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Interaction Between Two E3 ligases, NEDD8ylated Cullin and HHARI
Get PDFRBR (RING1-in between RING-RING2) is a special type of E3 ubiquitin ligase containing three zinc-binding RING (Really Interesting New Gene) domains, while adopting mechanisms of HECT (Homologous to E6-AP Carboxyl Terminus) for substrate ubiquitination. Most well known RBRs include Parkin and HOIP, which are associated with Parkinson’s disease and innate immune deficiency. However, it is not well known how the RBR proteins gain activity, as they are known to be autoinhibited. Here I show that a specific F430A, E431A, E503A triple mutation of RBR protein HHARI (Human homologue of Ariadne) and its interaction with NEDD8ylated cullin RING ligase can both boost its activity and stabilize complex formation. Analytical size-exclusion chromatography, autoubiquitination, and electron microscopy reveal consistent behavior for this triple-mutant. Future structure-based studies will help elucidate the mechanism of the unsolved mystery of RBR activation and its interaction with NEDD8ylated cullin RING ligases
Multipronged Approach to Study Glaucoma-Associated Phenotypes
Get PDFGlaucoma refers to a group of conditions characterized by death of RGCs, increase in intraocular pressure is usually a precursor to glaucoma and irreversible optic neuropathy leads to visual impairment and blindness. Recent advances have seen a surge of new ideas and technologies to aid in the early detection, efficacious treatments and neuroprotection. Despite advances we face several challenges in understanding the pathophysiology of glaucoma. One of the many challenges scientists and ophthalmologists are facing is, to better understand IOP, its role in glaucomatous damage and design safer, more predictable IOP-lowering therapies. Another challenge is to find a practical method or develop tools to understand the molecular pathways to study RGC function and health to develop novel therapies for vision loss. This work leads to
three complementary insights on how to address these challenges. First, we used a novel systems genetics approach to identify and validate genetic modifiers of IOP using the enlarged BXD family of strains in combination with human GWAS glaucoma cohorts. This will pave the way for improved drug development tailored to individual genotypes for POAG. Second, we have combined systems genetics, bidirectional studies using multiple species, meta-analyses, immunohistochemistry, FACS sorting and gene knockdown studies to identify and validate the identity of a genetic modulator of Sncg, a gene that has been previously implicated in RGC death in glaucoma. Outcomes of the investigation may provide clues to understanding the molecular mechanisms that account for the degenerative changes in RGCs in glaucoma. Third, we optimized a feasible, reproducible, standardized flow cytometry-based protocol for the isolation and enrichment of homogeneous RGC. This will allow for future careful assessment of important cell specific pathways in RGC to provide mechanistic insights into the declining of visual acuity in aged populations and those suffering from retinal neurodegenerative diseases. Taken together these studies offer a concise outlook on use of integrated systems science, molecular and imaging technologies as a needs-led innovation in ophthalmology and visual health
Endosomal Trafficking as a Determinant of Antifungal Tolerance in the Pathogenic Fungus Candida albicans
Get PDFSeveral important antifungal drugs inhibit the synthesis of ergosterol, a lipid that modulates the thickness, fluidity and permeability of fungal cell membranes. These include the azole antifungals, which block ergosterol biosynthesis by inhibiting lanosterol demethylase (Erg11p). The resulting depletion of cellular ergosterol and the accumulation of ‘toxic’ sterol intermediates are both thought to cause plasma membrane dysfunction and ultimately growth arrest. However, the effects of ergosterol depletion upon the function of intracellular membranes and organelles are not well described. The purpose of this study was to characterize the effects of azole treatment upon the integrity of the Candida albicans vacuole, and determine if, in turn, vacuolar trafficking influences azole susceptibility. Profound fragmentation of the C. albicans vacuole was observed as an early consequence of azole treatment, before significant growth inhibition was observed. Using a genetic approach, we determined that vacuole fragmentation was a consequence of Erg11p inhibition rather than an off-target effect of azole treatment. Moreover, the degree of vacuole fragmentation following azole treatment was influenced by Erg3p, an enzyme in the ergosterol biosynthetic pathway involved in the production of toxic sterol intermediaries upon Erg11p inhibition. We also determined that vacuolar trafficking significantly impacts C. albicans susceptibility to azole antifungals and other ergosterol biosynthesis inhibitors. For instance, a vps21∆/∆ mutant, blocked in membrane trafficking through the pre-vacuolar compartment (PVC), grew significantly more than wild-type controls in the presence of several azole antifungals under standard susceptibility testing conditions. Furthermore, the vps21∆/∆ mutant was able to grow in the presence of the azoles despite depletion of cellular ergosterol. This phenotype resembles an exaggerated form of azole tolerance known as ‘trailing growth’, which has been described for some clinical isolates. In contrast, the vps21∆/∆ mutant is hypersensitive to drugs that block alternate steps in ergosterol biosynthesis. The azole tolerance phenotype of the C. albicans vps21Δ/Δ mutant was independent of known azole resistance mechanisms such as the efflux pumps Cdr1p and Mdr1p. Moreover, the azole tolerance of the vps21Δ/Δ mutant was influenced by both pH and incubation temperature, consistent with trailing growth phenotypes. The C. albicans vps21Δ/Δ mutant exhibits less plasma membrane permeabilization upon azole treatment, as determined by the release of a cytoplasmic luciferase reporter into the culture supernatant. Our results also reveal that the vps21Δ/Δ mutant has elevated levels of intracellular calcium and enhanced calcineurin activity, as evidenced by increased expression of a calcineurin responsive RTA2-GFP reporter construct in response to fluconazole. Furthermore, the azole tolerant phenotype of the vps21Δ/Δ mutant is dependent upon both calcium and calcineurin signaling. These findings underscore the importance of endosomal trafficking in determining the cellular consequences of azole treatment through modulation of intracellular calcium levels and calcineurin dependent responses. While we determined that deletion of VPS21 alone was not sufficient to confer a survival advantage upon C. albicans following azole treatment in the mouse model of vaginal candidiasis, the azole susceptibility of the vps21Δ/Δ mutant in the mouse model disseminated infection is yet to be tested. Moreover, it is unclear how, or if the azole tolerant phenotype of the vps21Δ/Δ mutant relates to that of trailing clinical isolates, or if these clinical isolates have abnormal endosomal trafficking
The Copy and Paste Function and the Quality of the Electronic Health Record (EHR)
Get PDFThe copy and paste (c/p) function is utilized by many clinical and administrative staff in the healthcare organization. A survey comprising of 10 multiple choice questions was randomly administered to 60 employees at a skilled nursing facility to determine if the utilization of the copy and paste function leads to errors in the electronic health record (EHR) and ultimately reduced the quality of the record. After reviewing and analyzing the results it was found errors have been noticed in the record due to the c/p function, however, the respondents believed although these errors existed, the c/p function actually enhanced the quality of the EHR
A Qualitative Analysis of the Health Informatics Role in Addressing the Public Health Crisis of Addiction and Overall Effectiveness of Treatment
Get PDFIn recent years, the implementation of Health Information Management (HIM) data systems has had significant positive impacts in nearly all aspects of both health and healthcare delivery. However, during this time, there has also been a dramatic increase in the demand for addiction and mental health treatment. The rising number of individuals requiring mental health treatment for addiction has resulted in its declaration as an urgent public health epidemic by the Department of Health and Human Services. The all-encompassing effects of addiction are prevalent in all areas of society, seen in the form of across-the-board rising healthcare costs, addiction related crime rate increases, overcrowded prisons, and an inefficient system of communication among overwhelmed mental healthcare professionals and law enforcement officials. A qualitative survey was conducted in the form of a questionnaire provided to voluntary participants currently employed as medical professionals. Participants were selected based on their relevant experience with either treating addiction, and/or having firsthand experience with battling addiction themselves. Participants were encouraged to take into consideration the HIM systems in which they currently utilize when providing treatment, and provide evaluative input with emphasis on areas of desired future improvements and enhancements that may assist future HIM professionals envision more efficient mental health and addiction treatment database structures
Dissecting the Novel Roles of TRIM56 in Antiviral Innate Immunity
Get PDFThe tripartite motif-containing (TRIM) proteins have emerged as a new class of host antiviral restriction factors, with several demonstrating roles in regulating innate antiviral responses. Of \u3e70 known TRIMs, TRIM56 inhibits replication of bovine viral diarrhea virus, a ruminant pestivirus of the family Flaviviridae, but has no appreciable effect on VSV, a rhabdovirus. We have also shown that TRIM56 forms a complex with the Toll-like receptor-3 (TLR3) adaptor, TRIF, via its C-terminal residues 621-750, and augments TLR3-mediated interferon (IFN) induction and establishment of an antiviral state. Yet, TRIM56’s antiviral spectrum and the precise underlying mechanisms by which TRIM56 executes its direct antiviral functions and modulates TLR3 signaling remain undefined. Also unclear are the molecular determinants governing the direct and indirect antiviral activities of TRIM56.
Herein, in Chapter 3, I show that TRIM56 poses a barrier to infections by yellow fever virus (YFV), dengue virus serotype-2 (DENV2), and human coronavirus virus (HCoV)-OC43. Moreover, I demonstrate that TRIM56’s anti-flavivirus effects required both the E3 ligase activity that lies in the N-terminal RING domain and the integrity of its C-terminal portion, while the restriction of HCoV-OC43 relied upon the TRIM56 E3 ligase activity alone. Furthermore, TRIM56 was revealed to impair YFV and DENV2 propagation by suppressing intracellular viral RNA accumulation but to compromise HCoV-OC43 infection at a later step in the viral life cycle, suggesting that distinct TRIM56 domains accommodate differing antiviral mechanisms. Next, in Chapter 4, I show TRIM56 puts a check on replication of influenza A and B viruses in cell culture. Interestingly, the anti-influenza activity was independent of the E3 ligase activity, B-box, or coiled-coil domains. Rather, deletion of a 63-residue long, C-terminal tail portion of TRIM56 abrogated the antiviral function. Moreover, expression of this short C-terminal segment curtailed the replication of influenza viruses as effectively as that of full-length TRIM56. Mechanistically, TRIM56 was found to specifically impede intracellular influenza virus RNA synthesis. Altogether, TRIM56 is a versatile antiviral host factor that confers resistance to YFV, DENV2, HCoV-OC43 and influenza viruses through overlapping and distinct molecular determinants. Last, in Chapter 5, I report TRIM56 over-expression promoted activation of NF-κB following TLR3 engagement but not that induced by TNF-α- or IL-1β. Next, I observed that the coiled-coil domain and residues 431-610, but not the B-box or residues 355-433, were required for TRIM56 augmentation of TLR3-dependent IFN-β promoter activation. Furthermore, alanine screening mutagenesis suggested the S469A+S471A+S475A triple mutant and S471A, S475A and S710A single mutant failed to enhance TLR3 signaling. In line with this, S471A, S475A and S710A, as well as the coiled-coil deletion mutant lost the ability to enhance poly-I:C-mediated establishment of an antiviral state compared with wild-type TRIM56. Collectively, these data reveal novel insights into the mechanism of TRIM56 augmentation of TLR3-dependent antiviral response and highlight a role for TRIM56 scaffolding and phosphorylation in positive regulation of TLR3 signaling
Factors Associated with African American Women’s Sexual Health and Risk Behavior: A Mixed Methods Study
Get PDFIntroduction: When looking at the health status of our nation, an examination of sexual and reproductive health reveals that there are multiple conditions related to sexual behavior posing a burden on the health of African American women (AAW). Disproportionate rates of sexually transmitted infections, unintended/teen pregnancy, and sexual violence in this group reveal the need to integrate a broader construct to address the multiple factors known to contribute to disparities in sexual and reproductive health affecting AAW. Current health promotion efforts including the encouragement of abstinence and condom use fail to address the multiple components involved in sexual health and/or risk behavior and have been unsuccessful in curtailing disparities in this population. Recently, the concept of sexual health has increasingly been recognized for its potential to improve population health for various groups by addressing pertinent emotional, relational, and sexual aspects involved in individuals’ sexual behavior, decision making, and negotiation. The Sexual Health Model is used in this study, to identify multiple dimensions involved in AAW’s sexual health and participation in behaviors known to increase their sexual risk.
Methods: A convergent parallel mixed methods design was used in this study to better understand AAW’s sexual health and risk behavior. Quantitative (QUAN) methods using questionnaires were carried out on the full study sample (N=50) to describe associations between selected emotional and relational factors (spirituality/religiosity, depressive symptomology, and gender inequality) and AAWs sexual health and risk behavior. In addition, qualitative (QUAL) methods, which included the completion of focus group interviews, were conducted for a subsample (n=19) of study participants to examine AAWs perceptions of sexual health. Respondent validation procedures were also carried out on a subset of the QUAL study sample to ensure the accuracy of thematic content analysis. Analysis for QUAN data was conducted using SAS 9.4 and for QUAL data using NVivo 10. Individual analyses were integrated to corroborate findings for a mixed methods interpretation.
Results: Study participants (mean age = 38.5) reported having good overall sexual health and low sexual risk. Significant associations were identified among specified emotional and relational factors and both outcome variables. Sexual health was negatively correlated with depressive symptomology (r = -.19, p \u3c .20) and gender inequality (r=-.25, p \u3c .10). Additionally, lower depressive symptomology and gender inequality demonstrate predictive properties for AAWs sexual health (R2 = .12, p \u3c 0.05). Examination of sexual risk behavior reveal a positive association with gender inequality (r = .19, p \u3c .20). Spirituality/religiosity were not correlated with either outcome variable. QUAL study results reveal five organizing themes that emerged from focus group data, including: 1) “The new normal”; 2) having to compromise; 3) taking responsibility; 4) securing an attachment; and 5) resolving ambiguity.
Discussion/conclusion: By taking a holistic approach to better understand AAW’s sexual behavior, this study provided important insights into the multiple factors involved in achieving an optimal level of sexual health for these women. In this study, women’s personal accounts acknowledged participation in risky sexual behavior that was motivated by their intuitive feelings or the existing dynamic within their intimate relationships. Additionally, for women having the ability to make decisions within relationships, did not consistently translate to making sexually healthy decisions. Taking a broader approach toward sexual health may significantly contribute to the improvement of disparate health outcomes among AAW. Pertinent emotional, relational, and sexual factors involved in women’s sexual health and behavior must be addressed in strategies to reduce AAWs sexual risk and yield sustainable behavior change
Antibiotic Drug Discovery with an Eye Towards Overcoming Drug Resistance
Get PDFAs a species, humans have become ever reliant on the use of antibiotics to facilitate our everyday lives. The widespread emergence of resistance to currently used antibiotics is commonly attributed to an over use in our society. Such resistance, coupled with a lack of innovation and production of novel antibiotic drugs, threatens to return humanity to an era similar to one before the discovery of the first antibiotics. The need to find new agents to be used in this fight is paramount, as well as learning from our recent failures to produce such compounds. This document will highlight my efforts to contribute to the field of antibiotic drug discovery from a medicinal chemist’s perspective. Chapter one will be a brief survey of the current state of antibiotics. There will be a brief description of various classes of drugs used and some of their pharmacology. The second chapter will focus on the specific field of drug discovery for the pathogen Mycobacterium tuberculosis, and some of the difficulties associated with targeting this particular organism. This includes two classes of inhibitors that have distinct mechanisms of action from commonly used anti-tubercular compounds. These series have
distinctly different paths to clinical relevance; one seeks improved drug-like properties and the other seeks unique potency at an old target. The third chapter highlights a structure guided design of new anti-folate compounds, a reinvestigation of known inhibitors of this biosynthetic pathway and exploratory repurposing of compounds active against malaria. This chapter also holds a section on chemical repurposing, a technique becoming increasingly useful in the field of antibiotic discovery. The fourth and final chapter will be a synopsis and recollection of my graduate work and on the field of antibiotic drug discovery in general
Examining the Roles of GABAA Receptor Subtypes in Anxiety and Anxiolysis: Focusing on the Basolateral Amygdala
Get PDFThe investigation of the differential roles GABAA receptor (GABAAR) subtypes play in mediating various behaviors such as fear and anxiety was an intriguing research topic over the past decade. At present, most evidence suggests that benzodiazepine (BZ)-induced anxiolysis is primarily mediated by GABAARs containing the α2-subunit (α2-subtype). However, there is conflicting evidence as to whether α1- and α3-subtypes might also be involved in BZ-induced anxiolysis. In an attempt to further discern the role played by different α-subtype GABAARs in BZ-induced anxiolysis both systemically and within the basolateral amygdala (BLA), a brain region crucial for anxiety-like behaviors, we examined the anxiolytic-like effects, as measured by elevated-plus maze test (EPM), of several subtype selective and non-selective GABAAR positive allosteric modulators (PAMs) both in wild type mice and in mutant mice that express BZ-insensitive GABAARs of specific α-subtypes.
In our experiments, systemic injections of the α1-selective PAM zolpidem in WT mice produced slight anxiolytic-like effects with a narrow therapeutic window that overlapped with prominent motor-inhibiting effects. Systemic injection of the α3-selective PAM TP003 produced marked anxiolytic-like effects in WT mice that were accompanied by motor-stimulating effects. Systemic injection of the α2-, α3-, and α5-selective PAM L-838417 elicited significant anxiolytic-like effects in WT, and the effects were weakened in the α3(H126R) mice. Similarly, anxiolytic-like effects were observed when these selective PAMs were administered via microinjection into the BLA; however, these local injections did not significantly affect motor activity at the doses tested. In the experiment examining systemic injections of the non-selective BZ chlordiazepoxide (CDP), we found that CDP induced robust anxiolytic-like effects in both male and female WT mice. These effects were potentiated in female α1(H101R) mice, and were reduced in α2(H101R) mice of both sexes, as well as male α3(H126R) mice. Interestingly, intra-BLA microinjection of CDP produced few effects in WT, α1(H101R), or α2(H101R) mice, but showed some anxiolytic-like effects in α3(H126R) mice.
Taken together, our results suggests (i) all three (α1-, α2-, and α3-) GABAAR subtypes are involved in BZ-induced anxiolysis, but subtle differences do exist; (ii) augmentation of the α1-subtype GABAARs exerts anxiolytic-like effects; however, the therapeutic window is narrow; (iii) augmentation of the α2-, α3-, (and α5-) subtype GABAARs exerts anxiolytic-like effects and motor-stimulating effects, and these effects are weakened in α3(H126R) mice at doses tested, (iv) augmentation of the α3-subtype GABAARs exerts anxiolytic-like effects, accompanied by motor-stimulating effects; (v) BLA is an important brain region that is sufficient to mediate the anxiolytic-like effects, but not the motor-stimulating or inhibiting effects of subtype selective GABAAR PAMs; and (vi) intra-BLA microinjection of CDP yielded an inconclusive behavioral outcome, possibly due to the complex GABAergic intra-amygdaloidal microcircuitries which might antagonize each other when multiple subtypes of GABAARs are simultaneously modulated by BZs. Taken together, our results provide novel evidence that may benefit the current development of subtype selective drugs for treating clinical anxiety disorders
Environmental Signaling through the Target of Rapamycin Complex 1 (TORC1) and the Regulation of Epigenetic Mechanisms
Get PDFThe gene expression profile of a eukaryotic cell is responsive to a variety of extracellular stimuli, including nutrient availability, which allows cells to toggle between anabolism and catabolism based on the favorability of their environment. Much of this information is relayed through signaling complexes, such as the target of rapamycin complex 1 (TORC1), to downstream chromatin modifying enzymes. These enzymes impact the gene regulatory process through altered histone post-translation modifications, changes in chromatin structure, and docking of chromatin regulatory complexes. Yet, despite preliminary studies suggesting that TORC1 affects epigenetic mechanisms, including histone H3 lysine 56 acetylation (H3K56ac), almost nothing is known about how the complex functions in this regard. In this report, we demonstrate that inhibition of TORC1 results in a site-specific reduction in acetylation on N-terminal residues of both histone H3 and H4. This effect is dependent on sirtuin histone deacetylases (HDACs), as inactivation of these enzymes, specifically Hst4, rescues the acetylation defect. We also find that this sirtuin-mediated deacetylation response requires a functional protein phosphatase 6 complex (PP6). PP6 is under direct negative regulation of TORC1, and relief of this inhibition initiates a rapid cytoplasmic to nuclear redistribution of Hst4 which correlates temporally with our observed loss of histone acetylation. The nuclear accumulation of Hst4 precedes an increase in Hst4 protein levels that occurs due to a reduction in Hst4 turnover. Notably, deletion of a subset of sirtuins (hst3Δ or hst4Δ) rescued the sensitivity of a non-essential TORC1 mutant (tco89Δ) to an array of TORC1 inhibitors. This result suggests the link between TORC1 and acetylation may play an essential role in cell cycle regulation and the DNA damage response. We further evaluated whether these TORC1-mediated acetylation marks contribute to the chromatin association of high mobility group proteins (HMGs). And while TORC1-dependent displacement of the HMGs coincides with vacuolar acidification, hyperactivation of TORC1, and significant cell death, it appears to occur independently of TORC1’s regulation of Hst4. We conclude by investigating mitochondrial function in a tco89Δ mutant and mapping the functional domains of Tco89 necessary to sustain TORC1 activity and respond to extracellular stress