An expression tag toolbox for microbial production of membrane bound plant cytochromes P450

Membrane-associated Cytochromes P450 (P450s) are one of the most important enzyme families for biosynthesis of plant-derived medicinal compounds. However, the hydrophobic nature of P450s makes their use in robust cell factories a challenge. Here we explore a small library of N-terminal expression tag chimeras of the model plant P450 CYP79A1 in different Escherichia coli strains. Using a high-throughput screening platform based on C-terminal GFP fusions, we identify several highly expressing and robustly performing chimeric designs. Analysis of long-term cultures by flow cytometry showed homogeneous populations for some of the conditions. Three chimeric designs were chosen for a more complex combinatorial assembly of a multigene pathway consisting of two P450s and a redox partner. Cells expressing these recombinant enzymes catalysed the conversion of the substrate to highly different ratios of the intermediate and the final product of the pathway. Finally, the effect of a robustly performing expression tag was explored with a library of 49 different P450s from medicinal plants and nearly half of these were improved in expression by more than 2-fold. The developed toolbox serves as platform to tune P450 performance in microbial cells, thereby facilitating recombinant production of complex plant P450-derived biochemicals

Using serum urate as a validated surrogate end point for flares in patients with gout:protocol for a systematic review and meta-regression analysis

INTRODUCTION: Gout is the most common inflammatory arthritis in men over 40 years of age. Long-term urate-lowering therapy is considered a key strategy for effective gout management. The primary outcome measure for efficacy in clinical trials of urate-lowering therapy is serum urate levels, effectively acting as a surrogate for patient-centred outcomes such as frequency of gout attacks or pain. Yet it is not clearly demonstrated that the strength of the relationship between serum urate and clinically relevant outcomes is sufficiently strong for serum urate to be considered an adequate surrogate. Our objective is to investigate the strength of the relationship between changes in serum urate in randomised controlled trials and changes in clinically relevant outcomes according to the ‘Biomarker-Surrogacy Evaluation Schema version 3’ (BSES3), documenting the validity of selected instruments by applying the ‘OMERACT Filter 2.0’. METHODS AND ANALYSIS: A systematic review described in terms of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) reporting guidelines will identify all relevant studies. Standardised data elements will be extracted from each study by 2 independent reviewers and disagreements are resolved by discussion. The data will be analysed by meta-regression of the between-arm differences in the change in serum urate level (independent variable) from baseline to 3 months (or 6 and 12 months if 3-month values are not available) against flare rate, tophus size and number and pain at the final study visit (dependent variables). ETHICS AND DISSEMINATION: This study will not require specific ethics approval since it is based on analysis of published (aggregated) data. The intended audience will include healthcare researchers, policymakers and clinicians. Results of the study will be disseminated by peer-reviewed publications. TRIAL REGISTRATION NUMBER: CRD42016026991

Tophaceous Gout in an Anorectic Patient Visualized by Dual Energy Computed Tomography (DECT)

BACKGROUND Gout is characterized by deposition of uric acid crystals (monosodium urate) in tissues and fluids. This can cause acute inflammatory arthritis. The 2015 ACR/EULAR criteria for the diagnosis of gout include dual energy computed tomography (DECT)-demonstrated monosodium urate crystals as a new criterion. DECT is a spectral decomposition that permits recognition of different types of tissues based on their characteristic energy-dependent photon attenuation. A positive scan is defined as the presence of urate at articular or periarticular sites. CASE REPORT We describe a 51-year-old woman known to have anorexia nervosa. During our clinical examination, we detected plenty of tophi on both hands, but no swollen joints. The diagnosis of gout was made by visualizing crystals in a biopsy from a tophus. The first line of treatment was allopurinol, the second line was rasburicase, and the current treatment is febuxostat 80 mg/day, allopurinol 300 mg twice a day, and colchicine 0.5 mg twice a day. The patient has unchanged arthralgia and the size and number of tophi remain the same as before treatment in spite of active treatment for 3 years. Previously the patient had problems with adherence, but now she claims that she follows the proposed treatment. The last plasma urate (P-urate) was 0.57 mmol/L. Following two years of treatment, DECT of hands visualized monosodium urate crystal deposits in the tophi, as seen on the clinical photos, but also crystals in relation to the tendons and soft tissue. CONCLUSIONS DECT is an imaging modality useful to assess urate crystal deposits at diagnosis of gout and could be considered during treatment evaluation. Lack of adherence to treatment should be considered when P-urate values vary significantly and when DECT scans over years persistently visualize monosodium urate crystals.</p

Hospital physicians' experiences with procalcitonin - implications for antimicrobial stewardship; a qualitative study.

Background. Procalcitonin is an inflammatory biomarker that is sensitive for bacterial infections and a promising clinical decision aid in antimicrobial stewardship programs. However, there are few studies of physicians’ experiences concerning the use of PCT. The objective of this study was to investigate whether hospital physicians’ experience with procalcitonin after 18 months of use can inform the PCT implementation in antimicrobial stewardship programs. Materials/methods. We deployed a qualitative approach using semi-structured interviews with 14 hospital physicians who had experience with procalcitonin in clinical practice. Interviews were audio-taped, transcribed verbatim and analysed using thematic analysis. Results. Physicians reported a knowledge gap, which made them uncertain about the appropriate procalcitonin use, interpretation, and trustworthiness. Simultaneously, the physicians experienced procalcitonin as a useful clinical decision aid but emphasised that their clinical evaluation of the patient was the most important factor when deciding on antibiotic treatment. Conclusions. Procalcitonin was regarded a helpful clinical tool, but the physicians called for more knowledge about its appropriate uses. Active implementation of unambiguous procalcitonin algorithms and physician education may enhance the utility of the test as an antimicrobial stewardship adjunct.publishedVersio

The Role of Procalcitonin as an Antimicrobial Stewardship Tool in Patients Hospitalized with Seasonal Influenza

Background: Up to 60% of the antibiotics prescribed to patients hospitalized with seasonal influenza are unnecessary. Procalcitonin (PCT) has the potential as an antimicrobial stewardship program (ASP) tool because it can differentiate between viral and bacterial etiology. We aimed to explore the role of PCT as an ASP tool in hospitalized seasonal influenza patients. Methods: We prospectively included 116 adults with seasonal influenza from two influenza seasons, 2018–2020. All data was obtained from a single clinical setting and analyzed by descriptive statistics and regression models. Results: In regression analyses, we found a positive association of PCT with 30 days mortality and the amount of antibiotics used. Influenza diagnosis was associated with less antibiotic use if the PCT value was low. Patients with a low initial PCT (<0.25 µg/L) had fewer hospital and intensive care unit (ICU) days and fewer positive chest X-rays. PCT had a negative predictive value of 94% for ICU care stay, 98% for 30 days mortality, and 88% for bacterial coinfection. Conclusion: PCT can be a safe rule-out test for bacterial coinfection. Routine PCT use in seasonal influenza patients with an uncertain clinical picture, and rapid influenza PCR testing, may be efficient as ASP tools.publishedVersio

Exchange of CO₂in Arctic tundra:impacts of meteorological variations and biological disturbance

Abstract. An improvement in our process-based understanding of carbon (C) exchange in the Arctic, and its climate sensitivity, is critically needed for understanding the response of tundra ecosystems to a changing climate. In this context, we analyzed the net ecosystem exchange (NEE) of CO2 in West Greenland tundra (64° N) across eight snow-free periods in eight consecutive years, and characterized the key processes of net ecosystem exchange, and its two main modulating components: gross primary production (GPP) and ecosystem respiration (Reco). Overall, the ecosystem acted as a consistent sink of CO2, accumulating −30 g C m−2 on average (range −17 to −41 g C m−2) during the years 2008–2015, except 2011 that was associated with a major pest outbreak. The results do not reveal a marked meteorological effect on the net CO2 uptake despite the high inter-annual variability in the timing of snowmelt, start and duration of the growing season. The ranges in annual GPP (−182 to −316 g C m−2) and Reco (144 to 279 g C m−2) were &gt; 5 fold larger and they were also more variable (Coefficients of variation are 3.6 and 4.1 % respectively) than for NEE (0.7 %). GPP and Reco were sensitive to insolation and temperatures; and there was a tendency towards larger GPP and Reco during warmer and wetter years. The relative lack of sensitivity of NEE to climate was a result of the correlated meteorological response of GPP and Reco. During the 2011 anomalous year, the studied ecosystem released 41 g C m−2 as biological disturbance reduced GPP more strongly than Reco. With continued warming temperatures and longer growing seasons, tundra systems will increase rates of C cycling although shifts in sink strength will likely be triggered by factors such as biological disturbances, events that will challenge the forecast of upcoming C states. </jats:p