolic pathways affected by stromal IL6 can make pancreatic tumors amenable to checkpoint inhibitor therapy.

cancer biology↗

bioRxiv2020

Rapamycin-PLGA microspheres induce autophagy and prevent senescence in chondrocytes and exhibit long in vivo residence

Osteoarthritis (OA) is a joint disease that results in progressive destruction of articular cartilage and the adjoining subchondral bone. The current treatment is focused on symptomatic relief due to the absence of disease-modifying drugs. The primary cells of the cartilage, chondrocytes, have limited regenerative capacity and when they undergo stress due to trauma or with aging, they senesce or become apoptotic. Autophagy, a cellular homeostasis mechanism has a protective role in OA during stress but gets downregulated in OA. Rapamycin, a potent immunomodulator, has shown promise in OA treatment by autophagy activation and is known to prevent senescence. However, its clinical translation for OA is hampered due to systemic toxicity as high and frequent doses are required. Hence, there is a need to develop suitable delivery carriers that can result in sustained and controlled release of the drug in the joint. In this study, we have fabricated rapamycin encapsulated poly (lactic-co-glycolic acid) (PLGA) based carriers that induced autophagy and prevented cellular senescence in human chondrocytes. The microparticle (MP) delivery system showed sustained release of drug for several weeks. Rapamycin-microparticles protected in-vitro cartilage mimics from degradation, allowing sustained production of sGAG, and demonstrated a prolonged senescence preventive effect in vitro under oxidative and genomic stress conditions. These microparticles also exhibited a long residence time of more than 19 days in the joint after intra-articular injections in murine knee joints. Such particulate systems are a promising candidate for intra-articular delivery of rapamycin for treatment of osteoarthritis. Statement of SignificanceCurrent OA treatment is symptomatic and does not change the disease progression. Many drugs have failed as they are rapidly cleared and are unable to maintain therapeutic concentration in the OA joint. Direct joint administration of drugs using sustained-release systems offer several advantages, which includes increased bioavailability, fewer off-target effects, and lower total drug cost. We have engineered a suitable drug carrier which provides a tunable drug release pattern. This study provides evidence that PLGA encapsulated rapamycin remained potent and prevented OA like changes in chondrocytes under genomic and oxidative stress. The particle formulation also had a longer residence time in the knee joint of mice which can be translated in clinics for intra-articular therapeutic injections for increased patient compliance. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/027136v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1c5901borg.highwire.dtl.DTLVardef@177fa3eorg.highwire.dtl.DTLVardef@1ce2a55org.highwire.dtl.DTLVardef@3d5d6f_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2020

Single-Cell Mass Cytometry on Peripheral Blood Identifies Immune Cell Subsets Associated with Primary Biliary Cholangitis

The relationship between Primary Biliary Cholangitis (PBC), a chronic cholestatic autoimmune liver disease, and the peripheral immune system remains to be fully understood. Herein, we performed the first mass cytometry (CyTOF)-based, immunophenotyping analysis of the peripheral immune system in PBC at single-cell resolution. CyTOF was performed on peripheral blood mononuclear cells (PBMCs) from PBC patients (n=33) and age-/sex-matched healthy controls (n=33) to obtain immune cell abundance and marker expression profiles. Hiearchical clustering methods were applied to identify immune cell types and subsets significantly associated with PBC. Subsets of gamma-delta T cells (CD3+TCRgd+), CD8+ T cells (CD3+CD8+CD161+PD1+), and memory B cells (CD3-CD19+CD20+CD24+CD27+) were found to have lower abundance in PBC than in control. In contrast, higher abundance of subsets of monocytes and naive B cells were observed in PBC compared to control. Furthermore, several naive B cell (CD3-CD19+CD20+CD24-CD27-) subsets were significantly higher in PBC patients with cirrhosis (indicative of late-stage disease) than in those without cirrhosis. Alternatively, subsets of CD8+CD161+ T cells and memory B cells were lower in abundance in cirrhotic relative to non-cirrhotic PBC patients. Future immunophenotyping investigations could lead to better understanding of PBC pathogenesis and progression, and also to the discovery of novel biomarkers and treatment strategies.

bioinformatics↗

bioRxiv2020

A Novel Index for Predicting Health Status Using Species-level Gut Microbiome Profiling

The development of a biologically-interpretable and robust metric that provides clear insight into the general health status (i.e. healthy or non-healthy) of ones gut microbiome remains an important target in human microbiome research. We introduce the Gut Microbiome Health Index (GMHI), a mathematical formula that determines the degree to which a gut microbiome profile reflects good or adverse health. GMHI was formulated based on microbial species specific to healthy gut ecosystems. These species were identified through a multi-study, integrative analysis on 4,347 human stool metagenomes from 34 published studies across healthy and 12 different disease or abnormal bodyweight conditions. When demonstrated on our population-scale meta-dataset, GMHI is the most robust and consistent predictor of general health compared to -diversity indices commonly considered as markers for gut health. Validation of GMHI on 679 samples from 9 additional studies resulted in remarkable reproducibility in distinguishing healthy and non-healthy groups. Our findings suggest that gut taxonomic signatures can indeed serve as robust predictors of general health, and highlight the importance of how data sharing efforts can provide broadly-applicable novel discoveries.

bioinformatics↗

bioRxiv2019

Long non-coding RNA GAS5 acts as proliferation brakes in CD133+ cells responsible for tumor recurrence

Presence of quiescent, therapy evasive population often described as cancer stem cells (CSC) or tumor initiating cells (TIC) is often attributed to extreme metastasis and tumor recurrence. This population is typically enriched in a tumor as a result of microenvironment or chemotherapy induced stress. The TIC population adapts to this stress by turning on cell cycle arrest programs that is a \"fail-safe\" mechanism to prevent expansion of malignant cells to prevent further injury. Upon removal of the \"stress\" conditions, these cells restart their cell cycle and regain their proliferative nature thereby resulting in tumor relapse. Growth Arrest Specific 5 (GAS5) is a long-noncoding RNA that plays a vital role in this process. In pancreatic cancer, CD133+ population is a typical representation of the TIC population that is responsible for tumor relapse. In this study, we show for the first time that emergence of CD133+ population coincides with upregulation of GAS5, that reprograms the cell cycle to slow proliferation by inhibiting GR mediated cell cycle control. The CD133+ population further routed metabolites like glucose to shunt pathways like pentose phosphate pathway, that were predominantly biosynthetic in spite of being quiescent in nature but did not use it immediately for nucleic acid synthesis. Upon inhibiting GAS5, these cells were released from their growth arrest and restarted the nucleic acid synthesis and proliferation. Our study thus showed that GAS5 acts as a molecular switch for regulating quiescence and growth arrest in CD133+ population, that is responsible for aggressive biology of pancreatic tumors.

cancer biology↗

bioRxiv2019

Targeting tumor intrinsic metabolic node in pancreatic cancer causes tumor regression, remodels extracellular matrix and sensitizes to anti-PD1 therapy.

Pancreatic ductal adenocarcinoma (PDAC) is considered to be a highly immunosuppressive and heterogenous neoplasm. Despite improved knowledge regarding the genetic background of the tumor and better understanding of the tumor microenvironment, immune checkpoint inhibitor therapy (targeting CTLA-4, PD-1, PD-L1) has not been very successful against PDAC.\n\nThe robust desmoplastic stroma, along with an extensive extracellular matrix (ECM) that is rich in hyaluronan, plays an integral role in this immune evasion. Hexosamine biosynthesis pathway (HBP), a shunt pathway of glycolysis, is a metabolic node in cancer cells that can promote survival pathways on one hand and influence the hyaluronan synthesis in the ECM on the other. The rate-limiting enzyme of the pathway, glutamine-fructose amidotransferase (GFAT1), uses glutamine and fructose 6-phosphate to eventually synthesize UDP-GlcNAc.\n\nIn the current manuscript, we target this glutamine-utilizing enzyme by a small molecule glutamine analog (6-diazo-5-oxo-L-norleucine or DON). Our results show that DON decreases the self-renewal potential and metastatic ability of tumor cell. Further, treatment with DON results in a decrease in hyaluronan and collagen in the tumor microenvironment, leading to an extensive remodeling of the ECM. this in turn, increases CD8+ cytotoxic T-cells infiltration, and makes the tumors tumors more amenable and sensitive to anti-PD1 therapy.

cancer biology↗

bioRxiv2018

O-GlcNAc modification of oncogenic transcription factor Sox2 promotes protein stability and regulates self-renewal in pancreatic cancer

Pancreatic cancer is among the 3rd leading cause of cancer related deaths in the United States along with a 5-year survival rate of 7%. The aggressive biology of the disease is responsible for such dismal outcome and is manifested by an increase in self-renewal capacity of the cancer cells, which leads to an increased rate of tumor-recurrence, contributing to poor prognosis. Transcription factor SOX2 maintains a critical balance between differentiation and \"stemness\" and is thus tightly regulated within a cell. In cancer, SOX2 is aberrantly \"turned-on\" leading to activation of self-renewal pathways in cancer. Regulation of Sox2 in cancer is poorly understood. In the current study, we show for the first time that in pancreatic cancer, Sox2 is modified by addition of O-GlcNAc moiety, catalyzed by OGT (O-GlcNAc Transferase) at S246. This activates Sox2 transcriptional activity by stabilizing the protein in the nucleus. A CRISPR-OGT knockout in pancreatic cancer cell line S2VP10 resulted in a delayed tumor initiation. We further showed that mutation of this site (S246A) prevents the modification of Sox2 and its downstream activity. Our study also demonstrated that targeting OGT in vivo with a small molecule inhibitor OSMI, results in decreased tumor burden, delayed tumor progression and a decreased expression of SOX2 in pancreatic cancer cells. Our study highlights for the first time that that the O-GlcNAc transferase dependent SOX2 glycosylation has a profound effect on the transcriptional activity of SOX2 and is instrumental in determining self-renewal in pancreatic cancer.\n\nSignificanceOur study highlights for the first time that that the O-GlcNAc transferase dependent SOX2 glycosylation determines self-renewal in pancreatic cancer which is responsible for tumor initiation.

cancer biology↗