GFI1 proteins regulate stem cell formation in the AGM

In vertebrates, the first haematopoietic stem cells (HSCs) with multi-lineage and long-term repopulating potential arise in the AGM (aorta-gonad-mesonephros) region. These HSCs are generated from a rare and transient subset of endothelial cells, called haemogenic endothelium (HE), through an endothelial-to-haematopoietic transition (EHT). Here, we establish the absolute requirement of the transcriptional repressors GFI1 and GFI1B (growth factor independence 1 and 1B) in this unique trans-differentiation process. We first demonstrate that Gfi1 expression specifically defines the rare population of HE that generates emerging HSCs. We further establish that in the absence of GFI1 proteins, HSCs and haematopoietic progenitor cells are not produced in the AGM, revealing the critical requirement for GFI1 proteins in intra-embryonic EHT. Finally, we demonstrate that GFI1 proteins recruit the chromatin-modifying protein LSD1, a member of the CoREST repressive complex, to epigenetically silence the endothelial program in HE and allow the emergence of blood cells.We thank the staff at the Advanced Imaging, animal facility, Molecular Biology Core facilities and Flow Cytometry of CRUK Manchester Institute for technical support and Michael Lie-A-Ling and Elli Marinopoulou for initiating the DamID-PIP bioinformatics project. We thank members of the Stem Cell Biology group, the Stem Cell Haematopoiesis groups and Martin Gering for valuable advice and critical reading of the manuscript. Work in our laboratory is supported by the Leukaemia and Lymphoma Research Foundation (LLR), Cancer Research UK (CRUK) and the Biotechnology and Biological Sciences Research Council (BBSRC). SC is the recipient of an MRC senior fellowship (MR/J009202/1).This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/ncb327

Using Prior Information from the Medical Literature in GWAS of Oral Cancer Identifies Novel Susceptibility Variant on Chromosome 4 - the AdAPT Method

Background: Genome-wide association studies (GWAS) require large sample sizes to obtain adequate statistical power, but it may be possible to increase the power by incorporating complementary data. In this study we investigated the feasibility of automatically retrieving information from the medical literature and leveraging this information in GWAS. Methods: We developed a method that searches through PubMed abstracts for pre-assigned keywords and key concepts, and uses this information to assign prior probabilities of association for each single nucleotide polymorphism (SNP) with the phenotype of interest - the Adjusting Association Priors with Text (AdAPT) method. Association results from a GWAS can subsequently be ranked in the context of these priors using the Bayes False Discovery Probability (BFDP) framework. We initially tested AdAPT by comparing rankings of known susceptibility alleles in a previous lung cancer GWAS, and subsequently applied it in a two-phase GWAS of oral cancer. Results: Known lung cancer susceptibility SNPs were consistently ranked higher by AdAPT BFDPs than by p-values. In the oral cancer GWAS, we sought to replicate the top five SNPs as ranked by AdAPT BFDPs, of which rs991316, located in the ADH gene region of 4q23, displayed a statistically significant association with oral cancer risk in the replication phase (per-rare-allele log additive p-value [p(trend)] = 2.5 x 10(-3)). The combined OR for having one additional rare allele was 0.83 (95% CI: 0.76-0.90), and this association was independent of previously identified susceptibility SNPs that are associated with overall UADT cancer in this gene region. We also investigated if rs991316 was associated with other cancers of the upper aerodigestive tract (UADT), but no additional association signal was found. Conclusion: This study highlights the potential utility of systematically incorporating prior knowledge from the medical literature in genome-wide analyses using the AdAPT methodology. AdAPT is available online (url: http://services.gate.ac.uk/lld/gwas/service/config)

Study on Hydrodynamics of a New Comb-type Floating Breakwater Fixed on the Water Surface

Through the physical model cross-section experiment, the effects of the relative width and groove depth on the transmission coefficient, horizontal wave force and vertical wave force of the new comb-type floating breakwater (FBW) model under fixed condition are observed. The results show that the hydrodynamic parameters of the new comb-type FBW are mainly influenced by its relative width under the action of regular wave, and the transmission coefficient decreases with the increase of its relative width. Especially when the relative width is 0.139 to 0.188, the transmission coefficient of the new comb-type FBW decreases rapidly with the increase of the relative width, and the horizontal wave force and the vertical wave force change slowly. This indicates that the new comb-type FBW has obvious effect on wave dissipation about short and medium waves. In addition, numerical investigations of selected experiment cases are conducted using RANS based commercial CFD code Flow3D. The numerical results show a good ability to capture the hydrodynamic interaction effect of the fixed FBW

Study on Hydrodynamics of a New Comb-type Floating Breakwater Fixed on the Water Surface

Through the physical model cross-section experiment, the effects of the relative width and groove depth on the transmission coefficient, horizontal wave force and vertical wave force of the new comb-type floating breakwater (FBW) model under fixed condition are observed. The results show that the hydrodynamic parameters of the new comb-type FBW are mainly influenced by its relative width under the action of regular wave, and the transmission coefficient decreases with the increase of its relative width. Especially when the relative width is 0.139 to 0.188, the transmission coefficient of the new comb-type FBW decreases rapidly with the increase of the relative width, and the horizontal wave force and the vertical wave force change slowly. This indicates that the new comb-type FBW has obvious effect on wave dissipation about short and medium waves. In addition, numerical investigations of selected experiment cases are conducted using RANS based commercial CFD code Flow3D. The numerical results show a good ability to capture the hydrodynamic interaction effect of the fixed FBW

Competing endogenous RNA network analysis of Turner syndrome patient-specific iPSC-derived cardiomyocytes reveals dysregulation of autosomal heart development genes by altered dosages of X-inactivation escaping non-coding RNAs

Abstract Background A 45,X monosomy (Turner syndrome, TS) is the only chromosome haploinsufficiency compatible with life. Nevertheless, the surviving TS patients still suffer from increased morbidity and mortality, with around one-third of them subjecting to heart abnormalities. How loss of one X chromosome drive these conditions remains largely unknown. Methods Here, we have generated cardiomyocytes (CMs) from wild-type and TS patient-specific induced pluripotent stem cells and profiled the mRNA, lncRNA and circRNA expression in these cells. Results We observed lower beating frequencies and higher mitochondrial DNA copies per nucleus in TS-CMs. Moreover, we have identified a global transcriptome dysregulation of both coding and non-coding RNAs in TS-CMs. The differentially expressed mRNAs were enriched of heart development genes. Further competing endogenous RNA network analysis revealed putative regulatory circuit of autosomal genes relevant with mitochondrial respiratory chain and heart development, such as COQ10A, RARB and WNT2, mediated by X-inactivation escaping lnc/circRNAs, such as lnc-KDM5C-4:1, hsa_circ_0090421 and hsa_circ_0090392. The aberrant expressions of these genes in TS-CMs were verified by qPCR. Further knockdown of lnc-KDM5C-4:1 in wild-type CMs exhibited significantly reduced beating frequencies. Conclusions Our study has revealed a genomewide ripple effect of X chromosome halpoinsufficiency at post-transcriptional level and provided insights into the molecular mechanisms underlying heart abnormalities in TS patients

Integrative Analysis of CRISPR/Cas9 Target Sites in the Human HBB Gene

Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) system has emerged as a powerful customizable artificial nuclease to facilitate precise genetic correction for tissue regeneration and isogenic disease modeling. However, previous studies reported substantial off-target activities of CRISPR system in human cells, and the enormous putative off-target sites are labor-intensive to be validated experimentally, thus motivating bioinformatics methods for rational design of CRISPR system and prediction of its potential off-target effects. Here, we describe an integrative analytical process to identify specific CRISPR target sites in the human β-globin gene (HBB) and predict their off-target effects. Our method includes off-target analysis in both coding and noncoding regions, which was neglected by previous studies. It was found that the CRISPR target sites in the introns have fewer off-target sites in the coding regions than those in the exons. Remarkably, target sites containing certain transcriptional factor motif have enriched binding sites of relevant transcriptional factor in their off-target sets. We also found that the intron sites have fewer SNPs, which leads to less variation of CRISPR efficiency in different individuals during clinical applications. Our studies provide a standard analytical procedure to select specific CRISPR targets for genetic correction

Determination of Formulae for the Hydrodynamic Performance of a Fixed Box-Type Free Surface Breakwater in the Intermediate Water

A two-dimensional viscous numerical wave tank coded mass source function in a computational fluid dynamics (CFD) software Flow-3D 11.2 is built and validated. The effect of the core influencing factors (draft, breakwater width, wave period, and wave height) on the hydrodynamic performance of a fixed box-type free surface breakwater (abbreviated to F-BW in the following texts) are highlighted in the intermediate waters. The results show that four influence factors, except wave period, impede wave transmission; the draft and breakwater width boost wave reflection, and the wave period and wave height are opposite; the draft impedes wave energy dissipation, and the wave height is opposite; the draft and wave height boost the horizontal extreme wave force; four influence factors, except the draft, boost the vertical extreme wave force. Finally, new formulas are provided to determine the transmission, reflection, and dissipation coefficients and extreme wave forces of the F-BW by applying multiple linear regression. The new formulas are verified by comparing with existing literature observation datasets. The results show that it is in good agreement with previous datasets

Generation of an induced pluripotent stem cell line from an adult male with 45,X/46,XY mosaicism

Turner syndrome (TS) with 45,X/46,XY mosaic karyotype is a rare sex chromosome disorder with an occurrence of 0.15‰ at birth. We report the generation of an induced pluripotent stem cell (iPSC) line from peripheral blood mononuclear cells of a Chinese adult male with 45,X/46,XY mosaicism. The iPSC line retains the original 45,X/46,XY mosaic karyotype, expresses pluripotency markers and undergoes trilineage differentiation. Therefore, it offers an unprecedented cellular model to investigate the profound symptoms like infertility of TS in the male, and serve as a useful tool to develop therapies for the disease