Spotlight on zebrafish:translational impact

In recent years, the zebrafish has emerged as an increasingly prominent model in biomedical research. To showcase the translational impact of the model across multiple disease areas, Disease Models & Mechanisms has compiled a Special Issue that includes thought-provoking reviews, original research reporting new and important insights into disease mechanisms, and novel resources that expand the zebrafish toolkit. This Editorial provides a summary of the issue’s contents, highlighting the diversity of zebrafish disease models and their clinical applications

Gα16, a G Protein α Subunit Specifically Expressed in Hematopoietic Cells

Signal-transduction pathways mediated by guanine nucleotide-binding regulatory proteins (G proteins) determine many of the responses of hematopoietic cells. A recently identified gene encoding a G protein α subunit, Gα16, is specifically expressed in human cells of the hematopoietic lineage. The Gα16 cDNA encodes a protein with predicted Mr of 43,500, which resembles the Gq class of α subunits and does not include a pertussis toxin ADP-ribosylation site. In comparison with other G protein α subunits, the Gα16 predicted protein has distinctive amino acid sequences in the amino terminus, the region A guanine nucleotide-binding domain, and in the carboxyl-terminal third of the protein. Cell lines of myelomonocytic and T-cell phenotype express the Gα16 gene, but no expression is detectable in two B-cell lines or in nonhematopoietic cell lines. Gα16 gene expression is down-regulated in HL-60 cells induced to differentiate to neutrophils with dimethyl sulfoxide. Antisera generated from synthetic peptides that correspond to two regions of Gα16 specifically react with a protein of 42- to 43-kDa in bacterial strains that overexpress Gα16 and in HL-60 membranes. This protein is decreased in membranes from dimethyl sulfoxide-differentiated HL-60 cells and is not detectable in COS cell membranes. The restricted expression of this gene suggests that Gα16 regulates cell-type-specific signal-transduction pathways, which are not inhibited by pertussis toxin

Distinct forms of the ß subunit of GTP-binding regulatory proteins identified by molecular cloning

Two distinct β subunits of guanine nucleotide-binding regulatory proteins have been identified by cDNA cloning and are referred to as β 1 and β 2 subunits. The bovine transducin β subunit (β 1) has been cloned previously. We have now isolated and analyzed cDNA clones that encode the β 2 subunit from bovine adrenal, bovine brain, and a human myeloid leukemia cell line, HL-60. The 340-residue Mr 37,329 β 2 protein is 90% identical with β 1 in predicted amino acid sequence, and it is also organized as a series of repetitive homologous segments. The major mRNA that encodes the bovine β 2 subunit is 1.7 kilobases in length. It is expreβed at lower levels than β 1 subunit mRNA in all tiβues examined. The β 1 and β 2 meβages are expreβed in cloned human cell lines. Hybridization of cDNA probes to bovine DNA showed that β 1 and β 2 are encoded by separate genes. The amino acid sequences for the bovine and human β 2 subunit are identical, as are the amino acid sequences for the bovine and human β 1 subunit. This evolutionary conservation suggests that the two β subunits have different roles in the signal transduction process

Sviluppo di un sistema tridimensionale di coltura come modello preclinico di carcinoma del colon

Negli ultimi anni lo studio della biologia tumorale ha subito un progressivo sviluppo e molti aspetti fisiologici del microambiente tumorale sono stati chiariti, ma molto resta ancora da analizzare circa le modalità con cui il tumore interagisce con l’ambiente circostante e quali siano le caratteristiche molecolari di questo microambiente. Dal punto di vista tecnico, le colture cellulari bidimensionali (2D) riflettono solo parzialmente il pattern morfo-molecolare delle cellule tumorali umane ed inoltre non rispecchiano la complessità del microambiente in vivo. Nei sistemi in vivo, inoltre, lo sviluppo neoplastico differisce significativamente rispetto alle cellule coltivate in 2D, soprattutto per quanto riguarda la morfologia, la cinetica di crescita, l’espressione genica ed il grado di differenziazione. In questo scenario, le colture cellulari tridimensionali (3D) costituiscono un approccio alternativo e/o parallelo al 2D, esse sono dunque il punto di legame tra la coltura cellulare tradizionale e i modelli in vivo. L’utilizzo di sferoidi uni-cellulari e multi-cellulari si è rivelato un sistema efficiente per ottimizzare e superare le limitazioni legate ai sistemi convenzionali in vitro. Lo sferoide è costituito da cellule proliferanti nello strato periferico, che rispecchiano l’attivo turn-over delle cellule tumorali vicino ai capillari, e da cellule degli strati più interni che diventano, invece, quiescenti ed eventualmente muoiono, via apoptosi o necrosi, a causa della presenza di ipossia, man mano che il diametro dello sferoide aumenta. Al di sopra di 400-500µm di diametro si forma, infatti, un core necrotico, principalmente a causa della limitata diffusione di ossigeno e/o di nutrienti e all’accumulo di cataboliti e tossine. Tuttavia, nello studio di alcune tipologie neoplastiche, come ad esempio il carcinoma del colon, tale caratteristica potrebbe rappresentare un punto a favore più che un ostacolo, dal momento che la normale fisiologia del carcinoma del colon prevede la presenza di aree di tessuto altamente ipossiche e di tessuto necrotico. Al fine di creare un avanzato sistema 3D per la coltura di cellule del cancro del colon-retto in vitro, abbiamo comparato diversi metodi di coltura basati sulla tecnica “hanging drops”, cercando di ottimizzarli fino ad ottenere quello più efficiente per le nostre necessità e che mantenesse dimensioni e fenotipo costanti ed omogenee. Abbiamo inoltre valutato l’impatto di tali tecniche su linee cellulari di CRC inserite nel pannello di 60 linee cellulari del NCI, comparando fenotipo ed espressione genica delle colture in 3D rispetto alle tecniche di coltura tradizionali. Inoltre abbiamo analizzato il contributo della componente stromale sulla formazione e crescita degli sferoidi, utilizzando BM-MSC in co-coltura tridimensionale con le linee cellulari di CRC. I nostri risultati mettono in luce come l'istituzione di sistemi di coltura per una migliore integrazione tra le strutture in 3D, e maggiormente tra le cellule tumorali e la componente stromale, risulti fondamentale per uno studio più accurato della progressione tumorale, ma soprattutto per lo sviluppo e la validazione di nuovi farmaci antitumorali, che in questo modo possono esser testati anche su porzioni di tessuto che presentano ipossia e necrosi, andandone così a valutare la capacità di penetrazione in tutto il tessuto e la reale efficacia

Exploring the role of hypoxia in neuroinflammatory disease and the use of oxygen as a therapy

Multiple sclerosis (MS) is an inflammatory-demyelinating disease of the central nervous system (CNS). Pathological studies have revealed that MS lesions can have hypoxia- like properties, raising the possibility that the inflamed CNS may suffer an energy deficit. We recently demonstrated that the spinal cord of rats with active experimental autoimmune encephalomyelitis (EAE, a model of MS) is hypoxic, and that hypoxia can be reversed by inspiring oxygen-enriched air. This thesis examines the contribution of hypoxia to the neurological deficits, and the use of oxygen as a therapy. Demyelinating and non-demyelinating models of EAE in rats were evaluated, namely active and passive EAE respectively. Room air controls were used for comparison. Assessment of neurological deficits in active EAE revealed that oxygen (95%) promptly improved neurological function in paralysed rats, within only 1 hour of exposure. Furthermore, prolonged administration of oxygen (75%) applied either prophylactically (from the day of immunisation for 23 days) or therapeutically from the onset of disease (for 24, 48 or 72 hours) produced a greater and long-lasting amelioration of disease severity. Interestingly, oxygen treatment from disease onset reduced oligodendrocyte cell-stress and death, demyelination, microglial activation and macrophage infiltration in the spinal cord, without exacerbating oxidative damage. The protective effect was proportional to the duration of the treatment and significant in rats treated for 72 hours. Other experiments have revealed that the spinal cord of rats with passive EAE is also hypoxic and oxygen treatment significantly ameliorated disease progression when administered prophylactically. We also tested polynitroxylated pegylated hemoglobin (PNPH) as an alternative oxygen-based treatment in active EAE, revealing an acute improvement of the neurological function 1 hour after the injection of PNPH. We conclude that hypoxia contributes to neurological deficits and demyelination in inflammatory autoimmune demyelinating disease, and that oxygen therapy can reduce both the deficits and the demyelination

Small Fish, Big Science

The European Union recently awarded 12 million Euros to the ZF-MODELS research consortium to study zebrafish models for human development and diseas

Multiscale modelling of stent/vessel interactions

Angioplasty with stenting re-opens stenosed arteries, but in-stent restenosis remains a common negative outcome. Correlations between local mechanical stimuli and ISR have been reported, explained by mechanotransduction mechanisms that influence cell behaviour. This thesis investigates the loads imposed on the coronary artery following stent implantation. The changes in mechanical stimuli in a vessel following stent deployment were initially considered using a simple MATLAB model, followed by analysis of a 2D cross-section model to represent variation of stress with stent strut distribution. This model revealed the distribution of the stress through the thickness and around the circumference varied significantly for high expansion ratios and uneven strut distributions. A 3D continuum model of stent geometry post-expansion, obtained from micro-CT images, was used to analyse stent interaction with an idealised vessel geometry. The structural stress at the level of individual struts was compared to histological data and to fluid dynamic simulations of the same stent/vessel geometry. When structural and fluid dynamic stimuli were considered together, correlations with the amount of neointimal growth became more significant than when they were considered individually. This suggests that both stimuli contribute to the development of neointimal growth and their combination may accelerate the progression of ISR. Finally, the thesis describes a model of the evolution of in-stent restenosis. A cellular model of growth was developed to include feedback from a finite element model of the vessel and neointima. Change in the lumen geometry was captured with neointimal growth, an updated geometry was passed to the finite element model to compute the subsequent change of stress direction on cells during their growth. The results show encouraging resemblance with histological images of ISR, especially for the early phases of growth. The thesis concludes with a summary of the modelling results and a review of opportunities for further research

Validation of Neural Network-based Fault Diagnosis for Multi-stack Fuel Cell Systems: Stack Voltage Deviation Detection☆

Abstract This paper presents (i) an algorithm for the detection of unexpected stack voltage deviations in an Solid Oxide Fuel Cells (SOFC)-based power system with multiple stacks and (ii) its validation in a simulated online environment. The algorithm is based on recurrent neural networks (RNNs) and is validated by using operating data from the Wartsila WFC20 multi-stack SOFC system. The voltage deviation detection is based on statistical testing. Instead of a hardware implementation in the actual power plant, the algorithm is validated in a simulated online environment that provides data I/O communication based on the OPC (i.e. Object Linking and Embedding (OLE) for Process Control) protocol, which is also the technology utilized in the real hardware environment. The validation tests show that the RNN-based algorithm effectively detects unwanted stack voltage deviations and also that it is online-viable

The second European interdisciplinary Ewing sarcoma research summit – A joint effort to deconstructing the multiple layers of a complex disease

Despite multimodal treatment, long term outcome for patients with Ewing sarcoma is still poor. The second “European interdisciplinary Ewing sarcoma research summit” assembled a large group of scientific experts in the field to discuss their latest unpublished findings on the way to the identification of novel therapeutic targets and strategies. Ewing sarcoma is characterized by a quiet genome with presence of an EWSR1-ETS gene rearrangement as the only and defining genetic aberration. RNA sequencing of recently described Ewing-like sarcomas with variant translocations identified them as biologically distinct diseases. Various presentations adressed mechanisms of EWS-ETS fusion protein activities with a focus on EWS-FLI1. Data were presented shedding light on the molecular underpinnings of genetic permissiveness to this disease uncovering interaction of EWS-FLI1 with recently discovered susceptibility loci. Epigenetic context as a consequence of the interaction between the oncoprotein, cell type, developmental stage, and tissue microenvironment emerged as dominant theme in the discussion of the molecular pathogenesis and inter- and intratumor heterogeneity of Ewing sarcoma, and the difficulty to generate animal models faithfully recapitulating the human disease. The problem of preclinical development of biologically targeted therapeutics was discussed and promising perspectives were offered from the study of novel in vitro models. Finally, it was concluded that in order to facilitate rapid pre-clinical and clinical development of novel therapies in Ewing sarcoma, the community needs a platform to maintain knowledge of unpublished results, systems and models used in drug testing and to continue the open dialogue initiated at the first two Ewing sarcoma summits.Unión Europea 261743(ENCCA)Unión Europea 259348 (ASSET