Project description:MicroRNAs are important negative regulators of protein coding gene expression, and have been studied intensively over the last few years. To this purpose, different measurement platforms to determine their RNA abundance levels in biological samples have been developed. In this study, we have systematically compared 12 commercially available microRNA expression platforms by measuring an identical set of 20 standardized positive and negative control samples, including human universal reference RNA, human brain RNA and titrations thereof, human serum samples, and synthetic spikes from homologous microRNA family members. We developed novel quality metrics in order to objectively assess platform performance of very different technologies such as small RNA sequencing, RT-qPCR and (microarray) hybridization. We assessed reproducibility, sensitivity, quantitative performance, and specificity. The results indicate that each method has its strengths and weaknesses, which helps guiding informed selection of a quantitative microRNA gene expression platform in function of particular study goals.
Project description:Resistance training promotes metabolic health and stimulates muscle hypertrophy, but the precise routes by which resistance exercise (RE) conveys these health benefits are largely unknown.AimTo investigate how acute RE affects human skeletal muscle metabolism.MethodsWe collected vastus lateralis biopsies from six healthy male untrained volunteers at rest, before the first of 13 RE training sessions, and 45 min after the first and last bouts of RE. Biopsies were analysed using untargeted mass spectrometry-based metabolomics.ResultsWe measured 617 metabolites covering a broad range of metabolic pathways. In the untrained state RE altered 33 metabolites, including increased 3-methylhistidine and N-lactoylvaline, suggesting increased protein breakdown, as well as metabolites linked to ATP (xanthosine) and NAD (N1-methyl-2-pyridone-5-carboxamide) metabolism; the bile acid chenodeoxycholate also increased in response to RE in muscle opposing previous findings in blood. Resistance training led to muscle hypertrophy, with slow type I and fast/intermediate type II muscle fibre diameter increasing by 10.7% and 10.4%, respectively. Comparison of post-exercise metabolite levels between trained and untrained state revealed alterations of 46 metabolites, including decreased N-acetylated ketogenic amino acids and increased beta-citrylglutamate which might support growth. Only five of the metabolites that changed after acute exercise in the untrained state were altered after chronic training, indicating that training induces multiple metabolic changes not directly related to the acute exercise response.ConclusionThe human skeletal muscle metabolome is sensitive towards acute RE in the trained and untrained states and reflects a broad range of adaptive processes in response to repeated stimulation.
Project description:Cryotherapy is commonly used in the treatment of skeletal muscle injuries. However, the data to support the use of cryotherapy is inconclusive, and the biochemical etiology of cryotherapy in human skeletal muscle remains largely unknown. We therefore sought to determine how a clinically-relevant dose of cryotherapy would impact the transcriptome and metabolome of skeletal muscle. Eight healthy male subjects (age 24.7 ± 4.5 years, BMI 22.2 ± 1.6) received a 15 minute bout of local cryotherapy, delivered via ice cup massage over the anterolateral thigh. This resulted in an 85% decrease in skin temperature and a predicted 27% reduction in intramuscular temperature. The contralateral side served as a non-treated control. Two hours after cryotherapy, muscle biopsies were obtained to analyze changes in the transcriptome, metabolome, and activation of p38 MAPK, ERK1/2, Akt, and p70S6K proteins. No changes were detected in the transcriptome between control and cooled muscles. Cryotherapy reduced levels of hexose sugars and hypoxanthine by 1.3%, but no statistically different changes were observed in 60 additional metabolites. Overall, no differences in phosphorylated p38 MAPK, ERK1/2, Akt, and p70S6K were observed. A clinically relevant dose of cryotherapy produced negligible acute biochemical and molecular changes in the skeletal muscle of human subjects.
Project description:The transcriptional activator MyoD serves as a master controller of myogenesis. Often in partnership with Mef2, MyoD binds to the promoters of hundreds of muscle genes in proliferating myoblasts, yet activates these targets only upon receiving cues that launch differentiation. What regulates this off/on switch of MyoD function has been incompletely understood, although known to reflect the action of chromatin modifiers. Here, we identify KAP1/TRIM28 as a key regulator of MyoD function. In myoblasts, KAP1 is present with MyoD and Mef2 at many muscle genes, where it acts as a scaffold to recruit not only co-activators such as p300 and LSD1, but also co-repressors such as G9a and HDAC1, with promoter silencing as net outcome. Upon differentiation, MSK1-mediated phosphorylation of KAP1 releases the co-repressors from the scaffold, unleashing transcriptional activation by MyoD/Mef2 and their positive cofactors. Thus, our results reveal KAP1 as a previously unappreciated interpreter of cell signaling, which modulates the ability of MyoD to drive myogenesis. Transcriptome profiling of control and Kap1KD in C2C12 during proliferation or at 48hours differentiation stage
Project description:In an effort to identify the compendium of regulatory elements that govern myogenic differentiation, we generated chromatin state maps based on the recruitment of factors (p300 and PolII) and histone modifications (H3K4me1, H3K27ac) that typify enhancers in myoblasts and myotubes. We found a remarkable concordance between the location of these newly defined, active enhancers and MyoD1 binding events. In addition, we found a striking association between the position of enhancers and RNA polymerase II recruitment, which resulted in the expression of previously disclosed non-coding RNAs, as well as potentially new transcripts identified ab initio, throughout the enhancer region. Mapping of H3K27ac and p300 in growing myoblasts (MB) and fully differentiated myotubes (MT). Mapping of c-Jun in growing myoblasts (MB).
Project description:Cancer-associated fibroblasts (CAFs) have been recognized as important contributors to cancer development and progression. However, opposing evidence has been published whether CAFs, in addition to epigenetic, also undergo somatic genetic alterations and whether these changes contribute to carcinogenesis and tumour progression. We combined multiparameter DNA flow cytometry, flow-sorting and 6K SNP-arrays to study DNA aneuploidy, % S-phase, loss of heterozygosity (LOH) and copy number alterations (CNAs) to study somatic genetic alterations in cervical cancer-associated stromal cell fractions (n = 58) from formalin-fixed, paraffin-embedded (FFPE) samples. Tissue sections were examined for the presence of CAFs. Microsatellite analysis was used to study LOH. By flow cytometry we found no proof for DNA aneuploidy in the vimentin-positive stromal cell fractions of any samples (CV G0G1 population 3.7% +/- 1.2; S-phase 1.4% +/- 1.8). The genotype concordance between the stromal cells and the paired normal endometrium samples was > 99.9%. No evidence for CNAs or LOH was found in the stromal cell fractions. In contrast, high frequencies of DNA content abnormalities (43/57), a significant higher S-phase (14.6% +/- 8.5)(p = 0.0001) and substantial numbers of CNAs and LOH were identified in the keratin-positive epithelial cell fractions (CV G0G1 population 4.1% +/- 1.0). Smooth muscle actin and vimentin immunohistochemistry verified the presence of CAFs in all cases tested. LOH hot-spots on chromosomes 3p, 4p and 6p were confirmed by microsatellite analysis but the stromal cell fractions showed retention of heterozygosity only. From our study we conclude that stromal cell fractions from cervical carcinomas are DNA diploid, have a genotype undistinguishable from patient-matched normal tissue and are genetically stable. Stromal genetic changes do not seem to play a role during cervical carcinogenesis and progression. In addition, the stromal cell fraction of cervical carcinomas can be used as reference allowing large retrospective studies of archival FFPE tissues for which no normal reference tissue is available. Paired experiment, Endometrial (non-tumor) cells vs stromal cells from cervical tumors. Biological replicates: 58 patients. From 5 tumors also the tumor fraction was profiled.
Project description:Recurrent non-medullary thyroid carcinoma (NMTC) is a rare disease. We initially characterized 27 recurrent NMTC: 13 papillary thyroid cancers (PTC), 10 oncocytic follicular carcinomas (FTC-OV), and 4 non-oncocytic follicular carcinomas (FTC). A validation cohort composed of benign and malignant (both recurrent and non-recurrent) thyroid tumours was subsequently analysed (n = 20). Methods Data from genome-wide SNP arrays and flow cytometry were combined to determine the chromosomal dosage (allelic state) in these tumours, including mutation analysis of components of PIK3CA/AKT and MAPK pathways. Results All FTC-OVs showed a very distinct pattern of genomic alterations. Ten out of 10 FTC-OV cases showed near-haploidisation with or without subsequent genome endoreduplication. Near-haploidisation was seen in 5/10 as extensive chromosome-wide monosomy (allelic state [A]) with near-haploid DNA indices and retention of especially chromosome 7 (seen as a heterozygous allelic state [AB]). In the remaining 5/10 chromosomal allelic states AA with near diploid DNA indices were seen with allelic state AABB of chromosome 7, suggesting endoreduplication after preceding haploidisation. The latter was supported by the presence of both near-haploid and endoreduplicated tumour fractions in some of the cases. Results were confirmed using FISH analysis. Relatively to FTC-OV limited numbers of genomic alterations were identified in other types of recurrent NMTC studied, except for chromosome 22q which showed alterations in 6 of 13 PTCs. Only two HRAS, but no mutations of EGFR or BRAF were found in FTC-OV. The validation cohort showed two additional tumours with the distinct pattern of genomic alterations (both with oncocytic features and recurrent). Conclusions We demonstrate that recurrent FTC-OV is frequently characterised by genome-wide DNA haploidisation, heterozygous retention of chromosome 7, and endoreduplication of a near-haploid genome. Whether normal gene dosage on especially chromosome 7 (containing EGFR, BRAF, cMET) is crucial for FTC-OV tumour survival is an important topic for future research. 28 thyroid tumors from 27 patients were profiled by SNP array. Comparisons between different types were made.
Project description:Polyglutamine (polyQ) expansions in the androgen receptor (AR) gene cause spinal and bulbar muscular atrophy (SBMA), a neuromuscular disease characterized by lower motor neuron (MN) loss and skeletal muscle atrophy, with an unknown mechanism. We generated new mouse models of SBMA for constitutive and inducible expression of mutant AR and performed biochemical, histological and functional analyses of phenotype. We show that polyQ-expanded AR causes motor dysfunction, premature death, IIb-to-IIa/IIx fiber-type change, glycolytic-to-oxidative fiber-type switching, upregulation of atrogenes and autophagy genes and mitochondrial dysfunction in skeletal muscle, together with signs of muscle denervation at late stage of disease. PolyQ expansions in the AR resulted in nuclear enrichment. Within the nucleus, mutant AR formed 2% sodium dodecyl sulfate (SDS)-resistant aggregates and inclusion bodies in myofibers, but not spinal cord and brainstem, in a process exacerbated by age and sex. Finally, we found that two-week induction of expression of polyQ-expanded AR in adult mice was sufficient to cause premature death, body weight loss and muscle atrophy, but not aggregation, metabolic alterations, motor coordination and fiber-type switch, indicating that expression of the disease protein in the adulthood is sufficient to recapitulate several, but not all SBMA manifestations in mice. These results imply that chronic expression of polyQ-expanded AR, i.e. during development and prepuberty, is key to induce the full SBMA muscle pathology observed in patients. Our data support a model whereby chronic expression of polyQ-expanded AR triggers muscle atrophy through toxic (neomorphic) gain of function mechanisms distinct from normal (hypermorphic) gain of function mechanisms.