p21-Activated kinase-1 and its role in integrated regulation of cardiac contractility

We review here a novel concept in the regulation of cardiac contractility involving variations in the activity of the multifunctional enzyme, p21-activated kinase 1 (Pak1), a member of a family of proteins in the small G protein-signaling pathway that is activated by Cdc42 and Rac1. There is a large body of evidence from studies in noncardiac tissue that Pak1 activity is key in regulation of a number of cellular functions, including cytoskeletal dynamics, cell motility, growth, and proliferation. Although of significant potential impact, the role of Pak1 in regulation of the heart has been investigated in only a few laboratories. In this review, we discuss the structure of Pak1 and its sites of posttranslational modification and molecular interactions. We assemble an overview of the current data on Pak1 signaling in noncardiac tissues relative to similar signaling pathways in the heart, and we identify potential roles of Pak1 in cardiac regulation. Finally, we discuss the current state of Pak1 research in the heart in regard to regulation of contractility through functional myofilament and Ca(2+)-flux modification. An important aspect of this regulation is the modulation of kinase and phosphatase activity. We have focused on Pak1 regulation of protein phosphatase 2A (PP2A), which is abundant in cardiac muscle, thereby mediating dephosphorylation of sarcomeric proteins and sensitizing the myofilaments to Ca(2+). We present a model for Pak1 signaling that provides a mechanism for specifically affecting cardiac cellular processes in which regulation of protein phosphorylation states by PP2A dephosphorylation predominates.     

Analysis of oocyte physiology to improve cryopreservation procedures

In contrast to the preimplantation mammalian embryo, it has been notoriously difficult to cryopreserve the metaphase II oocyte. The ability to store oocytes successfully at -196 degrees C has numerous practical and financial advantages, together with ethical considerations, and will positively impact animal breeding programs and assisted conception in the human. Differences in membrane permeability and in physiology are two main reasons why successful oocyte cryopreservation has remained elusive. It is proposed, therefore, that rather than relying on technologies already established for the preimplantation embryo, the development of cryopreservation techniques suitable for the mammalian oocyte needs to take into account the idiosyncratic physiology of this cell. Analysis of intracellular calcium, for example, has revealed that exposure to conventional permeating cryoprotectants, such as propanediol, ethylene glycol and DMSO, all independently result in an increase in calcium, which in turn has the potential to initiate oocyte activation, culminating in zona hardening. Quantification of the metabolome and proteome of the oocyte has revealed that whereas slow freezing has a dramatic effect on cell physiology, vitrification appears to have limited effect. This is plausibly achieved by the limited exposure to cryoprotectants. Analysis of meiotic spindle dynamics and embryo development following IVF, also indicate that vitrification is less traumatic than slow freezing, and therefore has the greatest potential for successful oocyte cryopreservation.     

The efficacy of highway wildlife collision mitigation in preventing elk mortality in central Ontario

In the Burwash area of north-central Ontario, Canada, expansion of the Trans-Canada highway from 2 to 4 lanes was accompanied by installation of a range of wildlife collision-mitigation infrastructure (e.g., exclusion fencing, underpasses). To assess the overall effectiveness of these measures, we monitored the spatial distribution and mortality rates of elk (Cervus canadensis) prior to and following highway expansion, distinguished by season (winter, snowfree) and corridor-type (highway, railway). We measured herd-level risk by the proportion of positions falling within 200-m railway and highway buffer zones using Bayesian methods. Spatial analysis confirmed that there was a distinct northward shift in the winter distribution of elk following construction, situating the elk past the north end of the exclusion fence. This increased the herd's exposure to highway traffic by 3.6 times (proportion of points before = 0.0041 ± 0.002 [SE], after = 0.0147 ± 0.003, P = 0.005), and resulted in a more than 2-fold increase in elk road mortality from 0.6 elk/yr/20 km during 8 years prior to implementation to 1.5 elk/yr/20 km during 8 years after implementation. Exposure to railways remained unchanged and consistently higher than highway exposure regardless of season (e.g., post-mitigation, winter proportion of points = 0.0453 ± 0.005), matched by consistently high mortality counts (proportion of points before = 6.4 elk/yr/20 km, after = 6.6 elk/yr/20 km). Our results demonstrate that while wildlife-vehicle collision mitigation is generally beneficial to wildlife and humans, failure to account for the local characteristics of wildlife populations can lead to suboptimal mitigation designs that reduce their effectiveness and lead to unintended wildlife impacts.     

Passage of Wolbachia pipientis through Mutant Drosophila melanogaster Induces Phenotypic and Genomic Changes

Wolbachia pipientis is a nearly ubiquitous, maternally transmitted bacterium that infects the germ line of insect hosts. Estimates are that Wolbachia infects 40 to 60% of insect species on the planet, making it one of the most prevalent infections on Earth. However, we know surprisingly little about the molecular mechanisms used by Wolbachia to infect its hosts. We passaged Wolbachia through normally restrictive Drosophila melanogaster hosts, bottlenecking Wolbachia through stochastic segregation while simultaneously selecting for mutants that could recolonize these previously restrictive hosts. Here, we show that Wolbachia alters its behavior when passaged through heterozygous mutant flies. After only three generations, Wolbachia was able to colonize the previously restrictive hosts at control titers. Additionally, the Wolbachia organisms passaged through heterozygous mutant D. melanogaster alter their pattern of tissue-specific Wsp protein production, suggesting a behavioral response to the host genotype. Using whole-genome resequencing, we identified the mutations accumulated by these lineages of Wolbachia and confirmed the existence and persistence of the mutations through clone library Sanger sequencing. Our results suggest that Wolbachia can quickly adapt to new host contexts, with genomic mutants arising after only two generations.     

Rapid phylogenetic analysis of large samples of recombinant bacterial whole genome sequences using Gubbins

The emergence of new sequencing technologies has facilitated the use of bacterial whole genome alignments for evolutionary studies and outbreak analyses. These datasets, of increasing size, often include examples of multiple different mechanisms of horizontal sequence transfer resulting in substantial alterations to prokaryotic chromosomes. The impact of these processes demands rapid and flexible approaches able to account for recombination when reconstructing isolates’ recent diversification. Gubbins is an iterative algorithm that uses spatial scanning statistics to identify loci containing elevated densities of base substitutions suggestive of horizontal sequence transfer while concurrently constructing a maximum likelihood phylogeny based on the putative point mutations outside these regions of high sequence diversity. Simulations demonstrate the algorithm generates highly accurate reconstructions under realistically parameterized models of bacterial evolution, and achieves convergence in only a few hours on alignments of hundreds of bacterial genome sequences. Gubbins is appropriate for reconstructing the recent evolutionary history of a variety of haploid genotype alignments, as it makes no assumptions about the underlying mechanism of recombination. The software is freely available for download at github.com/sanger-pathogens/Gubbins, implemented in Python and C and supported on Linux and Mac OS X.     

AdmixKJump: identifying population structure in recently diverged groups

Motivation

Correctly modeling population structure is important for understanding recent evolution and for association studies in humans. While pre-existing knowledge of population history can be used to specify expected levels of subdivision, objective metrics to detect population structure are important and may even be preferable for identifying groups in some situations. One such metric for genomic scale data is implemented in the cross-validation procedure of the program ADMIXTURE, but it has not been evaluated on recently diverged and potentially cryptic levels of population structure. Here, I develop a new method, AdmixKJump, and test both metrics under this scenario.

Findings

I show that AdmixKJump is more sensitive to recent population divisions compared to the cross-validation metric using both realistic simulations, as well as 1000 Genomes Project European genomic data. With two populations of 50 individuals each, AdmixKJump is able to detect two populations with 100% accuracy that split at least 10KYA, whereas cross-validation obtains this 100% level at 14KYA. I also show that AdmixKJump is more accurate with fewer samples per population. Furthermore, in contrast to the cross-validation approach, AdmixKJump is able to detect the population split between the Finnish and Tuscan populations of the 1000 Genomes Project.

Conclusion

AdmixKJump has more power to detect the number of populations in a cohort of samples with smaller sample sizes and shorter divergence times.

Availability

A java implementation can be found at https://sites.google.com/site/igsevolgenomicslab/home/downloads     

Decellularized Cartilage May Be a Chondroinductive Material for Osteochondral Tissue Engineering

Extracellular matrix (ECM)-based materials are attractive for regenerative medicine in their ability to potentially aid in stem cell recruitment, infiltration, and differentiation without added biological factors. In musculoskeletal tissue engineering, demineralized bone matrix is widely used, but recently cartilage matrix has been attracting attention as a potentially chondroinductive material. The aim of this study was thus to establish a chemical decellularization method for use with articular cartilage to quantify removal of cells and analyze the cartilage biochemical content at various stages during the decellularization process, which included a physically devitalization step. To study the cellular response to the cartilage matrix, rat bone marrow-derived mesenchymal stem cells (rBMSCs) were cultured in cell pellets containing cells only (control), chondrogenic differentiation medium (TGF-β), chemically decellularized cartilage particles (DCC), or physically devitalized cartilage particles (DVC). The chemical decellularization process removed the vast majority of DNA and about half of the glycosaminoglycans (GAG) within the matrix, but had no significant effect on the amount of hydroxyproline. Most notably, the DCC group significantly outperformed TGF-β in chondroinduction of rBMSCs, with collagen II gene expression an order of magnitude or more higher. While DVC did not exhibit a chondrogenic response to the extent that DCC did, DVC had a greater down regulation of collagen I, collagen X and Runx2. A new protocol has been introduced for cartilage devitalization and decellularization in the current study, with evidence of chondroinductivity. Such bioactivity along with providing the ‘raw material’ building blocks of regenerating cartilage may suggest a promising role for DCC in biomaterials that rely on recruiting endogenous cell recruitment and differentiation for cartilage regeneration.     

A Cluster Randomised Trial on the Impact of Integrating Early Infant HIV Diagnosis with the Expanded Programme on Immunization on Immunization and HIV Testing Rates in Rural Health Facilities in Southern Zambia

Background

We assessed the integration of early infant HIV diagnosis with the expanded programme for immunization in a rural Zambian setting with the aim of determining whether infant and postpartum maternal HIV testing rates would increase without harming immunization uptake.

Methods

In an unblinded, location stratified, cluster randomised controlled trial, 60 facilities in Zambia’s Southern Province were equally allocated to a control group, Simple Intervention group that received a sensitization meeting and the resupply of HIV testing commodities in the event of a stock-out, and a Comprehensive Intervention group that received the Simple Intervention as well as on-site operational support to facilitate the integration of HIV testing services with EPI.

Findings

The average change in number of first dose diphtheria, pertussis, and tetanus vaccine (DPT1) provided per month, per facility was approximately 0.86 doses higher [90% confidence interval (CI) -1.40, 3.12] in Comprehensive Intervention facilities compared to the combined average change in the Simple Intervention and control facilities. The interventions resulted in a 16.6% [90% CI: -7%, 46%, P-value = 0.26] and 10% [90% CI: -10%, 36%, P-value = 0.43] greater change in average monthly infant DBS testing compared to control for the Simple and Comprehensive facilities respectively. We also found 15.76 (90% CI: 7.12, 24.41, P-value < 0.01) and 10.93 (90% CI: 1.52, 20.33, P-value = 0.06) additional total maternal re-tests over baseline for the Simple and Comprehensive Facilities respectively.

Conclusions

This study provides strong evidence to support Zambia’s policy of integration of HIV testing and EPI services. Actions in line with the interventions, including HIV testing material supply reinforcement, can increase HIV testing rates without harming immunization uptake. In response, Zambia’s Ministry of Health issued a memo to remind health facilities to provide HIV testing at under-five clinics and to include under-five HIV testing as part of district performance assessments.

Trial Registration

ClinicalTrials.gov Registration Number: {"type":"clinical-trial","attrs":{"text":"NCT02479659","term_id":"NCT02479659"}}NCT02479659     

A Phase I,Open-Label Trial,Evaluating the Safety and Immunogenicity of Candidate Tuberculosis Vaccines AERAS-402 and MVA85A,Administered by Prime-Boost Regime in BCG-Vaccinated Healthy Adults

Background

MVA85A and AERAS-402 are two clinically advanced viral vectored TB vaccine candidates expressing Mycobacterium tuberculosis antigens designed to boost BCG-induced immunity. Clinical trials with candidate malaria vaccines have demonstrated that adenoviral vector based priming immunisation, followed by MVA vector boost, induced high levels of immunity. We present the safety and immunogenicity results of the first clinical trial to evaluate this immunisation strategy in TB.

Methods

In this phase 1, open-label trial, 40 healthy previously BCG-vaccinated participants were enrolled into three treatment groups and vaccinated with 1 or 2 doses of AERAS-402 followed by MVA85A; or 3 doses of AERAS-402.

Results

Most related adverse events (AEs) were mild and there were no vaccine related serious AEs. Boosting AERAS-402 with MVA85A significantly increased Ag85A-specific T-cell responses from day of vaccination. Two priming doses of AERAS-402 followed by MVA85A boost, resulted in a significantly higher AUC post-peak Ag85A response compared to three doses of AERAS-402 and historical data with MVA85A vaccination alone. The frequency of CD8+ T-cells producing IFN-γ, TNF-α and IL-2 was highest in the group receiving two priming doses of AERAS-402 followed by MVA85A.

Conclusions

Vaccination with AERAS-402 followed by MVA85A was safe and increased the durability of antigen specific T-cell responses and the frequency and polyfunctionality of CD8+ T-cells, which may be important in protection against TB. Further clinical trials with adenoviral prime-MVA85A boost regimens are merited to optimise vaccination intervals, dose and route of immunisation and to evaluate this strategy in the target population in TB high burden countries.

Trial Registration

ClinicalTrials.gov NCT01683773.