The liver is tasked with managing the concentration of various metabolites in the blood, and of particular importance is the uptake of free fatty-acid (FFA), as elevated concentrations of FFA are toxic to cells. FFAs are transported across the cell membrane by CD36 and distributed by LFABP to the endoplasmic reticulum (ER), where they are esterified to glycerol, yielding more chemically inert triglyceride (TAG), which is essential to the process of VLDL assembly. VLDL secretion distributes energy rich TAG to peripheral tissues, and its dysfunction leads to hepatic steatosis, which may progress into hepatocellular carcinoma. The present study examined the role of cathepsin B (CatB) in regulating very-low density lipoprotein (VLDL) secretion through liver fatty-acid binding protein (LFABP) cleavage as well as CD36 expression in response to 0.5 mM oleic acid:BSA treatment, which has been reported to redistribute CatB from the lysosome to the cytosol, where the majority of cellular LFABP is localized. Genetic knock-down of CatB in McA-RH7777 cells resulted in increased VLDL secretion as measured by 3H TAG DPM counting and immunoblot for ApoB in cell culture media, due to increased expression of LFABP and CD36 and increased FFA uptake. Knock-down of CatB also resulted in decreased cellular TAG as measured by 3H DPM counting due to increased VLDL secretion. CatB over-expression in McA-RH7777 cells resulted in decreased FFA uptake leading to decreased VLDL secretion, which was due to increased cleavage of LFABP. Co-localization of LFABP and CatB was observed exclusively under conditions of 0.5 mM oleic acid:BSA treatment. Based on these results, we can conclude that CatB plays a distinct physiological role in the turnover of LFABP and CD36 protein, which leads to suppressed uptake of FFA, and thus, reduced TAG synthesis and VLDL secretion.

Atherosclerosis (ATH) is an inflammation-mediated disease in which cell death underlies the formation of lesions along the intima layer of vascular walls resulting in vessel narrowing, decreased blood flow, and increased risk of lesion rupture leading to myocardial infarction and stroke. The current study was undertaken to investigate whether inflammation in ATH can induce pyroptosis in vascular smooth muscle cells (SMC's). We therefore hypothesized that pyroptosis occurs and is inhibited by bone morphogenetic protein 7 (BMP7). We examined SMC pyroptosis at acute (D5) and midstage (D28) following disturbed flow-induced hemodynamic injury to the vascular wall using our partial left carotid artery ligation (PLCA) model. ApoE -/- mice (11±1 week old) were divided into three groups: Sham, PLCA, PLCA+BMP7 (200μg/kg; i.v) and arterial tissue was collected for immunohistochemical staining (IHC) and western blot (WB) analysis. At D5 and D28, IHC data demonstrated that PLCA significantly upregulated Toll-like receptor 4 (TLR4) and NLRP3 inflammasome components (NLRP3 and Caspase-1), indicating the initiation and activation of pyroptosis in SMC's (p < 0.05). Further, maturation of pro-IL-1β and pro-IL-18 released through cell membrane pores mediated by Caspase-11 were investigated. Our data shows a significant increase at D5 and D28 in IL-1β, IL-18, and Caspase-11 expression following PLCA, which was significantly improved upon treatment with BMP7 (p < 0.05). Western blot analysis supported these findings demonstrating initiation of pyroptosis via TLR4, upregulation of inflammasome components (Caspase-1 and NLRP3), and release of proinflammatory cytokines, IL-1β and IL-18 at D28, but not at D5. Overall, this study demonstrates that pyroptosis occurs in vascular smooth muscle cells in our PLCA model and that BMP7 administration attenuates pyroptosis significantly.

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.

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.

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.

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.

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.

Alzheimer's disease (AD) involves progressive neurodegeneration leading to the loss of normal neuronal function. Extracellular accumulation of amyloid-beta (Aß), through the abnormal cleavage of the amyloid precursor protein (APP) by ß- and γ-secretases, is one of the hallmarks of AD. Current research focuses on finding potential candidates for biomarkers and techniques with improved sensitivity for early disease detection. Extracellular vesicles (EVs) found in body fluids are a source of biomarkers for AD diagnosis. EVs transport pathologically significant biomolecules, like nucleic acids and proteins, across the blood-brain barrier, mediating local and distant cell-to-cell communication. Therefore, this study evaluated EV-associated DNA and a novel immuno-qPCR (iqPCR) technique for their prospective use in AD diagnosis. In the first part of the study, EVs secreted by AD iPS-derived neural cells (iPS-NCs) were analyzed for deviant sequences of APP DNA. Results indicate that AD EVs carry two nucleotide deletions in the sequence located upstream of the γ-secretase cleavage site, which could affect APP processing. For the second part of the study, various conditions were set up and optimized to test a novel iqPCR model for the detection of Aß. Results confirm the immunocapture of Aß and suggest that the proposed iqPCR model could detect and quantify Aß at concentrations as low as 10 picogram/mL. The differential sequences of EV-associated APP DNA and the highly sensitive iqPCR technique for the detection of Aß presented in this study create a crucial groundwork for research on early diagnosis, prognosis, and assessment of therapy response in AD.

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.

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.