Sistema di diagnosi collaborativa per cardiologia pediatrica

In questo rapporto viene dimostrato come è possibile costruire, partendo da tecnologie COTS, un sistema di telemedicina in grado di permettere ad un gruppo di esperti localizzati centralmente di fornire a personale clinico remoto supporto e guida in tempo reale per procedure ecografiche complesse. La prima applicazione del sistema è nella ecocardiologia neonatale, in particolare per la valutazione della pericolosità di potenziali patologie cardiache in neonati immediatamente dopo il parto

The combination of transcriptomics and informatics identifies pathways targeted by miR-204 during neurogenesis and axon guidance

Vertebrate organogenesis is critically sensitive to gene dosage and even subtle variations in the expression levels of key genes may result in a variety of tissue anomalies. MicroRNAs (miRNAs) are fundamental regulators of gene expression and their role in vertebrate tissue patterning is just beginning to be elucidated. To gain further insight into this issue, we analysed the transcriptomic consequences of manipulating the expression of miR-204 in the Medaka fish model system. We used RNA-Seq and an innovative bioinformatics approach, which combines conventional differential expression analysis with the behavior expected by miR-204 targets after its overexpression and knockdown. With this approach combined with a correlative analysis of the putative targets, we identified a wider set of miR-204 target genes belonging to different pathways. Together, these approaches confirmed that miR-204 has a key role in eye development and further highlighted its putative function in neural differentiation processes, including axon guidance as supported by in vivo functional studies. Together, our results demonstrate the advantage of integrating next-generation sequencing and bioinformatics approaches to investigate miRNA biology and provide new important information on the role of miRNAs in the control of axon guidance and more broadly in nervous system development. \uc2\ua9 The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research

The BioMart community portal: an innovative alternative to large, centralized data repositories.

The BioMart Community Portal (www.biomart.org) is a community-driven effort to provide a unified interface to biomedical databases that are distributed worldwide. The portal provides access to numerous database projects supported by 30 scientific organizations. It includes over 800 different biological datasets spanning genomics, proteomics, model organisms, cancer data, ontology information and more. All resources available through the portal are independently administered and funded by their host organizations. The BioMart data federation technology provides a unified interface to all the available data. The latest version of the portal comes with many new databases that have been created by our ever-growing community. It also comes with better support and extensibility for data analysis and visualization tools. A new addition to our toolbox, the enrichment analysis tool is now accessible through graphical and web service interface. The BioMart community portal averages over one million requests per day. Building on this level of service and the wealth of information that has become available, the BioMart Community Portal has introduced a new, more scalable and cheaper alternative to the large data stores maintained by specialized organizations

Mutant uromodulin expression leads to altered homeostasis of the endoplasmic reticulum and activates the unfolded protein response

<div><p>Uromodulin is the most abundant urinary protein in physiological conditions. It is exclusively produced by renal epithelial cells lining the thick ascending limb of Henle’s loop (TAL) and it plays key roles in kidney function and disease. Mutations in <i>UMOD</i>, the gene encoding uromodulin, cause autosomal dominant tubulointerstitial kidney disease uromodulin-related (ADTKD-<i>UMOD</i>), characterised by hyperuricemia, gout and progressive loss of renal function. While the primary effect of <i>UMOD</i> mutations, retention in the endoplasmic reticulum (ER), is well established, its downstream effects are still largely unknown. To gain insight into ADTKD-<i>UMOD</i> pathogenesis, we performed transcriptional profiling and biochemical characterisation of cellular models (immortalised mouse TAL cells) of robust expression of wild type or mutant GFP-tagged uromodulin. In this model mutant uromodulin accumulation in the ER does not impact on cell viability and proliferation. Transcriptional profiling identified 109 genes that are differentially expressed in mutant cells relative to wild type ones. Up-regulated genes include several ER resident chaperones and protein disulphide isomerases. Consistently, pathway enrichment analysis indicates that mutant uromodulin expression affects ER function and protein homeostasis. Interestingly, mutant uromodulin expression induces the Unfolded Protein Response (UPR), and specifically the IRE1 branch, as shown by an increased splicing of XBP1. Consistent with UPR induction, we show increased interaction of mutant uromodulin with ER chaperones Bip, calnexin and PDI. Using metabolic labelling, we also demonstrate that while autophagy plays no role, mutant protein is partially degraded by the proteasome through ER-associated degradation. Our work demonstrates that ER stress could play a central role in ADTKD-<i>UMOD</i> pathogenesis. This sets the bases for future work to develop novel therapeutic strategies through modulation of ER homeostasis and associated protein degradation pathways.</p></div

Whole transcriptome characterization of aberrant splicing events induced by lentiviral vector integrations

Gamma-retroviral/lentiviral vectors (γRV/LV) with self-inactivating (SIN) long terminal repeats (LTRs) and internal moderate cellular promoters pose a reduced risk of insertional mutagenesis when compared with vectors with active LTRs. Yet, in a recent LV-based clinical trial for β-thalassemia, vector integration within the HMGA2 gene induced the formation of an aberrantly spliced mRNA form that appeared to cause clonal dominance. Using a method that we developed, cDNA linear amplification-mediated PCR, in combination with high-throughput sequencing, we conducted a whole transcriptome analysis of chimeric LV-cellular fusion transcripts in transduced human lymphoblastoid cells and primary hematopoietic stem/progenitor cells. We observed a surprising abundance of read-through transcription originating outside and inside the provirus and identified the vector sequences contributing to the aberrant splicing process. We found that SIN LV has a sharply reduced propensity to engage in aberrant splicing compared with that of vectors carrying active LTRs. Moreover, by recoding the identified vector splice sites, we reduced residual read-through transcription and demonstrated an effective strategy for improving vectors. Characterization of the mechanisms and genetic features underlying vector-induced aberrant splicing will enable the generation of safer vectors, with low impact on the cellular transcriptome

Transcriptome analysis in mTAL cells expressing wild type or C150S mutant uromodulin.

<p>(A) Heat map showing differentially expressed genes in mTAL cells expressing C150S uromodulin compared to wild type ones. Cut off: fold change > 1.5; P adjusted < 0.05. (B) STRING analysis showing networks formed by proteins encoded by up-regulated genes in mTAL cells expressing C150S uromodulin. Edges represent protein-protein association (physical or functional); their thickness is proportional to confidence. (C) Same analysis as in panel B for down-regulated genes. The proteins encoded by these genes are not forming relevant networks.</p

Characterisation of mTAL cells expressing wild type or mutant uromodulin isoforms.

<p>(A) Live imaging showing GFP signal in mTAL cells expressing GFP-tagged WT or C150S mutant uromodulin isoform. Bar = 40 μm. (B) Uromodulin expression assessed by real-time RT-qPCR. Expression is normalised to <i>Hprt1</i>. Cells expressing GFP alone are shown as negative control (n = 5 independent experiments) (C) Western-blot analysis of mTAL cells expressing WT or C150S mutant uromodulin isoform. * indicates the ER glycosylated form that is Endo H sensitive (see panel below). (D) Immunofluorescence analysis of mTAL cells expressing GFP-tagged uromodulin isoforms. GFP signal is shown in green. Calreticulin, used as an ER marker, is shown in red. Merged pictures show ER localisation of mutant uromodulin while the wild type protein is trafficked to the membrane. Bar = 40 μm.</p

UPR induction in MDCK cells expressing wild type or C150S uromodulin isoforms.

<p>(A) <i>BIP</i> and <i>XBP1S</i> expression assessed by real-time RT-qPCR. Expression is normalised to <i>HPRT1</i>. *P<0.05, **P<0.01 (Student t test) (n = 5 independent experiments). (B) Western blot analysis of PERK in MDCK cells expressing wild type or C150S mutant uromodulin. A shift in PERK migration is seen upon tunicamycin treatment (2 μg/mL for 14 h), but not at baseline. (C) ATF6 activation assessed through the use of an ATF6 reporter construct. No ATF6 activation is observed in mutant uromodulin expressing cells. Activation can be observed in all cell lines upon tunicamycin treatment. ***P<0.005 (control <i>vs</i> tunicamycin-treated, Student t test) (n = 6 independent experiments).</p