Synthesis and Properties of RNA Modified with Thioamide Internucleoside Linkage.

Recent success of RNA therapeutics has reinvigorated interest in chemical modifications of RNA. As exemplified by the phosphorothioates, modifications of sugar-phosphate backbone have been remarkably impactful but relatively underexplored in therapeutic RNAs. The present study reports synthesis, thermal stability, and RNA interference activity of RNAs modified with thioamide linkages. Compared to the previously studied amide-modified RNA, thioamide linkages strongly destabilized a short self-complementary RNA model duplex. However, in short interfering RNAs amides and thioamides had a similar effect on duplex stability and target RNA cleavage activity and specificity. Hence, the thioamide may be added to the toolbox of chemical biologist as a useful backbone modification well tolerated by the RNA interference machinery.

Secretion of glucagon, GLP-1 and GIP may be affected by circadian rhythm in healthy males.

BACKGROUND: Glucagon is secreted from pancreatic alpha cells in response to low blood glucose and increases hepatic glucose production. Furthermore, glucagon enhances hepatic protein and lipid metabolism during a mixed meal. Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are secreted from gut endocrine cells during meals and control glucose homeostasis by potentiating insulin secretion and inhibiting food intake. Both glucose homeostasis and food intake have been reported to be affected by circadian rhythms and vice versa. In this study, we investigated whether the secretion of glucagon, GLP-1 and GIP was affected by circadian rhythms. METHODS: A total of 24 healthy men with regular sleep schedules were examined for 24 h at the hospital ward with 15 h of wakefulness and 9 h of sleep. Food intake was standardized, and blood samples were obtained every third hour. Plasma concentrations of glucagon, GLP-1 and GIP were measured, and data were analyzed by rhythmometric statistical methods. Available data on plasma glucose and plasma C-peptide were also included. RESULTS: Plasma concentrations of glucagon, GLP-1, GIP, C-peptide and glucose fluctuated with a diurnal 24-h rhythm, with the highest levels during the day and the lowest levels during the night: glucagon (p < 0.0001, peak time 18:26 h), GLP-1 (p < 0.0001, peak time 17:28 h), GIP (p < 0.0001, peak time 18:01 h), C-peptide (p < 0.0001, peak time 17.59 h), and glucose (p < 0.0001, peak time 23:26 h). As expected, we found significant correlations between plasma concentrations of C-peptide and GLP-1 and GIP but did not find correlations between glucose concentrations and concentrations of glucagon, GLP-1 and GIP. CONCLUSIONS: Our results demonstrate that under meal conditions that are similar to that of many free-living individuals, plasma concentrations of glucagon, GLP-1 and GIP were observed to be higher during daytime and evening than overnight. These findings underpin disturbed circadian rhythm as a potential risk factor for diabetes and obesity. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT06166368. Registered 12 December 2023.

In situ H2 O2 Generation by Choline Oxidase and Its Application in Amino Polysaccharide Degradation by Coupling to Lytic Polysaccharide Monooxygenase.

Chitin, the most abundant amino polysaccharide in Nature, has many applications in different fields. However, processing of this recalcitrant biopolymer in an environmentally friendly manner remains a major challenge. In this context, lytic polysaccharide monooxygenases (LPMOs) are of interest, as they can act on the most recalcitrant parts of chitin and related insoluble biopolymers such as cellulose. Efficient LPMO catalysis can be achieved by feeding reactions with H2 O2 , but careful control of H2 O2 is required to avoid autocatalytic enzyme inactivation. Herein, we present a coupled enzyme system in which a choline oxidase from Arthrobacter globiformis is employed for controlled in situ generation of H2 O2 that fuels LPMO-catalyzed oxidative degradation of chitin. We show that the rate, stability and extent of the LPMO reaction can be manipulated by varying the amount of choline oxidase and/or its substrate, choline chloride, and that efficient peroxygenase reactions may be achieved using sub-µM concentrations of the H2 O2 -generating enzyme. This coupled system requires only sub-stoichiometric amounts of the reductant that is needed to keep the LPMO in its active, reduced state. It is conceivable that this enzyme system may be used for bioprocessing of chitin in choline-based natural deep eutectic solvents.

Widespread Proprioceptive Acuity Impairment in Chronic Back Pain: A Cross-sectional Study.

OBJECTIVE: To investigate whether proprioceptive accuracy measured with the Joint Position Sense (JPS) in patients with chronic neck and low back pain is impaired exclusively in affected areas or also in distant areas, not affected by pain. DESIGN: Cross-sectional study. SETTING: Interdisciplinary outpatient rehabilitation clinic for back and neck pain. PARTICIPANTS: Patients with chronic neck pain (n=30), patients with chronic low back pain (n=30), and age- and sex-matched asymptomatic control subjects (n=30; N=90). INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Patients and asymptomatic control subjects completed a test procedure for the JPS of the cervical spine, lumbar spine, and ankle in a randomized order. Between group differences were analyzed with the univariate analysis of variance and associations of the JPS with clinical features using the Pearson's correlation coefficient. RESULTS: Both patients with chronic neck pain (P<.001) and patients with chronic low back pain (P<.01) differed significantly from asymptomatic controls in the JPS of the cervical spine, lumbar spine and ankle joint, regardless of the painful area. No difference was shown between patient groups (P>.05). An association of the JPS with clinical characteristics, however, could not be shown. CONCLUSION: These results suggest widespread impairment of proprioceptive accuracy in patients with chronic and low back pain and a role for central sensorimotor processes in musculoskeletal pain conditions.

Preanalytical impact on the accuracy of measurements of glucagon, GLP-1 and GIP in clinical trials.

BACKGROUND: Plasma concentrations of glucagon, GLP-1 and GIP are reported in numerous clinical trials as outcome measures but preanalytical guidelines are lacking. We addressed the impact of commonly used blood containers in metabolic research on measurements of glucagon, GLP-1 and GIP in humans. METHODS: Seventeen overweight individuals were subjected to an overnight fast followed by an intravenous infusion of amino acids to stimulate hormonal secretion. Blood was sampled into five containers: EDTA-coated tubes supplemented with DMSO (control), a neprilysin inhibitor, aprotinin (a kallikrein inhibitor) or a DPP-4 inhibitor, and P800 tubes. Plasma was kept on ice before and after centrifugation and stored at -80 Celsius until batch analysis using validated sandwich ELISAs or radioimmunoassays (RIA). RESULTS: Measures of fasting plasma glucagon did not depend on sampling containers, whether measured by ELISA or RIA. Amino acid-induced hyperglucagonemia was numerically higher when blood was collected into P800 tubes or tubes with aprotinin. The use of p800 tubes resulted in higher concentrations of GLP-1 by RIA compared to control tubes but not for measurements with sandwich ELISA. Plasma concentrations of GIP measured by ELISA were higher in control tubes and negatively affected by P800 and the addition of aprotinin. CONCLUSIONS: The choice of blood containers impacts on measurements of plasma concentrations of glucagon, GLP-1 and GIP, and based on this study, we recommend using EDTA-coated tubes without protease inhibitors or P800 tubes for measurements of glucagon, GLP-1 and GIP in clinical trials.

Arginine-induced glucagon secretion and glucagon-induced enhancement of amino acid catabolism are not influenced by ambient glucose levels in mice.

Amino acids stimulate the secretion of glucagon, and glucagon receptor signaling regulates amino acid catabolism via ureagenesis, together constituting the liver-α cell axis. Impairment of the liver-α cell axis is observed in metabolic diseases such as diabetes. It is, however, unknown whether glucose affects the liver-α cell axis. We investigated the role of glucose on the liver-α cell axis in vivo and ex vivo. The isolated perfused mouse pancreas was used to evaluate the direct effect of low (3.5 mmol/L) and high (15 mmol/L) glucose levels on amino acid (10 mmol/L arginine)-induced glucagon secretion. High glucose levels alone lowered glucagon secretion, but the amino acid-induced glucagon responses were similar in high and low glucose conditions (P = 0.38). The direct effect of glucose on glucagon and amino acid-induced ureagenesis was assessed using isolated perfused mouse livers stimulated with a mixture of amino acids (VaminR, 10 mmol/L) and glucagon (10 nmol/L) during high and low glucose conditions. Urea production increased robustly but was independent of glucose levels (P = 0.95). To investigate the whole body effects of glucose on the liver-α cell axis, four groups of mice received intraperitoneal injections of glucose-Vamin (2 g/kg, + 3.5 µmol/g, respectively, G/V), saline-Vamin (S/V), glucose-saline (G/S), or saline-saline (S/S). Blood glucose did not differ significantly between G/S and G/V groups. Levels of glucagon and amino acids were similar in the G/V and S/V groups (P = 0.28). Amino acids may overrule the inhibitory effect of glucose on glucagon secretion and the liver-α cell axis may operate independently of glucose in mice.NEW & NOTEWORTHY Glucagon is an essential regulator of our metabolism. Recent evidence suggests that the physiological actions of glucagon reside in amino acid catabolism in the so-called liver-α cell axis, in which amino acids stimulate glucagon secretion and glucagon enhances hepatic amino acid catabolism. Here, it is demonstrated that this feedback system is independent of glycemia possibly explaining why hyperglycemia in diabetes may not suppress α cell secretion.

A High-Throughput Continuous Spectroscopic Assay to Measure the Activity of Natural Product Methyltransferases.

Natural product methyltransferases (NPMTs) represent an emerging class of enzymes that can be of great use for the structural and functional diversification of bioactive compounds, such as the strategic modification of C-, N-, O- and S-moieties. To assess the activity and the substrate scope of the ever-expanding repertoire of NPMTs, a simple, fast, and robust assay is needed. Here, we report a continuous spectroscopic assay, in which S-adenosyl-L-methionine-dependent methylation is linked to NADH oxidation through the coupled activities of S-adenosyl-L-homocysteine (SAH) deaminase and glutamate dehydrogenase. The assay is highly suitable for a high-throughput evaluation of small molecule methylation and for determining the catalytic parameters of NPMTs under conditions that remove the potent inhibition by SAH. Through the modular design, the assay can be extended to match the needs of different aspects of methyltransferase cascade reactions and respective applications.

Methanosaeta and "Candidatus Velamenicoccus archaeovorus".

The phylum "Candidatus Omnitrophica" (candidate division OP3) is ubiquitous in anaerobic habitats but is currently characterized only by draft genomes from metagenomes and single cells. We had visualized cells of the phylotype OP3 LiM in methanogenic cultures on limonene as small epibiotic cells. In this study, we enriched OP3 cells by double density gradient centrifugation and obtained the first closed genome of an apparently clonal OP3 cell population by applying metagenomics and PCR for gap closure. Filaments of acetoclastic Methanosaeta, the largest morphotype in the culture community, contained empty cells, cells devoid of rRNA or of both rRNA and DNA, and dead cells according to transmission electron microscopy (TEM), thin-section TEM, scanning electron microscopy (SEM), catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH), and LIVE/DEAD imaging. OP3 LiM cells were ultramicrobacteria (200 to 300 nm in diameter) and showed two physiological stages in CARD-FISH fluorescence signals: strong signals of OP3 LiM cells attached to Bacteria and to Archaea indicated many rRNA molecules and an active metabolism, whereas free-living OP3 cells had weak signals. Metaproteomics revealed that OP3 LiM lives with highly expressed secreted proteins involved in depolymerization and uptake of macromolecules and an active glycolysis and energy conservation by the utilization of pyruvate via a pyruvate:ferredoxin oxidoreductase and an Rnf complex (ferredoxin:NAD oxidoreductase). Besides sugar fermentation, a nucleotidyl transferase may contribute to energy conservation by phosphorolysis, the phosphate-dependent depolymerization of nucleic acids. Thin-section TEM showed distinctive structures of predation. Our study demonstrated a predatory metabolism for OP3 LiM cells, and therefore, we propose the name "Candidatus Velamenicoccus archaeovorus" gen. nov., sp. nov., for OP3 LiM. IMPORTANCE Epibiotic bacteria are known to live on and off bacterial cells. Here, we describe the ultramicrobacterial anaerobic epibiont OP3 LiM living on Archaea and Bacteria. We detected sick and dead cells of the filamentous archaeon Methanosaeta in slowly growing methanogenic cultures. OP3 LiM lives as a sugar fermenter, likely on polysaccharides from outer membranes, and has the genomic potential to live as a syntroph. The predatory lifestyle of OP3 LiM was supported by its genome, the first closed genome for the phylum "Candidatus Omnitrophica," and by images of cell-to-cell contact with prey cells. We propose naming OP3 LiM "Candidatus Velamenicoccus archaeovorus." Its metabolic versatility explains the ubiquitous presence of "Candidatus Omnitrophica" 3 in anoxic habitats and gives ultramicrobacterial epibionts an important role in the recycling and remineralization of microbial biomass. The removal of polysaccharides from outer membranes by ultramicrobacteria may also influence biological interactions between pro- and eukaryotes.

Extract from Cucurbita pepo improves BPH symptoms without affecting sexual function: a 24-month noninterventional study.

PURPOSE: To assess the symptoms, quality of life and sexual well-being in patients with lower urinary tract symptoms due to benign prostatic hyperplasia LUTS/BPH treated with pumpkin seed soft extract (PSE) in routine practice. METHODS: This noninterventional study included 130 men treated for up to 24 months. The International Prostate Symptom Score (IPSS) and related quality of life, Aging Males' Symptoms Scale (AMS), and International Index of Erectile Function (IIEF-5) were recorded. Descriptive statistical methods were applied. The mean with 95% confidence interval (CI) was calculated for the primary end point (change in IPSS after 12-month treatment). RESULTS: Analysis at 12 months included 83 patients [mean (SD) age 65.2 (8.7) years and IPSS (15.6 (3.4), IPSS-QoL 3.4 (0.9)]. AMS and IIEF-5 indicated mild or mild to moderate disorder regarding sexual well-being and erectile dysfunction, respectively. After 12 months, the mean IPSS change from baseline was - 4.7 (95% CI - 5.4 to - 3.9), with 83% (95% CI 65.3 to 84.1) and 53% (95% CI 42.3 to 63.7) of the patients achieving reductions by at least 3 and 5 points, respectively. The proportion of patients with IPSS-QoL below 3 points (mostly satisfied) was 11% (9/83) at baseline and rose to 62% (51/83) and 73% (40/55) at 12 and 24 months, respectively. AMS and IIEF-5 scores did not indicate a negative impact on sexual function during treatment. CONCLUSION: In men with a moderate LUTS suggestive of BPH, a low progression risk and an active sex life, treatment with pumpkin seed soft extract provided symptomatic relief, improved IPSS-QoL, and maintained sexual well-being. TRIAL REGISTRATION: DRKS00010729, June 22, 2016.

Acute effects on glucose tolerance by neprilysin inhibition in patients with type 2 diabetes.

AIMS: Sacubitril/valsartan is a neprilysin-inhibitor/angiotensin II receptor blocker used for the treatment of heart failure. Recently, a post-hoc analysis of a 3-year randomized controlled trial showed improved glycaemic control with sacubitril/valsartan in patients with heart failure and type 2 diabetes. We previously reported that sacubitril/valsartan combined with a dipeptidyl peptidase-4 inhibitor increases active glucagon-like peptide-1 (GLP-1) in healthy individuals. We now hypothesized that administration of sacubitril/valsartan with or without a dipeptidyl peptidase-4 inhibitor would lower postprandial glucose concentrations (primary outcome) in patients with type 2 diabetes via increased active GLP-1. METHODS: We performed a crossover trial in 12 patients with obesity and type 2 diabetes. A mixed meal was ingested following five respective interventions: (a) a single dose of sacubitril/valsartan; (b) sitagliptin; (c) sacubitril/valsartan + sitagliptin; (d) control (no treatment); and (e) valsartan alone. Glucose, gut and pancreatic hormone responses were measured. RESULTS: Postprandial plasma glucose increased by 57% (incremental area under the curve 0-240 min) (p = .0003) and increased peak plasma glucose by 1.7 mM (95% CI: 0.6-2.9) (p = .003) after sacubitril/valsartan compared with control, whereas postprandial glucose levels did not change significantly after sacubitril/valsartan + sitagliptin. Glucagon, GLP-1 and C-peptide concentrations increased after sacubitril/valsartan, but insulin and glucose-dependent insulinotropic polypeptide did not change. CONCLUSIONS: The glucose-lowering effects of long-term sacubitril/valsartan treatment reported in patients with heart failure and type 2 diabetes may not depend on changes in entero-pancreatic hormones. Neprilysin inhibition results in hyperglucagonaemia and this may explain the worsen glucose tolerance observed in this study. CLINICALTRIALS: gov (NCT03893526).

Thermophilic archaea activate butane via alkyl-coenzyme M formation.

The anaerobic formation and oxidation of methane involve unique enzymatic mechanisms and cofactors, all of which are believed to be specific for C1-compounds. Here we show that an anaerobic thermophilic enrichment culture composed of dense consortia of archaea and bacteria apparently uses partly similar pathways to oxidize the C4 hydrocarbon butane. The archaea, proposed genus 'Candidatus Syntrophoarchaeum', show the characteristic autofluorescence of methanogens, and contain highly expressed genes encoding enzymes similar to methyl-coenzyme M reductase. We detect butyl-coenzyme M, indicating archaeal butane activation analogous to the first step in anaerobic methane oxidation. In addition, Ca. Syntrophoarchaeum expresses the genes encoding ß-oxidation enzymes, carbon monoxide dehydrogenase and reversible C1 methanogenesis enzymes. This allows for the complete oxidation of butane. Reducing equivalents are seemingly channelled to HotSeep-1, a thermophilic sulfate-reducing partner bacterium known from the anaerobic oxidation of methane. Genes encoding 16S rRNA and methyl-coenzyme M reductase similar to those identifying Ca. Syntrophoarchaeum were repeatedly retrieved from marine subsurface sediments, suggesting that the presented activation mechanism is naturally widespread in the anaerobic oxidation of short-chain hydrocarbons.

Efficacy and safety of systemic, high-dose glucocorticoid therapy for idiopathic sudden sensorineural hearing loss : Study protocol for a three-armed, randomized, triple-blind, multicenter trial (HODOKORT).

BACKGROUND: Systemic glucocorticosteroids ("steroids") are widely used worldwide as a standard of care for primary therapy of idiopathic sudden sensorineural hearing loss (ISSHL). The German ISSHL guideline recommends high-dose steroids without evidence from randomized controlled trials (RCTs) and refers solely to retrospective cohort studies. This RCT aims to assess the efficacy (improvement in hearing) and safety (especially systemic side effects) of high-dose steroids versus standard of care (standard dose systemic steroids) for the treatment of unilateral ISSHL, when given as a primary therapy. METHODS: The study is designed as a multicenter (approximately 40 centers), randomized, triple-blind, three-armed, parallel group, clinical trial with 312 adult patients. The interventions consist of 5 days of 250 mg/day intravenous prednisolone (intervention 1) + oral placebo, or 5 days of 40 mg/day oral dexamethasone (intervention 2) + intravenous placebo. The control intervention consists of 60 mg oral prednisolone for 5 days followed by five tapering doses + intravenous placebo. The primary efficacy endpoint is the change in hearing threshold in the three most affected contiguous frequencies between 0.25 and 8 kHz 1 month after ISSHL. Secondary endpoints include further measures of hearing improvement including speech audiometry, tinnitus, quality of life, blood pressure, and altered glucose tolerance. DISCUSSION: There is an unmet medical need for an effective medical therapy of ISSHL. Although sensorineural hearing impairment can be partially compensated by hearing aids or cochlear implants (CI), generic hearing is better than using hearing aids or CIs. Since adverse effects of a short course of high-dose systemic corticosteroids have not been documented with good evidence, the trial will improve knowledge on possible side effects in the different treatment arms with a focus on hyperglycemia and hypertension. TRIAL REGISTRATION: EudraCT (European Union Drug Regulating Authorities Clinical Trials Database) Nr. 2015-002602-36; Sponsor code: KKSH-127.

Ramucirumab in combination with dacarbazine in patients with progressive well-differentiated metastatic pancreatic neuroendocrine tumors (RamuNET): study protocol for a multicenter single-arm trial.

BACKGROUND: Cytotoxic chemotherapy combinations and targeted agents represent established treatment concepts in advanced pancreatic neuroendocrine tumors (PNETs). However, response rates, side effects and outcome data strongly vary among these therapeutic approaches. Head-to-head comparisons between chemo- and molecular therapies are missing and secondary resistances frequently occur. The RamuNET trial aims to identify the effectiveness of dual treatment with DTIC and ramucirumab in progressive advanced PNET patients. METHODS: The RamuNET study is an investigator-initiated multicenter prospective single-arm trial to evaluate the efficacy of ramucirumab in combination with dacarbazine (DTIC) over a period of at least 6 months. Patients with progressive well-differentiated and metastatic pancreatic neuroendocrine tumors are eligible. The study aims to include 45 patients over a period of 24 months with a minimum follow-up of 24 months. The primary endpoint is disease control after 6 months. Secondary endpoints include progression-free survival, biochemical response, overall survival, quality of life and toxicity. Based on the hypothesis that 80% of the patients can achieve a disease control after 6 months, the sample size calculation follows an exact binomial single-stage design. H0: p < =p0 = 60% versus H1: p > =p1 = 80%, alpha = 0.05, beta = 0.1. DISCUSSION: This study investigates a new therapeutic approach using the combination of cytotoxic and targeted antiangiogenic therapy in advanced PNET. If positive, this trial will be the basis for a randomized two-arm study to investigate the combination of ramucirumab and DTIC against other established therapies in PNET. TRIAL REGISTRATION: EudraCT: 2017-001207-68 . Date of registration: 2018.01.03.

Investigating the Influences of Task Demand and Reward on Cardiac Pre-Ejection Period Reactivity During a Speech-in-Noise Task.

OBJECTIVES: Effort investment during listening varies as a function of task demand and motivation. Several studies have manipulated both these factors to elicit and measure changes in effort associated with listening. The cardiac pre-ejection period (PEP) is a relatively novel measure in the field of cognitive hearing science. This measure, which reflects sympathetic nervous system activity on the heart, has previously been implemented during a tone discrimination task but not during a speech-in-noise task. Therefore, the primary goal of this study was to explore the influences of signal to noise ratio (SNR) and monetary reward level on PEP reactivity during a speech-in-noise task. DESIGN: Thirty-two participants with normal hearing (mean age = 22.22 years, SD = 3.03) were recruited at VU University Medical Center. Participants completed a Dutch speech-in-noise test with a single-interfering-talker masking noise. Six fixed SNRs, selected to span the entire psychometric performance curve, were presented in a block-wise fashion. Participants could earn a low (€0.20) or high (€5.00) reward by obtaining a score of ≥70% of words correct in each block. The authors analyzed PEP reactivity: the change in PEP measured during the task, relative to the baseline during rest. Two separate methods of PEP analysis were used, one including data from the whole task block and the other including data obtained during presentation of the target sentences only. After each block, participants rated their effort investment, performance, tendency to give up, and the perceived difficulty of the task. They also completed the need for recovery questionnaire and the reading span test, which are indices of additional factors (fatigue and working memory capacity, respectively) that are known to influence listening effort. RESULTS: Average sentence perception scores ranged from 2.73 to 91.62%, revealing a significant effect of SNR. In addition, an improvement in performance was elicited by the high, compared to the low reward level. A linear relationship between SNR and PEP reactivity was demonstrated: at the lower SNRs PEP reactivity was the most negative, indicating greater effort investment compared to the higher SNRs. The target stimuli method of PEP analysis was more sensitive to this effect than the block-wise method. Contrary to expectations, no significant impact of reward on PEP reactivity was found in the present dataset. Also, there was no physiological evidence that participants were disengaged, even when performance was poor. A significant correlation between need for recovery scores and average PEP reactivity was demonstrated, indicating that a lower need for recovery was associated with less effort investment. CONCLUSIONS: This study successfully implemented the measurement of PEP during a standard speech-in-noise test and included two distinct methods of PEP analysis. The results revealed for the first time that PEP reactivity varies linearly with task demand during a speech-in-noise task, although the effect size was small. No effect of reward on PEP was demonstrated. Finally, participants with a higher need for recovery score invested more effort, as shown by average PEP reactivity, than those with a lower need for recovery score.

Bioelectrocatalytic Cofactor Regeneration Coupled to CO2 Fixation in a Redox-Active Hydrogel for Stereoselective C-C Bond Formation.

The sustainable capture and conversion of carbon dioxide (CO2 ) is key to achieving a circular carbon economy. Bioelectrocatalysis, which aims at using renewable energies to power the highly specific, direct transformation of CO2 into value added products, holds promise to achieve this goal. However, the functional integration of CO2 -fixing enzymes onto electrode materials for the electrosynthesis of stereochemically complex molecules remains to be demonstrated. Here, we show the electricity-driven regio- and stereoselective incorporation of CO2 into crotonyl-CoA by an NADPH-dependent enzymatic reductive carboxylation. Co-immobilization of a ferredoxin NADP+ reductase and crotonyl-CoA carboxylase/reductase within a 2,2'-viologen-modified hydrogel enabled iterative NADPH recycling and stereoselective formation of (2S)-ethylmalonyl-CoA, a prospective intermediate towards multi-carbon products from CO2 , with 92±6 % faradaic efficiency and at a rate of 1.6±0.4 µmol cm-2 h-1 . This approach paves the way for realizing even more complex bioelectrocatalyic cascades in the future.

Multienzyme One-Pot Cascades Incorporating Methyltransferases for the Strategic Diversification of Tetrahydroisoquinoline Alkaloids.

The tetrahydroisoquinoline (THIQ) ring system is present in a large variety of structurally diverse natural products exhibiting a wide range of biological activities. Routes to mimic the biosynthetic pathways to such alkaloids, by building cascade reactions in vitro, represents a successful strategy and can offer better stereoselectivities than traditional synthetic methods. S-Adenosylmethionine (SAM)-dependent methyltransferases are crucial in the biosynthesis and diversification of THIQs; however, their application is often limited in vitro by the high cost of SAM and low substrate scope. In this study, we describe the use of methyltransferases in vitro in multi-enzyme cascades, including for the generation of SAM in situ. Up to seven enzymes were used for the regioselective diversification of natural and non-natural THIQs on an enzymatic preparative scale. Regioselectivites of the methyltransferases were dependent on the group at C-1 and presence of fluorine in the THIQs. An interesting dual activity was also discovered for the catechol methyltransferases used, which were found to be able to regioselectively methylate two different catechols in a single molecule.

Accounting for multiple ecosystem services in a simulation of land-use decisions: Does it reduce tropical deforestation?

Conversion of tropical forests is among the primary causes of global environmental change. The loss of their important environmental services has prompted calls to integrate ecosystem services (ES) in addition to socio-economic objectives in decision-making. To test the effect of accounting for both ES and socio-economic objectives in land-use decisions, we develop a new dynamic approach to model deforestation scenarios for tropical mountain forests. We integrate multi-objective optimization of land allocation with an innovative approach to consider uncertainty spaces for each objective. These uncertainty spaces account for potential variability among decision-makers, who may have different expectations about the future. When optimizing only socio-economic objectives, the model continues the past trend in deforestation (1975-2015) in the projected land-use allocation (2015-2070). Based on indicators for biomass production, carbon storage, climate and water regulation, and soil quality, we show that considering multiple ES in addition to the socio-economic objectives has heterogeneous effects on land-use allocation. It saves some natural forest if the natural forest share is below 38%, and can stop deforestation once the natural forest share drops below 10%. For landscapes with high shares of forest (38%-80% in our study), accounting for multiple ES under high uncertainty of their indicators may, however, accelerate deforestation. For such multifunctional landscapes, two main effects prevail: (a) accelerated expansion of diversified non-natural areas to elevate the levels of the indicators and (b) increased landscape diversification to maintain multiple ES, reducing the proportion of natural forest. Only when accounting for vascular plant species richness as an explicit objective in the optimization, deforestation was consistently reduced. Aiming for multifunctional landscapes may therefore conflict with the aim of reducing deforestation, which we can quantify here for the first time. Our findings are relevant for identifying types of landscapes where this conflict may arise and to better align respective policies.

Predominant archaea in marine sediments degrade detrital proteins.

Half of the microbial cells in the Earth's oceans are found in sediments. Many of these cells are members of the Archaea, single-celled prokaryotes in a domain of life separate from Bacteria and Eukaryota. However, most of these archaea lack cultured representatives, leaving their physiologies and placement on the tree of life uncertain. Here we show that the uncultured miscellaneous crenarchaeotal group (MCG) and marine benthic group-D (MBG-D) are among the most numerous archaea in the marine sub-sea floor. Single-cell genomic sequencing of one cell of MCG and three cells of MBG-D indicated that they form new branches basal to the archaeal phyla Thaumarchaeota and Aigarchaeota, for MCG, and the order Thermoplasmatales, for MBG-D. All four cells encoded extracellular protein-degrading enzymes such as gingipain and clostripain that are known to be effective in environments chemically similar to marine sediments. Furthermore, we found these two types of peptidase to be abundant and active in marine sediments, indicating that uncultured archaea may have a previously undiscovered role in protein remineralization in anoxic marine sediments.

The effect of increased parasympathetic activity on perceived duration.

Theories of human temporal perception suggest that changes in physiological arousal distort the perceived duration of events. Behavioural manipulations of sympathetic nervous system (SNS) activity support this suggestion, however the effects of behavioural manipulations of parasympathetic (PSNS) activity on time perception are unclear. The current study examined the effect of a paced respiration exercise known to increase PSNS activity on sub-second duration estimates. Participants estimated the duration of negatively and neutrally valenced images following a period of normal and paced breathing. PSNS and SNS activity were indexed by high-frequency heart-rate variability and pre-ejection period respectively. Paced breathing increased PSNS activity and reduced the perceived duration of the negative and neutrally valenced stimuli relative to normal breathing. The results show that manipulations of PSNS activity can distort time in the absence of a change in SNS activity. They also suggest that activities which increase PSNS activity may be effective in reducing the perceived duration of short events.

[German version of the Neurophysiology of Pain Questionnaire : Translation, cross-cultural adaptation, reliability and validity]. / Die deutsche Version des Neurophysiology of Pain Questionnaire : Übersetzung, transkulturelle Adaptation, Reliabilität und Validität.

BACKGROUND: Excellence in Pain Education was the motto of the International Association for the Study of Pain (IASP) Global Year Campaign for 2018. Explaining pain neurobiology to patients is one part of pain education. To assess patient's pain knowledge, the Neurophysiology of Pain Questionnaire (NPQ) has been extensively used internationally. The present study describes the translation, cross-cultural adaptation and psychometric properties of the German version of the NPQ (NPQ­D). MATERIALS AND METHODS: The questionnaire was translated and adapted following established recommendations. A total of 169 patients and 122 physiotherapists completed the NPQ­D. The patients also completed the SF12 and the FFbH-R for the purpose of construct validation of the NPQ­D. Furthermore, 55 patients repeated the NPQ­D after 10 days. The NPQ­D was tested for dimensionality, internal consistency, construct validity and test-retest reliability. RESULTS: Internal consistency was α = 0.52. An exploratory factor analysis revealed four different factors. Therapists did show significantly higher NPQ­D scores than patients (p < 0.001) and therapist who have been educated in pain neurophysiology scored significantly higher than therapists without this education (p < 0.01). Test-retest reliability was high (ICC = 0.88; 95% CI = 0.82-0.93). There were no correlations between NPQ­D and SF12 or FFbH-R indicating construct validity of the NPQ­D. CONCLUSIONS: The NPQ has been translated and adapted into German. The psychometric properties show satisfactory results and the questionnaire can be recommended for clinicians and researchers alike to assess pain knowledge and changes in pain knowledge after pain education interventions.