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Applicazione della genomica funzionale allo studio di specie di interesse in acquacoltura
Full text linkIn the last 10-15 years, the production of farmed marine fish species has increased rapidly, mainly as a consequence of improved breeding and husbandry methods and technologies. However, severe production bottlenecks, such as high larval mortality, skeletal malformations, susceptibility to stress and disease, remain to be solved. The application of genomic technologies offers much promise to rapidly increase our knowledge on molecular mechanisms underlying key productive traits in farmed species.
In the present study, many efforts were invested in order to provide genomic tools for the study of two fish species of great importance for the European aquaculture; the gilthead seabream (Sparus aurata) and the European sea bass (Dicentrarchus labrax¬). To this end, we report here on the development of two oligo DNA microarray for both S. aurata and D. labrax.
All the available ESTs from the gilthead sea bream were used to design two longer (60mer) probes for each transcript using Agilent SurePrintTM technology. This microarray platform was then validated to assess its reproducibility and accuracy on two early stages of gilthead sea bream development, respectively one-day and four days old larvae. Correlation between technical replicates was always > 0.99, with strong positive correlation between paired probes. Cross-validation of microarray data was then carried out using quantitative (q)RT-PCR; a statistically significant positive correlation was obtained comparing expression levels for each target gene across all biological replicates.
For the European sea bass, probe design was positively completed for 19,035 target transcripts; high levels of reliability, and reproducibility of the microarray platform were confirmed by qPCR validation and biological replicates analysis. The oligo microarray was then applied to profile gene expression in mandibles and whole-heads of fish affected by prognathism, a skeletal malformation that strongly affect sea bass production. Statistical analysis identified 242 transcripts that are significantly down-regulated in deformed individuals compared to normal fish. Functional annotation of significant genes gives new insights on putative mechanisms involved on mandibular prognathism development.
In this study we describe also the identification and characterisation of a NITR gene complex D. labrax, encoded in a single genomic region spanning 270 kilo bases (kb). In total, 27 NITR genes and 3 pseudogenes, organized in a tandemly arrayed cluster, were identified. Comparison of the predicted peptide structure across D. labrax NITRs shows considerable variations; these genes maintain the three major genomic organizations that appear to be essentially conserved among fish species along with new features presumably involving processes of intron loss, exon deletion, and acquisition on new exons. Comparative analyses of all published NITR genes suggest that these receptors follow birth-and-death model of gene evolution in which duplication events together with lineage-specific gain and loss of individual members contributed to the rapid diversification of individual gene familiesNegli ultimi 10-15 anni, la produzione di specie marine in acquacoltura è cresciuta molto rapidamente; diverse limitazioni, tuttavia, non sono ancora state superate (per es. elevata mortalità larvale, suscettibilità a stress e patologie, sviluppo di malformazioni scheletriche). La genomica funzionale può fornire gli strumenti per una migliore conoscenza dei meccanismi molecolari responsabili di tratti produttivi di forte interesse economico nelle specie allevate.
Nel presente studio, numerosi sforzi sono stati investiti allo scopo di sviluppare e sfruttare strumenti di genomica funzionale per lo studio di due specie di primaria importanza per l’acquacoltura europea, l'orata (Sparus aurata) e il branzino (Dicentrarchus labrax¬); di seguito viene riportato lo sviluppo di due piattaforme microarray a oligonucleotidi per entrambe le specie.
Tutti i trascritti disponibili di orata sono stati utilizzati per la sintesi di due sonde a oligonucleotidi (60meri) per ciascun trascritto utilizzando la tecnologia Agilent SurePrintTM. La riproducibilità e l’accuratezza della piattaforma microarray sono state quindi testate in due stadi precoci di sviluppo di orata: due e quattro giorni dalla schiusa. La correlazione tra le repliche tecniche si è sempre rivelata elevata (r >0,99) e forte correlazione è stata osservata anche tra le coppie di sonde per ciascun trascritto. La Real-time RT-PCR è stata quindi utilizzata per cross-validare i dati di espressione del microarray; anche in questo caso è stata ottenuta una correlazione statisticamente significativa per ciascun gene target in tutte le repliche biologiche.
Per il branzino, il disegno delle sonde è stato effettuato per 19.035 trascritti target; l’elevata affidabilità e riproducibilità della piattaforma è stata confermata tramite Real-time RT-PCR e analisi delle repliche biologiche. Il microarray è stato quindi impiegato per analizzare i profili di espressione genica in mandibole e teste di esemplari di branzino affetti da prognatismo, una malformazione scheletrica che ne compromette pesantemente la produzione e la commerciabilità. Analisi statistiche hanno permesso di identificare 242 trascritti sottoespressi negli esemplari prognati rispetto ai controlli; una annotazione funzionale di questi fornisce nuove informazioni sui potenziali meccanismi coinvolti nello sviluppo di questa malformazione.
Nel presente studio viene anche descritta l’identificazione e la caratterizzazione, in branzino, dei recettori Novel Immune-type Receptor (NITR). In totale sono stati identificati 27 geni e 3 pseudogeni, organizzati in cluster in una regione genomica di circa 270 kb. I membri di questo cluster sono caratterizzati da una elevata variabilità; i NITR, infatti, mantengono tre organizzazioni genomiche principali, che appaiono conservate in diverse specie di teleostei, oltre a forme nuove che coinvolgono processi di perdita di introni, delezioni di esoni o loro nuova acquisizione. Analisi comparative tra tutti i geni NITR finora noti sembrano suggerire che questi seguano il modello evolutivo noto col nome di “birth-and-death”, nel quale eventi di duplicazione, assieme alle perdite/acquisizioni di singoli membri, contribuiscono alla rapida evoluzione di diverse famiglie geniche
LESIONI INDOTTE DA CMV A LIVELLO DEL TRATTO GASTROINTESTINALE IN 45 PRIMATI XENOTRAPIANTATI
No full textIntroduzione: Lo xenotrapianto potrebbe rappresentare un’opzione terapeutica alla grave carenza di organi e studi sono in corso per minimizzare il rischio di infezioni legato alla immunosoppressione. In particolare, il cytomegalovirus (CMV) nei primati xenotrapiantati e immunosoppressi è una causa ben nota di patologia che coinvolge soprattutto l’apparato gastroenterico.
Materiali e metodi: I tessuti provenienti da 45 Macaca fascicularis immunosoppresse e xenotrapiantate sono stati analizzati istologicamente e tramite esame immunoistochimico (IHC) utilizzando un anticorpo specifico per una proteina della fase immediate-early 1 del CMV di Macaca Rhesus (RhCMV), una specie filogenicamente molto vicina a Macaca fascicularis. Altre analisi hanno compreso i test sierologici per RhCMV e l’analisi molecolare dei tessuti colpiti dall’infezione.
Risultati: 5/45 primati hanno evidenziato le caratteristiche lesioni da CMV del tratto gastroenterico, tra cui enterite neutrofilica con corpi inclusi. Altri 3 primati invece hanno mostrato una positività specifica per CMV solo a livello IHC. L’esame sierologico ha evidenziato positività in tutti gli animali per la presenza di anticorpi per CMV, in seguito all’utilizzo di un test specifico per RHCMV. Lo studio molecolare dei tessuti con lesioni istopatologiche da CMV ha confermato, attraverso amplificazione e sequenziamento del DNA, la presenza di CMV della specie ricevente, Macaca fascicularis.
Discussione e conclusioni: nei primati immunosoppressi e trapiantati con diarrea, le infezioni da CMV non devono essere sottovalutate. Le infezioni devono essere indagate con strumenti appropriati e specifici per primati non umani, e quindi trattate con terapia antivirale mirata
Real time RT-PCR analysis of inflammatory mediator expression in recurrent airway obstruction-affected horses
No full textEvaluation of epigenetic mechanisms regulating HOXD10, FGFR2 and ITIH5 gene expression in canine B-cell lymphoma: an in vitro approach.
No full textIntroduction. Despite B-cell Lymphoma (BCL) is the most common canine hematological cancer, there is still a lack of knowledge on molecular events contributing to its development and progression. Aim of this work was to investigate the epigenetic regulation of HOXD10, FGFR2 and ITIH5 genes, whose promoters were recently shown to be methylated, by genome-wide methylation profiling, in canine diffuse large BCL. To understand the role of methylation in silencing aforementioned genes, the CLBL-1 cell line was treated with two hypomethylating drugs (HDs). Owing to complexity of epigenetic mechanisms, histone deacetylase inhibitors (HDACis), alone or in combination with HDs, were also tested.
Materials and methods. CLBL-1 cells were incubated with two HDs (azacytidine and decitabine), alone or in combination with HDACis (valproic acid, trichostatin and vorinostat), at concentrations corresponding to their IC50 and IC20 values (Alamar blue test), respectively. Then, gene methylation status and mRNA levels were measured using methyl sensitive PCR (MSP) and quantitative Real Time PCR (qPCR).
Results. MSP showed an overall decrease of gene methylation status following the incubation with both HDs; meantime, qPCR highlighted a reversion of gene expression, confirming a methylation-dependent gene silencing mechanism. Interestingly, a higher pattern of gene re-expression was observed following the exposure to HDs combined with HDACis (and, mostly, with valproic acid). Moreover, HDACis alone increased the expression of FGFR2, demonstrating the important contribution of histone deacetylation, above methylation, in the regulation of this gene
Evolutionary analysis of Antarctic teleost Toll-like receptor 2.
No full textThe nucleotide sequence of TLR2 has been determined in both species, encoding 20 leucine-rich repeats (LRRs) in the extracellular region and a classical Toll/IL-1R (TIR) domain in the intracellular region. High expression level of T. bernacchii TLR2 was found in spleen and skin. Using different methods we identified six codons that underwent Darwinian selection while 20 were found to be negatively selected. Molecular models of C. hamatus and T bernacchii TLR2 ectodomain as well as of the TIR domain were built by Homology Modeling. Molecular Dynamics simulations were performed in water for 15 ns. The sites under positive selection were residing on the convex side of the solenoid, four out of six were in a 35-residue-long region including the central/N-terminal domain boundary: two in the external loop of LRR11 and the other two in the LRR12 loop. This region has been demonstrated to be the functional site of ligand interaction in human TLR2 structure. Antarctic TLR2 models showed more flexibility than TLR2 from the temperate species Gasterosteus aculeatus. These results suggest that the selective pressure has shaped TLR2 molecule in such a way that increased its activity under the peculiar Antarctic environmental conditions
Global analysis of gene expression in mineralizing fish vertebra-derived cell lines: new insights into anti-mineralogenic effect of vanadate
Full text linkBackground: Fish has been deemed suitable to study the complex mechanisms of vertebrate skeletogenesis and gilthead seabream (Sparus aurata), a marine teleost with acellular bone, has been successfully used in recent years to study the function and regulation of bone and cartilage related genes during development and in adult animals. Tools recently developed for gilthead seabream, e. g. mineralogenic cell lines and a 4 x 44K Agilent oligo array, were used to identify molecular determinants of in vitro mineralization and genes involved in anti-mineralogenic action of vanadate.
Results: Global analysis of gene expression identified 4,223 and 4,147 genes differentially expressed (fold change - FC > 1.5) during in vitro mineralization of VSa13 (pre-chondrocyte) and VSa16 (pre-osteoblast) cells, respectively. Comparative analysis indicated that nearly 45% of these genes are common to both cell lines and gene ontology (GO) classification is also similar for both cell types. Up-regulated genes (FC > 10) were mainly associated with transport, matrix/membrane, metabolism and signaling, while down-regulated genes were mainly associated with metabolism, calcium binding, transport and signaling. Analysis of gene expression in proliferative and mineralizing cells exposed to vanadate revealed 1,779 and 1,136 differentially expressed genes, respectively. Of these genes, 67 exhibited reverse patterns of expression upon vanadate treatment during proliferation or mineralization.
Conclusions: Comparative analysis of expression data from fish and data available in the literature for mammalian cell systems (bone-derived cells undergoing differentiation) indicate that the same type of genes, and in some cases the same orthologs, are involved in mechanisms of in vitro mineralization, suggesting their conservation throughout vertebrate evolution and across cell types. Array technology also allowed identification of genes differentially expressed upon exposure of fish cell lines to vanadate and likely involved in its anti-mineralogenic activity. Many were found to be unknown or they were never associated to bone homeostasis previously, thus providing a set of potential candidates whose study will likely bring insights into the complex mechanisms of tissue mineralization and bone formation
Molecular biomarkers of phospholipidosis in rat blood and heart after amiodarone treatment
No full textPhospholipidosis (PLD) is characterized by an intracellular accumulation of phospholipids in lysosomes and concurrent
development of concentric lamellar bodies. It is induced in humans and in animals by drugs with a cationic amphiphilic
structure. The purpose of the present study was to identify a set of molecular biomarkers of PLD in rat blood and heart, hypothetically applicable in preclinical screens within the drug development process. A toxicological study was set up in rats orally treated up to 11 days with 300 mg/kg/day amiodarone (AMD). Light and transmission electronmicroscopy investigations
were performed to confirm the presence of lamellar bodies indicative of phospholipid accumulation. The effects ofAMD upon the transcriptome of these tissues were estimated using DNA microarray technology. Microarray data analysis showed that a total of 545 and 8218 genes weremodulated byAMD treatment in heart and blood, respectively. Some genes implicated in the phospholipid accumulation in cells, such as phospholipase A2, showed similar alterations of gene expression. After transcriptome criteria of analysis and target selection, including also the involvement in the onset of PLD, 7 genes (Pla2g2a, Pla2g7, Gal, Il1b, Cebpb, Fcgr2b, Acer 2) were selected as candidate biomarkers of PLD in heart and blood tissues, and their potential usefulness as a sensitive screening test was screened and confirmed by quantitative Real-Time PCR analysis. Collectively, these data underscore the importance of transcriptional profiling in drug discovery and development, and suggest blood as a surrogate tissue for possible phospholipid accumulation in cardiomyocytes
CARATTERIZZAZIONE IMMUNOISTOCHIMICA E MOLECOLARE DI DISORDINI LINFOPROLIFERATIVI IN PRIMATI XENOTRAPIANTATI CON PRECURSORI NEURONALI
No full textIntroduzione: I disordini linfoproliferativi post-trapianto (PTLD) rappresentano un gruppo di malattie che insorgono in corso d’immunosoppressione dopo il trapianto (Tx). La maggior parte dei PTLD nell’uomo e nel primate sono dovuti alla presenza di Epstein-Barr virus e Lymphocryptovirus (MacLCV).
Materiali e Metodi: 28 adulti di Macaca fascicularis con malattia di Parkinson farmacologicamente indotta hanno ricevuto precursori neuronali di suini transgenici per CTLA4Ig (n=7) e wild-type (n=2).. La caratterizzazione di PTLD ha riguardato aspetti morfologici, immunoistochimici (IHC) e molecolari.
Risultati: i PTLD sono stati diagnosticati in 9 animali, ad una distanza media di 172,7 giorni dal tx.. Le neoplasie erano localizzate nelle cavità nasali (n=3), nell’intestino (n=4) e in entrambe le sedi (n=2). Istologia e IHC hanno rivelato un PTLD monomorfo (linfoma a grandi cellule B) di grado elevato. IHC a doppia marcatura per EBNA2 e CD20 ha dimostrato un’elevata percentuale di cellule neoplastiche CD20+ e EBNA2+. RT-PCR e sequenziamento dei trascritti di EBNA-1, EBNA-2 e LMP-1 eseguiti su 5 primati PTLD+ hanno evidenziato che EBNA-1 e EBNA-2 sono sempre presenti, mentre l’espressione di LMP1 è assente. Il DNA di MacLCV è stato isolato in tutti i primati riceventi ela viremia è stata evidenziata circa 60 gg prima dei sintomi clinici.
Conclusioni: lo studio identifica un pattern di espressione dei trascritti virali nel PTLD associato a MacLCV: EBNA-1+ EBNA-2+ LMP-1-. Nel ricevente si evidenzia un picco viremico di MacLCV prima dell’insorgenza del PLTD. Tale test potrebbe essere utilizzato come strumento precoce per la diagnosi di PTLD
Expression profiling of skeletal muscle in young bulls treated with steroidal growth promoters
No full textDexamethasone (Dex), alone or in association with estrogens, is often illegally administered per os at very low dosage as a growth promoter in beef cattle, with effects that are opposite to the muscle wasting and atrophy induced by repeated administration at therapeutic dosages. In vitro and in vivo studies have investigated the catabolic effects of Dex at therapeutic doses on skeletal muscle, demonstrating an increase in the expression of GDF8 (myostatin) gene, a well-known negative regulator of skeletal muscle mass, in a dose-dependent way. This suggested a direct role of myostatin in Dex-induced muscle wasting. In the present study, an oligonucleotide microarray platform was used to compare expression profiles of beef cattle muscle in animals treated with either Dex or Dex plus 17-beta estradiol (Estr) administered at subtherapeutic dosage, against untreated controls. Data analysis demonstrates that the expression profiles were strongly affected by Dex treatment with hundreds of genes upregulated with relevant fold-change, whereas seven genes were downregulated including the myostatin gene. On the contrary, the number of differentially regulated genes was lower in response to the addition of Estr to the Dex treatment. Differentially regulated genes were analyzed to describe the effects of these treatments on muscle physiology, highlighting the importance of specific pathways (e.g., Wnt or cytokine signaling) and cellular processes (e.g., cell shape and motility). Finally, the observed differences in the expression profile will allow the development of indirect bio-markers to detect illegal Dex treatments in beef cattle using quantitative RT-PCR
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