ABSTRACT
The specific nature of CRISPR-Cas12a makes it a desirable RNA-guided endonuclease for biotechnology and therapeutic applications. To understand how R-loop formation within the compact Cas12a enables target recognition and nuclease activation, we used cryo-electron microscopy to capture wild-type Acidaminococcussp. Cas12a R-loop intermediates and DNA delivery into the RuvC active site. Stages of Cas12a R-loop formation-starting from a 5-bp seed-are marked by distinct REC domain arrangements. Dramatic domain flexibility limits contacts until nearly complete R-loop formation, when the non-target strand is pulled across the RuvC nuclease and coordinated domain docking promotes efficient cleavage. Next, substantial domain movements enable target strand repositioning into the RuvC active site. Between cleavage events, the RuvC lid conformationally resets to occlude the active site, requiring re-activation. These snapshots build a structural model depicting Cas12a DNA targeting that rationalizes observed specificity and highlights mechanistic comparisons to other class 2 effectors.
ABSTRACT
INTRODUCTION: Chronic obstructive pulmonary disease (COPD) is frequently associated with a reduced functional exercise performance. Even if this parameter is routinely evaluated using 6-minute walking test (6MWT), new field tests are regularly investigated as alternative tests. The aim of this study was to compare functional exercise performance evaluation by sit-to-stand test (STST) and 6MWT and to evaluate reliability and repeatability of the STST in COPD patients. METHOD: Forty-two COPD patients performed randomly two tests: 6MWT and STST. Each test was repeated two times. Distance (6MWD) and number of repetitions were measured. Cardiorespiratory parameters, dyspnea and lower limb fatigue (Borg) were recorded before and after the tests. RESULTS: Sit-to-stand repetitions (19 ± 6) and 6MWD (441 ± 104 m) were correlated (r = .716; P < .001). Good repeatability was found for STST and 6MWT. Good reliability was observed for STST (ICC = 0.902). Variations of heart rate and pulsed oxygen saturation were significantly different between these two tests (23% ± 17% vs 13% ± 11%; P = .022 and -7.6% ± 4.6% vs -0.7% ± 2.7%; P < .001 for 6MWT and STST, respectively). Variations of dyspnea and lower limb fatigue were similar between both tests (P = .827 and P = .467). CONCLUSION: The one minute sit-to-stand test is a valuable alternative to 6MWT to estimate functional exercise performance in COPD patients. The cardiorespiratory demand is different between both tests although the variation of dypsnea is similar. No learning effect was observed for STST.
Subject(s)
Blood Pressure/physiology , Exercise Test/methods , Exercise Tolerance/physiology , Heart Rate/physiology , Muscle Strength/physiology , Pulmonary Disease, Chronic Obstructive/diagnosis , Walking/physiology , Aged , Female , Humans , Male , Pulmonary Disease, Chronic Obstructive/physiopathology , Quality of Life , Reproducibility of Results , Respiratory Function Tests/methods , Severity of Illness IndexABSTRACT
BACKGROUND: The validity and reproducibility of the 6-minute stepper test (6MST) have already been demonstrated in patients with chronic obstructive pulmonary disease (COPD). OBJECTIVES: The aim of this study was to evaluate the responsiveness of the 6MST to pulmonary rehabilitation (PR) in patients with COPD, to determine a minimal important difference (MID) for the 6MST, and to compare the 6MST and the 6-minute walk test (6MWT). METHODS: Sixty-two patients with COPD were included in a prospective experimental study. Participants underwent a 3-week inpatient PR program. The primary outcome was the change in the number of steps during the 6MST measured before and after PR. The secondary outcome included the change in the 6-minute walking distance (6MWD) pulse oximetry, heart rate, dyspnea, and leg discomfort during the tests measured before and after PR. MID was determined by anchor-based and distribution approaches. RESULTS: After PR, we observed a significant increase in the number of steps during the 6MST (22.5 steps; 95% CI 13.8-31.3; p < 0.0001) and in the 6MWD (26.6 m; 95% CI 17.6-35.5; p < 0.0001). The 6MST and 6MWT were improved by 10.1 and 6.5%, respectively. The number of steps during the 6MST was significantly correlated with the 6MWD before (r = 0.72; p < 0.0001) and after PR (r = 0.66; p < 0.0001). MID was estimated to be around 20 steps. CONCLUSION: The 6MST appears to be as responsive as the 6MWT in assessing functional improvement during PR in patients with COPD. The 6MST is a low-cost assessment and requires limited space.
