Subtyping Cryptosporidium ubiquitum,a Zoonotic Pathogen Emerging in Humans

Cryptosporidium ubiquitum is an emerging zoonotic pathogen. In the past, it was not possible to identify an association between cases of human and animal infection. We conducted a genomic survey of the species, developed a subtyping tool targeting the 60-kDa glycoprotein (gp60) gene, and identified 6 subtype families (XIIa–XIIf) of C. ubiquitum. Host adaptation was apparent at the gp60 locus; subtype XIIa was found in ruminants worldwide, subtype families XIIb–XIId were found in rodents in the United States, and XIIe and XIIf were found in rodents in the Slovak Republic. Humans in the United States were infected with isolates of subtypes XIIb–XIId, whereas those in other areas were infected primarily with subtype XIIa isolates. In addition, subtype families XIIb and XIId were detected in drinking source water in the United States. Contact with C. ubiquitum–infected sheep and drinking water contaminated by infected wildlife could be sources of human infections.Key words: Cryptosporidiosis, Cryptosporidium, zoonoses, epidemiology, molecular typing,whole genome sequencing, genomics, ruminants, rodents, horse, raccoon, sifaka, parasites, humansCryptosporidium infection is a leading cause of diarrhea in humans (1). Five Cryptosporidium species—C. hominis, C. parvum, C. meleagridis, C. felis, and C. canis—are responsible for most cases of cryptosporidiosis in humans. Among them, C. hominis and C. parvum are the most common etiologic agents, and the latter is responsible for most zoonotic infections (2). In recent years, C. ubiquitum, previously known as the cervine genotype, has been emerging as another major zoonotic species that infects persons. It has been found in humans worldwide, primarily in industrialized nations (3–11). In the United Kingdom, more human cases of cryptosporidiosis have been attributed to C. ubiquitum than to C. canis (9).C. ubiquitum is of public health concern because of its wide geographic distribution and broad host range. Of all Cryptosporidium spp. identified by molecular diagnostic tools, it infects the greatest variety of host species (12). C. ubiquitum has been commonly detected in domestic and wild ruminants (sheep, goats, mouflon sheep, blesboks, nyalas, white-tailed deer, Père David’s deer, sika deer, ibexes, buffalos, and yaks), rodents (squirrels, chipmunks, woodchucks, beavers, porcupines, deer mice, house mice, and gerbils), carnivores (raccoons), and primates (lemurs and humans) (12–16). It has also been found in drinking source water, storm water runoff, stream sediment, and wastewater in various geographic locations (17–22).Thus far, showing an association between human and animal cases of C. ubiquitum infection has not been possible because of the lack of suitable genetic markers for subtyping. For C. parvum, C. hominis, and some genetically related species, the most commonly used marker for subtyping is the 60-kDa glycoprotein gene (gp60, also called gp40/15). Sequence analysis of the gp60 gene has been used in studies of the genetic diversity, host adaptation, infection sources, and transmission dynamics of these Cryptosporidium spp. (2). However, it has been suggested that a single locus, such as gp60, is not a reliable marker of C. parvum and C. hominis population structure because genetic recombination may occur (23).Because C. ubiquitum is genetically distant from C. hominis and C. parvum, its homologue of the gp60 gene has thus far not been identified (24). In this study, we identified the gp60 gene of C. ubiquitum by whole-genome sequencing and used it to develop a subtyping technique to characterize specimens from humans, various animals, and drinking source water.     

Detecting Maternal‐Fetal Genotype Interactions Associated With Conotruncal Heart Defects: A Haplotype‐Based Analysis With Penalized Logistic Regression

Nonsyndromic congenital heart defects (CHDs) develop during embryogenesis as a result of a complex interplay between environmental exposures, genetics, and epigenetic causes. Genetic factors associated with CHDs may be attributed to either independent effects of maternal or fetal genes, or the intergenerational interactions between maternal and fetal genes. Detecting gene‐by‐gene interactions underlying complex diseases is a major challenge in genetic research. Detecting maternal‐fetal genotype (MFG) interactions and differentiating them from the maternal/fetal main effects has presented additional statistical challenges due to correlations between maternal and fetal genomes. Traditionally, genetic variants are tested separately for maternal/fetal main effects and MFG interactions on a single‐locus basis. We conducted a haplotype‐based analysis with a penalized logistic regression framework to dissect the genetic effect associated with the development of nonsyndromic conotruncal heart defects (CTD). Our method allows simultaneous model selection and effect estimation, providing a unified framework to differentiate maternal/fetal main effect from the MFG interaction effect. In addition, the method is able to test multiple highly linked SNPs simultaneously with a configuration of haplotypes, which reduces the data dimensionality and the burden of multiple testing. By analyzing a dataset from the National Birth Defects Prevention Study (NBDPS), we identified seven genes (GSTA1, SOD2, MTRR, AHCYL2, GCLC, GSTM3, and RFC1) associated with the development of CTDs. Our findings suggest that MFG interactions between haplotypes in three of seven genes, GCLC, GSTM3, and RFC1, are associated with nonsyndromic conotruncal heart defects.     

Inhibition of matrix metalloproteinase-2 improves endothelial function and prevents hypertension in insulin-resistant rats

BACKGROUND AND PURPOSE

Insulin resistance is often found to be associated with high blood pressure. We propose that in insulin-resistant hypertension, endothelial dysfunction is the consequence of increased activity of vascular MMP-2. As MMP-2 proteolytically cleaves a number of extracellular matrix proteins, we hypothesized that MMP-2 impairs endothelial function by proteolytic degradation of endothelial NOS (eNOS) or its cofactor, heat shock protein 90 (HSP90).

EXPERIMENTAL APPROACH

We tested our hypothesis in bovine coronary artery endothelial cells and fructose-fed hypertensive rats (FHR), a model of acquired systolic hypertension and insulin resistance.

KEY RESULTS

Treatment of FHRs with the MMP inhibitor doxycycline, preserved endothelial function as well as prevented the development of hypertension, suggesting that MMPs impair endothelial function. Furthermore, incubating endothelial cells in vitro with a recombinant MMP-2 decreased NO production in a dose-dependent manner. Using substrate cleavage assays and immunofluorescence microscopy studies, we found that MMP-2 not only cleaves and degrades HSP90, an eNOS cofactor but also co-localizes with both eNOS and HSP90 in endothelial cells, suggesting that MMPs functionally interact with the eNOS system. Treatment of FHRs with doxycycline attenuated the decrease in eNOS and HSP90 expression but did not improve insulin sensitivity.

CONCLUSIONS AND IMPLICATIONS

Our data suggest that increased activity of MMP-2 in FHRs impairs endothelial function and promotes hypertension. Inhibition of MMP-2 could be a potential therapeutic strategy for the management of hypertension.     

Aldehyde oxidase and its contribution to the metabolism of a structurally novel, functionally selective GABAA alpha5-subtype inverse agonist

(3-Tert-butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4-triazol-5-ylmethoxy)pyrazolo[1,5-d] [1,2,4]triazine was recently identified as a functionally selective, inverse agonist at the benzodiazepine site of GABA(A) alpha5-containing receptors, which enhances performance in animal models of cognition. The routes of metabolism of this compound in rat, dog, rhesus monkey and human in vitro systems, and in vivo in rat, dog and rhesus monkey have been characterized. The current study demonstrates that both a cytosolic oxidative reaction and cytochrome P450 play important roles in the metabolism of the compound. Chemical inhibition studies showed the oxidation in human cytosol to be catalysed predominantly by aldehyde oxidase rather than the related enzyme, xanthine oxidase. The aldehyde oxidase-mediated metabolites were present in vitro and in vivo in both rat and rhesus monkey, and also in vitro in man. They were absent both in vitro and in vivo in dog.     

Pharmacokinetic profile of single and repeated oral doses of MDMA in squirrel monkeys: relationship to lasting effects on brain serotonin neurons.

A large body of data indicates that (+/-)3,4-methylenedioxymethamphetamine (MDMA, 'ecstasy') can damage brain serotonin neurons in animals. However, the relevance of these preclinical data to humans is uncertain, because doses and routes of administration used in animals have generally differed from those used by humans. Here, we examined the pharmacokinetic profile of MDMA in squirrel monkeys after different routes of administration, and explored the relationship between acute plasma MDMA concentrations after repeated oral dosing and subsequent brain serotonin deficits. Oral MDMA administration engendered a plasma profile of MDMA in squirrel monkeys resembling that seen in humans, although the half-life of MDMA in monkeys is shorter (3 vs 6-9 h). MDMA was biotransformed into MDA, and the plasma ratio of MDA to MDMA was 3-5 / 100, similar to that in humans. MDMA accumulation in squirrel monkeys was nonlinear, and plasma levels were highly correlated with regional brain serotonin deficits observed 2 weeks later. The present results indicate that plasma concentrations of MDMA shown here to produce lasting serotonergic deficits in squirrel monkeys overlap those reported by other laboratories in some recreational 'ecstasy' consumers, and are two to three times higher than those found in humans administered a single 100-150 mg dose of MDMA in a controlled setting. Additional studies are needed on the relative sensitivity of brain serotonin neurons to MDMA toxicity in humans and non-human primates, the pharmacokinetic parameter(s) of MDMA most closely linked to the neurotoxic process, and metabolites other than MDA that may play a role.     

Self-reported stress and subsequent hospital admissions as a result of hypertension,varicose veins and haemorrhoids

BACKGROUND: This study examines a cohort in which individuals of privileged socio-economic position report greater psychological stress. We have previously shown in this cohort that stress is unrelated to coronary heart disease as measured by hospital discharge diagnosis and cause-specific death. In contrast, stress and hospitalization for cardiovascular conditions not requiring mandatory admission were associated. We hypothesized that psychosocial factors, in particular reporting tendency, are the likely mediator of this association, and the present study considers this further. METHODS: A total of 5,596 men underwent a health screening during which they completed the Reeder Stress Inventory. Details of hospital admissions were retrieved from the Scottish Morbidity Records over a 21 year follow-up. Relationships between stress and admission were evaluated using proportional hazards regression. RESULTS: Compared with low stress, reported high stress was found to be associated with increased numbers of admissions for each of three most common cardiovascular causes of non-mandatory admission: adjusted hazard ratios were 3.43 for essential hypertension (95 per cent confidence interval (CI) 1.36-8.65), 1.91 for lower limb varicose veins (95 per cent CI 1.12-3.24), and 2.01 for haemorrhoids (95 per cent CI 1.16-3.51). Stress and blood pressure at baseline were not associated. CONCLUSION: The association between stress and admissions as a result of hypertension appears unlikely to be mediated by blood pressure. More likely is a mechanism based upon the reporting of symptoms, or the recording of discharge diagnoses. There is no obvious medical explanation for associations between stress and hospitalization as a result of varicose veins or haemorrhoids, and again it is likely that psychosocial factors provide the mechanism.     

Presentation and interpretation of food intake data: Factors affecting comparability across studies

Non-uniform, unclear, or incomplete presentation of food intake data limits interpretation, usefulness, and comparisons across studies. In this contribution, we discuss factors affecting uniform reporting of food intake across studies. The amount of food eaten can be reported as mean portion size, number of servings or total amount of food consumed per day; the absolute intake value for the specific study depends on the denominator used because food intake data can be presented as per capita intake or for consumers only. To identify the foods mostly consumed, foods are reported and ranked according to total number of times consumed, number of consumers, total intake, or nutrient contribution by individual foods or food groups. Presentation of food intake data primarily depends on a study's aim; reported data thus often are not comparable across studies. Food intake data further depend on the dietary assessment methodology used and foods in the database consulted; and are influenced by the inherent limitations of all dietary assessments. Intake data can be presented as either single foods or as clearly defined food groups. Mixed dishes, reported as such or in terms of ingredients and items added during food preparation remain challenging. Comparable presentation of food consumption data is not always possible; presenting sufficient information will assist valid interpretation and optimal use of the presented data. A checklist was developed to strengthen the reporting of food intake data in science communication.     

In the cut and thrust of apoptosis, serine proteases come of age

Proteolysis is central to the systematic cellular degradation that occurs during apoptosis. Predominantly, caspases have been studied in this regard. However, increasing evidence suggests that certain serine proteases may also play a significant role in apoptosis. Not only are these serine proteases involved in apoptosis signalling pathways independently, but they may also interact with more classical mediators of apoptosis such as the caspases or Bcl-2 family proteins. Isolation of apoptosis-associated serine proteases and the use of specific inhibitors have helped to shed light on potential pathways in which they are involved. Despite the recent developments in the field, knowledge regarding the role of serine proteases in apoptosis remains limited, but it is clear that investigations are gathering momentum and such studies may herald a new and exciting departure in apoptosis research.