Assembly of polymerase chain reactions at room temperature can sometimes lead to low yields or unintentional products due to mispriming. Mutation of isoleucine 707 to leucine in DNA polymerase I from Thermus aquaticus substantially decreases its activity at room temperature without compromising its ability to amplify DNA. To understand why a conservative change to the enzyme over 20 Å from the active site can have a large impact on its activity at low temperature, we solved the X-ray crystal structure of the large (5′-to-3′ exonuclease-deleted) fragment of Taq DNA polymerase containing the cold-sensitive mutation in the ternary (E–DNA–ddNTP) and binary (E–DNA) complexes. The I707L KlenTaq1 ternary complex was identical to the wild-type in the closed conformation except for the mutation and a rotamer change in nearby phenylalanine 749, suggesting that the enzyme should remain active. However, soaking out of the nucleotide substrate at low temperature results in an altered binary complex made possible by the rotamer change at F749 near the tip of the polymerase O-helix. Surprisingly, two adenosines in the 5′-template overhang fill the vacated active site by stacking with the primer strand, thereby blocking the active site at low temperature. Replacement of the two overhanging adenosines with pyrimidines substantially increased activity at room temperature by keeping the template overhang out of the active site, confirming the importance of base stacking. These results explain the cold-sensitive phenotype of the I707L mutation in KlenTaq1 and serve as an example of a large conformational change affected by a conservative mutation.

BACKGROUND: Heart failure (HF) is a leading cause of hospitalization in older adults. Medicare data have been used to assess HF outcomes. However, the validity of ICD-10 diagnosis codes (used since 2015) to identify acute HF hospitalization or distinguish reduced (heart failure with reduced ejection fraction) versus preserved ejection fraction (HFpEF) is unknown in Medicare data. METHODS: Using Medicare data (2015–2017), we randomly sampled 200 HF hospitalizations with ICD-10 diagnosis codes for HF in the first/second claim position in a 1:1:2 ratio for systolic HF (I50.2), diastolic HF (I50.3), and other HF (I50.X). The primary gold standards included recorded HF diagnosis by a treating physician for HF hospitalization, ejection fraction (EF)≤50 for heart failure with reduced ejection fraction, and EF>50 for HFpEF. If the quantitative EF was not present, then qualitative descriptions of EF were used for heart failure with reduced ejection fraction/HFpEF gold standards. Multiple secondary gold standards were also tested. Gold standard data were extracted from medical records using standardized forms and adjudicated by cardiology fellows/staff. We calculated positive predictive values with 95% CIs. RESULTS: The 200-chart validation sample included 50 systolic, 50 diastolic, 47 combined dysfunction, and 53 unspecified HF patients. The positive predictive values of acute HF hospitalization was 98% [95% CI, 95–100] for first-position ICD-10 HF diagnosis and 66% [95% CI, 58–74] for first/second-position diagnosis. Quantitative EF was available for ≥80% of patients with systolic, diastolic, or combined dysfunction ICD-10 codes. The positive predictive value of systolic HF codes was 90% [95% CI, 82–98] for EFs≤50% and 72% [95% CI, 60–85] for EFs≤40%. The positive predictive value was 92% [95% CI, 85–100] for HFpEF for EFs>50%. The ICD-10 codes for combined or unspecified HF poorly predicted heart failure with reduced ejection fraction or HFpEF. CONCLUSIONs: ICD-10 principal diagnosis identified acute HF hospitalization with a high positive predictive value. Systolic and diastolic ICD-10 diagnoses reliably identified heart failure with reduced ejection fraction and HFpEF when EF 50% was used as the cutoff.

Introduction The impact of transcatheter aortic valve replacement (TAVR) on sex disparities has not been well established. This study sought to examine the impact of sex on outcomes following aortic valve replacement (AVR) for aortic stenosis (AS) in the era of routine TAVR. Methods We performed a cross-sectional analysis of the National Inpatient Sample (2009-18) to study AS visits for all AVR, and in-hospital outcomes as a function of sex. Survey estimation commands were used to provide national estimates. Results There were an estimated 431,344 surgical aortic valve replacement (SAVR) and 189,137 TAVR inpatient visits. Mortality was higher in women after SAVR (3.8%±0.1 vs 2.7%±0.07, p<0.001) and TAVR (2.4%±0.1 vs 1.7%±0.1, p<0.001) compared to men. Female patients undergoing SAVR had higher rates of permanent pacemaker (PPM) implantation, stroke, and bleeding (5.9%±0.1 vs 5%±0.1, 2.8%±0.1 vs 2.3%±0.07, and 37.8%±0.8 vs 29.8%±0.6; p<0.001, respectively) but lower rates of acute kidney injury (AKI) (16.4%±0.3 vs 20.3%±0.3, p<0.001). Women undergoing TAVR had higher rates of stroke and bleeding (2.4%±0.1 vs 1.6%±0.09 and 28.7%±0.6 vs 22%±0.5; p<0.001 respectively) but lower rates of PPM and AKI (9.5%±0.3 vs 10.7%±0.2 and 11.3%±0.3 vs 13.4%±0.3; p<0.001, respectively). Compared with isolated SAVR, isolated TAVR was associated with lower mortality in women during 2016-18, both after multivariable adjustment (OR = 0.40, 95% CI 0.27-0.60) and propensity matching (mean difference 0.66%±0.2), however there was no difference in men. Conclusion Although women continue to have higher in-hospital mortality following both TAVR and SAVR as compared to men, TAVR is associated with a lower in-hospital mortality in women compared to SAVR. Thus, TAVR may represent a potential intervention to narrow the sex-based disparities in the management of AS.