Multisite Phosphorylation of the Guanine Nucleotide Exchange Factor Cdc24 during Yeast Cell Polarization

BACKGROUND:Cell polarization is essential for processes such as cell migration and asymmetric cell division. A common regulator of cell polarization in most eukaryotic cells is the conserved Rho GTPase, Cdc42. In budding yeast, Cdc42 is activated by a single guanine nucleotide exchange factor, Cdc24. The mechanistic details of Cdc24 activation at the onset of yeast cell polarization are unclear. Previous studies have suggested an important role for phosphorylation of Cdc24, which may regulate activity or function of the protein, representing a key step in the symmetry breaking process. METHODOLOGY/PRINCIPAL FINDINGS:Here, we directly ask whether multisite phosphorylation of Cdc24 plays a role in its regulation. We identify through mass spectrometry analysis over thirty putative in vivo phosphorylation sites. We first focus on sites matching consensus sequences for cyclin-dependent and p21-activated kinases, two kinase families that have been previously shown to phosphorylate Cdc24. Through site-directed mutagenesis, yeast genetics, and light and fluorescence microscopy, we show that nonphosphorylatable mutations of these consensus sites do not lead to any detectable consequences on growth rate, morphology, kinetics of polarization, or localization of the mutant protein. We do, however, observe a change in the mobility shift of mutant Cdc24 proteins on SDS-PAGE, suggesting that we have indeed perturbed its phosphorylation. Finally, we show that mutation of all identified phosphorylation sites does not cause observable defects in growth rate or morphology. CONCLUSIONS/SIGNIFICANCE:We conclude that lack of phosphorylation on Cdc24 has no overt functional consequences in budding yeast. Yeast cell polarization may be more tightly regulated by inactivation of Cdc42 by GTPase activating proteins or by alternative methods of Cdc24 regulation, such as conformational changes or oligomerization

Compensating control participants when the intervention is of significant value: experience in Guatemala, India, Peru and Rwanda

The Household Air Pollution Intervention Network (HAPIN) trial is a randomised controlled trial in Guatemala, India, Peru and Rwanda to assess the health impact of a clean cooking intervention in households using solid biomass for cooking. The HAPIN intervention—a liquefied petroleum gas (LPG) stove and 18-month supply of LPG—has significant value in these communities, irrespective of potential health benefits. For control households, it was necessary to develop a compensation strategy that would be comparable across four settings and would address concerns about differential loss to follow-up, fairness and potential effects on household economics. Each site developed slightly different, contextually appropriate compensation packages by combining a set of uniform principles with local community input. In Guatemala, control compensation consists of coupons equivalent to the LPG stove’s value that can be redeemed for the participant’s choice of household items, which could include an LPG stove. In Peru, control households receive several small items during the trial, plus the intervention stove and 1 month of fuel at the trial’s conclusion. Rwandan participants are given small items during the trial and a choice of a solar kit, LPG stove and four fuel refills, or cash equivalent at the end. India is the only setting in which control participants receive the intervention (LPG stove and 18 months of fuel) at the trial’s end while also being compensated for their time during the trial, in accordance with local ethics committee requirements. The approaches presented here could inform compensation strategy development in future multi-country trials

The Cardiac Transcription Network Modulated by Gata4, Mef2a, Nkx2.5, Srf, Histone Modifications, and MicroRNAs

The transcriptome, as the pool of all transcribed elements in a given cell, is regulated by the interaction between different molecular levels, involving epigenetic, transcriptional, and post-transcriptional mechanisms. However, many previous studies investigated each of these levels individually, and little is known about their interdependency. We present a systems biology study integrating mRNA profiles with DNA–binding events of key cardiac transcription factors (Gata4, Mef2a, Nkx2.5, and Srf), activating histone modifications (H3ac, H4ac, H3K4me2, and H3K4me3), and microRNA profiles obtained in wild-type and RNAi–mediated knockdown. Finally, we confirmed conclusions primarily obtained in cardiomyocyte cell culture in a time-course of cardiac maturation in mouse around birth. We provide insights into the combinatorial regulation by cardiac transcription factors and show that they can partially compensate each other's function. Genes regulated by multiple transcription factors are less likely differentially expressed in RNAi knockdown of one respective factor. In addition to the analysis of the individual transcription factors, we found that histone 3 acetylation correlates with Srf- and Gata4-dependent gene expression and is complementarily reduced in cardiac Srf knockdown. Further, we found that altered microRNA expression in Srf knockdown potentially explains up to 45% of indirect mRNA targets. Considering all three levels of regulation, we present an Srf-centered transcription network providing on a single-gene level insights into the regulatory circuits establishing respective mRNA profiles. In summary, we show the combinatorial contribution of four DNA–binding transcription factors in regulating the cardiac transcriptome and provide evidence that histone modifications and microRNAs modulate their functional consequence. This opens a new perspective to understand heart development and the complexity cardiovascular disorders

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Genome-wide associations for birth weight and correlations with adult disease

Birth weight (BW) has been shown to be influenced by both fetal and maternal factors and in observational studies is reproducibly associated with future risk of adult metabolic diseases including type 2 diabetes (T2D) and cardiovascular disease1. These life-course associations have often been attributed to the impact of an adverse early life environment. Here, we performed a multi-ancestry genome-wide association study (GWAS) meta-analysis of BW in 153,781 individuals, identifying 60 loci where fetal genotype was associated with BW (P < 5 × 10−8). Overall, approximately 15% of variance in BW was captured by assays of fetal genetic variation. Using genetic association alone, we found strong inverse genetic correlations between BW and systolic blood pressure (Rg = −0.22, P = 5.5 × 10−13), T2D (Rg = −0.27, P = 1.1 × 10−6) and coronary artery disease (Rg = −0.30, P = 6.5 × 10−9). In addition, using large -cohort datasets, we demonstrated that genetic factors were the major contributor to the negative covariance between BW and future cardiometabolic risk. Pathway analyses indicated that the protein products of genes within BW-associated regions were enriched for diverse processes including insulin signalling, glucose homeostasis, glycogen biosynthesis and chromatin remodelling. There was also enrichment of associations with BW in known imprinted regions (P = 1.9 × 10−4). We demonstrate that life-course associations between early growth phenotypes and adult cardiometabolic disease are in part the result of shared genetic effects and identify some of the pathways through which these causal genetic effects are mediated

A principal component meta-analysis on multiple anthropometric traits identifies novel loci for body shape

Large consortia have revealed hundreds of genetic loci associated with anthropometric traits, one trait at a time. We examined whether genetic variants affect body shape as a composite phenotype that is represented by a combination of anthropometric traits. We developed an approach that calculates averaged PCs (AvPCs) representing body shape derived from six anthropometric traits (body mass index, height, weight, waist and hip circumference, waist-to-hip ratio). The first four AvPCs explain >99% of the variability, are heritable, and associate with cardiometabolic outcomes. We performed genome-wide association analyses for each body shape composite phenotype across 65 studies and meta-analysed summary statistics. We identify six novel loci: LEMD2 and CD47 for AvPC1, RPS6KA5/C14orf159 and GANAB for AvPC3, and ARL15 and ANP32 for AvPC4. Our findings highlight the value of using multiple traits to define complex phenotypes for discovery, which are not captured by single-trait analyses, and may shed light onto new pathways.Peer reviewe

Sex-stratified Genome-wide Association Studies Including 270,000 Individuals Show Sexual Dimorphism in Genetic Loci for Anthropometric Traits

Peer reviewe

Xenotransplantation of decellularized pig heart valves-Regulatory aspects in Europe

BACKGROUND: The lack of human donors for allotransplantation forces the development of other strategies to circumvent the existing organ shortage documented on the waiting lists. Here, xenotransplantation offers a suitable option since the genetic modification of animals has become an established method that allows the generation of animals as donors of cells, tissues, and organs with reduced antigenicity. METHODS: Focus is given on the generation of decellularized matrix scaffolds, for example, for valve transplantation and/or repair, that have the potential being fully assimilated by the recipient as they are no longer a mechanical implant with risk of calcification and related failure. RESULTS: This new class of products is transplants that will be regulated either as medical devices or as cell-based medicinal products, that is, advanced therapy medicinal products, according to the regulations in the European Union. CONCLUSIONS: In this review, we compile relevant regulatory aspects and point out the possibilities of how these products for human use may be regulated in the future

Test-Enabled Architecture for IoT Service Creation and Provisioning

The information generated from the Internet of Things (IoT) potentially enables a better understanding of the physical world for humans and supports creation of ambient intelligence for a wide range of applications in different domains. A semantics-enabled service layer is a promising approach to facilitate seamless access and management of the information from the large, distributed and heterogeneous sources. This paper presents the efforts of the IoT.est project towards developing a framework for service creation and testing in an IoT environment. The architecture design extends the existing IoT reference architecture and enables a test-driven, semantics-based management of the entire service lifecycle. The validation of the architecture is shown though a dynamic test case generation and execution scenario