I MICRORNA NELLE DISTROFIE MIOTONICHE

Myotonic dystrophy (DM), the most common form of muscular dystrophy in adults, is a dominantly inherited disorder with a peculiar and rare pattern of multisystemic clinical features affecting skeletal muscle, the heart, the eye, and the endocrine system. Classical DM (first described by Steinert and called Steinert\u2019s disease or DM1) has been identified as an autosomal dominant disorder associated with the presence of an abnormal expansion of a CTG trinucleotide repeat in the 3\u2019 untranslated region of the dystrophia myotonica-protein kinase (DMPK) gene on chromosome 19q13.3. Recently, the expansion of a CCTG tetranucleotide repeat located in the intron of the zinc finger 9 (ZNF9) gene on chromosome 19q13.3 was identified as the mutation responsible for DM2. Both mutations lead to the production of mRNA transcripts containing expanded tri- or tetranucleotide repeats (CUG/CCUG) that are retained in muscle nuclei as ribonuclear inclusions and interact with RNA-binding proteins. These interaction are supposed to disrupt the regulation of alternative splicing of several transcripts. Clinical and molecular parallels strongly support that DM1 and DM2 physiopathology is in part the pathogenic consequence of an RNA gain of toxic function. MicroRNAs (miRNAs) are short non-coding RNAs (~22 nucleotides) regulating gene expression post-trascriptionally either via the degradation of target mRNAs or the inhibition of protein translation. MicroRNAs have been shown to be involved in a range of biological processes, including myogenesis and muscle regeneration. miRNAs are expressed in cardiac and skeletal muscle, and dysregulated miRNA expression has been correlated with muscle-related diseases, including cardiac hypertrophy, cardiac arrhythmias and muscular dystrophy. Given the emerging roles of microRNAs, we have performed miRNAs expression profiling in DM1 and DM2 patients on muscle biopsies and primary cell culture line. Using fast real time PCR, we report here the differences in miRNAs expression profiles between DM1 (n=15), DM2 (n=9) and control subjects (n=14) of 24 specific miRNAs. miRNAs expression profiles in muscle biopsies of DM1 showed up-regulation of miR-1 and miR-335 and down-regulation of miR-29b, miR-29c, miR-33, establishing a provisional DM1 miRNA signature. A similar trend in miRNA modulation was observed in DM2 patients. However, none of the differences reached statistical significance. In order to assess whether DM1 signature miRNA deregulations and DM2 were cell autonomous events, primary cultures of skeletal muscle satellite cells obtained from either DM1 patients (n=5), DM2 patients (n=5) or controls (n=5) were examined. Myoblasts were cultured in growth factor rich medium and then switched to differentiation medium for five days. DM1 and DM2 myoblasts did not display overt morphological alterations of differentiation. When DM1 miRNA signature was examined, we found that miR-29b was strongly down-modulated in differentiated DM1 myotubes. Conversely, miR-335 was enhanced in DM1 myoblasts in growth medium whereas, upon switching to differentiation medium, it increased to a similar level both in DM1 and control myoblasts. When DM2 myoblasts and myotubes was examined, we not found significance statistical differences in miRNAs expression compared with control myoblasts and myotubes. Furthermore, The cellular localization of DM1 signature miRNAs was assayed by in situ hybridization on cryostat muscle sections derived from DM1 (n=5) and control (n=5) biopsies using digoxigenin labelled LNA probes. We found that miR-29b, -29c, -33 and -335 were either barely detectable or did not show any overt abnormal localization in DM1 compared to control biopsies. Conversely, miR-1 was readily detectable and its intracellular distribution was disrupted. Specifically, in control samples, miR-1 displayed a peculiar enrichment in the perinuclear area. In DM1 sections, centrally nucleated myofibers, a hallmark of DM1, also exhibited a centralization of miR-1 localization. Very small fibers with nuclear clumps, a typical histopathological DM1 alteration, displayed intense miR-1 staining. Certain myofibers displayed an extremely intense and polarized miR-1 accumulation. Atrophic fibers in DM1 muscle are predominantly type I fibers (slow fibers). Aberrant miR-1 distribution was present both in type I and type II myofibers, as assessed by the myosin heavy chain slow isoform counterstaining. We also tested the cellular localization of two more muscle specific microRNAs, miR-133b and -206, albeit no overt deregulation of their expression was found in whole skeletal muscle RNAs. In control biopsies we found that miR-133b displayed a perinuclear distribution similar to that of miR-1; in keeping with previous findings, miR-206 was barely detectable. In DM1 patients, both miR-133b and -206 exhibited centralization in centrally nucleated myofibers and accumulated in association to small myofibers nuclear clumps. Finally, miRNAs have been shown not only to inhibit protein translation, but also to induce mRNA degradation, at least for certain targets. Thus, in order to assess whether miRNA deregulation was functionally relevant, we examined the impact of the identified miRNAs deregulation on the expression of their potential target genes in DM1 patients. Specifically, we focused on miR-29, that displayed the strongest deregulation, and miR-1, that plays a crucial role in muscle differentiation. Search of the potential targets was performed using Pictar and Targetscan prediction algorithms, given their reported specificity. Indeed, to maximize the accuracy, only targets identified by both softwares were considered. A sub-pool of the identified targets were analyzed, selected among these with a potential link to DM1 physio-pathology. Specifically, selected genes were previously demonstrated to be expressed in skeletal muscle and to be involved in events such as muscle development, atrophy, arrhythmia and splicing. Potential targets were assayed by qPCR and shows that both miR-29 and miR-1 targets were significantly up-regulated in DM1 patients. In conclusion, we identified a small subset of miRNA whose expression and/or localization were deregulated in DM. These findings may improve our understanding of the molecular mechanisms linking (CTG)n/(CCTG)n expansion to disease and may serve as potential prognostic/diagnostic markers

A quantitative study on the growth variability of tumour cell clones in vitro

Objectives: In this study, we quantify the growth variability of tumour cell clones from a human leukemia cell line. Materials and methods: We have used microplate spectrophotometry to measure the growth kinetics of hundreds of individual cell clones from the Molt3 cell line. The growth rate of each clonal population has been estimated by fitting experimental data with the logistic equation. Results: The growth rates were observed to vary among different clones. Up to six clones with a growth rate above or below the mean growth rate of the parent population were further cloned and the growth rates of their offsprings were measured. The distribution of the growth rates of the subclones did not significantly differ from that of the parent population thus suggesting that growth variability has an epigenetic origin. To explain the observed distributions of clonal growth rates we have developed a probabilistic model assuming that the fluctuations in the number of mitochondria through successive cell cycles are the leading cause of growth variability. For fitting purposes, we have estimated experimentally by flow cytometry the maximum average number of mitochondria in Molt3 cells. The model fits nicely the observed distributions of growth rates, however, cells in which the mitochondria were rendered non functional (rho-0 cells) showed only a 30% reduction in the clonal growth variability with respect to normal cells. Conclusions: A tumor cell population is a dynamic ensemble of clones with highly variable growth rate. At least part of this variability is due to fluctuations in the number of mitochondria.Comment: 31 pages, 5 figure

Usporedba dinamičke analize para iznad otopine i mikroekstrakcije analita na čvrstoj fazi za plinskokromatografsko određivanje BTEX-a u urinu

The aim of this study was to compare two extraction procedures: dynamic headspace-purge and trap (PT) and headspace solid-phase microextraction (HS-SPME) for gas chromatographic determination of benzene, toluene, ethylbenzene, and isomeric xylenes (BTEX) in urine with photoionization (PID) and mass spectrometric (MS) detection, respectively. Both methods showed linearity in the range of interest [(50-2000) ng L-1], good accuracy (80% to 100 %), and repeatability (RSD≤11 %). Detection limits were in the low ng L-1 level for both methods, although slightly greater sensitivity was found for the PT method. In comparison with PT, HS-SPME was simpler and required less time for analysis. Although the analytical features of both examined methods are appropriate for biomonitoring of environmental exposure to BTEX, only the HS-SPME-GC-MS method is recommended for routine analysis of BTEX in urine. The method was applied for the quantitative analysis of BTEX in urine samples collected from non-smokers (n=10) and smokers (n=10).Cilj ovog rada bio je usporediti dva postupka ekstrakcije za plinskokromatografsko određivanje benzena, toluena, etilbenzena i izomera ksilena u urinu. Uspoređene su dinamička analiza para iznad otopine (tzv. purge and trap) uz fotoionizacijski detektor i mikroekstrakcija analita na čvrstoj fazi uz detektor spektrometar masa. Rezultati upućuju na linearnost odziva detektora u ispitivanome koncentracijskom području [(50- 2000) ng L-1], zadovoljavajuću točnost (80 %-100 %) i ponovljivost (RSD ≤11 %). Postignute su niske granice detekcije za obje metode. Mikroekstrakcija analita na čvrstoj fazi uz detektor spektrometar masa pokazala se jednostavnijom i bržom za izvođenje pa se preporučuje za rutinsko određivanje BTEX-a u urinu. Metoda je primijenjena za analizu tih spojeva u uzorcima urina nepušača (n=10) i pušača (n=10)

Physiological Biomimetic Culture System for Pig and Human Heart Slices

RATIONALE: Preclinical testing of cardiotoxicity and efficacy of novel heart failure therapies faces a major limitation: the lack of an in situ culture system that emulates the complexity of human heart tissue and maintains viability and functionality for a prolonged time. OBJECTIVE: To develop a reliable, easily reproducible, medium-throughput method to culture pig and human heart slices under physiological conditions for a prolonged period of time. METHODS AND RESULTS: Here, we describe a novel, medium-throughput biomimetic culture system that maintains viability and functionality of human and pig heart slices (300 µm thickness) for 6 days in culture. We optimized the medium and culture conditions with continuous electrical stimulation at 1.2 Hz and oxygenation of the medium. Functional viability of these slices over 6 days was confirmed by assessing their calcium homeostasis, twitch force generation, and response to β-adrenergic stimulation. Temporal transcriptome analysis using RNAseq at day 2, 6, and 10 in culture confirmed overall maintenance of normal gene expression for up to 6 days, while over 500 transcripts were differentially regulated after 10 days. Electron microscopy demonstrated intact mitochondria and Z-disc ultra-structures after 6 days in culture under our optimized conditions. This biomimetic culture system was successful in keeping human heart slices completely viable and functionally and structurally intact for 6 days in culture. We also used this system to demonstrate the effects of a novel gene therapy approach in human heart slices. Furthermore, this culture system enabled the assessment of contraction and relaxation kinetics on isolated single myofibrils from heart slices after culture. CONCLUSIONS: We have developed and optimized a reliable medium-throughput culture system for pig and human heart slices as a platform for testing the efficacy of novel heart failure therapeutics and reliable testing of cardiotoxicity in a 3D heart model

An Interleukin 13 polymorphism is associated with symptom severity in adult subjects with ever asthma

Different genes are associated with categorical classifications of asthma severity. However, continuous outcomes should be used to catch the heterogeneity of asthma phenotypes and to increase the power in association studies. Accordingly, the aim of this study was to evaluate the association between single nucleotide polymorphisms (SNPs) in candidate gene regions and continuous measures of asthma severity, in adult patients from the general population. In the Gene Environment Interactions in Respiratory Diseases (GEIRD) study (www.geird.org), 326 subjects (aged 20-64) with ever asthma were identified from the general population in Verona (Italy) between 2007 and 2010. A panel of 236 SNPs tagging 51 candidate gene regions (including one or more genes) was analysed. A symptom and treatment score (STS) and pre-bronchodilator FEV1% predicted were used as continuous measures of asthma severity. The association of each SNP with STS and FEV1% predicted was tested by fitting quasi-gamma and linear regression models, respectively, with gender, body mass index and smoking habits as potential confounders. The Simes multiple-test procedure was used for controlling the false discovery rate (FDR). SNP rs848 in the IL13 gene region (IL5/RAD50/IL13/IL4) was associated with STS (TG/GG vs TT genotype: uncorrected p-value = 0.00006, FDR-corrected p-value = 0.04), whereas rs20541 in the same gene region, in linkage disequilibrium with rs848 (r2 = 0.94) in our sample, did not reach the statistical significance after adjusting for multiple testing (TC/CC vs TT: uncorrected p-value = 0.0003, FDR-corrected p-value = 0.09). Polymorphisms in other gene regions showed a non-significant moderate association with STS (IL12B, TNS1) or lung function (SERPINE2, GATA3, IL5, NPNT, FAM13A) only. After adjusting for multiple testing and potential confounders, SNP rs848 in the IL13 gene region is significantly associated with a continuous measure of symptom severity in adult subjects with ever asthma

Deregulated MicroRNAs in Myotonic Dystrophy Type 2

Myotonic Dystrophy Type-2 (DM2) is an autosomal dominant disease caused by the expansion of a CCTG tetraplet repeat. It is a multisystemic disorder, affecting skeletal muscles, the heart, the eye, the central nervous system and the endocrine system. Since microRNA (miRNA) expression is disrupted in Myotonic Dystrophy Type-1 and many other myopathies, miRNAs deregulation was studied in skeletal muscle biopsies of 13 DM2 patients and 13 controls. Eleven miRNAs were deregulated: 9 displayed higher levels compared to controls (miR-34a-5p, miR-34b-3p, miR-34c-5p, miR-146b-5p, miR-208a, miR-221-3p and miR-381), while 4 were decreased (miR-125b-5p, miR-193a-3p, miR-193b-3p and miR-378a-3p). To explore the relevance of DM2 miRNA deregulation, the predicted interactions between miRNA and mRNA were investigated. Global gene expression was analyzed in DM2 and controls and bioinformatic analysis identified more than 1,000 miRNA/mRNA interactions. Pathway and function analysis highlighted the involvement of the miRNA-deregulated mRNAs in multiple aspects of DM2 pathophysiology. In conclusion, the observed miRNA dysregulations may contribute to DM2 pathogenetic mechanisms

Isoform Diversity and Regulation in Peripheral and Central Neurons Revealed through RNA-Seq

To fully understand cell type identity and function in the nervous system there is a need to understand neuronal gene expression at the level of isoform diversity. Here we applied Next Generation Sequencing of the transcriptome (RNA-Seq) to purified sensory neurons and cerebellar granular neurons (CGNs) grown on an axonal growth permissive substrate. The goal of the analysis was to uncover neuronal type specific isoforms as a prelude to understanding patterns of gene expression underlying their intrinsic growth abilities. Global gene expression patterns were comparable to those found for other cell types, in that a vast majority of genes were expressed at low abundance. Nearly 18% of gene loci produced more than one transcript. More than 8000 isoforms were differentially expressed, either to different degrees in different neuronal types or uniquely expressed in one or the other. Sensory neurons expressed a larger number of genes and gene isoforms than did CGNs. To begin to understand the mechanisms responsible for the differential gene/isoform expression we identified transcription factor binding sites present specifically in the upstream genomic sequences of differentially expressed isoforms, and analyzed the 3′ untranslated regions (3′ UTRs) for microRNA (miRNA) target sites. Our analysis defines isoform diversity for two neuronal types with diverse axon growth capabilities and begins to elucidate the complex transcriptional landscape in two neuronal populations