Exploring the Relationship Between Efpeglenatide Dose and Cardiovascular Outcomes in Type 2 Diabetes: Insights From the AMPLITUDE-O Trial.

BACKGROUND: In the AMPLITUDE-O (Effect of Efpeglenatide on Cardiovascular Outcomes) cardiovascular outcomes trial, adding either 4 mg or 6 mg weekly of the glucagon-like peptide-1 receptor agonist efpeglenatide to usual care reduced major adverse cardiovascular events (MACE) in people with type 2 diabetes at high cardiovascular risk. Whether these benefits are dose related remains uncertain. METHODS: Participants were randomly assigned in a 1:1:1 ratio to placebo, 4 mg or 6 mg of efpeglenatide. The effect of 6 mg versus placebo and of 4 mg versus placebo on MACE (a nonfatal myocardial infarction, nonfatal stroke, or death from cardiovascular or unknown causes) and on all the secondary composite cardiovascular and kidney outcomes was assessed. A dose-response relationship was assessed using the log-rank test and χ2 statistic for trend. RESULTS: During a median follow-up of 1.8 years, MACE occurred in 125 (9.2%) participants assigned to placebo, 84 (6.2%) participants assigned to 6 mg of efpeglenatide (hazard ratio [HR], 0.65 [95% CI, 0.5-0.86]; P=0.0027), and 105 (7.7%) assigned to 4 mg of efpeglenatide (HR, 0.82 [95% CI, 0.63-1.06]; P=0.14). Participants receiving high-dose efpeglenatide also experienced fewer secondary outcomes, including the composite of MACE, coronary revascularization, or hospitalization for unstable angina (HR, 0.73 for 6 mg, P=0.011; HR, 0.85 for 4 mg, P=0.17), a kidney composite outcome comprising sustained new macroalbuminuria, a ≥40% decline in estimated glomerular filtration rate or renal failure (HR, 0.63 for 6 mg, P<0.0001; HR, 0.73 for 4 mg, P=0.0009), MACE or any death (HR, 0.67 for 6 mg, P=0.0021; HR, 0.81 for 4 mg, P=0.08), a kidney function outcome comprising a sustained ≥40% decline in estimated glomerular filtration rate, renal failure, or death (HR, 0.61 for 6 mg, P=0.0072; HR, 0.97 for 4 mg, P=0.83), and the composite of MACE, any death, heart failure hospitalization, or the kidney function outcome (HR, 0.63 for 6 mg, P=0.0002; HR, 0.81 for 4 mg, P=0.067). A clear dose-response was noted for all primary and secondary outcomes (all P for trend ≤0.018). CONCLUSIONS: The graded salutary relationship between efpeglenatide dose and cardiovascular outcomes suggests that titrating efpeglenatide and potentially other glucagon-like peptide-1 receptor agonists to high doses may maximize their cardiovascular and renal benefits. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT03496298.

Effect of colchicine on perioperative atrial fibrillation and myocardial injury after non-cardiac surgery in patients undergoing major thoracic surgery (COP-AF): an international randomised trial.

BACKGROUND: Higher levels of inflammatory biomarkers are associated with an increased risk of perioperative atrial fibrillation and myocardial injury after non-cardiac surgery (MINS). Colchicine is an anti-inflammatory drug that might reduce the incidence of these complications. METHODS: COP-AF was a randomised trial conducted at 45 sites in 11 countries. Patients aged 55 years or older and undergoing major non-cardiac thoracic surgery were randomly assigned (1:1) to receive oral colchicine 0·5 mg twice daily or matching placebo, starting within 4 h before surgery and continuing for 10 days. Randomisation was done with use of a computerised, web-based system, and was stratified by centre. Health-care providers, patients, data collectors, and adjudicators were masked to treatment assignment. The coprimary outcomes were clinically important perioperative atrial fibrillation and MINS during 14 days of follow-up. The main safety outcomes were a composite of sepsis or infection, and non-infectious diarrhoea. The intention-to-treat principle was used for all analyses. This trial is registered with ClinicalTrials.gov, NCT03310125. FINDINGS: Between Feb 14, 2018, and June 27, 2023, we enrolled 3209 patients (mean age 68 years [SD 7], 1656 [51·6%] male). Clinically important atrial fibrillation occurred in 103 (6·4%) of 1608 patients assigned to colchicine, and 120 (7·5%) of 1601 patients assigned to placebo (hazard ratio [HR] 0·85, 95% CI 0·65 to 1·10; absolute risk reduction [ARR] 1·1%, 95% CI -0·7 to 2·8; p=0·22). MINS occurred in 295 (18·3%) patients assigned to colchicine and 325 (20·3%) patients assigned to placebo (HR 0·89, 0·76 to 1·05; ARR 2·0%, -0·8 to 4·7; p=0·16). The composite outcome of sepsis or infection occurred in 103 (6·4%) patients in the colchicine group and 83 (5·2%) patients in the placebo group (HR 1·24, 0·93-1·66). Non-infectious diarrhoea was more common in the colchicine group (134 [8·3%] events) than the placebo group (38 [2·4%]; HR 3·64, 2·54-5·22). INTERPRETATION: In patients undergoing major non-cardiac thoracic surgery, administration of colchicine did not significantly reduce the incidence of clinically important atrial fibrillation or MINS but increased the risk of mostly benign non-infectious diarrhoea. FUNDING: Canadian Institutes of Health Research, Accelerating Clinical Trials Consortium, Innovation Fund of the Alternative Funding Plan for the Academic Health Sciences Centres of Ontario, Population Health Research Institute, Hamilton Health Sciences, Division of Cardiology at McMaster University, Canada; Hanela Foundation, Switzerland; and General Research Fund, Research Grants Council, Hong Kong.

Impact of baseline FIB-4 score on efpeglenatide benefits on cardiovascular outcomes in people with type 2 diabetes: a participant-level exploratory analysis of the AMPLITUDE-O trial.

AIMS: To estimate the incidence of major adverse cardiovascular events (MACE), expanded MACE, and MACE or Death across Fibrosis- 4 score (FIB-4) categories in people with type 2 diabetes and to determine whether efpeglenatide's effect varies with increasing FIB-4 severity. MATERIALS AND METHODS: AMPLITUDE-O trial data were used to estimate the relationship of FIB-4 score categories to the hazard of MACE, expanded MACE, and MACE or death. Interactions on these outcomes between baseline FIB-4 score, and between FIB-4 score and efpeglenatide were also assessed. RESULTS: Baseline FIB-4 score was available for 4059 participants (99.6%) allowing subdivision of the population in tertiles. During a median follow-up of 1.8 years, numerical increases in the incidence of all 3 outcomes did not change significantly across tertiles of FIB-4 score (P for trend ≥ 0.25) with negligible relationship of the score to incident outcomes (MACE HR, per 1 SD higher score, 95% CI: 1.00, 0.89-1.13). Efpeglenatide's effect on all MACE outcomes did not vary across FIB-4 tertiles (all interaction p values ≥ 0.64). CONCLUSIONS: In high-risk people with type 2 diabetes, the degree of liver fibrosis, as estimated by FIB-4 score, was not related to incident cardiovascular outcomes. The beneficial effect of efpeglenatide on these outcomes is independent of FIB-4 category.

Rationale and design of the colchicine for the prevention of perioperative atrial fibrillation in patients undergoing major noncardiac thoracic surgery (COP-AF) trial.

BACKGROUND: Perioperative atrial fibrillation (AF) and myocardial injury after noncardiac surgery (MINS) are common complications after noncardiac surgery. Inflammation has been implicated in the pathogenesis of both disorders. The COP-AF trial tests the hypothesis that colchicine reduces the incidence of perioperative AF and MINS in patients undergoing major noncardiac thoracic surgery. METHODS AND RESULTS: The 'COlchicine for the Prevention of Perioperative Atrial Fibrillation' (COP-AF) trial is an international, blinded, randomized trial that compares colchicine to placebo in patients aged at least 55 years and undergoing major noncardiac thoracic surgery with general anesthesia. Exclusion criteria include a history of AF and a contraindication to colchicine (eg, severe renal dysfunction). Oral colchicine at a dose of 0.5 mg or matching placebo is given within 4 hours before surgery. Thereafter, patients receive colchicine 0.5 mg or placebo twice daily for a total of 10 days. The 2 independent co-primary outcomes are clinically important perioperative AF (including atrial flutter) and MINS during 14 days of follow-up. The main safety outcomes are sepsis or infection and non-infectious diarrhea. We aim to enroll 3,200 patients from approximately 40 sites across 11 countries to have at least 80% power for the independent evaluation of the 2 co-primary outcomes. The COP-AF main results are expected in 2023. CONCLUSIONS: COP-AF is a large randomized and blinded trial designed to determine whether colchicine reduces the risk of perioperative AF or MINS in patients who have major noncardiac thoracic surgery.

Harnessing Multiplex crRNA in the CRISPR/Cas12a System Enables an Amplification-Free DNA Diagnostic Platform for ASFV Detection.

CRISPR-associated (Cas) protein systems have been increasingly incorporated in nucleic-acid diagnosis. CRISPR/Cas12a can cleave single-stranded DNA (ssDNA) after being guided to the target double-stranded DNA (dsDNA) with crRNA, making it a specific tool for dsDNA detection. Assisted by nucleic acid preamplification, CRISPR/Cas12a enables dsDNA detection at the attomolar level. However, such mandatory preamplification in CRISPR/Cas12a also accompanies the extra step of transferring preamplification products into the CRISPR/Cas12a system, which is not only cumbersome and time-consuming but also induces the risk of cross-contamination. Herein, we demonstrate a multiplex-crRNA strategy to enhance the sensitivity of the CRISPR/Cas12a system without any preamplification. This multiplex-crRNA strategy harnesses multiple sequences of crRNA which target different regions of the same dsDNA substrate in the same CRISPR/Cas12a system. Therefore, detection signals are accumulated without amplification, which augments the conventional detection limit. For application demonstration, the B646L gene from the African swine fever virus (ASFV), which is a dsDNA virus, is exemplified. The detection limit of the multiplex-crRNA system can be improved to ∼1 picomolar (pM) without amplification, which is ∼64 times stronger than the conventional single-crRNA system. The multiplex-crRNA system presented in this study, with slight modifications, can be generalized to other biosensing settings where preamplification is not readily available.

Condensation of 2-((Alkylthio)(aryl)methylene)malononitrile with 1,2-Aminothiol as a Novel Bioorthogonal Reaction for Site-Specific Protein Modification and Peptide Cyclization.

Site-specific modification of peptides and proteins has wide applications in probing and perturbing biological systems. Herein we report that 1,2-aminothiol can react rapidly, specifically and efficiently with 2-((alkylthio)(aryl)methylene)malononitrile (TAMM) under biocompatible conditions. This reaction undergoes a unique mechanism involving thiol-vinyl sulfide exchange, cyclization, and elimination of dicyanomethanide to form 2-aryl-4,5-dihydrothiazole (ADT) as a stable product. An 1,2-aminothiol functionality can be introduced into a peptide or a protein as an N-terminal cysteine or an unnatural amino acid. The bioorthogonality of this reaction was demonstrated by site-specific labeling of not only synthetic peptides and a purified recombinant protein but also proteins on mammalian cells and phages. Unlike other reagents in bioorthogonal reactions, the chemical and physical properties of TAMM can be easily tuned. TAMM can also be applied to generate phage-based ADT-cyclic peptide libraries without reducing phage infectivity. Using this approach, we identified ADT-cyclic peptides with high affinity to different protein targets, providing valuable tools for biological studies and potential therapeutics. Furthermore, the mild reaction conditions of TAMM condensation warrant its use with other bioorthogonal reactions to simultaneously achieve multiple site-specific modifications.

Chemical and Ribosomal Synthesis of Topologically Controlled Bicyclic and Tricyclic Peptide Scaffolds Primed by Selenoether Formation.

Bicyclic and tricyclic peptides have emerged as promising candidates for the development of protein binders and new therapeutics. However, convenient and efficient strategies that can generate topologically controlled bicyclic and tricyclic peptide scaffolds from fully-unprotected peptides are still much in demand, particularly for those amenable to the design of biosynthetic libraries. In this work, we report a reliable chemical and ribosomal synthesis of topologically controlled bicyclic and tricyclic peptide scaffolds. Our strategy involves the combination of selenoether cyclization followed by disulfide or thioether cyclization, yielding desirable bicyclic and tricyclic peptides. This work thus lays the foundation for developing peptide libraries with controlled topology of multicyclic scaffolds for in vitro display techniques.

Tunable accessibility of dye-doped liposomes towards gold nanoparticles for fluorescence sensing of lipopolysaccharide.

Inspired by the primitive role of lipopolysaccharide (LPS) and taking advantage of the membrane-philic properties of amphiphilic gold nanoparticles (AuNPs), we established a facile and efficient fluorescence turn-on detection strategy for LPS. Upon binding onto the surface of liposomes, LPS can tailor the accessibility of liposomes towards AuNPs, reminiscent of its primitive function on the surface of bacteria. Thus, while the fluorescence of the dyes labeled on liposomes can be markedly quenched by the membrane-philic AuNPs, the quenching effect can be efficiently prevented by the surface-bound LPS. The de-quenching effect is highly selective to LPS, relative to other negatively charged bio-analytes, which is due to not only the extremely high affinity of LPS to lipid bilayers, but also the unique molecular structure of LPS. Furthermore, this easy-to-construct method offers a limit of detection of ∼0.65 nM, which is comparable to that obtained from the superb synthetic sensors for LPS reported in the literature. This study would open up a new route for the design of sensing systems for LPS exploiting its unique structural pattern and primitive function.

[Survival and prognosis of patients with polycythemia vera: a study of 816 patients in a single Chinese center].

OBJECTIVE: To explore the survival and the risk factors of poor prognosis in Chinese patients with polycythemia vera (PV). METHODS: A total of 816 patients with a definite diagnosis of PV were enrolled from August 1983 to June 2013 into this study. The standardized mortality ratio (SMR) was calculated by comparing the cumulative survival of 816 PV patients with age- and sex- and calendar year-matched healthy Chinese population from the national bureau of statistics of the People's Republic of China. The clinical features of diagnosis and prognosis of PV patients were analyzed by Cox regression to identify risk factors for the poor prognosis of PV and to develop a dynamic prognostic model in Chinese patients. The effects of different treatments on the development of acute myelocytic leukemia (AML) and post-PV myelofibrosis (post-PV MF) were determined by Kaplan-Meier analysis. JAK2 V617F allele burden (V617F%) was determined by quantitative real-time PCR in 104 patients. RESULTS: The median follow-up time was 6 (1-42) years. The 10-, 15- and 20-year overall survival (OS) was 89.50%, 76.70% and 64.70%, respectively. The SMR was 17.40 (95% CI: 13.71-21.78). Cox regression analysis revealed that white blood cell (WBC) count>10×10(9)/L (HR=3.10, 95% CI: 1.47-6.53, P=0.003), age>60 years (HR=2.89, 95% CI: 1.84-4.53, P<0.001) and prior thrombosis (HR=2.66, 95% CI: 1.65-4.29, P<0.001) were significant predictors for the poor prognosis of PV. Based on the hazard radio, 816 patents were allocated into 4 categories with significantly different survival: low (sum of points=0; median survival no reached), intermediate 1 (sum of points=1; median survival 33.10 (28.20-38.00) years), intermediate 2 (sum of points=2; median survival 23.00 (16.08-29.92) years), high (sum of points=3; median survival 13.00 (10.58-15.42) years). The mortality of high risk group was 5.37 fold higher than low risk patients. The 10- and 20-year survival of no post-PV MF were 89.50% and 79.60%, respectively, for interferon α (IFN-α); 73.80% and 43.50%, respectively, for hydroxyurea treatment; 82.20% and 71.40%, respectively, for alkylating agent treatment; and 80.00% and 38.20%, respectively, for no cytoreductive treatment. The treatment of exposure to IFN-α associated with a higher rate of no-post-PV MF survival (Log-rank=9.79, P=0.020). There were more post-PV MF patients with V617F%≥50% compared with those V617F%<50% (P<0.001). CONCLUSIONS: The mortality of PV patients is significantly higher than that of healthy Chinese population. The WBC count>10×10(9)/L, age>60 years, and prior thrombosis are identified as significant predictors for the prognosis of PV. The risk of post-PV MF transformation may be ameliorated by IFN-α via decreasing the burden of JAK2 V617F mutation.

One-Pot Visual Detection of African Swine Fever Virus Using CRISPR-Cas12a.

African swine fever virus (ASFV) is a leading cause of worldwide agricultural loss. ASFV is a highly contagious and lethal disease for both domestic and wild pigs, which has brought enormous economic losses to a number of countries. Conventional methods, such as general polymerase chain reaction and isothermal amplification, are time-consuming, instrument-dependent, and unsatisfactorily accurate. Therefore, rapid, sensitive, and field-deployable detection of ASFV is important for disease surveillance and control. Herein, we created a one-pot visual detection system for ASFV with CRISPR/Cas12a technology combined with LAMP or RPA. A mineral oil sealing strategy was adopted to mitigate sample cross-contamination between parallel vials during high-throughput testing. Furthermore, the blue fluorescence signal produced by ssDNA reporter could be observed by the naked eye without any dedicated instrument. For CRISPR-RPA system, detection could be completed within 40 min with advantageous sensitivity. While CRISPR-LAMP system could complete it within 60 min with a high sensitivity of 5.8 × 102 copies/µl. Furthermore, we verified such detection platforms display no cross-reactivity with other porcine DNA or RNA viruses. Both CRISPR-RPA and CRISPR-LAMP systems permit highly rapid, sensitive, specific, and low-cost Cas12a-mediated visual diagnostic of ASFV for point-of-care testing (POCT) applications.

Artificial disulfide-rich peptide scaffolds with precisely defined disulfide patterns and a minimized number of isomers.

Disulfide-rich peptides are emerging as potential templates for drug design applications. However, the synthesis and reengineering of disulfide-rich peptides are challenging, owing to the complexity of the oxidative folding process involving a number of diverse isomeric structures. Novel disulfide-rich peptide scaffolds that are not besieged by their disulfide isomers are still greatly desired. In this work, we report the design and synthesis of a novel class of artificial disulfide-rich peptide scaffolds with precisely defined disulfide patterns and a minimized number of isomers. In theory, natural peptides with three disulfide bonds have 15 possible isomers. By rationally engineering the thiol-framework of a peptide containing six cysteines with penicillamines and a dithiol amino acid, we demonstrated, for the first time, that the total number of isomers formed after oxidative folding can be decreased to a minimum of two (i.e., from 15 to 2). As fewer isomeric folds are involved in the oxidative folding, the pathway of the folding becomes more concise and the yield of the artificial scaffolds is substantially increased compared to that of its six-cysteine-containing analogue, which makes the artificial disulfide-rich scaffolds (with only 2 predefined isomeric folds) extremely promising for being exploited as structurally complex templates for the design of peptide therapeutics and ligands.