Migraine is a neurological disorder characterized by recurrent attacks of severe headache, affecting around 12% of Caucasian populations. It is well known that migraine has a strong genetic component, although the number and type of genes involved is still unclear. Prior linkage studies have reported mapping of a migraine gene to chromosome Xq 24-28, a region containing a cluster of genes for GABA A receptors (GABRE, GABRA3, GABRQ), which are potential candidate genes for migraine. The GABA neurotransmitter has been implicated in migraine pathophysiology previously; however its exact role has not yet been established, although GABA receptors agonists have been the target of therapeutic developments. The aim of the present research is to investigate the role of the potential candidate genes reported on chromosome Xq 24-28 region in migraine susceptibility. In this study, we have focused on the subunit GABA A receptors type epsilon (GABRE) and type theta (GABRQ) genes and their involvement in migraine.

Neurodegenerative disorders and cancer are severe diseases threatening human health. The glaring differences between neurons and cancer cells mask the processes involved in their pathogenesis. Defects in cell cycle, DNA repair, and cell differentiation can determine unlimited proliferation in cancer, or conversely, compromise neuronal plasticity, leading to cell death and neurodegeneration. Alteration in regulatory networks affecting gene expression contribute to human diseases onset, including neurodegenerative disorders, and deregulation of non-coding RNAs - particularly microRNAs (miRNAs) - is supposed to have a significant impact. Recently, competitive endogenous RNAs (ceRNAs) - acting as sponges - have been identified in cancer, indicating a new and intricate regulatory network. Given that neurodegenerative disorders and cancer share altered genes and pathways, and considering the emerging role of miRNAs in neurogenesis, we hypothesize ceRNAs may be implicated in neurodegenerative diseases. Here we propose, and computationally predict, such regulatory mechanism may be shared between the diseases. It is predictable that similar regulation occurs in other complex diseases, and further investigation is needed.

SEMA6B is a member of the semaphorins axon-guidance family. A growing body of evidence has been accumulated describing the role of semaphorin molecules in cancer development and the involvement of SEMA6B in cancer progression has recently been proposed.

The E2 multimeric scaffold represents a powerful delivery system able to elicit robust humoral and cellular immune responses upon systemic administrations. Here recombinant E2 scaffold displaying the third variable loop of HIV-1 Envelope gp120 glycoprotein was administered via mucosa, and the mucosal and systemic immune responses were analysed. To gain further insights into the molecular mechanisms that orchestrate the immune response upon E2 vaccination, we analysed the transcriptome profile of dendritic cells (DCs) exposed to the E2 scaffold with the aim to define a specific gene expression signature for E2-primed immune responses.

Type 2 diabetes and obesity are negative prognostic factors in patients with breast cancer (BC). We found that sensitivity to tamoxifen was reduced by 2-fold by 25 mM glucose (High Glucose; HG) compared to 5.5 mM glucose (Low Glucose; LG) in MCF7 BC cells. Shifting from HG to LG ameliorated MCF7 cell responsiveness to tamoxifen. RNA-Sequencing of MCF7 BC cells revealed that cell cycle-related genes were mainly affected by glucose. Connective Tissue Growth Factor (CTGF) was identified as a glucose-induced modulator of cell sensitivity to tamoxifen. Co-culturing MCF7 cells with human adipocytes exposed to HG, enhanced CTGF mRNA levels and reduced tamoxifen responsiveness of BC cells. Inhibition of adipocyte-released IL8 reverted these effects. Interestingly, CTGF immuno-detection in bioptic specimens from women with estrogen receptor positive (ER+) BC correlated with hormone therapy resistance, distant metastases, reduced overall and disease-free survival. Thus, glucose affects tamoxifen responsiveness directly modulating CTGF in BC cells, and indirectly promoting IL8 release by adipocytes.

Low-grade chronic inflammation and reduced differentiation capacity are hallmarks of hypertrophic adipose tissue (AT) and key contributors of insulin resistance. We identified PPARGΔ5 as a dominant-negative splicing isoform overexpressed in the AT of obese/diabetic patients able to impair adipocyte differentiation and PPARγ activity in hypertrophic adipocytes. Herein, we investigate the impact of macrophage-secreted pro-inflammatory factors on PPARG splicing, focusing on PPARGΔ5. We report that the epididymal AT of LPS-treated mice displays increased PpargΔ5/cPparg ratio and reduced expression of Pparg-regulated genes. Interestingly, pro-inflammatory factors secreted from murine and human pro-inflammatory macrophages enhance the PPARGΔ5/cPPARG ratio in exposed adipogenic precursors. TNFα is identified herein as factor able to alter PPARG splicing-increasing PPARGΔ5/cPPARG ratio-through PI3K/Akt signaling and SRp40 splicing factor. In line with in vitro data, TNFA expression is higher in the SAT of obese (vs. lean) patients and positively correlates with PPARGΔ5 levels. In conclusion, our results indicate that inflammatory factors secreted by metabolically-activated macrophages are potent stimuli that modulate the expression and splicing of PPARG. The resulting imbalance between canonical and dominant negative isoforms may crucially contribute to impair PPARγ activity in hypertrophic AT, exacerbating the defective adipogenic capacity of precursor cells.

Type 2 diabetes (T2D) is one of the most frequent mortality causes in western countries, with rapidly increasing prevalence. Anti-diabetic drugs are the first therapeutic approach, although many patients develop drug resistance. Most drug responsiveness variability can be explained by genetic causes. Inter-individual variability is principally due to single nucleotide polymorphisms, and differential drug responsiveness has been correlated to alteration in genes involved in drug metabolism (CYP2C9) or insulin signaling (IRS1, ABCC8, KCNJ11 and PPARG). However, most genome-wide association studies did not provide clues about the contribution of DNA variations to impaired drug responsiveness. Thus, characterizing T2D drug responsiveness variants is needed to guide clinicians toward tailored therapeutic approaches. Here, we extensively investigated polymorphisms associated with altered drug response in T2D, predicting their effects in silico. Combining different computational approaches, we focused on the expression pattern of genes correlated to drug resistance and inferred evolutionary conservation of polymorphic residues, computationally predicting the biochemical properties of polymorphic proteins. Using RNA-Sequencing followed by targeted validation, we identified and experimentally confirmed that two nucleotide variations in the CAPN10 gene-currently annotated as intronic-fall within two new transcripts in this locus. Additionally, we found that a Single Nucleotide Polymorphism (SNP), currently reported as intergenic, maps to the intron of a new transcript, harboring CAPN10 and GPR35 genes, which undergoes non-sense mediated decay. Finally, we analyzed variants that fall into non-coding regulatory regions of yet underestimated functional significance, predicting that some of them can potentially affect gene expression and/or post-transcriptional regulation of mRNAs affecting the splicing.

Bartonella henselae is able to internalize endothelial progenitor cells (EPCs), which are resistant to the infection of other common pathogens. Bacteroides fragilis is a gram-negative anaerobe belonging to the gut microflora. It protects from experimental colitis induced by Helicobacter hepaticus through the polysaccharide A (PSA). The aim of our study was to establish: 1) whether B. fragilis colonization could protect from B. henselae infection; if this event may have beneficial effects on EPCs, vascular system and tissues. Our in vitro results establish for the first time that B. fragilis can internalize EPCs and competes with B. henselae during coinfection. We observed a marked activation of the inflammatory response by Real-time PCR and ELISA in coinfected cells compared to B. henselae-infected cells (63 vs 23 up-regulated genes), and after EPCs infection with mutant B. fragilis ΔPSA (≅90% up-regulated genes) compared to B. fragilis. Interestingly, in a mouse model of coinfection, morphological and ultrastructural analyses by hematoxylin-eosin staining and electron microscopy on murine tissues revealed that damages induced by B. henselae can be prevented in the coinfection with B. fragilis but not with its mutant B. fragilis ΔPSA. Moreover, immunohistochemistry analysis with anti-Bartonella showed that the number of positive cells per field decreased of at least 50% in the liver (20±4 vs 50±8), aorta (5±1 vs 10±2) and spleen (25±3 vs 40±6) sections of mice coinfected compared to mice infected only with B. henselae. This decrease was less evident in the coinfection with ΔPSA strain (35±6 in the liver, 5±1 in the aorta and 30±5 in the spleen). Finally, B. fragilis colonization was also able to restore the EPC decrease observed in mice infected with B. henselae (0.65 vs 0.06 media). Thus, our data establish that B. fragilis colonization is able to prevent B. henselae damages through PSA.

By means of computational methods, we identified an uncharacterized human transcript, Chromosome 1 open reading frame 36 (C1orf36), that is expressed in the retina and that maps to 1q32.3. The cDNA contains an open reading frame of 585bp that encodes a 195-aminoacid protein with a predicted mass of 22.7kDa. An alternatively spliced transcript in a retinoblastoma cell line, encoding for a truncated peptide, was also identified. PCR experiments performed using human cDNA from several sources indicate that C1orf36 has a preferential expression in the retina. Accordingly, in situ hybridization experiments, performed using as probe a murine C1orf36 cDNA fragment, detected a hybridization signal on mouse retinal adult sections. The C1orf36 protein shares homology with putative proteins in Mus musculus and Fugu rubripes, suggesting evolutionary conservation of its function. Additional sequence analysis of the C1orf36 gene product predicts its subcellular mitochondrial localization and the presence of both evolutionary conserved phosphorylation sites and regions adopting a coiled-coil conformation. We also defined the genomic structure of the gene. This enabled us to perform a mutational analysis of the C1orf36 coding region of about 300 patients affected by retinitis pigmentosa. No pathological mutations were detected in this analysis.

Pathological angiogenesis represents a critical issue in the progression of many diseases. Down syndrome is postulated to be a systemic anti-angiogenesis disease model, possibly due to increased expression of anti-angiogenic regulators on chromosome 21. The aim of our study was to elucidate some features of circulating endothelial progenitor cells in the context of this syndrome.

The integrator complex has been recently identified as a key regulator of RNA Polymerase II-mediated transcription, with many functions including the processing of small nuclear RNAs, the pause-release and elongation of polymerase during the transcription of protein coding genes, and the biogenesis of enhancer derived transcripts. Moreover, some of its components also play a role in genome maintenance. Thus, it is reasonable to hypothesize that their functional impairment or altered expression can contribute to malignancies. Indeed, several studies have described the mutations or transcriptional alteration of some Integrator genes in different cancers. Here, to draw a comprehensive pan-cancer picture of the genomic and transcriptomic alterations for the members of the complex, we reanalyzed public data from The Cancer Genome Atlas. Somatic mutations affecting Integrator subunit genes and their transcriptional profiles have been investigated in about 11,000 patients and 31 tumor types. A general heterogeneity in the mutation frequencies was observed, mostly depending on tumor type. Despite the fact that we could not establish them as cancer drivers, INTS7 and INTS8 genes were highly mutated in specific cancers. A transcriptome analysis of paired (normal and tumor) samples revealed that the transcription of INTS7, INTS8, and INTS13 is significantly altered in several cancers. Experimental validation performed on primary tumors confirmed these findings.

Although the number of RNA-Seq datasets deposited publicly has increased over the past few years, incomplete annotation of the associated metadata limits their potential use. Because of the importance of RNA splicing in diseases and biological processes, we constructed a database called SFMetaDB by curating datasets related with RNA splicing factors. Our effort focused on the RNA-Seq datasets in which splicing factors were knocked-down, knocked-out or over-expressed, leading to 75 datasets corresponding to 56 splicing factors. These datasets can be used in differential alternative splicing analysis for the identification of the potential targets of these splicing factors and other functional studies. Surprisingly, only ∼15% of all the splicing factors have been studied by loss- or gain-of-function experiments using RNA-Seq. In particular, splicing factors with domains from a few dominant Pfam domain families have not been studied. This suggests a significant gap that needs to be addressed to fully elucidate the splicing regulatory landscape. Indeed, there are already mouse models available for ∼20 of the unstudied splicing factors, and it can be a fruitful research direction to study these splicing factors in vitro and in vivo using RNA-Seq. Database URL:http://sfmetadb.ece.tamu.edu/

The subtelomeric regions of human chromosomes exhibit an extraordinary plasticity. To date, due to the high GC content and to the presence of telomeric repeats, the subtelomeric sequences are underrepresented in the genomic libraries and consequently their sequences are incomplete in the finished human genome sequence, and still much remains to be learned about subtelomere organization, evolution and function. Indeed, only in recent years, several studies have disclosed, within human subtelomeres, novel gene family members.

Breast cancer is the most common tumor and the second leading cause of cancer death among woman, mainly caused by the metastatic spread. Tumor invasiveness is due to an altered expression of adhesion molecules. Among them, semaphorins are of peculiar interest. Cancer cells can manipulate alternative splicing patterns to modulate the expression of adhesion- and motility-related molecules, also at the isoform level. In this study, combining RNA-Sequencing on MCF-7 to targeted experimental validations-in human breast cell lines and breast tumor biopsies-we identified 12 new alternative splicing transcripts in genes encoding adhesion- and motility-related molecules, including semaphorins, their receptors and co-receptors. Among them, a new SEMA3F transcript is expressed in all breast cell lines and breast cancer biopsies, and is translated into a new semaphorin 3F isoform. In silico analysis predicted that most of the new putative proteins lack functional domains, potentially missing some functions and acquiring new ones. Our findings better describe the extent of alternative splicing in breast cancer and highlight the need to further investigate adhesion- and motility-related molecules to gain insights into breast cancer progression.

Thyroid hormones are normally involved in glycaemic control, but their excess can lead to altered glucose metabolism and insulin resistance (IR). Since hyperthyroidism-linked increase in ROS results in tissue oxidative stress that is considered a hallmark of conditions leading to IR, it is conceivable a role of ROS in the onset of IR in hyperthyroidism. To verify this hypothesis, we evaluated the effects of vitamin E on thyroid hormone-induced oxidative damage, insulin resistance, and on gene expression of key molecules involved in IR in the rat liver. The factors involved in oxidative damage, namely the total content of ROS, the mitochondrial production of ROS, the activity of antioxidant enzymes, the in vitro susceptibility to oxidative stress, have been correlated to insulin resistance indices, such as insulin activation of hepatic Akt and plasma level of glucose, insulin and HOMA index. Our results indicate that increased levels of oxidative damage ROS content and production and susceptibility to oxidative damage, parallel increased fasting plasma level of glucose and insulin, reduced activation of Akt and increased activation of JNK. This last result suggests a role for JNK in the insulin resistance induced by hyperthyroidism. Furthermore, the variation of the genes Pparg, Ppara, Cd36 and Slc2a2 could explain, at least in part, the observed metabolic phenotypes.

The PR/SET domain gene family (PRDM) encodes 19 different transcription factors that share a subtype of the SET domain [Su(var)3-9, enhancer-of-zeste and trithorax] known as the PRDF1-RIZ (PR) homology domain. This domain, with its potential methyltransferase activity, is followed by a variable number of zinc-finger motifs, which likely mediate protein⁻protein, protein⁻RNA, or protein⁻DNA interactions. Intriguingly, almost all PRDM family members express different isoforms, which likely play opposite roles in oncogenesis. Remarkably, several studies have described alterations in most of the family members in malignancies. Here, to obtain a pan-cancer overview of the genomic and transcriptomic alterations of PRDM genes, we reanalyzed the Exome- and RNA-Seq public datasets available at The Cancer Genome Atlas portal. Overall, PRDM2, PRDM3/MECOM, PRDM9, PRDM16 and ZFPM2/FOG2 were the most mutated genes with pan-cancer frequencies of protein-affecting mutations higher than 1%. Moreover, we observed heterogeneity in the mutation frequencies of these genes across tumors, with cancer types also reaching a value of about 20% of mutated samples for a specific PRDM gene. Of note, ZFPM1/FOG1 mutations occurred in 50% of adrenocortical carcinoma patients and were localized in a hotspot region. These findings, together with OncodriveCLUST results, suggest it could be putatively considered a cancer driver gene in this malignancy. Finally, transcriptome analysis from RNA-Seq data of paired samples revealed that transcription of PRDMs was significantly altered in several tumors. Specifically, PRDM12 and PRDM13 were largely overexpressed in many cancers whereas PRDM16 and ZFPM2/FOG2 were often downregulated. Some of these findings were also confirmed by real-time-PCR on primary tumors.

Peroxisome-proliferator-activated receptor γ (PPARγ) regulates glucose and lipid homeostasis, insulin signaling, and adipocyte differentiation. Here, we report the skipping of exon 5 as a legitimate splicing event generating PPARγΔ5, a previously unidentified naturally occurring truncated isoform of PPARγ, which lacks the entire ligand-binding domain. PPARγΔ5 is endogenously expressed in human adipose tissue and, during adipocyte differentiation, lacks ligand-dependent transactivation ability and acts as a dominant-negative isoform reducing PPARγ activity. Ligand-mediated PPARγ activation induces exon 5 skipping in a negative feedback loop, suggesting alternative splicing as a mechanism regulating PPARγ activity. PPARγΔ5 overexpression modifies the PPARγ-induced transcriptional network, significantly impairing the differentiation ability of adipocyte precursor cells. Additionally, PPARγΔ5 expression in subcutaneous adipose tissue positively correlates with BMI in two independent cohorts of overweight or obese and type 2 diabetic patients. From a functional perspective, PPARγΔ5 mimics PPARG dominant-negative mutated receptors, possibly contributing to adipose tissue dysfunction. These findings open an unexplored scenario in PPARG regulation and PPARγ-related diseases.

Filamentous bacteriophage fd particles delivering antigenic determinants via DEC-205 (fdsc-αDEC) represent a powerful delivery system that induces CD8(+) T-cell responses even when administered in the absence of adjuvants or maturation stimuli for dendritic cells. In order to investigate the mechanisms of this activity, RNA-Sequencing of fd-pulsed dendritic cells was performed. A significant differential expression of genes involved in innate immunity, co-stimulation and cytokine production was observed. In agreement with these findings, we demonstrate that induction of proinflammatory cytokines and type I interferon by fdsc-αDEC was MYD88 mediated and TLR9 dependent. We also found that fdsc-αDEC is delivered into LAMP-1-positive compartments and co-localizes with TLR9. Thus, phage particles containing a single-strand DNA genome rich in CpG motifs delivered via DEC-205 are able to intercept and trigger the active TLR9 innate immune receptor into late endosome/lysosomes and to enhance the immunogenicity of the displayed antigenic determinants. These findings make fd bacteriophage a valuable tool for immunization without administering exogenous adjuvants.

Alternative splicing is a pervasive mechanism of RNA maturation in higher eukaryotes, which increases proteomic diversity and biological complexity. It has a key regulatory role in several physiological and pathological states. The diffusion of Next Generation Sequencing, particularly of RNA-Sequencing, has exponentially empowered the identification of novel transcripts revealing that more than 95% of human genes undergo alternative splicing. The highest rate of alternative splicing occurs in transcription factors encoding genes, mostly in Krüppel-associated box domains of zinc finger proteins. Since these molecules are responsible for gene expression, alternative splicing is a crucial mechanism to "regulate the regulators". Indeed, different transcription factors isoforms may have different or even opposite functions. In this work, through a targeted re-analysis of our previously published RNA-Sequencing datasets, we identified nine novel transcripts in seven transcription factors genes. In silico analysis, combined with RT-PCR, cloning and Sanger sequencing, allowed us to experimentally validate these new variants. Through computational approaches we also predicted their novel structural and functional properties. Our findings indicate that alternative splicing is a major determinant of transcription factor diversity, confirming that accurate analysis of RNA-Sequencing data can reliably lead to the identification of novel transcripts, with potentially new functions.

Transcriptome studies have shown the pervasive nature of transcription, demonstrating almost all the genes undergo alternative splicing. Accurately annotating all transcripts of a gene is crucial. It is needed to understand the impact of mutations on phenotypes, to shed light on genetic and epigenetic regulation of mRNAs and more generally to widen our knowledge about cell functionality and tissue diversity. RNA-sequencing (RNA-Seq), and the other applications of the next-generation sequencing, provides precious data to improve annotations' accuracy, simultaneously creating issues related to the variety, complexity and the size of produced data. In this 'scenario', the lack of user-friendly resources, easily accessible to researchers with low skills in bioinformatics, makes difficult to retrieve complete information about one or few genes without browsing a jungle of databases. Concordantly, the increasing amount of data from 'omics' technologies imposes to develop integrated databases merging different data formats coming from distinct but complementary sources. In light of these considerations, and given the wide interest in studying Down syndrome-a genetic condition due to the trisomy of human chromosome 21 (HSA21)-we developed an integrated relational database and a web interface, named ALE-HSA21 (AnaLysis of Expression on HSA21), accessible at http://bioinfo.na.iac.cnr.it/ALE-HSA21. This comprehensive and user-friendly web resource integrates-for all coding and noncoding transcripts of chromosome 21-existing gene annotations and transcripts identified de novo through RNA-Seq analysis with predictive computational analysis of regulatory sequences. Given the role of noncoding RNAs and untranslated regions of coding genes in key regulatory mechanisms, ALE-HSA21 is also an interesting web-based platform to investigate such processes. The 'transcript-centric' and easily-accessible nature of ALE-HSA21 makes this resource a valuable tool to rapidly retrieve data at the isoform level, rather than at gene level, useful to investigate any disease, molecular pathway or cell process involving chromosome 21 genes. Database URL: http://bioinfo.na.iac.cnr.it/ALE-HSA21/.