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Metabolic Effects of 17a-Estradiol are Growth Hormone Independent and Sex SpecificSidhom, Silvana 01 January 2019 (has links)
Aging is a major risk factor for metabolic syndromes and type two diabetes. With growing elderly populations worldwide and increasing incidence of age-related diseases there is a great need to develop pharmacological interventions that would delay aging and protect from age-related diseases. 17-alpha estradiol (17α-E2) is an epimer of the primary female sex hormone estradiol and has been shown to extend lifespan and downregulate markers of age-related metabolic dysfunction in male mice. Because 17α-E2 does not induce feminization in males it holds potential as a novel therapeutic in humans for age-related metabolic dysfunction. Importantly, we have previously shown that 17α-E2 causes an increase of circulating and hepatic IGF-1 in aged mice, without any changes in GH release in treated animals. Based on this we propose a new hypothesis that 17α-E2 acts through a novel, GH-independent pathway stimulating production of IGF-1 and positively modulating metabolic function in a sex-specific manner. Here we studied 17α-E2 treated long-lived growth hormone receptor knockout (GHRKO) mice, characterized by severely reduced circulating and hepatic IGF-1 due to GH-resistance. We found increases in circulating IGF-1 after treatment in normal and GHRKO male mice, with no effect in female mice, which supports our hypothesis that 17α-E2 induces GH independent IGF-1 production. To determine novel genetic pathways activated by 17α-E2 we performed sequencing of hepatic RNA. Our analysis indicated differential regulation of steroid biosynthesis and insulin signaling pathways. The validation of our sequencing data using qPCR showed significant upregulation of genes involved in insulin action. Importantly, differential regulation of these pathways was present in normal male mice, with no changes in normal females or either male or female GHRKO animals. In summary, this new data supports our hypothesis of a sex-specific effect of 17α-E2 treatment and differing mechanisms of action by which 17α-E2 upregulates IGF-1 independently of GH action.
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Competence Transcriptome Changes in Streptococcus PneumoniaeShambhu, Smitha 01 January 2021 (has links)
Streptococcus pneumoniae is a human commensal and the causative agent of pneumococcal disease. Pneumococci are naturally competent – able to uptake exogenous DNA from the environment and incorporate it into their genome through homologous and non-homologous recombination. Recombination has significantly shaped the evolutionary history of S. pneumoniae, as it allows pneumococci to rapidly adapt to interventions such as antibiotic therapy or vaccine introduction. Recombination frequencies vary considerably across pneumococcal populations; yet the underlying mechanisms for these variations are not well understood. Entry and exit into competence, a state in which the cell can uptake DNA, is tightly regulated through transcriptional changes. To elucidate differences in transformation frequency among strains as well as the underlying genetic mechanisms, we carried out in-vitro competence assays and measured gene expression changes during the competent state using RNA sequencing of strains belonging to Serotype 3 clonal complex (CC) 180 and a non-CC180 comparison. We observed consistent differences in transformation frequencies among groups, which correlated with variation in differentially expressed genes during competence. While all strains exhibited a similar response to competence stimulating peptide (CSP) for early competence genes, we observed variation in expression of late competence genes, which encode the DNA uptake apparatus, DNA repair and recombination proteins needed for recombination. We also observed differences in expression of genes linked to bacteriocin production, which may partially explain observed population-level differences. Further genomic analysis suggests variation in promoter sequences governing late competence genes may be slowing transition from early to late components of the competence pathway. Additional studies are needed to assess the phenotypic impact of genomic variations. Overall, we show that there is considerable variation in competence even among closely related strains and that this variation may be the result of subtle genomic differences.
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Impact of R300 and C343 in the B' Domain of Protein Disulfide Isomerase on its Disaggregase Activity Against Cholera ToxinVincent, Evie 15 August 2023 (has links) (PDF)
Proteostasis in the endoplasmic reticulum (ER) is maintained, in part, through the activity of protein disulfide isomerase (PDI). This essential protein exhibits a modular domain arrangement of abb'xa'c that contributes to both its oxidoreductase and chaperone activities, with substrate binding primarily located in the b and b' domains and oxidoreductase activity located in the a and a' domains. During prolonged nitrosative stress conditions, PDI is post-translationally modified with nitric oxide at its cysteine residues in both the active site domains (a and a') and the substrate binding b' domain. This S-nitrosylation (SNO) event inactivates PDI activity by a mechanism thought to involve the reactive CGHC motifs in the a and a' domains. However, recent evidence suggests that cysteine 343 in the b' domain is stably S-nitrosylated and resistant to reversal compared to the active site cysteines. In addition, arginine 300 in the b' domain contributes to the redox-regulated conformational flexibility of PDI that allows it to act upon a wide range of substrates. Here, we used cholera toxin (CT) as a model substrate to examine the roles of C343 and R300 in PDI-substrate interactions. In the ER, PDI facilitates cholera intoxication by acting as a disaggregase to physically separate the enzymatically active CTA1 subunit from the rest of the holotoxin. The free CTA1 is then exported out of the ER to the cytosol where it alters cellular signaling through its ADP-ribosyltransferase activity. Using site-direct mutagenesis, we generated two PDI variants with single C343S or R300A substitutions. We then examined the effect of these mutations on PDI-CT interactions and the inactivation of PDI by S-nitrosylation. Although the R300A variant had a slightly altered secondary structure, neither C343S or R300A inhibited the binding or disassembly of CT by PDI. These results suggest a unique mechanism of action for PDI's disaggregase activity against CT. Current experiments are exploring if C343S is resistant to the inactivation of PDI's disaggregase activity that results from S-nitrosylation. This work also provides a possible molecular basis to understanding why SNO-PDI is linked to amyloid fibril formation in neurodegenerative diseases.
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Comprehensive Imaging And Quantitative Analysis Techniques For Nociceptive Afferent Innervation Of The Stomach And Development Of A Micro Liquid Thermal Regulator (MLTR)Madas, Jazune 15 August 2023 (has links) (PDF)
Understanding the innervation of nociceptive afferents in the stomach is pivotal for elucidating the mechanisms underlying pain perception and gastrointestinal disorders. Immunohistochemical labeling of common pain markers, including substance P (SP), calcitonin gene-related peptide (CGRP), and transient receptor potential vanilloid 1 (TRPV1), has been widely employed to visualize nociceptive afferents. However, existing studies predominantly rely on sectioned tissue, limiting the holistic assessment of axonal networks and presenting challenges in terms of time and labor. To address these limitations, we introduce an automated imaging and analysis pipeline tailored specifically for investigating nociceptive afferent innervation in flat-mount preparations of the stomach. This innovative pipeline combines advanced imaging techniques and sophisticated image analysis algorithms to facilitate precise quantification and characterization of nerve fibers. Through the implementation of this automated approach, we have successfully examined the distribution, density, and morphology of nociceptive afferent fibers in the stomach. Notably, our methodology enables the first-ever comprehensive imaging of the entire nociceptive afferent innervation in a rat stomach. Furthermore, we employed the advanced neuron tracing software, Neurolucida 360, to characterize spinal afferent fibers and to achieve full tracing of CGRP-IR axon fibers in the mouse stomach. This comprehensive tracing facilitates the identification of key structures involved in nociceptive processing within the gastric system. In the context of pain suppression, we propose an alternative approach by targeting the cooling of spinal dorsal root ganglia (DRG; the origin of nociceptive axons in the stomach). We've designed and fabricated a compact micro-liquid thermo-regulator (MLTR) device utilizing 3D printing technology to address this. Potentially, The MLTR device can be directly implanted into the rat DRG, enabling precise temperature control through microfluidic and thermo-regulation methods. In the future, we will test whether the MLTR device effectively modulates nociceptive transmission by lowering the local temperature, providing a potential alternative to opioid-based pain suppression.
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Polyamine Blockade Therapy: Modulating Pancreatic Tumor Microenvironment and Role in MDSC BiologyGandhi, Manav 15 August 2023 (has links) (PDF)
Pancreatic cancer (PC) is a challenging cancer to treat, with a 5 year survival of < 13% in the United States. This is attributed to multiple histologic subtypes, extensive desmoplastic reactions, resistance to chemotherapy, profound immunosuppression and crosstalk between tumor, immune and stromal cells in the microenvironment. Alternative modalities are needed to treat this aggressive tumor. Our laboratory has shown that targeting polyamines using a polyamine blockade therapy (PBT), which is a combination of Difluoromethylornithine (polyamine synthesis inhibitor) and Trimer44NMe (polyamine transport inhibitor), is effective against PC. In the present study, we used a KPC genetic model of PC, as it mimics human tumors from spontaneous tumor conception to metastasis. Despite tumor heterogeneity, PBT improved outcomes in KPC mice. Histopathology revealed decreased tumor size, variable decrease in tumor weights, and significant stromal alterations. Stromal alterations were driven by reduced collagen deposition. PBT was found to variably inhibit markers associated with cancer-associated fibroblasts and activated pancreatic stellate cells, which are key producers of collagen. Also, M1 macrophage associated markers were upregulated in the microenvironment. To elucidate the effect of PBT on cells known to contribute to the immunosuppressive environment in PC, we treated bone marrow-derived myeloid derived suppressor cells (MDSC). We found using flow cytometry that PBT inhibited the abundance of PMN (polymorphonuclear)-MDSC phenotype. Finally, RNA sequencing revealed that PBT inhibited genes involved in chemotaxis and inflammation associated with MDSC biology. Collectively, this work provides the basis for feasibility and utility of testing PBT in larger cohorts of the KPC model.
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Mechanisms of Early-Stage Mutant EGFR-Driven Lung Cancer: The Role of KynurenineRodriguez-Fuguet, Alice 15 August 2023 (has links) (PDF)
Lung cancer initiation and progression driven by epidermal growth factor receptor (EGFR) mutation has been extensively studied in the past decade. EGFR mutation is a significant driver of tumorigenesis and inflammation. This receptor is directly related to many downstream pathways that influence cancer metabolism and the ability of tumor cells to adapt to changes in the tumor microenvironment. Our study finds that cancer metabolism plays a large role in the progression of tumorigenesis in early-stage lung cancer. Kynurenine (KYN), a metabolite from the tryptophan pathway, was found to be highly upregulated in early-stage, EGFR-mutation driven lung cancer by metabolomic analysis. The connection between EGFR mutation and the upregulation of kynurenine remains largely unknown. Our data also shows that a significant pathway related to precancer initiation is systemic lupus erythematosus (SLE). We hypothesize that kynurenine may have a dual role in early tumorigenesis. We propose that high levels of kynurenine may be a part of metabolic reprogramming caused by genetic regulation of indoleamine 2,3-dioxygenase 1/2 (IDO1/2). Additionally, kynurenine may be used as a signaling molecule in the tumor microenvironment to allow the early tumor cells to evade immune detection. Our study has implications for the early treatment of lung cancer and provides insight into a potential clinical target in the tryptophan pathway.
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GATM is Involved in the Promotion of Drug Resistance in Lung CancerMurugavel, Nikitha 15 August 2023 (has links) (PDF)
The most prevalent subtype of lung cancer is non-small cell lung cancer (NSCLC), which has a low survival rate. Despite epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors being used as first-line treatment for patients with EGFR mutations, drug resistance frequently occurs in most patients. This is due to the involvement of a myriad of genes that help cancer cells survive against EGFR inhibitors, which are still poorly understood. Here, we focus on the molecular profiles that cause drug resistance. Our previous study has shown that the involvement of the arginine metabolic pathway contributes to drug resistance, as revealed by the whole transcriptomics analysis. Our study found that glycine amidinotransferase (GATM) and guanidinoacetate methyltransferase (GAMT) were top-upregulated genes, which catalyze creatine synthesis. GATM is the rate-limiting enzyme in this process. We also discovered that MYC and C15orf48, along with GATM, were upregulated in lung cancer cell lines treated with gefitinib. This suggests that MYC and C15orf48 may regulate GATM to acquire drug resistance. Further analysis showed that GATM promotes drug resistance by upregulating miR-147b, the mature microRNA of its host gene C15orf48. This upregulation generates reactive oxygen species and pseudohypoxia response, promoting drug resistance among lung cancer cell lines. Our study has implications for identifying a potential new target to overcome drug resistance in lung cancer.
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Identification of an Endogenous Multiprotein Complex Required to Generate the VLDL Transport Vesicle from the Hepatic ERMishra, Rutuparnna 15 August 2023 (has links) (PDF)
Lipids are essential for cellular functions, and the liver plays a crucial role in regulating lipid balance through processes like fatty acid metabolism and very low-density lipoprotein (VLDL) biosynthesis. Disruptions in these mechanisms contribute to diseases such as atherosclerotic cardiovascular diseases, diabetes, obesity, and metabolic syndromes. VLDL transport vesicles (VTVs) are specialized vesicles responsible for transporting nascent VLDL from the endoplasmic reticulum (ER) to the Golgi apparatus. VTVs, like the canonical protein transport vesicles (PTVs), rely on coatomer complex II (COPII) proteins for their formation from the hepatic ER membrane. Proteomic analysis and western blot identified proteins unique to the VTV proteome, namely cell death-inducing DFF45-like effector (CideB), small valosin-containing protein-interacting protein (SVIP), and reticulon 3 (RTN3). While CideB and SVIP have been identified previously as VLDL-sorting protein and intracellular trafficking protein respectively, the specific function of RTN3 and the factors affecting its expression remain unclear. This study aimed to investigate the effect of dietary fat on RTN3 expression, validate its association with COPII proteins, and identify the endogenous multiprotein complex involved in VTV biogenesis. Western blot and confocal analysis revealed that RTN3 protein expression was significantly elevated in hepatocytes (HepG2 cells) in response to dose and time-dependent oleic acid treatment. In contrast, SVIP and CideB levels remained stable under similar nutrient conditions, highlighting the unique role of RTN3. Coimmunoprecipitation experiments confirmed an interaction between RTN3 and COPII complex marker Sar1B, providing evidence that RTN3 regulates VTV formation through its interaction with the COPII component of VTV. Further analysis revealed the presence of an endogenous VTV complex in HepG2 cells composed of CideB, SVIP, RTN3, and the COPII complex marker Sar1B. These findings provide valuable insights into the assembly process of VTVs and underscore the critical role of RTN3 in VTV formation and lipid trafficking. Comprehending these processes can contribute to a better understanding of lipid-related diseases and potentially guide the development of therapeutic interventions.
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Mechanisms by Which LMAN1 Regulates HDM-induced Allergic InflammationSwaby, Lindsay 01 January 2023 (has links) (PDF)
Allergic asthma is a chronic inflammatory disease of the airways characterized by a type 2-high adaptive immune response towards common aeroantigens such as dust mite, pollen and animal dander. Despite the advances made towards translation of various biologics into the clinic, the limited efficacy of these therapies in certain populations, combined with the ineligibility of some patients for treatment (clinically or economically), have led to the continued need for the development of more widely effective allergic asthma therapies. Our lab has identified lectin mannose-binding 1 (LMAN1) as a novel receptor for house dust mite (HDM) and showed that in vitro, LMAN1 downregulated inflammatory NF-κB signaling in response to this allergen. Mechanistically, we demonstrated that LMAN1 interacted with FcRγ in dendritic cells (DCs) and recruited the negative regulator SHP1. These data suggest that LMAN1 acts as a negative regulator of the inflammatory response to HDM. To understand the in vivo relevance of this finding, we subjected LMAN1 knockout (KO) mice and wild type (WT) littermate controls to a model of HDM-induced allergic asthma. Surprisingly, we found that loss of LMAN1 led to opposing effects on airway hyperreactivity which were dependent on the sex of the mice. Female LMAN1 KO mice showed increased AHR, while male KO mice showed the opposite trend compared with their WT counterparts. We further characterized features of the HDM-induced inflammatory response which could explain these sex-dependent differences. Overall, this work provides the first mechanistic insights into potential signaling functions mediated by LMAN1 and also illuminates the complexity of the loss of LMAN1 in vivo.
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Identification and Functional Characterization of a Long Non-coding RNA associated with Prostate CancerHasan, Md Faqrul 01 January 2019 (has links) (PDF)
Prostate cancer is the most common cancer in men in the western world. Although early stage prostate cancer is treatable late stage, more specifically, metastatic and drug resistant prostate cancers are mostly incurable. The failure of current treatments obligates the research community to explore novel areas in prostate cancer biology and find better therapeutic targets. Emerging evidences show that non-coding RNAs specifically long non-coding RNAs (lncRNAs) play regulatory roles in various cellular processes and are frequently dysregulated in cancer including prostate cancer. These aberrantly expressed lncRNAs mostly with unexplored genetic information may drive cancer progression. Previous studies done in our laboratory showed a tumor suppressor role of a cluster of small non-coding RNAs or microRNA (miRNA) miR-17-92a in PC-3 prostate cancer cells. To learn the underlying mechanism, transcriptome analysis with or without expression of miR-17-92a was conducted in our laboratory. RNA-sequencing data analysis identified reduced expression of a set of lncRNAs and oncogenes, and up regulation of several tumor suppressor genes upon expression of miR-17-92a cluster miRNAs. One of the down regulated intergenic lncRNAs, PAINT (Prostate Cancer Associated Intergenic Non-coding Transcript) (LINC00888), was selected for determining its functional role in prostate cancer. TCGA and GEO profiles analyses revealed up regulation of PAINT in prostate tumors with higher Gleason Scores, in highly aggressive metastatic prostate cancer cell lines, and upon androgen deprivation therapy of prostate cancer cells. This observation was supported by our studies on expression analysis of PAINT in prostate tumor tissues using RNA in-situ hybridization in tissue microarrays (TMA) containing tissues from different stages of prostate cancer and normal prostate tissues, which showed higher expression of PAINT in prostate cancer tissues compared to normal tissues. Furthermore, late stage (stage III and stage IV) prostate tumors showed significant overexpression of PAINT compared to early stage (stage II) prostate cancer tissues. We examined the functional relevance of PAINT in promoting tumor progression next using different prostate cancer cell lines. Silencing of PAINT using siRNAs showed decreased cell proliferation, reduced S-phase progression and activation of pro-apoptotic proteins PARP and Caspase-3. Silencing of PAINT also showed decreased cell migration and increased expression of the epithelial marker, E-cadherin while reduced expression of mesenchymal markers Slug and Vimentin. Ectopic expression of PAINT reversed the effects observed upon silencing of PAINT. Increased cell proliferation, cell cycle progression and cell migration were noted in prostate cancer cells overexpressing PAINT. Additionally, cancer promoting phenotype such as larger colony formation and higher expression of mesenchymal marker Slug, was detected upon overexpression of PAINT. Our study also determined the therapeutic benefit of inhibition of expression showing an increased sensitivity of metastatic prostate cancer cells to the chemotherapeutic agent docetaxel (DTX) and selective Aurora kinase inhibitor VX-680. Taken together, our study establishes an oncogenic function of PAINT, its clinical relevance as a marker for advanced stage prostate cancer and its potential as a therapeutic target for metastatic prostate cancer.
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