Preclinical Evaluation of a Food-Derived Functional Ingredient to Address Skeletal Muscle Atrophy.

Skeletal muscle is the metabolic powerhouse of the body, however, dysregulation of the mechanisms involved in skeletal muscle mass maintenance can have devastating effects leading to many metabolic and physiological diseases. The lack of effective solutions makes finding a validated nutritional intervention an urgent unmet medical need. In vitro testing in murine skeletal muscle cells and human macrophages was carried out to determine the effect of a hydrolysate derived from vicia faba (PeptiStrong: NPN_1) against phosphorylated S6, atrophy gene expression, and tumour necrosis factor alpha (TNF-α) secretion, respectively. Finally, the efficacy of NPN_1 on attenuating muscle waste in vivo was assessed in an atrophy murine model. Treatment of NPN_1 significantly increased the phosphorylation of S6, downregulated muscle atrophy related genes, and reduced lipopolysaccharide-induced TNF-α release in vitro. In a disuse atrophy murine model, following 18 days of NPN_1 treatment, mice exhibited a significant attenuation of muscle loss in the soleus muscle and increased the integrated expression of Type I and Type IIa fibres. At the RNA level, a significant upregulation of protein synthesis-related genes was observed in the soleus muscle following NPN_1 treatment. In vitro and preclinical results suggest that NPN_1 is an effective bioactive ingredient with great potential to prolong muscle health.

Atomic force microscopy study of the conformational change in immobilized calmodulin.

Maintaining the biological functionality of immobilized proteins is the key to the success of numerous protein-based biomedical devices. To that end, we studied the conformational change in calmodulin (CaM) immobilized on chemical patterns. 1-Cysteine-mutated calmodulin was immobilized on a mercapto-terminated surface through cysteine-Hg-mercapto coupling. Utilizing atomic force microscopy (AFM), the average height of immobilized calmodulin was determined to be 1.87 ± 0.19 nm. After incubation in EGTA solution, the average height of the protein changed to 2.26 ± 0.21 nm, indicating the conformational change of CaM to Apo-CaM. Immobilized CaM also demonstrated a conformational change upon the reaction with known calmodulin antagonist chlorpromazine (CPZ). After incubation in CPZ solution, the average height of CPZ-bound CaM increased to 2.32 ± 0.20 nm, demonstrating that immobilized CaM has a similar response to that in bulk solution. These results show that the immobilization of calmodulin on a solid support does not interfere with the ability of the protein to bind calcium and calmodulin antagonists. Our results demonstrate the feasibility of employing AFM to probe and understand protein conformational changes.