Human Biology and Astrophysics

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Molecular characterization of novel transcription of antisense toxin-antitoxin RNA in regulating Mycobacterium tuberculosis

2020 Spring.Includes bibliographical references.Despite more than seventy years of available anti-tuberculosis (TB) treatments, Mycobacterium tuberculosis (Mtb) remains the deadliest human pathogen. Novel short-course therapies are needed that effectively treat latent TB infection (LTBI), which is like a major source for new infections. However, the molecular determinants of LTBI, including a large repertoire of regulators encoded by Mtb that mediate survival, are largely uncharacterized. Gene expression studies have implicated numerous regulators and particularly toxin-antitoxin (TA) systems in Mtb pathogenesis. Whole genome sequencing (i.e. WGS) studies have linked the massive genomic expansion of TA systems along with other pathogen-specific gene families to the emergence of TB-causing mycobacteria. In addition, a multitude of TA systems show genotypic differences that distinguish between ancient and modern lineages of Mtb. These predominantly include lineage-specific changes in amino acids, altering antitoxin DNA-binding, and nucleotides, generating new promoters. These mutations have led to an overrepresentation of differentially expressed Mtb TA genes responsible for mediating epigenetic changes that are associated with gains in virulence of modern lineages. Thus, the work presented in this dissertation begins to define the novel co-regulation of TA systems that underlie Mtb pathogenesis. Unraveling of more complex regulation of Mtb TA systems will provide keen insights into the phenotypic changes responsible for Mtb survival and persistence in vivo. This will ultimately help to streamline research and development of novel antibiotics as well as host directed immunotherapies against hard-to-treat tubercle bacilli, effectively shortening the duration of TB treatment. TA systems are ubiquitous among bacteria, especially pathogens, and increasingly found to be essential for adaptation to host immune defenses and in vivo drug pressures, resulting in the development of persistent or chronic infections. Phylogenomics comparisons have revealed that Mtb encodes a significantly expanded repertoire of TA systems that are solely conserved by tubercle bacilli, including homologous ParDE/RelBE systems like RelBE2 (i.e. Rv2865-Rv2866). Herein, we report a novel antisense (as)RNA, we call asRelE2, which is uniquely encoded by Mtb and involved in differentially post-transcriptionally regulating relE2 mRNA levels as part of the response to host-associated stress such as low pH in a cAMP-dependent manner. This dynamic regulation of the tripartite relBE2/asrelE2 TA locus appear to be essential for long-term survival under acidic stress in vitro. In addition, the overexpression of relE2 is found to mediate phenotypic development of a persistent state in Mtb associated with increasing tolerance towards frontline anti-TB drugs isoniazid (Inh) and rifampicin (Rif). In mice, asRelE2 acts in differentially regulating bi-cistronic relB2 and relE2 mRNA levels in a host tissue-specific manner dependent upon the downstream effector functions of interferon gamma (i.e. IFN-γ) in murine TB. Specifically, relE2 and relB2 mRNA levels are found to steadily increase in lungs and in spleens, respectively, in the development of the chronic phase of Mtb infection. To our knowledge, this is the first time a Mtb TA system has been shown to be co-regulated by an asRNA antitoxin. Furthermore, this is linked with the development of the adaptive host immune response to Mtb, demonstrating that the post-transcriptional regulation of TA systems is an important mechanism, coordinating the epigenetic changes that are a hallmark of Mtb persistence and pathogenesis

Engagement with care, substance use, and adherence to therapy in HIV/AIDS

Engagement with care for those living with HIV is aimed at establishing a strong relationship between patients and their health care provider and is often associated with greater adherence to therapy and treatment (Flickinger, Saha, Moore, and Beach, 2013). Substance use behaviors are linked with lower rates of engagement with care and medication adherence (Horvath, Carrico, Simoni, Boyer, Amico, and Petroli, 2013). This study is a secondary data analysis using a cross-sectional design from a larger randomized controlled trial (n = 775) that investigated the efficacy of a self-care symptom management manual for participants living with HIV. Participants were recruited from countries of Africa and the US. This study provides evidence that substance use is linked with lower self-reported engagement with care and adherence to therapy. Data on substance use and engagement are presented. Clinical implications of the study address the importance of utilizing health care system and policy factors to improve engagement with care

Research Priorities for Children's Nursing in Ireland: A Delphi Study

This paper is a report of a study which identified research priorities for children's nursing in an acute care setting in Ireland. A limited number of studies have examined research priorities for children's nursing. This study was undertaken against the backdrop of significant proposed changes to the delivery of of children's healthcare. A three round Delphi survey design was used to identify and rate the importance of research priorities for children's nursing. In round 1 participants were asked to identify five of the most important research priorities for children's nursing. Participants in round 2 were asked to rate the importance of each of each research priority on a seven point Likert scale. In round 3 participants were presented with the mean scoreof each research priority from the second questionaire, and again asked to consider the importance of each topic on a 7 point Likert scale. The aim was to reach a consensus on the priorities. The top three priorities identified were recognition and care of the deteriorating child, safe transfer of the critically ill child between acute health care facilities, and the child and families perceptions of care at end-of life. The wide variation of priorities reflects the scope of care delivery of children's nurses and mirrors many global care concerns in caring for children

Engagement with Care, Substance Use, and Adherence to Therapy in HIV/AIDS

Engagement with care for those living with HIV is aimed at establishing a strong relationship between patients and their health care provider and is often associated with greater adherence to therapy and treatment (Flickinger, Saha, Moore, and Beach, 2013). Substance use behaviors are linked with lower rates of engagement with care and medication adherence (Horvath, Carrico, Simoni, Boyer, Amico, and Petroli, 2013). This study is a secondary data analysis using a cross-sectional design from a larger randomized controlled trial (n = 775) that investigated the efficacy of a self-care symptom management manual for participants living with HIV. Participants were recruited from countries of Africa and the US. This study provides evidence that substance use is linked with lower self-reported engagement with care and adherence to therapy. Data on substance use and engagement are presented. Clinical implications of the study address the importance of utilizing health care system and policy factors to improve engagement with care

A field investigation of phreatophyte-induced fluctuations in the water table

This is the published version. Copyright American Geophysical Union[1] Hydrographs from shallow wells in vegetated riparian zones frequently display a distinctive pattern of diurnal water table fluctuations produced by variations in plant water use. A multisite investigation assessed the major controls on these fluctuations and the ecohydrologic insights that can be gleaned from them. Spatial and temporal variations in the amplitude of the fluctuations are primarily a function of variations in (1) the meteorological drivers of plant water use, (2) vegetation density, type, and vitality, and (3) the specific yield of sediments in the vicinity of the water table. Past hydrologic conditions experienced by the riparian zone vegetation, either in previous years or earlier within the same growing season, are also an important control. Diurnal water table fluctuations can be considered a diagnostic indicator of groundwater consumption by phreatophytes at most sites, so the information embedded within these fluctuations should be more widely exploited in ecohydrologic studies

Costs and Consequences: Hepatitis C Seroprevalence in the Military and Its Impact on Potential Screening Strategies

UNLABELLED: Knowledge of the contemporary epidemiology of hepatitis C viral (HCV) infection among military personnel can inform potential Department of Defense screening policy. HCV infection status at the time of accession and following deployment was determined by evaluating reposed serum from 10,000 service members recently deployed to combat operations in Iraq and Afghanistan in the period 2007-2010. A cost model was developed from the perspective of the Department of Defense for a military applicant screening program. Return on investment was based on comparison between screening program costs and potential treatment costs avoided. The prevalence of HCV antibody-positive and chronic HCV infection at accession among younger recently deployed military personnel born after 1965 was 0.98/1000 (95% confidence interval 0.45-1.85) and 0.43/1000 (95% confidence interval 0.12-1.11), respectively. Among these, service-related incidence was low; 64% of infections were present at the time of accession. With no screening, the cost to the Department of Defense of treating the estimated 93 cases of chronic HCV cases from a single year\u27s accession cohort was $9.3 million. Screening with the HCV antibody test followed by the nucleic acid test for confirmation yielded a net annual savings and a$3.1 million dollar advantage over not screening. CONCLUSIONS: Applicant screening will reduce chronic HCV infection in the force, result in a small system costs savings, and decrease the threat of transfusion-transmitted HCV infection in the battlefield blood supply and may lead to earlier diagnosis and linkage to care; initiation of an applicant screening program will require ongoing evaluation that considers changes in the treatment cost and practice landscape, screening options, and the epidemiology of HCV in the applicant/accession and overall force populations

Hepatitis B Seroprevalence in the U.S. Military and its Impact on Potential Screening Strategies

INTRODUCTION: Knowledge of the contemporary epidemiology of hepatitis B virus (HBV) infection among military personnel can inform potential Department of Defense (DoD) screening policy and infection and disease control strategies. MATERIALS AND METHODS: HBV infection status at accession and following deployment was determined by evaluating reposed serum from 10,000 service members recently deployed to combat operations in Iraq and Afghanistan in the period from 2007 to 2010. A cost model was developed from the perspective of the Department of Defense for a program to integrate HBV infection screening of applicants for military service into the existing screening program of screening new accessions for vaccine-preventable infections. RESULTS: The prevalence of chronic HBV infection at accession was 2.3/1,000 (95% CI: 1.4, 3.2); most cases (16/21, 76%) identified after deployment were present at accession. There were 110 military service-related HBV infections identified. Screening accessions who are identified as HBV susceptible with HBV surface antigen followed by HBV surface antigen neutralization for confirmation offered no cost advantage over not screening and resulted in a net annual increase in cost of $5.78 million. However, screening would exclude as many as 514 HBV cases each year from accession. CONCLUSIONS: Screening for HBV infection at service entry would potentially reduce chronic HBV infection in the force, decrease the threat of transfusion-transmitted HBV infection in the battlefield blood supply, and lead to earlier diagnosis and linkage to care; however, applicant screening is not cost saving. Service-related incident infections indicate a durable threat, the need for improved laboratory-based surveillance tools, and mandate review of immunization policy and practice

Farnesol-Induced Apoptosis in Candida albicans Is Mediated by Cdr1-p Extrusion and Depletion of Intracellular Glutathione

Farnesol is a key derivative in the sterol biosynthesis pathway in eukaryotic cells previously identified as a quorum sensing molecule in the human fungal pathogen Candida albicans. Recently, we demonstrated that above threshold concentrations, farnesol is capable of triggering apoptosis in C. albicans. However, the exact mechanism of farnesol cytotoxicity is not fully elucidated. Lipophilic compounds such as farnesol are known to conjugate with glutathione, an antioxidant crucial for cellular detoxification against damaging compounds. Glutathione conjugates act as substrates for ATP-dependent ABC transporters and are extruded from the cell. To that end, this current study was undertaken to validate the hypothesis that farnesol conjugation with intracellular glutathione coupled with Cdr1p-mediated extrusion of glutathione conjugates, results in total glutathione depletion, oxidative stress and ultimately fungal cell death. The combined findings demonstrated a significant decrease in intracellular glutathione levels concomitant with up-regulation of CDR1 and decreased cell viability. However, addition of exogenous reduced glutathione maintained intracellular glutathione levels and enhanced viability. In contrast, farnesol toxicity was decreased in a mutant lacking CDR1, whereas it was increased in a CDR1-overexpressing strain. Further, gene expression studies demonstrated significant up-regulation of the SOD genes, primary enzymes responsible for defense against oxidative stress, with no changes in expression in CDR1. This is the first study describing the involvement of Cdr1p-mediated glutathione efflux as a mechanism preceding the farnesol-induced apoptotic process in C. albicans. Understanding of the mechanisms underlying farnesol-cytotoxicity in C. albicans may lead to the development of this redox-cycling agent as an alternative antifungal agent