Impact of smoking in patients with suspected coronary artery disease in the randomised DISCHARGE trial.

OBJECTIVES: To investigate if the effect of cardiac computed tomography (CT) vs. invasive coronary angiography (ICA) on cardiovascular events differs based on smoking status. MATERIALS AND METHODS: This pre-specified subgroup analysis of the pragmatic, prospective, multicentre, randomised DISCHARGE trial (NCT02400229) involved 3561 patients with suspected coronary artery disease (CAD). The primary endpoint was major adverse cardiovascular events (MACE: cardiovascular death, non-fatal myocardial infarction, or stroke). Secondary endpoints included an expanded MACE composite (MACE, transient ischaemic attack, or major procedure-related complications). RESULTS: Of 3445 randomised patients with smoking data (mean age 59.1 years + / - 9.7, 1151 men), at 3.5-year follow-up, the effect of CT vs. ICA on MACE was consistent across smoking groups (p for interaction = 0.98). The percutaneous coronary intervention rate was significantly lower with a CT-first strategy in smokers and former smokers (p = 0.01 for both). A CT-first strategy reduced the hazard of major procedure-related complications (HR: 0.21, 95% CI: 0.03, 0.81; p = 0.045) across smoking groups. In current smokers, the expanded MACE composite was lower in the CT- compared to the ICA-first strategy (2.3% (8) vs 6.0% (18), HR: 0.38; 95% CI: 0.17, 0.88). The rate of non-obstructive CAD was significantly higher in all three smoking groups in the CT-first strategy. CONCLUSION: For patients with stable chest pain referred for ICA, the clinical outcomes of CT were consistent across smoking status. The CT-first approach led to a higher detection rate of non-obstructive CAD and fewer major procedure-related complications in smokers. CLINICAL RELEVANCE STATEMENT: This pre-specified sub-analysis of the DISCHARGE trial confirms that a CT-first strategy in patients with stable chest pain referred for invasive coronary angiography with an intermediate pre-test probability of coronary artery disease is as effective as and safer than invasive coronary angiography, irrespective of smoking status. TRIAL REGISTRATION: ClinicalTrials.gov NCT02400229. KEY POINTS: • No randomised studies have assessed smoking status on CT effectiveness in symptomatic patients referred for invasive coronary angiography. • A CT-first strategy results in comparable adverse events, fewer complications, and increased coronary artery disease detection, irrespective of smoking status. • A CT-first strategy is safe and effective for stable chest pain patients with intermediate pre-test probability for CAD, including never smokers.

Quantitative phosphoproteome analysis of Streptomyces coelicolor by immobilized zirconium (IV) affinity chromatography and mass spectrometry reveals novel regulated protein phosphorylation sites and sequence motifs.

Streptomycetes are multicellular gram-positive bacteria that produce many bioactive compounds, including antibiotics, antitumorals and immunosuppressors. The Streptomyces phosphoproteome remains largely uncharted even though protein phosphorylation at Ser/Thr/Tyr is known to modulate morphological differentiation and specialized metabolic processes. We here expand the S. coelicolor phosphoproteome by optimised immobilized zirconium (IV) affinity chromatography and mass spectrometry to identify phosphoproteins at the vegetative and sporulating stages. We mapped 361 phosphorylation sites (41% pSer, 56.2% pThr, 2.8% pTyr) and discovered four novel Thr phosphorylation motifs ("Kxxxx(pT)xxxxK", "DxE(pT)", "D(pT)" and "Exxxxx(pT)") in 351 phosphopeptides derived from 187 phosphoproteins. We identified 154 novel phosphoproteins, thereby almost doubling the number of experimentally verified Streptomyces phosphoproteins. Novel phosphoproteins included cell division proteins (FtsK, CrgA) and specialized metabolism regulators (ArgR, AfsR, CutR and HrcA) that were differentially phosphorylated in the vegetative and in the antibiotic producing sporulating stages. Phosphoproteins involved in primary metabolism included 27 novel ribosomal proteins that were phosphorylated during the vegetative stage. Phosphorylation of these proteins likely participate in the intricate and incompletely understood regulation of Streptomyces development and secondary metabolism. We conclude that Zr(IV)-IMAC is an efficient and sensitive method to study protein phosphorylation and regulation in bacteria and enhance our understanding of bacterial signalling. SIGNIFICANCE: Two thirds of the secondary metabolites used in clinic, especially antibiotics, were discovered in Streptomyces strains. Antibiotic resistance became one of the major challenges in clinic, and new antibiotics are urgently required in clinic. Next-generation sequencing analyses revealed that streptomycetes harbour many cryptic secondary metabolite pathways, i.e. pathways not expressed in the laboratory. Secondary metabolism is tightly connected with hypha differentiation and sporulation, and understanding Streptomyces differentiation is one of the main challenges in industrial microbiology, in order to activate the expression of cryptic pathways in the laboratory. Protein phosphorylation at Ser/Thr/Tyr modulates development and secondary metabolism, but the Streptomyces phosphoproteome is still largely uncharted. Previous S. coelicolor phosphoproteomic studies used TiO2 affinity enrichment and LC-MS/MS identifying a total of 184 Streptomyces phosphoproteins. Here, we used by first time zirconium (IV) affinity chromatography and mass spectrometry, identifying 186 S. coelicolor phosphoproteins. Most of these phosphoproteins (154) were not identified in previous phosphoproteomic studies using TiO2 affinity enrichment. Thereby we almost doubling the number of experimentally verified Streptomyces phosphoproteins. Zr(IV)-IMAC affinity chromatography also worked in E. coli, allowing the identification of phosphoproteins that were not identified by TiO2 affinity chromatography. We conclude that Zr(IV)-IMAC is an efficient and sensitive method for studies of protein phosphorylation and regulation in bacteria to enhance our understanding of bacterial signalling networks. Moreover, the new Streptomyces phosphoproteins identified will contribute to design further works to understand and modulate Streptomyces secondary metabolism activation.

Selective Enrichment of Histidine Phosphorylated Peptides Using Molecularly Imprinted Polymers.

Protein histidine phosphorylation (pHis) is involved in molecular signaling networks in bacteria, fungi, plants, and higher eukaryotes including mammals and is implicated in human diseases such as cancer. Detailed investigations of the pHis modification are hampered due to its acid-labile nature and consequent lack of tools to study this post-translational modification (PTM). We here demonstrate three molecularly imprinted polymer (MIP)-based reagents, MIP1-MIP3, for enrichment of pHis peptides and subsequent characterization by chromatography and mass spectrometry (LC-MS). The combination of MIP1 and ß-elimination provided some selectivity for improved detection of pHis peptides. MIP2 was amenable to larger pHis peptides, although with poor selectivity. Microsphere-based MIP3 exhibited improved selectivity and was amenable to enrichment and detection by LC-MS of pHis peptides in tryptic digests of protein mixtures. These MIP protocols do not involve any acidic solvents during sample preparation and enrichment, thus preserving the pHis modification. The presented proof-of-concept results will lead to new protocols for highly selective enrichment of labile protein phosphorylations using molecularly imprinted materials.

The Effect of a Family-Based Lifestyle Education Program on Dietary Habits, Hepatic Fat and Adiposity Markers in 8-12-Year-Old Children with Overweight/Obesity.

Healthy lifestyle education programs are recommended for obesity prevention and treatment. However, there is no previous information on the effects of these programs on the reduction of hepatic fat percentage. The aims were (i) to examine the effectiveness of a 22-week family-based lifestyle education program on dietary habits, and (ii) to explore the associations of changes in dietary intake with percent hepatic fat reduction and adiposity in children with overweight/obesity. A total of 81 children with overweight/obesity (aged 10.6 ± 1.1 years, 53.1% girls) and their parents attended a 22-week family based healthy lifestyle and psychoeducational program accompanied with (intensive group) or without (control) an exercise program. Hepatic fat (magnetic resonance imaging), adiposity (dual energy X-ray absorptiometry) and dietary habits (two non-consecutive 24 h-recalls) were assessed before and after the intervention. Energy (p < 0.01) fat (p < 0.01) and added sugar (p < 0.03) intake were significantly reduced in both groups at the end of the program, while, in addition, carbohydrates intake (p < 0.04) was reduced exclusively in the control group, and simple sugar (p < 0.05) and cholesterol (p < 0.03) intake was reduced in the exercise group. Fruit (p < 0.03) and low-fat/skimmed dairy consumption (p < 0.02), the adherence to the Mediterranean Diet Quality Index for children and teenagers (KIDMED, p < 0.01) and breakfast quality index (p < 0.03) were significantly higher in both control and intervention groups after the intervention. Moreover, participants in the exercise group increased the adherence to the Dietary Approaches to Stop Hypertension (DASH) diet (p < 0.001), whereas the ratio of evening-morning energy intake was significantly lower exclusively in the control group after the program (p < 0.02). Changes in energy intake were significantly associated with changes in fat mass index (FMI) in the exercise group, whereas changes in sugar-sweetened beverages (SSB) consumption was associated with percent hepatic fat reduction (p < 0.05) in the control group. A 22-week family-based healthy lifestyle program seems to be effective on improving diet quality and health in children with overweight/obesity and these should focus on SSB avoidance and physical activity.

[Stress echocardiography in the pre-operative evaluation of patients undergoing major vascular surgery. Are results comparable with dypiridamole versus dobutamine stress echo?]. / Ecocardiografía de estrés en el preoperatorio de cirugía vascular: son comparables los resultados con dipiridamol y dobutamina?

INTRODUCTION: [corrected] Perioperative cardiovascular complications are an important cause of post-surgical morbility and mortality in patients undergoing major vascular surgery. Dobutamine Stress Echo is considered one of the methods of choice in the detection of coronary artery disease in this subgroup of patients. OBJECTIVES: . Our aim was to analyze if dipyridamole stress echocardiography could be used as an alternative to Dobutamine Stress Echo in the perioperative evaluation of patients in need of major vascular surgery. PATIENTS AND METHOD: The result of consecutives dypiridamole and dobutamine stress exams prior to vascular surgery were reviewed. We analyzed if those patients with a positive stress echo presented a higher number of cardiac events during and after surgery than those with negative stress echo. The negative and positive predictive values were calculated for both techniques. RESULTS: 133 stress exams were analysed: 39 with dobutamine and 94 with dipyridamole. Of the 39 dobutamine studies 2 were positive, 29 negatives and 8 non conclusive. Of the 94 dypiridamole studies 13 were positive and 81 negatives. None of the patients with a positive dobutamine echo underwent surgery. The negative predictive value for dobutamine echo was 96.5%, quite similar to that of dypiridamole stress echo (97.5%). CONCLUSION: Dipyridamole stress echocardiography is a valid alternative to dobutamine echocardiography in the pre-surgical evaluation of patients undergoing major vascular surgery.