Methods in molecular biology
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DNA methylation plays an important role in the regulation of the expression of transposons and genes. Various methods have been developed to assay DNA methylation levels. Bisulfite sequencing is considered to be the "gold standard" for single-base resolution measurement of DNA methylation levels. ⋯ Here, we described a protocol for WGBS in plant species with large genomes. This protocol has been successfully applied to assay genome-wide DNA methylation levels in maize and barley. This protocol has also been successfully coupled with sequence capture technology to assay DNA methylation levels in a targeted set of genomic regions.
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Duchenne muscular dystrophy (DMD) is a devastating muscle disorder caused by mutations in the DMD gene. Antisense-mediated exon skipping is a promising strategy to treat DMD. The approval of Exondys 51 (eteplirsen) targeting exon 51 was the most noteworthy accomplishment in 2016. ⋯ In addition, an invasive muscle biopsy is required to obtain muscle cells from patients. Furthermore, many DMD mutations are very rare and it is hard to find a patient with a specific mutation for muscle biopsy in many cases. Here, we describe a novel approach to create an immortalized muscle cell line with a DMD deletion mutation using the human rhabdomyosarcoma (RD) cell line and the CRISPR/Cas9 system that can be used to test the efficacy of exon skipping.
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Comparative Study
Comparison of Mitochondrial Incubation Media for Measurement of Respiration and Hydrogen Peroxide Production.
High-Resolution FluoRespirometry is a well-established and versatile approach to study mitochondrial oxygen uptake amperometrically in combination with measurement of fluorescence signals. One of the most frequently applied fluorescent dyes is Amplex UltraRed for monitoring rates of hydrogen peroxide production. Selection of an appropriate mitochondrial respiration medium is of crucial importance, the primary role of which is to support and preserve optimum mitochondrial function. ⋯ Stability of assay sensitivity over experimental time was highest in MiR05 but slightly less in MiR07. Taken together, MiR05 and Buffer Z yield comparable results on respiration and H2O2 production. Despite the lower sensitivity, MiR05 was selected as the medium of choice for FluoRespirometry due to the highest stability of the sensitivity or calibration constant observed in experiments over periods of up to 2 h.
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Traumatic brain injury (TBI) diagnoses have increased in frequency during the past decade, becoming a silent epidemic. The pathophysiology of TBI involves pathophysiological processes affecting the brain, induced by traumatic biomechanical forces resulting in temporary impairment of neurological function. Preclinical models have been generated to recapitulate the mechanical, neuroinflammatory, and behavioral outcomes observed in the clinical setting. ⋯ The model is reproducible and can be adjusted to produce a mild to moderate and severe injury, as reflected by mortality and return of reflexes, by adjusting the amount of force applied. The histopathological changes achieved with this model reproduce that seen in human TBI including focal contusion in the cortex, with accompanying intraparenchymal punctate hemorrhage, followed by inflammation and neuronal degeneration. This chapter describes the LFP model, which produces a mixed model of focal and diffuse brain injury that progresses over time affecting predominantly the cortical parenchyma.
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After conducting systematic and quantitative comparisons of different sample preparation techniques regarding their capability to efficiently and reproducibly recover proteins from biopsies, we present here our superior protocol for extracting proteins from low amounts of adipose tissue. Adipose tissue as a matrix in bottom-up proteomics is challenging due to the extremely high lipid content. The lysis buffer utilized contains the detergent sodium deoxycholate, which does not impair the activity of trypsin and therefore enables direct digestion without detergent removal steps. The resulting workflow is time saving, cost efficient, easy to perform, and it can also be applied to other hydrophobic samples.