Site-specific Phosphorylation of Histone H3K36 Methyltransferase Set2p and Demethylase Jhd1p is Required for Stress Responses in Saccharomyces cerevisiae

Histone lysine methylation is a key epigenetic modification that regulates eukaryotic transcription. In Saccharomyces cerevisiae, it is controlled by a reduced but evolutionarily conserved suite of methyltransferase (Set1p, Set2p, Dot1p, and Set5p) and demethylase (Jhd1p, Jhd2p, Rph1p, and Gis1p) enzymes. Many of these enzymes are extensively phosphorylated in vivo; however, the functions of almost all phosphosites remain unknown. Here, we comprehensively analyse the phosphoregulation of the yeast histone methylation network by functionally investigating 40 phosphosites on six enzymes. A total of 82 genomically-edited S. cerevisiae strains were generated through mutagenesis of sites to aspartate as a phosphomimetic or alanine as a phosphonull. These phosphosite mutants were screened for changes in native H3K4, H3K36, and H3K79 methylation levels, and for sensitivity to environmental stress conditions. For methyltransferase Set2p, we found that phosphorylation at threonine 127 significantly decreased H3K36 methylation in vivo, and that an N-terminal phosphorylation cluster at serine residues 6, 8, and 10 is required for the diamide stress response. Proteomic analysis of Set2p phosphosite mutants revealed a specific downregulation of membrane-associated proteins and processes, consistent with changes brought about by SET2 deletion and the sensitivity of mutants to diamide. For demethylase Jhd1p, we found that its sole phosphorylation site at serine 44 is required for the cold stress response. This study represents the first systematic investigation into the phosphoregulation of the epigenetic network in any eukaryote, and shows that phosphosites on histone methylation enzymes are required for a normal cellular response to stress in S. cerevisiae

Response to pulmonary arterial hypertension drug therapies in patients with pulmonary arterial hypertension and cardiovascular risk factors.

The age at diagnosis of pulmonary arterial hypertension (PAH) and the prevalence of cardiovascular (CV) risk factors are increasing. We sought to determine whether the response to drug therapy was influenced by CV risk factors in PAH patients. We studied consecutive incident PAH patients (n = 146) between January 1, 2008, and July 15, 2011. Patients were divided into two groups: the PAH-No CV group included patients with no CV risk factors (obesity, systemic hypertension, type 2 diabetes mellitus, permanent atrial fibrillation, mitral and/or aortic valve disease, and coronary artery disease), and the PAH-CV group included patients with at least one. The response to PAH treatment was analyzed in all the patients who received PAH drug therapy. The PAH-No CV group included 43 patients, and the PAH-CV group included 69 patients. Patients in the PAH-No CV group were younger than those in the PAH-CV group (P < 0.0001). In the PAH-No CV group, 16 patients (37%) improved on treatment and 27 (63%) did not improve, compared with 11 (16%) and 58 (84%) in the PAH-CV group, respectively (P = 0.027 after adjustment for age). There was no difference in survival at 30 months (P = 0.218). In conclusion, in addition to older age, CV risk factors may predict a reduced response to PAH drug therapy in patients with PAH

The application of 'omics' to pulmonary arterial hypertension

Recent genome-wide analyses of rare and common sequence variations have brought greater clarity to the genetic architecture of pulmonary arterial hypertension and implicated novel genes in disease development. Transcriptional signatures have been reported in whole lung tissue, pulmonary vascular cells and peripheral circulating cells. High-throughput platforms for plasma proteomics and metabolomics have identified novel biomarkers associated with clinical outcomes and provided molecular instruments for risk assessment. There are methodological challenges to integrating these datasets, coupled to statistical power limitations inherent to the study of a rare disease, but the expectation is that this strategy will reveal novel druggable targets and biomarkers that will open the way to personalised medicine. Here we review the current state-of-the-art and future promise of "omics" in the field of translational medicine in pulmonary arterial hypertension

Descemet Membrane Endothelial Keratoplasty: Intraoperative and Postoperative Imaging Spectral-Domain Optical Coherence Tomography

We describe a case report of using the same handheld spectral-domain anterior segment optical coherence tomography (ASOCT) for rapid intraoperative and postoperative imaging in a case of Descemet membrane endothelial keratoplasty (DMEK). A 67-year-old woman, with Fuchs dystrophy and corneal decompensation, underwent DMEK with intraoperative ASOCT imaging using the handheld Envisu spectral domain ASOCT system (Bioptigen, Inc., Morrisville, NC, USA). We found that this easy-to-use portable system with handheld probe allowed for rapid imaging of the anterior segment during donor manipulation to visualize the orientation of the DMEK donor, as well as to confirm the initial adhesion of the DMEK donor. Moreover, the same system may be used for postoperative monitoring of graft adhesion, corneal thickness, and stromal remodeling in the clinic with very high-definition images

Riociguat: Mode of action and clinical development in pulmonary hypertension

Pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTEPH) are progressive and debilitating diseases characterized by gradual obstruction of the pulmonary vasculature, leading to elevated pulmonary artery pressure and increased pulmonary vascular resistance. If untreated, they can result in death due to right heart failure. Riociguat is a novel soluble guanylate cyclase (sGC) stimulator that is approved for the treatment of PAH and CTEPH. Here we describe in detail the role of the nitric oxide-sGC-cyclic guanosine monophosphate (cGMP) signaling pathway in the pathogenesis of PAH and CTEPH, and the mode of action of riociguat. We also review the preclinical data associated with the development of riociguat, along with the efficacy and safety data of riociguat from initial clinical trials and the pivotal Phase III randomized clinical trials in PAH and CTEPH

Pyomelanin produced by Vibrio cholerae confers resistance to predation by Acanthamoeba castellanii

© FEMS 2017. Protozoan predation is one of the main environmental factors constraining bacterial growth in aquatic environments, and thus has led to the evolution of a number of defence mechanisms that protect bacteria from predation. These mechanisms may also function as virulence factors in infection of animal and human hosts. Whole transcriptome shotgun sequencing of Vibrio cholerae biofilms during predation by the amoebae, Acanthamoeba castellanii, revealed that 131 transcripts were significantly differentially regulated when compared to the non-grazed control. Differentially regulated transcripts included those involved in biosynthetic and metabolic pathways. The transcripts of genes involved in tyrosine metabolism were down-regulated in the grazed population, which indicates that the tyrosine metabolic regulon may have a role in the response of V. cholerae biofilms to A. castellanii predation. Homogentisate 1, 2-dioxygenase (HGA) is the main intermediate of the normal L-tyrosine catabolic pathway which is known to auto-oxidize, leading to the formation of the pigment, pyomelanin. Indeed, a pigmented mutant, disrupted in hmgA, was more resistant to amoebae predation than the wild type. Increased grazing resistance was correlated with increased production of pyomelanin and thus reactive oxygen species (ROS), suggesting that ROS production is a defensive mechanism used by bacterial biofilms against predation by amoebae A. castellanii

Evidence for a Putative Isoprene Reductase in Acetobacterium wieringae

Recent discoveries of isoprene-metabolizing microorganisms suggest they might play an important role in the global isoprene budget. Under anoxic conditions, isoprene can be used as an electron acceptor and is reduced to methylbutene. This study describes the proteogenomic profiling of an isoprene-reducing bacterial culture to identify organisms and genes responsible for the isoprene hydrogenation reaction. A metagenome-assembled genome (MAG) of the most abundant (89% relative abundance) lineage in the enrichment, Acetobacterium wieringae, was obtained. Comparative proteogenomics and reverse transcription-PCR (RT-PCR) identified a putative five-gene operon from the A. wieringae MAG upregulated during isoprene reduction. The operon encodes a putative oxidoreductase, three pleiotropic nickel chaperones (2 × HypA, HypB), and one 4Fe-4S ferredoxin. The oxidoreductase is proposed as the putative isoprene reductase with a binding site for NADH, flavin adenine dinucleotide (FAD), two pairs of canonical [4Fe-4S] clusters, and a putative iron-sulfur cluster site in a Cys6bonding environment. Well-studied Acetobacterium strains, such as A. woodii DSM 1030, A. wieringae DSM 1911, or A. malicum DSM 4132, do not encode the isoprene-regulated operon but encode, like many other bacteria, a homolog of the putative isoprene reductase (;47 to 49% amino acid sequence identity). Uncharacterized homologs of the putative isoprene reductase are observed across the Firmicutes, Spirochaetes, Tenericutes, Actinobacteria, Chloroflexi, Bacteroidetes, and Proteobacteria, suggesting the ability of biohydrogenation of unfunctionalized conjugated doubled bonds in other unsaturated hydrocarbons

Riociguat for the treatment of chronic thromboembolic pulmonary hypertension.

BACKGROUND: Riociguat, a member of a new class of compounds (soluble guanylate cyclase stimulators), has been shown in previous clinical studies to be beneficial in the treatment of chronic thromboembolic pulmonary hypertension. METHODS: In this phase 3, multicenter, randomized, double-blind, placebo-controlled study, we randomly assigned 261 patients with inoperable chronic thromboembolic pulmonary hypertension or persistent or recurrent pulmonary hypertension after pulmonary endarterectomy to receive placebo or riociguat. The primary end point was the change from baseline to the end of week 16 in the distance walked in 6 minutes. Secondary end points included changes from baseline in pulmonary vascular resistance, N-terminal pro-brain natriuretic peptide (NT-proBNP) level, World Health Organization (WHO) functional class, time to clinical worsening, Borg dyspnea score, quality-of-life variables, and safety. RESULTS: By week 16, the 6-minute walk distance had increased by a mean of 39 m in the riociguat group, as compared with a mean decrease of 6 m in the placebo group (least-squares mean difference, 46 m; 95% confidence interval [CI], 25 to 67; P<0.001). Pulmonary vascular resistance decreased by 226 dyn · sec · cm-5in the riociguat group and increased by 23 dyn · sec · cm-5in the placebo group (least-squares mean difference, -246 dyn · sec · cm-5; 95% CI, -303 to -190; P<0.001). Riociguat was also associated with significant improvements in the NT-proBNP level (P<0.001) and WHO functional class (P = 0.003). The most common serious adverse events were right ventricular failure (in 3% of patients in each group) and syncope (in 2% of the riociguat group and in 3% of the placebo group). CONCLUSIONS: Riociguat significantly improved exercise capacity and pulmonary vascular resistance in patients with chronic thromboembolic pulmonary hypertension. (Funded by Bayer HealthCare; CHEST-1 and CHEST-2 ClinicalTrials.gov numbers, NCT00855465 and NCT00910429, respectively.) Copyright © 2013 Massachusetts Medical Society

RNA-Seq analysis and comparison of corneal epithelium in keratoconus and myopia patients

© 2017 The Author(s). Keratoconus is a common degenerative corneal disease that can lead to significant visual morbidity, and both genetic and environmental factors have been implicated in its pathogenesis. We compared the transcriptome of keratoconus and control epithelium using RNA-Seq. Epithelial tissues were obtained prior to surgery from keratoconus and myopia control patients, undergoing collagen cross-linking and photorefractive keratectomy, respectively. We identified major differences in keratoconus linked to cell-cell communication, cell signalling and cellular metabolism. The genes associated with the Hedgehog, Wnt and Notch1 signaling pathways were down-regulated in keratoconus. We also identified plasmolipin and Notch1 as being significantly reduced in keratoconus for both gene and protein expression (p < 0.05). Plasmolipin is a novel protein identified in human corneal epithelium, and has been demonstrated to have a key role in epithelial cell differentiation in other tissues. This study shows altered gene and protein expression of these three proteins in keratoconus, and further studies are clearly warranted to confirm the functional role of these proteins in the pathogenesis of keratoconus