RNA isolation method for single embryo transcriptome analysis in zebrafish

Background: Transcriptome analysis during embryogenesis usually requires pooling of embryos to obtain sufficient RNA. Hence, the measured levels of gene-expression represent the average mRNA levels of pooled samples and the biological variation among individuals is confounded. This can irreversibly reduce the robustness, resolution, or expressiveness of the experiment. Therefore, we developed a robust method to isolate abundant high-quality RNA from individual embryos to perform single embryo transcriptome analyses using zebrafish as a model organism. Available methods for embryonic zebrafish RNA isolation minimally utilize ten embryos. Further downscaling of these methods to one embryo is practically not feasible. Findings: We developed a single embryo RNA extraction method based on sample homogenization in liquid nitrogen, RNA extraction with phenol and column purification. Evaluation of this method showed that: the quality of the RNA was very good with an average RIN value of 8.3-8.9; the yield was always ≥ 200 ng RNA per embryo; the method was applicable to all stages of zebrafish embryogenesis; the success rate was almost 100%; and the extracted RNA performed excellent in microarray experiments in that the technical variation was much lower than the biological variation. Conclusions: Presented is a high-quality, robust RNA isolation method. Obtaining sufficient RNA from single embryos eliminates the necessity of sample pooling and its associated drawbacks. Although our RNA isolation method has been setup for transcriptome analysis in zebrafish, it can also be used for other model systems and other applications like (q)PCR and transcriptome sequencing

θ13\theta_{13}, δ\delta and the neutrino mass hierarchy at a γ=350\gamma=350 double baseline Li/B β\beta-Beam

We consider a $\beta$-Beam facility where $^8$Li and $^8$B ions are accelerated at $\gamma = 350$, accumulated in a 10 Km storage ring and let decay, so as to produce intense $\bar \nu_e$ and $\nu_e$ beams. These beams illuminate two iron detectors located at $L \simeq 2000$ Km and $L \simeq 7000$ Km, respectively. The physics potential of this setup is analysed in full detail as a function of the flux. We find that, for the highest flux ($10 \times 10^{18}$ ion decays per year per baseline), the sensitivity to $\theta_{13}$ reaches $\sin^2 2 \theta_{13} \geq 2 \times10^{-4}$; the sign of the atmospheric mass difference can be identified, regardless of the true hierarchy, for $\sin^2 2 \theta_{13} \geq 4\times10^{-4}$; and, CP-violation can be discovered in 70% of the $\delta$-parameter space for $\sin^2 2 \theta_{13} \geq 10^{-3}$, having some sensitivity to CP-violation down to $\sin^2 2 \theta_{13} \geq 10^{-4}$ for $|\delta| \sim 90^\circ$.Comment: 35 pages, 20 figures. Minor changes, matches the published versio

MicroRNAs in pulmonary arterial remodeling

Pulmonary arterial remodeling is a presently irreversible pathologic hallmark of pulmonary arterial hypertension (PAH). This complex disease involves pathogenic dysregulation of all cell types within the small pulmonary arteries contributing to vascular remodeling leading to intimal lesions, resulting in elevated pulmonary vascular resistance and right heart dysfunction. Mutations within the bone morphogenetic protein receptor 2 gene, leading to dysregulated proliferation of pulmonary artery smooth muscle cells, have been identified as being responsible for heritable PAH. Indeed, the disease is characterized by excessive cellular proliferation and resistance to apoptosis of smooth muscle and endothelial cells. Significant gene dysregulation at the transcriptional and signaling level has been identified. MicroRNAs are small non-coding RNA molecules that negatively regulate gene expression and have the ability to target numerous genes, therefore potentially controlling a host of gene regulatory and signaling pathways. The major role of miRNAs in pulmonary arterial remodeling is still relatively unknown although research data is emerging apace. Modulation of miRNAs represents a possible therapeutic target for altering the remodeling phenotype in the pulmonary vasculature. This review will focus on the role of miRNAs in regulating smooth muscle and endothelial cell phenotypes and their influence on pulmonary remodeling in the setting of PAH

Uncertainty estimation for operational ocean forecast products-a multi-model ensemble for the North Sea and the Baltic Sea

Multi-model ensembles for sea surface temperature (SST), sea surface salinity (SSS), sea surface currents (SSC), and water transports have been developed for the North Sea and the Baltic Sea using outputs from several operational ocean forecasting models provided by different institutes. The individual models differ in model code, resolution, boundary conditions, atmospheric forcing, and data assimilation. The ensembles are produced on a daily basis. Daily statistics are calculated for each parameter giving information about the spread of the forecasts with standard deviation, ensemble mean and median, and coefficient of variation. High forecast uncertainty, i.e., for SSS and SSC, was found in the Skagerrak, Kattegat (Transition Area between North Sea and Baltic Sea), and the Norwegian Channel. Based on the data collected, longer-term statistical analyses have been done, such as a comparison with satellite data for SST and evaluation of the deviation between forecasts in temporal and spatial scale. Regions of high forecast uncertainty for SSS and SSC have been detected in the Transition Area and the Norwegian Channel where a large spread between the models might evolve due to differences in simulating the frontal structures and their movements. A distinct seasonal pattern could be distinguished for SST with high uncertainty between the forecasts during summer. Forecasts with relatively high deviation from the multi-model ensemble (MME) products or the other individual forecasts were detected for each region and each parameter. The comparison with satellite data showed that the error of the MME products is lowest compared to those of the ensemble members

An improved algorithm for the retrieval of ocean wave spectra from synthetic aperture radar image spectra

An earlier algorithm for retrieving two-dimensional wave spectra from synthetic aperture radar (SAR) image spectra is improved by using a modified cost function and introducing an additional iteration loop in which the first-guess input spectrum is systematically updated. For this purpose a spectral partitioning scheme is applied in which the spectrum is decomposed into a finite number of distinct wave systems. At each iteration step, the individual wave systems of the partitioned nth-guess wave spectrum are adjusted to agree in mean energy, frequency, and direction with the corresponding mean values of the associated wave systems of the SAR-inverted wave spectrum. The algorithm retrieves smooth wave spectra, avoiding the discontinuities which tended to arise in the previous algorithm in the transition region near the azimuthal wavenumber cutoff of the SAR image spectrum. The azimuthal cutoff of the SAR spectrum is also reproduced more accurately. The greatest improvement of the new retrieval algorithm is obtained when the discrepancies between the initial first-guess wave spectrum and the observed SAR spectrum are large. In this case the additional updating loop for the input spectrum enables the retrieved spectrum to adjust such that the simulated SAR spectrum matches more closely the observed SAR spectrum. The overall correlation of a large set of simulated SAR spectra with the measured SAR spectra is found to be significantly higher than with the previous algorithm, indicating that the algorithm not only overcomes isolated shortcomings of the earlier algorithm but also yields retrieved wave spectra which are generally more consistent with the input SAR data. An additional practical advantage of the new algorithm is that it returns spectral partioning parameters which dan be used in SAR wave data assimilation schemes

Loss-of-function mutations in SIM1 contribute to obesity and Prader-Willi-like features

Sim1 haploinsufficiency in mice induces hyperphagic obesity and developmental abnormalities of the brain. In humans, abnormalities in chromosome 6q16, a region that includes SIM1, were reported in obese children with a Prader-Willi–like syndrome; however, SIM1 involvement in obesity has never been conclusively demonstrated. Here, SIM1 was sequenced in 44 children with Prader-Willi–like syndrome features, 198 children with severe early-onset obesity, 568 morbidly obese adults, and 383 controls. We identified 4 rare variants (p.I128T, p.Q152E, p.R581G, and p.T714A) in 4 children with Prader-Willi–like syndrome features (including severe obesity) and 4 other rare variants (p.T46R, p.E62K, p.H323Y, and p.D740H) in 7 morbidly obese adults. By assessing the carriers’ relatives, we found a significant contribution of SIM1 rare variants to intra-family risk for obesity. We then assessed functional effects of the 8 substitutions on SIM1 transcriptional activities in stable cell lines using luciferase gene reporter assays. Three mutations showed strong loss-of-function effects (p.T46R, p.H323Y, and p.T714A) and were associated with high intra-family risk for obesity, while the variants with mild or no effects on SIM1 activity were not associated with obesity within families. Our genetic and functional studies demonstrate a firm link between SIM1 loss of function and severe obesity associated with, or independent of, Prader-Willi–like features.Amélie Bonnefond, Anne Raimondo, Fanny Stutzmann, Maya Ghoussaini, Shwetha Ramachandrappa, David C. Bersten, Emmanuelle Durand, Vincent Vatin, Beverley Balkau, Olivier Lantieri, Violeta Raverdy, François Pattou, Wim Van Hul, Luc Van Gaal, Daniel J. Peet, Jacques Weill, Jennifer L. Miller, Fritz Horber, Anthony P. Goldstone, Daniel J. Driscoll, John B. Bruning, David Meyre, Murray L. Whitelaw and Philippe Frogue

Hormonal Signal Amplification Mediates Environmental Conditions during Development and Controls an Irreversible Commitment to Adulthood

Many animals can choose between different developmental fates to maximize fitness. Despite the complexity of environmental cues and life history, different developmental fates are executed in a robust fashion. The nematode Caenorhabditis elegans serves as a powerful model to examine this phenomenon because it can adopt one of two developmental fates (adulthood or diapause) depending on environmental conditions. The steroid hormone dafachronic acid (DA) directs development to adulthood by regulating the transcriptional activity of the nuclear hormone receptor DAF-12. The known role of DA suggests that it may be the molecular mediator of environmental condition effects on the developmental fate decision, although the mechanism is yet unknown. We used a combination of physiological and molecular biology techniques to demonstrate that commitment to reproductive adult development occurs when DA levels, produced in the neuroendocrine XXX cells, exceed a threshold. Furthermore, imaging and cell ablation experiments demonstrate that the XXX cells act as a source of DA, which, upon commitment to adult development, is amplified and propagated in the epidermis in a DAF-12 dependent manner. This positive feedback loop increases DA levels and drives adult programs in the gonad and epidermis, thus conferring the irreversibility of the decision. We show that the positive feedback loop canalizes development by ensuring that sufficient amounts of DA are dispersed throughout the body and serves as a robust fate-locking mechanism to enforce an organism-wide binary decision, despite noisy and complex environmental cues. These mechanisms are not only relevant to C. elegans but may be extended to other hormonal-based decision-making mechanisms in insects and mammals