The metabolome regulates the epigenetic landscape during naive-to-primed human embryonic stem cell transition.

For nearly a century developmental biologists have recognized that cells from embryos can differ in their potential to differentiate into distinct cell types. Recently, it has been recognized that embryonic stem cells derived from both mice and humans exhibit two stable yet epigenetically distinct states of pluripotency: naive and primed. We now show that nicotinamide N-methyltransferase (NNMT) and the metabolic state regulate pluripotency in human embryonic stem cells (hESCs).  Specifically, in naive hESCs, NNMT and its enzymatic product 1-methylnicotinamide are highly upregulated, and NNMT is required for low S-adenosyl methionine (SAM) levels and the H3K27me3 repressive state. NNMT consumes SAM in naive cells, making it unavailable for histone methylation that represses Wnt and activates the HIF pathway in primed hESCs. These data support the hypothesis that the metabolome regulates the epigenetic landscape of the earliest steps in human development

Analyses of pig genomes provide insight into porcine demography and evolution

For 10,000 years pigs and humans have shared a close and complex relationship. From domestication to modern breeding practices, humans have shaped the genomes of domestic pigs. Here we present the assembly and analysis of the genome sequence of a female domestic Duroc pig (Sus scrofa) and a comparison with the genomes of wild and domestic pigs from Europe and Asia. Wild pigs emerged in South East Asia and subsequently spread across Eurasia. Our results reveal a deep phylogenetic split between European and Asian wild boars ∼1 million years ago, and a selective sweep analysis indicates selection on genes involved in RNA processing and regulation. Genes associated with immune response and olfaction exhibit fast evolution. Pigs have the largest repertoire of functional olfactory receptor genes, reflecting the importance of smell in this scavenging animal. The pig genome sequence provides an important resource for further improvements of this important livestock species, and our identification of many putative disease-causing variants extends the potential of the pig as a biomedical model

microRNAs and metabolites in naïve to primed human embryonic stem cell transition

Thesis (Ph.D.)--University of Washington, 2014This dissertation research is focused on the small molecules of the cell: metabolites and miRNAs. The purpose is to gain a deeper understanding of how they control the cell by investigating the role of secondary structure during miRNA biogenesis and of the changes that occur in the metabolome during naïve to primed human embryonic stem cell (hESC) transition. The first chapter of the thesis focuses on miRNA regulation: Through analysis of miRNA secondary structure and miRNA expression levels in cells with different levels of the enzyme Drosha I have found that miRNAs with mismatches in the precursor region of the miRNA, 9-12 nucleotides from the Drosha cutting site show a higher sensitivity to changes in Drosha levels than miRNAs that lack mismatches in said regions. Through mutagenesis experiments I have shown that by altering the miRNA secondary structure its sensitivity to changes in Drosha levels can be changed. This shows how the cell can selectively regulate a group of miRNAs relative to another by changing Drosha expression and may explain the impact of point mutations outside of the seed region of certain miRNAs. miRNA therapies are also being developed, where the small size of miRNAs makes them excellent candidates for gene therapy. The results of this research can be applied to miRNA therapies by increasing the potency of select miRNAs in their target tissues through structural modifications. In cases where the target tissue has different levels of Drosha compared to surrounding tissues (such as in many types of cancer) the miRNA can also be designed to minimize side effects in surrounding healthy tissues. The second chapter of the thesis focuses on the metabolome in human embryonic stem cells: With the use of mass spectrometry combined with gene expression data I have analyzed the metabolome of naïve and primed hESCs. I show that the two developmental stages have distinct profiles in metabolite set up and I have identified pathways that show a change in activity during naïve to primed hESC transition. I show that tryptophan degradation is increased in primed cells leading to an accumulation of the tryptophan degradation product kynurenine, which is known to inhibit the antitumor response of the immune system by activting the transcription factor AhR and could be a mechanism which prevents rejection of the recently implanted embryo. I also show that primed hESCs display a decrease in activity of the enzyme NNMT, which uses the metabolite S-adenosyl methionine (SAM) to convert nicotinamide into 1-methyl nicotinamide. The decrease of NNMT activity makes SAM available as a substrate for other methylation mechanisms and increases H3K27me3 methylation. When knocking down NNMT the H3K27me3 marks are increased, supporting the hypothesis that the metabolome regulates the epigenetic landscape during naïve to primed hESC transition

A method for MR quantification of flow velocities in blood and CSF using interleaved gradient-echo pulse sequences

The aim of this study was to establish a rapid method for in vivo quantification of a large range of flow velocities using phase information. A basic gradient-echo sequence was constructed, in which flow was encoded along the slice selection direction by variation of the amplitude of a bipolar gradient without changes in sequence timings. The influence of field inhomogeneities and eddy currents was studied in a 1.5 T scanner. From the basic sequence, interleaved sequences for calibration and in vivo flow determination were constructed, and flow information was obtained by pairwise subtraction of velocity-encoded from velocity non-encoded phase images. Calibration was performed in a nongated mode using flow phantoms, and the results were compared with theoretically calculated encoding efficiencies. In vivo flow was studied in healthy volunteers in three different areas using cardiac gating; central blood flow in the great thoracic vessels, peripheral blood flow in the popliteal vessels, and flow of cerebrospinal fluid (CSF) in the cerebral aqueduct. The results show good agreement with results obtained with other techniques. The proposed method for flow determination was shown to be rapid and flexible, and we thus conclude that it seems well suited for routine clinical MR examinations

Molecular mechanism of sphingosine-1-phosphate action in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a lethal muscle-wasting disease. Studies in Drosophila showed that genetic increase of the levels of the bioactive sphingolipid sphingosine-1-phosphate (S1P) or delivery of 2-acetyl-5-tetrahydroxybutyl imidazole (THI), an S1P lyase inhibitor, suppresses dystrophic muscle degeneration. In the dystrophic mouse (mdx), upregulation of S1P by THI increases regeneration and muscle force. S1P can act as a ligand for S1P receptors and as a histone deacetylase (HDAC) inhibitor. Because Drosophila has no identified S1P receptors and DMD correlates with increased HDAC2 levels, we tested whether S1P action in muscle involves HDAC inhibition. Here we show that beneficial effects of THI treatment in mdx mice correlate with significantly increased nuclear S1P, decreased HDAC activity and increased acetylation of specific histone residues. Importantly, the HDAC2 target microRNA genes miR-29 and miR-1 are significantly upregulated, correlating with the downregulation of the miR-29 target Col1a1 in the diaphragm of THI-treated mdx mice. Further gene expression analysis revealed a significant THI-dependent decrease in inflammatory genes and increase in metabolic genes. Accordingly, S1P levels and functional mitochondrial activity are increased after THI treatment of differentiating C2C12 cells. S1P increases the capacity of the muscle cell to use fatty acids as an energy source, suggesting that THI treatment could be beneficial for the maintenance of energy metabolism in mdx muscles

Dissecting the Contributions of Cooperating Gene Mutations to Cancer Phenotypes and Drug Responses with Patient-Derived iPSCs

Summary: Connecting specific cancer genotypes with phenotypes and drug responses constitutes the central premise of precision oncology but is hindered by the genetic complexity and heterogeneity of primary cancer cells. Here, we use patient-derived induced pluripotent stem cells (iPSCs) and CRISPR/Cas9 genome editing to dissect the individual contributions of two recurrent genetic lesions, the splicing factor SRSF2 P95L mutation and the chromosome 7q deletion, to the development of myeloid malignancy. Using a comprehensive panel of isogenic iPSCs—with none, one, or both genetic lesions—we characterize their relative phenotypic contributions and identify drug sensitivities specific to each one through a candidate drug approach and an unbiased large-scale small-molecule screen. To facilitate drug testing and discovery, we also derive SRSF2-mutant and isogenic normal expandable hematopoietic progenitor cells. We thus describe here an approach to dissect the individual effects of two cooperating mutations to clinically relevant features of malignant diseases. : Papapetrou and colleagues develop a comprehensive panel of isogenic iPSC lines with SRSF2 P95L mutation and chr7q deletion. They use these cells to identify cellular phenotypes contributed by each genetic lesion and therapeutic vulnerabilities specific to each one and develop expandable hematopoietic progenitor cell lines to facilitate drug discovery. Keywords: induced pluripotent stem cells, myelodysplastic syndrome, CRISPR/Cas9, gene editing, mutational cooperation, splicing factor mutations, spliceosomal mutations, SRSF2, chr7q deletio

Identification of myocardial injury using perioperative troponin surveillance in major noncardiac surgery and net benefit over the Revised Cardiac Risk Index

Background: Patients with perioperative myocardial injury are at risk of death and major adverse cardiovascular and cerebrovascular events (MACCE). The primary aim of this study was to determine optimal thresholds of preoperative and perioperative changes in high-sensitivity cardiac troponin T (hs-cTnT) to predict MACCE and mortality. Methods: Prospective, observational, cohort study in patients &amp;gt;= 50 yr of age undergoing elective major noncardiac surgery at seven hospitals in Sweden. The exposures were hs-cTnT measured before and days 0-3 after surgery. Two previously published thresholds for myocardial injury and two thresholds identified using receiver operating characteristic analyses were evaluated using multivariable logistic regression models and externally validated. The weighted comparison net benefit method was applied to determine the additional value of hs-cTnT thresholds when compared with the Revised Cardiac Risk Index (RCRI). The primary outcome was a composite of 30-day all-cause mortality and MACCE. Results: We included 1291 patients between April 2017 and December 2020. The primary outcome occurred in 124 patients (9.6%). Perioperative increase in hs-cTnT &amp;gt;= 14 ng L-1 above preoperative values provided statistically optimal model performance and was associated with the highest risk for the primary outcome (adjusted odds ratio 2.9, 95% confidence interval 1.8-4.7). Validation in an independent, external cohort confirmed these findings. A net benefit over RCRI was demonstrated across a range of clinical thresholds. Conclusions: Perioperative increases in hsTnT &amp;gt;= 14 ng L-1 above baseline values identifies acute perioperative myocardial injury and provides a net prognostic benefit when added to RCRI for the identification of patients at high risk of death and MACCE.Funding Agencies|Swedish Research CouncilSwedish Research CouncilEuropean Commission [2019-02833]; South Eastern Sweden Research Council [746981, 712291]; Linkoping University-Region Ostergotland ALF [687681, 792291]; Swiss National Science FoundationSwiss National Science Foundation (SNSF)European Commission [320030-179362]; Swiss Heart Foundation; University Hospital of Basel; Roche Diagnostics; University of Basel; AstraZenecaAstraZeneca</p