Wide-Ranging Analysis of MicroRNA Profiles in Sporadic Amyotrophic Lateral Sclerosis Using Next-Generation Sequencing.

MicroRNA (miRNA) has emerged as an important regulator of gene expression in neurodegenerative disease as amyotrophic lateral sclerosis (ALS). In the nervous system, dysregulation in miRNA-related pathways is subordinated to neuronal damage and cell death, which contributes to the expansion of neurodegenerative disorders, such as ALS. In the present research, we aimed to profile dysregulation of miRNAs in ALS blood and neuromuscular junction as well as healthy blood control by next-generation sequencing (NGS). The expression of three upregulated miRNAs, as miR-338-3p, miR-223-3p, and miR-326, in the ALS samples compared to healthy controls, has been validated by qRT-PCR in a cohort of 45 samples collected previously. Bioinformatics tools were used to perform ALS miRNAs target analysis and to predict novel miRNAs secondary structure. The analysis of the NGS data identified 696 and 49 novel miRNAs which were differentially expressed in ALS tissues. In particular, in neuromuscular junction the differential expression of miR-338-3p, which we previously found upregulated in different types of ASL tissues, miR-223-3p, and miR-326 was elevated compared to normal control. ALS miRNAs gene target were significantly involved in neuronal related pathway as BDFN1 and HIF-1genes. This study presents the direct experimental evidence that, overall, miR-338-3p is highly expressed in ALS tissues including neuromuscular junction characterizing ALS from normal tissues. Beside, our analysis identified, for the first time, novel miRNAs highly expressed in ALS tissues. In conclusion, the results indicate that miRNAs has an important role in the diagnosis and treatment of ALS.

Rapid methods to create a positive control and identify the PAX8/PPARγ rearrangement in FNA thyroid samples by molecular biology.

Thyroid cancer is the most common malignancy of the endocrine system and includes well-differentiated forms, namely papillary and follicular carcinomas, and the poorly differentiated and undifferentiated forms that result from the transformation of thyroid follicular cells (anaplastic carcinomas). Notably, 5-10% of all thyroid cancers are medullary thyroid cancers that arise from parafollicular cells also known as C cells. The most common genetic mutations in papillary and follicular thyroid cancers are point mutations of the BRAF or RAS genes, while the most common chromosomal alterations are RET/PTC and PAX8/PPARγ rearrangements. The most frequent initial manifestation of thyroid cancer is the appearance of a nodule most of which are benign; indeed, less than 5% are malignant. However, some cases are misdiagnosed, and many patients undergo unnecessary surgery. Therefore, an accurate pre-surgery evaluation is crucial. The most reliable diagnostic test for thyroid nodules is fine needle aspiration (FNA) cytology, which accurately distinguishes between a benign and malignant lesion in most cases. However, cytological discrimination between malignant and benign follicular cancer is often difficult because of poor quality samples. Here we describe rapid methods to create a positive control and identify the PAX8/PPARγ rearrangement in FNA thyroid samples by molecular biology.

Molecular phylogeny of Oncaeidae (Copepoda) using nuclear ribosomal internal transcribed spacer (ITS rDNA).

Copepods belonging to the Oncaeidae family are commonly and abundantly found in marine zooplankton. In the Mediterranean Sea, forty-seven oncaeid species occur, of which eleven in the Gulf of Naples. In this Gulf, several Oncaea species were morphologically analysed and described at the end of the XIX century by W. Giesbrecht. In the same area, oncaeids are being investigated over seasonal and inter-annual scales at the long-term coastal station LTER-MC. In the present work, we identified six oncaeid species using the nuclear ribosomal internal transcribed spacers (ITS rDNA) and the mitochondrial cytochrome c oxidase subunit I (mtCOI). Phylogenetic analyses based on these two genomic regions validated the sisterhood of the genera Triconia and the Oncaea sensu stricto. ITS1 and ITS2 phylogenies produced incongruent results about the position of Oncaea curta, calling for further investigations on this species. We also characterised the ITS2 region by secondary structure predictions and found that all the sequences analysed presented the distinct eukaryotic hallmarks. A Compensatory Base Change search corroborated the close relationship between O. venusta and O. curta and between O. media and O. venusta already identified by ITS phylogenies. The present results, which stem from the integration of molecular and morphological taxonomy, represent an encouraging step towards an improved knowledge of copepod biodiversity: The two complementary approaches, when applied to long-term copepod monitoring, will also help to better understanding their genetic variations and ecological niches of co-occurring species.

Neuronal fate specification by the Dbx1 transcription factor is linked to the evolutionary acquisition of a novel functional domain.

BACKGROUND: Dbx1 is a homeodomain transcription factor involved in neuronal fate specification belonging to a widely conserved family among bilaterians. In mammals, Dbx1 was proposed to act as a transcriptional repressor by interacting with the Groucho corepressors to allow the specification of neurons involved in essential biological functions such as locomotion or breathing. RESULTS: Sequence alignments of Dbx1 proteins from different species allowed us to identify two conserved domains related to the Groucho-dependent Engrailed repressor domain (RD), as well as a newly described domain composed of clusterized acidic residues at the C-terminus (Cter) which is present in tetrapods but also several invertebrates. Using a heterologous luciferase assay, we showed that the two putative repressor domains behave as such in a Groucho-dependent manner, whereas the Cter does not bear any intrinsic transcriptional activity. Consistently with in vitro data, we found that both RDs are involved in cell fate specification using in vivo electroporation experiments in the chick spinal cord. Surprisingly, we show that the Cter domain is required for Dbx1 function in vivo, acting as a modulator of its repressive activity and/or imparting specificity. CONCLUSION: Our results strongly suggest that the presence of a Cter domain among tetrapods is essential for Dbx1 to regulate neuronal diversity and, in turn, nervous system complexity.

Small non-coding RNA signature in multiple sclerosis patients after treatment with interferon-ß.

BACKGROUND: Non-coding small RNA molecules play pivotal roles in cellular and developmental processes by regulating gene expression at the post-transcriptional level. In human diseases, the roles of the non-coding small RNAs in specific degradation or translational suppression of the targeted mRNAs suggest a potential therapeutic approach of post-transcriptional gene silencing that targets the underlying disease etiology. The involvement of non-coding small RNAs in the pathogenesis of neurodegenerative diseases such as Alzheimer's , Parkinson's disease and Multiple Sclerosis has been demonstrated. Multiple sclerosis (MS) is an autoimmune disease of the central nervous system, characterized by chronic inflammation, demyelination and scarring as well as a broad spectrum of signs and symptoms. The current standard treatment for SM is interferon ß (IFNß) that is less than ideal due to side effects. In this study we administered the standard IFN-ß treatment to Relapsing-Remitting MS patients, all responder to the therapy; then examined their sncRNA expression profiles in order to identify the ncRNAs that were associated with MS patients' response to IFNß. METHODS: 40 IFNß treated Relapsing-Remitting MS patients were enrolled. We analyzed the composition of the entire small transcriptome by a small RNA cloning method, using peripheral blood from Relapsing-Remitting MS patients at baseline and 3 and 6 months after the start of IFNß therapy. Real-time qPCR from the same patients group and from 20 additional patients was performed to profile miRNAs expression. RESULTS: Beside the altered expression of several miRNAs, our analyses revealed the differential expression of small nucleolar RNAs and misc-RNAs.For the first time, we found that the expression level of miR-26a-5p changed related to INF-ß response. MiR-26a-5p expression was significantly higher in IFN-ß treated RRMS patients at 3 months treatment, keeping quite stable at 6 months treatments. CONCLUSIONS: Our results might provide insights into the mechanisms of action of IFN-ß treatment in MS and provide fundamentals for the development of new biomarkers and/or therapeutic tools.