Biochemical and Biological Characterization of D. Discoideum TLP4, a Non-Canonical 3'-5' Polymerase

Post-transcriptional modification is an important step in the generation of many RNAs. For example tRNAHis requires the addition of a 5'-guanosine (G-1), a reaction that is catalyzed by the tRNAHis guanylyltransferase (Thg1). Thg1 and related enzymes, Thg1-like proteins (TLPs), make up the Thg1/TLP enzyme family which spans all three domains of life and whose family members act as unusual RNA polymerases. While other polymerases act to extend a growing polynucleotide chain in the 5'-3' direction, Thg1/TLP family members act as non-canonical 3'-5' polymerases by adding one or more base-paired nucleotides to the 5'-ends of RNA. Despite their shared chemistry, several features distinguish these two types of enzymes. While TLPs are capable of adding nucleotides to a variety of substrates, Thg1 enzymes selectively recognize a single tRNA (tRNAHis) for G-1 addition. Neither the molecular basis for Thg1’s selectivity nor the basis for the observed differences between Thg1 and TLP functions are fully understood. In addition, while the biological function of Thg1 in tRNAHis maturation is well established, the role(s) of most TLPs remain poorly understood. This project aims to address both of those questions through the study of an essential TLP found in the slime mold Dictyostelium discoideum, DdiTLP4. DdiTLP4 exhibits distinct substrate specificity from Thg1, catalyzing nucleotide addition to non-tRNA substrates such as 5S rRNA and class I noncoding RNA (ncRNA) in vitro, although the biological substrates for the enzyme in vivo remain unknown. Site-directed mutagenesis and enzyme assays with purified proteins have been used to investigate the unique features of DdiTLP4 that allow it to recognize ncRNA, and to assess its activity with model stem-loop substrates, in order to compare its activities to the prototypical Thg1 enzyme. Genetic methods have also been used to determine the effects of depletion of DdiTLP4 on D. discoideum development, to assess the effects of this unusual enzyme in biology.No embargoAcademic Major: Molecular Genetic

The Enemy Within: An Investigation of the Intracellular Bacteria in Urinary Tract Infections

Urinary tract infections (UTIs) are common diseases that are associated with significant morbidities. Multiple studies have indicated that multiple species of uropathogenesis bacteria invade and persist within bladder epithelial cells as a necessary step of uropathogenesis. Interestingly, many of these species are not canonically associated with intracellular infections. Although the first study describing bacteria within the urothelium was published two decades ago, this critical step of uropathogenesis remains relatively understudied. I established a murine model of community-acquired A. baumannii UTI, a previously unstudied manifestation of the disease. While immunocompetent mice resolved their infections quickly, immunocompromised mice displayed high bacterial burdens throughout their urinary tracts for several weeks. I found that mice infected using this model retained A. baumannii intracellular reservoirs (ABIRs) in their urothelium long after the resolution of the initial colonization event. Inserting a catheter into the bladders of these resolved mice triggered a same-strain UTI in over 50% of the mice. Stringent experimental controls suggest that these resurgences came from bacterial reservoirs within the resolved host. Further testing implicates the ABIRs as the most likely source. I have also characterized the intracellular phenotypes of multiple uropathogenic E. coli (UPEC) clinical isolates. While each isolate had unique strain-specific characteristics, all three of the phylogroup A strains proved to be incapable of properly undergoing the intracellular steps of uropathogenesis. I have also studied an important process used by intracellular UPEC, lactose metabolism. UPEC isolates with mutations in their lac operons formed significantly smaller intracellular bacterial communities, and were unable to complete IBC development. However, I found that UPEC strains with deficient lactose permeases were still capable of importing lactose-like sugars. Overall, my dissertation contributes to field of intracellular uropathogenesis in multiple ways. With E. coli, I have identified a potential link between bacterial genetics, specifically when a bacterium is a member of phylogroup A, and intracellular phenotype in the bladder. I have also established that lacY-deficient UPEC isolates are capable of internalizing lactose-like sugars, which suggests the presence of a secondary mechanism. I have also developed a murine model of community-acquired UTI for A. baumannii and have investigated both the pathogenesis and the prevalence of this manifestation. I have also developed the first murine model of resurgent infections for A. baumannii and have identified a potential reservoir bringing novel strains into the hospital in the form of host reservoirs. Previous Acinetobacter UTI research has focused primarily on complicated UTIs. Together, my findings expand our knowledge of Acinetobacter uropathogenesis in the previously unstudied, community-acquired model of infection

Giving Parents a Voice: A Case Study of a Family Treatment Drug Court Track in Lancaster County, Nebraska

Family drug courts (FDCs) were first established in 1994 as one judge’s response to substance abuse in the majority of his dependency-court cases.1 Since then, hundreds of similar specialized dependency courts have been established around the country. FDCs are based on an adult-drug-court model established in response to the apparent revolving door of drug offenders in criminal court. Drug courts and other problem-solving courts seek to identify the social and psychological dysfunction that brought the individuals before the court. Problem-solving-court judges adopt therapeutic jurisprudence to assess the dysfunction, prescribe appropriate services, and provide support, encouragement, and accountability. Procedural justice, characterized by judicial leadership and participant autonomy, is one of the psychological tools used to successfully adopt therapeutic jurisprudence. Successful problem-solving courts rely on judicial leadership for the network of providers and to engage with the participants. Additionally, the voluntary nature of problem-solving courts ensures participants are given autonomy and allowed to exercise voice and control in the process

Prognostic Value of Elevated Levels of Intestinal Microbe-Generated Metabolite Trimethylamine-N-Oxide in Patients With Heart Failure: Refining the Gut Hypothesis

Background: Altered intestinal function is prevalent in patients with heart failure (HF), but its role in adverse outcomes is unclear. Objectives: This study investigated the potential pathophysiological contributions of intestinal microbiota in HF. Methods: We examined the relationship between fasting plasma trimethylamine-N-oxide (TMAO) and all-cause mortality over a 5-year follow-up in 720 patients with stable HF. Results:The median TMAO level was 5.0 μM, which was higher than in subjects without HF (3.5 μM; p \u3c 0.001). There was modest but significant correlation between TMAO concentrations and B-type natriuretic peptide (BNP) levels (r = 0.23; p \u3c 0.001). Higher plasma TMAO levels were associated with a 3.4-fold increased mortality risk. Following adjustments for traditional risk factors and BNP levels, elevated TMAO levels remained predictive of 5-year mortality risk (hazard ratio [HR]: 2.2; 95% CI: 1.42 to 3.43; p \u3c 0.001), as well as following the addition of estimated glomerular filtration rate to the model (HR: 1.75; 95% CI: 1.07 to 2.86; p \u3c 0.001). Conclusions: High TMAO levels were observed in patients with HF, and elevated TMAO levels portended higher long-term mortality risk independent of traditional risk factors and cardiorenal indexes

An Abundant Dysfunctional Apolipoprotein A1 in Human Atheroma

Recent studies have indicated that high-density lipoproteins (HDLs) and their major structural protein, apolipoprotein A1 (apoA1), recovered from human atheroma are dysfunctional and are extensively oxidized by myeloperoxidase (MPO). In vitro oxidation of either apoA1 or HDL particles by MPO impairs their cholesterol acceptor function. Here, using phage display affinity maturation, we developed a high-affinity monoclonal antibody that specifically recognizes both apoA1 and HDL that have been modified by the MPO-H2O2-Cl− system. An oxindolyl alanine (2-OH-Trp) moiety at Trp72 of apoA1 is the immunogenic epitope. Mutagenesis studies confirmed a critical role for apoA1 Trp72 in MPO-mediated inhibition of the ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol acceptor activity of apoA1 in vitro and in vivo. ApoA1 containing a 2-OH-Trp72 group (oxTrp72-apoA1) is in low abundance within the circulation but accounts for 20% of the apoA1 in atherosclerosis-laden arteries. OxTrp72-apoA1 recovered from human atheroma or plasma is lipid poor, virtually devoid of cholesterol acceptor activity and demonstrated both a potent proinflammatory activity on endothelial cells and an impaired HDL biogenesis activity in vivo. Elevated oxTrp72-apoA1 levels in subjects presenting to a cardiology clinic (n = 627) were associated with increased cardiovascular disease risk. Circulating oxTrp72-apoA1 levels may serve as a way to monitor a proatherogenic process in the artery wall

An Abundant Dysfunctional Apolipoprotein A1 in Human Atheroma

Recent studies have indicated that high-density lipoproteins (HDLs) and their major structural protein, apolipoprotein A1 (apoA1), recovered from human atheroma are dysfunctional and are extensively oxidized by myeloperoxidase (MPO). In vitro oxidation of either apoA1 or HDL particles by MPO impairs their cholesterol acceptor function. Here, using phage display affinity maturation, we developed a high-affinity monoclonal antibody that specifically recognizes both apoA1 and HDL that have been modified by the MPO-H2O2-Cl− system. An oxindolyl alanine (2-OH-Trp) moiety at Trp72 of apoA1 is the immunogenic epitope. Mutagenesis studies confirmed a critical role for apoA1 Trp72 in MPO-mediated inhibition of the ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol acceptor activity of apoA1 in vitro and in vivo. ApoA1 containing a 2-OH-Trp72 group (oxTrp72-apoA1) is in low abundance within the circulation but accounts for 20% of the apoA1 in atherosclerosis-laden arteries. OxTrp72-apoA1 recovered from human atheroma or plasma is lipid poor, virtually devoid of cholesterol acceptor activity and demonstrated both a potent proinflammatory activity on endothelial cells and an impaired HDL biogenesis activity in vivo. Elevated oxTrp72-apoA1 levels in subjects presenting to a cardiology clinic (n = 627) were associated with increased cardiovascular disease risk. Circulating oxTrp72-apoA1 levels may serve as a way to monitor a proatherogenic process in the artery wall

An Abundant Dysfunctional Apolipoprotein A1 in Human Atheroma

Recent studies have indicated that high-density lipoproteins (HDLs) and their major structural protein, apolipoprotein A1 (apoA1), recovered from human atheroma are dysfunctional and are extensively oxidized by myeloperoxidase (MPO). In vitro oxidation of either apoA1 or HDL particles by MPO impairs their cholesterol acceptor function. Here, using phage display affinity maturation, we developed a high-affinity monoclonal antibody that specifically recognizes both apoA1 and HDL that have been modified by the MPO-H2O2-Cl− system. An oxindolyl alanine (2-OH-Trp) moiety at Trp72 of apoA1 is the immunogenic epitope. Mutagenesis studies confirmed a critical role for apoA1 Trp72 in MPO-mediated inhibition of the ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol acceptor activity of apoA1 in vitro and in vivo. ApoA1 containing a 2-OH-Trp72 group (oxTrp72-apoA1) is in low abundance within the circulation but accounts for 20% of the apoA1 in atherosclerosis-laden arteries. OxTrp72-apoA1 recovered from human atheroma or plasma is lipid poor, virtually devoid of cholesterol acceptor activity and demonstrated both a potent proinflammatory activity on endothelial cells and an impaired HDL biogenesis activity in vivo. Elevated oxTrp72-apoA1 levels in subjects presenting to a cardiology clinic (n = 627) were associated with increased cardiovascular disease risk. Circulating oxTrp72-apoA1 levels may serve as a way to monitor a proatherogenic process in the artery wall

Superhumps in Cataclysmic Binaries. XXIV. Twenty More Dwarf Novae

We report precise measures of the orbital and superhump period in twenty more dwarf novae. For ten stars, we report new and confirmed spectroscopic periods - signifying the orbital period P_o - as well as the superhump period P_sh. These are GX Cas, HO Del, HS Vir, BC UMa, RZ Leo, KV Dra, KS UMa, TU Crt, QW Ser, and RZ Sge. For the remaining ten, we report a medley of P_o and P_sh measurements from photometry; most are new, with some confirmations of previous values. These are KV And, LL And, WX Cet, MM Hya, AO Oct, V2051 Oph, NY Ser, KK Tel, HV Vir, and RX J1155.4-5641. Periods, as usual, can be measured to high accuracy, and these are of special interest since they carry dynamical information about the binary. We still have not quite learned how to read the music, but a few things are clear. The fractional superhump excess epsilon [=(P_sh-P_o)/P_o] varies smoothly with P_o. The scatter of the points about that smooth curve is quite low, and can be used to limit the intrinsic scatter in M_1, the white dwarf mass, and the mass-radius relation of the secondary. The dispersion in M_1 does not exceed 24%, and the secondary-star radii scatter by no more than 11% from a fixed mass-radius relation. For the well-behaved part of epsilon(P_o) space, we estimate from superhump theory that the secondaries are 18+-6% larger than theoretical ZAMS stars. This affects some other testable predictions about the secondaries: at a fixed P_o, it suggests that the secondaries are (compared with ZAMS predictions) 40+-14% less massive, 12+-4% smaller, 19+-6% cooler, and less luminous by a factor 2.5(7). The presence of a well-defined mass-radius relation, reflected in a well-defined epsilon(P_o) relation, strongly limits effects of nuclear evolution in the secondaries.Comment: PDF, 62 pages, 7 tables, 21 figures; accepted, in press, to appear November 2003, PASP; more info at http://cba.phys.columbia.edu

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Genome-wide association identifies nine common variants associated with fasting proinsulin levels and provides new insights into the pathophysiology of type 2 diabetes.

OBJECTIVE: Proinsulin is a precursor of mature insulin and C-peptide. Higher circulating proinsulin levels are associated with impaired β-cell function, raised glucose levels, insulin resistance, and type 2 diabetes (T2D). Studies of the insulin processing pathway could provide new insights about T2D pathophysiology. RESEARCH DESIGN AND METHODS: We have conducted a meta-analysis of genome-wide association tests of ∼2.5 million genotyped or imputed single nucleotide polymorphisms (SNPs) and fasting proinsulin levels in 10,701 nondiabetic adults of European ancestry, with follow-up of 23 loci in up to 16,378 individuals, using additive genetic models adjusted for age, sex, fasting insulin, and study-specific covariates. RESULTS: Nine SNPs at eight loci were associated with proinsulin levels (P < 5 × 10(-8)). Two loci (LARP6 and SGSM2) have not been previously related to metabolic traits, one (MADD) has been associated with fasting glucose, one (PCSK1) has been implicated in obesity, and four (TCF7L2, SLC30A8, VPS13C/C2CD4A/B, and ARAP1, formerly CENTD2) increase T2D risk. The proinsulin-raising allele of ARAP1 was associated with a lower fasting glucose (P = 1.7 × 10(-4)), improved β-cell function (P = 1.1 × 10(-5)), and lower risk of T2D (odds ratio 0.88; P = 7.8 × 10(-6)). Notably, PCSK1 encodes the protein prohormone convertase 1/3, the first enzyme in the insulin processing pathway. A genotype score composed of the nine proinsulin-raising alleles was not associated with coronary disease in two large case-control datasets. CONCLUSIONS: We have identified nine genetic variants associated with fasting proinsulin. Our findings illuminate the biology underlying glucose homeostasis and T2D development in humans and argue against a direct role of proinsulin in coronary artery disease pathogenesis