Replacing Lectures with Small Groups: The Impact of Flipping the Residency Conference Day.

The flipped classroom, an educational alternative to the traditional lecture, has been widely adopted by educators at all levels of education and across many disciplines. In the flipped classroom, learners prepare in advance of the face-to-face meeting by learning content material on their own. Classroom time is reserved for application of the learned content to solving problems or discussing cases. Over the past year, we replaced most residency program lectures with small-group discussions using the flipped-classroom model, case-based learning, simulation and procedure labs. In the new model, residents prepared for conference by reviewing a patient case and studying suggested learning materials. Conference day was set aside for facilitated small-group discussions about the case. This is a cross-cohort study of emergency medicine residents who experienced the lecture-based curriculum to residents in the new flipped-classroom curriculum using paired comparisons (independent t-tests) on in-training exam scores while controlling for program year level. We also compared results of the evaluation of various program components. We observed no differences between cohorts on in-training examination scores. Small-group methods were rated the same across program years. Two program components in the new curriculum, an updated format of both adult and pediatric case conferences, were rated significantly higher on program quality. In preparation for didactics, residents in the new curriculum report spending more time on average with outside learning materials, including almost twice as much time reviewing textbooks. Residents found the new format of the case conferences to be of higher quality because of the inclusion of rapid-fire case discussions with targeted learning points.

Using the Association of American Medical Colleges Standardized Video Interview in a Holistic Residency Application Review.

Each year, residency programs work diligently to identify the best applicants for their respective programs, given the increasing volume of applications. Interview offers are often based on a mix of subjective and objective measures, with different programs relying more or less on each. A holistic application review involves a flexible and individualized way of assessing an applicant's capabilities through a balanced consideration of experiences, attributes, and academic metrics. When considered collectively, these attributes may define how an individual may perform as a physician. One particular tool developed by the American Association of Medical Colleges (AAMC), the Standardized Video Interview (SVI), provides an objective measure of an applicant's professional behavior and interpersonal communication skills. The SVI may provide applicants with a chance to showcase the intangibles about themselves that are neither entered on their application nor reflected by their standardized examination scores.

Pro-Coagulant Endothelial Dysfunction Results from EHEC Shiga Toxins and Host Damage-Associated Molecular Patterns.

Hemolytic uremic syndrome (HUS) from enterohemorrhagic Escherichia coli infection is a leading cause of kidney failure in otherwise healthy U.S. children. The bacterial Shiga toxins (Stx) induce the characteristic coagulopathy of HUS, but the damage to toxin-receptor expressing cells and organ injury due to ischemia likely also releases inflammatory damage-associated molecular patterns (DAMPs), which may exacerbate injury along with the toxins. To examine this, human aortic and renal glomerular cell anti-coagulant and barrier functions were studied after in vitro challenge with Stx1, Stx2, and DAMPs. There was significant loss of surface anti-coagulant protein C pathway molecules, increased expression of pro-thrombotic PAR1 and reduced protein C activation capability by 15-27%. Histones nearly completely prevented the activated protein C protection of endothelial cells from thrombin-induced permeability. In mice, lethal Stx2 challenge elevated plasma HMGB1 (day 2, 321 ± 118%; p < 0.01) and extracellular histones (day 3, 158 ± 62%; p < 0.01). Mice colonized with Stx2-expressing Citrobacter rodentium developed increased HMGB1 (day 5, 155 ± 55%; p < 0.01) and histones (day 3, 378 ± 188%; p < 0.01). Anti-histone antibody reduced both DAMPs to baseline, but was not sufficient to improve survival outcome or kidney function. Together, these data suggest a potential role Stx to produce DAMPs, and DAMPs to produce endothelial injury and a pro-thrombotic environment.

Shiga toxin 2-induced endoplasmic reticulum stress is minimized by activated protein C but does not correlate with lethal kidney injury.

Enterohemorrhagic Escherichia coli produce ribotoxic Shiga toxins (Stx), which are responsible for kidney injury and development of hemolytic uremic syndrome. The endoplasmic reticulum (ER) stress response is hypothesized to induce apoptosis contributing to organ injury; however, this process has been described only in vitro. ER stress marker transcripts of spliced XBP1 (1.78-fold), HSP40 (4.45-fold) and CHOP (7.69-fold) were up-regulated early in kidneys of Stx2 challenged mice compared to saline controls. Anti-apoptotic Bcl2 decreased (-2.41-fold vs. saline) and pro-apoptotic DR5 increased (6.38-fold vs. saline) at later time points. Cytoprotective activated protein C (APC) reduced early CHOP expression (-3.3-fold vs. untreated), increased later Bcl2 expression (5.8-fold vs. untreated), and had early effects on survival but did not alter DR5 expression. Changes in kidney ER stress and apoptotic marker transcripts were observed in Stx2-producing C. rodentium challenged mice compared to mice infected with a non-toxigenic control strain. CHOP (4.14-fold) and DR5 (2.81-fold) were increased and Bcl2 (-1.65-fold) was decreased. APC reduced CHOP expression and increased Bcl2 expression, but did not alter mortality. These data indicate that Stx2 induces renal ER stress and apoptosis in murine models of Stx2-induced kidney injury, but decreasing these processes alone was not sufficient to alter survival outcome.

Quiescent complement in nonhuman primates during E coli Shiga toxin-induced hemolytic uremic syndrome and thrombotic microangiopathy.

Enterohemorrhagic Escherichia coli (EHEC) produce ribosome-inactivating Shiga toxins (Stx1, Stx2) responsible for development of hemolytic uremic syndrome (HUS) and acute kidney injury (AKI). Some patients show complement activation during EHEC infection, raising the possibility of therapeutic targeting of complement for relief. Our juvenile nonhuman primate (Papio baboons) models of endotoxin-free Stx challenge exhibit full spectrum HUS, including thrombocytopenia, hemolytic anemia, and AKI with glomerular thrombotic microangiopathy. There were no significant increases in soluble terminal complement complex (C5b-9) levels after challenge with lethal Stx1 (n = 6) or Stx2 (n = 5) in plasma samples from T0 to euthanasia at 49.5 to 128 hours post-challenge. d-dimer and cell injury markers (HMGB1, histones) confirmed coagulopathy and cell injury. Thus, complement activation is not required for the development of thrombotic microangiopathy and HUS induced by EHEC Shiga toxins in these preclinical models, and benefits or risks of complement inhibition should be studied further for this infection.

Shiga toxins and the pathophysiology of hemolytic uremic syndrome in humans and animals.

Food-borne diseases are estimated at 76 million illnesses and 5000 deaths every year in the United States with the greatest burden on young children, the elderly and immunocompromised populations. The impact of efficient food distribution systems and a truly global food supply ensures that outbreaks, previously sporadic and contained locally, are far more widespread and emerging pathogens have far more frequent infection opportunities. Enterohemorrhagic E. coli is an emerging food- and water-borne pathogen family whose Shiga-like toxins induce painful hemorrhagic colitis with potentially lethal complications of hemolytic uremic syndrome (HUS). The clinical manifestations of Shiga toxin-induced HUS overlap with other related syndromes yet molecular mechanisms differ considerably. As discussed herein, understanding these differences and the novel properties of the toxins is imperative for clinical management decisions, design of appropriate animal models, and choices of adjunctive therapeutics. The emergence of new strains with rapidly aggressive virulence makes clinical and research initiatives in this field a high public health priority.

X-ray structure determination of the glycine cleavage system protein H of Mycobacterium tuberculosis using an inverse Compton synchrotron X-ray source.

Structural genomics discovery projects require ready access to both X-ray diffraction and NMR spectroscopy which support the collection of experimental data needed to solve large numbers of novel protein structures. The most productive X-ray crystal structure determination laboratories make extensive use of tunable synchrotron X-ray light to solve novel structures by anomalous diffraction methods. This requires that frozen cryo-protected crystals be shipped to large multi acre synchrotron facilities for data collection. In this paper we report on the development and use of the first laboratory-scale synchrotron light source capable of performing many of the state-of-the-art synchrotron applications in X-ray science. This Compact Light Source is a first-in-class device that uses inverse Compton scattering to generate X-rays of sufficient flux, tunable wavelength and beam size to allow high-resolution X-ray diffraction data collection from protein crystals. We report on benchmarking tests of X-ray diffraction data collection with hen egg white lysozyme, and the successful high-resolution X-ray structure determination of the Glycine cleavage system protein H from Mycobacterium tuberculosis using diffraction data collected with the Compact Light Source X-ray beam.

Size-exclusion chromatography can identify faster-associating protein complexes and evaluate design strategies.

BACKGROUND: We previously developed a set of rationally designed mutant MICA protein ligands for the NKG2D immunoreceptor in which MICA was mutated at residues that do not contact NKG2D. Some of these MICA mutants, predicted by RosettaDesign to be destabilized, bound NKG2D with affinities enhanced by more than an order of magnitude when evaluated by surface plasmon resonance (SPR). FINDINGS: Small-zone size-exclusion chromatography (SEC) detected persistent high-affinity MICA mutant-NKG2D complexes in solution as early-eluting peaks. The SEC binding assay used standard protein purification instrumentation to evaluate complex stability, qualitatively paralleled the SPR results, and successfully discriminated among complexes that differed only in on-rates. We used the SEC binding assay, along with SPR, to assess the results of a follow-up design strategy targeting the non-interfacial redesigned region. Both SEC and SPR agreed that these mutations did not enhance affinity as much as previous mutants. When the SEC binding assay was run in 1 M urea, only the highest affinity complex was detected. CONCLUSION: This SEC binding assay provides a correlation with SPR results for protein complex affinities, detecting changes in complex on-rates, and tunable to lower sensitivity with 1 M urea. The SEC binding assay is complementary to other protein design evaluation methods, can be adapted to the undergraduate research laboratory, and may provide additional structural information about changes in hydrodynamic radii from elution times. Our assay allowed us to conclude that further alteration of MICA at non-contacting residues is unlikely to further enhance NKG2D affinity.

Serratia marcescens osteomyelitis in an infant.

Neutrophil dysfunction can result from oxidative burst defect or from glucose-6-phosphate dehydrogenase (G6PD) deficiency; we noted both in the same patient. A 4-month-old male infant with G6PD deficiency presented with swelling of the left middle finger, left leg, and right big toe. At 5 weeks of age he was hospitalized for fever for 2 days. A maternal uncle died at 5 years of age and a male maternal cousin died at the age of 21 months, both reportedly diagnosed with chronic granulomatous disease (CGD). On physical examination, he had a swollen erythematous left third finger, left distal leg swelling, and right big toe abscess. None of these areas was significantly tender. WBC was 18.7 x 10(3)/mm(3) with 37% PMN and 5% bands. The x-ray films showed osteomyelitis in the left third proximal phalanx and the distal right first metatarsal. Culture from the toe abscess grew Serratia marcescens. His neutrophil oxidative burst was tested by the dihydrorhodamine-123 assay and was markedly suppressed, typical of CGD. The mother and maternal grandmother were found to be CGD carriers. He was treated with i.v. antibiotics for 4 weeks and was discharged on prophylactic trimethoprim, itraconazole and interferon gamma, with substantial reduction in infections. Infection in this infant was unusual in its nature, in affecting multiple sites, and in its causative organism. Immune deficiency was suspected, particularly of the phagocytic component, but could not be attributed to his moderate degree of primary G6PD deficiency. Additional immunologic evaluation and the family history led to the diagnosis of X-linked CGD.

Food allergy: manifestations, diagnosis and management.

Adverse reactions to food are common but only a fraction of them are due to hypersensitivity (or allergy). Of the latter, immunoglobulin (Ig)E-mediated reactions appear to be the most common and best understood. A wide variety of manifestations may affect various body systems, particularly the gastrointestinal tract, skin and respiratory tract. Diagnosis depends primarily on thorough medical history, often supplemented by skin testing or specific serum IgE antibody measurement. Verification would require appropriately designed challenge testing. At present, treatment is primarily strict avoidance of the offending food, together with crossreacting other foods. Several immunomodulatory therapeutic agents are being explored. A few studies demonstrated a potential benefit of probiotics for prophylaxis and treatment. A multicenter study is currently investigating the efficacy and safety of anti-IgE in subjects with peanut allergy. Experimental studies in mice revealed preliminary findings that promise vaccine development using novel approaches, such as modified food allergen epitopes, immunostimulatory sequence-conjugated allergens and chimeric proteins capable of complexing with IgE or its receptors.