Refolding by High Pressure of a Toxin Containing Seven Disulfide Bonds: Bothropstoxin-1 from Bothrops jararacussu

Aggregation is a serious obstacle for recovery of biologically active heterologous proteins from inclusion bodies (IBs) produced by recombinant bacteria. E. coli transformed with a vector containing the cDNA for Bothropstoxin-1 (BthTx-1) expressed the recombinant product as IBs. In order to obtain the native toxin, insoluble and aggregated protein was refolded using high hydrostatic pressure (HHP). IBs were dissolved and refolded (2 kbar, 16 h), and the effects of protein concentration, as well as changes in ratio and concentration of oxido-shuffling reagents, guanidine hydrochloride (GdnHCl), and pH in the refolding buffer, were assayed. A 32% yield (7.6 mg per liter of bacterial culture) in refolding of the native BthTx-1 was obtained using optimal conditions of the refolding buffer (Tris–HCl buffer, pH 7.5, containing 3 mM of a 2:3 ratio of GSH/GSSG, and 1 M GdnHCl). Scanning electron microscopy (SEM) showed that that disaggregation of part of IBs particles occurred upon compression and that the morphology of the remaining IBs, spherical particles, was not substantially altered. Dose-dependent cytotoxic activity of high-pressure refolded BthTx-1 was shown in C2C12 muscle cells

Engaging with community researchers for exposure science: lessons learned from a pesticide biomonitoring study

A major challenge in biomonitoring studies with members of the general public is ensuring their continued involvement throughout the necessary length of the research. The paper presents evidence on the use of community researchers, recruited from local study areas, as a mechanism for ensuring effective recruitment and retention of farmer and resident participants for a pesticides biomonitoring study. The evidence presented suggests that community researchers' abilities to build and sustain trusting relationships with participants enhanced the rigour of the study as a result of their on-the-ground responsiveness and flexibility resulting in data collection beyond targets expected

The Waiting Time for Inter-Country Spread of Pandemic Influenza

BACKGROUND: The time delay between the start of an influenza pandemic and its subsequent initiation in other countries is highly relevant to preparedness planning. We quantify the distribution of this random time in terms of the separate components of this delay, and assess how the delay may be extended by non-pharmaceutical interventions. METHODS AND FINDINGS: The model constructed for this time delay accounts for: (i) epidemic growth in the source region, (ii) the delay until an infected individual from the source region seeks to travel to an at-risk country, (iii) the chance that infected travelers are detected by screening at exit and entry borders, (iv) the possibility of in-flight transmission, (v) the chance that an infected arrival might not initiate an epidemic, and (vi) the delay until infection in the at-risk country gathers momentum. Efforts that reduce the disease reproduction number in the source region below two and severe travel restrictions are most effective for delaying a local epidemic, and under favourable circumstances, could add several months to the delay. On the other hand, the model predicts that border screening for symptomatic infection, wearing a protective mask during travel, promoting early presentation of cases arising among arriving passengers and moderate reduction in travel volumes increase the delay only by a matter of days or weeks. Elevated in-flight transmission reduces the delay only minimally. CONCLUSIONS: The delay until an epidemic of pandemic strain influenza is imported into an at-risk country is largely determined by the course of the epidemic in the source region and the number of travelers attempting to enter the at-risk country, and is little affected by non-pharmaceutical interventions targeting these travelers. Short of preventing international travel altogether, eradicating a nascent pandemic in the source region appears to be the only reliable method of preventing country-to-country spread of a pandemic strain of influenza

Phylogenetic Distribution of Fungal Sterols

BACKGROUND: Ergosterol has been considered the "fungal sterol" for almost 125 years; however, additional sterol data superimposed on a recent molecular phylogeny of kingdom Fungi reveals a different and more complex situation. METHODOLOGY/PRINCIPAL FINDINGS: The interpretation of sterol distribution data in a modern phylogenetic context indicates that there is a clear trend from cholesterol and other Delta(5) sterols in the earliest diverging fungal species to ergosterol in later diverging fungi. There are, however, deviations from this pattern in certain clades. Sterols of the diverse zoosporic and zygosporic forms exhibit structural diversity with cholesterol and 24-ethyl -Delta(5) sterols in zoosporic taxa, and 24-methyl sterols in zygosporic fungi. For example, each of the three monophyletic lineages of zygosporic fungi has distinctive major sterols, ergosterol in Mucorales, 22-dihydroergosterol in Dimargaritales, Harpellales, and Kickxellales (DHK clade), and 24-methyl cholesterol in Entomophthorales. Other departures from ergosterol as the dominant sterol include: 24-ethyl cholesterol in Glomeromycota, 24-ethyl cholest-7-enol and 24-ethyl-cholesta-7,24(28)-dienol in rust fungi, brassicasterol in Taphrinales and hypogeous pezizalean species, and cholesterol in Pneumocystis. CONCLUSIONS/SIGNIFICANCE: Five dominant end products of sterol biosynthesis (cholesterol, ergosterol, 24-methyl cholesterol, 24-ethyl cholesterol, brassicasterol), and intermediates in the formation of 24-ethyl cholesterol, are major sterols in 175 species of Fungi. Although most fungi in the most speciose clades have ergosterol as a major sterol, sterols are more varied than currently understood, and their distribution supports certain clades of Fungi in current fungal phylogenies. In addition to the intellectual importance of understanding evolution of sterol synthesis in fungi, there is practical importance because certain antifungal drugs (e.g., azoles) target reactions in the synthesis of ergosterol. These findings also invalidate use of ergosterol as an indicator of biomass of certain fungal taxa (e.g., Glomeromycota). Data from this study are available from the Assembling the Fungal Tree of Life (AFTOL) Structural and Biochemical Database: http://aftol.umn.edu

The Pochonia chlamydosporia Serine Protease Gene vcp1 Is Subject to Regulation by Carbon, Nitrogen and pH: Implications for Nematode Biocontrol

The alkaline serine protease VCP1 of the fungus Pochonia chlamydosporia belongs to a family of subtilisin-like enzymes that are involved in infection of nematode and insect hosts. It is involved early in the infection process, removing the outer proteinaceous vitelline membrane of nematode eggs. Little is known about the regulation of this gene, even though an understanding of how nutrients and other factors affect its expression is critical for ensuring its efficacy as a biocontrol agent. This paper provides new information on the regulation of vcp1 expression. Sequence analysis of the upstream regulatory region of this gene in 30 isolates revealed that it was highly conserved and contained sequence motifs characteristic of genes that are subject to carbon, nitrogen and pH-regulation. Expression studies, monitoring enzyme activity and mRNA, confirmed that these factors affect VCP1 production. As expected, glucose reduced VCP1 expression and for a few hours so did ammonium chloride. Surprisingly, however, by 24 h VCP1 levels were increased in the presence of ammonium chloride for most isolates. Ambient pH also regulated VCP1 expression, with most isolates producing more VCP1 under alkaline conditions. There were some differences in the response of one isolate with a distinctive upstream sequence including a variant regulatory-motif profile. Cryo-scanning electron microscopy studies indicated that the presence of nematode eggs stimulates VCP1 production by P. chlamydosporia, but only where the two are in close contact. Overall, the results indicate that readily-metabolisable carbon sources and unfavourable pH in the rhizosphere/egg-mass environment may compromise nematode parasitism by P. chlamydosporia. However, contrary to previous indications using other nematophagous and entomopathogenic fungi, ammonium nitrate (e.g. from fertilizers) may enhance biocontrol potential in some circumstances

The transcriptional response of Caenorhabditis elegans to ivermectin exposure identifies novel genes involved in the response to reduced food intake

We have examined the transcriptional response of Caenorhabditis elegans following exposure to the anthelmintic drug ivermectin (IVM) using whole genome microarrays and real-time QPCR. Our original aim was to identify candidate molecules involved in IVM metabolism and/or excretion. For this reason the IVM tolerant strain, DA1316, was used to minimise transcriptomic changes related to the phenotype of drug exposure. However, unlike equivalent work with benzimidazole drugs, very few of the induced genes were members of xenobiotic metabolising enzyme families. Instead, the transcriptional response was dominated by genes associated with fat mobilization and fatty acid metabolism including catalase, esterase, and fatty acid CoA synthetase genes. This is consistent with the reduction in pharyngeal pumping, and consequential reduction in food intake, upon exposure of DA1316 worms to IVM. Genes with the highest fold change in response to IVM exposure, cyp-37B1, mtl-1 and scl-2, were comparably up-regulated in response to short–term food withdrawal (4 hr) independent of IVM exposure, and GFP reporter constructs confirm their expression in tissues associated with fat storage (intestine and hypodermis). These experiments have serendipitously identified novel genes involved in an early response of C. elegans to reduced food intake and may provide insight into similar processes in higher organisms

Acceptability of Condom Promotion and Distribution Among 10-19 Year-Old Adolescents in Mpwapwa and Mbeya Rural Districts, Tanzania.

\ud The HIV/AIDS pandemic remains a leading challenge for global health. Although condoms are acknowledged for their key role on preventing HIV transmission, low and inappropriate use of condoms persists in Tanzania and elsewhere in Africa. This study assesses factors affecting acceptability of condom promotion and distribution among adolescents in Mpwapwa and Mbeya rural districts of Tanzania. Data were collected in 2011 as part of a larger cross-sectional survey on condom use among 10-19 year-olds in Mpwapwa and Mbeya rural districts of Tanzania using a structured questionnaire. Associations between acceptability of condom promotion and distribution and each of the explanatory variables were tested using Chi Square. Multivariate logistic regression model was used to examine independent predictors of the acceptability of condom promotion and distribution using STATA (11) statistical software at 5% significance level. Mean age of the 1,327 adolescent participants (50.5% being males) was 13.5 years (SD = 1.4). Acceptance of condom promotion and distribution was found among 37% (35% in Mpwapwa and 39% in Mbeya rural) of the adolescents. Being sexually active and aged 15-19 was the strongest predictor of the acceptability of condom promotion and distribution (OR = 7.78, 95% CI 4.65-12.99). Others were; not agreeing that a condom is effective in preventing transmissions of STIs including HIV (OR = 0.34, 95% CI 0.20-0.56), being a resident of Mbeya rural district (OR = 1.67, 95% CI 1.28-2.19), feeling comfortable being seen by parents/guardians holding/buying condoms (OR = 2.20, 95% CI 1.40-3.46) and living with a guardian (OR = 1.48, 95% CI 1.08-2.04). Acceptability of condom promotion and distribution among adolescents in Mpwapwa and Mbeya rural is low. Effect of sexual activity on the acceptability of condom promotion and distribution is age-dependent and was the strongest. Feeling comfortable being seen by parents/guardians buying or holding condoms, perceived ability of condoms to offer protection against HIV/AIDS infections, district of residence and living arrangements also offered significant predictive effect. Knowledge of these factors is vital in designing successful and sustainable condom promotion and distribution programs in Tanzania.\u

B Cells Regulate Neutrophilia during Mycobacterium tuberculosis Infection and BCG Vaccination by Modulating the Interleukin-17 Response

We have previously demonstrated that B cells can shape the immune response to Mycobacterium tuberculosis, including the level of neutrophil infiltration and granulomatous inflammation at the site of infection. The present study examined the mechanisms by which B cells regulate the host neutrophilic response upon exposure to mycobacteria and how neutrophilia may influence vaccine efficacy. To address these questions, a murine aerosol infection tuberculosis (TB) model and an intradermal (ID) ear BCG immunization mouse model, involving both the μMT strain and B cell-depleted C57BL/6 mice, were used. IL (interleukin)-17 neutralization and neutrophil depletion experiments using these systems provide evidence that B cells can regulate neutrophilia by modulating the IL-17 response during M. tuberculosis infection and BCG immunization. Exuberant neutrophilia at the site of immunization in B cell-deficient mice adversely affects dendritic cell (DC) migration to the draining lymph nodes and attenuates the development of the vaccine-induced Th1 response. The results suggest that B cells are required for the development of optimal protective anti-TB immunity upon BCG vaccination by regulating the IL-17/neutrophilic response. Administration of sera derived from M. tuberculosis-infected C57BL/6 wild-type mice reverses the lung neutrophilia phenotype in tuberculous μMT mice. Together, these observations provide insight into the mechanisms by which B cells and humoral immunity modulate vaccine-induced Th1 response and regulate neutrophila during M. tuberculosis infection and BCG immunization. © 2013 Kozakiewicz et al

Sequential biventricular pacing improves regional contractility, longitudinal function and dyssynchrony in patients with heart failure and prolonged QRS

<p>Abstract</p> <p>Aims</p> <p>Biventricular pacing (BiP) is an effective treatment in systolic heart failure (HF) patients with prolonged QRS. However, approximately 35% of the patients receiving BiP are classified as non-responders. The aim of this study is to evaluate the acute effects of VV-optimization on systolic heart function.</p> <p>Methods</p> <p>Twenty-one HF patients aged 72 (46-88) years, QRS 154 (120-190) ms, were studied with echocardiography, Tissue Doppler Imaging (TDI) and 3D-echo the first day after receiving a BiP device. TDI was performed; during simultaneous pacing (LV-lead pacing 4 ms before the RV-lead) and during sequential pacing (LV 20 and 40 ms before RV and RV 20 and 40 ms before LV-lead pacing). Systolic heart function was studied by tissue tracking (TT) for longitudinal function and systolic maximal velocity (SMV) for regional contractility and signs of dyssynchrony assessed by time-delays standard deviation of aortic valve opening to SMV, AVO-SMV/SD and tissue synchronization imaging (TSI).</p> <p>Results</p> <p>The TT mean value preoperatively was 4,2 ± 1,5 and increased at simultaneous pacing to 5,0 ± 1,2 mm (p < 0,05), and at best VV-interval to 5,4 ± 1,2 (p < 0,001). Simultaneous pacing achieved better TT distance compared with preoperative in 16 patients (76%). However, it was still higher after VV-optimization in 12 patients 57%. Corresponding figures for SMV were 3,0 ± 0,7, 3,5 ± 0,8 (p < 0,01), and 3,6 ± 0,8 (p < 0,001). Also dyssynchrony improved.</p> <p>Conclusions</p> <p>VV-optimization in the acute phase improves systolic heart function more than simultaneous BiP pacing. Long-term effects should be evaluated in prospective randomized trials.</p

Thermal Image Scanning for Influenza Border Screening: Results of an Airport Screening Study

Background: Infrared thermal image scanners (ITIS) appear an attractive option for the mass screening of travellers for influenza, but there are no published data on their performance in airports. Methods: ITIS was used to measure cutaneous temperature in 1275 airline travellers who had agreed to tympanic temperature measurement and respiratory sampling. The prediction by ITIS of tympanic temperature (37.8uC and 37.5uC) and of influenza infection was assessed using Receiver Operating Characteristic (ROC) curves and estimated sensitivity, specificity and positive predictive value (PPV). Findings: Using front of face ITIS for prediction of tympanic temperature $37.8uC, the area under the ROC curve was 0.86 (95$37.8uC at screening (95%CI 0 % to 12%); three had no influenza symptoms. Conclusion: ITIS performed moderately well in detecting fever but in this study, during a seasonal epidemic of predominantly influenza type B, the proportion of influenza-infected travellers who were febrile was low and ITIS were no