Genetic and Pharmacological YAP Activation Induces Proliferation and Improves Survival in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Cardiomyocyte loss following myocardial infarction cannot be addressed with current clinical therapies. Cell therapy with induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) is a potential approach to replace cardiomyocyte loss. However, engraftment rates in pre-clinical studies have been low, highlighting a need to refine current iPSC-CM technology. In this study, we demonstrated that inducing Yes-associated protein (YAP) by genetic and pharmacological approaches resulted in increased iPSC-CM proliferation and reduced apoptosis in response to oxidative stress. Interestingly, iPSC-CM maturation was differently affected by each strategy, with genetic activation of YAP resulting in a more immature cardiomyocyte-like phenotype not witnessed upon pharmacological YAP activation. Overall, we conclude that YAP activation in iPSC-CMs enhances cell survival and proliferative capacity. Therefore, strategies targeting YAP, or its upstream regulator the Hippo signalling pathway, could potentially be used to improve the efficacy of iPSC-CM technology for use as a future regenerative therapy in myocardial infarction

RNA extraction from self-assembling peptide hydrogels to allow qPCR analysis of encapsulated cells

Self-assembling peptide hydrogels offer a novel 3-dimensional platform for many applications in cell culture and tissue engineering but are not compatible with current methods of RNA isolation; owing to interactions between RNA and the biomaterial. This study investigates the use of two techniques based on two different basic extraction principles: solution-based extraction and direct solid-state binding of RNA respectively, to extract RNA from cells encapsulated in four β-sheet forming self-assembling peptide hydrogels with varying net positive charge. RNA-peptide fibril interactions, rather than RNA-peptide molecular complexing, were found to interfere with the extraction process resulting in low yields. A column-based approach relying on RNA-specific binding was shown to be more suited to extracting RNA with higher purity from these peptide hydrogels owing to its reliance on strong specific RNA binding interactions which compete directly with RNA-peptide fibril interactions. In order to reduce the amount of fibrils present and improve RNA yields a broad spectrum enzyme solution—pronase—was used to partially digest the hydrogels before RNA extraction. This pre-treatment was shown to significantly increase the yield of RNA extracted, allowing downstream RT-qPCR to be performed

Understanding community health worker employment preferences in Malang district, Indonesia, using a discrete choice experiment

Background Community health workers (CHWs) play a critical role in supporting health systems, and in improving accessibility to primary healthcare. In many settings CHW programmes do not have formalised employment models and face issues of high attrition and poor performance. This study aims to determine the employment preferences of CHWs in Malang district, Indonesia, to inform policy interventions. Methods A discrete choice experiment was conducted with 471 CHWs across 28 villages. Attributes relevant to CHW employment were identified through a multistage process including literature review, focus group discussions and expert consultation. Respondents' choices were analysed with a mixed multinomial logit model and latent class analyses. Results Five attributes were identified: (1) supervision; (2) training; (3) monthly financial benefit; (4) recognition; and (5) employment structure. The most important influence on choice of job was a low monthly financial benefit (US∼2) (β=0.53, 95% CI=0.43 to 0.63), followed by recognition in the form of a performance feedback report (β=0.13, 95% CI=0.07 to 0.20). A large monthly financial benefit (US∼20) was most unappealing to respondents (β=-0.13, 95% CI=-0.23 to -0.03). Latent class analysis identified two groups of CHWs who differed in their willingness to accept either job presented and preferences over specific attributes. Preferences diverged based on respondent characteristics including experience, hours' worked per week and income. Conclusion CHWs in Malang district, Indonesia, favour a small monthly financial benefit which likely reflects the unique cultural values underpinning the programme and a desire for remuneration that is commensurate with the limited number of hours worked. CHWs also desire enhanced methods of performance feedback and greater structure around training and their rights and responsibilities. Fulfilling these conditions may become increasingly important should CHWs work longer hours

Relation of exaggerated cytokine responses of CF airway epithelial cells to PAO1 adherence

In many model systems, cystic fibrosis (CF) phenotype airway epithelial cells in culture respond to P. aeruginosa with greater interleukin (IL)-8 and IL-6 secretion than matched controls. In order to test whether this excess inflammatory response results from the reported increased adherence of P. aeruginosa to the CF cells, we compared the inflammatory response of matched pairs of CF and non CF airway epithelial cell lines to the binding of GFP-PAO1, a strain of pseudomonas labeled with green fluorescent protein. There was no clear relation between GFP-PAO1 binding and cytokine production in response to PAO1. Treatment with exogenous aGM1 resulted in greater GFP-PAO1 binding to the normal phenotype compared to CF phenotype cells, but cytokine production remained greater from the CF cell lines. When cells were treated with neuraminidase, PAO1 adherence was equalized between CF and nonCF phenotype cell lines, but IL-8 production in response to inflammatory stimuli was still greater in CF phenotype cells. The polarized cell lines 16HBEo-Sense (normal phenotype) and Antisense (CF phenotype) cells were used to test the effect of disrupting tight junctions, which allows access of PAO1 to basolateral binding sites in both cell lines. IL-8 production increased from CF, but not normal, cells. These data indicate that increased bacterial binding to CF phenotype cells cannot by itself account for excess cytokine production in CF airway epithelial cells, encourage investigation of alternative hypotheses, and signal caution for therapeutic strategies proposed for CF that include disruption of tight junctions in the face of pseudomonas infection

The Plasma Membrane Calcium ATPases and Their Role as Major New Players in Human Disease.

The Ca2+ extrusion function of the four mammalian isoforms of the plasma membrane calcium ATPases (PMCAs) is well established. There is also ever-increasing detail known of their roles in global and local Ca2+ homeostasis and intracellular Ca2+ signaling in a wide variety of cell types and tissues. It is becoming clear that the spatiotemporal patterns of expression of the PMCAs and the fact that their abundances and relative expression levels vary from cell type to cell type both reflect and impact on their specific functions in these cells. Over recent years it has become increasingly apparent that these genes have potentially significant roles in human health and disease, with PMCAs1-4 being associated with cardiovascular diseases, deafness, autism, ataxia, adenoma, and malarial resistance. This review will bring together evidence of the variety of tissue-specific functions of PMCAs and will highlight the roles these genes play in regulating normal physiological functions and the considerable impact the genes have on human disease