Designing Efficient Enzymes: Eight Predicted Mutations Convert a Hydroxynitrile Lyase into an Efficient Esterase.

Hydroxynitrile lyase from rubber tree (HbHNL) shares 45% identical amino acid residues with the homologous esterase from tobacco, SABP2, but the two enzymes catalyze different reactions. The x-ray structures reveal a serine-histidine-aspartate catalytic triad in both enzymes along with several differing amino acid residues within the active site. Previous exchange of three amino acid residues in the active site of HbHNL with the corresponding amino acid residue in SABP2 (T11G-E79H-K236M) created variant HNL3, which showed low esterase activity toward p-nitrophenyl acetate. Further structure comparison reveals additional differences surrounding the active site. HbHNL contains an improperly positioned oxyanion hole residue and differing solvation of the catalytic aspartate. We hypothesized that correcting these structural differences would impart good esterase activity on the corresponding HbHNL variant. To predict the amino acid substitutions needed to correct the structure, we calculated shortest path maps for both HbHNL and SABP2, which reveal correlated movements of amino acids in the two enzymes. Replacing four amino acid residues (C81L-N104T-V106F-G176S) whose movements are connected to the movements of the catalytic residues yielded variant HNL7TV (stabilizing substitution H103V was also added), which showed an esterase catalytic efficiency comparable to that of SABP2. The x-ray structure of an intermediate variant, HNL6V, showed an altered solvation of the catalytic aspartate and a partially corrected oxyanion hole. This dramatic increase in catalytic efficiency demonstrates the ability of shortest path maps to predict which residues outside the active site contribute to catalytic activity.

Lifetime Risk of Heart Failure and Trends in Incidence Rates Among Individuals With Type 2 Diabetes Between 1995 and 2018.

Background There are limited data on the lifetime risk of heart failure (HF) in people with type 2 diabetes and how incidence has changed over time. We estimated the cumulative incidence and incidence rates of HF among Danish adults with type 2 diabetes between 1995 and 2018 using nationwide data. Methods and Results In total, 398 422 patients (49% women) with type 2 diabetes were identified. During follow-up, 36 400 (9%) were diagnosed with HF and 121 459 (30%) were censored due to death. Using the Aalen-Johansen estimators, accounting for the risk of death, the estimated residual lifetime risk of HF at age 50 years was calculated as 24% (95% CI 22%-27%) in women and 27% (25%-28%) in men. During the observational period, the proportion of patients treated with statins, angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers, and metformin increased from <30% to >60%. Similarly, the annual incidence rates of HF decreased significantly, with declines being greater in older versus younger individuals (5% versus 2% in age >50 versus ≤50 years, respectively; P<0.0001) and in women versus men (5% versus 4%, P=0.02), but similar in patients with and without IHD (4% versus 4%, P=0.53). Conclusions The current lifetime risk of HF in type 2 diabetes approximates 1 in 4 for men and women. Paralleled by an increase in use of evidence-based pharmacotherapy over the past decades, the risk of developing HF has declined across several subgroups and regardless of underlying IHD, suggesting that optimal diabetes treatment can mitigate HF risk.

Long-Term Mortality Associated With Use of Carvedilol Versus Metoprolol in Heart Failure Patients With and Without Type 2 Diabetes: A Danish Nationwide Cohort Study.

Background Carvedilol may have favorable glycemic properties compared with metoprolol, but it is unknown if carvedilol has mortality benefit over metoprolol in patients with type 2 diabetes (T2D) and heart failure with reduced ejection fraction (HFrEF). Methods and Results Using Danish nationwide databases between 2010 and 2018, we followed patients with new-onset HFrEF treated with either carvedilol or metoprolol for all-cause mortality until the end of 2018. Follow-up started 120 days after initial HFrEF diagnosis to allow initiation of guideline-directed medical therapy. There were 39 260 patients on carvedilol or metoprolol at baseline (mean age 70.8 years, 35% women), of which 9355 (24%) had T2D. Carvedilol was used in 2989 (32%) patients with T2D and 10 411 (35%) of patients without T2D. Users of carvedilol had a lower prevalence of atrial fibrillation (20% versus 35%), but other characteristics appeared well-balanced between the groups. Totally 11 306 (29%) were deceased by the end of follow-up. We observed no mortality differences between carvedilol and metoprolol, multivariable-adjusted hazard ratio (HR) 0.97 (0.90-1.05) in patients with T2D versus 1.00 (0.95-1.05) for those without T2D, P for difference =0.99. Rates of new-onset T2D were lower in users of carvedilol versus metoprolol; age, sex, and calendar year adjusted HR 0.83 (0.75-0.91), P<0.0001. Conclusions In a contemporary clinical cohort of HFrEF patients with and without T2D, carvedilol was not associated with a reduction in long-term mortality compared with metoprolol. However, carvedilol was associated with lowered risk of new-onset T2D supporting the assertion that carvedilol has a more favorable metabolic profile than metoprolol.

Artificial Intelligence for Histology-Based Detection of Microsatellite Instability and Prediction of Response to Immunotherapy in Colorectal Cancer.

Microsatellite instability (MSI) is a molecular marker of deficient DNA mismatch repair (dMMR) that is found in approximately 15% of colorectal cancer (CRC) patients. Testing all CRC patients for MSI/dMMR is recommended as screening for Lynch Syndrome and, more recently, to determine eligibility for immune checkpoint inhibitors in advanced disease. However, universal testing for MSI/dMMR has not been uniformly implemented because of cost and resource limitations. Artificial intelligence has been used to predict MSI/dMMR directly from hematoxylin and eosin (H&E) stained tissue slides. We review the emerging data regarding the utility of machine learning for MSI classification, focusing on CRC. We also provide the clinician with an introduction to image analysis with machine learning and convolutional neural networks. Machine learning can predict MSI/dMMR with high accuracy in high quality, curated datasets. Accuracy can be significantly decreased when applied to cohorts with different ethnic and/or clinical characteristics, or different tissue preparation protocols. Research is ongoing to determine the optimal machine learning methods for predicting MSI, which will need to be compared to current clinical practices, including next-generation sequencing. Predicting response to immunotherapy remains an unmet need.

Larger active site in an ancestral hydroxynitrile lyase increases catalytically promiscuous esterase activity.

Hydroxynitrile lyases (HNL's) belonging to the α/ß-hydrolase-fold superfamily evolved from esterases approximately 100 million years ago. Reconstruction of an ancestral hydroxynitrile lyase in the α/ß-hydrolase fold superfamily yielded a catalytically active hydroxynitrile lyase, HNL1. Several properties of HNL1 differ from the modern HNL from rubber tree (HbHNL). HNL1 favors larger substrates as compared to HbHNL, is two-fold more catalytically promiscuous for ester hydrolysis (p-nitrophenyl acetate) as compared to mandelonitrile cleavage, and resists irreversible heat inactivation to 35 °C higher than for HbHNL. We hypothesized that the x-ray crystal structure of HNL1 may reveal the molecular basis for the differences in these properties. The x-ray crystal structure solved to 1.96-Å resolution shows the expected α/ß-hydrolase fold, but a 60% larger active site as compared to HbHNL. This larger active site echoes its evolution from esterases since related esterase SABP2 from tobacco also has a 38% larger active site than HbHNL. The larger active site in HNL1 likely accounts for its ability to accept larger hydroxynitrile substrates. Site-directed mutagenesis of HbHNL to expand the active site increased its promiscuous esterase activity 50-fold, consistent with the larger active site in HNL1 being the primary cause of its promiscuous esterase activity. Urea-induced unfolding of HNL1 indicates that it unfolds less completely than HbHNL (m-value = 0.63 for HNL1 vs 0.93 kcal/mol·M for HbHNL), which may account for the ability of HNL1 to better resist irreversible inactivation upon heating. The structure of HNL1 shows changes in hydrogen bond networks that may stabilize regions of the folded structure.