Standardized patient outcomes trial (SPOT) in neurology

Background: The neurologic examination is a challenging component of the physical examination for medical students. In response, primarily based on expert consensus, medical schools have supplemented their curricula with standardized patient (SP) sessions that are focused on the neurologic examination. Hypothesis-driven quantitative data are needed to justify the further use of this resource-intensive educational modality, specifically regarding whether using SPs to teach the neurological examination effects a long-term benefit on the application of neurological examination skills. Methods: This study is a cross-sectional analysis of prospectively collected data from medical students at Weill Cornell Medical College. The control group (n=129) received the standard curriculum. The intervention group (n=58) received the standard curriculum and an additional SP session focused on the neurologic examination during the second year of medical school. Student performance on the neurologic examination was assessed in the control and intervention groups via an OSCE administered during the fourth year of medical school. A Neurologic Physical Exam (NPE) score of 0.0 to 6.0 was calculated for each student based on a neurologic examination checklist completed by the SPs during the OSCE. Composite NPE scores in the control and intervention groups were compared with the unpaired t-test. Results: In the fourth year OSCE, composite NPE scores in the intervention group (3.5±1.1) were statistically significantly greater than those in the control group (2.2±1.1) (p<0.0001). Conclusions: SP sessions are an effective tool for teaching the neurologic examination. We determined that a single, structured SP session conducted as an adjunct to our traditional lectures and small groups is associated with a statistically significant improvement in student performance measured 2 years after the session

A microfluidic device with fluorimetric detection for intracellular components analysis

An integrated microfluidic system that coupled lysis of two cell lines: L929 fibroblasts and A549 epithelial cells, with fluorescence-based enzyme assay was developed to determine β-glucocerebrosidase activity. The microdevice fabricated in poly(dimethylsiloxane) consists of three main parts: a chemical cell lysis zone based on the sheath flow geometry, a micromeander and an optical fibers detection zone. Unlike many methods described in literature that are designed to analyse intracellular components, the presented system enables to perform enzyme assays just after cell lysis process. It reduces the effect of proteases released in lysis process on determined enzymes. Glucocerebrosidase activity, the diagnostic marker for Gaucher’s disease, is the most commonly measured in leukocytes and fibroblasts using 4-methylumbelliferyl-β-D-glucopyranoside as synthetic β-glucoside. The enzyme cleavage releases the fluorescent product, i.e. 4-methylumbelliferone, and its fluorescence is measured as a function of time. The method of enzyme activity determination described in this paper was adapted for flow measurements in the microdevice. The curve of the enzymatic reaction advancement was prepared for three reaction times obtained from application of different flow rates of solutions introduced to the microsystem. Afterwards, determined β-glucocerebrosidase activity was recalculated with regard to 105 cells present in samples used for the tests. The obtained results were compared with a cuvette-based measurements. The lysosomal β-glucosidase activities determined in the microsystem were in good correlation with the values determined during macro-scale measurements

A Comparative Chemogenomics Strategy to Predict Potential Drug Targets in the Metazoan Pathogen, Schistosoma mansoni

Schistosomiasis is a prevalent and chronic helmintic disease in tropical regions. Treatment and control relies on chemotherapy with just one drug, praziquantel and this reliance is of concern should clinically relevant drug resistance emerge and spread. Therefore, to identify potential target proteins for new avenues of drug discovery we have taken a comparative chemogenomics approach utilizing the putative proteome of Schistosoma mansoni compared to the proteomes of two model organisms, the nematode, Caenorhabditis elegans and the fruitfly, Drosophila melanogaster. Using the genome comparison software Genlight, two separate in silico workflows were implemented to derive a set of parasite proteins for which gene disruption of the orthologs in both the model organisms yielded deleterious phenotypes (e.g., lethal, impairment of motility), i.e., are essential genes/proteins. Of the 67 and 68 sequences generated for each workflow, 63 were identical in both sets, leading to a final set of 72 parasite proteins. All but one of these were expressed in the relevant developmental stages of the parasite infecting humans. Subsequent in depth manual curation of the combined workflow output revealed 57 candidate proteins. Scrutiny of these for ‘druggable’ protein homologs in the literature identified 35 S. mansoni sequences, 18 of which were homologous to proteins with 3D structures including co-crystallized ligands that will allow further structure-based drug design studies. The comparative chemogenomics strategy presented generates a tractable set of S. mansoni proteins for experimental validation as drug targets against this insidious human pathogen

Diagnosing Hunter syndrome in pediatric practice: practical considerations and common pitfalls

Mucopolysaccharidosis II (MPS II), or Hunter syndrome, is an X-linked lysosomal storage disorder caused by a deficiency in the enzyme iduronate-2-sulfatase. Affected patients suffer progressive damage to multiple organ systems and early mortality. Two thirds of patients also manifest cognitive impairment and developmental delays. MPS II can be extremely difficult to diagnose before irreversible organ and tissue damage has occurred because of an insidious onset and the overlap in signs and symptoms with common childhood complaints. This is particularly true of patients without cognitive impairment (attenuated phenotype). Although not curative, early treatment with enzyme replacement therapy before irreversible organ damage has occurred may result in the greatest clinical benefit. Here, the signs, symptoms, and surgical history that should trigger suspicion of MPS II are described, and the diagnostic process is reviewed with a focus on practical considerations and the avoidance of common diagnostic pitfalls. Once a diagnosis is made, multidisciplinary management with an extended team of pediatric specialists is essential and should involve the pediatrician or family practice physician as facilitator and medical home for the patient and family. Conclusion: Because routine newborn screening is not yet available for MPS II, the involvement and awareness of pediatricians, family practice physicians, and pediatric specialists is critical for early identification, diagnosis, and referral in order to help optimize patient outcomes

The Replicase Gene of Avian Coronavirus Infectious Bronchitis Virus Is a Determinant of Pathogenicity

We have previously demonstrated that the replacement of the S gene from an avirulent strain (Beaudette) of infectious bronchitis virus (IBV) with an S gene from a virulent strain (M41) resulted in a recombinant virus (BeauR-M41(S)) with the in vitro cell tropism of the virulent virus but that was still avirulent. In order to investigate whether any of the other structural or accessory genes played a role in pathogenicity we have now replaced these from the Beaudette strain with those from M41. The recombinant IBV was in effect a chimaeric virus with the replicase gene derived from Beaudette and the rest of the genome from M41. This demonstrated that it is possible to exchange a large region of the IBV genome, approximately 8.4 kb, using our transient dominant selection method. Recovery of a viable recombinant IBV also demonstrated that it is possible to interchange a complete replicase gene as we had in effect replaced the M41 replicase gene with the Beaudette derived gene. Analysis of the chimaeric virus showed that it was avirulent indicating that none of the structural or accessory genes derived from a virulent isolate of IBV were able to restore virulence and that therefore, the loss of virulence associated with the Beaudette strain resides in the replicase gene

Selective Inhibitors of Protozoan Protein N-myristoyltransferases as Starting Points for Tropical Disease Medicinal Chemistry Programs

Inhibition of N-myristoyltransferase has been validated pre-clinically as a target for the treatment of fungal and trypanosome infections, using species-specific inhibitors. In order to identify inhibitors of protozoan NMTs, we chose to screen a diverse subset of the Pfizer corporate collection against Plasmodium falciparum and Leishmania donovani NMTs. Primary screening hits against either enzyme were tested for selectivity over both human NMT isoforms (Hs1 and Hs2) and for broad-spectrum anti-protozoan activity against the NMT from Trypanosoma brucei. Analysis of the screening results has shown that structure-activity relationships (SAR) for Leishmania NMT are divergent from all other NMTs tested, a finding not predicted by sequence similarity calculations, resulting in the identification of four novel series of Leishmania-selective NMT inhibitors. We found a strong overlap between the SARs for Plasmodium NMT and both human NMTs, suggesting that achieving an appropriate selectivity profile will be more challenging. However, we did discover two novel series with selectivity for Plasmodium NMT over the other NMT orthologues in this study, and an additional two structurally distinct series with selectivity over Leishmania NMT. We believe that release of results from this study into the public domain will accelerate the discovery of NMT inhibitors to treat malaria and leishmaniasis. Our screening initiative is another example of how a tripartite partnership involving pharmaceutical industries, academic institutions and governmental/non-governmental organisations such as Medical Research Council and Wellcome Trust can stimulate research for neglected diseases

Using a Non-Image-Based Medium-Throughput Assay for Screening Compounds Targeting N-myristoylation in Intracellular Leishmania Amastigotes

We have refined a medium-throughput assay to screen hit compounds for activity against N-myristoylation in intracellular amastigotes of Leishmania donovani. Using clinically-relevant stages of wild type parasites and an Alamar blue-based detection method, parasite survival following drug treatment of infected macrophages is monitored after macrophage lysis and transformation of freed amastigotes into replicative extracellular promastigotes. The latter transformation step is essential to amplify the signal for determination of parasite burden, a factor dependent on equivalent proliferation rate between samples. Validation of the assay has been achieved using the anti-leishmanial gold standard drugs, amphotericin B and miltefosine, with EC50 values correlating well with published values. This assay has been used, in parallel with enzyme activity data and direct assay on isolated extracellular amastigotes, to test lead-like and hit-like inhibitors of Leishmania Nmyristoyl transferase (NMT). These were derived both from validated in vivo inhibitors of Trypanosoma brucei NMT and a recent high-throughput screen against L. donovani NMT. Despite being a potent inhibitor of L. donovani NMT, the activity of the lead T. brucei NMT inhibitor (DDD85646) against L. donovani amastigotes is relatively poor. Encouragingly, analogues of DDD85646 show improved translation of enzyme to cellular activity. In testing the high-throughput L. donovani hits, we observed macrophage cytotoxicity with compounds from two of the four NMT-selective series identified, while all four series displayed low enzyme to cellular translation, also seen here with the T. brucei NMT inhibitors. Improvements in potency and physicochemical properties will be required to deliver attractive lead-like Leishmania NMT inhibitors