The Stroke Riskometer(TM) App: validation of a data collection tool and stroke risk predictor.

BACKGROUND: The greatest potential to reduce the burden of stroke is by primary prevention of first-ever stroke, which constitutes three quarters of all stroke. In addition to population-wide prevention strategies (the 'mass' approach), the 'high risk' approach aims to identify individuals at risk of stroke and to modify their risk factors, and risk, accordingly. Current methods of assessing and modifying stroke risk are difficult to access and implement by the general population, amongst whom most future strokes will arise. To help reduce the burden of stroke on individuals and the population a new app, the Stroke Riskometer(TM) , has been developed. We aim to explore the validity of the app for predicting the risk of stroke compared with current best methods. METHODS: 752 stroke outcomes from a sample of 9501 individuals across three countries (New Zealand, Russia and the Netherlands) were utilized to investigate the performance of a novel stroke risk prediction tool algorithm (Stroke Riskometer(TM) ) compared with two established stroke risk score prediction algorithms (Framingham Stroke Risk Score [FSRS] and QStroke). We calculated the receiver operating characteristics (ROC) curves and area under the ROC curve (AUROC) with 95% confidence intervals, Harrels C-statistic and D-statistics for measure of discrimination, R(2) statistics to indicate level of variability accounted for by each prediction algorithm, the Hosmer-Lemeshow statistic for calibration, and the sensitivity and specificity of each algorithm. RESULTS: The Stroke Riskometer(TM) performed well against the FSRS five-year AUROC for both males (FSRS = 75.0% (95% CI 72.3%-77.6%), Stroke Riskometer(TM) = 74.0(95% CI 71.3%-76.7%) and females [FSRS = 70.3% (95% CI 67.9%-72.8%, Stroke Riskometer(TM) = 71.5% (95% CI 69.0%-73.9%)], and better than QStroke [males - 59.7% (95% CI 57.3%-62.0%) and comparable to females = 71.1% (95% CI 69.0%-73.1%)]. Discriminative ability of all algorithms was low (C-statistic ranging from 0.51-0.56, D-statistic ranging from 0.01-0.12). Hosmer-Lemeshow illustrated that all of the predicted risk scores were not well calibrated with the observed event data (P < 0.006). CONCLUSIONS: The Stroke Riskometer(TM) is comparable in performance for stroke prediction with FSRS and QStroke. All three algorithms performed equally poorly in predicting stroke events. The Stroke Riskometer(TM) will be continually developed and validated to address the need to improve the current stroke risk scoring systems to more accurately predict stroke, particularly by identifying robust ethnic/race ethnicity group and country specific risk factors.

NLRP3 inflammasome is a target for development of broad-spectrum anti-infective drugs.

We describe the molecular mode of action and pharmacodynamics of a new molecular entity (NME) that induces the NLRP3 inflammasome-mediated innate immune response. This innate response reduces the pathogen load in an experimentally induced methicillin-resistant Staphylococcos aureus infection, enhances survival in an experimentally induced Gram-negative bacteremia, and overrides the escape mechanism of an obligate intracellular pathogen, viz. Chlamydia pneumoniae. Furthermore, the NME is more effective than standard-of-care antibiotic therapy in a clinically established multifactorial bacterial infection. Analysis of transcriptional regulation of inflammasome signaling genes and innate/adaptive immune genes revealed consistent and significant host changes responsible for the improved outcomes in these infections. These studies pave the way for the development of first-in-class drugs that enhance inflammasome-mediated pathogen clearance and identify the NLRP3 inflammasome as a drug target to address the global problem of emerging new infectious diseases and the reemergence of old diseases in an antibiotic-resistant form.

Rapid sporulation of Bacillus anthracis in a high iron, glucose-free medium.

Spores are the infectious form of Bacillus anthracis (BA), causing cutaneous, inhalation and gastrointestinal anthrax. Because of the possible use of BA spores in a bioterrorism attack, there is considerable interest in studying spore biology. In the laboratory, however, it takes a number of days to prepare spores. Standard sporulation protocols, such as the use of 'PA broth', allow sporulation of BA to occur in 3 to 5 days. Another method employs growth of BA on plates in the dark for several days until they have efficiently sporulated. In efforts to determine the effect of iron on gene expression in BA, we grew BA Sterne strain 7702 in a minimal defined medium (CDM; Koppisch et al., 2005) with various concentrations of iron and glucose. As part of our initial observations, we monitored BA sporulation in CDM via light microscopy. In glucose-free CDM containing 1.5mM Fe(NO(3))(3) (CDM-Fe), >95% of the BA sporulated by 30 h; a far shorter time period than expected. We pursued this observation and we further characterized spores derived from PA and CDM-Fe media. Purified spores derived from PA or CDM-Fe had similar morphologies when viewed by light or electron microscopy, and were equally resistant to harsh conditions including heat (65 degrees C), ice and fresh 30% H(2)O(2). Spore viability in long term cold storage in water was similar for the two spore preparations. Extracted spore coat proteins were evaluated by SDS-PAGE and silver staining, which revealed distinct protein profiles for PA and CDM-Fe spore coat extracts. ELISA assays were done to compare the interaction of the two spore preparations with rabbit antiserum raised against UV-killed Sterne strain 7702 spores prepared in PA medium. Spores from both media reacted identically with this antiserum. Finally, the interaction and fate of spores incubated with macrophages in vitro was very similar. In summary, BA spores induced in CDM-Fe or in PA medium are similar by several criteria, but show distinct extractable coat proteins. CDM-Fe liquid medium can be used for rapid production of BA spores, and could save considerable time in spore research studies.

1-Peptidyl-2-arachidonoyl-3-stearoyl-sn-glyceride: an immunologically active lipopeptide from goat serum (Capra hircus) is an endogenous damage-associated molecular pattern.

Experiments were undertaken to isolate a component of the serum of goat (Capra hircus) that is effective at mediating an innate immune response. This report describes the isolation and structure elucidation of 1-(N-acetyl-ALYDKGYTSKEQKDCVGI)-2-arachidonoyl-3-stearoyl glyceride (1) and its immunomodulatory activity. A dose-response relationship for inflammatory cytokine and chemokine production and release from human fibroblasts incubated with nanomolar concentrations of 1 was shown. Moreover, the membrane transport role of the diacylglycerol moiety in 1 is demonstrated with nanomolar quantities of the transfected N-acetyl peptide moiety of 1 also inducing inflammatory cytokine and chemokine production and release. The apparent EC99 for 1 was 3 ng/mL (1 nM). The likely biological role for naturally occurring 1 as a damage-associated molecular pattern is postulated.

Array lead zirconate titanate/glass piezoelectric microcantilevers for real-time detection of Bacillus anthracis with 10 spores/ml sensitivity and 1/1000 selectivity in bacterial mixtures.

An array of three identical piezoelectric microcantilever sensors (PEMSs) consisting of a lead zirconate titanate layer bonded to a glass layer was fabricated and examined for simultaneous, in situ, real-time, all-electrical detection of Bacillus anthracis (BA) spores in an aqueous suspension using the first longitudinal extension mode of resonance. With anti-BA antibody immobilized on the sensor surfaces all three PEMS exhibited identical BA detection resonance frequency shifts at all tested concentrations, 10-10(7) spores/ml with a standard deviation of less than 10%. The detection concentration limit of 10 spores/ml was about two orders of magnitude lower than would be permitted by flexural peaks. In blinded-sample testing, the array PEMS detected BA in three samples containing BA: (1) 3.3x10(3) spores/ml, (2) a mixture of 3.3x10(3) spores/ml and 3.3x10(5) S. aureus (SA) and P. aeruginosa (PA) per ml, and (3) a mixture of 3.3x10(3) spores/ml with 3.3x10(6) SA+PA/ml. There was no response to a sample containing only 3.3x10(6) SA+PA/ml. These results illustrate the sensitivity, specificity, reusability, and reliability of array PEMS for in situ, real-time detection of BA spores.

Label-free flow-enhanced specific detection of Bacillus anthracis using a piezoelectric microcantilever sensor.

Differentiation between species of similar biological structure is of critical importance in biosensing applications. Here, we report specific detection of Bacillus anthracis (BA) spores from that of close relatives, such as B. thuringiensis (BT), B. cereus (BC), and B. subtilis (BS) by varying the flow speed of the sampling liquid over the surface of a piezoelectric microcantilever sensor (PEMS). Spore binding to the anti-BA spore IgG coated PEMS surface is determined by monitoring the resonance frequency change in the sensor's impedance vs. frequency spectrum. Flow increases the resonance frequency shift at lower flow rates until the impingement force from the flow overcomes the binding strength of the antigen and decreases the resonance frequency shift at higher flow rates. We showed that the change from increasing to decreasing resonance frequency shift occurred at a lower fluid flow speed for BT, BC, and BS spores than for BA spores. This trend reduces the cross reactivity ratio of BC, BS, and BT to the anti-BA spore IgG immobilized PEMS from around 0.4 at low flow velocities to less than 0.05 at 3.8 mm s(-1). This cross reactivity ratio of 0.05 was essentially negligible considering the experimental uncertainty. The use of the same flow that is used for detection to further distinguish the specific binding (BA to anti-BA spore antibody) from nonspecific binding (BT, BC, and BS to anti-BA spore antibody) is unique and has great potential in the detection of general biological species.

Modeling the interactions of herbal drugs to beta-ketoacyl ACP synthase of Mycobacterium tuberculosis H37Rv.

The present paper reports a bio-computational study carried out with the aim of understanding the binding mode of anti-TB herbal ligands onto the homology modeled structure of fatty acid synthase of Mycobacterium tuberculosis (M.tb) H37Rv. Sequence alignment of beta-ketoacyl ACP synthase (KAS) domain of the protein with other related KAS sequences of PDB database revealed high degree of sequence variation. However, the catalytic triad comprising of CHH (cys150-his279-his320) was found to be conserved in the KAS sequence of M.tb H37Rv. The tertiary structure of this protein predicted using genetic algorithm operator in the MODELLER package appeared to give a satisfactory structure for the purpose of studying ligand and substrate binding pockets on the protein. PDB templates complexed with ligands (citric acid and lauric acid) were used for model building. Docking studies carried out with different herbal ligands suggest that, aloe-emodin and nimbin are the best herbal candidates to replace the synthetic drugs 'thiolactomycin/cerulenin'.