Two-component carnitine monooxygenase from Escherichia coli: functional characterization, inhibition and mutagenesis of the molecular interface.

Gut microbial production of trimethylamine (TMA) from l-carnitine is directly linked to cardiovascular disease. TMA formation is facilitated by carnitine monooxygenase, which was proposed as a target for the development of new cardioprotective compounds. Therefore, the molecular understanding of the two-component Rieske-type enzyme from Escherichia coli was intended. The redox cofactors of the reductase YeaX (FMN, plant-type [2Fe-2S] cluster) and of the oxygenase YeaW (Rieske-type [2Fe-2S] and mononuclear [Fe] center) were identified. Compounds meldonium and the garlic-derived molecule allicin were recently shown to suppress microbiota-dependent TMA formation. Based on two independent carnitine monooxygenase activity assays, enzyme inhibition by meldonium or allicin was demonstrated. Subsequently, the molecular interplay of the reductase YeaX and the oxygenase YeaW was addressed. Chimeric carnitine monooxygenase activity was efficiently reconstituted by combining YeaX (or YeaW) with the orthologous oxygenase CntA (or reductase CntB) from Acinetobacter baumannii. Partial conservation of the reductase/oxygenase docking interface was concluded. A structure guided mutagenesis approach was used to further investigate the interaction and electron transfer between YeaX and YeaW. Based on AlphaFold structure predictions, a total of 28 site-directed variants of YeaX and YeaW were kinetically analyzed. Functional relevance of YeaX residues Arg271, Lys313 and Asp320 was concluded. Concerning YeaW, a docking surface centered around residues Arg83, Lys104 and Lys117 was hypothesized. The presented results might contribute to the development of TMA-lowering strategies that could reduce the risk for cardiovascular disease.

Chimeric Interaction of Nitrogenase-Like Reductases with the MoFe Protein of Nitrogenase.

The engineering of transgenic organisms with the ability to fix nitrogen is an attractive possibility. However, oxygen sensitivity of nitrogenase, mainly conferred by the reductase component (NifH)2 , is an imminent problem. Nitrogenase-like enzymes involved in coenzyme F430 and chlorophyll biosynthesis utilize the highly homologous reductases (CfbC)2 and (ChlL)2 , respectively. Chimeric protein-protein interactions of these reductases with the catalytic component of nitrogenase (MoFe protein) did not support nitrogenase activity. Nucleotide-dependent association and dissociation of these complexes was investigated, but (CfbC)2 and wild-type (ChlL)2 showed no modulation of the binding affinity. By contrast, the interaction between the (ChlL)2 mutant Y127S and the MoFe protein was markedly increased in the presence of ATP (or ATP analogues) and reduced in the ADP state. Upon formation of the octameric (ChlL)2 MoFe(ChlL)2 complex, the ATPase activity of this variant is triggered, as seen in the homologous nitrogenase system. Thus, the described reductase(s) might be an attractive tool for further elucidation of the diverse functions of (NifH)2 and the rational design of a more robust reductase.

Expression, Purification, and Activity Analysis of Chlorophyllide Oxidoreductase and Ni2+-Sirohydrochlorin a,c-Diamide Reductase.

Nitrogenase-like enzymes play a vital role in the reduction of the conjugated ring systems of diverse tetrapyrrole molecules. The biosynthesis of all bacteriochlorophylls involves the two-electron reduction of the C7-C8 double bond of the green pigment chlorophyllide, which is catalyzed by the nitrogenase-like two-component metalloenzyme chlorophyllide oxidoreductase (COR); whereas in all methanogenic microbes, another nitrogenase-like system, CfbC/D, is responsible for the sophisticated six-electron reduction of Ni2+-sirohydrochlorin a,c-diamide in the course of coenzyme F430 biosynthesis. The first part of this chapter describes the production and purification of the COR components (BchY/BchZ)2 and BchX2, the measurement of COR activity, and the trapping of the ternary COR complex; and the second part describes the strategy for obtaining homogenous and catalytically active preparations of CfbC2 and CfbD2 and a suitable method for extracting the reaction product Ni2+-hexahydrosirohydrochlorin a,c-diamide.

Hydrothermal carbonization of biomass from landscape management - Influence of process parameters on soil properties of hydrochars.

Besides pyrolysis the technology of hydrothermal carbonization (HTC) is tested to produce hydrochars for soil improvement. The chemical and physical properties of the hydrochars mainly depend on the feedstock and the process parameters reaction time and process temperature. Systematic investigations on the influences of these process parameters on soil properties of hydrochars like water holding capacity (WHC) and cation exchange capacity (CEC) are missing. In this study, a rush-rich biomass was carbonized within defined HTC process conditions under variation of reaction time and process temperature to produce hydrochars. Analysis of WHC, CEC, the elemental composition and Fourier-transform infrared spectroscopy (FT-IR) were performed to evaluate the influence of HTC process conditions on the pedological hydrochar properties. The results indicated that at increasing reaction severity (reaction time and process temperature) WHC and CEC decreased as well as the elemental O/C ratio. The decrease of WHC and CEC is based on the decrease of the hydrochar surface polarity. However, even the lowest WHC and CEC of investigated hydrochars still exceeded those of pure quartz sand by factors of 5-10. An application of hydrochars produced at severe HTC conditions could improve WHC and CEC of sandy soils. This has to be investigated in further studies.

Pancreatic fistulae after pancreatic resections for neuroendocrine tumours compared with resections for other lesions.

BACKGROUND: Resection for pancreatic neuroendocrine tumours (PNET) is suggested to be associated with an increased risk of a post-operative pancreatic fistula (POPF). The aim of this study was to describe morbidity after resections for PNET, focusing on POPF. Outcomes were compared with resections for other lesions. METHODS: Patients undergoing an elective pancreatic resection during a 12-year period were retrospectively analysed. Morbidity was defined according to the International Study Group of Pancreatic Surgery (ISGPS) definitions. RESULTS: Eighty-eight out of 832 patients (10.6%) underwent a resection for PNET. Atypical pancreatic resections (enucleation and central pancreatectomy) and distal pancreatectomies were more frequently performed for PNET. The POPF rate was 22.7% in patients operated for PNET compared with 17.2% in other patients (P = 0.200). In univariate analysis, body mass index (BMI), pancreatic duct diameter, somatostatin analogue administration, type of resection and type of pathology were associated with a POPF. In multivariate analysis, BMI, a pancreatic duct diameter <3 mm and central pancreatectomy remained independent risk factors [odds ratio (OR) 1.93, 95% confidence interval (CI) 1.22-3.07 and OR 3.04, 95% CI 1.05-8.82, respectively]. CONCLUSIONS: High rates of POPF were found in patients operated for PNET. However, this was mainly owing to the fact that atypical resections, known to be associated with a higher fistula rate, were performed more frequently in these patients.

Gait adaptability training improves obstacle avoidance and dynamic stability in patients with cerebellar degeneration.

Balance and gait problems in patients with cerebellar degeneration lead to reduced mobility, loss of independence, and frequent falls. It is currently unclear, however, whether balance and gait capacities can be improved by training in this group of patients. Therefore, the aim of this study was to examine the effects of gait adaptability training on obstacle avoidance and dynamic stability during adaptive gait. Ten patients with degenerative cerebellar ataxia received 10 protocolized gait adaptability training sessions of 1 h each during 5 weeks. Training was performed on a treadmill with visual stepping targets and obstacles projected on the belt's surface. As the primary outcome, we used an obstacle avoidance task while walking on a treadmill. We determined avoidance success rates, as well as dynamic stability during the avoidance manoeuvre. Clinical ratings included the scale for the assessment of ataxia (SARA), 10 m walking test, timed up-and-go test, berg balance scale, and the obstacle subtask of the emory functional ambulation profile (EFAP). Following the intervention, success rates on the obstacle avoidance task had significantly improved compared to pre-intervention. For successful avoidance, participants allowed themselves smaller stability margins in the sagittal plane in the (shortened) pre-crossing step. However, in the subsequent steps they returned to baseline stability values more effectively than before training. SARA scores and the EFAP obstacle subtask improved significantly as well. This pilot study provides preliminary evidence of a beneficial effect of gait adaptability training on obstacle avoidance capacity and dynamic stability in patients with cerebellar degeneration.

Acute coronary syndrome in a patient with a single coronary artery arising from the right sinus of Valsalva.

Coronary artery anomalies are usually encountered as coincidental findings during coronary angiography or at autopsy. Life threatening symptoms, such as arrhythmias, syncope, myocardial infarction, or sudden death, can occur in up to 20% of patients. However, the majority of anomalies (80%) are benign and asymptomatic. A single coronary artery (SCA) is one of the most rarely seen coronary anomalies with an incidence of 0.05%. We report the case of a 55-year old male patient who presented with symptoms of chest pain associated with an acute myocardial infarction. Coronary angiography revealed an anomalous left main coronary artery (LMCA) originating from the right coronary ostium, and an occluded distal right coronary artery. The occluded distal right coronary artery was successfully treated by thrombosuction and stenting. In order to confirm the origin and course of the SCA, multi-slice computed tomography (MSCT) of the heart was performed after coronary angiography. MSCT showed that the anomalous LMCA originated from the right coronary artery ostium and then passed the interventricular septum, instead of being intra arterial, and under the right ventricular infundibulum. The anomalous LMCA was classified as R-II S subtype according to Lipton's classification.

Genetic determinants of treatment benefit of the angiotensin-converting enzyme-inhibitor perindopril in patients with stable coronary artery disease.

AIMS: The efficacy of angiotensin-converting enzyme (ACE)-inhibitors in stable coronary artery disease (CAD) may be increased by targeting the therapy to those patients most likely to benefit. However, these patients cannot be identified by clinical characteristics. We developed a genetic profile to predict the treatment benefit of ACE-inhibitors exist and to optimize therapy with ACE-inhibitors. METHODS AND RESULTS: In 8907 stable CAD patients participating in the randomized placebo-controlled EUROPA-trial, we analysed 12 candidate genes within the pharmacodynamic pathway of ACE-inhibitors, using 52 haplotype-tagging-single nucleotide polymorphisms (SNPs). The primary outcome was the reduction in cardiovascular mortality, non-fatal myocardial infarction, and resuscitated cardiac arrest during 4.2 years of follow-up. Multivariate Cox regression was performed with multiple testing corrections using permutation analysis. Three polymorphisms, located in the angiotensin-II type I receptor and bradykinin type I receptor genes, were significantly associated with the treatment benefit of perindopril after multivariate adjustment for confounders and correction for multiple testing. A pharmacogenetic score, combining these three SNPs, demonstrated a stepwise reduction of risk in the placebo group and a stepwise decrease in treatment benefit of perindopril with an increasing scores (interaction P < 0.0001). A pronounced treatment benefit was observed in a subgroup of 73.5% of the patients [hazard ratio (HR) 0.67; 95% confidence interval (CI) 0.56-0.79], whereas no benefit was apparent in the remaining 26.5% (HR 1.26; 95% CI 0.97-1.67) with a trend towards a harmful effect. In 1051 patients with cerebrovascular disease from the PROGRESS-trial, treated with perindopril or placebo, an interaction effect of similar direction and magnitude, although not statistically significant, was observed. CONCLUSION: The current study is the first to identify genetic determinants of treatment benefit of ACE-inhibitor therapy. We developed a genetic profile which predicts the treatment benefit of ACE-inhibitors and which could be used to optimize therapy.

Relation of periprocedural bleeding complications and long-term outcome in patients undergoing percutaneous coronary revascularization (from the Evaluation of Oral Xemilofiban in Controlling Thrombotic Events [EXCITE] Trial).

Several clinical trials have shown that antagonists of the glycoprotein IIb/IIIa receptor decreased the incidence of death, nonfatal myocardial infarction, and the need for urgent revascularization when administered immediately before or during the 24- to 48-hour period after percutaneous coronary intervention (PCI). However, these agents increased the risk of thrombocytopenia and periprocedural bleeding complications. Therefore, the relation between periprocedural bleeding complications during PCI and long-term outcome was assessed in 6,995 patients in the EXCITE trial. Periprocedural bleeding was classified as none, mild, moderate, and severe. Measured outcomes included the incidence of all-cause mortality or the composite end point (cardiovascular disease) of death, myocardial infarction, or stroke. Subjects were followed up for a median of 210 days (7 months). Mean patient age was 59.1 years, and 21.8% were women. Periprocedural bleeding complications occurred in 1,869 patients (26.7%), and blood transfusion was administered to 189 patients (2.7%). In multivariate analysis, periprocedural bleeding complications were significantly associated with increased risk of the composite outcome for mild (hazard ratio [HR] 0.80, 95% confidence interval [CI] 0.64 to 0.97), moderate (HR 2.38, 95% CI 1.78 to 3.20), and severe bleeding complications (HR 3.55, 95% CI 2.20 to 5.73) during follow-up. Also, the necessity of blood transfusion was an important predictor of the composite end point (HR 2.61, 95% CI 1.96 to 3.60). Patients in the United States were more likely to be administered a blood transfusion than non-US patients independently of cardiovascular risk factors. In conclusion, moderate and severe periprocedural bleeding complications increased the risk of mortality and incident cardiovascular events after PCI.