A survey of retaining faculty at a new medical school: opportunities, challenges and solutions.

BACKGROUND: At well-established academic university settings, retaining faculty remains a pressing challenge due to competing market forces, decreasing institutional support, and changing personal expectations. There is a paucity of information about the difficulties faced by new medical schools to maintain their academic workforce. The objective of this study was to determine the challenges facing the faculty at a newly developed medical school. METHODS: Twelve founding faculty were surveyed anonymously by a 32-item questionnaire. Their responses were independently analyzed by three researchers. RESULTS: The views of the faculty were categorized into in four inter-related themes: personal, support, institutional, and environmental. The constant sources of satisfaction among faculty were higher academic rank (75%), harmonious inter-collegial relationships (74%), healthy pecuniary rewards (58%), better professional growth (58%) along with greater autonomy, administrative independence, minimum groupism and excellent team work. Poor opportunities for promotion (68%), reduced support for scholarly activities (67%) and unsatisfactory support from the administration (55%) were detrimental to retaining faculty. CONCLUSION: By addressing specific issues facing its staff, every new medical school will not only manage to retain its academic faculty but also be able to attract well qualified academic staff from established medical institutions worldwide.

Curriculum mapping as a tool to facilitate curriculum development: a new School of Medicine experience.

BACKGROUND: Every curriculum needs to be reviewed, implemented and evaluated; it must also comply with the regulatory standards. This report demonstrates the value of curriculum mapping (CM), which shows the spatial relationships of a curriculum, in developing and managing an integrated medical curriculum. METHODS: A new medical school developed a clinical presentation driven integrated curriculum that incorporates the active-learning pedagogical practices of many educational institutions worldwide while adhering to the mandated requirements of the accreditation bodies. A centralized CM process was run in parallel as the curriculum was being developed. A searchable database, created after the CM data was uploaded into an electronic curriculum management system, was used to ensure placing, integrating, evaluating and revising the curricular content appropriately. RESULTS: CM facilitated in a) appraising the content integration, b) identifying gaps and redundancies, c) linking learning outcomes across all educational levels (i.e. session to course to program), c) organizing the teaching schedules, instruction methods, and assessment tools and d) documenting compliance with accreditation standards. CONCLUSIONS: CM is an essential tool to develop, review, improve and refine any integrated curriculum however complex. Our experience, with appropriate modifications, should help other medical schools efficiently manage their curricula and fulfill the accreditation requirements at the same time.

Brief heat treatment causes a structural change and enhances cytotoxicity of the Escherichia coli α-hemolysin.

α-Hemolysin (HLY) is an important virulence factor for uropathogenic Escherichia coli. HLY is a member of the RTX family of exotoxins secreted by a number of Gram-negative bacteria. Recently, it was reported that a related RTX toxin, the Mannheimia haemolytica leukotoxin, exhibits increased cytotoxicity following brief heat treatment. In this article, we show that brief heat treatment (1 min at 100°C) increases cytotoxicity of HLY for human bladder cells, kidney epithelial cells (A498) and neutrophils. Heat treatment also increased hemolysis of human red blood cells (RBCs). Furthermore, heat treatment of previously inactived HLY restored its cytotoxicity. Heat-activated and native HLY both required glycophorin A to lyse RBCs. Native and heat-activated HLY appeared to bind equally well to the surface of A498 cells; although, Western blot analyses demonstrated binding to different proteins on the surface. Confocal microscopy revealed that heat-activated HLY bound more extensively to internal structures of permeabilized A498 cells than did native HLY. Several lines of spectroscopic evidence demonstrate irreversible changes in the structure of heat activated compared to native HLY. We show changes in secondary structure, increased exposure of tryptophan residues to the aqueous environment, an increase in molecular dimension and an increase in hydrophobic surface area. These properties are among the most common characteristics described for the molten globule state, first identified as an intermediate in protein folding. We hypothesize that brief heat treatment of HLY causes a conformational change leading to significant differences in protein-protein interactions that result in increased cytotoxicity for target cells.

N-terminal region of Mannheimia haemolytica leukotoxin serves as a mitochondrial targeting signal in mammalian cells.

Mannheimia haemolytica leukotoxin (LktA) is a member of the RTX toxin family that specifically kills ruminant leukocytes. Previous studies have shown that LktA induces apoptosis in susceptible cells via a caspase-9-dependent pathway that involves binding of LktA to mitochondria. In this study, using the bioinformatics tool MitoProt II we identified an N-terminal amino acid sequence of LktA that represents a mitochondrial targeting signal (MTS). We show that expression of this sequence, as a GFP fusion protein within mammalian cells, directs GFP to mitochondria. By immunoprecipitation we demonstrate that LktA interacts with the Tom22 and Tom40 components of the translocase of the outer mitochondrial membrane (TOM), which suggests that import of this toxin into mitochondria involves a classical import pathway for endogenous proteins. We also analysed the amino acid sequences of other RTX toxins and found a MTS in the N-terminal region of Actinobacillus pleuropneumoniae ApxII and enterohaemorrhagic Escherichia coli EhxA, but not in A. pleuropneumoniae ApxI, ApxIII, Aggregatibacter actinomycetemcomitans LtxA or the haemolysin (HlyA) from uropathogenic strains of E. coli. These findings provide a new evidence for the importance of the N-terminal region in addressing certain RTX toxins to mitochondria.

Dynamin-2-dependent targeting of mannheimia haemolytica leukotoxin to mitochondrial cyclophilin D in bovine lymphoblastoid cells.

Exotoxins which belong to the family containing the RTX toxins (repeats in toxin) contribute to a variety of important human and animal diseases. One example of such a toxin is the potent leukotoxin (LKT) produced by the bovine respiratory pathogen Mannheimia haemolytica. LKT binds to CD18, resulting in the death of bovine leukocytes. In this study, we showed that internalized LKT binds to the outer mitochondrial membrane, which results in the release of cytochrome c and collapse of the mitochondrial membrane potential (psi(m)). Incubation of bovine lymphoblastoid cells (BL-3 cells) with the mitochondrial membrane-stabilizing agent cyclosporine (CSA) reduced LKT-mediated cytotoxicity, cytochrome c release, and collapse of the psi(m). Coimmunoprecipitation and intracellular binding studies suggested that LKT binds to the mitochondrial matrix protein cyclophilin D. We also demonstrated that LKT mobilizes the vesicle scission protein dynamin-2 from mitochondria to the cell membrane. Incubation with CSA depleted mitochondrial dynamin-2 in BL-3 cells, making it unavailable for vesicle scission and LKT internalization. The results of this study show that LKT trafficking and LKT-mediated cell death involve dynamin-2 and cyclophilin D, in a process that can be prevented by the mitochondrial membrane-protecting function of CSA.

Gene expression profiling of bovine bronchial epithelial cells exposed in vitro to bovine herpesvirus 1 and Mannheimia haemolytica.

Bovine respiratory disease (BRD) often occurs when active respiratory virus infections (BHV-1, etc.) impair resistance to Mannheimia haemolytica infection in the lower respiratory tract. The interactions that occur when the respiratory epithelium encounters these viral and bacterial pathogens are poorly understood. We used Agilent bovine gene microarray chips containing 44,000 transcripts to elucidate bovine bronchial epithelial cell (BBEC) responses following in vitro exposure to BHV-1 alone, M. haemolytica alone, or both BHV-1 and M. haemolytica. Microarray analysis revealed differential regulation (>2-fold) of 978 transcripts by BHV-1 alone, 2040 transcripts by M. haemolytica alone, and 2189 genes by BHV-1 and M. haemolytica in combination. M. haemolytica treatment produced significantly greater inductions (>10-fold) of several inflammation associated genes, such as CXCL2, IL-6, IL-1α, e-selectin, and IL-8, than to BHV-1 alone. Functional analysis of the microarray data revealed a significant upregulation of genes involved in important biological processes such as inflammation (TNF-α, IL-8, Tlr-2, IL-1, CXCL2, CSF2), vascular functions (VEGF, EDN2) and leukocyte migration (ICAM1, IL-16) during a co-infection with BHV-1 and M. haemolytica compared to either pathogen alone. This study provides evidence to support that lung epithelial cells are a source of mediators that may promote inflammatory changes observed during bovine respiratory disease.

Brief heat treatment increases cytotoxicity of Mannheimia haemolytica leukotoxin in an LFA-1 independent manner.

Mannheimia haemolytica is an important respiratory pathogen in cattle. Its predominant virulence factor is a leukotoxin (LKT) that is a member of the RTX family of exotoxins produced by a variety of Gram negative bacteria. LKT binds to the CD18 chain of beta(2) integrins on bovine leukocytes, resulting in cell death. In this study, we show that brief heat treatment of native LKT (95 degrees C for 3 min) results in increased cytotoxicity for BL-3 (bovine lymphoblastoid) cells. Similar heat treatment restored the activity of LKT that had been rendered inactive by incubation at 22 degrees C for 3 days. A hallmark of LKT is that its toxicity is restricted to leukocytes from cattle or other ruminant species. Surprisingly, heat treatment rendered LKT cytotoxic for human, porcine and canine leukocytes. Membrane binding studies suggested that heat-treated LKT binds to membrane proteins other than LFA-1, and is distributed diffusely along the BL-3 cell membrane. Circular Dichroism spectroscopy studies indicate that heat treatment induced a small change in the secondary structure of the LKT that was not reversed when the LKT was cooled to room temperature. Thus, we speculate that these structural changes might contribute to the altered biological properties of heat-treated LKT.

Mannheimia haemolytica leukotoxin binds to lipid rafts in bovine lymphoblastoid cells and is internalized in a dynamin-2- and clathrin-dependent manner.

Mannheimia haemolytica is the principal bacterial pathogen of the bovine respiratory disease complex. Its most important virulence factor is a leukotoxin (LKT), which is a member of the RTX family of exotoxins produced by many gram-negative bacteria. Previous studies demonstrated that LKT binds to the beta(2)-integrin LFA-1 (CD11a/CD18) on bovine leukocytes, resulting in cell death. In this study, we demonstrated that depletion of lipid rafts significantly decreases LKT-induced bovine lymphoblastoid cell (BL-3) death. After binding to BL-3 cells, some of the LKT relocated to lipid rafts in an LFA-1-independent manner. We hypothesized that after binding to LFA-1 on BL-3 cells, LKT moves to lipid rafts and clathrin-coated pits via a dynamic process that results in LKT internalization and cytotoxicity. Knocking down dynamin-2 by small interfering RNA reduced both LKT internalization and cytotoxicity. Similarly, expression of dominant negative Eps15 protein expression, which is required for clathrin coat formation, reduced LKT internalization and LKT-mediated cytotoxicity to BL-3 cells. Finally, we demonstrated that inhibiting actin polymerization reduced both LKT internalization and LKT-mediated cytotoxicity. These results suggest that both lipid rafts and clathrin-mediated mechanisms are important for LKT internalization and cytotoxicity in BL-3 cells and illustrate the complex nature of LKT internalization by the cytoskeletal network.

Mannheimia haemolytica leukotoxin induces apoptosis of bovine lymphoblastoid cells (BL-3) via a caspase-9-dependent mitochondrial pathway.

Mannheimia haemolytica is a key pathogen in the bovine respiratory disease complex. It produces a leukotoxin (LKT) that is an important virulence factor, causing cell death in bovine leukocytes. The LKT binds to the beta(2) integrin CD11a/CD18, which usually activates signaling pathways that facilitate cell survival. In this study, we investigated mechanisms by which LKT induces death in bovine lymphoblastoid cells (BL-3). Incubation of BL-3 cells with a low concentration of LKT results in the activation of caspase-3 and caspase-9 but not caspase-8. Similarly, the proapoptotic proteins Bax and BAD were significantly elevated, while the antiapoptotic proteins Bcl-2, Bcl(XL) and Akt-1 were downregulated. Following exposure to LKT, we also observed a reduction in mitochondrial cytochrome c and corresponding elevation of cytosolic cytochrome c, suggesting translocation from the mitochondrial compartment to the cytosol. Consistent with this observation, tetramethylrhodamine ethyl ester perchlorate staining revealed that mitochondrial membrane potential was significantly reduced. These data suggest that LKT induces apoptosis of BL-3 cells via a caspase-9-dependent mitochondrial pathway. Furthermore, scanning electron micrographs of mitochondria from LKT-treated BL-3 cells revealed lesions in the outer mitochondrial membrane, which are larger than previous reports of the permeability transition pore through which cytochrome c is usually released.

The assessment of the viability of the endospores of Rhinosporidium seeberi with MTT (3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide).

This report describes tests with Evan's Blue and MTT (3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide) for the assessment of the viability of rhinosporidial endospores. MTT stained a proportion of the spherical bodies that we regard as the Electron Dense Bodies (EDBs), and the cytoplasm of freshly prepared endospores or ones that were stored at 4 degrees. Slow-freezing at -20 degrees C, exposure to 10% formalin, or 0.1% sodium azide of the endospores abolished MTT-staining in both sites. Evan's Blue stained the EDBs and cytoplasm of fresh endospores or those stored at 4 degrees, and of sodium azide-treated or frozen (-20 degrees)-thawed endospores. TMRE (tetramethyl-rhodamine ethyl ester) specifically labelled the spherical bodies, supporting the conclusion that these spherical bodies have a mitochondrial-like structure. TMRE-staining was however retained in endospores after their treatment with formalin, sodium azide and slow-freezing while MTT-staining was abolished in all these treated endospores. These results indicate that EvB and TMRE were capable of revealing the morphological integrity of endospores but failed to identify the metabolic inactivation of the endospores after treatment with formalin, sodium azide or slow-freezing. Only MTT was capable of identifying metabolically active endospores and hence their viability and could prove to be of value in standardizing models of infection.