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Summary Acetogens such as Clostridium ljungdahlii can play a crucial role reducing the human CO2 footprint by converting industrial emissions containing CO2, CO and H2 into valuable products such as organic acids or alcohols. The quantitative understanding of cellular metabolism is a prerequisite to exploit the bacterial endowments and to fine‐tune the cells by applying metabolic engineering tools. Studying the three gas mixtures CO2 + H2, CO and CO + CO2 + H2 (syngas) by continuously gassed batch cultivation experiments and applying flux balance analysis, we identified CO as the preferred carbon and electron source for growth and producing alcohols. However, the total yield of moles of carbon (mol‐C) per electrons consumed was almost identical in all setups which underlines electron availability as the main factor influencing product formation. The Wood–Ljungdahl pathway (WLP) showed high flexibility by serving as the key NAD+ provider for CO2 + H2, whereas this function was strongly compensated by the transhydrogenase‐like Nfn complex when CO was metabolized. Availability of reduced ferredoxin (Fdred) can be considered as a key determinant of metabolic control. Oxidation of CO via carbon monoxide dehydrogenase (CODH) is the main route of Fdred formation when CO is used as substrate, whereas Fdred is mainly regenerated via the methyl branch of WLP and the Nfn complex utilizing CO2 + H2. Consequently, doubled growth rates, highest ATP formation rates and highest amounts of reduced products (ethanol, 2,3‐butanediol) were observed when CO was the sole carbon and electron source.

Abstract Background In vivo protein formation is a crucial part of cellular life. The process needs to adapt to growth conditions and is exploited for the production of technical and pharmaceutical proteins in microbes such as Escherichia coli. Accordingly, the elucidation of basic regulatory mechanisms controlling the in vivo translation machinery is of primary interest, not only to improve heterologous protein production but also to elucidate fundamental regulation regimens of cellular growth. Results The current modeling analysis elucidates the impact of diffusion for the stochastic supply of crucial substrates such as the elongation factor EFTu, and tRNA species, all regarded as key elements for ensuring optimum transcriptional elongation. Together with the consideration of cellular ribosome numbers, their impact on the proper functioning of the translation machinery was investigated under different in vivo and in vitro conditions and utilizing the formation of non-native GFP and native EFTu as target proteins. The results show that translational elongation was diffusion limited. However, this effect was much more pronounced for the translation of non-native proteins than for the formation of codon-optimized native proteins. Conclusions Cellular ATP requirements constrain the options of improving protein production. In the case of non-native protein sequences, an optimized tRNA supply may be the most economical solution, as cells necessarily have to invest in ATP-costly ribosome synthesis to boost translation and increase growth rates.

Clostridium ljungdahlii (C. ljungdahlii, CLJU) is natively endowed producing acetic acid, 2,3-butandiol, and ethanol consuming gas mixtures of CO2, CO, and H2 (syngas). Here, we present the syngas-based isobutanol formation using C. ljungdahlii harboring the recombinant amplification of the “Ehrlich” pathway that converts intracellular KIV to isobutanol. Autotrophic isobutanol production was studied analyzing two different strains in 3-L gassed and stirred bioreactors. Physiological characterization was thoroughly applied together with metabolic profiling and flux balance analysis. Thereof, KIV and pyruvate supply were identified as key “bottlenecking” precursors limiting preliminary isobutanol formation in CLJU[KAIA] to 0.02 g L–1. Additional blocking of valine synthesis in CLJU[KAIA]:ilvE increased isobutanol production by factor 6.5 finally reaching 0.13 g L–1. Future metabolic engineering should focus on debottlenecking NADPH availability, whereas NADH supply is already equilibrated in the current generation of strains.

Corynebacterium glutamicum is a commonly applied host for the industrial production of amino acids. While valued for its robustness, it is somewhat inferior to competing strains such as Escherichia coli because of the relatively low growth rate of 0.40 h−1 in synthetic, industrial media. Accordingly, adaptive laboratory evolution (ALE) experiments were performed in continuous cultivation mode to select for a growth-improved host. To ensure industrial attractiveness, this ALE study aimed at a reduction of dependency on costly growth-boosting additives such as protocatechuate (PCA) or complex media supplements. Consequently, double selection pressures were installed consisting of a steady increase in growth rate demands and a parallel reduction of complex medium fractions. Selection yielded C. glutamicum EVO5 achieving 0.54 h−1 and 1.03 gGlc gCDW−1 h−1 in minimal medium without abovementioned supplements. Sequencing revealed 10 prominent mutations, three of them in key regulator genes.

The growth rate (μ) of industrially relevant microbes, such as Corynebacterium glutamicum, is a fundamental property that indicates its production capacity. Therefore, understanding the mechanism underlying the growth rate is imperative for improving productivity and performance through metabolic engineering. Despite recent progress in the understanding of global regulatory interactions, knowledge of mechanisms directing cell growth remains fragmented and incomplete. The current study investigated RNA-Seq data of three growth rate transitions, induced by different pre-culture conditions, in order to identify transcriptomic changes corresponding to increasing growth rates. These transitions took place in minimal medium and ranged from 0.02 to 0.4 h-1 μ. This study enabled the identification of 447 genes as components of the growth modulon. Enrichment of genes within the growth modulon revealed 10 regulons exhibiting a significant effect over growth rate transition. In summary, central metabolism was observed to be regulated by a combination of metabolic and transcriptional activities orchestrating control over glycolysis, pentose phosphate pathway, and the tricarboxylic acid cycle. Additionally, major responses to changes in the growth rate were linked to iron uptake and carbon metabolism. In particular, genes encoding glycolytic enzymes and the glucose uptake system showed a positive correlation with the growth rate.

To ensure economic competitiveness, bioprocesses should achieve maximum productivities enabled by high growth rates (μ) and equally high substrate consumption rates (qS) as a prerequisite of sufficient carbon-to-product conversion. Both traits were investigated and improved via bioprocess engineering approaches studying the industrial work horse Corynebacterium glutamicum. Standard minimal medium CGXII with glucose as sole carbon source was supplemented with complex brain-heart-infusion (BHI) or amino acid (AA) cocktails. Maximum μ of 0.67 h-1 was exclusively observed in 37 g BHI L-1 whereas only minor growth stimulation was found after AA supplementation (μ = 0.468 h-1). Increasing glucose consumption rates (qGlc) were solely observed in certain dosages of BHI (1–10 g L-1), while 37 g BHI L-1 and AA addition revealed qGlc below the reference experiments. Moreover, BHI supplementation revealed Monod-type saturation kinetics of μ (KBHI = 2.73 g BHI L-1) referring to the preference of non-AAs as key boosting nutrients. ATP-demands under reference, 1 g BHI L-1, and AA conditions were nearly constant but halved in BHI concentrations above 5 g L-1 reflecting the energetic advantage of consuming complex nutrient components in addition to “simple” building blocks such as AAs. Furthermore, C. glutamicum revealed maximum biomass per carbon yields of about 18 gCDW C-mol-1 irrespective of the medium. In AA supplementation experiments, simultaneous uptake of 17 AAs was observed, maximum individual consumption rates determined, and L-asparagine and L-glutamine were distinguished as compounds with the highest consumption rates. Employment of the expanded stoichiometric model iMG481 successfully reproduced experimental results and revealed the importance of C. glutamicum’s transaminase network to compensate needs of limiting AA supply. Model-based sensitivity studies attributed the highest impact on μ to AAs with high ATP and NADPH demands such as L-tryptophan or L-phenylalanine.

Rapidly changing concentrations of substrates frequently occur during large-scale microbial cultivations. These changing conditions, caused by large mixing times, result in a heterogeneous population distribution. Here, we present a powerful and efficient modeling approach to predict the influence of varying substrate levels on the transcriptional and translational response of the cell. This approach consists of two parts, a single-cell model to describe transcription and translation for an exemplary operon (trp operon) and a second part to characterize cell distribution during the experimental setup. Combination of both models enables prediction of transcriptional patterns for the whole population. In summary, the resulting model is not only able to anticipate the experimentally observed short-term and long-term transcriptional response, it further allows envision of altered protein levels. Our model shows that locally induced stress responses propagate throughout the bioreactor, resulting in temporal, and spatial population heterogeneity. Stress induced transcriptional response leads to a new population steady-state shortly after imposing fluctuating substrate conditions. In contrast, the protein levels take more than 10 h to achieve steady-state conditions.

Abstract Aims We aim to explore the relationship of heart failure (HF) and diabetes with cardiovascular (CV) death or hospitalization for HF (HHF) and to study the clinical utility of N‐terminal pro‐B‐type natriuretic peptide (NT‐proBNP) in an unselected patient population with atrial fibrillation (AF). Methods and results Patients with AF admitted to a tertiary academic center between January 2005 and July 2019 were identified through a search of electronic health records. We used Cox regression models adjusted for age, sex, estimated glomerular filtration rate, diabetes, HF, body mass index, prior myocardial infarction, coronary artery disease, hypertension, smoking, C‐reactive protein, and low‐density lipoprotein cholesterol. To select the most informative variables, we performed a least absolute shrinkage and selection operator Cox regression with 10‐fold cross‐validation. In total, 7412 patients (median age 70 years, 39.7% female) were included in this analysis and followed over a median of 4.5 years. Both diabetes [adjusted (Adj.) HR 1.87, 95% CI 1.55–2.25] and HF (Adj. HR 2.57, 95% CI 2.22–2.98) were significantly associated with CV death/HHF after multivariable adjustment. Compared with patients with diabetes, HF patients had a higher risk of HHF but a similar risk of CV and all‐cause death. NT‐proBNP showed good discriminatory performance (area under the curve 0.78, 95% CI 0.77–0.80) and the addition of NT‐proBNP to the covariates used for adjustment resulted in a significant area under the curve improvement (Δ = 0.04, P

Background Fibroblast growth factor 23 (FGF‐23) is crucial in regulating phosphate and vitamin D metabolism and is moreover associated with an increased cardiovascular risk. The specific objective of this study was to investigate the influence of FGF‐23 on cardiovascular outcomes, including hospitalization for heart failure (HHF), postoperative atrial fibrillation, and cardiovascular death, in an unselected patient population after cardiac surgery. Methods and Results Patients undergoing elective coronary artery bypass graft and/or cardiac valve surgery were prospectively enrolled. FGF‐23 blood plasma concentrations were assessed before surgery. A composite of cardiovascular death/HHF was chosen as primary end point. A total of 451 patients (median age 70 years; 28.8% female) were included in the present analysis and followed over a median of 3.9 years. Individuals with higher FGF‐23 quartiles showed elevated incidence rates of the composite of cardiovascular death/HHF (quartile 1, 7.1%; quartile 2, 8.6%; quartile 3, 15.1%; and quartile 4, 34.3%). After multivariable adjustment, FGF‐23 modeled as a continuous variable (adjusted hazard ratio for a 1‐unit increase in standardized log‐transformed biomarker, 1.82 [95% CI, 1.34–2.46]) as well as using predefined risk groups and quartiles remained independently associated with the risk of cardiovascular death/HHF and the secondary outcomes, including postoperative atrial fibrillation. Reclassification analysis indicated that the addition of FGF‐23 to N‐terminal pro‐B‐type natriuretic peptide provides a significant improvement in risk discrimination (net reclassification improvement at the event rate, 0.58 [95% CI, 0.34–0.81]; P

Abstract Aims The HERMES (HEart failure Molecular Epidemiology for Therapeutic targetS) consortium aims to identify the genomic and molecular basis of heart failure. Methods and results The consortium currently includes 51 studies from 11 countries, including 68 157 heart failure cases and 949 888 controls, with data on heart failure events and prognosis. All studies collected biological samples and performed genome‐wide genotyping of common genetic variants. The enrolment of subjects into participating studies ranged from 1948 to the present day, and the median follow‐up following heart failure diagnosis ranged from 2 to 116 months. Forty‐nine of 51 individual studies enrolled participants of both sexes; in these studies, participants with heart failure were predominantly male (34–90%). The mean age at diagnosis or ascertainment across all studies ranged from 54 to 84 years. Based on the aggregate sample, we estimated 80% power to genetic variant associations with risk of heart failure with an odds ratio of ≥1.10 for common variants (allele frequency ≥ 0.05) and ≥1.20 for low‐frequency variants (allele frequency 0.01–0.05) at P

In cancer patients, thrombocytopenia can result from bone marrow infiltration or from anticancer medications and represents an important limitation for the use of antithrombotic treatments, including anticoagulant, antiplatelet, and fibrinolytic agents. These drugs are often required for prevention or treatment of cancer-associated thrombosis or for cardioembolic prevention in atrial fibrillation in an increasingly older cancer population. Data indicate that cancer remains an independent risk factor for thrombosis even in case of thrombocytopenia, since mild-to-moderate thrombocytopenia does not protect against arterial or venous thrombosis. In addition, cancer patients are at increased risk of antithrombotic drug-associated bleeding, further complicated by thrombocytopenia and acquired hemostatic defects. Furthermore, some anticancer treatments are associated with increased thrombotic risk and may generate interactions affecting the effectiveness or safety of antithrombotic drugs. In this complex scenario, the European Hematology Association in collaboration with the European Society of Cardiology has produced this scientific document to provide a clinical practice guideline to help clinicians in the management of patients with cancer and thrombocytopenia. The Guidelines focus on adult patients with active cancer and a clear indication for anticoagulation, single or dual antiplatelet therapy, their combination, or reperfusion therapy, who have concurrent thrombocytopenia because of either malignancy or anticancer medications. The level of evidence and the strength of the recommendations were discussed according to a Delphi procedure and graded according to the Oxford Centre for Evidence-Based Medicine.

Heart failure is a complex syndrome that is associated with many different underlying risk factors. Here, to increase power, the authors jointly analyse cases of heart failure of different aetiologies in a genome-wide association study and identify 11 loci of which ten had not been previously reported.

Meditation as a form of body–mind interaction for primary and secondary prevention in cardiovascular disease has been discussed critically in the past. However, data that aimed to link this intervention to a reduction of various aspects of cardiovascular disease, rendering it a potential part of a cost-effective treatment approach in patients at risk, remain scarce and inconclusive. This article aims to provide an overview of currently available evidence in the literature and the potential impact of meditation on cardiovascular health. However, the data highlighted in this article cannot render with certainty directly reproducible effects of meditation on patients’ cardiovascular disease profiles. Meditation may be suggested only as an additional link in the chain of primary and secondary prevention until future research provides sufficient data on this topic.

Background: The propensity of serum to calcify, as assessed by the T50-test, associates with mortality in patients with chronic kidney disease. In chronic heart failure, phosphate and fibroblast growth factor-23 (FGF-23), which are important components of the vascular calcification pathway, have been linked to patient survival. Here, we investigated whether T50 associates with overall and cardiovascular survival in patients with chronic heart failure with reduced ejection fraction (HFrEF).Methods: We measured T50, intact and c-terminal FGF-23 levels in a cohort of 306 HFrEF patients. Associations with overall and cardiovascular mortality were analyzed in survival analysis and Cox-regression models.Results: After a median follow-up time of 3.2 years (25th−75th percentile: 2.0–4.9 years), 114 patients (37.3%) died due to any cause and 76 patients (24.8%) died due to cardiovascular causes. 139 patients (45.4%) had ischemic and 167 patients (54.6%) had non-ischemic HFrEF. Patients with ischemic HFrEF in the lowest T50-tertile had significantly greater 2-year cardiovascular mortality compared to patients in higher tertiles (p = 0.011). In ischemic but not in non-ischemic HFrEF, T50 was significantly associated with cardiovascular mortality in univariate (p = 0.041) and fully adjusted (p = 0.046) Cox regression analysis. Significant associations of intact and c-terminal FGF-23 with all-cause and cardiovascular mortality in univariate Cox regression analysis did not remain significant after adjustment for confounding factors.Conclusion: T50 is associated with 2-year cardiovascular mortality in patients with ischemic HFrEF but not in non-ischemic HFrEF. More research on the role of T50 measurements in coronary artery disease is warranted.

Background. Heart failure with reduced ejection fraction (HFrEF) constitutes a global health issue. While proinflammatory cytokines proved to have a pivotal role in the development and progression of HFrEF, less attention has been paid to the cellular immunity. Regulatory T lymphocytes (Tregs) seem to have an important role in the induction and maintenance of immune homeostasis. Therefore, we aimed to investigate the impact of Tregs on the outcome in HFrEF. Methods. We prospectively enrolled 112 patients with HFrEF and performed flow cytometry for cell phenotyping. Individuals were stratified in ischemic (iHFrEF, n=57) and nonischemic etiology (niHFrEF, n=55). Cox regression hazard analysis was used to assess the influence of Tregs on survival. Results. Comparing patients with iHFrEF to niHFrEF, we found a significantly lower fraction of Tregs within lymphocytes in the ischemic subgroup (0.42% vs. 0.56%; p=0.009). After a mean follow-up time of 4.5 years, 32 (28.6%) patients died due to cardiovascular causes. We found that Tregs were significantly associated with cardiovascular survival in the entire study cohort with an adjusted HR per one standard deviation (1-SD) of 0.60 (95% CI: 0.39-0.92; p=0.017). A significant inverse association of Tregs and cardiovascular mortality in patients with iHFrEF with an adj. HR per 1-SD of 0.59 (95% CI: 0.36-0.96; p=0.034) has been observed, while this association was not evident in the nonischemic subgroup (adj. HR per 1-SD of 0.62 (95% CI: 0.17-2.31); p=0.486). Conclusion. Our results indicate a potential influence of Tregs in the pathogenesis and progression of iHFrEF, fostering the implication of cellular immunity in iHFrEF pathophysiology and proving Tregs as a predictor for long-term survival among iHFrEF patients. A preview of this study has been presented at a meeting of the European Society of Cardiology earlier this year.

Abstract Cardiovascular risk assessment in patients with diabetes relies on traditional risk factors. However, numerous novel biomarkers have been found to be independent predictors of cardiovascular disease, which might significantly improve risk prediction in diabetic patients. We aimed to improve prediction of cardiovascular risk in diabetic patients by investigating 135 evolving biomarkers. Based on selected biomarkers a clinically applicable prediction algorithm for long-term cardiovascular mortality was designed. We prospectively enrolled 864 diabetic patients of the LUdwigshafen RIsk and Cardiovascular health (LURIC) study with a median follow-up of 9.6 years. Independent risk factors were selected using bootstrapping based on a Cox regression analysis. The following seven variables were selected for the final multivariate model: NT-proBNP, age, male sex, renin, diabetes duration, Lp-PLA2 and 25-OH vitamin D3. The risk score based on the aforementioned variables demonstrated an excellent discriminatory power for 10-year cardiovascular survival with a C-statistic of 0.76 (P

BackgroundExtracorporeal membrane oxygenation following cardiac surgery safeguards end‐organ oxygenation but unfavorably alters cardiac hemodynamics. Along with the detrimental effects of cardiac surgery to the right heart, this might impact outcome, particularly in patients with preexisting right ventricular (RV) dysfunction. We sought to determine the prognostic impact of RV function and to improve established risk‐prediction models in this vulnerable patient cohort. Methods and ResultsOf 240 patients undergoing extracorporeal membrane oxygenation support following cardiac surgery, 111 had echocardiographic examinations at our institution before implantation of extracorporeal membrane oxygenation and were thus included. Median age was 67 years (interquartile range 60‐74), and 74 patients were male. During a median follow‐up of 27 months (interquartile range 16‐63), 75 patients died. Fifty‐one patients died within 30 days, 75 during long‐term follow‐up (median follow‐up 27 months, minimum 5 months, maximum 125 months). Metrics of RV function were the strongest predictors of outcome, even stronger than left ventricular function (P

BackgroundOxidative stress is mechanistically linked to the pathogenesis of chronic heart failure (CHF). Antioxidative functions of high‐density lipoprotein (HDL) particles have been found impaired in patients with ischemic cardiomyopathy; however, the impact of antioxidative HDL capacities on clinical outcome in CHF patients is unknown. We therefore investigated the predictive value of antioxidative HDL function on mortality in a representative cohort of patients with CHF. Methods and ResultsWe prospectively enrolled 320 consecutive patients admitted to our outpatient department for heart failure and determined antioxidative HDL function using the HDL oxidative index (HOI). During a median follow‐up time of 2.8 (IQR: 1.8‐4.9) years, 88 (27.5%) patients reached the combined cardiovascular endpoint defined as the combination of death due to cardiovascular events and heart transplantation. An HOI ≥1 was significantly associated with survival free of cardiovascular events in Cox regression analysis with a hazard ratio (HR) of 2.28 (95% CI 1.48‐3.51, P

The incidence of acute coronary syndrome (ACS) in young people (≤65 years) is continuously rising. While prognostic factors in ACS are well-investigated less attention has been paid to their age-dependent prognostic value and their particular relevance in younger patients. The aim of our study was to assess the age-dependent prognostic impact of butyrylcholinesterase (BChE).Retrospective cohort study including 624 patients with ACS. Patients were stratified by age into equal groups (n = 208) corresponding to "young patients" (45-64 years), "middle-aged patients" (65-84 years) and "old patients" (85-100 years). Cox regression hazard analysis was used to assess the influence of BChE on survival.After a mean follow-up time of 4.0 (interquartile range [IQR] 2.0-6.4) years, 154 patients (24.7%) died due to a cardiac cause. In the overall cohort, BChE was indirectly associated with cardiac mortality-free survival (adjusted hazard ratio (HR): 0.70 (95% confidence interval [CI] 0.53-0.93, p = 0.01). The primary-analysis of BChE by age strata showed the strongest effect in the age group 45-64 years with an adjusted HR per 1-SD of 0.28 (95% CI 0.12-0.64, p = 0.003), a weaker association with mortality in middle aged (65-84 years: adjusted HR per 1-SD 0.66 [95% CI: 0.41-1.06], p = 0.087), and no association in older patients (85-100 years: adjusted HR per 1-SD 0.89 [95% CI: 0.58-1.38], p = 0.613).BChE is a strong predictor for cardiac mortality specifically in younger patients with ACS aged between 45 and 64 years. No significant association of BChE with cardiac-mortality was detected in other age classes.

Atherosclerosis is considered to be an inflammatory disease in which monocytes and monocyte-derived macrophages play a key role. Circulating monocytes can be divided into three distinct subtypes, namely in classical monocytes (CM; CD14++CD16-), intermediate monocytes (IM; CD14++CD16+) and non-classical monocytes (NCM; CD14+CD16++). Low density lipoprotein particles are heterogeneous in size and density, with small, dense LDL (sdLDL) crucially implicated in atherogenesis. The aim of this study was to examine whether monocyte subsets are associated with sdLDL serum levels.We included 90 patients with angiographically documented stable coronary artery disease and determined monocyte subtypes by flow cytometry. sdLDL was measured by an electrophoresis method on polyacrylamide gel.Patients with sdLDL levels in the highest tertile (sdLDL≥4mg/dL;T3) showed the highest levels of pro-inflammatory NCM (15.2±7% vs. 11.4±6% and 10.9±4%, respectively; p