Gene Expression Commons: an open platform for absolute gene expression profiling.

Gene expression profiling using microarrays has been limited to comparisons of gene expression between small numbers of samples within individual experiments. However, the unknown and variable sensitivities of each probeset have rendered the absolute expression of any given gene nearly impossible to estimate. We have overcome this limitation by using a very large number (>10,000) of varied microarray data as a common reference, so that statistical attributes of each probeset, such as the dynamic range and threshold between low and high expression, can be reliably discovered through meta-analysis. This strategy is implemented in a web-based platform named "Gene Expression Commons" (https://gexc.stanford.edu/) which contains data of 39 distinct highly purified mouse hematopoietic stem/progenitor/differentiated cell populations covering almost the entire hematopoietic system. Since the Gene Expression Commons is designed as an open platform, investigators can explore the expression level of any gene, search by expression patterns of interest, submit their own microarray data, and design their own working models representing biological relationship among samples

Laser powder bed fusion of high-strength and corrosion-resistant Inconel alloy 725

The development of additive manufacturing, or three-dimensional (3D) printing, technologies has produced breakthroughs in the design and manufacturing of products by enhancing design freedom and minimising manufacturing steps. In addition, the complex, unique microstructures imparted by the additive processes offer prospects of unprecedented advances to produce high-performance metal alloys for high-temperature and corrosive environments. Here, we present the first additive manufacturing of Inconel alloy 725, an advanced nickel-base superalloy that is the widely accepted gold standard material of choice for oil and gas, chemical, and marine applications. We explore the printability of Inconel alloy 725 and identify a wide processing space to build material with a crack- and near-pore-free microstructure. The conventionally heat-treated Inconel alloy 725 has an equiaxed, near-fully recrystallised microstructure containing copious twin boundaries and nano-precipitates. It also displays promising tensile properties and corrosion resistance compared to its wrought counterpart. Our work opens the door toward additive manufacturing of Inconel alloy 725 components with optimised microstructure and topology geometry for applications in harsh environments

A high-throughput technique for determining grain boundary character non-destructively in microstructures with through-thickness grains

Grain boundaries (GBs) govern many properties of polycrystalline materials. However, because of their structural variability, our knowledge of GB constitutive relations is still very limited. We present a novel method to characterise the complete crystallography of individual GBs non-destructively, with high-throughput, and using commercially available tools. This method combines electron diffraction, optical reflectance and numerical image analysis to determine all five crystallographic parameters of numerous GBs in samples with through-thickness grains. We demonstrate the technique by measuring the crystallographic character of about 1,000 individual GBs in aluminum in a single run. Our method enables cost- and time-effective assembly of crystallography–property databases for thousands of individual GBs. Such databases are essential for identifying GB constitutive relations and for predicting GB-related behaviours of polycrystalline solids.United States. Department of Energy. Office of Basic Energy Sciences (award no DE-SC0008926)MIT International Science and Technology InitiativesNational Science Foundation (U.S.) (grant DMR-1003901

Laser powder bed fusion of high-strength and corrosion-resistant Inconel alloy 725

The development of additive manufacturing, or three-dimensional (3D) printing, technologies has produced breakthroughs in the design and manufacturing of products by enhancing design freedom and minimising manufacturing steps. In addition, the complex, unique microstructures imparted by the additive processes offer prospects of unprecedented advances to produce high-performance metal alloys for high-temperature and corrosive environments. Here, we present the first additive manufacturing of Inconel alloy 725, an advanced nickel-base superalloy that is the widely accepted gold standard material of choice for oil and gas, chemical, and marine applications. We explore the printability of Inconel alloy 725 and identify a wide processing space to build material with a crack- and near-pore-free microstructure. The conventionally heat-treated Inconel alloy 725 has an equiaxed, near-fully recrystallised microstructure containing copious twin boundaries and nano-precipitates. It also displays promising tensile properties and corrosion resistance compared to its wrought counterpart. Our work opens the door toward additive manufacturing of Inconel alloy 725 components with optimised microstructure and topology geometry for applications in harsh environments

Comprehensive methylome map of lineage commitment from haematopoietic progenitors.

Epigenetic modifications must underlie lineage-specific differentiation as terminally differentiated cells express tissue-specific genes, but their DNA sequence is unchanged. Haematopoiesis provides a well-defined model to study epigenetic modifications during cell-fate decisions, as multipotent progenitors (MPPs) differentiate into progressively restricted myeloid or lymphoid progenitors. Although DNA methylation is critical for myeloid versus lymphoid differentiation, as demonstrated by the myeloerythroid bias in Dnmt1 hypomorphs, a comprehensive DNA methylation map of haematopoietic progenitors, or of any multipotent/oligopotent lineage, does not exist. Here we examined 4.6 million CpG sites throughout the genome for MPPs, common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs), and thymocyte progenitors (DN1, DN2, DN3). Marked epigenetic plasticity accompanied both lymphoid and myeloid restriction. Myeloid commitment involved less global DNA methylation than lymphoid commitment, supported functionally by myeloid skewing of progenitors following treatment with a DNA methyltransferase inhibitor. Differential DNA methylation correlated with gene expression more strongly at CpG island shores than CpG islands. Many examples of genes and pathways not previously known to be involved in choice between lymphoid/myeloid differentiation have been identified, such as Arl4c and Jdp2. Several transcription factors, including Meis1, were methylated and silenced during differentiation, indicating a role in maintaining an undifferentiated state. Additionally, epigenetic modification of modifiers of the epigenome seems to be important in haematopoietic differentiation. Our results directly demonstrate that modulation of DNA methylation occurs during lineage-specific differentiation and defines a comprehensive map of the methylation and transcriptional changes that accompany myeloid versus lymphoid fate decisions

Epigenetic memory in induced pluripotent stem cells.

Somatic cell nuclear transfer and transcription-factor-based reprogramming revert adult cells to an embryonic state, and yield pluripotent stem cells that can generate all tissues. Through different mechanisms and kinetics, these two reprogramming methods reset genomic methylation, an epigenetic modification of DNA that influences gene expression, leading us to hypothesize that the resulting pluripotent stem cells might have different properties. Here we observe that low-passage induced pluripotent stem cells (iPSCs) derived by factor-based reprogramming of adult murine tissues harbour residual DNA methylation signatures characteristic of their somatic tissue of origin, which favours their differentiation along lineages related to the donor cell, while restricting alternative cell fates. Such an 'epigenetic memory' of the donor tissue could be reset by differentiation and serial reprogramming, or by treatment of iPSCs with chromatin-modifying drugs. In contrast, the differentiation and methylation of nuclear-transfer-derived pluripotent stem cells were more similar to classical embryonic stem cells than were iPSCs. Our data indicate that nuclear transfer is more effective at establishing the ground state of pluripotency than factor-based reprogramming, which can leave an epigenetic memory of the tissue of origin that may influence efforts at directed differentiation for applications in disease modelling or treatment

Reliability and importance of structural diversity of climate model ensembles

PublishedJournal ArticleWe investigate the performance of the newest generation multi-model ensemble (MME) from the Coupled Model Intercomparison Project (CMIP5). We compare the ensemble to the previous generation models (CMIP3) as well as several single model ensembles (SMEs), which are constructed by varying components of single models. These SMEs range from ensembles where parameter uncertainties are sampled (perturbed physics ensembles) through to an ensemble where a number of the physical schemes are switched (multi-physics ensemble). We focus on assessing reliability against present-day climatology with rank histograms, but also investigate the effective degrees of freedom (EDoF) of the fields of variables which makes the statistical test of reliability more rigorous, and consider the distances between the observation and ensemble members. We find that the features of the CMIP5 rank histograms, of general reliability on broad scales, are consistent with those of CMIP3, suggesting a similar level of performance for present-day climatology. The spread of MMEs tends towards being "over-dispersed" rather than "under-dispersed". In general, the SMEs examined tend towards insufficient dispersion and the rank histogram analysis identifies them as being statistically distinguishable from many of the observations. The EDoFs of the MMEs are generally greater than those of SMEs, suggesting that structural changes lead to a characteristically richer range of model behaviours than is obtained with parametric/physical-scheme-switching ensembles. For distance measures, the observations and models ensemble members are similarly spaced from each other for MMEs, whereas for the SMEs, the observations are generally well outside the ensemble. We suggest that multi-model ensembles should represent an important component of uncertainty analysis. © 2013 The Author(s).We acknowledge the World Climate Research Programme’s Working Group on Coupled Modelling, which is responsible for CMIP. For CMIP the US Department of Energy’s Pro- gram for Climate Model Diagnosis and Intercomparison provides coordinating support and led development of software infrastructure in partnership with the Global Organization for Earth System Science Portals. M.C. was partially supported by funding from NERC grants NE/I006524/1 and NE/I022841/1. MW is supported by the Joint DECC/Defra Met Office Hadley Centre Climate Programme (GA01101). T.Y., J.D.A, H.S., S.E., M.Y., J.C.H. were supported by the Global Environment Research Fund of the Ministry of the Environment of Japan (S-10, Integrated Climate Assessment – Risks,Uncertainties and Society, ICA-RUS)

Establishment of macaque trophoblast stem cell lines derived from cynomolgus monkey blastocysts

The placenta forms a maternal-fetal junction that supports many physiological functions such as the supply of nutrition and exchange of gases and wastes. Establishing an in vitro culture model of human and non-human primate trophoblast stem/progenitor cells is important for investigating the process of early placental development and trophoblast differentiation. In this study, we have established five trophoblast stem cell (TSC) lines from cynomolgus monkey blastocysts, named macTSC #1-5. Fibroblast growth factor 4 (FGF4) enhanced proliferation of macTSCs, while other exogenous factors were not required to maintain their undifferentiated state. macTSCs showed a trophoblastic gene expression profile and trophoblast-like DNA methylation status and also exhibited differentiation capacity towards invasive trophoblast cells and multinucleated syncytia. In a xenogeneic chimera assay, these stem cells contributed to trophectoderm (TE) development in the chimeric blastocysts. macTSC are the first primate trophoblast cell lines whose proliferation is promoted by FGF4. These cell lines provide a valuable in vitro culture model to analyze the similarities and differences in placental development between human and non-human primates

Transcriptional activation of hypoxia-inducible factor-1 (HIF-1) in myeloid cells promotes angiogenesis through VEGF and S100A8

Emerging evidence indicates that myeloid cells are essential for promoting new blood vessel formation by secreting various angiogenic factors. Given that hypoxia-inducible factor (HIF) is a critical regulator for angiogenesis, we questioned whether HIF in myeloid cells also plays a role in promoting angiogenesis. To address this question, we generated a unique strain of myeloid-specific knockout mice targeting HIF pathways using human S100A8 as a myeloid-specific promoter. We observed that mutant mice where HIF-1 is transcriptionally activated in myeloid cells (by deletion of the von Hippel-Lindau gene) resulted in erythema, enhanced neovascularization in matrigel plugs, and increased production of vascular endothelial growth factor (VEGF) in the bone marrow, all of which were completely abrogated by either genetic or pharmacological inactivation of HIF-1. We further found that monocytes were the major effector producing VEGF and S100A8 proteins driving neovascularization in matrigel. Moreover, by using a mouse model of hindlimb ischemia we observed significantly improved blood flow in mice intramuscularly injected with HIF-1-activated monocytes. This study therefore demonstrates that HIF-1 activation in myeloid cells promotes angiogenesis through VEGF and S100A8 and that this may become an attractive therapeutic strategy to treat diseases with vascular defects.X1137Ysciescopu

A Sensitive and Quantitative Polymerase Chain Reaction-Based Cell Free In Vitro Non-Homologous End Joining Assay for Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) are responsible for sustaining hematopoietic homeostasis and regeneration after injury for the entire lifespan of an organism. Maintenance of genomic stability is crucial for the preservation of HSCs, which depends on their efficient repair of DNA damage, particularly DNA double strand breaks (DSBs). Because of the paucity of HSCs and lack of sensitive assays, directly measuring the ability of HSCs to repair DSBs has been difficult. Therefore, we developed a sensitive and quantitative cell free in vitro non-homologous end joining (NHEJ) assay using linearized plasmids as the substrates and quantitative polymerase chain reaction (qPCR) technique. This assay can sensitively detect DSB repair via NHEJ in less than 1 µg 293T cell nuclear proteins or nuclear extracts from about 5,000 to 10,000 human BM CD34+ hematopoietic cells. Using this assay, we confirmed that human bone marrow HSCs (CD34+CD38− cells) are less proficient in the repair of DSBs by NHEJ than HPCs (CD34+CD38+ cells). In contrast, mouse quiescent HSCs (Pyronin-Ylow LKS+ cells) and cycling HSCs (Pyronin-Yhi LKS+ cells) repaired the damage more efficiently than HPCs (LKS− cells). The difference in the abilities of human and mouse HSCs and HPCs to repair DSBs through NHEJ is likely attributed to their differential expression of key NHEJ DNA damage repair genes such as LIG4. These findings suggest that the qPCR-based cell free in vitro NHEJ assay can be used to sensitively measure the ability of human and mouse HSCs to repair DSBs