Assessment of brain reference genes for RT-qPCR studies in neurodegenerative diseases

Evaluation of gene expression levels by reverse transcription quantitative real-time PCR (RT-qPCR) has for many years been the favourite approach for discovering disease-associated alterations. Normalization of results to stably expressed reference genes (RGs) is pivotal to obtain reliable results. This is especially important in relation to neurodegenerative diseases where disease-related structural changes may affect the most commonly used RGs. We analysed 15 candidate RGs in 98 brain samples from two brain regions from Alzheimer’s disease (AD), Parkinson’s disease (PD), Multiple System Atrophy, and Progressive Supranuclear Palsy patients. Using RefFinder, a web-based tool for evaluating RG stability, we identified the most stable RGs to be UBE2D2, CYC1, and RPL13 which we recommend for future RT-qPCR studies on human brain tissue from these patients. None of the investigated genes were affected by experimental variables such as RIN, PMI, or age. Findings were further validated by expression analyses of a target gene GSK3B, known to be affected by AD and PD. We obtained high variations in GSK3B levels when contrasting the results using different sets of common RG underlining the importance of a priori validation of RGs for RT-qPCR studies

Transcriptome analysis of the adult human Klinefelter testis and cellularity-matched controls reveals disturbed differentiation of Sertoli- and Leydig cells article

AbstractThe most common human sex chromosomal disorder is Klinefelter syndrome (KS; 47,XXY). Adult patients with KS display a diverse phenotype but are nearly always infertile, due to testicular degeneration at puberty. To identify mechanisms causing the selective destruction of the seminiferous epithelium, we performed RNA-sequencing of 24 fixed paraffin-embedded testicular tissue samples. Analysis of informative transcriptomes revealed 235 differentially expressed transcripts (DETs) in the adult KS testis showing enrichment of long non-coding RNAs, but surprisingly not of X-chromosomal transcripts. Comparison to 46,XY samples with complete spermatogenesis and Sertoli cell-only-syndrome allowed prediction of the cellular origin of 71 of the DETs. DACH2 and FAM9A were validated by immunohistochemistry and found to mark apparently undifferentiated somatic cell populations in the KS testes. Moreover, transcriptomes from fetal, pre-pubertal, and adult KS testes showed a limited overlap, indicating that different mechanisms are likely to operate at each developmental stage. Based on our data, we propose that testicular degeneration in men with KS is a consequence of germ cells loss initiated during early development in combination with disturbed maturation of Sertoli- and Leydig cells.</jats:p

Slackline Training in Children with Spastic Cerebral Palsy: A Randomized Clinical Trial

[EN] Objective: To assess whether a slackline intervention program improves postural control in children/adolescents with spastic cerebral palsy (CP). Design: Randomized controlled trial. Setting: Patients’ association. Participants: Twenty-seven children/adolescents with spastic CP (9–16 years) were randomly assigned to a slackline intervention (n = 14, 13 ± 3 years) or control group (n = 13, 12 ± 2 years ). Intervention: Three slackline sessions per week (30 min/session) for 6 weeks. Main outcome measures: The primary outcome was static posturography (center of pressure—CoP—parameters). The secondary outcomes were surface myoelectrical activity of the lower-limb muscles during the posturography test and jump performance (countermovement jump test and Abalakov test). Overall (RPE, >6–20 scale) rating of perceived exertion was recorded at the end of each intervention session. Results: The intervention was perceived as “very light” (RPE = 7.6 ± 0.6). The intervention yielded significant benefits on static posturography (a significant group by time interaction on Xspeed, p = 0.006) and jump performance (a significant group by time interaction on Abalakov test, p = 0.015). Conclusions: Slackline training improved static postural control and motor skills and was perceived as non-fatiguing in children/adolescents with spastic CP.S

The molecular evolution of spermatogenesis across mammals

The testis produces gametes through spermatogenesis and evolves rapidly at both the morphological and molecular level in mammals1-6, probably owing to the evolutionary pressure on males to be reproductively successful7. However, the molecular evolution of individual spermatogenic cell types across mammals remains largely uncharacterized. Here we report evolutionary analyses of single-nucleus transcriptome data for testes from 11 species that cover the three main mammalian lineages (eutherians, marsupials and monotremes) and birds (the evolutionary outgroup), and include seven primates. We find that the rapid evolution of the testis was driven by accelerated fixation rates of gene expression changes, amino acid substitutions and new genes in late spermatogenic stages, probably facilitated by reduced pleiotropic constraints, haploid selection and transcriptionally permissive chromatin. We identify temporal expression changes of individual genes across species and conserved expression programs controlling ancestral spermatogenic processes. Genes predominantly expressed in spermatogonia (germ cells fuelling spermatogenesis) and Sertoli (somatic support) cells accumulated on X chromosomes during evolution, presumably owing to male-beneficial selective forces. Further work identified transcriptomal differences between X- and Y-bearing spermatids and uncovered that meiotic sex-chromosome inactivation (MSCI) also occurs in monotremes and hence is common to mammalian sex-chromosome systems. Thus, the mechanism of meiotic silencing of unsynapsed chromatin, which underlies MSCI, is an ancestral mammalian feature. Our study illuminates the molecular evolution of spermatogenesis and associated selective forces, and provides a resource for investigating the biology of the testis across mammals