Enhancing a de novo enzyme activity by computationally-focused ultra-low-throughput screening

Directed evolution has revolutionized protein engineering. Still, enzyme optimization by random library screening remains sluggish, in large part due to futile probing of mutations that are catalytically neutral and/or impair stability and folding. FuncLib is a novel approach which uses phylogenetic analysis and Rosetta design to rank enzyme variants with multiple mutations, on the basis of predicted stability. Here, we use it to target the active site region of a minimalist-designed, de novo Kemp eliminase. The similarity between the Michaelis complex and transition state for the enzymatic reaction makes this system particularly challenging to optimize. Yet, experimental screening of a small number of active-site variants at the top of the predicted stability ranking leads to catalytic efficiencies and turnover numbers ( 2 104 M 1 s 1 and 102 s 1) for this anthropogenic reaction that compare favorably to those of modern natural enzymes. This result illustrates the promise of FuncLib as a powerful tool with which to speed up directed evolution, even on scaffolds that were not originally evolved for those functions, by guiding screening to regions of the sequence space that encode stable and catalytically diverse enzymes. Empirical valence bond calculations reproduce the experimental activation energies for the optimized eliminases to within 2 kcal mol 1 and indicate that the enhanced activity is linked to better geometric preorganization of the active site. This raises the possibility of further enhancing the stabilityguidance of FuncLib by computational predictions of catalytic activity, as a generalized approach for computational enzyme designKnut and Alice Wallenberg Foundation (Wallenberg Academy Fellowship) 2018.0140Human Frontier Science Program RGP0041/2017FEDER Funds/Spanish Ministry of Science, Innovation and Universities BIO2015-66426-R RTI2018-097142-B-100FEDER/Junta de Andalucia - Consejeria de Economia y Conocimiento E.FQM.113.UGR18Swedish National Infrastructure for computing (SNAC) 2018/2-3 2019/2-

Surgical treatment for colorectal cancer: Analysis of the influence of an enhanced recovery programme on long-term oncological outcomes-a study protocol for a prospective, multicentre, observational cohort study

Introduction The evidence currently available from enhanced recovery after surgery (ERAS) programmes concerns their benefits in the immediate postoperative period, but there is still very little evidence as to whether their correct implementation benefits patients in the long term. The working hypothesis here is that, due to the lower response to surgical aggression and lower rates of postoperative complications, ERAS protocols can reduce colorectal cancer-related mortality. The main objective of this study is to analyse the impact of an ERAS programme for colorectal cancer on 5-year survival. As secondary objectives, we propose to analyse the weight of each of the predefined items in the oncological results as well as the quality of life. Methods and analysis A multicentre prospective cohort study was conducted in patients older than 18 years of age who are scheduled to undergo surgery for colorectal cancer. The study involved 12 hospitals with an implemented enhanced recovery protocol according to the guidelines published by the Spanish National Health Service. The intervention group includes patients with a minimum implementation level of 70%, and the control group includes those who fail to reach this level. Compliance will be studied using 18 key performance indicators, and the results will be analysed using cancer survival indicators, including overall survival, cancer-specific survival and relapse-free survival. The time to recurrence, perioperative morbidity and mortality, hospital stay and quality of life will also be studied, the latter using the validated EuroQol Five questionnaire. The propensity index method will be used to create comparable treatment and control groups, and a multivariate regression will be used to study each variable. The Kaplan-Meier estimator will be used to estimate survival and the log-rank test to make comparisons. A p value of less than 0.05 (two-tailed) will be considered to be significant. Ethics and dissemination Ethical approval for this study was obtained from the Aragon Ethical Committee (C.P.-C.I. PI20/086) on 4 March 2020. The findings of this study will be submitted to peer-reviewed journals (BMJ Open, JAMA Surgery, Annals of Surgery, British Journal of Surgery). Abstracts will be submitted to relevant national and international meetings.The present research study was awarded a Ministerio de Ciencia e Innovación health research project grant (PI19/00291) from the Carlos III Institute of the Spanish National Health Service as part of the 2019 call for Strategic Action in Health

Primary prevention of cardiovascular disease with a Mediterranean diet

BACKGROUND: Observational cohort studies and a secondary prevention trial have shown an inverse association between adherence to the Mediterranean diet and cardiovascular risk. We conducted a randomized trial of this diet pattern for the primary prevention of cardiovascular events. METHODS: In a multicenter trial in Spain, we randomly assigned participants who were at high cardiovascular risk, but with no cardiovascular disease at enrollment, to one of three diets: a Mediterranean diet supplemented with extra-virgin olive oil, a Mediterranean diet supplemented with mixed nuts, or a control diet (advice to reduce dietary fat). Participants received quarterly individual and group educational sessions and, depending on group assignment, free provision of extra-virgin olive oil, mixed nuts, or small nonfood gifts. The primary end point was the rate of major cardiovascular events (myocardial infarction, stroke, or death from cardiovascular causes). On the basis of the results of an interim analysis, the trial was stopped after a median follow-up of 4.8 years. RESULTS: A total of 7447 persons were enrolled (age range, 55 to 80 years); 57% were women. The two Mediterranean-diet groups had good adherence to the intervention, according to self-reported intake and biomarker analyses. A primary end-point event occurred in 288 participants. The multivariable-adjusted hazard ratios were 0.70 (95% confidence interval [CI], 0.54 to 0.92) and 0.72 (95% CI, 0.54 to 0.96) for the group assigned to a Mediterranean diet with extra-virgin olive oil (96 events) and the group assigned to a Mediterranean diet with nuts (83 events), respectively, versus the control group (109 events). No diet-related adverse effects were reported. CONCLUSIONS: Among persons at high cardiovascular risk, a Mediterranean diet supplemented with extra-virgin olive oil or nuts reduced the incidence of major cardiovascular events

The Fourteenth Data Release of the Sloan Digital Sky Survey: First Spectroscopic Data from the extended Baryon Oscillation Spectroscopic Survey and from the second phase of the Apache Point Observatory Galactic Evolution Experiment

The fourth generation of the Sloan Digital Sky Survey (SDSS-IV) has been in operation since July 2014. This paper describes the second data release from this phase, and the fourteenth from SDSS overall (making this, Data Release Fourteen or DR14). This release makes public data taken by SDSS-IV in its first two years of operation (July 2014-2016). Like all previous SDSS releases, DR14 is cumulative, including the most recent reductions and calibrations of all data taken by SDSS since the first phase began operations in 2000. New in DR14 is the first public release of data from the extended Baryon Oscillation Spectroscopic Survey (eBOSS); the first data from the second phase of the Apache Point Observatory (APO) Galactic Evolution Experiment (APOGEE-2), including stellar parameter estimates from an innovative data driven machine learning algorithm known as "The Cannon"; and almost twice as many data cubes from the Mapping Nearby Galaxies at APO (MaNGA) survey as were in the previous release (N = 2812 in total). This paper describes the location and format of the publicly available data from SDSS-IV surveys. We provide references to the important technical papers describing how these data have been taken (both targeting and observation details) and processed for scientific use. The SDSS website (www.sdss.org) has been updated for this release, and provides links to data downloads, as well as tutorials and examples of data use. SDSS-IV is planning to continue to collect astronomical data until 2020, and will be followed by SDSS-V.Comment: SDSS-IV collaboration alphabetical author data release paper. DR14 happened on 31st July 2017. 19 pages, 5 figures. Accepted by ApJS on 28th Nov 2017 (this is the "post-print" and "post-proofs" version; minor corrections only from v1, and most of errors found in proofs corrected

Associations of the FTO rs9939609 and the MC4R rs17782313 polymorphisms with type 2 diabetes are modulated by diet, being higher when adherence to the mediterranean diet pattern is low

BACKGROUND: Although the fat mass and obesity (FTO) and melanocortin-4 receptor (MC4R) genes have been consistently associated with obesity risk, the association between the obesity-risk alleles with type 2 diabetes is still controversial. In some recent meta-analyses in which significant results have been reported, the associations disappeared after adjustment for body mass index (BMI). However gene-diet interactions with dietary patterns have not been investigated. Our main aim was to analyze whether these associations are modulated by the level of adherence to the Mediterranean Diet (MedDiet). METHODS: Case-control study in 7,052 high cardiovascular risk subjects (3,430 type 2 diabetes cases and 3,622 non-diabetic subjects) with no differences in BMI. Diet was assessed by validated questionnaires. FTO-rs9939609 and MC4R-rs17782313 were determined. An aggregate genetic score was calculated to test additive effects. Gene-diet interactions were analyzed. RESULTS: Neither of the polymorphisms was associated with type 2 diabetes in the whole population. However, we found consistent gene-diet interactions with adherence to the MedDiet both for the FTO-rs9939609 (P-interaction=0.039), the MC4R-rs17782313 (P-interaction=0.009) and for their aggregate score (P-interaction=0.006). When adherence to the MedDiet was low, carriers of the variant alleles had higher type 2 diabetes risk (OR=1.21, 95%CI: 1.03-1.40; P=0.019 for FTO-rs9939609 and OR=1.17, 95%CI:1.01-1.36; P=0.035 for MC4R-rs17782313) than wild-type subjects. However, when adherence to the MedDiet was high, these associations disappeared (OR=0.97, 95%CI: 0.85-1.16; P=0.673 for FTO-rs9939609 and OR=0.89, 95%CI:0.78-1.02; P=0.097 for MC4R-rs17782313). These gene-diet interactions remained significant even after adjustment for BMI. As MedDiet is rich in folate, we also specifically examined folate intake and detected statistically significant interaction effects on fasting plasma glucose concentrations in non-diabetic subjects. However these findings should be interpreted with caution because folate intake may simply reflect a healthy dietary pattern. CONCLUSIONS: These novel results suggest that the association of the FTO-rs9939609 and the MC4R-rs17782313 polymorphisms with type 2 diabetes depends on diet and that a high adherence to the MedDiet counteracts the genetic predisposition

Effectiveness of an mHealth intervention combining a smartphone app and smart band on body composition in an overweight and obese population: Randomized controlled trial (EVIDENT 3 study)

Background: Mobile health (mHealth) is currently among the supporting elements that may contribute to an improvement in health markers by helping people adopt healthier lifestyles. mHealth interventions have been widely reported to achieve greater weight loss than other approaches, but their effect on body composition remains unclear. Objective: This study aimed to assess the short-term (3 months) effectiveness of a mobile app and a smart band for losing weight and changing body composition in sedentary Spanish adults who are overweight or obese. Methods: A randomized controlled, multicenter clinical trial was conducted involving the participation of 440 subjects from primary care centers, with 231 subjects in the intervention group (IG; counselling with smartphone app and smart band) and 209 in the control group (CG; counselling only). Both groups were counselled about healthy diet and physical activity. For the 3-month intervention period, the IG was trained to use a smartphone app that involved self-monitoring and tailored feedback, as well as a smart band that recorded daily physical activity (Mi Band 2, Xiaomi). Body composition was measured using the InBody 230 bioimpedance device (InBody Co., Ltd), and physical activity was measured using the International Physical Activity Questionnaire. Results: The mHealth intervention produced a greater loss of body weight (–1.97 kg, 95% CI –2.39 to –1.54) relative to standard counselling at 3 months (–1.13 kg, 95% CI –1.56 to –0.69). Comparing groups, the IG achieved a weight loss of 0.84 kg more than the CG at 3 months. The IG showed a decrease in body fat mass (BFM; –1.84 kg, 95% CI –2.48 to –1.20), percentage of body fat (PBF; –1.22%, 95% CI –1.82% to 0.62%), and BMI (–0.77 kg/m2, 95% CI –0.96 to 0.57). No significant changes were observed in any of these parameters in men; among women, there was a significant decrease in BMI in the IG compared with the CG. When subjects were grouped according to baseline BMI, the overweight group experienced a change in BFM of –1.18 kg (95% CI –2.30 to –0.06) and BMI of –0.47 kg/m2 (95% CI –0.80 to –0.13), whereas the obese group only experienced a change in BMI of –0.53 kg/m2 (95% CI –0.86 to –0.19). When the data were analyzed according to physical activity, the moderate-vigorous physical activity group showed significant changes in BFM of –1.03 kg (95% CI –1.74 to –0.33), PBF of –0.76% (95% CI –1.32% to –0.20%), and BMI of –0.5 kg/m2 (95% CI –0.83 to –0.19). Conclusions: The results from this multicenter, randomized controlled clinical trial study show that compared with standard counselling alone, adding a self-reported app and a smart band obtained beneficial results in terms of weight loss and a reduction in BFM and PBF in female subjects with a BMI less than 30 kg/m2 and a moderate-vigorous physical activity level. Nevertheless, further studies are needed to ensure that this profile benefits more than others from this intervention and to investigate modifications of this intervention to achieve a global effect

Severe Asthma Standard-of-Care Background Medication Reduction With Benralizumab: ANDHI in Practice Substudy

Background: The phase IIIb, randomized, parallel-group, placebo-controlled ANDHI double-blind (DB) study extended understanding of the efficacy of benralizumab for patients with severe eosinophilic asthma. Patients from ANDHI DB could join the 56-week ANDHI in Practice (IP) single-arm, open-label extension substudy. Objective: Assess potential for standard-of-care background medication reductions while maintaining asthma control with benralizumab. Methods: Following ANDHI DB completion, eligible adults were enrolled in ANDHI IP. After an 8-week run-in with benralizumab, there were 5 visits to potentially reduce background asthma medications for patients achieving and maintaining protocol-defined asthma control with benralizumab. Main outcome measures for non-oral corticosteroid (OCS)-dependent patients were the proportions with at least 1 background medication reduction (ie, lower inhaled corticosteroid dose, background medication discontinuation) and the number of adapted Global Initiative for Asthma (GINA) step reductions at end of treatment (EOT). Main outcomes for OCS-dependent patients were reductions in daily OCS dosage and proportion achieving OCS dosage of 5 mg or lower at EOT. Results: For non-OCS-dependent patients, 53.3% (n = 208 of 390) achieved at least 1 background medication reduction, increasing to 72.6% (n = 130 of 179) for patients who maintained protocol-defined asthma control at EOT. A total of 41.9% (n = 163 of 389) achieved at least 1 adapted GINA step reduction, increasing to 61.8% (n = 110 of 178) for patients with protocol-defined EOT asthma control. At ANDHI IP baseline, OCS dosages were 5 mg or lower for 40.4% (n = 40 of 99) of OCS-dependent patients. Of OCS-dependent patients, 50.5% (n = 50 of 99) eliminated OCS and 74.7% (n = 74 of 99) achieved dosages of 5 mg or lower at EOT. Conclusions: These findings demonstrate benralizumab's ability to improve asthma control, thereby allowing background medication reduction

Dietary a-Linolenic acid, Marine x-3 fatty acids, and mortality in a population with high fish consumption: findings from the PREevención con DIeta MEDiterránea (PREDIMED) study

Epidemiological evidence suggests a cardioprotective role of α‐linolenic acid (ALA), a plant‐derived ω‐3 fatty acid. It is unclear whether ALA is beneficial in a background of high marine x-3 fatty acids (long-chain n-3 polyunsaturated fatty acids) intake. In persons at high cardiovascular risk from Spain, a country in which fish consumption is customarily high, we investigated whether meeting the International Society for the Study of Fatty Acids and Lipids recommendation for dietary ALA (0.7% of total energy) at baseline was related to all-cause and cardiovascular disease mortality. We also examined the effect of meeting the society’s recommendation for long-chain n-3 polyunsaturated fatty acids (≥500 mg/day). Methods and Results-—We longitudinally evaluated 7202 participants in the PREvenci on con DIeta MEDiterr anea (PREDIMED) trial. Multivariable-adjusted Cox regressionmodels were fitted to estimate hazard ratios. ALA intake correlated towalnut consumption (r=0.94). During a 5.9-y follow-up, 431 deaths occurred (104 cardiovascular disease, 55 coronary heart disease, 32 sudden cardiac death, 25 stroke). The hazard ratios formeeting ALArecommendation (n=1615, 22.4%) were 0.72 (95% CI 0.56–0.92) for all-causemortality and 0.95 (95% CI 0.58–1.57) for fatal cardiovascular disease. The hazard ratios formeeting the recommendation for long-chain n-3 polyunsaturated fatty acids (n=5452, 75.7%) were 0.84 (95% CI 0.67–1.05) for all-causemortality, 0.61 (95% CI 0.39–0.96) for fatal cardiovascular disease, 0.54 (95% CI 0.29–0.99) for fatal coronary heart disease, and 0.49 (95% CI 0.22–1.01) for sudden cardiac death. The highest reduction in all-cause mortality occurred in participants meeting both recommendations (hazard ratio 0.63 [95% CI 0.45–0.87]). Conclusions-—In participants without prior cardiovascular disease and high fish consumption, dietary ALA, supplied mainly by walnuts and olive oil, relates inversely to all-cause mortality, whereas protection from cardiac mortality is limited to fish-derived long-chain n-3 polyunsaturated fatty acids

Mercury exposure and risk of cardiovascular disease: a nested casecontrol study in the PREDIMED (PREvention with MEDiterranean Diet) study

Background: Substantial evidence suggests that consuming 1–2 servings of fish per week, particularly oily fish (e.g., salmon, herring, sardines) is beneficial for cardiovascular health due to its high n-3 polyunsaturated fatty acid content. However, there is some concern that the mercury content in fish may increase cardiovascular disease risk, but this relationship remains unclear. Methods: The PREDIMED trial included 7477 participants who were at high risk for cardiovascular disease at baseline. In this study, we evaluated associations between mercury exposure, fish consumption and cardiovascular disease. We randomly selected 147 of the 288 cases diagnosed with cardiovascular disease during follow-up and matched them on age and sex to 267 controls. Instrumental neutron activation analysis was used to assess toenail mercury concentration. In-person interviews, medical record reviews and validated questionnaires were used to assess fish consumption and other covariates. Information was collected at baseline and updated yearly during follow-up. We used conditional logistic regression to evaluate associations in the total nested case-control study, and unconditional logistic regression for population subsets. Results: Mean (±SD) toenail mercury concentrations (μg per gram) did not significantly differ between cases (0.63 (±0.53)) and controls (0.67 (±0.49)). Mercury concentration was not associated with cardiovascular disease in any analysis, and neither was fish consumption or n-3 fatty acids. The fully-adjusted relative risks for the highest versus lowest quartile of mercury concentration were 0.71 (95% Confidence Interval [CI], 0.34, 1.14; ptrend = 0.37) for the nested case-control study, 0.74 (95% CI, 0.32, 1.76; ptrend = 0.43) within the Mediterranean diet intervention group, and 0.50 (95% CI, 0.13, 1.96; ptrend = 0.41) within the control arm of the trial. Associations remained null when mercury was jointly assessed with fish consumption at baseline and during follow-up. Results were similar in different sensitivity analyses.Conclusions: We found no evidence that mercury exposure from regular fish consumption increases cardiovascular disease risk in a population of Spanish adults with high cardiovascular disease risk and high fish consumption. This implies that the mercury content in fish does not detract from the already established cardiovascular benefits of fish consumption