The type IIS restriction enzyme MmeI can cut across a double-strand break.

BACKGROUND: Type-IIS restriction enzymes cut outside their recognition sites, allowing them to remove their binding sites upon digestion. This feature has resulted in their wide application in molecular biology techniques, including seamless cloning methods, enzymatic CRISPR library generation, and others. We studied the ability of the Type-IIS restriction enzyme MmeI, which recognizes an asymmetric sequence TCCRAC and cuts 20 bp downstream, to cut across a double-strand break (DSB). METHODS AND RESULTS: We used synthetic double-stranded oligos with MmeI recognition sites close to 5' end and different overhang lengths to measure digestion after different periods of time and at different temperatures. We found that the MmeI binding and cutting sites can be situated on opposite sides of a DSB if the edges of the DNA molecules are held together by transient base-pairing interactions between compatible overhangs. CONCLUSION: We found that MmeI can cut across a DSB, and the efficiency of the cutting depends on both overhang length and temperature.

Agreement of the American Academy of Orthopaedic Surgeons Appropriate Use Criteria With Treatment Recommendations From Arthroplasty Surgeons.

Background: The American Academy of Orthopaedic Surgeons (AAOS) Appropriate Use Criteria (AUC) for Surgical Management of Osteoarthritis of the Knee (2016) and Management of Osteoarthritis of the Hip (2017) are intended to provide treatment recommendations for osteoarthritis (OA). This study examined the agreement of AUC appropriateness classifications with arthroplasty surgeon recommendations for total knee arthroplasty (TKA) and total hip arthroplasty (THA). Methods: The cohort included 558 OA patients (397 knee, 161 hip) referred to a specialty arthroplasty clinic. Surgeons completed the online AAOS AUC patient profiles to generate appropriateness ratings. Surgeons' recommendations for treatment were recorded. We performed univariate and bivariate analyses to evaluate relationships between AUC appropriateness and surgeon recommendations. Results: The knee OA AUC classified TKA as "appropriate" for 309 (77.8%) of the 397 knee OA patients. Surgeons recommended TKA for 123 (31.0%), resulting in 46.8% (n = 186) higher rate of "appropriate" classification by AUC than TKA recommendation by surgeons. Weighted Cohen's κ demonstrated slight agreement (κ = 0.06, 95% confidence interval: 0.04, 0.09) between AUC appropriateness and surgeon TKA recommendation. The hip OA AUC classified THA as "appropriate" for 98 (60.9%) of the 161 hip OA patients. Surgeons recommended THA for 76 (47.2%), resulting in 13.7% (n = 22) higher rate of "appropriate" classification by AUC than THA recommendation by surgeons. Weighted Cohen's κ demonstrated moderate agreement (κ = 0.47, 95% confidence interval: 0.37, 0.57) between the AUC appropriateness classification and the surgeon's THA recommendation. Conclusions: AAOS AUC guidelines indicated surgical appropriateness significantly more than arthroplasty surgeons. AUC agreed slightly with surgeons for TKA and moderately for THA.