Enzymatic biotransformation of adipic acid to 6-aminocaproic acid and 1,6- hexamethylenediamine using engineered carboxylic acid reductases and aminotransferases

Biocatalytic reduction of carboxylic acids is gaining importance for the production of polymer precursors and different chemicals. Carboxylic acid reductases (CARs) reduce carboxylic acids to aldehydes using ATP and NADPH as cofactors under mild conditions. Recently, we demonstrated that several bacterial CARs can reduce a broad range of bifunctional carboxylic acids containing amino group or second carboxylic group including adipic acid, which is a precursor for nylon-6-6 (Khusnutdinova et al., 2017). In this project, we demonstrate application of CARs and aminotransferases for further bioconversion of adipic acid to 6-aminocaproic acid and hexamethylenediamine, two other important precursors for nylon synthesis. Based on the crystal structure of the adenylating domain of the CAR enzyme MCH22995 from Mycobacterium chelonae, we generated a structural model of the CAR enzyme MAB4714 from M. abscessus, which is active toward adipic acid. Aiming at improving MAB4714 activity toward 6-aminocaproic acid, we used structure-based protein engineering and generated 16 MAB4714 mutant proteins. Screening of 16 purified MAB4714 variants against 6-aminocaproic acid,identified one protein, which was 10 times more active than the wild-type protein. We also identified several bacterial aminotransferases producing 6-aminocaproic acid from adipic acid in combination with CARs. Further optimization of reaction conditions and application of cofactor regeneration systems resulted in efficient biotransformation of adipic acid to 6-aminocaproic acid (88% conversion) and further to 1,6-hexamethylenediamine (78% conversion). Please click Additional Files below to see the full abstract

Pharmacological activation of SIRT6 triggers lethal autophagy in human cancer cells

Sirtuin 6 (SIRT6) is a member of the NAD+-dependent class III deacetylase sirtuin family, which plays a key role in cancer by controlling transcription, genome stability, telomere integrity, DNA repair, and autophagy. Here we analyzed the molecular and biological effects of UBCS039, the first synthetic SIRT6 activator. Our data demonstrated that UBCS039 induced a time-dependent activation of autophagy in several human tumor cell lines, as evaluated by increased content of the lipidated form of LC3B by western blot and of autophagosomal puncta by microscopy analysis of GFP-LC3. UBCS039-mediated activation of autophagy was strictly dependent on SIRT6 deacetylating activity since the catalytic mutant H133Y failed to activate autophagy. At the molecular level, SIRT6-mediated autophagy was triggered by an increase of ROS levels, which, in turn, resulted in the activation of the AMPK-ULK1-mTOR signaling pathway. Interestingly, antioxidants were able to completely counteract UBCS039-induced autophagy, suggesting that ROS burst had a key role in upstream events leading to autophagy commitment. Finally, sustained activation of SIRT6 resulted in autophagy-related cell death, a process that was markedly attenuated using either a pan caspases inhibitor (zVAD-fmk) or an autophagy inhibitor (CQ). Overall, our results identified UBCS039 as an efficient SIRT6 activator, thereby providing a proof of principle that modulation of the enzyme can influence therapeutic strategy by enhancing autophagy-dependent cell death

Prevalence and associated factors of obesity in inflammatory bowel disease: A case-control study

BACKGROUND In recent years, an increasing prevalence of obesity in inflammatory bowel disease (IBD) has been observed. Obesity, moreover, has been directly correlated with a more severe clinical course and loss of response to treatment. AIM To assess the prevalence and associated factors of obesity in IBD. METHODS We collected data about IBD disease pattern and activity, drugs and laboratory investigations in our center. Anthropometric measures were retrieved and obesity defined as a body mass index (BMI) > 30. Then, we compared characteristics of obese vs non obese patients, and Chi-squared test and Student’s t test were used for discrete and continuous variables, respectively, at univariate analysis. For multivariate analysis, we used binomial logistic regression and estimated odd ratios (OR) and 95% confidence intervals (CI) to ascertain factors associated with obesity. RESULTS We enrolled 807 patients with IBD, either ulcerative colitis (UC) or Crohn’s disease (CD). Four hundred seventy-four patients were male (58.7%); the average age was 46.2 ± 13.2 years; 438 (54.2%) patients had CD and 369 (45.8%) UC. We enrolled 378 controls, who were comparable to IBD group for age, sex, BMI, obesity, diabetes and abdominal circumference, while more smokers and more subjects with hypertension were observed among controls. The prevalence of obesity was 6.9% in IBD and 7.9% in controls (not statistically different; P = 0.38). In the comparison of obese IBD patients and obese controls, we did not find any difference regarding diabetes and hypertension prevalence, nor in sex or smoking habits. Obese IBD patients were younger than obese controls (51.2 ± 14.9 years vs 60.7 ± 12.1 years, P = 0.03). At univariate analysis, obese IBD were older than normal weight ones (51.2 ± 14.9 vs 44.5 ± 15.8, P = 0.002). IBD onset age was earlier in obese population (44.8 ± 13.6 vs 35.6 ± 15.6, P = 0.004). We did not detect any difference in disease extension. Obese subjects had consumed more frequently long course of systemic steroids (66.6% vs 12.5%, P = 0.02) as well as antibiotics such as metronidazole or ciprofloxacin (71.4% vs 54.7%, P = 0.05). No difference about other drugs (biologics, mesalazine or thiopurines) was observed. Disease activity was similar between obese and non obese subjects both for UC and CD. Obese IBD patients suffered more frequently from arterial hypertension, type 2 diabetes, non-alcoholic fatty liver disease. Regarding laboratory investigations, obese IBD patients had higher levels of triglyceridemia, fasting blood glucose, gamma-glutamyl-transpeptidase. On multivariate analysis, however, the only factor that appeared to be independently linked to obesity in IBD was the high abdominal circumference (OR = 16.3, 95%CI: 1.03-250, P = 0.04). CONCLUSION Obese IBD patients seem to have features similar to general obese population, and there is no disease-specific factor (disease activity, extension or therapy) that may foster obesity in IBD

Boosting plant food polyphenol concentration by saline eustress as supplement strategies for the prevention of metabolic syndrome: an example of randomized interventional trial in the adult population

IntroductionPhenolic compounds in lettuce can increase by the application of positive stress (eustress) such as moderate saline stress. Phenolic compounds possess antioxidant capacity that is a key factor in the detoxification of excess reactive oxygen species. A double-blinded randomized interventional and placebo- controlled study design was carried out to compare the effect of daily dietary eustress lettuce ingestion in hepatic, lipid, bone, glucose, and iron metabolism.MethodsForty-two healthy volunteers, 19 female and 23 male participants, were divided into two groups. Participants were randomized into a polyphenol-enriched treatment (PET) arm or control arm. Each arm consumed 100 g/day of control or eustress (polyphenols enriched treatment = PET) lettuce for 12 days. Primary study outcomes were serological analysis for assessing hepatic, lipid, bone, iron, and glucose markers at baseline and after 12 days. Secondary outcomes assessed body composition.ResultsSalinity stress reduced plant yield but increased caffeic acid (+467%), chlorogenic acid (+320%), quercetin (+538%), and rutin (+1,095%) concentrations. The intake of PET lettuce reduced PTH, low-density lipoprotein (LDL), cholesterol, alanine transaminase (ALT), and aspartate transaminase (AST) enzyme levels and increased vitamin D and phosphate levels, while iron and glucose metabolism were unaffected.DiscussionSupplementation with eustress lettuce by increasing polyphenols concentration ameliorates hepatic, lipid, and bone homeostasis. Body composition was not affected.Clinical trial registrationhttps://classic.clinicaltrials.gov/ct2/show/NCT06002672, identifier: NCT06002672

Structural characterization of the family GH115 alpha-glucuronidase from Amphibacillus xylanus yields insight into its coordinated action with alpha-arabinofuranosidases

The coordinated action of carbohydrate-active enzymes has mainly been evaluated for the purpose of complete saccharification of plant biomass (lignocellulose) to sugars. By contrast, the coordinated action of accessory hemicellulases on xylan debranching and recovery is less well characterized. Here, the activity of two family GH115 alpha-glucuronidases (SdeAgu115A from Saccharophagus degradans, and AxyAgu115A from Amphibacillus xylanus) on spruce arabinoglucuronoxylan (AGX) was evaluated in combination with an alpha-arabinofuranosidase from families GH51 (AniAbf51A, aka E-AFASE from Aspergillus niger) and GH62 (SthAbf62A from Streptomyces thermoviolaceus). The alpha-arabinofuranosidases boosted (methyl)-glucuronic acid release by SdeAgu115A by approximately 50 % and 30 %, respectively. The impact of the alpha-arabinofuranosidases on AxyAgu115A activity was comparatively low, motivating its structural characterization. The crystal structure of AxyAgu115A revealed increased length and flexibility of the active site loop compared to SdeAgu115A. This structural difference could explain the ability of AxyAgu115A to accommodate more highly substituted arabinoglucuronoxylan, and inform enzyme selections for improved AGX recovery and use.Peer reviewe

725 Assessment of myocardial work for the evaluation of patients undergoing transcatheter aortic valve implantation (TAVI)

Abstract Aims A growing number of patients is undergoing transcatheter treatment of severe Aortic Stenosis. Changes in cardiac mechanics after removal of afterload in these patients are under-investigated. Myocardial Work (MW) is emerging as a useful non-invasive correlate of invasively measured myocardial performance and oxygen consumption. Aim of this study was to assess the usefulness of non-invasive MW indices in the clinical assessment of patients with severe aortic stenosis undergoing transcatheter aortic valve implantation (TAVI). Methods and results Consecutive patients with severe aortic stenosis referred for TAVI in a single tertiary centre were included. Echocardiography recordings (GE-Healthcare) with systolic and diastolic non-invasive pressures, were obtained immediately before TAVI and after TAVI to measure myocardial work index (MWI), myocardial constructive work (MCW), myocardial wasted work (MWW), and myocardial work efficiency (MWE). Consecutive patients with severe AS (n = 73) undergoing TAVI and matched controls (n = 50) were included. Mean transaortic gradient, AV area, and peak transvalvular velocity were significantly improved (all P < 0.05). No changes in left ventricular ejection fraction nor in global longitudinal strain (GLS) were observed. GWI (P < 0.001) and GCW (P < 0.001) were significantly reduced after TAVI. On the contrary, we observed no significant change in GWW (P = 0.241) nor GWE (P = 0.854). Women had higher GWI (P = 0.007) and GCW (P = 0.014) compared to men, with a larger delta change of GCW. Patients with a low flow low gradient (LF-LG) AS had lower LVEF (P < 0.001), worse GLS (P < 0.001) and lower baseline GWI (P < 0.001), GCW (P < 0.001), and GWE (P = 0.003). The improvement in GWI and GCW observed after TAVI in the general study population were abolished among LF-LG patients. Conclusions The use of non-invasive myocardial work might be useful to further classify patients with AS and could be useful to predict non responders

Echocardiographic Normal Reference Ranges for Non-invasive Myocardial Work Parameters in Pediatric Age: Results From an International Multi-Center Study

This international multi-center study aimed to demonstrate the feasibility and reliability of non-invasive myocardial work (MW) parameters in the pediatric population, and to provide normal reference ranges for this useful echocardiographic tool in this specific subset of patients