Innate immune and inflammatory responses contribute to myocardial and cerebral ischemia/reperfusion (I/R) injury. Toll-like receptors (TLRs) play a critical role in the induction of innate immune and inflammatory responses via activation of nuclear factor kappa B (NF-κB). We have shown that activation of NF-κB contributes to myocardial and cerebral I/R injury. Indeed, inhibition of TLR4-mediated NF-κB activation significantly decreased myocardial and cerebral I/R injury via activation of PI3K/Akt signaling. PI3K/Akt signaling is an important pathway in regulating cellular survival and inflammatory responses. Therefore, an important question is how to differentially modulate PI3K/Akt signaling and TLR/NF-κB-mediated signaling pathway during I/R injury? We demonstrated that pretreatment of mice with Pam3CSK4, a specific TLR2 ligand, significantly decreased cerebral I/R injury and improved neuronal functional recovery. Importantly, therapeutic administration of Pam3CSK4 also markedly decreased cerebral I/R injury. The mechanisms involved suppression of NF-κB binding activity and activation of PI3K/Akt signaling. We also demonstrated that CpG-ODN, a specific TLR9 ligand, induced protection against cerebral I/R injury via activation of PI3K/Akt signaling. Our findings were consistent with our previous reports showing that administration of Pam3CSK4 or CpG-ODN protected against myocardial I/R injury via a PI3K/Akt-dependent mechanism. In addition, we demonstrated for the first time that TLR3 located in endosomes played a deleterious role in myocardial I/R injury via activation of NF-κB. To investigate how to activate PI3K/Akt signaling during I/R injury, we examined the role of microRNA (miRs) in regulating PI3K/Akt signaling during myocardial ischemic injury. We discovered that Pam3CSK4 or CpG-ODN treatment significantly increased the expression of miR-130a in the myocardium, while myocardial infarction markedly decreased the levels of miR-130a in the myocardium. The data indicated that miR-130a served a protective role in myocardial ischemic injury. Indeed, we demonstrated for the first time that increased expression of miR-130a significantly attenuated cardiac dysfunction and promoted angiogenesis after myocardial infarction. The mechanisms involved activation of PI3K/Akt signaling via targeting PTEN expression by microRNA-130a. This dissertation discovers novel mechanisms of cerebral and myocardial ischemic injury and provides solid basis for developing new approaches for the treatment and management of stroke and heart attack patients.

Metabolic dysfunction in skeletal muscle is a major contributor to the development of type 2 diabetes. Endurance exercise training has long been established as an effective means to directly restore skeletal muscle glucose and lipid uptake and metabolism. However, in addition to the direct effects of skeletal muscle on glucose and lipids, there is renewed interest in the ability of skeletal muscle to coordinate metabolic activity of other tissues, such as adipose tissue and liver. The purpose of this study was to examine the effects of endurance exercise on the expression level of two novel muscle-derived secreted factors, or myokines, Myonectin and Fibronectin type III domain containing 5 (FNDC5), the precursor for Irisin. Methods. We performed immunoblot analysis and quantitative real-time PCR analysis of Myonectin and FNDC5 in the diaphragm muscles of obese Zucker rat (OZR) and lean Zucker rat (LZR) with 9 weeks of aerobic training on a motorized treadmill. Results. We show that myonectin gene expression is increased in the OZR model of obesity and decreases with exercise in both lean and obese Zucker rats. Conversely, myonectin protein concentration was elevated with exercise. Similarly, FNDC5 mRNA levels are significantly higher in the OZR, however exercise training had no effect on the expression level of FNDC5 in either the LZR or OZR. We did not observe any difference in muscle protein content of Irisin with obesity or exercise. Conclusion. Our data shows that exercise training does not increase either FNDC5 or myonectin gene expression, indicating that increased transcriptional regulation of these myokines is not induced by exercise. However, our data also indicates a yet to be explored disconnect between myonectin gene expression and protein content. Further, this report highlights the importance of verifying reference genes when completing gene expression analysis. We found that many commonly used reference genes varied significantly by obesity and/or exercise and would have skewed the results of this study if used to normalize gene expression data. The unstable reference genes include: beta-Actin, beta-2-microglobulin, Non-POU domain containing, octamer-binding, Peptidylprolyl isomerase H, 18S ribosomal RNA, TATA box binding protein and Transferrin receptor.

Antimicrobial resistance is becoming an increasingly serious problem accompanied by relatively few studies examining the relationship between use and resistance. The present study undertakes a twenty year analysis of antimicrobial production and factors affecting antimicrobial use for a particular microorganism (Stp. faecalis)/antimicrobial agent (Cephalothin) combination. The period is inclusive of the market introduction of the agent and considerate of prescribing practices to the present time. The accumulated data reveal that there is indeed a relationship between total drug availability (medicinal, agricultural) and increased antimicrobial resistance. The data also suggest that national (or global) use changes would likely have a long term beneficial effect on the deteriorating circumstances surrounding microbial resistance to antimicrobial chemotherapeutic agents The methodology utilized includes analysis of primary historical data and graphical representation of indices derived from these data. A literature review examines the impact on antimicrobial resistance by historical duration of use, various mechanisms of resistance, non-medical uses of antimicrobial agents and clinical misuse.

Significant advances in the efficacy of cancer therapy have been accompanied by an escalation of side effects that result from therapy-induced injury to normal tissues. Patients with high grade cancer or metastasis are often treated with chemotherapy, 50% of which are associated with reactive oxygen species generation and cellular oxidative stress. Heart is the normal tissue most susceptible to chemotherapy-induced oxidative stress and heart disease is the most common leading cause of death in cancer survivors. However, early and sensitive biomarkers to identify heart disease are still lacking. Extracellular vesicles (EVs) are released from cells during oxidative stress and send oxidized proteins into the circulation as a compensatory mechanism that prevents cellular proteotoxicity. Thus, the protein contents of EVs released during the pre-degeneration stage reveal that oxidative stress is occurring early in the damaged tissue. Using a mouse model of doxorubicin (DOX)-induced cardiac injury, we demonstrated that EVs can be used as an early diagnostic tool for tissue injury as they are oxidatively modified with 4-hydroxynonenal and contain tissue specific proteins—glycogen phosphorylase brain/heart, muscle, and liver isoforms—that indicate their origins. These biomarkers increased early, before the changes of conventional biomarkers occurred. EVs also mediate intercellular communication by transferring bioactive molecules between cells. In the cell culture system, EVs play an important role in oxidative stress response by inducing macrophage polarization. EVs from cardiomyocytes promoted both proinflammatory (M1) and anti-inflammatory (M2) macrophage polarization evidenced by higher pro- and anti-inflammatory cytokines and nitric oxide generation, as well as mitochondrial oxidative phosphorylation suppression and glycolysis enhancement. In contrast, EVs from the hepatocytes supported anti-inflammatory macrophage (M2) by enhancing oxidative phosphorylation and anti-oxidant proteins. DOX promoted the immunostimulatory effects of cardiomyocyte EVs but not hepatocyte EVs. The differential functions of EVs on macrophage phenotype switching are due to their different effects on Thioredoxin 1 redox state, which regulates activities of redox sensitive transcription factors NFκB and Nrf-2. Our findings shed light on the role of EVs as a redox active mediator of immune response during chemotherapy.

Our laboratory delineated a role for alternative pre-mRNA splicing (AS) in triple negative breast cancer (TNBC). We found the translational regulator cytosolic polyadenylation element binding protein 2 (CPEB2) which has two isoforms, CPEB2A and CPEB2B, is alternatively spliced during acquisition of anoikis resistance (AnR) and metastasis. The splicing event which determines the CPEB2 isoform is via inclusion/ exclusion of exon four in the mature mRNA transcript. The loss of CPEB2A with a concomitant increase in CPEB2B is required for TNBC cells to metastasize in vivo. We examined RNAseq profiles of TNBC cells which had CPEB2 isoforms specifically downregulated to examine the mechanism by which CPEB2 isoforms mediate opposing effects on cancer-related phenotypes. Downregulation of the CPEB2B isoform inhibited pathways driving the epithelial-to-mesenchymal transition (EMT) and hypoxic response, whereas downregulation of the CPEB2A isoform did not have this effect. Specifically, CPEB2B functioned as a translational activator of TWIST1 and HIF1a. Functional studies showed that specific downregulation of either HIF1α or TWIST1 inhibited the ability of CPEB2B to induce AnR and drive metastasis. The mechanism governing inclusion/ exclusion of exon 4 was determined to be serine/ arginine-rich splicing factor 3 (SRSF3). Binding of SRSF3 to a consensus sequence within CPEB2 exon 4 promoted its inclusion in the mature mRNA, and mutation of this sequence abolished association of SRSF3 with exon 4. SRSF3 expression was upregulated in TNBC cells upon acquisition of AnR correlating with a reduction in the CPEB2A/B ratio. Importantly, downregulation of SRSF3 by siRNA in these cells induced the exclusion of exon 4. Downregulation of SRSF3 also reversed the CPEB2A/B ratio in a wild-type CPEB2 exon 4 minigene construct, but not a mutant CPEB2 minigene with the SRSF3 RNA cis-element ablated. Physiologic studies demonstrated SRSF3 downregulation ablated AnR in TNBC cells, and was “rescued” by ectopic expression of CPEB2B. Importantly, biostatistical analysis of The Cancer Genome Atlas database showed a positive relationship between alterations in SRSF3 expression and lower overall survival in TNBC. Overall, this study demonstrates that SRSF3 modulates CPEB2 AS to induce the expression of the CPEB2B isoform that drives TNBC phenotypes correlating with aggressive human breast cancer.

Obesity has reached an epidemic level in the United States. A number of epidemiological studies have established obesity as a critical risk factor for postmenopausal breast cancer (post-BC), whereas a reverse association holds prior to menopause. A significant scientific gap exists in understanding the mechanism(s) underpinning this epidemiological phenomenon, particularly the reverse association between obesity and premenopausal breast cancer (pre-BC). This study aimed to understand how folate metabolism and DNA methylation informs the association between obesity and pre-BC. Fifty normal breast tissue samples were collected from premenopausal women who underwent reduction mammoplasty. We developed and measured the breast tissue folate by a Lactobacillus Casei microbiological assay, and the DNA methylation of LINE-1, a biomarker of genome-wide methylation, and the promoter methylation and gene expression of SFRP1, a tumor suppressor, were measured by pyrosequencing and real-time PCR. We found a high BMI is associated with increased folate level in the mammary tissue, with an increase of 2.65 ng/g of folate per every 5-unit increase of BMI (p < 0.05). The LINE-1 DNA methylation was significantly associated with BMI (p < 0.05), and marginally associated with folate concentration (p = 0.087). For the 8 CpG sites analyzed in the promoter region of the SFRP1 gene, no associations were observed for either BMI or tissue folate (p > 0.05), although a high expression of SFRP1 was observed in subjects with high BMI or high folate (p < 0.05). This study demonstrated that, in premenopausal women, obesity is associated with an increased mammary folate status, genome-wide DNA methylation and SFRP1 gene expression, indicating that the improved folate and epigenetic status is potentially responsible for the reverse association between obesity and pre-BC. More studies are warranted to further understand how obesity mediates pre-BC via altering folate metabolism and DNA methylation.

Apurinic/apyrimidinic DNA repair endonuclease-1 (APE1), first recognized as an important DNA excision repair enzyme, is also known as Redox Factor-1 (Ref-1) involved in the activation of many nuclear transcription factors in both redox-dependent and independent manner. It has been well-documented that the overexpression of APE/Ref-1 contributes to the development of chemo-resistance and is associated with tumor progression in many human malignancies [1]. Our previous study in melanoma demonstrated that the development of novel inhibitors targeting the redox regulation domain of APE/Ref-1 is a promising strategy for melanoma treatment. To date, limited successes have been reported in developing novel APE/Ref-1 inhibitors for cancer treatment. Utilizing a structure-based approach, our study identified and characterized small molecular inhibitors of APE/Ref-1. First, N-terminally truncated APE/Ref-1 protein lacking the first 40 amino acid residues (∆40APE-1wt) was cloned into the pGEX-6P1 vector to express the GST-∆40APE-1wtprotein. After cleavage of GST-tag, the concentrated ∆40APE-1wt protein was subjected to protein crystallization study. We have successfully diffracted ∆40APE-1wt crystals and collected data with a resolution of 1.57Å. The crystal structure was further determined by molecular replacement in Molrep using the already available human APE-1 structure (PDB: 5CFG). For the first time, we observed the dimerization of APE/Ref-1 protein formed under oxidative conditions, which may contribute to the redox regulation of APE/Ref-1. Such structural transformation of APE/Ref-1 protein under distinct redox conditions may pave the way for future drug development and optimization. The binding affinity of the candidate compounds with ∆40APE-1wt protein was also determined using Surface Plasmon Resonance (SPR), and the Ki values were analyzed. One of the potent inhibitors developed by our group by structure-based approach, exhibited promising anti-melanoma activities both in vitro and in vivo. Future studies on the structure-activity association are warranted.

Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections induce a myriad of disturbances to CD4 T cell functions, including mitochondrial compromise, excessive inflammation, increased telomeric DNA damage and attrition, cellular exhaustion and senescence, and accelerated aging. In this dissertation, the mechanisms underlying metabolic failure, accelerated aging, and cellular dysfunctions were evaluated in CD4 T cells from healthy subjects (HS) treated with a telomere-targeting drug (KML001) or HCV-infected individuals or people living with HIV (PLHIV) compared to HS. We observed that KML001-induced telomere injury resulted in mitochondrial swelling and decreased mitochondrial membrane potential, cellular respiration, mitochondrial DNA (mtDNA) copy number, and ATP production mediated by p53-mediated repression of the master mitochondrial regulators peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and nuclear respiratory factor 1 (NRF-1). We then investigated the mechanisms responsible for T cell dysfunction and metabolic failure during chronic viral infections (HCV, HIV). We observed that chronic HCV infection leads to elevated production of cellular and mitochondrial reactive oxygen species (ROS), impaired mtDNA, and altered levels of proteins responsible for mediating oxidative stress, apoptosis, and mtDNA maintenance, as well as mitochondrial regulators PGC-1α and mitochondrial transcription factor A (mtTFA), contributing to impaired cellular respiration and mtDNA content. Similarly, we demonstrated that latent HIV infection induced disruptions to CD4 T cell homeostasis and increased cellular exhaustion, senescence, and apoptosis and reduced proliferation. We also observed significant repression of mitochondrial respiration, mtDNA content, and mtTFA levels in CD4 T cells from PLHIV, which was reversed via ectopic expression of mtTFA. Finally, we observed elevated cellular and mitochondria ROS production in CD4 T cells from PLHIV, along with significant deregulation of levels of antioxidant defense (superoxide dismutase 1, SOD1) and oxidative stress-induced DNA damage repair (apurinic/apyrimidinic endonuclease 1, APE1) proteins, which were shown to be essential for cellular respiration independently of mtDNA content. Taken together, this research highlights novel multi-leveled mechanisms by which chronic viral infection induces accelerated T cell aging and mitochondrial compromise via deregulating master mitochondrial regulators and provides a diverse collection of novel therapeutic targets that may be applied to various infectious diseases.

Human Immunodeficiency Virus (HIV) infection initiates major metabolic and cell- survival complications. Anti-retroviral therapy (ART) is the current approach to suppress active HIV replication to a level of undetected viral load, but it is not a curative approach. Newer and sophisticated gene editing technologies could indeed be a potent antiviral therapy to achieve a clinical sterilization/cure of HIV infection. Chronic HIV patients, even under a successful ART regimen, exhibit a low-grade inflammation, immune senescence, premature aging, telomeric DNA attrition, T cell apoptosis, and cellular homeostasis. In this dissertation, we investigated CD4 T cell homeostasis, degree of T cell apoptosis, an associated telomeric DNA damage, DNA damage repair signaling, and the apoptotic pathways in CD4 T cells during HIV infection with or without ART treatment. Our data support a DNA damage accumulation, and impaired DNA damage repair in chromosome ends via recruitment of 53BP1 protein to the damaged foci. We found that a key player of DNA damage and repair enzyme, ATM, and its associated checkpoint proteins (CHK1, CKH2) are affected by HIV infection. HIV infection also altered another multifunctional master regulator protein AKT that is crucial in maintaining cellular homeostasis. Curing HIV is the ultimate redemption against HIV-associated complications. To explore the possibility of a functional cure, we investigated the use of a transient and a non-viral CRISPR/Cas9-based gene editing technology targeting the latently incorporated HIV provirus. After performing a nucleofection/electroporation using an in vitro formulated ribonucleoprotein (RNP) constituting a synthetic guide RNA (gRNA) and Cas9 nuclease protein, we demonstrated a significant (maximum 97%) reduction of HIV-mRNA and p24-capsid protein expression, upon stimulation (using PMA) and latency reactivation of latently HIV-infected CD4 T cells and latent-monocytes. Notably, the RNP treatment did not induce any cytotoxic effects, without affecting the abilility of cell proliferation. A sequence specific cleavage of HIV-provirus in two crucial gene locations (targeting vpr/tat genes) showed the most significant suppression of HIV reactivation or latency reversal. We have used DNA sequencing, and T7EI assay to confirm the target-site-specific cleavage of the HIV-proviral genome. Our data confirm the activation of non- homologous end joining (NHEJ) pathway to repair the double-stranded DNA break created by the CRISPR/Cas9 treatment. Taken together, this study provides a new gene therapeutic approach using synthetic gRNA/Cas9 targeting HIV genome, which warrant further in vivo animal and human studies.

Preterm birth is the number one cause of death in neonates, accounting for 35% of neonatal mortality. The preterm birth rate in the U.S. in 2021 was 10.5%, disproportionally affected by race and ethnicity. Obese women have an increased risk of preterm pregnancy, and if delivered before 37 weeks of gestation, the offspring have higher rates of complications that extend from the neonatal period into life-long metabolic and immune adverse outcomes. In the early months after delivery, preterm infants have higher rates of adverse health outcomes than term infants, including infections, extrauterine growth restrictions, respiratory, metabolic, and neurological complications, necrotizing enterocolitis (NEC), and bronchopulmonary dysplasia (BPD). Diet for the preterm infant is crucial for infection prevention, and maternal breast milk is most beneficial when given in the first few days after birth. Expectant and breastfeeding mothers should consume appropriate food and supplements to optimize their nutrition. In addition to nutrients, bioactive components, vitamins, and minerals found in breast milk (BM), there is evidence that microbes (microbiome) are a significant factor in infant development, contributing to protection against pathogens and playing a role in the development of the immune and nervous systems. Maternal BM composition and microbiome are affected by many factors, including maternal body mass index (BMI), maternal health, antibiotics use, mode of delivery, maternal parity, gestational age, and time and duration of lactation. Maternal body composition, however, and not maternal nutritional status, is associated with breast milk nutritional composition. Altogether, these maternal factors may modify the premature infant gut microbiome. We examined the role of maternal adiposity and how it impacts the composition of human breast milk, specifically hormones, nutrient composition, short and long chain fatty acids, and microbiome. We also examined the role of maternal adiposity and how it impacts the short-chain fatty acids and microbiome in infant fecal samples. We found that maternal adiposity affects breast milk hormones, potentially modulating infant metabolism. Additionally, we found that maternal adiposity does not alter the nutrient composition of breast milk; however, differences in both short and long chain fatty acids and maternal adiposity were detected. In our small cross-sectional cohort of preterm infants, we did not observe differences in short-chain fatty acids in the preterm infant stool samples compared to maternal adiposity. Concerning maternal adiposity and its impact on the microbiomes of breast milk and infants, we observed differences in phyla and genera between the maternal BMI groups on the outcome of breast milk and preterm infant microbiomes but no statistical significance in alpha and beta diversities between the groups. Thus, our results indicate that maternal adiposity impacts hormonal, microbial composition, and short-chain fatty acid profiles in breast milk, which tremendously influences infant growth and development.