Effect of testosterone metabolites on ABC half-transporter relative gene expression in X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is an inherited neurodegenerative disorder associated with reduced very long-chain fatty acid beta-oxidation, mainly affecting the nervous system, the adrenal cortex and the testes. The clinical manifestations of hypogonadism, alopecia and the impairment of the enzyme 5alpha-reductase, which converts testosterone into dihydrotestosterone, clearly point to an involvement of androgens in this pathology. The disease is characterized by mutations in the ABCD1 gene, which codes for the peroxisomal ABC half-transporter ALDP, and by a broad range of clinical manifestations. The altered function of ALDP can be compensated by the overexpression of proteins belonging to the same family of ABC half-transporters. A promising therapeutic approach is represented by the activation of these proteins by specific agonists. In this study we evaluated the effect of the testosterone metabolite dihydrotestosterone (DHT) and 5alpha-androstan-3alpha,17beta-diol (3alpha-diol) on the expression of the ABC half-transporters encoded by the ABCD2 and ABCD3 genes, in fibroblasts drawn from controls and from two affected brothers. The two patients presented the same mutation in exon 9 but had different clinical manifestations, one patient being asymptomatic and the second one severely affected. When the cells were stimulated with testosterone metabolites, only the severely affected patient showed a significant increase in ABCD2 mRNA levels, while the ABCD3 expression remained unchanged in both patients

Ribonucleoprotein Assembly Defects Correlate with Spinal Muscular Atrophy Severity and Preferentially Affect a Subset of Spliceosomal snRNPs

Spinal muscular atrophy (SMA) is a motor neuron disease caused by reduced levels of the survival motor neuron (SMN) protein. SMN together with Gemins2-8 and unrip proteins form a macromolecular complex that functions in the assembly of small nuclear ribonucleoproteins (snRNPs) of both the major and the minor splicing pathways. It is not known whether the levels of spliceosomal snRNPs are decreased in SMA. Here we analyzed the consequence of SMN deficiency on snRNP metabolism in the spinal cord of mouse models of SMA with differing phenotypic severities. We demonstrate that the expression of a subset of Gemin proteins and snRNP assembly activity are dramatically reduced in the spinal cord of severe SMA mice. Comparative analysis of different tissues highlights a similar decrease in SMN levels and a strong impairment of snRNP assembly in tissues of severe SMA mice, although the defect appears smaller in kidney than in neural tissue. We further show that the extent of reduction in both Gemin proteins expression and snRNP assembly activity in the spinal cord of SMA mice correlates with disease severity. Remarkably, defective SMN complex function in snRNP assembly causes a significant decrease in the levels of a subset of snRNPs and preferentially affects the accumulation of U11 snRNP—a component of the minor spliceosome—in tissues of severe SMA mice. Thus, impairment of a ubiquitous function of SMN changes the snRNP profile of SMA tissues by unevenly altering the normal proportion of endogenous snRNPs. These findings are consistent with the hypothesis that SMN deficiency affects the splicing machinery and in particular the minor splicing pathway of a rare class of introns in SMA

Expression profile of microRNAs in young stroke patients

10.1371/journal.pone.0007689PLoS ONE41

In Vitro Identification and Characterization of CD133pos Cancer Stem-Like Cells in Anaplastic Thyroid Carcinoma Cell Lines

Background: Recent publications suggest that neoplastic initiation and growth are dependent on a small subset of cells, termed cancer stem cells (CSCs). Anaplastic Thyroid Carcinoma (ATC) is a very aggressive solid tumor with poor prognosis, characterized by high dedifferentiation. The existence of CSCs might account for the heterogeneity of ATC lesions. CD133 has been identified as a stem cell marker for normal and cancerous tissues, although its biological function remains unknown. Methodology/Principal Findings: ATC cell lines ARO, KAT-4, KAT-18 and FRO were analyzed for CD133 expression. Flow cytometry showed CD133pos cells only in ARO and KAT-4 (6469% and 57612%, respectively). These data were confirmed by qRT-PCR and immunocytochemistry. ARO and KAT-4 were also positive for fetal marker oncofetal fibronectin and negative for thyrocyte-specific differentiating markers thyroglobulin, thyroperoxidase and sodium/iodide symporter. Sorted ARO/ CD133pos cells exhibited higher proliferation, self-renewal, colony-forming ability in comparison with ARO/CD133neg. Furthermore, ARO/CD133pos showed levels of thyroid transcription factor TTF-1 similar to the fetal thyroid cell line TAD-2, while the expression in ARO/CD133neg was negligible. The expression of the stem cell marker OCT-4 detected by RT-PCR and flow cytometry was markedly higher in ARO/CD133pos in comparison to ARO/CD133neg cells. The stem cell markers c- KIT and THY-1 were negative. Sensitivity to chemotherapy agents was investigated, showing remarkable resistance to chemotherapy-induced apoptosis in ARO/CD133pos when compared with ARO/CD133neg cells. Conclusions/Significance: We describe CD133pos cells in ATC cell lines. ARO/CD133pos cells exhibit stem cell-like features - such as high proliferation, self-renewal ability, expression of OCT-4 - and are characterized by higher resistance to chemotherapy. The simultaneous positivity for thyroid specific factor TTF-1 and onfFN suggest they might represent putative thyroid cancer stem-like cells. Our in vitro findings might provide new insights for novel therapeutic approaches

Comparative Expression Profile of miRNA and mRNA in Primary Peripheral Blood Mononuclear Cells Infected with Human Immunodeficiency Virus (HIV-1)

Host cells respond to exogenous infectious agents such as viruses, including HIV-1. Studies have evaluated the changes associated with virus infection at the transcriptional and translational levels of the cellular genes involved in specific pathways. While this approach is useful, in our view it provides only a partial view of genome-wide changes. Recently, technological advances in the expression profiling at the microRNA (miRNA) and mRNA levels have made it possible to evaluate the changes in the components of multiple pathways. To understand the role of miRNA and its interplay with host cellular gene expression (mRNA) during HIV-1 infection, we performed a comparative global miRNA and mRNA microarray using human PBMCs infected with HIV-1. The PBMCs were derived from multiple donors and were infected with virus generated from the molecular clone pNL4-3. The results showed that HIV-1 infection led to altered regulation of 21 miRNAs and 444 mRNA more than 2-fold, with a statistical significance of p<0.05. Furthermore, the differentially regulated miRNA and mRNA were shown to be associated with host cellular pathways involved in cell cycle/proliferation, apoptosis, T-cell signaling, and immune activation. We also observed a number of inverse correlations of miRNA and mRNA expression in infected PBMCs, further confirming the interrelationship between miRNA and mRNA regulation during HIV-1 infection. These results for the first time provide evidence that the miRNA profile could be an early indicator of host cellular dysfunction induced by HIV-1

Emergency hernia repair in the elderly. multivariate analysis of morbidity and mortality from an Italian registry

Purpose The incidence of inguinal hernia is higher in elderly because of aging-related diseases like prostatism, bronchitis, collagen laxity. A conservative management is common in elderly to reduce surgery-related risks, however watchful waiting can expose to obstruction and strangulation. The aim of the present study was to assess the impact of emergency surgery in a large series of elderly with complicated groin hernia and to identify the independent risk factors for postoperative morbidity and mortality. The predictive performance of prognostic risk scores has been also assessed. Methods This is a prospective observational study carried out between January 2017 and June 2018 in elderly patients who underwent emergency surgery for complicated hernia in 38 Italian hospitals. Pre-operative, surgical and postoperative data were recorded for each patient. ASA score, Charlson's comorbidity index, P-POSSUM and CR-POSSUM were assessed. Results 259 patients were recruited, mean age was 80 years. A direct repair without mesh was performed in 62 (23.9%) patients. Explorative laparotomy was performed in 56 (21.6%) patients and bowel resection was necessary in 44 (17%). Mortality occurred in seven (2.8%) patients. Fifty-five (21.2%) patients developed complications, 12 of whom had a major one. At univariate and multivariate analyses, Charlson's comorbidity index &gt;= 6, altered mental status, and need for laparotomy were associated with major complications and mortality Conclusion Emergency surgery for complicated hernia is burdened by high morbidity and mortality in elderly patients. Preoperative comorbidity played a pivotal role in predicting complications and mortality and therefore Charlson's comorbidity index could be adopted to select patients for elective operation