Electroporation of cDNA/Morpholinos to targeted areas of embryonic CNS in <Emphasis Type="Italic">Xenopus</Emphasis>

Background  

Blastomere injection of mRNA or antisense oligonucleotides has proven effective in analyzing early gene function in Xenopus. However, functional analysis of genes involved in neuronal differentiation and axon pathfinding by this method is often hampered by earlier function of these genes during development. Therefore, fine spatio-temporal control of over-expression or knock-down approaches is required to specifically address the role of a given gene in these processes.     

Bioinformatic discovery of novel bioactive peptides

Short synthetic oligopeptides based on regions of human proteins that encompass functional motifs are versatile reagents for understanding protein signaling and interactions. They can either mimic or inhibit the parent protein's activity and have been used in drug development. Peptide studies typically either derive peptides from a single identified protein or (at the other extreme) screen random combinatorial peptides, often without knowledge of the signaling pathways targeted. Our objective was to determine whether rational bioinformatic design of oligopeptides specifically targeted to potentially signaling-rich juxtamembrane regions could identify modulators of human platelet function. High-throughput in vitro platelet function assays of palmitylated cell-permeable oligopeptides corresponding to these regions identified many agonists and antagonists of platelet function. Many bioactive peptides were from adhesion molecules, including a specific CD226-derived inhibitor of inside-out platelet signaling. Systematic screens of this nature are highly efficient tools for discovering short signaling motifs in molecular signaling pathways.     

Orthosis-Shaped Sandals Are as Efficacious as In-Shoe Orthoses and Better than Flat Sandals for Plantar Heel Pain: A Randomized Control Trial

Objective

To investigate efficacy of a contoured sandal being marketed for plantar heel pain with comparison to a flat flip-flop and contoured in-shoe insert/orthosis.

Method

150 volunteers aged 50 (SD: 12) years with plantar heel pain (>4 weeks) were enrolled after responding to advertisements and eligibility determined by telephone and at first visit. Participants were randomly allocated to receive commercially available contoured sandals (n = 49), flat flip-flops (n = 50) or over the counter, pre-fabricated full-length foot orthotics (n = 51). Primary outcomes were a 15-point Global Rating of Change scale (GROC: 1 = a very great deal worse, 15 = a very great deal better), 13 to 15 representing an improvement and the 20-item Lower Extremity Function Scale (LEFS) on which participants rate 20 common weight bearing activities and activities of daily living on a 5-point scale (0 = extreme difficulty, 4 = no difficulty). Secondary outcomes were worst level of heel pain in the preceding week, and the foot and ankle ability measure. Outcomes were collected blind to allocation. Analyses were done on an intention to treat basis with 12 weeks being the primary outcome time of interest.

Results

The contoured sandal was 68% more likely to report improvement in terms of GROC compared to flat flip-flop. On the LEFS the contoured sandal was 61% more likely than flat flip-flop to report improvement. The secondary outcomes in the main reflected the primary outcomes, and there were no differences between contoured sandal and shoe insert.

Conclusions and Relevance

Physicians can have confidence in supporting a patient''s decision to wear contoured sandals or in-shoe orthoses as one of the first and simple strategies to manage their heel pain.

Trial Registration

The Australian New Zealand Clinical Trials Registry ACTRN12612000463875     

Targeting AML through DR4 with a novel variant of rhTRAIL

Despite progress in the treatment of acute myelogenous leukaemia (AML) the outcome often remains poor. Tumour necrosis factor related apoptosis-inducing ligand (TRAIL) is a promising therapeutic agent in many different types of tumours, but AML cells are relatively insensitive to TRAIL-induced apoptosis. Here we show that TRAIL-induced apoptosis in AML cells is predominantly mediated by death receptor 4 (DR4) and not DR5. Therefore, we constructed a variant of TRAIL (rhTRAIL-C3) that is a strong inducer of DR4-mediated apoptosis. TRAIL-C3 demonstrated much stronger pro-apoptotic activity than wild-type (WT) TRAIL in a panel of AML cell lines as well as in primary AML blasts. The higher pro-apoptotic potential was further enhanced when the TRAIL mutant was used in combination with BMS-345541, a selective inhibitor of inhibitor-κB kinases. It illustrates that combination of this TRAIL variant with chemotherapeutics or other targeted agents can kill AML with high efficacy. This may represent a major advantage over the currently used therapies that have serious toxic side effects. The high efficacy of rhTRAIL-C3 containing therapies may enable the use of lower drug doses to reduce the toxic side effects and improve patient outcome. Our findings suggest that the rational design of TRAIL variants that target DR4 potentiate the death-inducing activity of TRAIL and offer a novel therapeutic strategy for the treatment of AML.     

The long-term impact of the MEMA kwa Vijana adolescent sexual and reproductive health intervention: effect of dose and time since intervention exposure

Background

Despite recent decreases in HIV incidence in many sub-Saharan African countries, there is little evidence that specific behavioural interventions have led to a reduction in HIV among young people. Further and wider-scale decreases in HIV require better understanding of when behaviour change occurs and why. The MEMA kwa Vijana adolescent sexual and reproductive health intervention has been implemented in rural Mwanza, Tanzania since 1999. A long-term evaluation in 2007/8 found that the intervention improved knowledge, attitudes to sex and some reported risk behaviours, but not HIV or HSV2 prevalence. The aim of this paper was to assess the differential impact of the intervention according to gender, age, marital status, number of years of exposure and time since last exposure to the intervention.

Methods

In 2007, a cross-sectional survey was conducted in the 20 trial communities among 13,814 young people (15–30 yrs) who had attended intervention or comparison schools between 1999 and 2002. Outcomes for which the intervention had an impact in 2001 or 2007 were included in this subgroup analysis. Data were analysed using cluster-level methods for stratified cluster-randomised trials, using interaction tests to determine if intervention impact differed by subgroup.

Results

Taking into account multiplicity of testing, concurrence with a priori hypotheses and consistency within the results no strong effect-modifiers emerged. Impact on pregnancy knowledge and reported attitudes to sex increased with years of exposure to high-quality intervention.

Conclusions

The desirable long-term impact of the MEMA kwa Vijana intervention did not vary greatly according to the subgroups examined. This suggests that the intervention can have an impact on a broad cross-section of young people in rural Mwanza.

Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00248469","term_id":"NCT00248469"}}NCT00248469     

Evaluation and identification of markers of damage in mushrooms (Agaricus bisporus) postharvest using a GC/MS metabolic profiling approach

The aim of this research was to use the gas chromatography-mass spectrometry (GC/MS) profiling method coupled with chemometric tools to profile mechanically damaged and undamaged mushrooms during storage and to identify specific metabolites that may be used as markers of damage. Mushrooms grown under controlled conditions were bruise damaged by vibration to simulate damage during normal transportation. Three damage levels were evaluated; undamaged, damaged for 20 min and damaged for 40 min and two time levels studied; day zero and day one after storage at 4oC. Applying this technique over 100 metabolites were identified, quantified and compiled in a library. Random forest classification models were used to predict damage in mushrooms producing models with error rates of >10% using cap and stipe tissue. Fatty acids were found to be the most important group of metabolites for predicting damage in mushrooms. PLS models were also developed producing models with low error rates. With a view to exploring biosynthetic links between metabolites, a pairwise correlation analysis was performed for all polar and non-polar metabolites. The appearance of high correlation between linoleic acid and pentadecanoic acid in the non-polar phase of damaged mushrooms indicated the switching on of a metabolic pathway when a mushroom is damaged.     

Rapid increase in prevalence of male circumcision in rural Tanzania in the absence of a promotional campaign

Objectives

To estimate the prevalence of circumcision among young men in rural Mwanza, North-Western Tanzania, and document trends in circumcision prevalence over time. To investigate associations of circumcision with socio-demographic characteristics, reported sexual behaviours and sexually transmitted infections (STIs).

Design

A cross-sectional survey in communities which had previously participated in a cluster-randomized trial of an adolescent sexual health intervention that did not include male circumcision in 20 rural communities.

Methods

In 2007/08, 7300 young men (age 16–23 years) were interviewed and examined by a clinician. The prevalence of circumcision by age was compared with data collected during the trial in 1998–2002. Odds ratios (OR) and 95% confidence intervals (CI) for the association of circumcision with socio-demographic characteristics, reported sexual behaviours and with HIV and other STIs were estimated using multivariable conditional logistic regression.

Results

The prevalence of male circumcision was 40.6%, and age-specific prevalence had more than doubled since 2001/2002. Circumcised men reported less risky sexual behaviours, being more likely to report having ever used a condom (adjusted OR = 2.62, 95%CI:2.32–2.95). Men circumcised before sexual debut were at reduced risk of being HIV seropositive compared with non-circumcised men (adjusted OR = 0.50, 95%CI:0.25–0.97), and also had reduced risks of HSV-2 infection and genital ulcer syndrome in the past 12 months compared with non-circumcised men.

Conclusions

There was a steep increase in circumcision prevalence between 2001/02 and 2007/08 in the absence of a promotional campaign. Circumcised men reported safer sexual practices than non-circumcised men and had lower prevalence of HIV and HSV-2 infection.     

Time-domain representation of ventricular-arterial coupling as a windkessel and wave system

The differences in shape between central aortic pressure (P(Ao)) and flow waveforms have never been explained satisfactorily in that the assumed explanation (substantial reflected waves during diastole) remains controversial. As an alternative to the widely accepted frequency-domain model of arterial hemodynamics, we propose a functional, time-domain, arterial model that combines a blood conducting system and a reservoir (i.e., Frank's hydraulic integrator, the windkessel). In 15 anesthetized dogs, we measured P(Ao), flows, and dimensions and calculated windkessel pressure (P(Wk)) and volume (V(Wk)). We found that P(Wk) is proportional to thoracic aortic volume and that the volume of the thoracic aorta comprises 45.1 +/- 2.0% (mean +/- SE) of the total V(Wk). When we subtracted P(Wk) from P(Ao), we found that the difference (excess pressure) was proportional to aortic flow, thus resolving the differences between P(Ao) and flow waveforms and implying that reflected waves were minimal. We suggest that P(Ao) is the instantaneous summation of a time-varying reservoir pressure (i.e., P(Wk)) and the effects of (primarily) forward-traveling waves in this animal model.     

Monitoring of complex industrial bioprocesses for metabolite concentrations using modern spectroscopies and machine learning: application to gibberellic acid production

Two rapid vibrational spectroscopic approaches (diffuse reflectance-absorbance Fourier transform infrared [FT-IR] and dispersive Raman spectroscopy), and one mass spectrometric method based on in vacuo Curie-point pyrolysis (PyMS), were investigated in this study. A diverse range of unprocessed, industrial fed-batch fermentation broths containing the fungus Gibberella fujikuroi producing the natural product gibberellic acid, were analyzed directly without a priori chromatographic separation. Partial least squares regression (PLSR) and artificial neural networks (ANNs) were applied to all of the information-rich spectra obtained by each of the methods to obtain quantitative information on the gibberellic acid titer. These estimates were of good precision, and the typical root-mean-square error for predictions of concentrations in an independent test set was <10% over a very wide titer range from 0 to 4925 ppm. However, although PLSR and ANNs are very powerful techniques they are often described as "black box" methods because the information they use to construct the calibration model is largely inaccessible. Therefore, a variety of novel evolutionary computation-based methods, including genetic algorithms and genetic programming, were used to produce models that allowed the determination of those input variables that contributed most to the models formed, and to observe that these models were predominantly based on the concentration of gibberellic acid itself. This is the first time that these three modern analytical spectroscopies, in combination with advanced chemometric data analysis, have been compared for their ability to analyze a real commercial bioprocess. The results demonstrate unequivocally that all methods provide very rapid and accurate estimates of the progress of industrial fermentations, and indicate that, of the three methods studied, Raman spectroscopy is the ideal bioprocess monitoring method because it can be adapted for on-line analysis.