A microbial supply chain for production of the anti-cancer drug vinblastine.

Monoterpene indole alkaloids (MIAs) are a diverse family of complex plant secondary metabolites with many medicinal properties, including the essential anti-cancer therapeutics vinblastine and vincristine1. As MIAs are difficult to chemically synthesize, the world's supply chain for vinblastine relies on low-yielding extraction and purification of the precursors vindoline and catharanthine from the plant Catharanthus roseus, which is then followed by simple in vitro chemical coupling and reduction to form vinblastine at an industrial scale2,3. Here, we demonstrate the de novo microbial biosynthesis of vindoline and catharanthine using a highly engineered yeast, and in vitro chemical coupling to vinblastine. The study showcases a very long biosynthetic pathway refactored into a microbial cell factory, including 30 enzymatic steps beyond the yeast native metabolites geranyl pyrophosphate and tryptophan to catharanthine and vindoline. In total, 56 genetic edits were performed, including expression of 34 heterologous genes from plants, as well as deletions, knock-downs and overexpression of ten yeast genes to improve precursor supplies towards de novo production of catharanthine and vindoline, from which semisynthesis to vinblastine occurs. As the vinblastine pathway is one of the longest MIA biosynthetic pathways, this study positions yeast as a scalable platform to produce more than 3,000 natural MIAs and a virtually infinite number of new-to-nature analogues.

Cervical intraepithelial neoplasia and fecundability: a prospective cohort study.

STUDY QUESTION: Is cervical intraepithelial neoplasia (CIN) associated with reduced fecundability, defined as the probability of conceiving per menstrual cycle? SUMMARY ANSWER: Overall, we observed no meaningful association between CIN and fecundability, regardless of surgical status, although a recent diagnosis of moderate or severe CIN might be associated with slightly reduced fecundability for 2 years after diagnosis. WHAT IS KNOWN ALREADY: About 15% of couples experience infertility. Few studies have examined the influence of CIN on fertility, and the results have been inconsistent. No study has investigated the association between fecundability and pathologist-reported CIN diagnoses, particularly with respect to the recency of the specific CIN diagnoses. STUDY DESIGN, SIZE, DURATION: This prospective cohort study included 9586 women trying to conceive. The women were enrolled from 1 June 2007 to 3 February 2020. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women were invited to complete a baseline questionnaire and bimonthly follow-up questionnaires for up to 12 months or until pregnancy occurred. Data on cervical cytologies and biopsies were retrieved from The National Pathology Registry (DNPR), which holds records of all cervical specimens examined in Denmark. Women were categorized based on their most severe diagnosis of CIN: no lesion, other cervical changes, mild CIN (CIN1), or moderate/severe CIN (CIN2+) with or without surgery. To investigate the association between CIN and fecundability, we computed fecundability ratios (FR) and 95% confidence intervals (CI) using a proportional probabilities regression model. We adjusted for age at study entry, partner age, body mass index, smoking status, timing of intercourse, parity, education, number of sexual partners, and household income. MAIN RESULTS AND THE ROLE OF CHANCE: Compared with no lesion, the adjusted FRs (95% CI) for the association between CIN and fecundability were: other cervical lesions, 0.97 (0.91-1.04); CIN1, 1.04 (0.96-1.13); CIN2+ no surgery, 1.00 (0.82-1.22); and CIN2+ with surgery 0.99 (0.89-1.10). The FRs (95% CI) for a recent diagnosis (<2 years) of CIN were 0.98 (0.86-1.11) for other cervical lesions; 1.13 (0.99-1.29) for CIN1; 0.89 (0.62-1.26) for CIN2+ no surgery and 0.91 (0.75-1.10) for CIN2+ with surgery compared with the no lesion group. LIMITATIONS, REASONS FOR CAUTION: In the analyses, we adjusted for several covariates related to the women. However, we had little information on the male partners which could lead to unmeasured confounding as fecundability is a couple-based measure of fertility. Furthermore, a CIN diagnosis may not be constant as it may regress or progress spontaneously; therefore, it is possible that we have misclassified some women, especially women categorized as having normal cells or CIN1. WIDER IMPLICATIONS OF THE FINDINGS: Our results contribute important knowledge to women who are concerned about their future fertility after receiving a CIN diagnosis. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by The Danish Cancer Society (R167-A11036-17-S2). The overall cohorts were funded by the National Institute of Child Health and Human Development (R01-HD086742 and R03-HD094117). The authors report no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Targeting the gut-lung axis by synbiotic feeding to infants in a randomized controlled trial.

BACKGROUND: Formula-fed infants are at increased risk of infections. Due to the cross-talk between the mucosal systems of the gastrointestinal and respiratory tracts, adding synbiotics (prebiotics and probiotics) to infant formula may prevent infections even at distant sites. Infants that were born full term and weaned from breast milk were randomized to prebiotic formula (fructo- and galactooligosaccharides) or the same prebiotic formula with Lactobacillus paracasei ssp. paracasei F19 (synbiotics) from 1 to 6 months of age. The objective was to examine the synbiotic effects on gut microbiota development. RESULTS: Fecal samples collected at ages 1, 4, 6, and 12 months were analyzed using 16S rRNA gene sequencing and a combination of untargeted gas chromatography-mass spectrometry/liquid chromatography-mass spectrometry. These analyses revealed that the synbiotic group had a lower abundance of Klebsiella, a higher abundance of Bifidobacterium breve compared to the prebiotic group, and increases in the anti-microbial metabolite d-3-phenyllactic acid. We also analyzed the fecal metagenome and antibiotic resistome in the 11 infants that had been diagnosed with lower respiratory tract infection (cases) and 11 matched controls using deep metagenomic sequencing. Cases with lower respiratory tract infection had a higher abundance of Klebsiella species and antimicrobial resistance genes related to Klebsiella pneumoniae, compared to controls. The results obtained using 16S rRNA gene amplicon and metagenomic sequencing were confirmed in silico by successful recovery of the metagenome-assembled genomes of the bacteria of interest. CONCLUSIONS: This study demonstrates the additional benefit of feeding specific synbiotics to formula-fed infants over prebiotics only. Synbiotic feeding led to the underrepresentation of Klebsiella, enrichment of bifidobacteria, and increases in microbial degradation metabolites implicated in immune signaling and in the gut-lung and gut-skin axes. Our findings support future clinical evaluation of synbiotic formula in the prevention of infections and associated antibiotic treatment as a primary outcome when breastfeeding is not feasible. TRIAL REGISTRATION: ClinicalTrials.gov NCT01625273 . Retrospectively registered on 21 June 2012.

Human Blood Lipoprotein Predictions from 1H NMR Spectra: Protocol, Model Performances, and Cage of Covariance.

Lipoprotein subfractions are biomarkers for the early diagnosis of cardiovascular diseases. The reference method, ultracentrifugation, for measuring lipoproteins is time-consuming, and there is a need to develop a rapid method for cohort screenings. This study presents partial least-squares regression models developed using 1H nuclear magnetic resonance (NMR) spectra and concentrations of lipoproteins as measured by ultracentrifugation on 316 healthy Danes. This study explores, for the first time, different regions of the 1H NMR spectrum representing signals of molecules in lipoprotein particles and different lipid species to develop parsimonious, reliable, and optimal prediction models. A total of 65 lipoprotein main and subfractions were predictable with high accuracy, Q2 of >0.6, using an optimal spectral region (1.4-0.6 ppm) containing methylene and methyl signals from lipids. The models were subsequently tested on an independent cohort of 290 healthy Swedes with predicted and reference values matching by up to 85-95%. In addition, an open software tool was developed to predict lipoproteins concentrations in human blood from standardized 1H NMR spectral recordings.

Production of abscisic acid in the oleaginous yeast Yarrowia lipolytica.

Abscisic acid (ABA) is a phytohormone with applications in agriculture and human health. ABA can be produced by Botrytis cinerea, a plant pathogenic filamentous fungus. However, the cultivation process is lengthy and strain improvement by genetic engineering is difficult. Therefore, we engineered the oleaginous yeast Yarrowia lipolytica as an alternative host for ABA production. First, we expressed five B. cinerea genes involved in ABA biosynthesis (BcABA1,BcABA2,BcABA3,BcABA4 and BcCPR1) in a Y. lipolytica chassis with optimized mevalonate flux. The strain produced 59.2 mg/L of ABA in small-scale cultivation. Next, we expressed an additional copy of each gene in the strain, but only expression of additional copy of BcABA1 gene increased the ABA titer to 168.5 mg/L. We then integrated additional copies of the mevalonate pathway and ABA biosynthesis encoding genes, and we expressed plant ABA transporters resulting in an improved strain producing 263.5 mg/L and 9.1 mg/g dry cell weight (DCW) ABA. Bioreactor cultivation resulted in a specific yield of 12.8 mg/g DCW ABA; however, surprisingly, the biomass level obtained in bioreactors was only 10.5 g DCW/L, with a lower ABA titer of 133.6 mg/L. While further optimization is needed, this study confirms Y. lipolytica as a potential alternative host for the ABA production.

Coupling S-adenosylmethionine-dependent methylation to growth: Design and uses.

We present a selection design that couples S-adenosylmethionine-dependent methylation to growth. We demonstrate its use in improving the enzyme activities of not only N-type and O-type methyltransferases by 2-fold but also an acetyltransferase of another enzyme category when linked to a methylation pathway in Escherichia coli using adaptive laboratory evolution. We also demonstrate its application for drug discovery using a catechol O-methyltransferase and its inhibitors entacapone and tolcapone. Implementation of this design in Saccharomyces cerevisiae is also demonstrated.

Tailored biosynthesis of gibberellin plant hormones in yeast.

The application of small amounts of natural plant growth hormones, such as gibberellins (GAs), can increase the productivity and quality of many vegetable and fruit crops. However, gibberellin growth hormones usage is limited by the high cost of their production, which is currently based on fermentation of a natural fungal producer Fusarium fujikuroi that produces a mix of several GAs. We explored the potential of the oleaginous yeast Yarrowia lipolytica to produce specific profiles of GAs. Firstly, the production of the GA-precursor ent-kaurenoic acid (KA) at 3.75 mg/L was achieved by expression of biosynthetic enzymes from the plant Arabidopsis thaliana and upregulation of the mevalonate (MVA) pathway. We then built a GA4-producing strain by extending the GA-biosynthetic pathway and upregulating the MVA-pathway further, resulting in 17.29 mg/L GA4. Additional expression of the F. fujikoroi GA-biosynthetic enzymes resulted in the production of GA7 (trace amounts) and GA3 (2.93 mg/L). Lastly, through protein engineering and the expression of additional KA-biosynthetic genes, we increased the GA3-production 4.4-fold resulting in 12.81 mg/L. The developed system presents a promising resource for the recombinant production of specific gibberellins, identifying bottlenecks in GA biosynthesis, and discovering new GA biosynthetic genes. CLASSIFICATION: Biological Sciences, Applied Biological Sciences.

Sagittal abdominal diameter and waist circumference appear to be equally good as identifiers of cardiometabolic risk.

BACKGROUND AND AIMS: Body mass index (BMI) and waist circumference (WC) are commonly used markers of cardiometabolic risk. However, sagittal abdominal diameter (SAD) has been proposed as a possibly more sensitive marker of intra-abdominal obesity. We investigated differences in how SAD, WC, and BMI were correlated with cardiometabolic risk markers. METHODS AND RESULTS: This cross-sectional study investigated anthropometric and metabolic baseline measurements of individuals from six trials. Multiple linear regression and (partial) correlation coefficients were used to investigate associations between SAD, WC, and BMI and cardiometabolic risk markers, including components of the metabolic syndrome as well as insulin resistance, blood lipids, and lowgrade inflammation. In total 1516 mostly overweight or obese individuals were included in the study. SAD was significantly more correlated with TG than WC for all studies, and overall increase in correlation was 0.05 (95% CI (0.02; 0.08). SAD was significantly more correlated with the markers TG and DBP 0.11 (95% CI (0.08, 0.14)) and 0.04 (95% CI (0.006, 0.07), respectively compared to BMI across all or most studies. CONCLUSION: This study showed that no single anthropometric indicator was consistently more strongly correlated across all markers of cardiometabolic risk. However, SAD was significantly more strongly correlated with TG than WC and significantly more strongly correlated with DBP and TG than BMI.

SmartPeak Automates Targeted and Quantitative Metabolomics Data Processing.

Technological advances in high-resolution mass spectrometry (MS) vastly increased the number of samples that can be processed in a life science experiment, as well as volume and complexity of the generated data. To address the bottleneck of high-throughput data processing, we present SmartPeak (https://github.com/AutoFlowResearch/SmartPeak), an application that encapsulates advanced algorithms to enable fast, accurate, and automated processing of capillary electrophoresis-, gas chromatography-, and liquid chromatography (LC)-MS(/MS) data and high-pressure LC data for targeted and semitargeted metabolomics, lipidomics, and fluxomics experiments. The application allows for an approximate 100-fold reduction in the data processing time compared to manual processing while enhancing quality and reproducibility of the results.

Systems Analysis of NADH Dehydrogenase Mutants Reveals Flexibility and Limits of Pseudomonas taiwanensis VLB120's Metabolism.

Obligate aerobic organisms rely on a functional electron transport chain for energy conservation and NADH oxidation. Because of this essential requirement, the genes of this pathway are likely constitutively and highly expressed to avoid a cofactor imbalance and energy shortage under fluctuating environmental conditions. We here investigated the essentiality of the three NADH dehydrogenases of the respiratory chain of the obligate aerobe Pseudomonas taiwanensis VLB120 and the impact of the knockouts of corresponding genes on its physiology and metabolism. While a mutant lacking all three NADH dehydrogenases seemed to be nonviable, the single or double knockout mutant strains displayed no, or only a weak, phenotype. Only the mutant deficient in both type 2 dehydrogenases showed a clear phenotype with biphasic growth behavior and a strongly reduced growth rate in the second phase. In-depth analyses of the metabolism of the generated mutants, including quantitative physiological experiments, transcript analysis, proteomics, and enzyme activity assays revealed distinct responses to type 2 and type 1 dehydrogenase deletions. An overall high metabolic flexibility enables P. taiwanensis to cope with the introduced genetic perturbations and maintain stable phenotypes, likely by rerouting of metabolic fluxes. This metabolic adaptability has implications for biotechnological applications. While the phenotypic robustness is favorable in large-scale applications with inhomogeneous conditions, the possible versatile redirecting of carbon fluxes upon genetic interventions can thwart metabolic engineering efforts.IMPORTANCE While Pseudomonas has the capability for high metabolic activity and the provision of reduced redox cofactors important for biocatalytic applications, exploitation of this characteristic might be hindered by high, constitutive activity of and, consequently, competition with the NADH dehydrogenases of the respiratory chain. The in-depth analysis of NADH dehydrogenase mutants of Pseudomonas taiwanensis VLB120 presented here provides insight into the phenotypic and metabolic response of this strain to these redox metabolism perturbations. This high degree of metabolic flexibility needs to be taken into account for rational engineering of this promising biotechnological workhorse toward a host with a controlled and efficient supply of redox cofactors for product synthesis.

Whole grain-rich diet reduces body weight and systemic low-grade inflammation without inducing major changes of the gut microbiome: a randomised cross-over trial.

OBJECTIVE: To investigate whether a whole grain diet alters the gut microbiome and insulin sensitivity, as well as biomarkers of metabolic health and gut functionality. DESIGN: 60 Danish adults at risk of developing metabolic syndrome were included in a randomised cross-over trial with two 8-week dietary intervention periods comprising whole grain diet and refined grain diet, separated by a washout period of ≥6 weeks. The response to the interventions on the gut microbiome composition and insulin sensitivity as well on measures of glucose and lipid metabolism, gut functionality, inflammatory markers, anthropometry and urine metabolomics were assessed. RESULTS: 50 participants completed both periods with a whole grain intake of 179±50 g/day and 13±10 g/day in the whole grain and refined grain period, respectively. Compliance was confirmed by a difference in plasma alkylresorcinols (p<0.0001). Compared with refined grain, whole grain did not significantly alter glucose homeostasis and did not induce major changes in the faecal microbiome. Also, breath hydrogen levels, plasma short-chain fatty acids, intestinal integrity and intestinal transit time were not affected. The whole grain diet did, however, compared with the refined grain diet, decrease body weight (p<0.0001), serum inflammatory markers, interleukin (IL)-6 (p=0.009) and C-reactive protein (p=0.003). The reduction in body weight was consistent with a reduction in energy intake, and IL-6 reduction was associated with the amount of whole grain consumed, in particular with intake of rye. CONCLUSION: Compared with refined grain diet, whole grain diet did not alter insulin sensitivity and gut microbiome but reduced body weight and systemic low-grade inflammation. TRIAL REGISTRATION NUMBER: NCT01731366; Results.

Microbial fermentation of flaxseed fibers modulates the transcriptome of GPR41-expressing enteroendocrine cells and protects mice against diet-induced obesity.

Dietary fibers, an integral part of the human diet, require the enzymatic activity of the gut microbiota for complete metabolism into short-chain fatty acids (SCFAs). SCFAs are important modulators of host metabolism and physiology and act in part as signaling molecules by activating G protein-coupled receptors (GPCRs), such as GPR41. Flaxseed fibers improve metabolism in rodents and mice, but their fermentation profiles, effects on enteroendocrine cells, and associated metabolic benefits are unknown. We fed GPR41-red fluorescent protein mice, an enteroendocrine reporter mouse strain, chow, high-fat diet (HFD), or HFD supplemented either with 10% nonfermentable fiber cellulose or fermentable flaxseed fibers for 12 wk to assess changes in cecal gut microbiota, enteroendocrine cell transcriptome in the ileum and colon, and physiological parameters. We observed that flaxseed fibers restructured the gut microbiota and promoted proliferation of the genera Bifidobacterium and Akkermansia compared with HFD. The shifts in cecal bacterial composition restored levels of the SCFAs butyrate similar to the chow diet, resulting in colonic but not ileal enteroendocrine cell transcriptional changes in genes related to cell cycle, mRNA, and protein transport compared with HFD. Consistent with the effects on enteroendocrine functions, flaxseed fibers also protected mice from diet-induced obesity, potentially by preventing a reduction in energy expenditure induced by an HFD. Our study shows that flaxseed fibers alter cecal microbial ecology, are fermented to SCFAs in the cecum, and modulate enteroendocrine cell transcriptome in the colon, which may contribute to their metabolically favorable phenotype.

CRISPR/Cas9-RNA interference system for combinatorial metabolic engineering of Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is widely used in industrial biotechnology for the production of fuels, chemicals, food ingredients, food and beverages, and pharmaceuticals. To obtain high-performing strains for such bioprocesses, it is often necessary to test tens or even hundreds of metabolic engineering targets, preferably in combinations, to account for synergistic and antagonistic effects. Here, we present a method that allows simultaneous perturbation of multiple selected genetic targets by combining the advantage of CRISPR/Cas9, in vivo recombination, USER assembly and RNA interference. CRISPR/Cas9 introduces a double-strand break in a specific genomic region, where multiexpression constructs combined with the knockdown constructs are simultaneously integrated by homologous recombination. We show the applicability of the method by improving cis,cis-muconic acid production in S. cerevisiae through simultaneous manipulation of several metabolic engineering targets. The method can accelerate metabolic engineering efforts for the construction of future cell factories.

Prevotella Abundance Predicts Weight Loss Success in Healthy, Overweight Adults Consuming a Whole-Grain Diet Ad Libitum: A Post Hoc Analysis of a 6-Wk Randomized Controlled Trial.

BACKGROUND: The key to effective weight loss may be to match diet and gut microbes, since recent studies have found that subjects with high Prevotella abundances in their gut microbiota lose more weight on diets rich in fiber than subjects with low Prevotella abundances. OBJECTIVES: We reanalyzed a 6-wk, parallel, randomized trial to investigate difference in body weight changes when participants, stratified by fecal microbiota composition, consumed ad libitum a whole-grain (WG) or a refined-wheat (RW) diet. METHODS: We stratified 46 (19 men, 27 women; ages 30-65 y) healthy, overweight adults by baseline Prevotella-to-Bacteroides ratios and Prevotella abundances. Subjects with no Prevotella were analyzed separately (n = 24). Compared with the RW diet (mean = 221 g/d), the WG diet (mean = 228 g/d) had a higher fiber content (33 g/d compared with 23 g/d). Linear mixed models and correlations were applied to link 6-wk changes in body weights and metabolic and microbiota markers, according to Prevotella groups and diets. RESULTS: The Prevotella abundances correlated inversely with weight changes (r = -0.34; P = 0.043). Consequently, subjects with high Prevotella abundances (n = 15) spontaneously lost 1.80 kg (95% CI: -3.23, -0.37 kg; P = 0.013) more on the WG diet than on the RW diet, whereas those with low Prevotella abundances (n = 31) were weight stable (-0.22 kg; 95% CI: -1.40, 0.96 kg; P = 0.72). Thus, the mean difference between the Prevotella groups was 2.02 kg (95% CI: -3.87, -0.17 kg; P = 0.032). Subjects with no Prevotella lost 1.59 kg (95% CI: -2.65, -0.52 kg; P = 0.004) more on the WG diet than on the RW diet. No 6-wk changes in appetite sensations, glucose metabolisms, or fecal SCFAs were associated with the Prevotella groups. CONCLUSIONS: Healthy, overweight adults with high Prevotella abundances lost more weight than subjects with low Prevotella abundances when consuming a diet rich in WG and fiber ad libitum for 6 wk. This further supports enterotypes as a potential biomarker in personalized nutrition for obesity management. This t rial was registered at clinicaltrials.gov as NCT02358122.

International travel as source of a hospital outbreak with an unusual meticillin-resistant Staphylococcus aureus clonal complex 398, Denmark, 2016.

In May 2016, an unusual outbreak with the Panton-Valentine leukocidin-positive human variant of meticillin-resistant Staphylococcus aureus (MRSA) clonal complex 398 occurred among mothers and infants in the maternity unit of a Danish hospital. MRSA sharing genotypic and phenotypic characteristics was confirmed in 36 cases, including 26 patients, nine household members and a healthcare worker (HCW) who had contact with all the patients. The national MRSA database contained 37 seemingly unlinked MRSA cases whose isolates shared the same genotypic and phenotypic characteristics as the outbreak strain. Whole genome sequencing showed that three of these isolates clustered together with the 36 outbreak isolates, suggesting spread outside the hospital. The HCW and 21 of 37 cases from the national MRSA database had links to south-eastern Asia, where the outbreak strain is endemic. These findings suggest that the HCW acquired the outbreak strain while travelling in south-eastern Asia and then introduced it into the hospital; from there, it spread within the patients' households and into the community. Screening of travellers returning from countries with high levels of MRSA could be an important intervention to prevent spread of these bacteria into hospitals via patients or HCWs.

Resistance towards nondepolarising muscle relaxants: prolonged onset time: A systematic review.

BACKGROUND: Nondepolarising muscle relaxants (NDMRs) provide optimal conditions for tracheal intubation and improve surgical conditions. Several clinical conditions, diseases and pharmacological interactions have been suggested to cause resistance towards NDMRs that may translate into difficult intubation or inadequate operating conditions during surgery. OBJECTIVE: The aim of this study was to evaluate the current evidence of patient groups with resistance towards NDMRs. A prolonged onset time was defined as a difference that exceeded 25% compared with controls. DESIGN: A systematic review of randomised controlled trials and cohort studies. DATA SOURCES: A comprehensive search was performed in 2016 in PubMed and EMBASE. ELIGIBILITY CRITERIA: Patients with conditions or diseases, or patients taking medication, which lead to resistance towards current NDMRs (rocuronium, vecuronium, cisatracurium, atracurium, mivacurium and pancuronium). Included outcomes were onset time defined as the time between administration of NDMR to maximal (90, 95 or 100%) depression of baseline twitch height of the first twitch in a train-of-four. RESULTS: Twenty-five studies were included. Strong evidence supports a prolonged onset time of rocuronium in patients with thermal injury and Duchenne muscular dystrophy. Moderate evidence supports a prolonged onset time of NDMRs during hypothermia and in patients with infection, oculopharyngeal muscular dystrophy, liver cirrhosis treated with ulinastatin, when remifentanil is administered prior to administration of an NDMR, in fasting patients being rehydrated intravenously prior to administration of NDMR, in children with end-stage renal failure and in patients with atrial or ventricular septal defects. CONCLUSION: A prolonged onset time should be suspected in patients with thermal injury and Duchenne's muscular dystrophy. Further, evidence supports a prolonged onset time in patients with infection, oculopharyngeal muscular dystrophy, congenital heart defects, kidney failure, liver cirrhosis treated with ulinastatin along with remifentanil or intravenous fluids administered prior to NDMR.

Exploring Perceptions of Continuity of Care Among People With Long-Term Mental Disorders in Denmark.

Continuity of mental health care is central to improve the treatment and rehabilitation of people with mental disorders. While most studies on continuity of care fail to take the perspectives of service users into account, the aim of this study was to explore the perceived meanings of continuity of care among people with long-term mental disorders. Fifteen service users participated in semi-structured in-depth interviews. We used template analysis to guide the analysis. The main transversal themes of continuity were "Navigating the system" and "Connecting to people and everyday life." While the first theme related to the participants' experiences of their interaction with the mental health care system, the latter related to their hopes and perceived opportunities for a good life as desired outcomes of mental health care. We conclude that efforts to improve continuity of mental health care should be tailored to the priorities of service users.

Addressing organizational barriers to continuity of care in the Danish mental health system - a comparative analysis of 14 national intervention projects.

BACKGROUND: Continuity of mental health care is central to improve the conditions of people with enduring mental disorders. In Denmark, several government-funded projects on the improvement of continuity of mental health care have been initiated since 2009. AIM: The aim of this study was to investigate how national intervention projects on continuity of mental health care have addressed major barriers for continuity of care and extract general learning points from the projects on the improvement of continuity of care. METHOD: The study was designed as a thematic document analysis of external evaluations of 14 major national projects on the improvement of continuity of routine mental health care from 2009 to 2017. The data material was processed through thematic coding and comparative analysis. RESULTS: The analysis was organized around four main barriers for continuity: Lack of models for collaboration, different professional cultures and methods, lack of channels of communication, and intersectoral differences in management, economy, and legislation. The first three barriers were addressed in a predominant part of the projects through development of collaborative models, common tools and communication systems. The latter structural barrier was not addressed in any of the projects. CONCLUSION: There is an ongoing need to address barriers for continuity of mental health care. So far, there has been a much larger focus on organizational, cultural and communicational aspects of continuity than on structural aspects. The study calls for an increased focus on how changes in existing managerial, economic and legislative structures can improve continuity of care.

Functional mining of transporters using synthetic selections.

Only 25% of bacterial membrane transporters have functional annotation owing to the difficulty of experimental study and of accurate prediction of their function. Here we report a sequence-independent method for high-throughput mining of novel transporters. The method is based on ligand-responsive biosensor systems that enable selective growth of cells only if they encode a ligand-specific importer. We developed such a synthetic selection system for thiamine pyrophosphate and mined soil and gut metagenomes for thiamine-uptake functions. We identified several members of a novel class of thiamine transporters, PnuT, which is widely distributed across multiple bacterial phyla. We demonstrate that with modular replacement of the biosensor, we could expand our method to xanthine and identify xanthine permeases from gut and soil metagenomes. Our results demonstrate how synthetic-biology approaches can effectively be deployed to functionally mine metagenomes and elucidate sequence-function relationships of small-molecule transport systems in bacteria.

Engineering prokaryotic transcriptional activators as metabolite biosensors in yeast.

Whole-cell biocatalysts have proven a tractable path toward sustainable production of bulk and fine chemicals. Yet the screening of libraries of cellular designs to identify best-performing biocatalysts is most often a low-throughput endeavor. For this reason, the development of biosensors enabling real-time monitoring of production has attracted attention. Here we applied systematic engineering of multiple parameters to search for a general biosensor design in the budding yeast Saccharomyces cerevisiae based on small-molecule binding transcriptional activators from the prokaryote superfamily of LysR-type transcriptional regulators (LTTRs). We identified a design supporting LTTR-dependent activation of reporter gene expression in the presence of cognate small-molecule inducers. As proof of principle, we applied the biosensors for in vivo screening of cells producing naringenin or cis,cis-muconic acid at different levels, and found that reporter gene output correlated with production. The transplantation of prokaryotic transcriptional activators into the eukaryotic chassis illustrates the potential of a hitherto untapped biosensor resource useful for biotechnological applications.