The development and implementation of a layperson trauma first responder course in La Paz, Bolivia: A pilot study.

BACKGROUND: Ninety percent of nearly five million annual global injury deaths occur in low- and middle-income countries (LMICs), where prehospital care systems are frequently rudimentary or nonexistent. The World Health Organization considers layperson first-responders as essential for emergency medical services in low-resource settings lacking more formalized systems. This study sought to develop and implement a layperson trauma first responder course (TFRC) in Bolivia. MATERIALS AND METHODS: In March and April 2013 nine sessions of the eight-hour TFRC were held in La Paz, Bolivia. The course charged a nominal fee, and was led by an American surgeon and medical student. The TFRC built upon existing models with local stakeholder input, and included both didactic and practical components. Participants completed a baseline survey, and pre and posttests. The primary outcome was test performance, with secondary outcomes including demographic sub-group test score analyses and exam question validation. Data were assessed using nonparametric and psychometric methods RESULTS: One hundred fifty-nine individuals met study inclusion criteria. Participant median age was 28 (IQR 24, 36), 49.1% were male, 59.1% worked in a medical field, most had secondary (35.2%) or university (56.0%) level educations, and 67.3% had prior first aid training. Median test scores improved after course completion (48% vs. 76%, p <0.001), along with skill confidence (4 vs. 4.5, p <0.001). Most questions had appropriate item difficulty indices, point bi-serial correlation coefficients, and positive Pretest Posttest Difference Indices. Cronbach alpha coefficients for pre and posttest scores were 0.72 and 0.78, respectively. CONCLUSIONS: This study presents data from the first offering of an original TFRC for laypeople in Bolivia. Increased participant knowledge and skill confidence after course completion, and acceptable overall psychometric test properties, indicate this model is valid and effective. Future aims include TFRC revision, and enrollment of more layperson first responders to increase population-level impacts.

Structural and biochemical analyses of alanine racemase from the multidrug-resistant Clostridium difficile strain 630.

Clostridium difficile, a Gram-positive, spore-forming anaerobic bacterium, is the leading cause of infectious diarrhea among hospitalized patients. C. difficile is frequently associated with antibiotic treatment, and causes diseases ranging from antibiotic-associated diarrhea to life-threatening pseudomembranous colitis. The severity of C. difficile infections is exacerbated by the emergence of hypervirulent and multidrug-resistant strains, which are difficult to treat and are often associated with increased mortality rates. Alanine racemase (Alr) is a pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes the reversible racemization of L- and D-alanine. Since D-alanine is an essential component of the bacterial cell-wall peptidoglycan, and there are no known Alr homologs in humans, this enzyme is being tested as an antibiotic target. Cycloserine is an antibiotic that inhibits Alr. In this study, the catalytic properties and crystal structures of recombinant Alr from the virulent and multidrug-resistant C. difficile strain 630 are presented. Three crystal structures of C. difficile Alr (CdAlr), corresponding to the complex with PLP, the complex with cycloserine and a K271T mutant form of the enzyme with bound PLP, are presented. The structures are prototypical Alr homodimers with two active sites in which the cofactor PLP and cycloserine are localized. Kinetic analyses reveal that the K271T mutant CdAlr has the highest catalytic constants reported to date for any Alr. Additional studies are needed to identify the basis for the high catalytic activity. The structural and activity data presented are first steps towards using CdAlr for the development of structure-based therapeutics for C. difficile infections.

Structure of 2-keto-3-deoxy-D-manno-octulosonate-8-phosphate synthase from Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a major cause of opportunistic infection and is resistant to most antibiotics. As part of efforts to generate much-needed new antibiotics, structural studies of enzymes that are critical for the virulence of P. aeruginosa but are absent in mammals have been initiated. 2-Keto-3-deoxy-D-manno-octulosonate-8-phosphate synthase (KDO8Ps), also known as 2-dehydro-3-deoxyphosphooctonate aldolase, is vital for the survival and virulence of P. aeruginosa. This enzyme catalyzes a key step in the synthesis of the lipopolysaccharide (LPS) of most Gram-negative bacteria: the condensation reaction between phosphoenolpyruvate (PEP) and arabinose 5-phosphate to produce 2-keto-3-deoxy-D-manno-octulosonate-8-phosphate (KDO8P). This step is vital for the proper synthesis and assembly of LPS and the survival of P. aeruginosa. Here, the recombinant expression, purification and crystal structure of KDO8Ps from P. aeruginosa are presented. Orthorhombic crystals were obtained by vapor diffusion in sitting drops in the presence of 1 mM phosphoenlpyruvate. The structure reveals the prototypical α/ß TIM-barrel structure expected from this family of enzymes and contains a tetramer in the asymmetric unit.

Vasopressin increases expression of UT-A1, UT-A3, and ER chaperone GRP78 in the renal medulla of mice with a urinary concentrating defect.

Activation of V2 receptors (V2R) during antidiuresis increases the permeability of the inner medullary collecting duct to urea and water. Extracellular osmolality is elevated as the concentrating capacity of the kidney increases. Osmolality is known to contribute to the regulation of collecting duct water (aquaporin-2; AQP2) and urea transporter (UT-A1, UT-A3) regulation. AQP1KO mice are a concentrating mechanism knockout, a defect attributed to the loss of high interstitial osmolality. A V2R-specific agonist, deamino-8-D-arginine vasopressin (dDAVP), was infused into wild-type and AQP1KO mice for 7 days. UT-A1 mRNA and protein abundance were significantly increased in the medullas of wild-type and AQP1KO mice following dDAVP infusion. The mRNA and protein abundance of UT-A3, the basolateral urea transporter, was significantly increased by dDAVP in both wild-type and AQP1KO mice. Semiquantitative immunoblots revealed that dDAVP infusion induced a significant increase in the medullary expression of the endoplasmic reticulum (ER) chaperone GRP78. Immunofluorescence studies demonstrated that GRP78 expression colocalized with AQP2 in principal cells of the papillary tip of the renal medulla. Using immunohistochemistry and immunogold electron microscopy, we demonstrate that vasopressin induced a marked apical targeting of GRP78 in medullary principal cells. Urea-sensitive genes, GADD153 and ATF4 (components of the ER stress pathway), were significantly increased in AQP1KO mice by dDAVP infusion. These findings strongly support an important role of vasopressin in the activation of an ER stress response in renal collecting duct cells, in addition to its role in activating an increase in UT-A1 and UT-A3 abundance.

Production of hyperpolarized [1,4-13C2]malate from [1,4-13C2]fumarate is a marker of cell necrosis and treatment response in tumors.

Dynamic nuclear polarization of (13)C-labeled cell substrates has been shown to massively increase their sensitivity to detection in NMR experiments. The sensitivity gain is sufficiently large that if these polarized molecules are injected intravenously, their spatial distribution and subsequent conversion into other cell metabolites can be imaged. We have used this method to image the conversion of fumarate to malate in a murine lymphoma tumor in vivo after i.v. injection of hyperpolarized [1,4-(13)C(2)]fumarate. In isolated lymphoma cells, the rate of labeled malate production was unaffected by coadministration of succinate, which competes with fumarate for transport into the cell. There was, however, a correlation with the percentage of cells that had lost plasma membrane integrity, suggesting that the production of labeled malate from fumarate is a sensitive marker of cellular necrosis. Twenty-four hours after treating implanted lymphoma tumors with etoposide, at which point there were significant levels of tumor cell necrosis, there was a 2.4-fold increase in hyperpolarized [1,4-(13)C(2)]malate production compared with the untreated tumors. Therefore, the formation of hyperpolarized (13)C-labeled malate from [1,4-(13)C(2)]fumarate appears to be a sensitive marker of tumor cell death in vivo and could be used to detect the early response of tumors to treatment. Given that fumarate is an endogenous molecule, this technique has the potential to be used clinically.