The authors show the direct in vitro action of thyroid hormones on RNA-polymerase activity in rat liver mitochondria. 3,5,3' L-triiodothyronine (L-T3) and 3,5,3',5' L-tetraiodothyronine (L-T4) stimulate mitochondrial RNA synthesis without either increasing the permeability of preswollen mitochondria or stimulating the synthesis of the triphosphate ribonucleotides (NTP's). Thyroid hormones do not directly depress mitochondrial RNA hydrolysis. Studies carried out with structural analogues of thyroid hormones indicate the structural specifications of the regulating system of the mitochondrial RNA-polymerase. L-T3 and L-T4 are also effective 'in vitro' on mitochondria obtained from animals undergoing different hormonal and dietary treatments, with the exceptions of those fed with a hypoprotein diet. Thus, the authors suggest the possible intervention of a specific mitochondrial receptor for L-T3 and L-T4.
Duchenne muscular dystrophy (DMD) is a devastating and debilitating muscle degenerative disease affecting 1 in every 3,500 male births worldwide. DMD is progressive and fatal; accumulated weakening of the muscle tissue leads to an inability to walk and eventual loss of life due to respiratory and cardiac failure. Importantly, there remains no effective cure for DMD. DMD is caused by defective expression of the <i>DMD</i> gene, which encodes for dystrophin, a component of the dystrophin glycoprotein complex. In muscle fibers, this protein complex plays a critical role in maintaining muscle membrane integrity. Emerging studies have shown that muscle stem cells, which are adult stem cells responsible for muscle repair, are also affected in DMD. DMD muscle stem cells do not function as healthy muscle stem cells, and their impairment contributes to disease progression. Deficiencies in muscle stem cell function include impaired establishment of cell polarity leading to defective asymmetric stem cell division, reduced myogenic commitment, impaired differentiation, altered metabolism, and enhanced entry into senescence. Altogether, these findings indicate that DMD muscle stem cells are dysfunctional and have impaired regenerative potential. Although recent advances in adeno-associated vector and antisense oligonucleotide-mediated mechanisms for gene therapy have shown clinical promise, the current therapeutic strategies for muscular dystrophy do not effectively target muscle stem cells and do not address the deficiencies in muscle stem cell function. Here, we discuss the merits of restoring endogenous muscle stem cell function in degenerating muscle as a viable regenerative medicine strategy to mitigate DMD.
A variety of brain disorders such as neural injury, brain dysfunction, vascular malformation, and neurodegenerative diseases are associated with abnormal levels of oxygen. Current methods to directly monitor tissue oxygenation in the brain are expensive and invasive, suffering from a lack of accuracy. Electrochemical detection has been used as an invasiveness and cost-effectiveness method, minimizing pain, discomfort, and injury to the patient. In this work, we developed a minimally invasive needle-sensor with a high surface area to monitor O2 levels in the brain using acupuncture needles. The approach was to directly etch the iron from stainless steel acupuncture needles via a controlled pitting corrosion process, obtaining a high microporous surface area. In order to increase the conductivity and selectivity, we designed and applied for the first time a low-cost coating process using non-toxic chemicals to deposit high surface area carbon nanoparticle, catalytically active laccase, and biocompatible polypyrrole. The physicochemical properties of the materials were characterized as well as their efficacy and viability as probes for the electrochemical detection of PO2. Our modified needles exhibited efficient electrocatalysis and high selectivity toward O2, with excellent repeatability. We well engineered a small diagnostic tool to monitor PO2, minimally invasive, able to monitor real-time O2 in vivo complex environments.
Fluorescence microscopy is a powerful tool enabling the visualization of protein localization within cells. In this article, we outline an automated and non‐biased way to detect and quantify subcellular particles using immunocytochemistry, fluorescence microscopy, and the program CellProfiler. We discuss the examination of two types of subcellular particles: messenger ribonucleoprotein (mRNP) granules, namely processing bodies and stress granules, and autophagosomes. Fluorescent microscopy Z‐stacks are acquired and deconvolved, and maximum intensity images are generated. The number of subcellular particles per cell is then quantified using the described CellProfiler pipeline. We also explain how to isolate primary myoblast progenitor cells from mice, which were used to obtain the presented results. Last, we discuss the critical parameters to be considered for each of these techniques. Both mRNP granules and autophagosomes play important roles in sequestering intracellular cargo, such as messenger RNAs and RNA‐binding proteins for mRNP granules and cytoplasmic waste for autophagosomes. The methods outlined in this article are widely applicable for studies relating to subcellular particle formation, localization, and flux during homeostasis, following stimuli, and during disease. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Immunofluorescence microscopy of messenger ribonucleoprotein granules in primary myoblasts Alternate Protocol: Immunofluorescence microscopy of autophagosomes in primary myoblasts Support Protocol: Isolation of primary myoblasts from mice Basic Protocol 2: Automated quantification of subcellular particles
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