Sex and age-biased nematode prevalence in reptiles.

Prevalence and intensity of parasitic infections are often higher in male than in female vertebrates. This bias may represent either differences between host sex in exposure or susceptibility to parasites. The former may be due to sex-specific behaviour of the host, including differential habitat use or diet. Differences in susceptibility are often regarded as a negative effect of male sex steroid hormones on the immune system. Host-parasite dynamics are of great interest in terms of reptile survival, ecology and conservation. We used, for the first time, molecular diagnostics to track nematode parasitism in wild populations of reptiles noninvasively. Using slow worms (Anguis fragilis) as a model species, we investigated the interacting effects of time of year, sex, length, weight and climatic variables on the prevalence of the gastroenterological parasitic nematode Neoxysomatium brevicaudatum. Faeces were collected from three sites over 2 years. There was an interaction between sex and time of year, with lower nematode prevalence in males than in females in July or August (different between years) but a high prevalence in males in April. As the latter is during the slow worm breeding season, this may be the result of testosterone-induced immunosuppression. A second-order interaction between slow worm length and weight was found to be significant, with a positive association between prevalence and body condition in young slow worms and a negative association in older slow worms. The convex pattern of nematode prevalence with age that emerged suggests an increase with age-related exposure and a decrease with age-related acquired immunity.

Molecular analysis of the diets of snakes: changes in prey exploitation during development of the rare smooth snake Coronella austriaca.

Reptiles are declining in many parts of the world, mainly due to habitat loss and environmental change. A major factor in this is availability of suitable food. For many animals, dietary requirements shift during developmental stages and a habitat will only be suitable for conserving a species if it supports all stages. Conventional methods for establishing diet often rely on visual recognition of morphologically identifiable features of prey in faeces, regurgitation or stomach contents, which suffer from biases and poor resolution of taxa. DNA-based techniques facilitate noninvasive analysis of diet from faeces without these constraints. We tested the hypothesis that diet changes during growth stages of smooth snakes (Coronella austriaca), which have a highly restricted distribution in the UK but are widespread in continental Europe. Small numbers of the sympatric grass snake (Natrix natrix) were analysed for comparison. Faecal samples were collected from snakes and prey DNA analysed using PCR, targeting amphibians, reptiles, mammals and invertebrates. Over 85% of smooth snakes were found to have eaten reptiles and 28% had eaten mammals. Predation on mammals increased with age and was entirely absent among juveniles and subadults. Predation on reptiles did not change ontogenetically. Smooth snakes may, therefore, be restricted to areas of sufficiently high reptile densities to support young snakes.

Molecular field analysis of trophic relationships in soil-dwelling invertebrates to identify mercury, lead and cadmium transmission through forest ecosystems.

Contamination pathways in complex food chains in soil ecosystems can be difficult to elucidate. Molecular analysis of predator gut content can, however, rapidly reveal previously unidentified trophic interactions between invertebrates and thereby uncover pathways of pollutant spread. Here, we measured concentrations of the toxic metals lead, cadmium and mercury in carabid beetle predators and their prey. Invertebrates were sampled at one control and four heavy metal-polluted sites to reveal the impact of diet composition and seasonal variation in prey availability on metal burden in carabids and metal transfer pathways through forest ecosystems. This is the first report, to our knowledge, of carabid diet composition based on PCR analysis of gut contents at the forest community level, rather than in cultivated fields. Extensive screening using group- and species-specific primers revealed that carabids ate primarily earthworms and slugs, as well as smaller numbers of woodlice and springtails. Metal concentrations in carabids correlated with seasonal changes in diet. Mercury accumulated in beetle predators more than in their slug prey. As earthworms, slugs and carabid beetles are the major prey of many birds and mammals, prey-predator transfer and associated toxicity are major risks at mercury-contaminated sites. Carabids may be useful bioindicators for assessing the impact of pollutants on soil ecosystems, as long as species and seasonal factors are taken into account.

Revisiting Ebola Virus Disease infection control protocols at an academic level I trauma center: Successes, challenges, and ways ahead.

In the decade since the largest Ebola Virus Disease (EVD) outbreak in history, hospitals within the United States have discovered deficiencies in EVD infection control protocols. A large academic level I trauma medical center and frontline EVD receiving hospital in northeast Florida conducted a large-scale review and revision of the facility's EVD infection control protocols to increase preparedness. The revision process revealed opportunities for improvement and highlighted the need for excellent resource management and interdepartmental communication.

Identification and infection control response to Candida auris at an academic level I trauma center.

Candida auris, an emerging fungal pathogen with significant morbidity and mortality, can be difficult for health care facilities to identify, isolate, and control. We present our identification and infection control response to Candida auris at a 695-bed academic level I trauma center in Florida.

Who is eating what: diet assessment using next generation sequencing.

The analysis of food webs and their dynamics facilitates understanding of the mechanistic processes behind community ecology and ecosystem functions. Having accurate techniques for determining dietary ranges and components is critical for this endeavour. While visual analyses and early molecular approaches are highly labour intensive and often lack resolution, recent DNA-based approaches potentially provide more accurate methods for dietary studies. A suite of approaches have been used based on the identification of consumed species by characterization of DNA present in gut or faecal samples. In one approach, a standardized DNA region (DNA barcode) is PCR amplified, amplicons are sequenced and then compared to a reference database for identification. Initially, this involved sequencing clones from PCR products, and studies were limited in scale because of the costs and effort required. The recent development of next generation sequencing (NGS) has made this approach much more powerful, by allowing the direct characterization of dozens of samples with several thousand sequences per PCR product, and has the potential to reveal many consumed species simultaneously (DNA metabarcoding). Continual improvement of NGS technologies, on-going decreases in costs and current massive expansion of reference databases make this approach promising. Here we review the power and pitfalls of NGS diet methods. We present the critical factors to take into account when choosing or designing a suitable barcode. Then, we consider both technical and analytical aspects of NGS diet studies. Finally, we discuss the validation of data accuracy including the viability of producing quantitative data.

The design, synthesis, and biological evaluation of PIM kinase inhibitors.

A series of substituted benzofuropyrimidinones with pan-PIM activities and excellent selectivity against a panel of diverse kinases is described. Initial exploration identified aryl benzofuropyrimidinones that were potent, but had cell permeability limitation. Using X-ray crystal structures of the bound PIM-1 complexes with 3, 5m, and 6d, we were able to guide the SAR and identify the alkyl benzofuropyrimidinone (6l) with good PIM potencies, permeability, and oral exposure.

Discovery of XL413, a potent and selective CDC7 inhibitor.

CDC7 is a serine/threonine kinase that has been shown to be required for the initiation and maintenance of DNA replication. Up-regulation of CDC7 is detected in multiple tumor cell lines, with inhibition of CDC7 resulting in cell cycle arrest. In this paper, we disclose the discovery of a potent and selective CDC7 inhibitor, XL413 (14), which was advanced into Phase 1 clinical trials. Starting from advanced lead 3, described in a preceding communication, we optimized the CDC7 potency and selectivity to demonstrate in vitro CDC7 dependent cell cycle arrest and in vivo tumor growth inhibition in a Colo-205 xenograft model.

SAR and in vivo evaluation of 4-aryl-2-aminoalkylpyrimidines as potent and selective Janus kinase 2 (JAK2) inhibitors.

We report the discovery of a series of 4-aryl-2-aminoalkylpyrimidine derivatives as potent and selective JAK2 inhibitors. High throughput screening of our in-house compound library led to the identification of hit 1, from which optimization resulted in the discovery of highly potent and selective JAK2 inhibitors. Advanced lead 10d demonstrated a significant dose-dependent pharmacodynamic and antitumor effect in a mouse xenograft model. Based upon the desirable profile of 10d (XL019) it was advanced into clinical trials.

Anatomic factors related to the cause of tennis elbow.

BACKGROUND: The pathogenesis of lateral epicondylitis remains unclear. Our purpose was to study the anatomy of the lateral aspect of the elbow under static and dynamic conditions in order to identify bone-to-tendon and tendon-to-tendon contact or rubbing that might cause abrasion of the tissues. METHODS: Eighty-five cadaveric elbows were examined to determine details related to the bone structure and musculotendinous origins. We identified the relative positions of the musculotendinous units and the underlying bone when the elbow was in different degrees of flexion. We also recorded the contact between the extensor carpi radialis brevis and the lateral edge of the capitellum as elbow motion occurred, and we sought to identify the areas of the capitellum and extensor carpi radialis brevis where contact occurs. RESULTS: The average site of origin of the extensor carpi radialis brevis on the humerus lay slightly medial and superior to the outer edge of the capitellum. As the elbow was extended, the undersurface of the extensor carpi radialis brevis rubbed against the lateral edge of the capitellum while the extensor carpi radialis longus compressed the brevis against the underlying bone. CONCLUSIONS: The extensor carpi radialis brevis tendon has a unique anatomic location that makes its undersurface vulnerable to contact and abrasion against the lateral edge of the capitellum during elbow motion.

Biomechanics of cantilever "plow" during anterior thoracic scoliosis correction.

BACKGROUND CONTEXT: Anterior instrumentation is often used for correction of thoracic scoliosis. Loss of spinal correction may occur after failure at the bone-implant interface, and forces on the bone-implant interface during scoliosis correction remain unclear. PURPOSE: Evaluate two different mechanisms of loading associated with anterior scoliosis correction. STUDY SETTING: In vitro biomechanics lab. METHODS: Polyurethane foam and human cadaveric thoracic vertebral bodies were instrumented with transvertebral body screws. Bone-implant interface failure loads were measured during constrained, fixed-angle screw translation, as well as unconstrained translation allowing coronal plane screw rotation. Vertebral body staples were randomly assigned to both conditions. RESULTS: Data were consistent across foam and cadaveric specimens. Failures occurred at significantly lower loads during unconstrained translation (with rotation) compared with constrained translation. Staple usage significantly increased the load to failure in both testing modes. In cadaveric bone, the constrained plowing load to failure was 562N+110N versus 188N+20N in the unconstrained testing. With a staple, these values increased to 694N+53N and 530N+100N, respectively. CONCLUSIONS: The 280% increase in cadaveric failure loads when a staple was added in the unconstrained testing method exceeds previous reports. The unconstrained method of plow simulated anterior scoliosis instrumentation when a rod was cantilevered and compressed into position. Supplemental vertebral body staples may be clinically indicated, particularly at the ends of the construct where residual deforming forces remain the greatest.

Pyrosequencing of prey DNA in reptile faeces: analysis of earthworm consumption by slow worms.

Little quantitative ecological information exists on the diets of most invertebrate feeding reptiles, particularly nocturnal or elusive species that are difficult to observe. In the UK and elsewhere, reptiles are legally required to be relocated before land development can proceed, but without knowledge of their dietary requirements, the suitability of receptor sites cannot be known. Here, we tested the ability of non-invasive DNA-based molecular diagnostics (454 pyrosequencing) to analyse reptile diets, with the specific aims of determining which earthworm species are exploited by slow worms (the legless lizard Anguis fragilis) and whether they feed on the deeper-living earthworm species that only come to the surface at night. Slow worm faecal samples from four different habitats were analysed using earthworm-specific PCR primers. We found that 86% of slow worms (N=80) had eaten earthworms. In lowland heath and marshy/acid grassland, Lumbricus rubellus, a surface-dwelling epigeic species, dominated slow worm diet. In two other habitats, riverside pasture and calciferous coarse grassland, diet was dominated by deeper-living anecic and endogeic species. We conclude that all species of earthworm are exploited by these reptiles and lack of specialization allows slow worms to thrive in a wide variety of habitats. Pyrosequencing of prey DNA in faeces showed promise as a practical, rapid and relatively inexpensive means of obtaining detailed and valuable ecological information on the diets of reptiles.

CD4+ regulatory T cells require CTLA-4 for the maintenance of systemic tolerance.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) plays a critical role in negatively regulating T cell responses and has also been implicated in the development and function of natural FOXP3(+) regulatory T cells. CTLA-4-deficient mice develop fatal, early onset lymphoproliferative disease. However, chimeric mice containing both CTLA-4-deficient and -sufficient bone marrow (BM)-derived cells do not develop disease, indicating that CTLA-4 can act in trans to maintain T cell self-tolerance. Using genetically mixed blastocyst and BM chimaeras as well as in vivo T cell transfer systems, we demonstrate that in vivo regulation of Ctla4(-/-) T cells in trans by CTLA-4-sufficient T cells is a reversible process that requires the persistent presence of FOXP3(+) regulatory T cells with a diverse TCR repertoire. Based on gene expression studies, the regulatory T cells do not appear to act directly on T cells, suggesting they may instead modulate the stimulatory activities of antigen-presenting cells. These results demonstrate that CTLA-4 is absolutely required for FOXP3(+) regulatory T cell function in vivo.