Development and utilization of a web-based application as a robust radiology teaching tool (radstax) for medical student anatomy teaching.

Rationale and Objectives: The primary role of radiology in the preclinical setting is the use of imaging to improve students' understanding of anatomy. Many currently available Web-based anatomy programs include either suboptimal or overwhelming levels of detail for medical students.Our objective was to develop a user-friendly software program that anatomy instructors can completely tailor to match the desired level of detail for their curriculum, meets the unique needs of the first- and the second-year medical students, and is compatible with most Internet browsers and tablets.Materials and Methods: RadStax is a Web-based application developed using free, open-source, ubiquitous software. RadStax was first introduced as an interactive resource for independent study and later incorporated into lectures. First- and second-year medical students were surveyed for quantitative feedback regarding their experience.Results: RadStax was successfully introduced into our medical school curriculum. It allows the creation of learning modules with labeled multiplanar (MPR) image sets, basic anatomic information, and a self-assessment feature. The program received overwhelmingly positive feedback from students. Of 115 students surveyed, 87.0% found it highly effective as a study tool and 85.2% reported high user satisfaction with the program.Conclusions: RadStax is a novel application for instructors wishing to create an atlas of labeled MPR radiologic studies tailored to meet the specific needs their curriculum. Simple and focused, it provides an interactive experience for students similar to the practice of radiologists.This program is a robust anatomy teaching tool that effectively aids in educating the preclinical medical student.

Molecular analysis of the prokaryotic ubiquitin-like protein (Pup) conjugation pathway in Mycobacterium tuberculosis.

Proteins targeted for degradation by the Mycobacterium proteasome are post-translationally tagged with prokaryotic ubiquitin-like protein (Pup), an intrinsically disordered protein of 64 residues. In a process termed 'pupylation', Pup is synthesized with a terminal glutamine, which is deamidated to glutamate by Dop (deamidase of Pup) prior to attachment to substrate lysines by proteasome accessory factor A (PafA). Importantly, PafA was previously shown to be essential to cause lethal infections by Mycobacterium tuberculosis (Mtb) in mice. In this study we show that Dop, like PafA, is required for the full virulence of Mtb. Additionally, we show that Dop is not only involved in the deamidation of Pup, but also needed to maintain wild-type steady state levels of pupylated proteins in Mtb. Finally, using structural models and site-directed mutagenesis our data suggest that Dop and PafA are members of the glutamine synthetase fold family of proteins.

Protection from Alzheimer's-like disease in the mouse by genetic ablation of inducible nitric oxide synthase.

Brains from subjects who have Alzheimer's disease (AD) express inducible nitric oxide synthase (iNOS). We tested the hypothesis that iNOS contributes to AD pathogenesis. Immunoreactive iNOS was detected in brains of mice with AD-like disease resulting from transgenic expression of mutant human beta-amyloid precursor protein (hAPP) and presenilin-1 (hPS1). We bred hAPP-, hPS1-double transgenic mice to be iNOS(+/+) or iNOS(-/-), and compared them with a congenic WT strain. Deficiency of iNOS substantially protected the AD-like mice from premature mortality, cerebral plaque formation, increased beta-amyloid levels, protein tyrosine nitration, astrocytosis, and microgliosis. Thus, iNOS seems to be a major instigator of beta-amyloid deposition and disease progression. Inhibition of iNOS may be a therapeutic option in AD.

Lysis-deficient bacteriophage therapy decreases endotoxin and inflammatory mediator release and improves survival in a murine peritonitis model.

BACKGROUND: Lysis-deficient (LyD) bacteriophages (phages) kill bacteria without endotoxin (Et) release. This may minimize systemic cytokine responses and limit inflammation in bacterial sepsis. We determined the effects of t amber A3 T4 LyD and virulent wild-type (WT) phages on mouse bacterial peritonitis. METHODS: Balb/c mice were injected with B40sul Escherichia coli, treated intraperitoneally with LyD, WT, or a beta-lactam antibiotic [latamoxef sodium (LMOX)], and followed for survival. We measured Et release, tumor necrosis factor (TNF)-alpha and interleukin (IL)-6, as well as bacterial counts and peritoneal exudative cells (PECs) in peritoneal lavage fluid at 6 and 12 hours after infection. RESULTS: LyD mice showed significantly greater survival compared with other groups. Et levels were significantly lower in the LyD mice at 6 and 12 hours after infection. TNF-alpha and IL-6 levels were lower in LyD mice compared with control (untreated) mice at 12 hours. Compared with controls, bacteria counts in peritoneal lavage fluid were lower in all treatment groups (LyD, WT, or LMOX) at 6 and 12 hours. PEC counts were highest in LyD mice at 6 hours but significantly lower than that in WT phage- and LMOX-treated mice at 12 hours. CONCLUSIONS: LyD phage therapy significantly improves survival and attenuates the systemic effects of bacterial sepsis by minimizing Et release and pro-inflammatory mediators in murine bacterial peritonitis. Further studies may find phage therapy useful in treating peritonitis and multidrug-resistant bacterial infections.

Enhanced expression of cyclooxygenase-2 and prostaglandin E2 in response to endotoxin after trauma is dependent on MAPK and NF-kappaB mechanisms.

Macrophage prostaglandin E2 (PGE2) production is important in cellular immune suppression and in affecting the potential development of sepsis after trauma. We hypothesized that macrophage PGE2 production after trauma is regulated by mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-kappaB). Mice were subjected to trauma and splenic macrophages isolated 7 days later. Macrophages from traumatized mice showed increased cyclooxygenase-2 (COX-2) mRNA, protein expression, and PGE2 production compared with controls. Increased phosphorylation of extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38 kinase was observed in macrophages from traumatized mice. Pharmacologic inhibition of MAPK blocked trauma-induced COX-2 expression, and PGE2 production. Trauma macrophages showed increased IkappaBalpha phosphorylation and NF-kappaB binding to DNA. Inhibiting IkappaBalpha blocked trauma-induced NF-kappaB activity, COX-2 expression and PGE2 production. This suggests that trauma-induced PGE2 production is mediated through MAPK and NF-kappaB activation and offers potential for modifying the macrophages' responses following injury.

MyD88 primes macrophages for full-scale activation by interferon-gamma yet mediates few responses to Mycobacterium tuberculosis.

Macrophages are activated from a resting state by a combination of cytokines and microbial products. Microbes are often sensed through Toll-like receptors signaling through MyD88. We used large-scale microarrays in multiple replicate experiments followed by stringent statistical analysis to compare gene expression in wild-type (WT) and MyD88-/- macrophages. We confirmed key results by quantitative reverse transcription polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay. Surprisingly, many genes, such as inducible nitric oxide synthase, IRG-1, IP-10, MIG, RANTES, and interleukin 6 were induced by interferon (IFN)-gamma from 5- to 100-fold less extensively in MyD88-/- macrophages than in WT macrophages. Thus, widespread, full-scale activation of macrophages by IFN-gamma requires MyD88. Analysis of the mechanism revealed that MyD88 mediates a process of self-priming by which resting macrophages produce a low level of tumor necrosis factor. This and other factors lead to basal activation of nuclear factor kappaB, which synergizes with IFN-gamma for gene induction. In contrast, infection by live, virulent Mycobacterium tuberculosis (Mtb) activated macrophages largely through MyD88-independent pathways, and macrophages did not need MyD88 to kill Mtb in vitro. Thus, MyD88 plays a dynamic role in resting macrophages that supports IFN-gamma-dependent activation, whereas macrophages can respond to a complex microbial stimulus, the tubercle bacillus, chiefly by other routes.

Critical role of the carboxyl terminus of proline-rich tyrosine kinase (Pyk2) in the activation of human neutrophils by tumor necrosis factor: separation of signals for the respiratory burst and degranulation.

Transduction of Tat-tagged fusion proteins confirmed a hypothesis based on pharmacologic inhibitors (Fuortes, M., M. Melchior, H. Han, G.J. Lyon, and C. Nathan. 1999. J. Clin. Invest. 104:327-335) that proline-rich tyrosine kinase (Pyk2) plays a critical role in the activation of adherent human neutrophils, and allowed an analysis of individual Pyk2 domains not possible with chemical inhibitors. Acting as a dominant negative, the COOH terminus of Pyk2 fused to a Tat peptide (Tat-CT), but not other regions of Pyk2, specifically inhibited the respiratory burst of cells responding to tumor necrosis factor (TNF), Salmonella, or Listeria, while sparing responses induced by phorbol ester. Tat-CT suppressed TNF-triggered cell spreading and the phosphorylation of endogenous Pyk2 and the associated tyrosine kinase Syk without blocking the ability of neutrophils to degranulate and kill bacteria. Thus, separate signals control the respiratory burst and degranulation, and a normal rate of killing of some bacteria can be sustained by granule products in conjunction with a minimal residual respiratory burst. Inhibition of select inflammatory functions without impairment of antibacterial activity may commend the Pyk2 pathway as a potential target for antiinflammatory therapy.

Membrane lipid organization is critical for human neutrophil polarization.

In response to chemoattractants neutrophils extend an actin-rich pseudopod, which imparts morphological polarity and is required for migration. Even when stimulated by an isotropic bath of chemoattractant, neutrophils exhibit persistent polarization and continued lamellipod formation at the front, suggesting that the cells establish an internal polarity. In this report, we show that perturbing lipid organization by depleting plasma membrane cholesterol levels reversibly inhibits cell polarization and migration. Among other receptor-mediated responses, beta(2) integrin up-regulation was unaffected, and initial calcium mobilization was only partially reduced by cholesterol depletion, indicating that this treatment did not abrogate initial receptor-mediated signal transduction. Interestingly, cholesterol depletion did not prevent initial activation of the GTPase Rac or an initial burst of actin polymerization, but rather it inhibited prolonged activation of Rac and sustained actin polymerization. Collectively, these findings support a model in which the plasma membrane is organized into domains that aid in amplifying the chemoattractant gradient and maintaining cell polarization.

Cholesterol-induced thrombogenicity of the vessel wall: inhibitory effect of fluvastatin.

High cholesterol levels are a known risk factor for coronary events. The molecular links between high serum cholesterol and the increased thrombogenicity of the arterial wall are still matter of investigation. In the present study we investigate the relationship between plasma cholesterol, thrombus formation and TF expression in a atherosclerotic rabbit model. Hypercholesterolemic rabbits showed a pronounced TF staining as well as NF-kappaB activation in the aortic arch. A consistent vessel wall platelet deposition was also observed. Treatment with fluvastatin reduced lipid accumulation, TF overexpression (-60%), NF-kappaB activation, and platelet deposition (-56%). In vitro studies showed that the drug upregulated IkappaB alpha in unstimulated as well as in TNFalpha-stimulated cells and also impaired the TNFalpha-induced Cdc42 prenylation, indicating that fluvastatin interferes with the transcriptional activation of TF gene. These results indicate that the prothrombotic phenotype of arterial wall, associated with elevated serum cholesterol levels, is mediated by TF overexpression. Fluvastatin treatment reduces the prothrombotic tendency by inhibiting TF synthesis.