Trans-heterozygosity for mutations enhances the risk of recurrent/chronic pancreatitis in patients with Cystic Fibrosis.

BACKGROUND: Recurrent (RP) and chronic pancreatitis (CP) may complicate Cystic Fibrosis (CF). It is still unknown if mutations in genes involved in the intrapancreatic activation of trypsin (IPAT) or in the pancreatic secretion pathway (PSP) may enhance the risk for RP/CP in patients with CF. METHODS: We enrolled: 48 patients affected by CF complicated by RP/CP and, as controls 35 patients with CF without pancreatitis and 80 unrelated healthy subjects. We tested a panel of 8 genes involved in the IPAT, i.e. PRSS1, PRSS2, SPINK1, CTRC, CASR, CFTR, CTSB and KRT8 and 23 additional genes implicated in the PSP. RESULTS: We found 14/48 patients (29.2%) with mutations in genes involved in IPAT in the group of CF patients with RP/CP, while mutations in such genes were found in 2/35 (5.7%) patients with CF without pancreatitis and in 3/80 (3.8%) healthy subjects (p < 0.001). Thus, we found mutations in 12 genes of the PSP in 11/48 (22.9%) patients with CF and RP/CP. Overall, 19/48 (39.6%) patients with CF and RP/CP showed one or more mutations in the genes involved in the IPAT and in the PSP while such figure was 4/35 (11.4%) for patients with CF without pancreatitis and 11/80 (13.7%) for healthy controls (p < 0.001). CONCLUSIONS: The trans-heterozygous association between CFTR mutations in genes involved in the pathways of pancreatic enzyme activation and the pancreatic secretion may be risk factors for the development of recurrent or chronic pancreatitis in patients with CF.

Extensive molecular analysis suggested the strong genetic heterogeneity of idiopathic chronic pancreatitis.

Rationale: Genetic features of Chronic Pancreatitis (CP) have been extensively investigated mainly testing genes associated to the trypsinogen activation pathway. However, different molecular pathways involving other genes may be implicated in CP pathogenesis. Objectives: 80 patients with Idiopathic CP were investigated using Next Generation Sequencing approach with a panel of 70 genes related to six different pancreatic pathways: premature activation of trypsinogen; modifier genes of Cystic Fibrosis phenotype; pancreatic secretion and ion homeostasis; Calcium signalling and zymogen granules exocytosis; autophagy; autoimmune pancreatitis related genes. Results: We detected mutations in 34 out of 70 genes examined; 64/80 patients (80.0%) were positive for mutations in one or more genes, 16/80 patients (20.0%) had no mutations. Mutations in CFTR were detected in 32/80 patients (40.0%) and 22 of them exhibited at least one mutation in genes of other pancreatic pathways. Of the remaining 48 patients, 13/80 (16.3%) had mutations in genes involved in premature activation of trypsinogen and 19/80 (23.8%) had mutations only in genes of the other pathways: 38/64 patients positive for mutations showed variants in two or more genes (59.3%). Conclusions: Our data, although to be extended with functional analysis of novel mutations, suggest a high rate of genetic heterogeneity in chronic pancreatitis and that trans-heterozygosity may predispose to the idiopathic CP phenotype.

HIV-1 gp120 influences the expression of microRNAs in human monocyte-derived dendritic cells via STAT3 activation.

BACKGROUND: MicroRNAs (miRs) are an abundant class of small non-coding RNAs (~22 nt) that reprogram gene expression by targeting mRNA degradation and translational disruption. An emerging concept implicates miR coupling with transcription factors in myeloid cell development and function, thus contributing to host defense and inflammation. The important role that these molecules play in the pathogenesis of HIV-1 is only now emerging. RESULTS: We provide evidence that exposure of monocyte-derived dendritic cells (MDDCs) to recombinant HIV-1 R5 gp120, but not to CCR5 natural ligand CCL4, influences the expression of a panel of miRs (i.e., miR-21, miR-155 and miR-181b) regulated by STAT3 and potentially targeting genes belonging to the STAT3 signaling pathway. The blockage of gp120-induced STAT3 activation impairs gp120 capacity to modulate the expression level of above mentioned miRs. Predictive analysis of miR putative targets emphasizes that these miRs share common target genes. Furthermore, gene ontology and pathway enrichment analysis outline that these genes mainly belong to biological processes related to regulation of transcription, in a complex network of interactions involving pathways relevant to HIV-DC interaction. CONCLUSIONS: Overall, these results point to gp120-triggered modulation of miR expression via STAT3 activation as a novel molecular mechanism exploited by HIV-1 to affect DC biology and thus modulate the immune response through complex regulatory loops involving, at the same time, miRs and transcription factors.

HIV-1 gp120 activates the STAT3/interleukin-6 axis in primary human monocyte-derived dendritic cells.

UNLABELLED: Dendritic cells (DCs) are fundamental for the initiation of immune responses and are important players in AIDS immunopathogenesis. The modulation of DC functional activities represents a strategic mechanism for HIV-1 to evade immune surveillance. Impairment of DC function may result from bystander effects of HIV-1 envelope proteins independently of direct HIV-1 infection. In this study, we report that exposure of immature monocyte-derived DCs (MDDCs) to HIV-1 R5 gp120 resulted in the CCR5-dependent production of interleukin-6 (IL-6) via mitogen-activated protein kinase (MAPK)/NF-κB pathways. IL-6 in turn activated STAT3 by an autocrine loop. Concomitantly, gp120 promoted an early activation of STAT3 that further contributed to IL-6 induction. This activation paralleled a concomitant upregulation of the STAT3 inhibitor PIAS3. Notably, STAT3/IL-6 pathway activation was not affected by the CCR5-specific ligand CCL4. These results identify STAT3 as a key signaling intermediate activated by gp120 in MDDCs and highlight the existence of a virus-induced dysregulation of the IL-6/STAT3 axis. HIV-1 gp120 signaling through STAT3 may provide an explanation for the impairment of DC function observed upon HIV exposure. IMPORTANCE: This study provides new evidence for the molecular mechanisms and signaling pathways triggered by HIV-1 gp120 in human DCs in the absence of productive infection, emphasizing a role of aberrant signaling in early virus-host interaction, contributing to viral pathogenesis. We identified STAT3 as a key component in the gp120-mediated signaling cascade involving MAPK and NF-κB components and ultimately leading to IL-6 secretion. STAT3 now is recognized as a key regulator of DC functions. Thus, the identification of this transcription factor as a signaling molecule mediating some of gp120's biological effects unveils a new mechanism by which HIV-1 may deregulate DC functions and contribute to AIDS pathogenesis.

Telomere shortening and telomere position effect in mild ring 17 syndrome.

BACKGROUND: Ring chromosome 17 syndrome is a rare disease that arises from the breakage and reunion of the short and long arms of chromosome 17. Usually this abnormality results in deletion of genetic material, which explains the clinical features of the syndrome. Moreover, similar phenotypic features have been observed in cases with complete or partial loss of the telomeric repeats and conservation of the euchromatic regions. We studied two different cases of ring 17 syndrome, firstly, to clarify, by analyzing gene expression analysis using real-time qPCR, the role of the telomere absence in relationship with the clinical symptoms, and secondly, to look for a new model of the mechanism of ring chromosome transmission in a rare case of familial mosaicism, through cytomolecular and quantitative fluorescence in-situ hybridization (Q-FISH) investigations. RESULTS: The results for the first case showed that the expression levels of genes selected, which were located close to the p and q ends of chromosome 17, were significantly downregulated in comparison with controls. Moreover, for the second case, we demonstrated that the telomeres were conserved, but were significantly shorter than those of age-matched controls; data from segregation analysis showed that the ring chromosome was transmitted only to the affected subjects of the family. CONCLUSIONS: Subtelomeric gene regulation is responsible for the phenotypic aspects of ring 17 syndrome; telomere shortening influences the phenotypic spectrum of this disease and strongly contributes to the familial transmission of the mosaic ring. Together, these results provide new insights into the genotype-phenotype relationships in mild ring 17 syndrome.

Rat mir-155 generated from the lncRNA Bic is 'hidden' in the alternate genomic assembly and reveals the existence of novel mammalian miRNAs and clusters.

MicroRNAs (miRNAs) are a class of small noncoding RNAs acting as post-transcriptional gene expression regulators in many physiological and pathological conditions. During the last few years, many novel mammalian miRNAs have been predicted experimentally with bioinformatics approaches and validated by next-generation sequencing. Although these strategies have prompted the discovery of several miRNAs, the total number of these genes still seems larger. Here, by exploiting the species conservation of human, mouse, and rat hairpin miRNAs, we discovered a novel rat microRNA, mir-155. We found that mature miR-155 is overexpressed in rat spleen myeloid cells treated with LPS, similarly to humans and mice. Rat mir-155 is annotated only on the alternate genome, suggesting the presence of other "hidden" miRNAs on this assembly. Therefore, we comprehensively extended the homology search also to mice and humans, finally validating 34 novel mammalian miRNAs (two in humans, five in mice, and up to 27 in rats). Surprisingly, 15 of these novel miRNAs (one for mice and 14 for rats) were found only on the alternate and not on the reference genomic assembly. To date, our findings indicate that the choice of genomic assembly, when mapping small RNA reads, is an important option that should be carefully considered, at least for these animal models. Finally, the discovery of these novel mammalian miRNA genes may contribute to a better understanding of already acquired experimental data, thereby paving the way to still unexplored investigations and to unraveling the function of miRNAs in disease models.

Recent insights and novel bioinformatics tools to understand the role of microRNAs binding to 5' untranslated region.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression through the binding of the 3' untranslated region (3'UTR) of specific mRNAs. MiRNAs are post-transcriptional regulators and determine the repression of translation processes or the degradation of mRNA targets. Recently, another kind of miRNA-mediated regulation of translation (repression or activation) involving the binding of miRNA to the 5'UTR of target gene has been reported. The possible interactions and the mechanism of action have been reported in many works that we reviewed here. Moreover, we discussed also the available bioinformatics tools for predicting the miRNA binding sites in the 5'UTR and public databases collecting this information.

Bioinformatics tools and novel challenges in long non-coding RNAs (lncRNAs) functional analysis.

The advent of next generation sequencing revealed that a fraction of transcribed RNAs (short and long RNAs) is non-coding. Long non-coding RNAs (lncRNAs) have a crucial role in regulating gene expression and in epigenetics (chromatin and histones remodeling). LncRNAs may have different roles: gene activators (signaling), repressors (decoy), cis and trans gene expression regulators (guides) and chromatin modificators (scaffolds) without the need to be mutually exclusive. LncRNAs are also implicated in a number of diseases. The huge amount of inhomogeneous data produced so far poses several bioinformatics challenges spanning from the simple annotation to the more complex functional annotation. In this review, we report and discuss several bioinformatics resources freely available and dealing with the study of lncRNAs. To our knowledge, this is the first review summarizing all the available bioinformatics resources on lncRNAs appeared in the literature after the completion of the human genome project. Therefore, the aim of this review is to provide a little guide for biologists and bioinformaticians looking for dedicated resources, public repositories and other tools for lncRNAs functional analysis.

FGF17, a gene involved in cerebellar development, is downregulated in a patient with Dandy-Walker malformation carrying a de novo 8p deletion.

Fibroblast growth factors (FGFs) are important signaling molecules which act during early vertebrate central nervous system development. FGF17, together with FGF8, is a key factor in the patterning of the mid-hindbrain region with a complex picture of spatiotemporal gene expression during the various stages of cerebellar development. Disruption or reduced expression of fgf17 in mice has been associated with cerebellar vermis abnormalities. We have identified a de novo 2.3-Mb deletion of chromosome 8p21.2-p21.3 in a girl with severe growth retardation, seizures, and classical Dandy-Walker malformation. Analysis of gene expression in blood lymphocytes and skin fibroblasts revealed markedly reduced levels of FGF17, which is located 1 Mb from the proximal deletion breakpoint. This is the first report of a human cerebellar malformation associated with transcriptional downregulation of the FGF17 gene.

Mirnome analysis reveals novel molecular determinants in the pathogenesis of diet-induced nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is an emerging disease with a broad spectrum of liver conditions. The complex molecular pathogenesis of NAFLD is still unclear. In this study, we conducted an analysis of microRNA (miRNA) expression profiles in liver of rats made NAFLD by different diets. To this aim, Sprague-Dawley rats were fed ad libitum for 3 months with different diets: standard diet (SD), diet enriched in fats and low in carbohydrates (HFD), SD with high fructose (SD-HF) and diet with high levels of fats and fructose (HFD-HF). Our results demonstrated that the treatment with different dietetic regimens caused a significant increase of the body weight and the alteration of some metabolic parameters compared with control animals, as well as various liver injuries. The miRNAs analysis showed the significant downregulation of three miRNAs (miR-122, miR-451 and miR-27) and the upregulation of miR-200a, miR-200b and miR-429 in HFD, SD-HF and HFD-HF rats. Besides, miR-21 expression was significantly decreased only in fructose-enriched diets. These miRNAs target molecules involved in the control of lipid and carbohydrate metabolism, signal transduction, cytokine and chemokine-mediated signaling pathway and apoptosis. Western blot analysis of PKCδ, LITAF, ALDOLASE-A, p38MAPK, PTEN, LIPIN1, EPHRIN-A1, EPHA2 and FLT1 showed a diet-induced deregulation of all these proteins. Interestingly, the expression pattern of LITAF, PTEN, LIPIN1, EPHRIN-A1, EPHA2 and FLT1 might be well explained by the trend of their specific mRNAs, by potentially regulatory miRNAs, or both. In conclusion, we highlight for the first time the potential involvement of novel determinants (miRNAs and proteins) in the molecular pathogenesis of diet-induced NAFLD.

Microarray technology: a promising tool in nutrigenomics.

Microarray technology is a powerful tool for the global evaluation of gene expression profiles in tissues and for understanding many of the factors controlling the regulation of gene transcription. This technique not only provides a considerable amount of information on markers and predictive factors that may potentially characterize a specific clinical picture, but also promises new applications for therapy. One of the most recent applications of microarrays concerns nutritional genomics. Nutritional genomics, known as nutrigenomics, aims to identify and understand mechanisms of molecular interaction between nutrients and/or other dietary bioactive compounds and the genome. Actually, many nutrigenomic studies utilize new approaches such as microarrays, genomics, and bioinformatics to understand how nutrients influence gene expression. The coupling of these new technologies with nutrigenomics promises to lead to improvements in diet and health. In fact, it may provide new information which can be used to ameliorate dietary regimens and to discover novel natural agents for the treatment of important diseases such as diabetes and cancer. This critical review gives an overview of the clinical relevance of a nutritional approach to several important diseases, and proposes the use of microarray for nutrigenomic studies.

Chitin and chitosan as multipurpose natural polymers for groundwater arsenic removal and AS2O3 delivery in tumor therapy.

Chitin and chitosan are natural polysaccharide polymers. These polymers have been used in several agricultural, food protection and nutraceutical applications. Moreover, chitin and chitosan have been also used in biomedical and biotechnological applications as drug delivery systems or in pharmaceutical formulations. So far, there are only few studies dealing with arsenic (As) removal from groundwater using chitin or chitosan and no evidence of the use of these natural polymers for arsenic trioxide (As(2)O(3)) delivery in tumor therapy. Here we suggest that chitin and/or chitosan might have the right properties to be employed as efficient polymers for such applications. Besides, nanotechnology offers suitable tools for the fabrication of novel nanostructured materials of natural origin. Since different nanostructured materials have already been employed successfully in various multidisciplinary fields, we expect that the integration of nanotechnology and natural polymer chemistry will further lead to innovative applications for environment and medicine.

Quantification of small non-coding RNAs allows an accurate comparison of miRNA expression profiles.

MicroRNAs (miRNAs) are highly conserved approximately 22-mer RNA molecules, encoded by plants and animals that regulate the expression of genes binding to the 3'-UTR of specific target mRNAs. The amount of miRNAs in a total RNA sample depends on the recovery efficiency that may be significantly affected by the different purification methods employed. Traditional approaches may be inefficient at recovering small RNAs, and common spectrophotometric determination is not adequate to quantify selectively these low molecular weight (LMW) species from total RNA samples. Here, we describe the use of qualitative and quantitative lab-on-a-chip tools for the analysis of these LMW RNA species. Our data emphasize the close correlation of LMW RNAs with the expression levels of some miRNAs. We therefore applied our result to the comparison of some miRNA expression profiles in different tissues. Finally, the methods we used in this paper allowed us to analyze the efficiency of extraction protocols, to study the small (but significant) differences among various preparations and to allow a proper comparison of some miRNA expression profiles in various tissues.

Computational challenges in miRNA target predictions: to be or not to be a true target?

All microRNA (miRNA) target--finder algorithms return lists of candidate target genes. How valid is that output in a biological setting? Transcriptome analysis has proven to be a useful approach to determine mRNA targets. Time course mRNA microarray experiments may reliably identify downregulated genes in response to overexpression of specific miRNA. The approach may miss some miRNA targets that are principally downregulated at the protein level. However, the high-throughput capacity of the assay makes it an effective tool to rapidly identify a large number of promising miRNA targets. Finally, loss and gain of function miRNA genetics have the clear potential of being critical in evaluating the biological relevance of thousands of target genes predicted by bioinformatic studies and to test the degree to which miRNA-mediated regulation of any "validated" target functionally matters to the animal or plant.

CC chemokine ligand 2 down-modulation by selected Toll-like receptor agonist combinations contributes to T helper 1 polarization in human dendritic cells.

Toll-like receptor (TLR) signaling activation by pathogens is critical to the induction of immune responses, and demands tight regulation. We describe in this study that CC chemokine ligand 2 (CCL2) secretion triggered by TLR4 or TLR8 engagement is strongly inhibited upon simultaneous activation of both TLRs in human monocyte-derived dendritic cells (DCs). Impaired CCL2 secretion occurs concomitantly to interleukin-12 up-regulation, being part of a complex regulatory circuit ensuring optimal T helper type 1 polarization. Interestingly, triggering selected TLRs or their combinations differently affects nuclear factor-kappaB p65 activation and microRNA expression. Overall, these results indicate that CCL2 supplies an important immunomodulatory role to DCs, and may contribute to dictate the cytokine profile in T helper type 1 responses induced by DCs.