Histone modifications are specifically relocated during gene activation and nuclear differentiation

BACKGROUND: Post-translational histone modifications (PTMs) and their specific distribution on genes play a crucial role in the control of gene expression, but the regulation of their dynamics upon gene activation and differentiation is still poorly understood. Here, we exploit the unique genome organization of ciliates to analyse PTM dynamics during gene activation in the differentiated cell and during nuclear differentiation. In the macronucleus of these cells the DNA is organized into nanochromosomes which represent independent functional units. Therefore, ciliated protozoa represent a simplistic model system to analyse the relevance of histone modifications and their localization for gene expression and differentiation. RESULTS: We analysed the distribution of three PTMs on six individual nanochromosomes, two of which are silenced in the vegetative cell and only activated during sexual reproduction. We show that a specific relocation of these PTMs correlates with gene activation. Moreover, macronuclear-destined sequences in the differentiating macronucleus display a distribution of PTMs which differs significantly from the PTM patterns of actively transcribed genes. CONCLUSION: We show for the first time that a relocation of specific histone modifications takes place during activation of genes. In addition, we demonstrate that genes in a differentiating nucleus are characterised by a specific distribution and composition of PTMs. This allows us to propose a mechanistic model about the relevance of PTMs for gene activation, gene silencing and nuclear differentiation. Results described here will be relevant for eukaryotic cells in general

Spatial and temporal plasticity of chromatin during programmed DNA-reorganization in Stylonychia macronuclear development

Background: In this study we exploit the unique genome organization of ciliates to characterize the biological function of histone modification patterns and chromatin plasticity for the processing of specific DNA sequences during a nuclear differentiation process. Ciliates are single-cell eukaryotes containing two morphologically and functionally specialized types of nuclei, the somatic macronucleus and the germline micronucleus. In the course of sexual reproduction a new macronucleus develops from a micronuclear derivative. During this process specific DNA sequences are eliminated from the genome, while sequences that will be transcribed in the mature macronucleus are retained. Results: We show by immunofluorescence microscopy, Western analyses and chromatin immunoprecipitation (ChIP) experiments that each nuclear type establishes its specific histone modification signature. Our analyses reveal that the early macronuclear anlage adopts a permissive chromatin state immediately after the fusion of two heterochromatic germline micronuclei. As macronuclear development progresses, repressive histone modifications that specify sequences to be eliminated are introduced de novo. ChIP analyses demonstrate that permissive histone modifications are associated with sequences that will be retained in the new macronucleus. Furthermore, our data support the hypothesis that a PIWI-family protein is involved in a transnuclear cross-talk and in the RNAi-dependent control of developmental chromatin reorganization. Conclusion: Based on these data we present a comprehensive analysis of the spatial and temporal pattern of histone modifications during this nuclear differentiation process. Results obtained in this study may also be relevant for our understanding of chromatin plasticity during metazoan embryogenesis

Control of Gastric H,K-ATPase Activity by Cations, Voltage and Intracellular pH Analyzed by Voltage Clamp Fluorometry in Xenopus Oocytes

Whereas electrogenic partial reactions of the Na,K-ATPase have been studied in depth, much less is known about the influence of the membrane potential on the electroneutrally operating gastric H,K-ATPase. In this work, we investigated site-specifically fluorescence-labeled H,K-ATPase expressed in Xenopus oocytes by voltage clamp fluorometry to monitor the voltage-dependent distribution between E1P and E2P states and measured Rb+ uptake under various ionic and pH conditions. The steady-state E1P/E2P distribution, as indicated by the voltage-dependent fluorescence amplitudes and the Rb+ uptake activity were highly sensitive to small changes in intracellular pH, whereas even large extracellular pH changes affected neither the E1P/E2P distribution nor transport activity. Notably, intracellular acidification by approximately 0.5 pH units shifted V0.5, the voltage, at which the E1P/E2P ratio is 50∶50, by −100 mV. This was paralleled by an approximately two-fold acceleration of the forward rate constant of the E1P→E2P transition and a similar increase in the rate of steady-state cation transport. The temperature dependence of Rb+ uptake yielded an activation energy of ∼90 kJ/mol, suggesting that ion transport is rate-limited by a major conformational transition. The pronounced sensitivity towards intracellular pH suggests that proton uptake from the cytoplasmic side controls the level of phosphoenzyme entering the E1P→E2P conformational transition, thus limiting ion transport of the gastric H,K-ATPase. These findings highlight the significance of cellular mechanisms contributing to increased proton availability in the cytoplasm of gastric parietal cells. Furthermore, we show that extracellular Na+ profoundly alters the voltage-dependent E1P/E2P distribution indicating that Na+ ions can act as surrogates for protons regarding the E2P→E1P transition. The complexity of the intra- and extracellular cation effects can be rationalized by a kinetic model suggesting that cations reach the binding sites through a rather high-field intra- and a rather low-field extracellular access channel, with fractional electrical distances of ∼0.5 and ∼0.2, respectively

Sex difference and intra-operative tidal volume: Insights from the LAS VEGAS study

BACKGROUND: One key element of lung-protective ventilation is the use of a low tidal volume (VT). A sex difference in use of low tidal volume ventilation (LTVV) has been described in critically ill ICU patients.OBJECTIVES: The aim of this study was to determine whether a sex difference in use of LTVV also exists in operating room patients, and if present what factors drive this difference.DESIGN, PATIENTS AND SETTING: This is a posthoc analysis of LAS VEGAS, a 1-week worldwide observational study in adults requiring intra-operative ventilation during general anaesthesia for surgery in 146 hospitals in 29 countries.MAIN OUTCOME MEASURES: Women and men were compared with respect to use of LTVV, defined as VT of 8 ml kg-1 or less predicted bodyweight (PBW). A VT was deemed 'default' if the set VT was a round number. A mediation analysis assessed which factors may explain the sex difference in use of LTVV during intra-operative ventilation.RESULTS: This analysis includes 9864 patients, of whom 5425 (55%) were women. A default VT was often set, both in women and men; mode VT was 500 ml. Median [IQR] VT was higher in women than in men (8.6 [7.7 to 9.6] vs. 7.6 [6.8 to 8.4] ml kg-1 PBW, P < 0.001). Compared with men, women were twice as likely not to receive LTVV [68.8 vs. 36.0%; relative risk ratio 2.1 (95% CI 1.9 to 2.1), P < 0.001]. In the mediation analysis, patients' height and actual body weight (ABW) explained 81 and 18% of the sex difference in use of LTVV, respectively; it was not explained by the use of a default VT.CONCLUSION: In this worldwide cohort of patients receiving intra-operative ventilation during general anaesthesia for surgery, women received a higher VT than men during intra-operative ventilation. The risk for a female not to receive LTVV during surgery was double that of males. Height and ABW were the two mediators of the sex difference in use of LTVV.TRIAL REGISTRATION: The study was registered at Clinicaltrials.gov, NCT01601223

Discovery and Optimization of 1‑Phenoxy-2-aminoindanes as Potent, Selective, and Orally Bioavailable Inhibitors of the Na⁺/H⁺ Exchanger Type 3 (NHE3)

The design, synthesis, and structure–activity relationship of 1-phenoxy-2-aminoindanes as inhibitors of the Na⁺/H⁺ exchanger type 3 (NHE3) are described based on a hit from high-throughput screening (HTS). The chemical optimization resulted in the discovery of potent, selective, and orally bioavailable NHE3 inhibitors with <b>13d</b> as best compound, showing high in vitro permeability and lacking CYP2D6 inhibition as main optimization parameters. Aligning 1-phenoxy-2-aminoindanes onto the X-ray structure of <b>13d</b> then provided 3D-QSAR models for NHE3 inhibition capturing guidelines for optimization. These models showed good correlation coefficients and allowed for activity estimation. In silico ADMET models for Caco-2 permeability and CYP2D6 inhibition were also successfully applied for this series. Moreover, docking into the CYP2D6 X-ray structure provided a reliable alignment for 3D-QSAR models. Finally <b>13d</b>, renamed as SAR197, was characterized in vitro and by in vivo pharmacokinetic (PK) and pharmacological studies to unveil its potential for reduction of obstructive sleep apneas

Discovery and Optimization of 1‑Phenoxy-2-aminoindanes as Potent, Selective, and Orally Bioavailable Inhibitors of the Na⁺/H⁺ Exchanger Type 3 (NHE3)

The design, synthesis, and structure–activity relationship of 1-phenoxy-2-aminoindanes as inhibitors of the Na⁺/H⁺ exchanger type 3 (NHE3) are described based on a hit from high-throughput screening (HTS). The chemical optimization resulted in the discovery of potent, selective, and orally bioavailable NHE3 inhibitors with <b>13d</b> as best compound, showing high in vitro permeability and lacking CYP2D6 inhibition as main optimization parameters. Aligning 1-phenoxy-2-aminoindanes onto the X-ray structure of <b>13d</b> then provided 3D-QSAR models for NHE3 inhibition capturing guidelines for optimization. These models showed good correlation coefficients and allowed for activity estimation. In silico ADMET models for Caco-2 permeability and CYP2D6 inhibition were also successfully applied for this series. Moreover, docking into the CYP2D6 X-ray structure provided a reliable alignment for 3D-QSAR models. Finally <b>13d</b>, renamed as SAR197, was characterized in vitro and by in vivo pharmacokinetic (PK) and pharmacological studies to unveil its potential for reduction of obstructive sleep apneas

Production of medically useful bromine isotopes via alpha-induced nuclear reactions

The cross sections of α-particle induced reactions on arsenic leading to the formation of 76,77,78Br were measured from their respective thresholds up to 37 MeV. Thin sediments of elemental arsenic powder were irradiated together with Al degrader and Cu monitor foils using the established stacked-foil technique. For determination of the effective α-particle energies and of the effective beam current through the stacks the cross-section ratios of the monitor nuclides 67Ga/66Ga were used. This should help resolve discrepancies in existing literature data. Comparison of the data with the available excitation functions shows some slight energy shifts as well as some differences in curve shapes. The calculated thick target yields indicate, that 77Br can be produced in the energy range Eα = 25 → 17 MeV free of isotopic impurities in quantities sufficient for medical application

Simulation-Based Training of the Rapid Evaluation and Management of Acute Stroke (STREAM)-A Prospective Single-Arm Multicenter Trial

Introduction: Acute stroke care delivered by interdisciplinary teams is time-sensitive. Simulation-based team training is a promising tool to improve team performance in medical operations. It has the potential to improve process times, team communication, patient safety, and staff satisfaction. We aim to assess whether a multi-level approach consisting of a stringent workflow revision based on peer-to-peer review and 2-3 one-day in situ simulation trainings can improve acute stroke care processing times in high volume neurocenters within a 6 months period. Methods and Analysis: The trial is being carried out in a pre-test-post-test design at 7 tertiary care university hospital neurocenters in Germany. The intervention is directed at the interdisciplinary multiprofessional stroke teams. Before and after the intervention, process times of all direct-to-center stroke patients receiving IV thrombolysis (IVT) and/or endovascular therapy (EVT) will be recorded. The primary outcome measure will be the door-to-needle time of all consecutive stroke patients directly admitted to the neurocenters who receive IVT. Secondary outcome measures will be intervention-related process times of the fraction of patients undergoing EVT and effects on team communication, perceived patient safety, and staff satisfaction via a staff questionnaire. Interventions: We are applying a multi-level intervention in cooperation with three STREAM multipliers from each center. First step is a central meeting of the multipliers at the sponsor's institution with the purposes of algorithm review in a peer-to-peer process that is recorded in a protocol and an introduction to the principles of simulation training and debriefing as well as crew resource management and team communication. Thereafter, the multipliers cooperate with the stroke team trainers from the sponsor's institution to plan and execute 2-3 one-day simulation courses in situ in the emergency department and CT room of the trial centers whereupon they receive teaching materials to perpetuate the trainings