PKC signaling regulates drug resistance of the fungal pathogen Candida albicans via circuitry comprised of Mkc1, calcineurin, and Hsp90.

Fungal pathogens exploit diverse mechanisms to survive exposure to antifungal drugs. This poses concern given the limited number of clinically useful antifungals and the growing population of immunocompromised individuals vulnerable to life-threatening fungal infection. To identify molecules that abrogate resistance to the most widely deployed class of antifungals, the azoles, we conducted a screen of 1,280 pharmacologically active compounds. Three out of seven hits that abolished azole resistance of a resistant mutant of the model yeast Saccharomyces cerevisiae and a clinical isolate of the leading human fungal pathogen Candida albicans were inhibitors of protein kinase C (PKC), which regulates cell wall integrity during growth, morphogenesis, and response to cell wall stress. Pharmacological or genetic impairment of Pkc1 conferred hypersensitivity to multiple drugs that target synthesis of the key cell membrane sterol ergosterol, including azoles, allylamines, and morpholines. Pkc1 enabled survival of cell membrane stress at least in part via the mitogen activated protein kinase (MAPK) cascade in both species, though through distinct downstream effectors. Strikingly, inhibition of Pkc1 phenocopied inhibition of the molecular chaperone Hsp90 or its client protein calcineurin. PKC signaling was required for calcineurin activation in response to drug exposure in S. cerevisiae. In contrast, Pkc1 and calcineurin independently regulate drug resistance via a common target in C. albicans. We identified an additional level of regulatory control in the C. albicans circuitry linking PKC signaling, Hsp90, and calcineurin as genetic reduction of Hsp90 led to depletion of the terminal MAPK, Mkc1. Deletion of C. albicans PKC1 rendered fungistatic ergosterol biosynthesis inhibitors fungicidal and attenuated virulence in a murine model of systemic candidiasis. This work establishes a new role for PKC signaling in drug resistance, novel circuitry through which Hsp90 regulates drug resistance, and that targeting stress response signaling provides a promising strategy for treating life-threatening fungal infections.

Dasatinib (BMS-35482) potentiates the activity of gemcitabine and docetaxel in uterine leiomyosarcoma cell lines.

BACKGROUND: To explore the activity of dasatinib alone and in combination with gemcitabine and docetaxel in uterine leiomyosarcoma (uLMS) cell lines, and determine if dasatinib inhibits the SRC pathway. METHODS: SK-UT-1 and SK-UT-1B uLMS cells were treated with gemcitabine, docetaxel and dasatinib individually and in combination. SRC and paxcillin protein expression were determined pre- and post-dasatinib treatment using Meso Scale Discovery (MSD) multi-array immunogenicity assay. Dose-response curves were constructed and the coefficient of drug interaction (CDI) and combination index (CI) for drug interaction calculated. RESULTS: Activated phosphorylated levels of SRC and paxillin were decreased after treatment with dasatinib in both cell lines (p < 0.001). The addition of a minimally active concentration of dasatinib (IC25) decreased the IC50 of each cytotoxic agent by 2-4 fold. The combination of gemcitabine-docetaxel yielded a synergistic effect in SK-UT-1 (CI = 0.59) and an antagonistic effect in SK-UT-1B (CI = 1.36). Dasatinib combined with gemcitabine or docetaxel revealed a synergistic anti-tumor effect (CDI < 1) in both cell lines. The triple drug combination and sequencing revealed conflicting results with a synergistic effect in SK-UT-1B and antagonistic in SK-UT-1. CONCLUSION: Dasatinib inhibits the SRC pathway and yields a synergistic effect with the two-drug combination with either gemcitabine or docetaxel. The value of adding dasatinib to gemcitabine and docetaxel in a triple drug combination is uncertain, but may be beneficial in select uLMS cell lines. Based on our pre-clinical data and known activity of gemcitabine and docetaxel, further evaluation of dasatinib in combination with these agents for the treatment of uLMS is warranted.

Cumulative exposure to traumatic events in older adults.

OBJECTIVES: The present study examined the impact of cumulative trauma exposure on current posttraumatic stress disorder (PTSD) symptom severity in a nonclinical sample of adults in their 60s. The predictive utility of cumulative trauma exposure was compared to other known predictors of PTSD, including trauma severity, personality traits, social support, and event centrality. METHOD: Community-dwelling adults (n = 2515) from the crest of the Baby Boom generation completed the Traumatic Life Events Questionnaire, the PTSD Checklist, the NEO Personality Inventory, the Centrality of Event Scale, and rated their current social support. RESULTS: Cumulative trauma exposure predicted greater PTSD symptom severity in hierarchical regression analyses consistent with a dose-response model. Neuroticism and event centrality also emerged as robust predictors of PTSD symptom severity. In contrast, the severity of individuals' single most distressing life event, as measured by self-report ratings of the A1 PTSD diagnostic criterion, did not add explanatory variance to the model. Analyses concerning event categories revealed that cumulative exposure to childhood violence and adulthood physical assaults were most strongly associated with PTSD symptom severity in older adulthood. Moreover, cumulative self-oriented events accounted for a larger percentage of variance in symptom severity compared to events directed at others. CONCLUSION: Our findings suggest that the cumulative impact of exposure to traumatic events throughout the life course contributes significantly to posttraumatic stress in older adulthood above and beyond other known predictors of PTSD.

Later Life Consequences of Subteratogenic Exposure to a Complex PAH Mixture in the Atlantic Killifish (Fundulus heteroclitus)

Subteratogenic and other low-level chronic exposures to toxicant mixtures are an understudied threat to environmental and human health. It is especially important to understand the effects of these exposures for contaminants, such as polycyclic aromatic hydrocarbons (PAHs) a large group of more than 100 individual compounds, which are important environmental (including aquatic) contaminants. Aquatic sediments constitute a major sink for hydrophobic pollutants, and studies show PAHs can persist in sediments over time. Furthermore, estuarine systems (namely breeding grounds) are of particular concern, as they are highly impacted by a wide variety of pollutants, and estuarine fishes are often exposed to some of the highest levels of contaminants of any vertebrate taxon. Acute embryonic exposure to PAHs results in cardiac teratogenesis in fish, and early life exposure to certain individual PAHs and PAH mixtures cause heart alterations with decreased swimming capacity in adult fish. Consequently, the heart and cardiorespiratory system are thought to be targets of PAH mixture exposure. While many studies have investigated acute, teratogenic PAH exposures, few studies have longitudinally examined the impacts of subtle, subteratogenic PAH mixture exposures, which are arguably more broadly applicable to environmental contamination scenarios. The goal of this dissertation was to highlight the later-life consequences of early-life exposure to subteratogenic concentrations of a complex, environmentally relevant PAH mixture.

A unique population of Fundulus heteroclitus (the Atlantic killifish or mummichog, hereafter referred to as killifish), has adapted to creosote-based polycyclic aromatic hydrocarbons (PAHs) found at the Atlantic Wood Industries (AW) Superfund site in the southern branch of the Elizabeth River, VA, USA. This killifish population survives in a site heavily contaminated with a mixture of PAHs from former creosote operations. They have developed resistance to the acute toxicity and teratogenic effects caused by the mixture of PAHs in sediment from the site. The primary goal of this dissertation was to compare and contrast later-life outcomes of early-life, subteratogenic PAH mixture exposure in both the Atlantic Wood killifish (AW) and a naïve reference population of killifish from King’s Creek (KC; a relatively uncontaminated tributary of the Severn River, VA). Killifish from both populations were exposed to subteratogenic concentrations of a complex PAH-sediment extract, Elizabeth River Sediment Extract (ERSE), made by collecting sediment from the AW site. Fish were reared over a 5-month period in the laboratory, during which they were examined for a variety of molecular, physiological and behavioral responses.

The central aims of my dissertation were to determine alterations to embryonic gene expression, larval swimming activity, adult behavior, heart structure, enzyme activity, and swimming/cardiorespiratory performance following subteratogenic exposure to ERSE. I hypothesized that subteratogenic exposure to ERSE would impair cardiac ontogenic processes in a way that would be detectable via gene expression in embryos, and that the misregulation of cardiac genes would help to explain activity changes, behavioral deficits, and later-life swimming deficiencies. I also hypothesized that fish heart structure would be altered. In addition, I hypothesized that the AW killifish population would be resistant to developmental exposures and perform normally in later life challenges. To investigate these hypotheses, a series of experiments were carried out in PAH-adapted killifish from Elizabeth River and in reference killifish. As an ancillary project to the primary aims of the dissertation, I examined the toxicity of weaker aryl hydrocarbon receptor (AHR) agonists in combination with fluoranthene (FL), an inhibitor of cytochrome P4501A1 (CYP1A1). This side project was conducted in both Danio rerio (zebrafish) and the KC and AW killifish.

Embryonic gene expression was measured in both killifish populations over an ERSE dose response with multiple time points (12, 24, 48, and 144 hours post exposure). Genes known to play critical roles in cardiac structure/development, cardiac function, and angiogenesis were elevated, indicating cardiac damage and activation of cardiovascular repair mechanisms. These data helped to inform later-life swimming performance and cardiac histology studies. Behavior was assessed during light and dark cycles in larvae of both populations following developmental exposure to ERSE. While KC killifish showed activity differences following exposure, AW killifish showed no significant changes even at concentrations that would cause overt cardiac toxicity in KC killifish. Juvenile behavior experiments demonstrated hyperactivity following ERSE exposure in KC killifish, but no significant behavioral changes in AW killifish. Adult swimming performance via prolonged critical swimming capacity (Ucrit) demonstrated performance costs in the AW killifish. Furthermore, swimming performance decline was observed in KC killifish following exposure to increasing dilutions of ERSE. Lastly, cardiac histology suggested that early-life exposure to ERSE could result in cardiac structural alteration and extravasation of blood into the pericardial cavity.

Responses to AHR agonists resulted in a ranking of relative potency for agonists, and determined which agonists, when combined with FL, caused cardiac teratogenesis. These experiments showed interesting species differences for zebrafish and killifish. To probe mechanisms responsible for cardiotoxicity, a CYP1A-morpholino and a AHR2-morpholino were used to mimic FL effects or attempt to rescue cardiac deformities respectively. Findings suggested that the cardiac toxicity elicited by weak agonist + FL exposure was likely driven by AHR-independent mechanisms. These studies stand in contrast to previous research from our lab showing that moderate AHR agonist + FL caused cardiac toxicity that can be partially rescued by AHR-morpholino knockdown.

My findings will form better characterization of mechanisms of PAH toxicity, and advance our understanding of how subteratogenic mixtures of PAHs exert their toxic action in naïve killifish. Furthermore, these studies will provide a framework for investigating how subteratogenic exposures to PAH mixtures can impact aquatic organismal health and performance. Most importantly, these experiments have the potential to help inform risk assessment in fish, mammals, and potentially humans. Ultimately, this research will help protect populations exposed to subtle PAH-contamination.

Different Mechanisms Confer Gradual Control and Memory at Nutrient- and Stress-Regulated Genes in Yeast.

Cells respond to environmental stimuli by fine-tuned regulation of gene expression. Here we investigated the dose-dependent modulation of gene expression at high temporal resolution in response to nutrient and stress signals in yeast. The GAL1 activity in cell populations is modulated in a well-defined range of galactose concentrations, correlating with a dynamic change of histone remodeling and RNA polymerase II (RNAPII) association. This behavior is the result of a heterogeneous induction delay caused by decreasing inducer concentrations across the population. Chromatin remodeling appears to be the basis for the dynamic GAL1 expression, because mutants with impaired histone dynamics show severely truncated dose-response profiles. In contrast, the GRE2 promoter operates like a rapid off/on switch in response to increasing osmotic stress, with almost constant expression rates and exclusively temporal regulation of histone remodeling and RNAPII occupancy. The Gal3 inducer and the Hog1 mitogen-activated protein (MAP) kinase seem to determine the different dose-response strategies at the two promoters. Accordingly, GAL1 becomes highly sensitive and dose independent if previously stimulated because of residual Gal3 levels, whereas GRE2 expression diminishes upon repeated stimulation due to acquired stress resistance. Our analysis reveals important differences in the way dynamic signals create dose-sensitive gene expression outputs.