(CCUG)n RNA toxicity in a Drosophila model of myotonic dystrophy type 2 (DM2) activates apoptosis

The myotonic dystrophies are prototypic toxic RNA gain-of-function diseases. Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are caused by different unstable, noncoding microsatellite repeat expansions - (CTG)DM1 in DMPK and (CCTG)DM2 in CNBP Although transcription of mutant repeats into (CUG)DM1 or (CCUG)DM2 appears to be necessary and sufficient to cause disease, their pathomechanisms remain incompletely understood. To study the mechanisms of (CCUG)DM2 toxicity and develop a convenient model for drug screening, we generated a transgenic DM2 model in the fruit fly Drosophila melanogaster with (CCUG)n repeats of variable length (n=16 and 106). Expression of noncoding (CCUG)106, but not (CCUG)16, in muscle and retinal cells led to the formation of ribonuclear foci and mis-splicing of genes implicated in DM pathology. Mis-splicing could be rescued by co-expression of human MBNL1, but not by CUGBP1 (CELF1) complementation. Flies with (CCUG)106 displayed strong disruption of external eye morphology and of the underlying retina. Furthermore, expression of (CCUG)106 in developing retinae caused a strong apoptotic response. Inhibition of apoptosis rescued the retinal disruption in (CCUG)106 flies. Finally, we tested two chemical compounds that have shown therapeutic potential in DM1 models. Whereas treatment of (CCUG)106 flies with pentamidine had no effect, treatment with a PKR inhibitor blocked both the formation of RNA foci and apoptosis in retinae of (CCUG)106 flies. Our data indicate that expression of expanded (CCUG)DM2 repeats is toxic, causing inappropriate cell death in affected fly eyes. Our Drosophila DM2 model might provide a convenient tool for in vivo drug screening

Absence of MutSβ leads to the formation of slipped-DNA for CTG/CAG contractions at primate replication forks

Typically disease-causing CAG/CTG repeats expand, but rare affected families can display high levels of contraction of the expanded repeat amongst offspring. Understanding instability is important since arresting expansions or enhancing contractions could be clinically beneficial. The MutSβ mismatch repair complex is required for CAG/CTG expansions in mice and patients. Oddly, by unknown mechanisms MutSβ-deficient mice incur contractions instead of expansions. Replication using CTG or CAG as the lagging strand template is known to cause contractions or expansions respectively; however, the interplay between replication and repair leading to this instability remains unclear. Towards understanding how repeat contractions may arise, we performed in vitro SV40-mediated replication of repeat-containing plasmids in the presence or absence of mismatch repair. Specifically, we separated repair from replication: Replication mediated by MutSβ- and MutSα-deficient human cells or cell extracts produced slipped-DNA heteroduplexes in the contraction- but not expansion-biased replication direction. Replication in the presence of MutSβ disfavoured the retention of replication products harbouring slipped-DNA heteroduplexes. Post-replication repair of slipped-DNAs by MutSβ-proficient extracts eliminated slipped-DNAs. Thus, a MutSβ-deficiency likely enhances repeat contractions because MutSβ protects against contractions by repairing template strand slip-outs. Replication deficient in LigaseI or PCNA-interaction mutant LigaseI revealed slipped-DNA formation at lagging strands. Our results reveal that distinct mechanisms lead to expansions or contractions and support inhibition of MutSβ as a therapeutic strategy to enhance the contraction of expanded repeats

Six Serum miRNAs Fail to Validate as Myotonic Dystrophy Type 1 Biomarkers

<div><p>Myotonic dystrophy type 1 (DM1) is an autosomal dominant genetic disease caused by expansion of a CTG microsatellite in the 3’ untranslated region of the <i>DMPK</i> gene. Despite characteristic muscular, cardiac, and neuropsychological symptoms, CTG trinucleotide repeats are unstable both in the somatic and germinal lines, making the age of onset, clinical presentation, and disease severity very variable. A molecular biomarker to stratify patients and to follow disease progression is, thus, an unmet medical need. Looking for a novel biomarker, and given that specific miRNAs have been found to be misregulated in DM1 heart and muscle tissues, we profiled the expression of 175 known serum miRNAs in DM1 samples. The differences detected between patients and controls were less than 2.6 fold for all of them and a selection of six candidate miRNAs, <i>miR-103</i>, <i>miR-107</i>, <i>miR-21</i>, <i>miR-29a</i>, <i>miR-30c</i>, and <i>miR-652</i> all failed to show consistent differences in serum expression in subsequent validation experiments.</p></div

Validation by q-PCR did not reveal differences in miRNA expression levels between controls and myotonic dystrophy type 1 patients.

<p>(A, B) Graphical representation of the results generated by two algorithms, geNorm and NormFinder, to identify the optimal normalisation miRNA from among all of the candidates (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150501#pone.0150501.s005" target="_blank">S4 Table</a>). (C) Analysis of the relative expression levels of <i>miR-103</i>, <i>miR-107</i>, <i>miR-21</i>, <i>miR-29a</i>, <i>miR-30c</i>, and <i>miR-652</i> by quantitative PCR on the serum samples of nine DM1 patients and nine healthy individuals. All data were normalised to <i>miR-15</i> expression levels but no significant differences were observed between either group. Graph bars represent average fold-changes of miRNA expression on a logarithmic scale, calculated using the 2^-∆∆Ct method, as well as their confidence intervals. Graph bars represent average fold changes of miRNA expression, calculated using the 2^-∆∆Ct method, along with their standard error.</p

Profiling of miRNA expression levels in myotonic dystrophy type 1 patients and controls.

<p>(A) Heat map graphical representation and clustering analysis of miRNA expression from 9 DM1 patients (P01-P10, excluding P03) and 9 healthy controls (C11-C20 excluding C18). Blue and yellow indicate statistically significant down- and upregulated miRNAs compared to controls, respectively (t-test α  =  0.05). Data is presented as a dendrogram, with the closest branches of the tree showing samples with less dissimilar expression patterns. (B) Statistical analysis of the miRNA profiling carried out with the G*Power tool. These miRNAs have the highest fold-change and Power∼1 statistics in the sample pool. (C) Graphical representation of the expression levels of the miRNAs selected via G*Power analysis. Only <i>miR-21</i> showed a statistically-significant difference when Bonferroni correction was applied. Graph bars represent average fold changes and their standard errors. <i>P</i> > 0.05.</p

Information about the samples used in the miRNA profiling.

<p>Information about the samples used in the miRNA profiling.</p

Information about the samples used in qPCR.

<p>Information about the samples used in qPCR.</p

The ratio of <i>miR-130a</i> and <i>miR-21</i> failed as a myotonic dystrophy type 1 biomarker.

<p>(A) The ratio of <i>miR-130a</i> and <i>miR-21</i> according to expression levels obtained from the profiling performed with serum samples from nine DM1 patients and nine healthy controls. (B) The same ratio was calculated after measuring <i>miR-130a</i> and <i>miR-21</i> expression levels by quantitative PCR on serum samples from 21 DM1 and 17 control individuals. No statistically-significant differences were observed. Graph bars represent the average ∆Cts (<i>miR-130a</i>--<i>miR-2</i>1) and their standard errors.</p

Contribuições da Sociologia na América Latina à imaginação sociológica: análise, crítica e compromisso social Sociology's contribution in Latin America to sociological imagination: analysis, critique, and social commitment

O artigo aborda o papel desempenhado pela Sociologia na análise dos processos de transformação das sociedades latino-americanas, no acompanhamento do processo de construção do Estado e da Nação, na problematização das questões sociais na América Latina. São analisados seis períodos na Sociologia na América Latina e no Caribe: I) a herança intelectual da Sociologia ; II) a sociologia da cátedra; III) O período da "Sociologia Científica" e a configuração da "Sociologia Crítica"; IV) a crise institucional, a consolidação da "Sociologia Crítica" e a diversificação da sociologia; V) a sociologia do autoritarismo, da democracia e da exclusão; VI) a consolidação institucional e a mundialização da sociologia da América Latina (desde o ano de 2000), podendo-se afirmar que os traços distintivos do saber sociológico no continente foram: o internacionalismo, o hibridismo, a abordagem crítica dos processos e conflitos das sociedades latino-americanas e o compromisso social do sociólogo.<br>The article focuses on the role played by Sociology in the analysis of processes of change in Latin American societies, in the process of construction of Nation and State, in the debate of social issues in Latin America and the Caribbean. Six periods in Sociology in Latin America and the Caribbean are examined: I) sociology's intellectual legacy; II) sociology as a cathedra; III) the period of "Scientific Sociology"; IV) the institutional crisis, the consolidation of "Critical Sociology", and the diversifying of sociology; V) sociology of authoritarianism, democracy and exclusion; VI) institutional consolidation and globalization of Latin American sociology (since 2000). It may be said that the distinctive features of sociological knowledge in the continent were: internationalism, hybridism, the critical approach to processes and conflicts of Latin American societies, and the sociologist social commitment