Prediction of Cellular Burden with Host--Circuit Models

Heterologous gene expression draws resources from host cells. These resources include vital components to sustain growth and replication, and the resulting cellular burden is a widely recognised bottleneck in the design of robust circuits. In this tutorial we discuss the use of computational models that integrate gene circuits and the physiology of host cells. Through various use cases, we illustrate the power of host-circuit models to predict the impact of design parameters on both burden and circuit functionality. Our approach relies on a new generation of computational models for microbial growth that can flexibly accommodate resource bottlenecks encountered in gene circuit design. Adoption of this modelling paradigm can facilitate fast and robust design cycles in synthetic biology

Refactoring of a Synthetic Raspberry Ketone Pathway with EcoFlex

Background: A key focus of synthetic biology is to develop microbial or cell-free based biobased routes to value-added chemicals such as fragrances. Originally, we developed the EcoFlex system, a Golden Gate toolkit, to study genes/pathways flexibly using Escherichia coli heterologous expression. In this current work, we sought to use EcoFlex to optimise a synthetic raspberry ketone biosynthetic pathway. Raspberry ketone is a high-value (~£20,000 kg-1) fine chemical farmed from raspberry (Rubeus rubrum) fruit.Results: By applying a synthetic biology led design-build-test-learn cycle approach, we refactor the raspberry ketone pathway from a low level of productivity (0.2 mg/L), to achieve a 65-fold (12.9 mg/L) improvement in production. We perform this optimisation at the prototype level (using microtiter plate cultures) with E. coli DH10, as a routine cloning host. The use of E. coli DH10 facilitates the Golden Gate cloning process for the screening of combinatorial libraries. In addition, we also newly establish a novel colour-based phenotypic screen to identify productive clones quickly from solid/liquid culture. Conclusions: Our findings provide a stable raspberry ketone pathway that relies upon a natural feedstock (L-tyrosine) and uses only constitutive promoters to control gene expression. In conclusion we demonstrate the capability of EcoFlex for fine-tuning a model fine chemical pathway and provide a range of newly characterised promoter tools gene expression in E. coli

Effectiveness of pulmonary rehabilitation in COPD with mild symptoms: a systematic review with meta-analyses

Mette Rugbjerg,1 Ulrik Winning Iepsen,1 Karsten Juhl Jørgensen,2 Peter Lange1,3,4 1The Centre of Inflammation and Metabolism and The Centre for Physical Activity Research, Department of Infectious Diseases, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark; 2The Nordic Cochrane Centre, Rigshospitalet, Copenhagen, Denmark; 3Department of Respiratory Medicine, University Hospital Hvidovre, Copenhagen, Denmark; 4Department of Public Health, Section of Social Medicine, University of Copenhagen, Copenhagen, Denmark Purpose: Most guidelines recommend pulmonary rehabilitation (PR) for patients with chronic obstructive pulmonary disease (COPD) and modified Medical Research Council dyspnea scale (mMRC) levels ≥2, but the effectiveness of PR in patients with less advanced disease is not well established. Our aim was to investigate the effects of PR in patients with COPD and mMRC ≤1.Methods: The methodology was developed as a part of evidence-based guideline development and is in accordance with the principles of the Grading of Recommendations Assessment, Development and Evaluation (GRADE) Working Group. We identified randomized controlled trials (RCTs) through a systematic, multidatabase literature search and selected RCTs comparing the effects of PR with usual care in patients with COPD and mMRC ≤1. Predefined critical outcomes were health-related quality of life (HRQoL), adverse effects and mortality, while walking distance, maximal exercise capacity, muscle strength, and dropouts were important outcomes. Two authors independently extracted data, assessed trial eligibility and risk of bias, and graded the evidence. Meta-analyses were performed when deemed feasible.Results: Four RCTs (489 participants) were included. On the basis of moderate-quality evidence, we found a clinically and statistically significant improvement in short-term HRQoL of 4.2 units (95% confidence interval [CI]: [-4.51 to -3.89]) on St George’s Respiratory Questionnaire, but not at the longest follow-up. We also found a statistically significant improvement of 25.71 m (95% CI: [15.76–35.65]) in the 6-minute walk test with PR; however, this improvement was not considered clinically relevant. No difference was found for mortality, and insufficient data prohibited meta-analysis for muscle strength and maximal exercise capacity. No adverse effects were reported.Conclusion: We found a moderate quality of evidence suggesting a small, significant improvement in short-term HRQoL and a clinically nonsignificant improvement in walking distance following PR in patients with COPD and mild symptoms. This resulted in a weak recommendation of routine PR in these patients using the GRADE approach. Keywords: pulmonary disease, COPD, COPD with mild symptoms, physical activity, pulmonary rehabilitation, health-related quality of life&nbsp

Effect of endurance versus resistance training on quadriceps muscle dysfunction in COPD: a pilot study

Ulrik Winning Iepsen,1 Gregers Druedal Wibe Munch,1 Mette Rugbjerg,1 Anders Rasmussen Rinnov,1 Morten Zacho,1 Stefan Peter Mortensen,1,2 Niels H Secher,3 Thomas Ringbaek,4 Bente Klarlund Pedersen,1 Ylva Hellsten,5 Peter Lange,1,4,6 Pia Thaning1,4 1The Centre of Inflammation and Metabolism and the Centre for Physical Activity Research, Rigshospitalet, University of Copenhagen, Denmark, 2Department of Cardiovascular and Renal Research, University of Southern Denmark, Odense, 3Department of Anesthesiology, University of Copenhagen, Rigshospitalet, Copenhagen, 4Department of Respiratory Medicine, University Hospital Hvidovre, Hvidovre, 5Department of Nutrition, Exercise and Sports, University of Copenhagen, 6Department of Public Health, Section of Social Medicine, University of Copenhagen, Copenhagen, Denmark Introduction: Exercise is an important countermeasure to limb muscle dysfunction in COPD. The two major training modalities in COPD rehabilitation, endurance training (ET) and resistance training (RT), may both be efficient in improving muscle strength, exercise capacity, and health-related quality of life, but the effects on quadriceps muscle characteristics have not been thoroughly described.Methods: Thirty COPD patients (forced expiratory volume in 1 second: 56% of predicted, standard deviation [SD] 14) were randomized to 8 weeks of ET or RT. Vastus lateralis muscle biopsies were obtained before and after the training intervention to assess muscle morphology and metabolic and angiogenic factors. Symptom burden, exercise capacity (6-minute walking and cycle ergometer tests), and vascular function were also assessed.Results: Both training modalities improved symptom burden and exercise capacity with no difference between the two groups. The mean (SD) proportion of glycolytic type IIa muscle fibers was reduced after ET (from 48% [SD 11] to 42% [SD 10], P<0.05), whereas there was no significant change in muscle fiber distribution with RT. There was no effect of either training modality on muscle capillarization, angiogenic factors, or vascular function. After ET the muscle protein content of phosphofructokinase was reduced (P<0.05) and the citrate synthase content tended increase (P=0.08) but no change was observed after RT.Conclusion: Although both ET and RT improve symptoms and exercise capacity, ET induces a more oxidative quadriceps muscle phenotype, counteracting muscle dysfunction in COPD. Keywords: muscle fibers, phosphofructokinase-1, vasodilation, rehabilitation, randomized controlled tria