Allosteric activation of CRISPR-Cas12a requires the concerted movement of the bridge helix and helix 1 of the RuvC II domain

Nucleases derived from the prokaryotic defense system CRISPR-Cas are frequently re-purposed for gene editing and molecular diagnostics. Hence, an indepth understanding of the molecular mechanisms of these enzymes is of crucial importance. We focused on Cas12a from Francisella novicida (Fn- Cas12a) and investigated the functional role of helix 1, a structural element that together with the bridge helix (BH) connects the recognition and the nuclease lobes of FnCas12a. Helix 1 is structurally connected to the lid domain that opens upon DNA target loading thereby activating the active site of FnCas12a.We probed the structural states of FnCas12a variants altered in helix 1 and/or the bridge helix using singlemolecule FRET measurements and assayed the precrRNA processing, cis- and trans-DNA cleavage activity. We show that helix 1 and not the bridge helix is the predominant structural element that confers conformational stability of FnCas12a. Even small perturbations in helix 1 lead to a decrease in DNA cleavage activity while the structural integrity is not affected. Our data, therefore, implicate that the concerted remodeling of helix 1 and the bridge helix upon DNA binding is structurally linked to the opening of the lid and therefore involved in the allosteric activation of the active site

The osteoarthritis prevention study (TOPS) - A randomized controlled trial of diet and exercise to prevent Knee Osteoarthritis:Design and rationale

Background: Osteoarthritis (OA), the leading cause of disability among adults, has no cure and is associated with significant comorbidities. The premise of this randomized clinical trial is that, in a population at risk, a 48-month program of dietary weight loss and exercise will result in less incident structural knee OA compared to control. Methods/design: The Osteoarthritis Prevention Study (TOPS) is a Phase III, assessor-blinded, 48-month, parallel 2 arm, multicenter randomized clinical trial designed to reduce the incidence of structural knee OA. The study objective is to assess the effects of a dietary weight loss, exercise, and weight-loss maintenance program in preventing the development of structural knee OA in females at risk for the disease. TOPS will recruit 1230 ambulatory, community dwelling females with obesity (Body Mass Index (BMI) ​≥ ​30 ​kg/m2) and aged ≥50 years with no radiographic (Kellgren-Lawrence grade ≤1) and no magnetic resonance imaging (MRI) evidence of OA in the eligible knee, with no or infrequent knee pain. Incident structural knee OA (defined as tibiofemoral and/or patellofemoral OA on MRI) assessed at 48-months from intervention initiation using the MRI Osteoarthritis Knee Score (MOAKS) is the primary outcome. Secondary outcomes include knee pain, 6-min walk distance, health-related quality of life, knee joint loading during gait, inflammatory biomarkers, and self-efficacy. Cost effectiveness and budgetary impact analyses will determine the value and affordability of this intervention. Discussion: This study will assess the efficacy and cost effectiveness of a dietary weight loss, exercise, and weight-loss maintenance program designed to reduce incident knee OA.Trial registration: ClinicalTrials.gov Identifier: NCT05946044.</p

Weight-loss and exercise for communities with arthritis in North Carolina (we-can): design and rationale of a pragmatic, assessor-blinded, randomized controlled trial

Background: Recently, we determined that in a rigorously monitored environment an intensive diet-induced weight loss of 10% combined with exercise was significantly more effective at reducing pain in men and women with symptomatic knee osteoarthritis (OA) than either intervention alone. Compared to previous long-term weight loss and exercise trials of knee OA, our intensive diet-induced weight loss and exercise intervention was twice as effective at reducing pain intensity. Whether these results can be generalized to less intensively monitored cohorts is unknown. Thus, the policy relevant and clinically important question is: Can we adapt this successful solution to a pervasive public health problem in real-world clinical and community settings? This study aims to develop a systematic, practical, cost-effective diet-induced weight loss and exercise intervention implemented in community settings and to determine its effectiveness in reducing pain and improving other clinical outcomes in persons with knee OA. Methods/Design: This is a Phase III, pragmatic, assessor-blinded, randomized controlled trial. Participants will include 820 ambulatory, community-dwelling, overweight and obese (BMI ≥ 27 kg/m2) men and women aged ≥ 50 years who meet the American College of Rheumatology clinical criteria for knee OA. The primary aim is to determine whether a community-based 18-month diet-induced weight loss and exercise intervention based on social cognitive theory and implemented in three North Carolina counties with diverse residential (from urban to rural) and socioeconomic composition significantly decreases knee pain in overweight and obese adults with knee OA relative to a nutrition and health attention control group. Secondary aims will determine whether this intervention improves self-reported function, health-related quality of life, mobility, and is cost-effective. Discussion: Many physicians who treat people with knee OA have no practical means to implement weight loss and exercise treatments as recommended by numerous OA treatment guidelines. This study will establish the effectiveness of a community program that will serve as a blueprint and exemplar for clinicians and public health officials in urban and rural communities to implement a diet-induced weight loss and exercise program designed to reduce knee pain and improve other clinical outcomes in overweight and obese adults with knee OA

I-CAN: INDEPENDENT WEIGHT-LOSS MAINTENANCE FOR COMMUNITIES WITH ARTHRITIS IN NORTH CAROLINA

BACKGROUND: Osteoarthritis (OA) is one of the chief forms of arthritis and a major reason for disability among older adults. Weight loss is a non-pharmacologic intervention that may help decrease the effects of OA while improving one’s quality of life. The objective of the I-CAN study was to determine whether the weight-loss maintenance (WLM) intervention had a long lasting impact on self-efficacy, maintained weight loss, and ultimately better clinical outcomes. METHODS: Participants who lost more than 5% of their base body weight in the parent study (Weight Loss and Exercise for Communities with Arthritis in North Carolina), were re-randomized and placed into a WLM or an attention control (AC) group. The WLM intervention was a 6-month facilitated maintenance phase that included exercise, as well as group and individual behavioral sessions grounded in social cognitive theory to increase self-efficacy. The AC group included sessions to provide social interaction and health education. The primary outcome was weight change from baseline to the end of intervention. This treatment effect was estimated using mixed linear models adjusted for site, sex, and baseline values of the outcome. Effect sizes were estimated using Cohen’s d. Additionally, the role played by self-efficacy for maintaining weight loss on one’s own was examined. RESULTS: At the start of I-CAN, the participants (n=104, 79 females, mean age=67 years) had a weight of 85.1 kg (SD 13.9). At the 6-month follow up the WLM group gained back less weight (mean=1.3 kg; 95% CI -0.25, 2.86) compared to the control group who regained at nearly twice the rate (mean=3.5 kg; 95% CI 1.80, 5.11; P=.037). Self-efficacy accounted for 22% of the total effect of the WLM group. Interviews at the end of the intervention suggest that the WLM was well-received, with all participants endorsing the statement “The program had a positive impact on my confidence that I can maintain my weight indefinitely” and one participant commenting that “it is possible to lose weight and keep it off and feel better about yourself with a simple and systematic approach.” CONCLUSION: These findings support a short-term WLM program, based on social cognitive theory, in the maintenance of weight loss, while also promoting an increase in self-efficacy. This program has the potential to sustain both the functional and symptomatic improvements achieved through weight loss for older adults with knee OA