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.     

New approach to capture and characterize synaptic proteome

Little is known regarding the identity of the population of proteins that are transported and localized to synapses. Here we describe a new approach that involves the isolation and systematic proteomic characterization of molecular motor kinesins to identify the populations of proteins transported to synapses. We used this approach to identify and compare proteins transported to synapses by kinesin (Kif) complexes Kif5C and Kif3A in the mouse hippocampus and prefrontal cortex. Approximately 40–50% of the protein cargos identified in our proteomics analysis of kinesin complexes are known synaptic proteins. We also found that the identity of kinesins and where they are expressed determine what proteins they transport. Our results reveal a previously unappreciated role of kinesins in regulating the composition of synaptic proteome.A number of researchers have reported specific roles of proteins that are localized to synapses, termed the “synaptic proteome,” in determining synaptic function and plasticity (1–6). Identifying the composition and dynamics of the synaptic proteome is key to understanding the remodeling of existing synapses and growth of new synapses, as well as identifying novel therapeutic targets for synaptopathies (7, 8). The composition of the synaptic proteome is determined by proteins transported from the cell body and local synthesis of proteins at the synapses. Several studies have shown the identification and localization of RNAs at the synapse (9–12). Comprehensive knowledge of the proteins that are actively targeted to synapses will help elucidate the dynamics of signal transduction at the synapse, as well as the molecules that are critical for the remodeling of synapses and formation of new synapses that accompany long-term memory storage. Despite this perceived significance, however, there are no currently available methodologies for studying populations of proteins targeted to synapses.To identify actively transported proteins, we focused on the protein complexes associated with kinesins, molecular motor proteins that move from the cell body to distal neuronal processes and mediate the transport of various gene products (9, 13). We considered that proteins associated with kinesins include both synaptic proteins and proteins localized to dendrites and axons. Our methodology involved isolating specific kinesin complexes by coimmunoprecipitation (co-IP) from different regions of the mouse brain and systematically identifying the protein cargos by mass spectrometry (Fig. 1A). Using this strategy, we have successfully isolated and characterized the Kif5C kinesin complex from hippocampus and prefrontal cortex (PFC) and the Kif3A kinesin complex from hippocampus. The cargos include organelles and proteins involved in translation, signaling, and ion channels. Several of these proteins are implicated in neuropsychiatric disorders (14).Open in a separate windowFig. 1.Identification of the Kif5C complex in mouse hippocampus. (A) Strategy used to analyze Kif5C-specific protein cargos in the mouse hippocampus. The schematic diagram shows the workflow for profiling Kif5C-transported proteins by proteomics. The processes include brain dissection, hippocampal tissue extraction and homogenization, Kif5C IP, complex separation by SDS/PAGE, isolation of specific bands followed by in-gel tryptic digestion, and LC-MS/MS analysis. (B) IP of Kif5C from mouse hippocampus. Kif5C complexes were immunoprecipitated from hippocampus using anti Kif5C antibody. (Upper) Protein complexes were then separated on SDS/PAGE and stained with silver. (Lower) Kif5C also was detected by Western blot analysis. Immunoprecipitation using CREB antibody and beads alone served as controls for IP. (C) Co-IP and Western blot analysis of Kif5C cargo proteins (PURα, DIC1, and GluR1) in mouse hippocampus. Total extract (input), IP, post-IP supernatant, beads-alone IP, and post-IP supernatant are shown. Arrows in B indicate the positions of Kif5C and antibody.     

Combining physical performance and Functional Movement Screen testing to identify elite junior Australian Football athletes at risk of injury

The Functional Movement Screen (FMS) and physical performance testing are often suggested to be related to sports injury risk. This study explored if the combination of FMS and physical performance testing improved identification of non-contact injury risk over FMS testing alone in an elite junior Australian football cohort. Over a 3-year period, 573 players completed pre-season injury history questionnaires, FMS, physical performance testing (20-m sprint, vertical jump, planned agility testing, and shuttle run test), and subsequent in-season injury surveillance. Results: Neither previous injury or FMS score <14 were related to an increased risk of subsequent injury in isolation. The combination of FMS composite score ≤14 and previous injury moderately increased the risk of injury (Hazard ratio [HR] = 2.22 [1.09-4.54]). None of the physical performance measures improved the ability to predict injuries based on FMS composite score. FMS asymmetry was only associated with injury when combined with previous injury and vertical jump performance. Players with ≥1 FMS asymmetry and history of previous injury experienced a large increase in injury risk when vertical jump was poor (HR = 4.26 [1.35-13.42]) or good (HR = 3.17 [1.08-9.29]). Players with a combination of a good vertical jump, no previous injury, and no FMS asymmetries were also at moderately increased risk of injury (HR = 3.41 [1.11-10.42]). No physical performance tests improved the ability to identify non-contact injury risk using an FMS composite score threshold. However, a U-shaped relationship between vertical jump and injury risk was identified with both poor and good vertical jump height associated with a moderate-large increase in non-contact injury risk in the presence of ≥1 asymmetrical FMS sub-test.     

A Nonionic Polyethylene Oxide (PEO) Surfactant Model: Experimental and Molecular Dynamics Studies of Kolliphor EL

Polyethoxylated, nonionic surfactants are important constituents of many drug formulations, including lipid-based formulations. In an effort to better understand the behavior of formulation excipients at the molecular level, we have developed molecular dynamics (MD) models for the widely used surfactant Kolliphor EL (KOL), a triricinoleate ester of ethoxylated glycerol. In this work, we have developed models based on a single, representative molecular component modeled with 2 force field variations based on the GROMOS 53A6_DBW and 2016H66 force field parameters for polyethoxylate chains. To compare the computational models to experimental measurements, we investigated the phase behavior of KOL using nephelometry, dynamic light scattering, cross-polarized microscopy, small-angle X-ray scattering, and cryogenic transmission electron microscopy. The potential for digestion of KOL was also evaluated using an in vitro digestion experiment. We found that the size and spherical morphology of the KOL colloids at low concentrations was reproduced by the MD models as well as the growing interactions between the aggregates to from rod-like structures at high concentrations. We believe that this model reproduces the phase behavior of KOL relevant to drug absorption and that it can be used in whole formulation simulations to accelerate the formulation development.     

Socioeconomic Position and Factors Associated With Use of a Nonsupine Infant Sleep Position: Findings From the Canadian Maternity Experiences Survey

Objectives. With a focus on socioeconomic position, we examined the association between maternal education and nonsupine infant sleep position, and examined patterns of effect modification with additional sociodemographic, maternal, infant, and health services predictors.Methods. Data were from the Maternity Experiences Survey, a national population-based sample of 76 178 new Canadian mothers (unweighted n = 6421) aged 15 years or older interviewed in 2006–2007. Using logistic regression, we developed multivariate models for 3 maternal education strata.Results. Level of maternal education was significantly and inversely related to nonsupine infant sleep position. Stratified analyses revealed different predictive factors for nonsupine infant sleep position across strata of maternal education. Postpartum home visits were not associated with use of this sleep position among new mothers with less than high school completion. Adequacy of postpartum information regarding sudden infant death syndrome was not associated with nonsupine infant sleep position in any of the educational strata.Conclusions. These findings suggest a need to revisit Back to Sleep health promotion strategies and to ensure that these interventions are tailored to match the information needs of all families, including mothers with lower levels of formal education.Optimizing infant sleep position in an effort to reduce the incidence of sudden infant death syndrome (SIDS) has been an important maternal child health promotion strategy since nonsupine sleep position was identified as an important modifiable risk factor for SIDS in the early 1990s.1 Dramatic decreases in SIDS rates in many countries over the subsequent decade2–5 were largely credited to the success of public health campaigns that initially advised that infants be placed in nonprone positions for sleep and later restricted the recommendation to the supine sleep position only.5–10 In Canada and the United States, SIDS rates dropped by 70% and 60%, respectively, between 1985 and 2004.2,5,6,11 These reductions in SIDS rates were accompanied by reductions in postneonatal mortality rates, suggesting that they were real reductions rather than artifacts of changes in diagnostic criteria or coding practices.5More recently, SIDS rates have stabilized in most countries. In some countries, such as the United States and New Zealand,5 and among socioeconomically disadvantaged, indigenous, and African American subpopulations,12,13 SIDS rates remain unacceptably high. These rates have been linked to the need for ongoing promotion of risk-reduction activities, such as supine sleep position, particularly in communities still experiencing a high SIDS burden.5 For example, despite efforts to promote parents’ and caregivers’ use of a supine position for infant sleep, longitudinal data from the National Infant Sleep Position Study in the United States have demonstrated that a sizable proportion of infants continue to be placed in nonsupine positions for usual sleep (27.9% in 2008).14 Moreover, following consistent increases in the proportion of infants being placed to sleep in supine positions throughout the 1990s, the rates have shown little change since approximately 2001.14Analysis of data from the US Pregnancy Risk Assessment Monitoring System survey has shown that socioeconomic position (SEP), measured by both education level and income, has been consistently, significantly, and positively associated with supine sleep position across states.15 Accordingly, nonsupine sleep position has been associated with socioeconomic deprivation in several other studies.16,17 For example, in a prospective cohort study of sleep position among inner-city mothers of infants aged 3 to 7 months in the United States, Brenner et al.16 found that mothers living below the poverty level were 1.81 times more likely to put their infant to sleep in a prone position than mothers living above the poverty level (95% confidence interval [CI] = 1.10, 2.99). Low SEP was not found to be significantly associated with nonsupine sleep position in an analysis of the National Infant Sleep Position Study; however, this data set underrepresented women with fewer than 12 years of education by more than 50% compared with other national surveys.18,19The size and scope of the population surveyed by the Canadian Maternity Experiences Survey (MES) provided a unique opportunity to pursue our study objectives, which were to (1) clarify the significance of the association between SEP and nonsupine sleep position, and (2) examine patterns of effect modification by SEP on the associations between nonsupine infant sleep position and additional sociodemographic, maternal, infant, and health services predictors.     

The CD6/ALCAM pathway promotes lupus nephritis via T cell–mediated responses

T cells are central to the pathogenesis of lupus nephritis (LN), a common complication of systemic lupus erythematosus (SLE). CD6 and its ligand, activated leukocyte cell adhesion molecule (ALCAM), are involved in T cell activation and trafficking. Previously, we showed that soluble ALCAM is increased in urine (uALCAM) of patients with LN, suggesting that this pathway contributes to disease. To investigate, uALCAM was examined in 1038 patients with SLE and LN from 5 ethnically diverse cohorts; CD6 and ALCAM expression was assessed in LN kidney cells; and disease contribution was tested via antibody blockade of CD6 in murine models of SLE and acute glomerulonephritis. Extended cohort analysis offered resounding validation of uALCAM as a biomarker that distinguishes active renal involvement in SLE, irrespective of ethnicity. ALCAM was expressed by renal structural cells whereas CD6 expression was exclusive to T cells, with elevated numbers of CD6⁺ and ALCAM⁺ cells in patients with LN. CD6 blockade in models of spontaneous lupus and immune-complex glomerulonephritis revealed significant decreases in immune cells, inflammatory markers, and disease measures. Our data demonstrate the contribution of the CD6/ALCAM pathway to LN and SLE, supporting its use as a disease biomarker and therapeutic target.