Tadalafil for Treatment of Combined Postcapillary and Precapillary Pulmonary Hypertension in Patients With Heart Failure and Preserved Ejection Fraction: A Randomized Controlled Phase 3 Study.

BACKGROUND: We assessed the efficacy and safety of tadalafil, a phosphodiesterase type 5 inhibitor, in patients with heart failure with preserved ejection fraction and combined postcapillary and precapillary pulmonary hypertension. METHODS: In the double-blind PASSION study (Phosphodiesterase-5 Inhibition in Patients With Heart Failure With Preserved Ejection Fraction and Combined Post- and Pre-Capillary Pulmonary Hypertension), patients with heart failure with preserved ejection fraction and combined postcapillary and precapillary pulmonary hypertension were randomized 1:1 to receive tadalafil at a target dose of 40 mg or placebo. The primary end point was the time to the first composite event of adjudicated heart failure hospitalization or all-cause death. Secondary end points included all-cause mortality and improvements in New York Heart Association functional class or ≥10% improvement in 6-minute walking distance from baseline. RESULTS: Initially targeting 372 patients, the study was terminated early because of disruption in study medication supply. At that point, 125 patients had been randomized (placebo: 63; tadalafil: 62,). Combined primary end-point events occurred in 20 patients (32%) assigned to placebo and 17 patients (27%) assigned to tadalafil (hazard ratio, 1.02 [95% CI, 0.52-2.01]; P=0.95). There was a possible signal of higher all-cause mortality in the tadalafil group (hazard ratio, 5.10 [95% CI, 1.10-23.69]; P=0.04). No significant between-group differences were observed in other secondary end points. Serious adverse events occurred in 29 participants (48%) in the tadalafil group and 35 (56%) in the placebo group. CONCLUSIONS: The PASSION trial, terminated prematurely due to study medication supply disruption, does not support tadalafil use in patients with heart failure with preserved ejection fraction and combined postcapillary and precapillary pulmonary hypertension, with potential safety concerns and no observed benefits in primary and secondary end points. REGISTRATION: URL: https://www.clinicaltrialsregister.eu/; Unique identifier: 2017-003688-37. URL: https://drks.de; Unique identifier: DRKS -DRKS00014595.

Treatment strategies and survival of patients with connective tissue disease and pulmonary arterial hypertension: a COMPERA analysis.

OBJECTIVES: Pulmonary arterial hypertension (PAH) occurs in various connective tissue diseases (CTDs). We sought to assess contemporary treatment patterns and survival of patients with various forms of CTD-PAH. METHODS: We analysed data from COMPERA, a European pulmonary hypertension registry, to describe treatment strategies and survival in patients with newly diagnosed PAH associated with SSc, SLE, MCTD, UCTD and other types of CTD. All-cause mortality was analysed according to the underlying CTD. For patients with SSc-PAH, we also assessed survival according to initial therapy with endothelin receptor antagonists (ERAs), phosphodiesterase type 5 inhibitors (PDE5is) or a combination of these two drug classes. RESULTS: This analysis included 607 patients with CTD-PAH. Survival estimates at 1, 3 and 5 years for SSc-PAH (n = 390) were 85%, 59% and 42%; for SLE-PAH (n = 34) they were 97%, 77% and 61%; for MCTD-PAH (n = 33) they were 97%, 70% and 59%; for UCTD-PAH (n = 60) they were 88%, 67% and 52%; and for other CTD-PAH (n = 90) they were 92%, 69% and 55%, respectively. After multivariable adjustment, the survival of patients with SSc-PAH was significantly worse compared with the other conditions (P = 0.001). In these patients, the survival estimates were significantly better with initial ERA-PDE5i combination therapy than with initial ERA or PDE5i monotherapy (P = 0.016 and P = 0.012, respectively). CONCLUSIONS: Mortality remains high in patients with CTD-PAH, especially for patients with SSc-PAH. However, for patients with SSc-PAH, our results suggest that long-term survival may be improved with initial ERA-PDE5i combination therapy compared with initial monotherapy.

Chronic thromboembolic pulmonary hypertension and impairment after pulmonary embolism: the FOCUS study.

AIMS: To systematically assess late outcomes of acute pulmonary embolism (PE) and to investigate the clinical implications of post-PE impairment (PPEI) fulfilling prospectively defined criteria. METHODS AND RESULTS: A prospective multicentre observational cohort study was conducted in 17 large-volume centres across Germany. Adult consecutive patients with confirmed acute symptomatic PE were followed with a standardized assessment plan and pre-defined visits at 3, 12, and 24 months. The co-primary outcomes were (i) diagnosis of chronic thromboembolic pulmonary hypertension (CTEPH), and (ii) PPEI, a combination of persistent or worsening clinical, functional, biochemical, and imaging parameters during follow-up. A total of 1017 patients (45% women, median age 64 years) were included in the primary analysis. They were followed for a median duration of 732 days after PE diagnosis. The CTEPH was diagnosed in 16 (1.6%) patients, after a median of 129 days; the estimated 2-year cumulative incidence was 2.3% (1.2-4.4%). Overall, 880 patients were evaluable for PPEI; the 2-year cumulative incidence was 16.0% (95% confidence interval 12.8-20.8%). The PPEI helped to identify 15 of the 16 patients diagnosed with CTEPH during follow-up (hazard ratio for CTEPH vs. no CTEPH 393; 95% confidence interval 73-2119). Patients with PPEI had a higher risk of re-hospitalization and death as well as worse quality of life compared with those without PPEI. CONCLUSION: In this prospective study, the cumulative 2-year incidence of CTEPH was 2.3%, but PPEI diagnosed by standardized criteria was frequent. Our findings support systematic follow-up of patients after acute PE and may help to optimize guideline recommendations and algorithms for post-PE care.

COMPERA 2.0: a refined four-stratum risk assessment model for pulmonary arterial hypertension.

BACKGROUND: Risk stratification plays an essential role in the management of patients with pulmonary arterial hypertension (PAH). The current European guidelines propose a three-stratum model to categorise risk as low, intermediate or high, based on the expected 1-year mortality. However, with this model, most patients are categorised as intermediate risk. We investigated a modified approach based on four risk categories, with intermediate risk subdivided into intermediate-low and intermediate-high risk. METHODS: We analysed data from the Comparative, Prospective Registry of Newly Initiated Therapies for Pulmonary Hypertension (COMPERA), a European pulmonary hypertension registry, and calculated risk at diagnosis and first follow-up based on World Health Organization functional class, 6-min walk distance (6MWD) and serum levels of brain natriuretic peptide (BNP) or N-terminal pro-BNP (NT-proBNP), using refined cut-off values. Survival was assessed using Kaplan-Meier analyses, log-rank testing and Cox proportional hazards models. RESULTS: Data from 1655 patients with PAH were analysed. Using the three-stratum model, most patients were classified as intermediate risk (76.0% at baseline and 63.9% at first follow-up). The refined four-stratum risk model yielded a more nuanced separation and predicted long-term survival, especially at follow-up assessment. Changes in risk from baseline to follow-up were observed in 31.1% of the patients with the three-stratum model and in 49.2% with the four-stratum model. These changes, including those between the intermediate-low and intermediate-high strata, were associated with changes in long-term mortality risk. CONCLUSIONS: Modified risk stratification using a four-stratum model based on refined cut-off levels for functional class, 6MWD and BNP/NT-proBNP was more sensitive to prognostically relevant changes in risk than the original three-stratum model.

Temporal trends in pulmonary arterial hypertension: results from the COMPERA registry.

BACKGROUND: Since 2015, the European pulmonary hypertension guidelines recommend the use of combination therapy in most patients with pulmonary arterial hypertension (PAH). However, it is unclear to what extent this treatment strategy is adopted in clinical practice and if it is associated with improved long-term survival. METHODS: We analysed data from COMPERA, a large European pulmonary hypertension registry, to assess temporal trends in the use of combination therapy and survival of patients with newly diagnosed PAH between 2010 and 2019. For survival analyses, we looked at annualised data and at cumulated data comparing the periods 2010-2014 and 2015-2019. RESULTS: A total of 2531 patients were included. The use of early combination therapy (within 3 months after diagnosis) increased from 10.0% in patients diagnosed with PAH in 2010 to 25.0% in patients diagnosed with PAH in 2019. The proportion of patients receiving combination therapy 1 year after diagnosis increased from 27.7% to 46.3%. When comparing the 2010-2014 and 2015-2019 periods, 1-year survival estimates were similar (89.0% (95% CI 87.2-90.9%) and 90.8% (95% CI 89.3-92.4%), respectively), whereas there was a slight but nonsignificant improvement in 3-year survival estimates (67.8% (95% CI 65.0-70.8%) and 70.5% (95% CI 67.8-73.4%), respectively). CONCLUSIONS: The use of combination therapy increased from 2010 to 2019, but most patients still received monotherapy. Survival rates at 1 year after diagnosis did not change over time. Future studies need to determine if the observed trend suggesting improved 3-year survival rates can be confirmed.

Backbone NMR reveals allosteric signal transduction networks in the ß1-adrenergic receptor.

G protein-coupled receptors (GPCRs) are physiologically important transmembrane signalling proteins that trigger intracellular responses upon binding of extracellular ligands. Despite recent breakthroughs in GPCR crystallography, the details of ligand-induced signal transduction are not well understood owing to missing dynamical information. In principle, such information can be provided by NMR, but so far only limited data of functional relevance on few side-chain sites of eukaryotic GPCRs have been obtained. Here we show that receptor motions can be followed at virtually any backbone site in a thermostabilized mutant of the turkey ß1-adrenergic receptor (ß1AR). Labelling with [(15)N]valine in a eukaryotic expression system provides over twenty resolved resonances that report on structure and dynamics in six ligand complexes and the apo form. The response to the various ligands is heterogeneous in the vicinity of the binding pocket, but gets transformed into a homogeneous readout at the intracellular side of helix 5 (TM5), which correlates linearly with ligand efficacy for the G protein pathway. The effect of several pertinent, thermostabilizing point mutations was assessed by reverting them to the native sequence. Whereas the response to ligands remains largely unchanged, binding of the G protein mimetic nanobody NB80 and G protein activation are only observed when two conserved tyrosines (Y227 and Y343) are restored. Binding of NB80 leads to very strong spectral changes throughout the receptor, including the extracellular ligand entrance pocket. This indicates that even the fully thermostabilized receptor undergoes activating motions in TM5, but that the fully active state is only reached in presence of Y227 and Y343 by stabilization with a G protein-like partner. The combined analysis of chemical shift changes from the point mutations and ligand responses identifies crucial connections in the allosteric activation pathway, and presents a general experimental method to delineate signal transmission networks at high resolution in GPCRs.

[Risk Stratification in Patients with Pulmonary Arterial Hypertension under Treatment - Results of Four German Centers]. / Risikoänderung bei Patienten mit Pulmonaler Arterieller Hypertonie unter medikamentöser Therapie ­ Ergebnisse aus vier deutschen Zentren.

Risk stratification plays an essential role in the management of patients with pulmonary arterial hypertension (PAH). According to the current European guidelines the expected 1-year risk of mortality for PAH patients can be categorized as low, intermediate, or high, based on clinical, non-invasive and hemodynamic data.Data from 131 patients with incident PAH (age 64 ±â€Š14) and frequent comorbidities (in 66.4 %) treated between 2016 and 2018 at 4 German PH centers were analyzed. At baseline, most patients were classified as intermediate risk (76 %), 13.8 % as high risk and only 9.9 % as low risk.During follow-up while on treatment after 12 ±â€Š3 months (range 9-16 months) 64.9 % were still classified as intermediate risk (76 %), 14.4 % as high risk and 20.7 % as low risk.Survival at 12 and 24 months was 96 % and 82 % in the intermediate risk group, while only 89 % and 51 % of the high risk patients were alive at these time points. In contrast, all patients in the low risk category were alive at 24 months.Despite the availability of various treatment options for patients with PAH even in specialized centers only a minority of patients can be stabilized in the low risk group associated with a good outcome.

[Invasive Cardiopulmonary Exercise Testing: A Review]. / Übersicht über die Entwicklung der invasiven Spiroergometrie (iCPET).

Right heart catheterization (RHC) is the internationally standardized reference method for measuring pulmonary hemodynamics under resting conditions. In recent years, increasing efforts have been made to establish the reliable assessment of exercise hemodynamics as well, in order to obtain additional diagnostic and prognostic data. Furthermore, cardiopulmonary exercise testing (CPET), as the most comprehensive non-invasive exercise test, is increasingly performed in combination with RHC providing detailed pathophysiological insights into the exercise response, so-called invasive cardiopulmonary exercise testing (iCPET).In this review, the accumulated experience with iCPET is presented and methodological details are discussed. This complex examination is especially helpful in differentiating the underlying causes of unexplained dyspnea. In particular, early forms of cardiac or pulmonary vascular dysfunction can be detected by integrated analysis of hemodynamic as well as ventilatory and gas exchange data. It is expected that with increasing validation of iCPET parameters, a more reliable differentiation of normal from pathological stress reactions will be possible.

Monotherapy in patients with pulmonary arterial hypertension at four German PH centres.

BACKGROUND: Although combination therapy is the gold standard for patients with pulmonary arterial hypertension (PAH), some of these patients are still being treated with monotherapy. METHODS: We conducted a retrospective analysis at four German PH centres to describe the prevalence and characteristics of patients receiving monotherapy. RESULTS: We identified 131 incident PAH patients, with a mean age of 64 ± 13.8 years and a varying prevalence of comorbidities, cardiovascular risk factors and targeted therapy. As in other studies, the extent of prescribed PAH therapy varied with age and coexisting diseases, and younger, so-called "typical" PAH patients were more commonly treated early with combination therapy (48% at 4-8 months). In contrast, patients with multiple comorbidities or cardiovascular risk factors were more often treated with monotherapy (69% at 4-8 months). Survival at 12 months was not significantly associated with the number of PAH drugs used (single, dual, triple therapy) and was not different between "atypical" and "typical" PAH patients (89% vs. 85%). CONCLUSION: Although "atypical" PAH patients with comorbidities or a more advanced age are less aggressively treated with respect to combination therapy, the outcome of monotherapy in these patients appears to be comparable to that of dual or triple therapy in "typical" PAH patients.

Deuterium induces a distinctive Escherichia coli proteome that correlates with the reduction in growth rate.

Substitution of protium (H) for deuterium (D) strongly affects biological systems. Whereas higher eukaryotes such as plants and mammals hardly survive a deuterium content of >30%, many microorganisms can grow on fully deuterated media, albeit at reduced rates. Very little is known about how the H/D replacement influences life at the systems level. Here, we used MS-based analysis to follow the adaptation of a large part of the Escherichia coli proteome from growth on a protonated full medium, over a protonated minimal medium, to a completely deuterated minimal medium. We could quantify >1800 proteins under all conditions, several 100 of which exhibited strong regulation during both adaptation processes. The adaptation to minimal medium strongly up-regulated amino acid synthesis and sugar metabolism and down-regulated translational proteins on average by 9%, concomitant with a reduction in growth rate from 1.8 to 0.67 h-1 In contrast, deuteration caused a very wide proteomic response over many cell functional categories, together with an additional down-regulation of the translational proteins by 5%. The latter coincided with a further reduction in growth rate to 0.37 h-1, revealing a clear linear correlation between growth rate and abundance of translational proteins. No significant morphological effects are observed under light and electron microscopies. Across all protein categories, about 80% of the proteins up-regulated under deuteration are enzymes with hydrogen transfer functions. Thus, the H/D kinetic isotope effect appears as the major limiting factor of cellular functions under deuteration.

Rapid determination of general cell status, cell viability, and optimal harvest time in eukaryotic cell cultures by impedance flow cytometry.

The determination of cell viability is essential to many areas of life sciences and biotechnology. Typically, cell viability measurements are based on the optical analysis of stained cells, which requires additional labeling steps and is hard to implement online. Frequency-dependent impedance flow cytometry (IFC) provides a label-free, fast, and reliable alternative to determine cell viability at the single cell level based on the Coulter principle. Here, we describe the application of IFC to eukaryotic cell cultures and compare the results to commonly used staining methods. Yeast cell parameters were assessed in normal and heat-inactivated cells as well as in alcoholic fermentation and long-term batch cultures providing a precise and fast determination of the cell viability and further quantitative measures of the cell culture status. As an important new application, we have investigated recombinant protein production in the widely used baculovirus insect cell expression system. The IFC analysis revealed the presence of a subpopulation of cells, which correlates with the protein expression yield, but it is not detectable with conventional optical cell counters. We tentatively identify this subpopulation as cells in the late phase of infection. Their detection can serve as a predictor for the optimal time point of harvest. The IFC technique should be generally applicable to many eukaryotic cell cultures in suspension, possibly also implemented online.

Production of isotope-labeled proteins in insect cells for NMR.

Baculovirus-infected insect cells have become a powerful tool to express recombinant proteins for structural and functional studies by NMR spectroscopy. This article provides an introduction into the insect cell/baculovirus expression system and its use for the production of recombinant isotope-labeled proteins. We discuss recent advances in inexpensive isotope-labeling methods using labeled algal or yeast extracts as the amino acid source and give examples of advanced NMR applications for proteins, which have become accessible by this eukaryotic expression host.

Risk assessment in medically treated chronic thromboembolic pulmonary hypertension patients.

Abbreviated versions of the risk stratification strategy of the European Society of Cardiology (ESC)/European Respiratory Society (ERS) pulmonary hypertension guidelines have been recently validated in patients with pulmonary arterial hypertension. We aimed to investigate their prognostic value in medically treated chronic thromboembolic pulmonary hypertension (CTEPH) patients from the COMPERA registry, which collects six variables of interest (World Health Organization Functional Class, 6-min walk distance, brain natriuretic peptide, right atrial pressure, cardiac index and mixed venous oxygen saturation).We included patients with at least one follow-up visit, no pulmonary endarterectomy and at least three of the six variables available, and classified the patients into low-, intermediate- and high-risk groups. As a secondary analysis, the number of noninvasive low-risk criteria was counted. The association between risk assessment and survival was evaluated.Data from inclusion and follow-up (median 7 months) visits were available for 561 and 231 patients, respectively. Baseline 1- and 5-year survival estimates were significantly different (p<0.0001) in the baseline low-risk (98.6% and 88.3%, respectively), intermediate-risk (94.9% and 61.8%, respectively) and high-risk (75.5% and 32.9%, respectively) cohorts. Follow-up data were even more discriminative, with 100%, 92% and 69% 1-year survival, respectively. The number of low-risk noninvasive criteria was also associated with survival.These analyses suggest that the ESC/ERS risk assessment may be applicable in patients with medically treated CTEPH.

Right Heart Catheterization During Exercise in Patients with COPD-An Overview of Clinical Results and Methodological Aspects.

While right heart catheterization (RHC) at rest is the gold standard to assess pulmonary hemodynamics in patients with chronic obstructive pulmonary disease (COPD) and pulmonary hypertension (PH), the invasive measurement of exercise hemodynamics is less well established in this group. Since exercise hemodynamics are increasingly recognized as important clinical information in patients with PH, our goal was to review the literature in this field to provide a basis for clinical use, further studies, and future recommendations. We identified 69 studies (published since 1968) reporting RHC data in 2819 patients with COPD, of whom 2561 underwent exercise testing. Few studies simultaneously measured gas exchange during exercise. Overall, these studies showed large variations in the patient populations and research questions studied and the methods and definitions employed. Despite these limitations, the data consistently demonstrated the presence of precapillary PH at rest in up to 38% of patients with COPD. With exercise, a relevant proportion of patients developed an abnormal hemodynamic response, depending on the definition used. Furthermore, some studies assessed right ventricular function during exercise and showed a blunted increase in right ventricular ejection fraction. Drug effects and the impact of interventional procedures were also studied. Again, due to large variations in the patients studied and the methods used, firm conclusions are difficult to derive. Despite the limitations of this dataset, several recommendations with respect to technical aspects (body position, exercise protocol, and data acquisition) can be inferred for this challenging patient population and may be helpful for further studies or recommendations.

Mortality in pulmonary arterial hypertension: prediction by the 2015 European pulmonary hypertension guidelines risk stratification model.

The 2015 European pulmonary hypertension (PH) guidelines propose a risk stratification strategy for patients with pulmonary arterial hypertension (PAH). Low-, intermediate- and high-risk strata are defined by estimated 1-year mortality risks of <5%, 5-10% and >10%, respectively. This risk assessment strategy awaits validation.We analysed data from patients with newly diagnosed PAH enrolled into COMPERA (Comparative, Prospective Registry of Newly Initiated Therapies for Pulmonary Hypertension), a European-based PH registry. An abbreviated version of the risk assessment strategy proposed by the European PH guidelines was applied, using the following variables: World Health Organization functional class, 6-min walking distance, brain natriuretic peptide or its N-terminal fragment, right atrial pressure, cardiac index and mixed venous oxygen saturation.Data from 1588 patients were analysed. Mortality rates were significantly different between the three risk strata (p<0.001 for all comparisons). In the entire patient population, the observed mortality rates 1 year after diagnosis were 2.8% in the low-risk cohort (n=196), 9.9% in the intermediate-risk cohort (n=1116) and 21.2% in the high-risk cohort (n=276). In addition, the risk assessment strategy proved valid at follow-up and in major PAH subgroups.An abbreviated version of the risk assessment strategy proposed by the current European PH guidelines provides accurate mortality estimates in patients with PAH.

Exercise training improves peak oxygen consumption and haemodynamics in patients with severe pulmonary arterial hypertension and inoperable chronic thrombo-embolic pulmonary hypertension: a prospective, randomized, controlled trial.

AIMS: The impact of exercise training on the right heart and pulmonary circulation has not yet been invasively assessed in patients with pulmonary hypertension (PH) and right heart failure. This prospective randomized controlled study investigates the effects of exercise training on peak VO2/kg, haemodynamics, and further clinically relevant parameters in PH patients. METHODS AND RESULTS: Eighty-seven patients with pulmonary arterial hypertension and inoperable chronic thrombo-embolic PH (54% female, 56 ± 15 years, 84% World Health Organization functional class III/IV, 53% combination therapy) on stable disease-targeted medication were randomly assigned to a control and training group. Medication remained unchanged during the study period. Non-invasive assessments and right heart catheterization at rest and during exercise were performed at baseline and after 15 weeks. Primary endpoint was the change in peak VO2/kg. Secondary endpoints included changes in haemodynamics. For missing data, multiple imputation and responder analyses were performed. The study results showed a significant improvement of peak VO2/kg in the training group (difference from baseline to 15 weeks: training +3.1 ± 2.7 mL/min/kg equals +24.3% vs. control -0.2 ± 2.3 mL/min/kg equals +0.9%, P < 0.001). Cardiac index (CI) at rest and during exercise, mean pulmonary arterial pressure, pulmonary vascular resistance, 6 min walking distance, quality of life, and exercise capacity significantly improved by exercise training. CONCLUSION: Low-dose exercise training at 4-7 days/week significantly improved peak VO2/kg, haemodynamics, and further clinically relevant parameters. The improvements of CI at rest and during exercise indicate that exercise training may improve the right ventricular function. Further, large multicentre trials are necessary to confirm these results.

Late outcomes after acute pulmonary embolism: rationale and design of FOCUS, a prospective observational multicenter cohort study.

Acute pulmonary embolism (PE) is a frequent cause of death and serious disability. The risk of PE-associated mortality and morbidity extends far beyond the acute phase of the disease. In earlier follow-up studies, as many as 30 % of the patients died during a follow-up period of up to 3 years, and up to 50 % of patients continued to complain of dyspnea and/or poor physical performance 6 months to 3 years after the index event. The most feared 'late sequela' of PE is chronic thromboembolic pulmonary hypertension (CTEPH), the true incidence of which remains obscure due to the large margin of error in the rates reported by mostly small, single-center studies. Moreover, the functional and hemodynamic changes corresponding to early, possibly reversible stages of CTEPH, have not been systematically investigated. The ongoing Follow-Up after acute pulmonary embolism (FOCUS) study will prospectively enroll and systematically follow, over a 2-year period and with a standardized comprehensive program of clinical, echocardiographic, functional and laboratory testing, a large multicenter prospective cohort of 1000 unselected patients (all-comers) with acute symptomatic PE. FOCUS will possess adequate power to provide answers to relevant remaining questions regarding the patients' long-term morbidity and mortality, and the temporal pattern of post-PE abnormalities. It will hopefully provide evidence for future guideline recommendations regarding the selection of patients for long-term follow-up after PE, the modalities which this follow-up should include, and the findings that should be interpreted as indicating progressive functional and hemodynamic post-PE impairment, or the development of CTEPH.

Monitoring Backbone Hydrogen-Bond Formation in ß-Barrel Membrane Protein Folding.

ß-barrel membrane proteins are key components of the outer membrane of bacteria, mitochondria and chloroplasts. Their three-dimensional structure is defined by a network of backbone hydrogen bonds between adjacent ß-strands. Here, we employ hydrogen-deuterium (H/D) exchange in combination with NMR spectroscopy and mass spectrometry to monitor backbone hydrogen bond formation during folding of the outer membrane protein X (OmpX) from E. coli in detergent micelles. Residue-specific kinetics of interstrand hydrogen-bond formation were found to be uniform in the entire ß-barrel and synchronized to formation of the tertiary structure. OmpX folding thus propagates via a long-lived conformational ensemble state in which all backbone amide protons exchange with the solvent and engage in hydrogen bonds only transiently. Stable formation of the entire OmpX hydrogen bond network occurs downhill of the rate-limiting transition state and thus appears cooperative on the overall folding time scale.

Anticoagulation and survival in pulmonary arterial hypertension: results from the Comparative, Prospective Registry of Newly Initiated Therapies for Pulmonary Hypertension (COMPERA).

BACKGROUND: For almost 30 years, anticoagulation has been recommended for patients with idiopathic pulmonary arterial hypertension (IPAH). Supporting evidence, however, is limited, and it is unclear whether this recommendation is still justified in the modern management era and whether it should be extended to patients with other forms of pulmonary arterial hypertension (PAH). METHODS AND RESULTS: We analyzed data from Comparative, Prospective Registry of Newly Initiated Therapies for Pulmonary Hypertension (COMPERA), an ongoing European pulmonary hypertension registry. Survival rates of patients with IPAH and other forms of PAH were compared by the use of anticoagulation. The sample consisted of 1283 consecutively enrolled patients with newly diagnosed PAH. Anticoagulation was used in 66% of 800 patients with IPAH and in 43% of 483 patients with other forms of PAH. In patients with IPAH, there was a significantly better 3-year survival (P=0.006) in patients on anticoagulation compared with patients who never received anticoagulation, albeit the patients in the anticoagulation group had more severe disease at baseline. The survival difference at 3 years remained statistically significant (P=0.017) in a matched-pair analysis of n=336 IPAH patients. The beneficial effect of anticoagulation on survival of IPAH patients was confirmed by Cox multivariable regression analysis (hazard ratio, 0.79; 95% confidence interval, 0.66-0.94). In contrast, the use of anticoagulants was not associated with a survival benefit in patients with other forms of PAH. CONCLUSIONS: The present data suggest that the use of anticoagulation is associated with a survival benefit in patients with IPAH, supporting current treatment recommendations. The evidence remains inconclusive for other forms of PAH. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT01347216.

An economic approach to efficient isotope labeling in insect cells using homemade 15N-, 13C- and 2H-labeled yeast extracts.

Heterologous expression of proteins in insect cells is frequently used for crystallographic structural studies due to the high yields even for challenging proteins requiring the eukaryotic protein processing capabilities of the host. However for NMR studies, the need for isotope labeling poses extreme challenges in eukaryotic hosts. Here, we describe a robust method to achieve uniform protein (15)N and (13)C labeling of up to 90 % in baculovirus-infected insect cells. The approach is based on the production of labeled yeast extract, which is subsequently supplemented to insect cell growth media. The method also allows deuteration at levels of >60 % without decrease in expression yield. The economic implementation of the labeling procedures into a standard structural biology laboratory environment is described in a step-by-step protocol. Applications are demonstrated for a variety of NMR experiments using the Abelson kinase domain, GFP, and the beta-1 adrenergic receptor as examples. Deuterated expression of the latter provides spectra of very high quality of a eukaryotic G-protein coupled receptor.