The bromodomain protein BRD4 regulates splicing during heat shock.

The cellular response to heat stress is an ancient and evolutionarily highly conserved defence mechanism characterised by the transcriptional up-regulation of cyto-protective genes and a partial inhibition of splicing. These features closely resemble the proteotoxic stress response during tumor development. The bromodomain protein BRD4 has been identified as an integral member of the oxidative stress as well as of the inflammatory response, mainly due to its role in the transcriptional regulation process. In addition, there are also several lines of evidence implicating BRD4 in the splicing process. Using RNA-sequencing we found a significant increase in splicing inhibition, in particular intron retentions (IR), following heat treatment in BRD4-depleted cells. This leads to a decrease of mRNA abundancy of the affected transcripts, most likely due to premature termination codons. Subsequent experiments revealed that BRD4 interacts with the heat shock factor 1 (HSF1) such that under heat stress BRD4 is recruited to nuclear stress bodies and non-coding SatIII RNA transcripts are up-regulated. These findings implicate BRD4 as an important regulator of splicing during heat stress. Our data which links BRD4 to the stress induced splicing process may provide novel mechanisms of BRD4 inhibitors in regard to anti-cancer therapies.

Efficacy and safety of nasal high-flow oxygen in COPD patients.

BACKGROUND: Nasal high-flow oxygen therapy (HFOT) is a novel treatment option for patients suffering from acute or chronic respiratory failure. Aim of our study was to compare safety and efficacy of HFOT with those of conventional oxygen treatment (COT) in normo- and hypercapnic COPD patients. METHODS: A single cohort of 77 clinically stable hypoxemic patients with an indication for long-term oxygen treatment (LTOT) with or without hypercapnia successively received COT and HFOT for 60 min each, including oxygen adaption and separated by a 30 min washout phase. RESULTS: HFOT was well-tolerated in all patients. A significant decrease in PaCO2 was observed during oxygen adaption of HFOT, and increased PaO2 coincided with significantly increased SpO2 and decreased AaDO2 during both treatment phases. Even at a flow rate of 15 L/min, oxygen requirement delivered as air mixture by HFOT tended to be lower than that of COT (2.2 L/min). Not only was no increase in static or dynamic lung volumes observed during HFOT, but even was a significant reduction of residual lung volume measured in 36 patients (28%). CONCLUSIONS: Thus, short-term use of HFOT is safe in both normocapnic and hypercapnic COPD patients. Lower oxygen levels were effective in correcting hypoxemic respiratory failure and reducing hypercapnia, leading to a reduced amount of oxygen consumption. Long-term studies are needed to assess safety, tolerability, and clinical efficacy of HFOT. TRIAL REGISTRATION: ClinicalTrials.gov NCT01686893 13.09.2012 retrospectively registered (STIT-1) and NCT01693146 14.09.2012 retrospectively registered (STIT-2). Studies were approved by the local ethics committee (Ethikkommission der Medizinischen Universität Innsbruck, Studienkennzahl UN3547, Sitzungsnummer 274/4.19).

PRMT1-mediated arginine methylation controls ATXN2L localization.

Arginine methylation is a posttranslational modification that is of importance in diverse cellular processes. Recent proteomic mass spectrometry studies reported arginine methylation of ataxin-2-like (ATXN2L), the paralog of ataxin-2, a protein that is implicated in the neurodegenerative disorder spinocerebellar ataxia type 2. Here, we investigated the methylation state of ATXN2L and its significance for ATXN2L localization. We first confirmed that ATXN2L is asymmetrically dimethylated in vivo, and observed that the nuclear localization of ATXN2L is altered under methylation inhibition. We further discovered that ATXN2L associates with the protein arginine-N-methyltransferase 1 (PRMT1). Finally, we showed that neither mutation of the arginine-glycine-rich motifs of ATXN2L nor methylation inhibition alters ATXN2L localization to stress granules, suggesting that methylation of ATXN2L is probably not mandatory.

5-Fluorouracil affects assembly of stress granules based on RNA incorporation.

The antimetabolite 5-fluorouracil is a widely used chemotherapeutic for the treatment of several solid cancers. However, resistance to 5-fluorouracil remains a major drawback in its clinical use. In this study we report that treatment of HeLa cells with 5-fluorouracil resulted in de novo assembly of stress granules. Moreover, we revealed that stress granule assembly under stress conditions as well as disassembly is altered in cells treated with 5-fluorouracil. Notably, we discovered that RACK1, a protein mediating cell survival and apoptosis, is a component of 5-fluorouracil-induced stress granules. To explore the mode of action of 5-fluorouracil accountable for de novo stress granule assembly, we analyzed 5-fluorouracil metabolites and noticed that stress granule assembly is caused by RNA, not DNA incorporating 5-fluorouracil metabolites. Interestingly, we observed that other RNA incorporating drugs also cause assembly of stress granules. Thus, our results suggest that incorporation of chemotherapeutics into RNA may result in stress granule assembly with potential significance in chemoresistance.

AMOT130 drives BMP-SMAD signaling at the apical membrane in polarized cells.

The large isoform of the transmembrane protein angiomotin (AMOT130) controls cell proliferation and migration of many cell types. AMOT130 associates to the actin cytoskeleton and regulates tight-junction maintenance and signaling often via endosomal uptake of polarity proteins at tight junctions. AMOT130 is highly polarized and present only at the apical side of polarized cells. Here we show that bone morphogenetic protein (BMP) growth factor signaling and AMOT function are interlinked in apical-basal polarized cells. BMP6 controls AMOT internalization and endosomal trafficking in epithelial cells. AMOT130 interacts with the BMP receptor BMPR2 and facilitates SMAD activation and target gene expression. We further demonstrate that this effect of AMOT on BMP-SMAD signaling is dependent on endocytosis and specific to the apical side of polarized epithelial and endothelial cells. Knockdown of AMOT reduces SMAD signaling only from the apical side of polarized cells, while basolateral BMP-SMAD signaling is unaffected. This allows for the first time interference with BMP signaling in a polarized manner and identifies AMOT130 as a novel BMP signaling regulator.

Unlocking pathology archives for microRNA-profiling.

BACKGROUND: MicroRNAs (miRNAs) are approximately 22 nucleotide long, non-coding RNAs that regulate gene expression by binding to the 3'-untranslated region of target mRNAs and also a variety of cellular processes. It has recently been established that dysregulation of miRNA expression can be detected in the majority of human cancers. A variety of high-throughput screening methods has been developed to identify dysregulated miRNA species in tumours. For retrospective clinical studies formalin-fixed, paraffin-embedded (FFPE) tissue is the most widely used material. MATERIALS AND METHODS: The miRNA expression profiles of freshly frozen (CRYO) and FFPE tissues of seven tonsil and four liver samples were compared, using a qPCR-based assay, profiling 157 miRNA species. RESULTS: The significance of miRNA-profiles was barely influenced by FFPE treatment in both tissues and the variance induced by FFPE treatment was much smaller than the variance caused by biologically based differential expression. CONCLUSION: FFPE material is well suited for miRNA profiling.

Effects of Arginine Vasopressin on Migration and Respiratory Burst Activity in Human Leukocytes.

Arginine vasopressin can bind to high-affinity vasopressin V1a receptors in human leukocytes. This study aims to investigate the effects of arginine vasopressin on migration and chemotaxis of neutrophils and oxygen free radical release by human leukocytes. Neutrophils and monocytes were obtained from peripheral blood samples of ten healthy volunteers. Leukocyte migration was microscopically assessed in a modified 48-blind well microchemotaxis chamber, and respiratory burst activity was estimated using 2',7'-dichlorofluorescin diacetate in descending concentrations of arginine vasopressin. Arginine vasopressin stimulates migration of monocytes and neutrophils depending on concentration and on interaction with other chemoattractants. The strongest chemotactic responses of monocytes to arginine vasopressin were observed in the micro and nanomolar range and in the nanomolar range for neutrophils (p<0.001). Pre-incubation of leukocytes with arginine vasopressin decreased migration of leukocytes in a dose-dependent manner. Arginine vasopressin did not stimulate release of oxygen free radicals by neutrophils. Arginine vasopressin stimulates in a dose-dependent manner the migration of monocytes and neutrophils. However, pre-incubation of leukocytes with arginine vasopressin decreased the migratory response of monocytes and neutrophils to other chemoattractants. These findings may be of importance in the treatment regimen of patients with septic shock.

Decompensated right heart failure, intensive care and perioperative management in patients with pulmonary hypertension: Updated recommendations from the Cologne Consensus Conference 2018.

In June 2016, members of the German Society of Cardiology (DGK), the German Society of Respiratory Medicine (DGP) and the German Society of Pediatric Cardiology (DGPK) met for a Consensus Conference in Cologne, Germany. Aim of this Conference was to compile consensus based practice recommendations based on the 2015 European Pulmonary Hypertension guidelines, aiming at their practical implementation, considering country-specific issues, and including new evidence, where available. This article summarizes the results and updated recommendations 2018 of the working group on decompensated right heart failure (RHF), intensive care and perioperative management in patients with pulmonary hypertension. The RHF section comprises definition and pathophysiology, diagnosis and monitoring, identification of triggering factors and supportive therapy of RHF, volume management as well as PAH targeting therapy, therapy with inotropic, inodilator and vasopressor drugs, extracorporeal support and transplantation. The second part of this article summarizes preoperative management, perioperative monitoring and choice of anesthesia.

Ataxin-2-like is a regulator of stress granules and processing bodies.

Paralogs for several proteins implicated in neurodegenerative disorders have been identified and explored to further facilitate the identification of molecular mechanisms contributing to disease pathogenesis. For the disease-causing protein in spinocerebellar ataxia type 2, ataxin-2, a paralog of unknown function, termed ataxin-2-like, has been described. We discovered that ataxin-2-like associates with known interaction partners of ataxin-2, the RNA helicase DDX6 and the poly(A)-binding protein, and with ataxin-2 itself. Furthermore, we found that ataxin-2-like is a component of stress granules. Interestingly, sole ataxin-2-like overexpression led to the induction of stress granules, while a reduction of stress granules was detected in case of a low ataxin-2-like level. Finally, we observed that overexpression of ataxin-2-like as well as its reduction has an impact on the presence of microscopically visible processing bodies. Thus, our results imply a functional overlap between ataxin-2-like and ataxin-2, and further indicate a role for ataxin-2-like in the regulation of stress granules and processing bodies.

FOX-2 dependent splicing of ataxin-2 transcript is affected by ataxin-1 overexpression.

Alternative splicing is a fundamental posttranscriptional mechanism for controlling gene expression, and splicing defects have been linked to various human disorders. The splicing factor FOX-2 is part of a main protein interaction hub in a network related to human inherited ataxias, however, its impact remains to be elucidated. Here, we focused on the reported interaction between FOX-2 and ataxin-1, the disease-causing protein in spinocerebellar ataxia type 1. In this line, we further evaluated this interaction by yeast-2-hybrid analyses and co-immunoprecipitation experiments in mammalian cells. Interestingly, we discovered that FOX-2 localization and splicing activity is affected in the presence of nuclear ataxin-1 inclusions. Moreover, we observed that FOX-2 directly interacts with ataxin-2, a protein modulating spinocerebellar ataxia type 1 pathogenesis. Finally, we provide evidence that splicing of pre-mRNA of ataxin-2 depends on FOX-2 activity, since reduction of FOX-2 levels led to increased skipping of exon 18 in ataxin-2 transcripts. Most striking, we observed that ataxin-1 overexpression has an effect on this splicing event as well. Thus, our results demonstrate that FOX-2 is involved in splicing of ataxin-2 transcripts and that this splicing event is altered by overexpression of ataxin-1.

Interleukin-9 (IL-9) and NPM-ALK each generate mast cell hyperplasia as single 'hit' and cooperate in producing a mastocytosis-like disease in mice.

Mast cell neoplasms are characterized by abnormal growth and focal accumulation of mast cells (MC) in one or more organs. Although several cytokines, including stem cell factor (SCF) and interleukin-9 (IL-9) have been implicated in growth of normal MC, little is known about pro-oncogenic molecules and conditions triggering differentiation and growth of MC far enough to lead to the histopathological picture of overt mastocytosis. The anaplastic lymphoma kinase (ALK) has recently been implicated in growth of neoplastic cells in malignant lymphomas. Here, we describe that transplantation of NPM-ALK-transplanted mouse bone marrow progenitors into lethally irradiated IL-9 transgenic mice not only results in lymphoma-formation, but also in the development of a neoplastic disease exhibiting histopathological features of systemic mastocytosis, including multifocal dense MC-infiltrates, occasionally with devastating growth in visceral organs. Transplantation of NPM-ALK-transduced progenitors into normal mice or maintenance of IL-9-transgenic mice without NPM-ALK each resulted in MC hyperplasia, but not in mastocytosis. Neoplastic MC in mice not only displayed IL-9, but also the IL-9 receptor, and the same was found to hold true for human neoplastic MC. Together, our data show that neoplastic MC express IL-9 receptors, that IL-9 and NPM-ALK upregulate MC-production in vivo, and that both'hits' act in concert to induce a mastocytosis-like disease in mice. These data may have pathogenetic and clinical implications and fit well with the observation that neoplastic MC in advanced SM strongly express NPM and multiple "lymphoid" antigens including CD25 and CD30.