Megan M. Shuey

Highly sensitive quantitative PCR for the detection and differentiation of Pseudogymnoascus destructans and other Pseudogymnoascus species

ABSTRACT White-nose syndrome is a fungal disease that has decimated bat populations across eastern North America. Identification of the etiologic agent, Pseudogymnoascus destructans (formerly Geomyces destructans), in environmental samples is essential to proposed management plans. A major challenge is the presence of closely related species, which are ubiquitous in many soils and cave sediments and often present in high abundance. We present a dual-probe real-time quantitative PCR assay capable of detecting and differentiating P. destructans from closely related fungi in environmental samples from North America. The assay, based on a single nucleotide polymorphism (SNP) specific to P. destructans, is capable of rapid low-level detection from various sampling media, including sediment, fecal samples, wing biopsy specimens, and skin swabs. This method is a highly sensitive, high-throughput method for identifying P. destructans, other Pseudogymnoascus spp., and Geomyces spp. in the environment, providing a fundamental component of research and risk assessment for addressing this disease, as well as other ecological and mycological work on related fungi.

Arg287Gln variant of EPHX2 and epoxyeicosatrienoic acids are associated with insulin sensitivity in humans

Epoxyeicosatrienoic acids (EETs) protect against the development of insulin resistance in rodents. EETs are hydrolyzed to less biologically active diols by soluble epoxide hydrolase (encoded for by EPHX2). Functional variants of EPHX2 encode for enzymes with increased (Lys55Arg) or decreased (Arg287Gln) hydrolase activity. This study tested the hypothesis that variants of EPHX2 are associated with insulin sensitivity or secretion in humans. Subjects participating in metabolic phenotyping studies were genotyped. Eighty-five subjects underwent hyperglycemic clamps. There was no relationship between the Lys55Arg genotype and insulin sensitivity or secretion. In contrast, the EPHX2 287Gln variant was associated with higher insulin sensitivity index (p=0.019 controlling for body mass index and metabolic syndrome). Also, there was an interactive effect of EPHX2 Arg287Gln genotype and body mass index on insulin sensitivity index (p=0.029). There was no relationship between EPHX2 Arg287Gln genotype and acute or late-phase glucose-stimulated insulin secretion, but disposition index was higher in 287Gln carriers compared with Arg/Arg (p=0.022). Plasma EETs correlated with insulin sensitivity index (r=0.64, p=0.015 for total EETs) and were decreased in the metabolic syndrome. A genetic variant that results in decreased soluble epoxide hydrolase activity is associated with increased insulin sensitivity, as are higher EETs.

THE INNATE IMMUNE RESPONSE MAY BE IMPORTANT FOR SURVIVING PLAGUE IN WILD GUNNISON'S PRAIRIE DOGS

Abstract Prairie dogs (Cynomys spp.) are highly susceptible to Yersinia pestis, with ≥99% mortality reported from multiple studies of plague epizootics. A colony of Gunnisons prairie dogs (Cynomys gunnisoni) in the Aubrey Valley (AV) of northern Arizona appears to have survived several regional epizootics of plague, whereas nearby colonies have been severely affected by Y. pestis. To examine potential mechanisms accounting for survival in the AV colony, we conducted a laboratory Y. pestis challenge experiment on 60 wild-caught prairie dogs from AV and from a nearby, large colony with frequent past outbreaks of plague, Espee (n = 30 per colony). Test animals were challenged subcutaneously with the fully virulent Y. pestis strain CO92 at three doses: 50, 5,000, and 50,000 colony-forming units (cfu); this range is lethal in black-tailed prairie dogs (Cynomys ludovicianus). Contrary to our expectations, only 40% of the animals died. Although mortality trended higher in the Espee colony (50%) compared with AV (30%), the differences among infectious doses were not statistically significant. Only 39% of the survivors developed moderate to high antibody levels to Y. pestis, indicating that mechanisms other than humoral immunity are important in resistance to plague. The ratio of neutrophils to lymphocytes was not correlated with plague survival in this study. However, several immune proteins with roles in innate immunity (VCAM-1, CXCL-1, and vWF) were upregulated during plague infection and warrant further inquiry into their role for protection against this disease. These results suggest plague resistance exists in wild populations of the Gunnisons prairie dog and provide important directions for future studies.

Association of gain-of-function EPHX2 polymorphism Lys55Arg with acute kidney injury following cardiac surgery

Twenty to thirty percent of patients undergoing cardiac surgery develop acute kidney injury (AKI). In mice, inhibition of soluble epoxide hydrolase (sEH) attenuates renal injury following ischemia-reperfusion. We tested the hypothesis that functional variants of EPHX2, encoding sEH, are associated with AKI after cardiac surgery. We genotyped patients in two independent cardiac surgery cohorts for functional EPHX2 polymorphisms, Lys55Arg and Arg287Gln, and determined AKI using Acute Kidney Injury Network criteria. The 287Gln variant was not associated with AKI. In the discovery cohort, the gain-of-function 55Arg variant was associated with an increased incidence of AKI in univariate (p = 0.03) and multivariable (p = 0.04) analyses. In white patients without chronic kidney disease (CKD), the 55Arg variant was independently associated with AKI with an OR of 2.04 (95% CI 0.95–4.42) for 55Arg heterozygotes and 31.53 (1.57–633.19) for homozygotes (p = 0.02), after controlling for age, sex, body mass index, baseline estimated glomerular filtration rate, and use of cardiopulmonary bypass. These findings were replicated in the second cardiac surgery cohort. 12,13- and total- dihydroxyoctadecanoic acids (DiHOME): epoxyoctadecanoic acids (EpOME) ratios were increased in EPHX2 55Arg variant carriers, consistent with increased hydrolase activity. The EPHX2 Lys55Arg polymorphism is associated with AKI following cardiac surgery in patients without preexisting CKD. Pharmacological strategies to decrease sEH activity might decrease postoperative AKI.

The Cytochrome P450 Slow Metabolizers CYP2C9*2 and CYP2C9*3 Directly Regulate Tumorigenesis via Reduced Epoxyeicosatrienoic Acid Production

Increased expression of cytochrome P450 CYP2C9, together with elevated levels of its products epoxyeicosatrienoic acids (EET), is associated with aggressiveness in cancer. Cytochrome P450 variants CYP2C9*2 and CYP2C9*3 encode proteins with reduced enzymatic activity, and individuals carrying these variants metabolize drugs more slowly than individuals with wild-type CYP2C9*1, potentially affecting their response to drugs and altering their risk of disease. Although genetic differences in CYP2C9-dependent oxidation of arachidonic acid (AA) have been reported, the roles of CYP2C9*2 and CYP2C9*3 in EET biosynthesis and their relevance to disease are unknown. Here, we report that CYP2C9*2 and CYP2C9*3 metabolize AA less efficiently than CYP2C9*1 and that they play a role in the progression of non-small cell lung cancer (NSCLC) via impaired EET biosynthesis. When injected into mice, NSCLC cells expressing CYP2C9*2 and CYP2C9*3 produced lower levels of EETs and developed fewer, smaller, and less vascularized tumors than cells expressing CYP2C9*1. Moreover, endothelial cells expressing these two variants proliferated and migrated less than cells expressing CYP2C*1. Purified CYP2C9*2 and CYP2C9*3 exhibited attenuated catalytic efficiency in producing EETs, primarily due to impaired reduction of these two variants by NADPH-P450 reductase. Loss-of-function SNPs within CYP2C9*2 and CYP2C9*3 were associated with improved survival in female cases of NSCLC. Thus, decreased EET biosynthesis represents a novel mechanism whereby CYPC29*2 and CYP2C9*3 exert a direct protective role in NSCLC development.Significance: These findings report single nucleotide polymorphisms in the human CYP2C9 genes, CYP2C9*2 and CYP2C9*3, exert a direct protective role in tumorigenesis by impairing EET biosynthesis. Cancer Res; 78(17); 4865-77. ©2018 AACR.

Characteristics and treatment of African-American and European-American patients with resistant hypertension identified using the electronic health record in an academic health centre: a case−control study

Objective To identify patients with hypertension with resistant and controlled blood pressure (BP) using electronic health records (EHRs) in order to elucidate practices in the real-world clinical treatment of hypertension and to enable future genetic studies. Design Using EHRs, we developed and validated algorithms to identify patients with resistant and controlled hypertension. Setting An academic medical centre in Nashville, Tennessee. Population European-American (EA) and African-American (AA) patients with hypertension. Main outcome measures Demographic characteristics: race, age, gender, body mass index, outpatient BPs and the history of diabetes mellitus, chronic kidney disease stage 3, ischaemic heart disease, transient ischaemic attack, atrial fibrillation and sleep apnoea. Medication treatment All antihypertensive medication classes prescribed to a patient at the time of classification and ever prescribed following classification. Results The algorithms had performance metrics exceeding 92%. The prevalence of resistant hypertension in the total hypertensive population was 7.3% in EA and 10.5% in AA. At diagnosis, AA were younger, heavier, more often female and had a higher incidence of type 2 diabetes and higher BPs than EA. AA with resistant hypertension were more likely to be treated with vasodilators, dihydropyridine calcium channel blockers and alpha-2 agonists while EA were more likely to be treated with angiotensin receptor blockers, renin inhibitors and beta blockers. Mineralocorticoid receptor antagonists use was increased in patients treated with more than four antihypertensive medications compared with patients treated with three (12.4% vs 2.6% in EA, p<0.001; 12.3% vs 2.8% in AA, p<0.001). The number of patients treated with a mineralocorticoid receptor antagonist increased to 37.4% in EA and 41.2% in AA over a mean follow-up period of 7.4 and 8.7 years, respectively. Conclusions Clinical treatment of resistant hypertension differs in EA and AA patients. These results demonstrate the feasibility of identifying resistant hypertension using an EHR.

Hypertension and Type 2 Diabetes Are Associated With Decreased Inhibition of Dipeptidyl Peptidase-4 by Sitagliptin

Context: Patients with diabetes often have comorbidities such as hypertension. It is not known how individual characteristics influence response to dipeptidyl peptidase-4 (DPP4) inhibitors. Objective: We tested the hypothesis that individual characteristics, sitagliptin dose, and genetic variability in DPP4 influence DPP4 activity during sitagliptin. Design and Setting: Post hoc analysis of clinical and laboratory data from individuals randomized to sitagliptin versus placebo in crossover studies. Patients and Interventions: Sixty-five subjects [27 with type 2 diabetes mellitus (T2DM) and hypertension, 38 healthy controls] were randomized to 100 mg/d sitagliptin or 200 mg sitagliptin and matching placebo in double-blind, crossover fashion. Fasting blood was obtained at baseline and 60 to 180 minutes after sitagliptin or placebo. Main Outcome Measure(s): DPP4 activity and antigen during placebo and sitagliptin and DPP4 inhibition during sitagliptin. Results: Sitagliptin 100 mg/d was less effective at inhibiting DPP4 activity in individuals with T2DM and hypertension than in healthy controls (P = 0.001, percent inhibition). In healthy controls, 100 mg/d sitagliptin was not as effective as single-dose 200 mg sitagliptin (P = 0.001, percent inhibition). DPP4 genotypes rs2909451 TT (P = 0.02) and rs759717 CC (P = 0.02) were associated with DPP4 activity during sitagliptin. In multivariable analyses, T2DM with hypertension, sitagliptin dose, age, systolic blood pressure, DPP4 activity during placebo, and rs2909451 genotype were significantly associated with DPP4 activity during sitagliptin. Conclusions: Sitagliptin is less effective in inhibiting DPP4 in individuals with T2DM and hypertension than in healthy controls. Higher doses of DPP4 inhibitors may be required in patients with the metabolic syndrome.

VNTR diversity in Yersinia pestis isolates from an animal challenge study reveals the potential for in vitro mutations during laboratory cultivation

Underlying mutation rates and other evolutionary forces shape the population structure of bacteria in nature. Although easily overlooked, similar forces are at work in the laboratory and may influence observed mutations. Here, we investigated tissue samples and Yersinia pestis isolates from a rodent laboratory challenge with strain CO92 using whole genome sequencing and multi-locus variable-number tandem repeat (VNTR) analysis (MLVA). We identified six VNTR mutations that were found to have occurred in vitro during laboratory cultivation rather than in vivo during the rodent challenge. In contrast, no single nucleotide polymorphism (SNP) mutations were observed, either in vivo or in vitro. These results were consistent with previously published mutation rates and the calculated number of Y. pestis generations that occurred during the in vitro versus the in vivo portions of the experiment. When genotyping disease outbreaks, the potential for in vitro mutations should be considered, particularly when highly variable genetic markers such as VNTRs are used.

Genetic variation at the MHC DRB1 locus is similar across Gunnison's prairie dog (Cynomys gunnisoni) colonies regardless of plague history.

Abstract Yersinia pestis was introduced to North America around 1900 and leads to nearly 100% mortality in prairie dog (Cynomys spp.) colonies during epizootic events, which suggests this pathogen may exert a strong selective force. We characterized genetic diversity at an MHC class II locus (DRB1) in Gunnisons prairie dog (C. gunnisoni) and quantified population genetic structure at the DRB1 versus 12 microsatellite loci in three large Arizona colonies. Two colonies, Seligman (SE) and Espee Ranch (ES), have experienced multiple plague‐related die‐offs in recent years, whereas plague has never been documented at Aubrey Valley (AV). We found fairly low allelic diversity at the DRB1 locus, with one allele (DRB1*01) at high frequency (0.67–0.87) in all colonies. Two other DRB1 alleles appear to be trans‐species polymorphisms shared with the black‐tailed prairie dog (C. ludovicianus), indicating that these alleles have been maintained across evolutionary time frames. Estimates of genetic differentiation were generally lower at the MHC locus (F ST = 0.033) than at microsatellite markers (F ST = 0.098). The reduced differentiation at DRB1 may indicate that selection has been important for shaping variation at MHC loci, regardless of the presence or absence of plague in recent decades. However, genetic drift has probably also influenced the DRB1 locus because its level of differentiation was not different from that of microsatellites in an F ST outlier analysis. We then compared specific MHC alleles to plague survivorship in 60 C. gunnisoni that had been experimentally infected with Y. pestis. We found that survival was greater in individuals that carried at least one copy of the most common allele (DRB1*01) compared to those that did not (60% vs. 20%). Although the sample sizes of these two groups were unbalanced, this result suggests the possibility that this MHC class II locus, or a nearby linked gene, could play a role in plague survival.