Acetyl-CoA-driven respiration in frozen muscle contributes to the diagnosis of mitochondrial disease.

BACKGROUND: Freezing human biopsies is common in clinical practice for storage. However, this technique disrupts mitochondrial membranes, hampering further analyses of respiratory function. To contribute to laboratorial diagnosis of mitochondrial diseases, this study sought to develop a respirometry approach using O2k (Oroboros Ins.) to measure the whole electron transport chain (ETC) activity in homogenates of frozen skeletal muscle biopsies. PATIENTS AND METHODS: We enrolled 16 patients submitted to muscle biopsy in the process of routine diagnostic investigation: four with mitochondrial disease and severe mitochondrial dysfunction; seven with exercise intolerance and multiple deletions of mitochondrial DNA, presenting mild to moderate mitochondrial dysfunction; five without mitochondrial disease, as controls. Whole homogenates of muscle fragments were prepared using grinder-type equipment. O2 consumption rates were normalized using citrate synthase activity. RESULTS: Transmission electron microscopy confirmed mitochondrial membrane discontinuation, indicating increased permeability of mitochondrial membranes in homogenates from frozen biopsies. O2 consumption rates in the presence of acetyl-CoA lead to maximum respiratory rates sensitive to rotenone, malonate and antimycin. This protocol of acetyl-CoA-driven respiration (ACoAR), applied in whole homogenates of frozen muscle, was sensitive enough to identify ETC abnormality, even in patients with mild to moderate mitochondrial dysfunction. We demonstrated adequate repeatability of ACoAR and found significant correlation between O2 consumption rates and enzyme activity assays of individual ETC complexes. CONCLUSIONS: We present preliminary data on a simple, low cost and reliable procedure to measure respiratory function in whole homogenates of frozen skeletal muscle biopsies, contributing to diagnosis of mitochondrial diseases in humans.

Bcl-2 knockdown by multifunctional lipid nanoparticle and its influence in apoptosis pathway regarding cutaneous melanoma: in vitro and ex vivo studies.

Multifunctional therapies have emerged as innovative strategies in cancer treatment. In this research article, we proposed a nanostructured lipid carrier (NLC) designed for the topical treatment of cutaneous melanoma, which simultaneously delivers 5-FU and Bcl-2 siRNA. The characterized nanoparticles exhibited a diameter of 259 ± 9 nm and a polydispersion index of 0.2, indicating a uniform size distribution. The NLCs were primarily localized in the epidermis, effectively minimizing the systemic release of 5-FU across skin layers. The ex vivo skin model revealed the formation of a protective lipid film, decreasing the desquamation process of the stratum corneum which can be associated to an effect of increasing permeation. In vitro assays demonstrated that A375 melanoma cells exhibited a higher sensitivity to the treatment compared to non-cancerous cells, reflecting the expected difference in their metabolic rates. The uptake of NLC by A375 cells reached approximately 90% within 4 h. The efficacy of Bcl-2 knockdown was thoroughly assessed using ELISA, Western blot, and qRT-PCR analyses, revealing a significant knockdown and synergistic action of the NLC formulation containing 5-FU and Bcl-2 siRNA (at low concentration --100 pM). Notably, the silencing of Bcl-2 mRNA also impacted other members of the Bcl-2 protein family, including Mcl-1, Bcl-xl, BAX, and BAK. The observed modulation of these proteins strongly indicated the activation of the apoptosis pathway, suggesting a successful inhibition of melanoma growth and prevention of its in vitro spread.