Abstract: Employing a new algorithm for identifying differentially methylated regions (DMRs) from reduced representation bisulfite sequencing profiles, we identified 1972 hypermethylated and 3250 hypomethylated myogenic DMRs in a comparison of myoblasts (Mb) and myotubes (Mt) with 16 types of nonmuscle cell cultures. DMRs co-localized with a variety of chromatin structures, as deduced from ENCODE whole-genome profiles. Myogenic hypomethylation was highly associated with both weak and strong enhancer-type chromatin, while hypermethylation was infrequently associated with enhancer-type chromatin. Both myogenic hypermethylation and hypomethylation often overlapped weak transcription-type chromatin and Polycomb-repressed-type chromatin. For representative genes, we illustrate relationships between DNA methylation, the local chromatin state, DNaseI hypersensitivity, and gene expression. For example, MARVELD2 exhibited myogenic hypermethylation in transcription-type chromatin that overlapped a silenced promoter in Mb and Mt while TEAD4 had myogenic hypomethylation in intronic subregions displaying enhancer-type or transcription-type chromatin in these cells. For LSP1, alternative promoter usage and active promoter-type chromatin were linked to highly specific myogenic or lymphogenic hypomethylated DMRs. Lastly, despite its myogenesis-associated expression, TBX15 had multiple hypermethylated myogenic DMRs framing its promoter region. This could help explain why TBX15 was previously reported to be underexpressed and, unexpectedly, its promoter undermethylated in placentas exhibiting vascular intrauterine growth restriction.
Abstract: Recent genome-wide analysis of C-phosphate-G (CpG) sites has shown that the DNA methylome changes with increasing age, giving rise to genome-wide hypomethylation with site‑specific incidences of hypermethylation. This notion has received a lot of attention, as it potentially explains why aged organisms generally have a higher risk of age-related diseases. However, very little is known about the mechanisms that could cause the occurrence of these changes. Moreover, there does not appear to be a clear link between popular theories of aging and alterations in the methylome. Some of the most fruitful of these theories attribute an important role to reactive oxygen species, which seem to be responsible for an increase in oxidative damage to macromolecules, such as DNA, during the lifetime of an organism. In this review, the connection between changes in DNA methylation and these reactive oxygen species is discussed, as well as the effect of these changes on health. Deeper insights into the nature, causes and consequences of the aging methylome might provide a deeper understanding of the molecular mechanisms of aging and eventually contribute to the development of new diagnostic and therapeutic tools.
Abstract: Multiple Reaction Monitoring (MRM) conducted on a triple quadrupole mass spectrometer allows researchers to quantify the expression levels of a set of target proteins. Each protein is often characterized by several unique peptides that can be detected by monitoring predetermined fragment ions, called transitions, for each peptide. Concatenating large numbers of MRM transitions into a single assay enables simultaneous quantification of hundreds of peptides and proteins. In recognition of the important role that MRM can play in hypothesis-driven research and its increasing impact on clinical proteomics, targeted proteomics such as MRM was recently selected as the Nature Method of the Year. However, there are many challenges in MRM applications, especially data pre‑processing where many steps still rely on manual inspection of each observation in practice. In this paper, we discuss an analysis pipeline to automate MRM data pre‑processing. This pipeline includes data quality assessment across replicated samples, outlier detection, identification of inaccurate transitions, and data normalization. We demonstrate the utility of our pipeline through its applications to several real MRM data sets.
Abstract: The muscle wasting and loss of specific force associated with Critical Illness Myopathy (CIM) is, at least in part, due to a preferential loss of the molecular motor protein myosin. This acquired myopathy is common in critically ill immobilized and mechanically ventilated intensive care patients (ICU). There is a growing understanding of the mechanisms underlying CIM, but the role of nutritional factors triggering this serious complication of modern intensive care remains unknown. This study aims at establishing the effect of nutritional status in the pathogenesis of CIM. An experimental ICU model was used where animals are mechanically ventilated, pharmacologically paralysed post-synaptically and extensively monitored for up to 14 days. Due to the complexity of the experimental model, the number of animals included is small. After exposure to this ICU condition, animals develop a phenotype similar to patients with CIM. The results from this study show that the preferential myosin loss, decline in specific force and muscle fiber atrophy did not differ between low vs. eucaloric animals. In both experimental groups, passive mechanical loading had a sparing effect of muscle weight independent on nutritional status. Thus, this study confirms the strong impact of the mechanical silencing associated with the ICU condition in triggering CIM, overriding any potential effects of caloric intake in triggering CIM. In addition, the positive effects of passive mechanical loading on muscle fiber size and force generating capacity was not affected by the nutritional status in this study. However, due to the small sample size these pilot results need to be validated in a larger cohort.
Abstract: Human cancer cell lines are an integral part of drug discovery practices. However, modeling the complexity of cancer utilizing these cell lines on standard plastic substrata, does not accurately represent the tumor microenvironment. Research into developing advanced tumor cell culture models in a three-dimensional (3D) architecture that more prescisely characterizes the disease state have been undertaken by a number of laboratories around the world. These 3D cell culture models are particularly beneficial for investigating mechanistic processes and drug resistance in tumor cells. In addition, a range of molecular mechanisms deconstructed by studying cancer cells in 3D models suggest that tumor cells cultured in two-dimensional monolayer conditions do not respond to cancer therapeutics/compounds in a similar manner. Recent studies have demonstrated the potential of utilizing 3D cell culture models in drug discovery programs; however, it is evident that further research is required for the development of more complex models that incorporate the majority of the cellular and physical properties of a tumor.
Abstract: High intensity training (HIT) is effective at improving health; however, it is unknown whether HIT also improves physical function. This study aimed to determine whether HIT improves metabolic health and physical function in untrained middle aged individuals. Fourteen (three male and eleven female) untrained individuals were recruited (control group n = 6: age 42 ± 8 y, weight 64 ± 10 kg, BMI 24 ± 2 kg·m−2 or HIT group n = 8: age 43 ± 8 y, weight 80 ± 8 kg, BMI 29 ± 5 kg·m−2). Training was performed twice weekly, consisting of 10 × 6-second sprints with a one minute recovery between each sprint. Metabolic health (oral glucose tolerance test), aerobic capacity (incremental time to exhaustion on a cycle ergometer) and physical function (get up and go test, sit to stand test and loaded 50 m walk) were determined before and after training. Following eight weeks of HIT there was a significant improvement in aerobic capacity (8% increase in VO2 peak; p < 0.001), physical function (11%–27% respectively; p < 0.05) and a reduction in blood glucose area under the curve (6% reduction; p < 0.05). This study demonstrates for the first time the potential of HIT as a training intervention to improve skeletal muscle function and glucose clearance as we age.