Genetic Association and Altered Gene Expression of CYBB in Multiple Sclerosis Patients

Multiple sclerosis (MS) is a chronic neurological disorder characterized by inflammation, demyelination, and axonal damage. Increased levels of reactive oxygen species (ROS), produced by macrophages and leading to oxidative stress, have been implicated as mediators of demyelination and axonal injury in both MS and experimental autoimmune encephalomyelitis, the murine model of the disease. On the other hand, reduced ROS levels can increase susceptibility to autoimmunity. In this work, we screened for association with MS 11 single nucleotide polymorphisms (SNPs) and two microsatellite markers in the five genes (NCF1, NCF2, NCF4, CYBA, and CYBB) of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX2) system, the enzymatic pathway producing ROS in the brain and neural tissues, in 347 Finnish patients with MS and 714 unaffected family members. This analysis showed suggestive association signals for NCF1 and CYBB (lowest p = 0.038 and p = 0.013, respectively). Functional relevance for disease predisposition was further supported for the CYBB gene, by microarray analysis in CD4+/− mononuclear cells of 21 individuals from five Finnish multiplex MS families, as well as by real-time RT-PCRs performed on RNA extracted from peripheral blood mononuclear cells of an Italian replication cohort of 21 MS cases and 21 controls. Our results showed a sex-specific differential expression of CYBB, suggesting that this gene, and more in general the NOX2 system, deserve to be further investigated for their possible role in MS.Peer reviewe

Non-random retention of protein-coding overlapping genes in Metazoa

<p>Abstract</p> <p>Background</p> <p>Although the overlap of transcriptional units occurs frequently in eukaryotic genomes, its evolutionary and biological significance remains largely unclear. Here we report a comparative analysis of overlaps between genes coding for well-annotated proteins in five metazoan genomes (human, mouse, zebrafish, fruit fly and worm).</p> <p>Results</p> <p>For all analyzed species the observed number of overlapping genes is always lower than expected assuming functional neutrality, suggesting that gene overlap is negatively selected. The comparison to the random distribution also shows that retained overlaps do not exhibit random features: antiparallel overlaps are significantly enriched, while overlaps lying on the same strand and those involving coding sequences are highly underrepresented. We confirm that overlap is mostly species-specific and provide evidence that it frequently originates through the acquisition of terminal, non-coding exons. Finally, we show that overlapping genes tend to be significantly co-expressed in a breast cancer cDNA library obtained by 454 deep sequencing, and that different overlap types display different patterns of reciprocal expression.</p> <p>Conclusion</p> <p>Our data suggest that overlap between protein-coding genes is selected against in Metazoa. However, when retained it may be used as a species-specific mechanism for the reciprocal regulation of neighboring genes. The tendency of overlaps to involve non-coding regions of the genes leads to the speculation that the advantages achieved by an overlapping arrangement may be optimized by evolving regulatory non-coding transcripts.</p

The circular RNA landscape in multiple sclerosis: Disease-specific associated variants and exon methylation shape circular RNA expression profile

BACKGROUND: Circular RNAs (circRNAs) are a class of non-coding RNAs increasingly emerging as crucial actors in the pathogenesis of human diseases, including autoimmune and neurological disorders as multiple sclerosis (MS). Despite several efforts, the mechanisms regulating circRNAs expression are still largely unknown and the circRNA profile and regulation in MS-relevant cell models has not been completely investigated. In this work, we aimed at exploring the global landscape of circRNA expression in MS patients, also evaluating a possible correlation with their genetic and epigenetic background. METHODS: We performed RNA-seq experiments on circRNA-enriched samples, derived from peripheral blood mononuclear cells (PBMCs) of 10 MS patients and 10 matched controls and performed differential circRNA expression. The genetic background was evaluated using array genotyping, and an expression quantitative trait loci (eQTL) analysis was carried out. RESULTS: Expression analysis revealed 166 differentially expressed circRNAs in MS patients, 125 of which are downregulated. One of the top dysregulated circRNAs, hsa_circ_0007990, derives from the PGAP3 gene, encoding a protein relevant for the control of autoimmune responses. The downregulation of this circRNA was confirmed in two independent replication cohorts, suggesting its implementation as a possible RNA-based biomarker. The eQTL analysis evidenced a significant association between 89 MS-associated loci and the expression of at least one circRNA, suggesting that MS-associated variants could impact on disease pathogenesis by altering circRNA profiles. Finally, we found a significant correlation between exon methylation and circRNA expression levels, supporting the hypothesis that epigenetic features may play an important role in the definition of the cell circRNA pool. CONCLUSION: We described the circRNA expression profile of PBMCs in MS patients, suggesting that MS-associated variants may tune the expression levels of circRNAs acting as circ-QTLs , and proposing a role for exon-based DNA methylation in regulating circRNA expression

Enhanced Uptake and Phototoxicity of C60@albumin Hybrids by Folate Bioconjugation

Fullerenes are considered excellent photosensitizers, being highly suitable for photodynamic therapy (PDT). A lack of water solubility and low biocompatibility are, in many instances, still hampering the full exploitation of their potential in nanomedicine. Here, we used human serum albumin (HSA) to disperse fullerenes by binding up to five fullerene cages inside the hydrophobic cavities. Albumin was bioconjugated with folic acid to specifically address the folate receptors that are usually overexpressed in several solid tumors. Concurrently, tetramethylrhodamine isothiocyanate, TRITC, a tag for imaging, was conjugated to C-60@HSA in order to build an effective phototheranostic platform. The in vitro experiments demonstrated that: (i) HSA disperses C-60 molecules in a physiological environment, (ii) HSA, upon C-60 binding, maintains its biological identity and biocompatibility, (iii) the C-60@HSA complex shows a significant visible-light-induced production of reactive oxygen species, and (iv) folate bioconjugation improves both the internalization and the PDT-induced phototoxicity of the C-60@HSA complex in HeLa cells

Usutu virus growth in human cell lines: induction of and sensitivity to type I and III interferons

The mechanisms of Usutu virus (USUV) pathogenesis are largely unknown. The aim of this study was to evaluate the sensitivity of USUV to interferon (IFN) and the capacity of USUV to stimulate IFN production. Initial experiments were conducted to characterize the susceptibility of human cell lines to USUV infection and to evaluate the single-growth cycle replication curve of USUV. Results indicate that USUV is able to infect a variety of human cell lines, completing the replication cycle in Hep-2 and Vero cells within 48 h. Pre-treatment of cells with types I and III IFNs significantly inhibited the replication of USUV. However, the inhibitory effects of IFNs were considerably less if IFN was added after viral infection had been initiated. Also, USUV weakly induced types I and III IFNs

Expression of distinct RNAs from 3′ untranslated regions

The 3′ untranslated regions (3′UTRs) of eukaryotic genes regulate mRNA stability, localization and translation. Here, we present evidence that large numbers of 3′UTRs in human, mouse and fly are also expressed separately from the associated protein-coding sequences to which they are normally linked, likely by post-transcriptional cleavage. Analysis of CAGE (capped analysis of gene expression), SAGE (serial analysis of gene expression) and cDNA libraries, as well as microarray expression profiles, demonstrate that the independent expression of 3′UTRs is a regulated and conserved genome-wide phenomenon. We characterize the expression of several 3′UTR-derived RNAs (uaRNAs) in detail in mouse embryos, showing by in situ hybridization that these transcripts are expressed in a cell- and subcellular-specific manner. Our results suggest that 3′UTR sequences can function not only in cis to regulate protein expression, but also intrinsically and independently in trans, likely as noncoding RNAs, a conclusion supported by a number of previous genetic studies. Our findings suggest novel functions for 3′UTRs, as well as caution in the use of 3′UTR sequence probes to analyze gene expression

A transcriptional sketch of a primary human breast cancer by 454 deep sequencing

Background: The cancer transcriptome is difficult to explore due to the heterogeneity of quantitative and qualitative changes in gene expression linked to the disease status. An increasing number of "unconventional" transcripts, such as novel isoforms, non-coding RNAs, somatic gene fusions and deletions have been associated with the tumoral state. Massively parallel sequencing techniques provide a framework for exploring the transcriptional complexity inherent to cancer with a limited laboratory and financial effort. We developed a deep sequencing and bioinformatics analysis protocol to investigate the molecular composition of a breast cancer poly(A)+ transcriptome. This method utilizes a cDNA library normalization step to diminish the representation of highly expressed transcripts and biology-oriented bioinformatic analyses to facilitate detection of rare and novel transcripts. Results: We analyzed over 132,000 Roche 454 high-confidence deep sequencing reads from a primary human lobular breast cancer tissue specimen, and detected a range of unusual transcriptional events that were subsequently validated by RT-PCR in additional eight primary human breast cancer samples. We identified and validated one deletion, two novel ncRNAs (one intergenic and one intragenic), ten previously unknown or rare transcript isoforms and a novel gene fusion specific to a single primary tissue sample. We also explored the non-protein-coding portion of the breast cancer transcriptome, identifying thousands of novel non-coding transcripts and more than three hundred reads corresponding to the non-coding RNA MALAT1, which is highly expressed in many human carcinomas. Conclusion: Our results demonstrate that combining 454 deep sequencing with a normalization step and careful bioinformatic analysis facilitates the discovery and quantification of rare transcripts or ncRNAs, and can be used as a qualitative tool to characterize transcriptome complexity, revealing many hitherto unknown transcripts, splice isoforms, gene fusion events and ncRNAs, even at a relatively low sequence sampling

A novel mutation within the MIR96 gene causes non-syndromic inherited hearing loss in an Italian family by altering pre-miRNA processing

The miR-96, miR-182 and miR-183 microRNA (miRNA) family is essential for differentiation and function of the vertebrate inner ear. Recently, point mutations within the seed region of miR-96 were reported in two Spanish families with autosomal dominant non-syndromic sensorineural hearing loss (NSHL) and in a mouse model of NSHL. We screened 882 NSHL patients and 836 normal-hearing Italian controls and identified one putative novel mutation within the miR-96 gene in a family with autosomal dominant NSHL. Although located outside the mature miR-96 sequence, the detected variant replaces a highly conserved nucleotide within the companion miR-96*, and is predicted to reduce the stability of the pre-miRNA hairpin. To evaluate the effect of the detected mutation on miR-96/mir-96* biogenesis, we investigated the maturation of miR-96 by transient expression in mammalian cells, followed by real-time reverse-transcription polymerase chain reaction (PCR). We found that both miR-96 and miR-96* levels were significantly reduced in the mutant, whereas the precursor levels were unaffected. Moreover, miR-96 and miR-96* expression levels could be restored by a compensatory mutation that reconstitutes the secondary structure of the pre-miR-96 hairpin, demonstrating that the mutation hinders precursor processing, probably interfering with Dicer cleavage. Finally, even though the mature miR-96 sequence is not altered, we demonstrated that the identified mutation significantly impacts on miR-96 regulation of selected targets. In conclusion, we provide further evidence of the involvement of miR-96 mutations in human deafness and demonstrate that a quantitative defect of this miRNA may contribute to NSHL

Genetic Association and Altered Gene Expression of Mir-155 in Multiple Sclerosis Patients

Multiple sclerosis (MS) is a complex autoimmune disease of the central nervous system characterized by chronic inflammation, demyelination, and axonal damage. As microRNA (miRNA)-dependent alterations in gene expression in hematopoietic cells are critical for mounting an appropriate immune response, miRNA deregulation may result in defects in immune tolerance. In this frame, we sought to explore the possible involvement of miRNAs in MS pathogenesis by monitoring the differential expression of 22 immunity-related miRNAs in peripheral blood mononuclear cells of MS patients and healthy controls, by using a microbead-based technology. Three miRNAs resulted >2 folds up-regulated in MS vs controls, whereas none resulted down-regulated. Interestingly, the most up-regulated miRNA (mir-155; fold change = 3.30; P = 0.013) was previously reported to be up-regulated also in MS brain lesions. Mir-155 up-regulation was confirmed by qPCR experiments. The role of mir-155 in MS susceptibility was also investigated by genotyping four single nucleotide polymorphisms (SNPs) mapping in the mir-155 genomic region. A haplotype of three SNPs, corresponding to a 12-kb region encompassing the last exon of BIC (the B-cell Integration Cluster non-coding RNA, from which mir-155 is processed), resulted associated with the disease status (P = 0.035; OR = 1.36, 95% CI = 1.05–1.77), suggesting that this locus strongly deserves further investigations

Implementation and experimental verification of a 3D-to-2D Visual Odometry Algorithm for real-time measurements of a vehicle pose

This work describes the implementation of a software for real-time vehicle pose reconstruction using stereo visual odometry algorithms. We firstly guess motion by a linear 2D-to-3D method solving a PnP problem embedded within a RANSAC process to remove outliers. Then, a maximum likelihood motion estimation is performed minimizing a non-linear problem. GPU implementations of feature extraction and matching algorithms are used. We demonstrate an accuracy of better than 1% over 1350 mm of trave