Modeling Gardnerella Host-Pathogen Interaction: Characterization of Vaginolysin and the Opacity Phenotype

Bacterial vaginosis (BV) describes a dysbiotic state of the vaginal mucosa, during which the dominance by beneficial lactobacilli species is lost, the vaginal pH increases, and the flora comprising the vaginal microbiome becomes more diverse. Bacterial taxa associated with this dysbiotic state interact to form a dense polymicrobial biofilm on the surface of the vaginal epithelium. This shift in the vaginal microflora is clinically important, even when symptoms are not noted, as women with BV are at increased risk for acquisition of other sexually transmitted infections, including HIV, and are more than two times as likely to experience preterm birth. Moreover, BV has been associated with numerous other adverse outcomes, including pelvic inflammatory disease, recurring urinary tract infections, chorioamnionitis, postpartum endometritis, infertility, and adverse neonatal outcomes, even among full term infants. The prevalence of BV, in the United States alone, is approximately 30% for women of reproductive age, and is significantly increased in Hispanic and African American women. Standard metronidazole treatment carries a recurrence rate of 52% within 12 months of treatment and recent reports indicate that BV recurrence can, at least in part, be attributed to the inability to reestablish the beneficial lactobacilli population after antibiotic treatment. Thus, it is clear that more targeted approaches to BV treatment are needed. While the last few decades of research have given us a better understanding of the organisms that comprise the vaginal microbiome, there are still a lot of gaps in our understanding of what they are doing and how they are interacting with the host and each other, both in the context of health and disease. This is due to inadequacies of animal models and difficulty in culturing and genetically manipulating vaginally relevant microorganisms. The overarching aim of this dissertation research was to characterize the factors that contribute to the establishment and maintenance of vaginal dysbiosis, focusing on the role(s) of one organism, Gardnerella vaginalis. Various tools were developed to further characterize putative virulence determinants produced by Gardnerella to better understand how the bacterium interacts with the human host, with emphasis on the cholesterol-dependent toxin, vaginolysin (VLY), and an opacity phenotype presumptively driven by differential pilus production. Through the development of a human vaginal epithelial model of Gardnerella infection, in which Gardnerella spp. grow to in vivo densities, we uncovered a previously undescribed influence of tissue polarity on VLY activity. This influence was mediated by differential expression of the proposed VLY co-receptor, CD59. Further investigation of VLY in different Gardnerella strains revealed conserved amino acid differences within regions of the toxin known to interact with host cells, allowing for the characterization of distinct VLY types. Two predominant VLY types exhibited differential interaction with vaginal epithelial cells, putatively related to CD59 availability. Bioinformatic analyses showed that certain VLY types are restricted to select Gardnerella species and the dominant VLY type in vaginal samples has implications for overall Gardnerella abundance and symptom frequency during BV. We then characterized a colony opacity phenotype potentially susceptible to phase variation. Both opaque and translucent variants were detected in all tested Gardnerella isolates, representing six different species. Opaque variants exhibited increased bacterial aggregation in suspension as well as increased surface biofilm formation, to suggest that opacity may promote resistance to host defenses. Finally, we report the development of the first method for performing site-specific mutagenesis in Gardnerella spp. After optimization, this oligonucleotide-based protocol yielded transformants at an efficiency similar to that observed in other bacterial species. Using these optimized methods, we were able to transform 3 strains of Gardnerella representing 3 different species. While this represents a significant advance in the field, continued optimization is necessary to further improve transformation efficiency in order to increase the practicality of using this tool to generate non-selectable transformants. In sum, continuing to build upon the knowledge gleaned from this work will allow us to develop more targeted therapies for BV and ultimately reduce its global health burden

Stroke Quality Measures in Mexican Americans and Non-Hispanic Whites

Mexican Americans (MAs) have been shown to have worse outcomes after stroke than non-Hispanic Whites (NHWs), but it is unknown if ethnic differences in stroke quality of care may contribute to these worse outcomes. We investigated ethnic differences in the quality of inpatient stroke care between MAs and NHWs within the population-based prospective Brain Attack Surveillance in Corpus Christi (BASIC) Project (February 2009- June 2012). Quality measures for inpatient stroke care, based on the 2008 Joint Commission Primary Stroke Center definitions were assessed from the medical record by a trained abstractor. Two summary measure of overall quality were also created (binary measure of defect-free care and the proportion of measures achieved for which the patient was eligible). 757 individuals were included (480 MAs and 277 NHWs). MAs were younger, more likely to have hypertension and diabetes, and less likely to have atrial fibrillation than NHWs. MAs were less likely than NHWs to receive tPA (RR: 0.72, 95% confidence interval (CI) 0.52, 0.98), and MAs with atrial fibrillation were less likely to receive anticoagulant medications at discharge than NHWs (RR 0.73, 95% CI 0.58, 0.94). There were no ethnic differences in the other individual quality measures, or in the two summary measures assessing overall quality. In conclusion, there were no ethnic differences in the overall quality of stroke care between MAs and NHWs, though ethnic differences were seen in the proportion of patients who received tPA and anticoagulant at discharge for atrial fibrillation

Quadratic associations between empathy and depression and the moderating influence of dysregulation

Empathic tendencies have been associated with interpersonal and psychological benefits, but empathy at extreme levels or in combination with certain personal characteristics may contribute to risk for depression. This study tested the moderating role of cognitive emotion regulation in depression’s association with empathy using nonlinear models. Young adults (N=304; 77% female; M=19 years) completed measures of cognitive emotion regulation strategies, depression, and affective and cognitive empathy. Individuals with good regulation had low levels of depression overall and their depression symptoms were lowest when levels of affective empathy were average. Individuals with poor regulation had high levels of depression overall, particularly when levels of empathy were moderate to high. Extremely high and low levels of cognitive empathy were associated with elevated depression, and this association was not moderated by regulation. These findings suggest tendencies to respond empathically to others’ needs is neither an adaptive nor maladaptive characteristic but rather moderate empathy, particularly in the context of good regulation, may offer the greatest protection against depression

X-ray/UVOIR Frequency-resolved Time Lag Analysis of Mrk 335 Reveals Accretion Disk Reprocessing

UV and optical continuum reverberation mapping is powerful for probing the accretion disk and inner broad-line region. However, recent reverberation mapping campaigns in the X-ray, UV, and optical have found lags consistently longer than those expected from the standard disk reprocessing picture. The largest discrepancy to-date was recently reported in Mrk 335, where UV/optical lags are up to 12 times longer than expected. Here, we perform a frequency-resolved time lag analysis of Mrk 335, using Gaussian processes to account for irregular sampling. For the first time, we compare the Fourier frequency-resolved lags directly to those computed using the popular Interpolated Cross-Correlation Function (ICCF) method applied to both the original and detrended light curves. We show that the anticipated disk reverberation lags are recovered by the Fourier lags when zeroing in on the short-timescale variability. This suggests that a separate variability component is present on long timescales. If this separate component is modeled as reverberation from another region beyond the accretion disk, we constrain a size-scale of roughly 15 light-days from the central black hole. This is consistent with the size of the broad line region inferred from H$\beta$ reverberation lags. We also find tentative evidence for a soft X-ray lag, which we propose may be due to light travel time delays between the hard X-ray corona and distant photoionized gas that dominates the soft X-ray spectrum below 2 keV.Comment: Accepted for publication in ApJ, 8 figure

The Soft-Excess in Mrk 509: Warm Corona or Relativistic Reflection?

We present the analysis of the first NuSTAR observations ($\sim 220$ ks), simultaneous with the last SUZAKU observations ($\sim 50$ ks), of the active galactic nucleus of the bright Seyfert 1 galaxy Mrk 509. The time-averaged spectrum in the $1-79$ keV X-ray band is dominated by a power-law continuum ($\Gamma\sim 1.8-1.9$), a strong soft excess around 1 keV, and signatures of X-ray reflection in the form of Fe K emission ($\sim 6.4$ keV), an Fe K absorption edge ($\sim 7.1$ keV), and a Compton hump due to electron scattering ($\sim 20-30$ keV). We show that these data can be described by two very different prescriptions for the soft excess: a warm ($kT\sim 0.5-1$ keV) and optically thick ($\tau\sim10-20$) Comptonizing corona, or a relativistically blurred ionized reflection spectrum from the inner regions of the accretion disk. While these two scenarios cannot be distinguished based on their fit statistics, we argue that the parameters required by the warm corona model are physically incompatible with the conditions of standard coronae. Detailed photoionization calculations show that even in the most favorable conditions, the warm corona should produce strong absorption in the observed spectrum. On the other hand, while the relativistic reflection model provides a satisfactory description of the data, it also requires extreme parameters, such as maximum black hole spin, a very low and compact hot corona, and a very high density for the inner accretion disk. Deeper observations of this source are thus necessary to confirm the presence of relativistic reflection, and to further understand the nature of its soft excess.Comment: Accepted for publication in ApJ, 18 pages, 7 figure

Impact of Age on Consent in a Geriatric Orthopaedic Trauma Patient Population.

Introduction: Persistent misconceptions of frailty and dementia in geriatric patients impact physician-patient communication and leave patients vulnerable to disempowerment. Physicians may inappropriately focus the discussion of treatment options to health care proxies instead of patients. Our study explores the consenting process in a decision-making capable orthogeriatric trauma patient population to determine if there is a relationship between increased patient age and surgical consent by health care proxy. Methods: Patients aged 65 and older who underwent operative orthopaedic fracture fixation between 1 of 2 Level 1 Trauma Centers were retrospectively reviewed. Decision-making capable status was defined as an absence of patient history of cognitive impairment and a negative patient pre-surgical Confusion Assessment Method (CAM) and Mini-Cog Assessment screen. Provider of surgical consent was the main outcome and was determined by signature on the consent form. Results: 510 patients were included, and 276 (54.1%) patients were deemed capable of consent. In 27 (9.8%) of 276 decision-capable patients, physicians obtained consent from health care proxies. 20 of these 27 patients (74.1%) were 80 years of age or older. However, in patients aged 70 to 79, only 7 health care proxies provided consent. (p = 0.07). For every unit increase in age, the log odds of proxy consent increased by .0008 (p \u3c 0.001). Age (p \u3c 0.001), income level (p = 0.03), and physical presence of proxy at consult (p \u3c 0.001) were factors associated with significantly increased utilization of health care proxy provided consent. Language other than English was a significant predictor of proxy-provided consent (p = 0.035). 48 (22%) decision-making incapable patients provided their own surgical consent. Discussion: The positive linear association between age and health care proxy provided consent in cognitively intact geriatric orthopaedic patients indicates that increased patient age impacts the consenting process. Increased physician vigilance and adoption of institutional consenting guidelines can reinforce appropriate respect of geriatric patients\u27 consenting capacity

MicroRNAs of Gallid and Meleagrid herpesviruses show generally conserved genomic locations and are virus-specific

AbstractMany herpesviruses, including Marek's disease viruses (MDV1 and MDV2), encode microRNAs. In this study, we report microRNAs of two related herpesviruses, infectious laryngotracheitis virus (ILTV) and herpesvirus of turkeys (HVT), as well as additional MDV2 microRNAs. The genome locations, but not microRNA sequences, are conserved among all four of these avian herpesviruses. Most are clustered in the repeats flanking the unique long region (I/TRL), except in ILTV which lacks these repeats. Two abundant ILTV microRNAs are antisense to the immediate early gene ICP4. A homologue of host microRNA, gga-miR-221, was found among the HVT microRNAs. Additionally, a cluster of HVT microRNAs was found in a region containing two locally duplicated segments, resulting in paralogous HVT microRNAs with 96–100% identity. The prevalence of microRNAs in the genomic repeat regions as well as in local repeats suggests the importance of genetic plasticity in herpesviruses for microRNA evolution and preservation of function

iPSCORE: A Resource of 222 iPSC Lines Enabling Functional Characterization of Genetic Variation across a Variety of Cell Types.

Large-scale collections of induced pluripotent stem cells (iPSCs) could serve as powerful model systems for examining how genetic variation affects biology and disease. Here we describe the iPSCORE resource: a collection of systematically derived and characterized iPSC lines from 222 ethnically diverse individuals that allows for both familial and association-based genetic studies. iPSCORE lines are pluripotent with high genomic integrity (no or low numbers of somatic copy-number variants) as determined using high-throughput RNA-sequencing and genotyping arrays, respectively. Using iPSCs from a family of individuals, we show that iPSC-derived cardiomyocytes demonstrate gene expression patterns that cluster by genetic background, and can be used to examine variants associated with physiological and disease phenotypes. The iPSCORE collection contains representative individuals for risk and non-risk alleles for 95% of SNPs associated with human phenotypes through genome-wide association studies. Our study demonstrates the utility of iPSCORE for examining how genetic variants influence molecular and physiological traits in iPSCs and derived cell lines

Increased SIRT3 combined with PARP inhibition rescues motor function of SBMA mice.

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disease with substantial mitochondrial and metabolic dysfunctions. SBMA is caused by polyglutamine (polyQ) expansion in the androgen receptor (AR). Activating or increasing the NAD+-dependent deacetylase, SIRT3, reduced oxidative stress and death of cells modeling SBMA. However, increasing diminished SIRT3 in AR100Q mice failed to reduce acetylation of the SIRT3 target/antioxidant, SOD2, and had no effect on increased total acetylated peptides in quadriceps. Yet, overexpressing SIRT3 resulted in a trend of motor recovery, and corrected TCA cycle activity by decreasing acetylation of SIRT3 target proteins. We sought to boost blunted SIRT3 activity by replenishing diminished NAD+ with PARP inhibition. Although NAD+ was not affected, overexpressing SIRT3 with PARP inhibition fully restored hexokinase activity, correcting the glycolytic pathway in AR100Q quadriceps, and rescued motor endurance of SBMA mice. These data demonstrate that targeting metabolic anomalies can restore motor function downstream of polyQ-expanded AR