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Simple Summary Current treatments for complex diseases, including cancer, are generally characterized by high toxicity due to their low selectivity for target cells. Moreover, patients often develop drug resistance, hence becoming less sensitive to the therapy. For this reason, novel, improved, and more specific pharmacological therapies are needed. The high cost and the time required to develop new drugs poses the attention on the development of computational methods for drug repositioning and combination therapy prediction. In this study, we developed an integrated network pharmacology framework that combines mechanistic and chemocentric approaches in order to predict potential drug combinations for cancer therapy. We applied our paradigm in five cancer types, which we used as case studies. Our strategy can be applied to the study of any complex disease by guiding the prioritization of drug combinations. Despite remarkable efforts of computational and predictive pharmacology to improve therapeutic strategies for complex diseases, only in a few cases have the predictions been eventually employed in the clinics. One of the reasons behind this drawback is that current predictive approaches are based only on the integration of molecular perturbation of a certain disease with drug sensitivity signatures, neglecting intrinsic properties of the drugs. Here we integrate mechanistic and chemocentric approaches to drug repositioning by developing an innovative network pharmacology strategy. We developed a multilayer network-based computational framework integrating perturbational signatures of the disease as well as intrinsic characteristics of the drugs, such as their mechanism of action and chemical structure. We present five case studies carried out on public data from The Cancer Genome Atlas, including invasive breast cancer, colon adenocarcinoma, lung squamous cell carcinoma, hepatocellular carcinoma and prostate adenocarcinoma. Our results highlight paclitaxel as a suitable drug for combination therapy for many of the considered cancer types. In addition, several non-cancer-related genes representing unusual drug targets were identified as potential candidates for pharmacological treatment of cancer.

Pancreatic stellate cells (PSCs) secrete high levels of transforming growth factor-β1 (TGF-β1) that contributes to the development of pancreatic ductal adenocarcinoma (PDAC). TGF-β1 modulates the expression of L1 cell adhesion molecule (L1CAM), but its role in tumour progression still remains controversial. To clarify L1 function in PDAC and cellular phenotypes, we performed L1CAM cell sorting, silencing and overexpression in several primary pancreatic cancer cells. PSCs silenced for TGF-β1 were used for crosstalk experiments. We found that TGF-β1 secreted by PSCs negatively regulates L1CAM expression, through canonical TGF-β-Smad2/3 signalling, leading to a more aggressive PDAC phenotype. Cells with reduced expression of L1CAM harboured enhanced stemness potential and tumourigenicity. Inactivation of TGF-β1 signalling in PSCs strongly reduced the aggressiveness of PDAC cells. Our data provide functional proof and mechanistic insights for the tumour-suppressive function of L1CAM via reducing stemness. Rescuing L1CAM expression in cancer cells through targeting of TGF-β1 reverses stemness and bears the potential to improve the still miserable prognosis of PDAC patients.

X-linked lissencephaly with abnormal genitalia (XLAG) and developmental epileptic encephalopathy-1 (DEE1) are caused by mutations in the Aristaless-related homeobox (ARX) gene, which encodes a transcription factor responsible for brain development. It has been unknown whether the phenotypically diverse XLAG and DEE1 phenotypes may converge on shared pathways. To address this question, a label-free quantitative proteomic approach was applied to the neonatal brain of Arx knockout (ArxKO/Y) and knock-in polyalanine (Arx(GCG)7/Y) mice that are respectively models for XLAG and DEE1. Gene ontology and protein–protein interaction analysis revealed that cytoskeleton, protein synthesis and splicing control are deregulated in an allelic-dependent manner. Decreased α-tubulin content was observed both in Arx mice and Arx/alr-1(KO) Caenorhabditis elegans ,and a disorganized neurite network in murine primary neurons was consistent with an allelic-dependent secondary tubulinopathy. As distinct features of Arx(GCG)7/Y mice, we detected eIF4A2 overexpression and translational suppression in cortex and primary neurons. Allelic-dependent differences were also established in alternative splicing (AS) regulated by PUF60 and SAM68. Abnormal AS repertoires in Neurexin-1, a gene encoding multiple pre-synaptic organizers implicated in synaptic remodelling, were detected in Arx/alr-1(KO) animals and in Arx(GCG)7/Y epileptogenic brain areas and depolarized cortical neurons. Consistent with a conserved role of ARX in modulating AS, we propose that the allelic-dependent secondary synaptopathy results from an aberrant Neurexin-1 repertoire. Overall, our data reveal alterations mirroring the overlapping and variant effects caused by null and polyalanine expanded mutations in ARX. The identification of these effects can aid in the design of pathway-guided therapy for ARX endophenotypes and NDDs with overlapping comorbidities.

Simple Summary Long non-coding RNAs (lncRNAs) are generally defined as transcripts longer than 200 nucleotides and not coding for proteins. This review provides an overview of the main functions of lncRNAs in the cells and their role in tumorigenesis. Additionally, key examples of oncogenic and/or tumor suppressive lncRNAs with a well-established role in tumor development and progression are discussed. Finally, the currently available technological approaches to target lncRNAs (e.g., by modifying their expression, stability, processing and/or binding capacity), with a specific focus on the pros and cons of their use as therapeutic targets are considered. Abstract Sequencing-based transcriptomics has significantly redefined the concept of genome complexity, leading to the identification of thousands of lncRNA genes identification of thousands of lncRNA genes whose products possess transcriptional and/or post-transcriptional regulatory functions that help to shape cell functionality and fate. Indeed, it is well-established now that lncRNAs play a key role in the regulation of gene expression through epigenetic and posttranscriptional mechanims. The rapid increase of studies reporting lncRNAs alteration in cancers has also highlighted their relevance for tumorigenesis. Herein we describe the most prominent examples of well-established lncRNAs having oncogenic and/or tumor suppressive activity. We also discuss how technical advances have provided new therapeutic strategies based on their targeting, and also report the challenges towards their use in the clinical settings.

Background: Next-generation sequencing (NGS) needs to be validated and standardized to ensure that cancer patients are reliably selected for target treatments. In Italy, NGS is performed in several institutions and harmonization of wet and dry procedures is needed. To this end, a consortium of five different laboratories, covering the most part of the Italian peninsula, was constituted. A narrow gene panel (SiRe®) covering 568 clinically relevant mutations in six different genes (EGFR, KRAS, NRAS, BRAF, cKIT, and PDGFRα) with a predictive role for therapy selection in non-small cell lung cancer (NSCLC), gastrointestinal stromal tumor, colorectal carcinoma (CRC), and melanoma was evaluated in each participating laboratory. Methods: To assess the NGS inter-laboratory concordance, the SiRe® panel, with a related kit and protocol for library preparation, was used in each center to analyze a common set of 20 NSCLC and CRC routine samples. Concordance rate, in terms of mutation detected and relative allelic frequencies, was assessed. Then, each institution prospectively analyzed an additional set of 40 routine samples (for a total of 160 specimens) to assess the reproducibility of the NGS run parameters in each institution. Results: An inter-laboratory agreement of 100% was reached in analyzing the data obtained from the 20 common sample sets; the concordance rate of allelic frequencies distribution was 0.989. The prospective analysis of the run metric parameters obtained by each center locally showed that the analytical performance of the SiRe® panel in the different institutions was highly reproducible. Conclusions: The SiRe® panel represents a robust diagnostic tool to harmonize the NGS procedure in different Italian laboratories.

Reduced neo-adipogenesis and dysfunctional lipid-overloaded adipocytes are hallmarks of hypertrophic obesity linked to insulin resistance. Identifying molecular features of hypertrophic adipocytes requires appropriate in vitro models. We describe the generation of a model of human hypertrophic-like adipocytes directly comparable to normal adipose cells and the pathologic evolution toward hypertrophic state. We generate in vitro hypertrophic cells from mature adipocytes, differentiated from human mesenchymal stem cells. Combining optical, confocal, and transmission electron microscopy with mRNA/protein quantification, we characterize this cellular model, confirming specific alterations also in subcutaneous adipose tissue. Specifically, we report the generation and morphological/molecular characterization of human normal and hypertrophic-like adipocytes. The latter displays altered morphology and unbalance between canonical and dominant negative (PPARG&Delta, 5) transcripts of PPARG, paralleled by reduced expression of PPAR&gamma, targets, including GLUT4. Furthermore, the unbalance of PPAR&gamma, isoforms associates with GLUT4 down-regulation in subcutaneous adipose tissue of individuals with overweight/obesity or impaired glucose tolerance/type 2 diabetes, but not with normal weight or glucose tolerance. In conclusion, the hypertrophic-like cells described herein are an innovative tool for studying molecular dysfunctions in hypertrophic obesity and the unbalance between PPAR&gamma, isoforms associates with down-regulation of GLUT4 and other PPAR&gamma, targets, representing a new hallmark of hypertrophic adipocytes.

RET rearrangements as well as BRAF and RAS mutations drive differential pathway activation in papillary thyroid carcinomas, leading to different tumor phenotypes and prognoses. Although The Cancer Genome Atlas Consortium has identified tumor subgroups based on protein-coding gene signatures, neither expression of long noncoding RNAs (lncRNA) nor their correlation with specific tumor-driving mutations and rearrangements have been systematically assessed. Here, we reanalyzed our RNA-sequencing data using a de novo discovery approach to identify lncRNAs and define tumor subtype-specific signatures of annotated lncRNAs. Among them, we identified COMET (Correlated-to-MET), a natural antisense transcript that was highly expressed in carcinomas harboring BRAFV600E mutation or RET gene rearrangements (i.e., BRAF-like tumors) and induced the downstream MAPK pathway. In papillary thyroid carcinomas, COMET was part of a coexpression network including different oncogenes belonging to the MAPK pathway, and its expression highly correlated with MET expression. Depletion of COMET resulted in reduced expression of genes within this network, including the MET oncogene. COMET repression inhibited viability and proliferation of tumor cells harboring BRAFV600E somatic mutation or RET oncogene rearrangement and dramatically reduced motility and invasiveness of tumor cells. Moreover, silencing COMET markedly increased sensitivity to vemurafenib, a common inhibitor of mutated B-raf. Collectively, our results suggest COMET as a new target to improve drug-based cancer therapies, especially in BRAF-mutated and MET-addicted papillary thyroid carcinomas. Significance: These results highlight the oncogenic role of lncRNA COMET in thyroid and indicate it as a potential new target to overcome vemurafenib resistance in BRAF-mutated and MET-addicted carcinomas.

RNA-binding proteins may play a critical role in gene regulation in various diseases or biological processes by controlling post-transcriptional events such as polyadenylation, splicing, and mRNA stabilization via binding activities to RNA molecules. Due to the importance of RNA-binding proteins in gene regulation, a great number of studies have been conducted, resulting in a large amount of RNA-Seq datasets. However, these datasets usually do not have structured organization of metadata, which limits their potentially wide use. To bridge this gap, the metadata of a comprehensive set of publicly available mouse RNA-Seq datasets with perturbed RNA-binding proteins were collected and integrated into a database called RBPMetaDB. This database contains 278 mouse RNA-Seq datasets for a comprehensive list of 163 RNA-binding proteins. These RNA-binding proteins account for only ∼10% of all known RNA-binding proteins annotated in Gene Ontology, indicating that most are still unexplored using high-throughput sequencing. This negative information provides a great pool of candidate RNA-binding proteins for biologists to conduct future experimental studies. In addition, we found that DNA-binding activities are significantly enriched among RNA-binding proteins in RBPMetaDB, suggesting that prior studies of these DNA- and RNA-binding factors focus more on DNA-binding activities instead of RNA-binding activities. This result reveals the opportunity to efficiently reuse these data for investigation of the roles of their RNA-binding activities. A web application has also been implemented to enable easy access and wide use of RBPMetaDB. It is expected that RBPMetaDB will be a great resource for improving understanding of the biological roles of RNA-binding proteins.Database URL: http://rbpmetadb.yubiolab.org

Papillary thyroid carcinoma (PTC) is the most frequent thyroid malignant neoplasia. Oncogene activation occurs in more than 70% of the cases. Indeed, about 40% of PTCs harbor mutations in BRAF gene, whereas RET rearrangements (RET/PTC oncogenes) are present in about 20% of cases. Finally, RAS mutations and TRK rearrangements account for about 5% each of these malignancies. We used RNA-Sequencing to identify fusion transcripts and mutations in cancer driver genes in a cohort of 18 PTC patients. Furthermore, we used targeted DNA sequencing to validate identified mutations. We extended the screening to 50 PTC patients and 30 healthy individuals. Using this approach we identified new missense mutations in CBL, NOTCH1, PIK3R4 and SMARCA4 genes. We found somatic mutations in DICER1, MET and VHL genes, previously found mutated in other tumors, but not described in PTC. We identified a new chimeric transcript generated by the fusion of WNK1 and B4GALNT3 genes, correlated with B4GALNT3 overexpression. Our data confirmed PTC genetic heterogeneity, revealing that gene expression correlates more with the mutation pattern than with tumor staging. Overall, this study provides new data about mutational landscape of this neoplasia, suggesting potential pharmacological adjuvant therapies against Notch signaling and chromatin remodeling enzymes.

Ultraconserved regions (UCRs) represent a relatively new class of non-coding genomic sequences highly conserved between human, rat and mouse genomes. These regions can reside within exons of protein-coding genes, despite the vast majority of them localizes within introns or intergenic regions. Several studies have undoubtedly demonstrated that most of these regions are actively transcribed in normal cells/tissues, where they contribute to regulate many cellular processes. Interestingly, these non-coding RNAs exhibit aberrant expression levels in human cancer cells and their expression profiles have been used as prognostic factors in human malignancies, as well as to unambiguously distinguish among distinct cancer types. In this review, we first describe their identification, then we provide some updated information about their genomic localization and classification. More importantly, we discuss about the available literature describing an overview of the mechanisms through which some transcribed UCRs (T-UCR) contribute to cancer progression or to the metastatic spread. To date, the interplay between T-UCRs and microRNAs is the most convincing evidence linking T-UCRs and tumorigenesis. The limitations of these studies and the future challenges to be addressed in order to understand the biological role of T-UCRs are also discussed herein. We envision that future efforts are needed to convincingly include this class of ncRNAs in the growing area of cancer therapeutics.

A novel two-step bioinformatics strategy was applied for identification of signatures with therapeutic implications in hepatitis-associated HCC. Transcriptional profiles from HBV- and HCV-associated HCC samples were compared with non-tumor liver controls. Resulting HCC modulated genes were subsequently compared with different non-tumor tissue samples. Two related signatures were identified, namely “HCC-associated” and “HCC-specific”. Expression data were validated by RNA-Seq analysis carried out on unrelated HCC samples and protein expression was confirmed according to The Human Protein Atlas" (http://proteinatlas.org/), a public repository of immunohistochemistry data. Among all, aldo-keto reductase family 1 member B10, and IGF2 mRNA-binding protein 3 were found strictly HCC-specific with no expression in 18/20 normal tissues. Target peptides for vaccine design were predicted for both proteins associated with the most prevalent HLA-class I and II alleles. The described novel strategy showed to be feasible for identification of HCC-specific proteins as highly potential target for HCC immunotherapy.

Background/Objectives:The genomic bases of the adipose tissue abnormalities induced by chronic positive calorie excess have been only partially elucidated. We adopted a genome-wide approach to directly test whether long-term high-fat diet (HFD) exposure affects the DNA methylation profile of the mouse adipose tissue and to identify the functional consequences of these changes.Subjects/Methods:We have used epididymal fat of mice fed either high-fat (HFD) or regular chow (STD) diet for 5 months and performed genome-wide DNA methylation analyses by methylated DNA immunoprecipitation sequencing (MeDIP-seq). Mouse Homeobox (Hox) Gene DNA Methylation PCR, RT-qPCR and bisulphite sequencing analyses were then performed.Results:Mice fed the HFD progressively expanded their adipose mass accompanied by a significant decrease in glucose tolerance (P

BACKGROUND: 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. RESULTS: The in vivo immunogenicity and the potential of E2 scaffold as a mucosal vaccine candidate were investigated in BALB/c mice vaccinated via the intranasal route. Fecal and systemic antigen-specific IgA antibodies, cytokine-producing CD4(+) and CD8(+) cells were induced assessing the immunogenicity of E2 particles via intranasal administration. The cytokine analysis identified a mixed T-helper cell response, while the systemic antibody response showed a prevalence of IgG1 isotype indicative of a polarized Th2-type immune response. RNA-Sequencing analysis revealed that E2 scaffold up-regulates in DCs transcriptional regulators of the Th2-polarizing cell response, defining a type 2 DC transcriptomic signature. CONCLUSIONS: The current study provides experimental evidence to the possible application of E2 scaffold as antigen delivery system for mucosal immunization and taking advantages of genome-wide approach dissects the type of response induced by E2 particles.

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.

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.

The availability of omic data produced from international consortia, as well as from worldwide laboratories, is offering the possibility both to answer long-standing questions in biomedicine/molecular biology and to formulate novel hypotheses to test. However, the impact of such data is not fully exploited due to a limited availability of multi-omic data integration tools and methods. In this paper, we discuss the interplay between gene expression and epigenetic markers/transcription factors. We show how integrating ChIP-seq and RNA-seq data can help to elucidate gene regulatory mechanisms. In particular, we discuss the two following questions: (i) Can transcription factor occupancies or histone modification data predict gene expression? (ii) Can ChIP-seq and RNA-seq data be used to infer gene regulatory networks? We propose potential directions for statistical data integration. We discuss the importance of incorporating underestimated aspects (such as alternative splicing and long-range chromatin interactions). We also highlight the lack of data benchmarks and the need to develop tools for data integration from a statistical viewpoint, designed in the spirit of reproducible research.

Background 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. Methods Our analysis, based on real-time PCR, focused on the expression of SEMA6B in a panel of breast cancer tissues, compared to the normal counterpart. Results In cancer tissues we found a significantly strong down-modulation of this transcript. Moreover we identified and characterized a novel SEMA6B isoform, named SEMA6B a. This isoform has a novel splice junction, created by the usage of alternative donor and acceptor splice sites internal to the exon 17. By in silico analysis we found that the new transcript 3′ UTR lacks some highly-conserved miRNA binding sites, suggesting possible consequences on both spatial and temporal expression of SEMA6Ba. The translated sequence of SEMA6Ba lacks the cytoplasmic tail, crucial for triggering the reverse signaling described for the transmembrane semaphorins. We also demonstrated, by immunofluorescence analysis of endogenous and overexpressed SEMA6Ba , that the protein clearly localized to the endoplasmic reticulum and plasma membrane. In conclusion, SEMA6B gene products are strongly down modulated in breast cancer tissues and a new isoform named SEMA6Ba has been described and characterized. General significance Our work states a clear relation among breast cancer and SEMA6B expression; moreover we describe for the first time the SEMA6Ba protein and report here the analysis of SEMA6Ba RNA messenger, the protein expression and the cellular localization.

The availability of the human genome sequence has allowed identification of disease-causing mutations in many Mendelian disorders, and detection of significant associations of nucleotide polymorphisms to complex diseases and traits. Despite these progresses, finding the causative variations for most of the common diseases remains a complex task. Several studies have shown gene expression analyses provide a quite unbiased way to investigate complex traits and common disorders' pathogenesis. Therefore, whole-transcriptome analysis is increasingly acquiring a key role in the knowledge of mechanisms responsible for complex diseases. Hybridization- and tag-based technologies have elucidated the involvement of multiple genes and pathways in pathological conditions, providing insights into the expression of thousand of coding and noncoding RNAs, such as microRNAs. However, the introduction of Next-Generation Sequencing, particularly of RNA-Seq, has overcome some drawbacks of previously used technologies. Identifying, in a single experiment, potentially novel genes/exons and splice isoforms, RNA editing, fusion transcripts and allele-specific expression are some of its advantages. RNA-Seq has been fruitfully applied to study cancer and host-pathogens interactions, and it is taking first steps for studying neurodegenerative diseases (ND) as well as neuropsychiatric diseases. In addition, it is emerging as a very powerful tool to study quantitative trait loci associated with gene expression in complex diseases. This paper provides an overview on gene expression profiling of complex diseases, with emphasis on RNA-Seq, its advantages over conventional technologies for studying cancer and ND, and for linking nucleotide variations to gene expression changes, also discussing its limitations.

Background 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. Methods Circulating endothelial progenitors of Down syndrome affected individuals were isolated, in vitro cultured and analyzed by confocal and transmission electron microscopy. ELISA was performed to measure SDF-1α plasma levels in Down syndrome and euploid individuals. Moreover, qRT-PCR was used to quantify expression levels of CXCL12 gene and of its receptor in progenitor cells. The functional impairment of Down progenitors was evaluated through their susceptibility to hydroperoxide-induced oxidative stress with BODIPY assay and the major vulnerability to the infection with human pathogens. The differential expression of crucial genes in Down progenitor cells was evaluated by microarray analysis. Results We detected a marked decrease of progenitors' number in young Down individuals compared to euploid, cell size increase and some major detrimental morphological changes. Moreover, Down syndrome patients also exhibited decreased SDF-1α plasma levels and their progenitors had a reduced expression of SDF-1α encoding gene and of its membrane receptor. We further demonstrated that their progenitor cells are more susceptible to hydroperoxide-induced oxidative stress and infection with Bartonella henselae. Further, we observed that most of the differentially expressed genes belong to angiogenesis, immune response and inflammation pathways, and that infected progenitors with trisomy 21 have a more pronounced perturbation of immune response genes than infected euploid cells. Conclusions Our data provide evidences for a reduced number and altered morphology of endothelial progenitor cells in Down syndrome, also showing the higher susceptibility to oxidative stress and to pathogen infection compared to euploid cells, thereby confirming the angiogenesis and immune response deficit observed in Down syndrome individuals.

Peroxisome proliferator-activated receptor gamma (PPARγ) is one of the most extensively studied ligand-inducible transcription factors (TFs), able to modulate its transcriptional activity through conformational changes. It is of particular interest because of its pleiotropic functions: it plays a crucial role in the expression of key genes involved in adipogenesis, lipid and glucid metabolism, atherosclerosis, inflammation, and cancer. Its protein isoforms, the wide number of PPARγtarget genes, ligands, and coregulators contribute to determine the complexity of its function. In addition, the presence of genetic variants is likely to affect expression levels of target genes although the impact ofPPARGgene variations on the expression of target genes is not fully understood. The introduction of massively parallel sequencing platforms—in the Next Generation Sequencing (NGS) era—has revolutionized the way of investigating the genetic causes of inherited diseases. In this context, DNA-Seq for identifying—within both coding and regulatory regions ofPPARGgene—novel nucleotide variations and haplotypes associated to human diseases, ChIP-Seq for defining a PPARγbinding map, and RNA-Seq for unraveling the wide and intricate gene pathways regulated by PPARG, represent incredible steps toward the understanding of PPARγin health and disease.

The recruitment of circulating endothelial progenitor cells (EPCs) might have a beneficial effect on the clinical course of several diseases. Endothelial damage and detachment of endothelial cells are known to occur in infection, tissue ischemia, and sepsis. These detrimental effects in EPCs are unknown. Here we elucidated whether human EPCs internalize Bartonella henselae constituting a circulating niche of the pathogen. B. henselae invades EPCs as shown by gentamicin protection assays and transmission electron microscopy (TEM). Dil-Ac-LDL/lectin double immunostaining and fluorescence-activated cell sorting (FACS) analysis of EPCs revealed EPC bioactivity after infection with B. henselae . Nitric oxide (NO) and its precursor l -arginine ( l -arg) exert a plethora of beneficial effects on vascular function and modulation of immune response. Therefore, we tested also the hypothesis that l -arg (1–30 mM) would affect the infection of B. henselae or tumor necrosis factor (TNF) in EPCs. Our data provide evidence that l -arg counteracts detrimental effects induced by TNF or Bartonella infections via NO (confirmed by DETA-NO and L-NMMA experiments) and by modulation of p38 kinase phosphorylation. Microarray analysis indicated several genes involved in immune response were differentially expressed in Bartonella -infected EPCs, whereas these genes returned in steady state when cells were exposed to sustained doses of l -arg. This mechanism may have broad therapeutic applications in tissue ischemia, angiogenesis, immune response, and sepsis.

Background Sensorineural hearing impairment is a common pathological manifestation in patients affected by X-linked intellectual disability. A few cases of interstitial deletions at Xq21 with several different phenotypic characteristics have been described, but to date, a complete molecular characterization of the deletions harboring disease-causing genes is still missing. Thus, the aim of this study is to realize a detailed clinical and molecular analysis of a family affected by syndromic X-linked hearing loss with intellectual disability. Results Clinical analyses revealed a very complex phenotype that included inner ear malformations, vestibular problems, choroideremia and hypotonia with a peculiar pattern of phenotypic variability. Genomic analysis revealed, for the first time, the presence of two close interstitial deletions in the Xq21.1-21.3, harboring 11 protein coding, 9 non-coding genes and 19 pseudogenes. Among these, 3 protein coding genes have already been associated with X-linked hearing loss, intellectual disability and choroideremia. Conclusions In this study we highlighted the presence of peculiar genotypic and phenotypic details in a family affected by syndromic X-linked hearing loss with intellectual disability. We identified two, previously unreported, Xq21.1-21.3 interstitial deletions. The two rearrangements, containing several genes, segregate with the clinical features, suggesting their role in the pathogenicity. However, not all the observed phenotypic features can be clearly associated with the known genes thus, further study is necessary to determine regions involved. Electronic supplementary material The online version of this article (doi:10.1186/s13039-015-0120-0) contains supplementary material, which is available to authorized users.