Constitutive Gs activation using a single-construct tetracycline-inducible expression system in embryonic stem cells and mice

Abstract Introduction The controlled expression of many genes, including G-protein coupled receptors (GPCRs), is important for delineating gene functions in complex model systems. Binary systems for inducible regulation of transgene expression are widely used in mice. One system is the tTA/TRE expression system, composed of a tetracycline-dependent DNA binding factor and a separate tetracycline operon. However, the requirement for two separate transgenes (one for each tTA or TRE component) makes this system less amenable to models requiring directed cell targeting, increases the risk of multiple transgene integration sites, and requires extensive screening for appropriately-functioning clones. Methods We developed a single, polycistronic tetracycline-inducible expression platform to control the expression of multiple cistrons in mammalian cells. This platform has three basic constructs: regulator, responder, and destination vectors. The modular platform is compatible with both the TetOff (tTA) and TetOn (rtTA) systems. The modular Gateway recombineering-compatible components facilitate rapidly generating vectors to genetically modify mammalian cells. We apply this system to use the elongation factor 1α (EF1α) promoter to drive doxycycline-regulated expression of both the fluorescent marker mCherry and an engineered Gs-coupled GPCR "Rs1" separated by a 2A ribosomal skip site. Results We show that our combined expression construct drives expression of both the mCherry and Rs1 transgenes in a doxycycline-dependent manner. We successfully target the expression construct into the Rosa26 locus of mouse embryonic stem (ES) cells. Rs1 expression in mouse ES cells increases cAMP accumulation via both basal and ligand-induced Gs mechanisms and is associated with increased embryoid body size. Heterozygous mice carrying the Rs1 expression construct showed normal growth and weight, and developed small increases in bone formation that could be observed in the calvaria. Conclusions Our results demonstrate the feasibility of a single-vector strategy that combines both the tTA and TRE tetracycline-regulated components for use in cells and mouse models. Although the EF1α promoter is useful for driving expression in pluripotent cells, a single copy of the EF1α promoter did not drive high levels of mCherry and Rs1 expression in the differentiated tissues of adult mice. These findings indicate that promoter selection is an important factor when developing transgene expression models

MicroRNA Regulation of Cell Lineages in Mouse and Human Embryonic Stem Cells

SummaryCell fate decisions of pluripotent embryonic stem (ES) cells are dictated by activation and repression of lineage-specific genes. Numerous signaling and transcriptional networks progressively narrow and specify the potential of ES cells. Whether specific microRNAs help refine and limit gene expression and, thereby, could be used to manipulate ES cell differentiation has largely been unexplored. Here, we show that two serum response factor (SRF)-dependent muscle-specific microRNAs, miR-1 and miR-133, promote mesoderm formation from ES cells but have opposing functions during further differentiation into cardiac muscle progenitors. Furthermore, miR-1 and miR-133 were potent repressors of nonmuscle gene expression and cell fate during mouse and human ES cell differentiation. miR-1's effects were in part mediated by translational repression of the Notch ligand Delta-like 1 (Dll-1). Our findings indicate that muscle-specific miRNAs reinforce the silencing of nonmuscle genes during cell lineage commitment and suggest that miRNAs may have general utility in regulating cell-fate decisions from pluripotent ES cells

L-Edge Spectroscopy of Dilute, Radiation-Sensitive Systems Using a Transition-Edge-Sensor Array

We present X-ray absorption spectroscopy and resonant inelastic X-ray scattering (RIXS) measurements on the iron L-edge of 0.5 mM aqueous ferricyanide. These measurements demonstrate the ability of high-throughput transition-edge-sensor (TES) spectrometers to access the rich soft X-ray (100-2000eV) spectroscopy regime for dilute and radiation-sensitive samples. Our low-concentration data are in agreement with high-concentration measurements recorded by conventional grating-based spectrometers. These results show that soft X-ray RIXS spectroscopy acquired by high-throughput TES spectrometers can be used to study the local electronic structure of dilute metal-centered complexes relevant to biology, chemistry and catalysis. In particular, TES spectrometers have a unique ability to characterize frozen solutions of radiation- and temperature-sensitive samples.Comment: 19 pages, 4 figure

Genetic Surveillance Detects Both Clonal and Epidemic Transmission of Malaria following Enhanced Intervention in Senegal

Using parasite genotyping tools, we screened patients with mild uncomplicated malaria seeking treatment at a clinic in Thiès, Senegal, from 2006 to 2011. We identified a growing frequency of infections caused by genetically identical parasite strains, coincident with increased deployment of malaria control interventions and decreased malaria deaths. Parasite genotypes in some cases persisted clonally across dry seasons. The increase in frequency of genetically identical parasite strains corresponded with decrease in the probability of multiple infections. Further, these observations support evidence of both clonal and epidemic population structures. These data provide the first evidence of a temporal correlation between the appearance of identical parasite types and increased malaria control efforts in Africa, which here included distribution of insecticide treated nets (ITNs), use of rapid diagnostic tests (RDTs) for malaria detection, and deployment of artemisinin combination therapy (ACT). Our results imply that genetic surveillance can be used to evaluate the effectiveness of disease control strategies and assist a rational global malaria eradication campaign.Human Evolutionary BiologyOrganismic and Evolutionary Biolog

Sequence-Based Association and Selection Scans Identify Drug Resistance Loci in the Plasmodium Falciparum Malaria Parasite

Through rapid genetic adaptation and natural selection, the Plasmodium falciparum parasite—the deadliest of those that cause malaria—is able to develop resistance to antimalarial drugs, thwarting present efforts to control it. Genome-wide association studies (GWAS) provide a critical hypothesis-generating tool for understanding how this occurs. However, in P. falciparum, the limited amount of linkage disequilibrium hinders the power of traditional array-based GWAS. Here, we demonstrate the feasibility and power improvements gained by using whole-genome sequencing for association studies. We analyzed data from 45 Senegalese parasites and identified genetic changes associated with the parasites’ in vitro response to 12 different antimalarials. To further increase statistical power, we adapted a common test for natural selection, XP-EHH (cross-population extended haplotype homozygosity), and used it to identify genomic regions associated with resistance to drugs. Using this sequence-based approach and the combination of association and selection-based tests, we detected several loci associated with drug resistance. These loci included the previously known signals at pfcrt, dhfr, and pfmdr1, as well as many genes not previously implicated in drug-resistance roles, including genes in the ubiquitination pathway. Based on the success of the analysis presented in this study, and on the demonstrated shortcomings of array-based approaches, we argue for a complete transition to sequence-based GWAS for small, low linkage-disequilibrium genomes like that of P. falciparum.Molecular and Cellular BiologyOrganismic and Evolutionary Biolog

The Atacama Cosmology Telescope: Two-Season ACTPol Spectra and Parameters

We present the temperature and polarization angular power spectra measured by the Atacama Cosmology Telescope Polarimeter (ACTPol). We analyze night-time data collected during 2013-14 using two detector arrays at 149 GHz, from 548 deg$^2$ of sky on the celestial equator. We use these spectra, and the spectra measured with the MBAC camera on ACT from 2008-10, in combination with Planck and WMAP data to estimate cosmological parameters from the temperature, polarization, and temperature-polarization cross-correlations. We find the new ACTPol data to be consistent with the LCDM model. The ACTPol temperature-polarization cross-spectrum now provides stronger constraints on multiple parameters than the ACTPol temperature spectrum, including the baryon density, the acoustic peak angular scale, and the derived Hubble constant. Adding the new data to planck temperature data tightens the limits on damping tail parameters, for example reducing the joint uncertainty on the number of neutrino species and the primordial helium fraction by 20%.Comment: 23 pages, 25 figure

Patient-reported outcomes in the aging population of adults with congenital heart disease: results from APPROACH-IS.

The congenital heart disease (CHD) population now comprises an increasing number of older persons in their 6th decade of life and beyond. We cross-sectionally evaluated patient-reported outcomes (PROs) in persons with CHD aged 60 years or older, and contrasted these with PROs of younger patients aged 40-59 years and 18-39 years. Adjusted for demographic and medical characteristics, patients ≥60 years had a lower Physical Component Summary, higher Mental Component Summary, and lower anxiety (Hospital Anxiety and Depression Scale-Anxiety) scores than patients in the two younger categories. For satisfaction with life, older persons had a higher score than patients aged 40-59 years. Registration: ClinicalTrials.gov NCT02150603

Engineering GPCR signaling pathways with RASSLs

We are creating families of designer G-protein-coupled receptors (GPCRs) to allow for precise spatiotemporal control of GPCR signaling in vivo. These engineered GPCRs, called receptors activated solely by synthetic ligands (RASSLs), are unresponsive to endogenous ligands but can be activated by nanomolar concentrations of pharmacologically inert, drug-like small molecules. Currently, RASSLs exist for the three major GPCR signaling pathways (Gs, Gi, Gq). These new advances are reviewed here to help facilitate the use of these powerful and diverse tools

The Role of Industry, Geography and Firm Heterogeneity in Credit Risk Diversification

In theory the potential for credit risk diversification for banks could be substantial. Portfolio diversification is driven broadly by two characteristics: the degree to which systematic risk factors are correlated with each other and the degree of dependence individual firms have to the different types of risk factors. We propose a model for exploring these dimensions of credit risk diversification: across industry sectors and across different countries or regions. We find that full firm-level parameter heterogeneity matters a great deal for capturing differences in simulated credit loss distributions. Imposing homogeneity results in overly skewed and fat-tailed loss distributions. These differences become more pronounced in the presence of systematic risk factor shocks: increased parameter heterogeneity greatly reduces shock sensitivity. Allowing for regional parameter heterogeneity seems to better approximate the loss distributions generated by the fully heterogeneous model than allowing just for industry heterogeneity. The regional model also exhibits less shock sensitivity

Fibrodysplasia Ossificans Progressiva: what have we achieved and where are we now? follow-up to the 2015 Lorentz Workshop

Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare progressive genetic disease effecting one in a million individuals. During their life, patients with FOP progressively develop bone in the soft tissues resulting in increasing immobility and early death. A mutation in the ACVR1 gene was identified as the causative mutation of FOP in 2006. After this, the pathophysiology of FOP has been further elucidated through the efforts of research groups worldwide. In 2015, a workshop was held to gather these groups and discuss the new challenges in FOP research. Here we present an overview and update on these topics