Development of an oligo DNA microarray for the European sea bass and its application to expression profiling of jaw deformity

<p>Abstract</p> <p>Background</p> <p>The European sea bass (<it>Dicentrarchus labrax</it>) is a marine fish of great importance for fisheries and aquaculture. Functional genomics offers the possibility to discover the molecular mechanisms underlying productive traits in farmed fish, and a step towards the application of marker assisted selection methods in this species. To this end, we report here on the development of an oligo DNA microarray for <it>D. labrax</it>.</p> <p>Results</p> <p>A database consisting of 19,048 unique transcripts was constructed, of which 12,008 (63%) could be annotated by similarity and 4,692 received a GO functional annotation. Two non-overlapping 60mer probes were designed for each unique transcript and <it>in-situ </it>synthesized on glass slides using Agilent SurePrint™ technology. Probe design was positively completed for 19,035 target clusters; 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 affects sea bass production. Statistical analysis identified 242 transcripts that are significantly down-regulated in deformed individuals compared to normal fish, with a significant enrichment in genes related to nervous system development and functioning. A set of genes spanning a wide dynamic range in gene expression level were selected for quantitative RT-PCR validation. Fold change correlation between microarray and qPCR data was always significant.</p> <p>Conclusions</p> <p>The microarray platform developed for the European sea bass has a high level of flexibility, reliability, and reproducibility. Despite the well known limitations in achieving a proper functional annotation in non-model species, sufficient information was obtained to identify biological processes that are significantly enriched among differentially expressed genes. New insights were obtained on putative mechanisms involved on mandibular prognathism, suggesting that bone/nervous system development might play a role in this phenomenon.</p

Identificazione di marcatori molecolari per la resistenza alla fotobatteriosi nell'orata di allevamento (Sparus aurata)

Fish photobacteriosis is an infectious disease that affects several fish species living in marine temperate waters. Its causative agent is the Gram-negative bacterium Photobacterium damselae subsp. piscicida (Phdp). Fish photobacteriosis represents a serious health problem for the majority of intensive sea bream hatcheries, with 90–100% mortality during disease outbreaks. Larvae and juveniles are the most susceptible stages. A potential strategy to prevent fish photobacteriosis is to select for animals that are genetically resistant to it. Resistance to Phdp infection has low medium hereditabilty (0.12-0.45) and it is costly to measure, thus the best option for selective breeding is marked assisted selection. Aim of this work is to identify genetic loci involved in disease resistance in the gilthead sea bream (Sparus aurata) through an integrated genomic approach. A QTL analysis for resistance to photobacteriosis was carried out on an experimental population of 500 offspring, originating from eight sires and six dams in a single mass-spawning event and experimentally infected with Phdp. A total of 151 microsatellite loci were genotyped in the experimental population, and half-sib regression QTL analysis was carried out on two continuous traits, body length at time of death and survival, and for two binary traits, survival at day 7 and survival at day 15, when the highest peaks of mortality were observed. Two significant QTLs were detected for disease resistance. The first one was located on linkage group LG3 affecting late survival (survival at day 15). The second one, for overall survival, was located on LG21, which allowed us to highlight a potential marker (Id13) linked to disease resistance. A significant QTL was also found for body length at death on LG6 explaining 5-8% of the phenotypic variation. Microarray-based experiments were used to analyse changes at the transcriptome level upon Phdp experimental infection in sea bream juvenile head kidney. An update of the oligo-DNA microarray developed by Ferraresso et al. (2008) was produced by adding 6,412 novel unique transcripts. Statistical analysis identified 293 transcripts significantly up-regulated and 123 transcripts down-regulated leading to an infection response mainly associated to the more immediate innate immune system. It was observed, however, a significant predominance of anti-inflammatory mediators/signals, which help controlling excessive collateral damage to host tissue and cells due to host response, but, in so doing, might also reduce the effectiveness of immune mechanisms responsible for the clearance of the pathogen. Independent testing of a selection of differentially expressed genes with real-time RT-PCR confirmed microarray results. Differentially expressed genes based on microarray analysis were mapped onto the stickleback genome, to find a possible co-localization of the loci contributing to disease resistance or susceptibility. These genes, which putatively co-localize with genome-wide significant QTLs, represent a starting point to refine the candidate regions for the already identified QTLs and might constitute potential markers for the implementation of selective breeding programs for photobacteriosis resistance.La fotobatteriosiosi ittica, causata dal batterio Gram negativo Photobacterium damselae subsp. piscicida (Phdp), è una patologia infettiva che colpisce diverse specie di pesci che vivono in acque marine temperate. La fotobatteriosi rappresenta un reale problema sanitario per gran parte degli allevamenti intesivi di orata (Sparus aurata), con tassi di mortalità che possono raggiungere il 90-100%; gli stadi larvali e giovanili sono i più suscettibili all’infezione. Una possibile strategia per prevenire la patologia prevede la selezione di animali geneticamente resistenti a essa. La resistenza alla fotobatteriosi presenta un’ereditabilità medio bassa (0.12-0.45) e la sua stima risulta dispendiosa, di conseguenza, la strategia migliore per l’attuazione di programmi di miglioramento genetico per questo tratto è la selezione assistita da marcatori. Scopo di questo progetto è l’identificazione di loci genetici coinvolti nella determinazione della resistenza all’infezione in orata, mediante un approccio genomico integrato. Un’analisi di QTL per la resistenza alla fotobatteriosi è stata effettuata considerando una popolazione di 500 individui, generati da 8 maschi e 5 femmine, infettati sperimentalmente con Phdp e genotipizzati utilizzando 151 loci microsatelliti. I dati ottenuti sono stati elaborati attraverso un’analisi di regressione half-sib per due caratteri con distribuzione continua, la lunghezza al momento del decesso e la saprovvivenza, e per due caratteri binari, la sopravvivenza al giorno 7 e al giorno 15, associati ai maggiori picchi di mortalità. Per la resistenza alla fotobatteriosi sono stati identificati due QTL significativi. Il primo, coinvolto nella sopravvivenza al giorno 15, è stato associato al LG3. Il secondo, per la sopravvivenza al termine del challenge, è stato collocato nel LG21, per cui è stato possibile anche identificare un potenziale marcatore (Id13) associato alla resistenza alla patologia. Per la lunghezza al momento del decesso è stato individuato un QTL significativo nel LG6, in grado di spiegare il 5-8% della varianza fenotipica. La tecnologia microarray è stata impiegata per analizzare i cambiamenti a livello trascrizionale nel rene cefalico di orate sottoposte a un’infezione sperimentale con Phdp. La piattaforma microarray a oligonucleotidi, sviluppata da Ferraresso e colleghi (2008), è stata aggiornata aggiungendo 6412 nuovi trascritti unici. Le analisi statistiche dei dati di espressione hanno identificato 293 trascritti significativamente sovraespressi e 123 trascritti significativamente sottoespressi, associati a una risposta all’infezione che coinvolge principalmente i più immediati meccanismi del sistema immunitario innato. È stata rilevata, inoltre, una significativa predominanza di molecole antinfiammatorie che aiutano a controllare gli eccessivi danni collaterali ai tessuti dovuti alla risposta dell’ospite, ma così facendo, porterebbero anche a una riduzione dell’efficacia dei meccanismi immunitari responsabili dell’eliminazione del patogeno. I saggi di espressione in Real time RT-PCR hanno confermato i risultati di microarray. I geni differenzialmente espressi sono stati localizzati nel genoma di Gasterosteus aculeatus, per trovare una possibile co-localizzazione dei loci che contribuiscono alla resistenza all’infezione o alla suscettibilità. Questi geni, che apparentemente si collocano nelle stesse regioni dei QTL significativi, rappresentano un punto di partenza per raffinare la localizzazione dei QTL qui identificati e potrebbero raprresentare dei potenziali marcatori per la selezione di linee di animali maggiormente resistenti alla fotobatterios

Sense making and information in an agent-based model of cooperation

This paper studies the profile of cooperation emerging in a context in which agents may choose to join one of two groups or stay on their own, in a world where similarity across peers matters. In particular, we investigate the role of heterogeneity in individual contributions, of the level of information and of in-group processes of convergence in values (sense making) in fostering higher levels of cooperation, assessed through higher participation rates to groups. Starting from the result that more heterogeneity reduces participation, we show that increasing the level of information available to subjects and activating sense-making dynamics are able to support higher cooperation levels, which, however, come at the cost of increased radicalization of agent types within grou

Evaluation of real-time PCR assays for detection and quantification of fraudulent addition of bovine milk to caprine and ovine milk for cheese manufacture

Many typical Italian cheeses made from ovine milk are certified as Protected Designation of Origin (PDO). Because caprine and ovine milk production is limited, the fraudulent addition of cows' milk is widespread. In addition, some compounds in bovine milk have high allergenic potential; therefore, such fraud also has implications for Consumer health. In this study, a real-time polymerase chain reaction (real-time PCR) test was developed to detect and quantify cow's milk in caprine and ovine cheeses, based on two target genes. The mitochondrial Cytochrome b gene (Cytb) of Has taurus was used to detect and quantify bovine DNA. The nuclear gene myostatin (Myo), nuclear ribosomal gene 18S, or mitochondrial gene 16S were used alternatively as universal reference markets. Caprine (n = 30) and ovine (it = 51) cheese samples were purchased and analyzed and most were shown to be contaminated by bovine milk. Pairwise analysis of quantification data using a Spearmann Rank Correlation test demonstrated a highly significant correlation between data obtained with the different reference assays

High mortality of juvenile gilthead sea bream (Sparus aurata) from photobacteriosis is associated with alternative macrophage activation and anti-inflammatory response: Results of gene expression profiling of early responses in the head kidney

The halophilic bacterium Photobacterium damselae subsp. piscicida (Phdp) represents a substantial health problem for several fish species in aquaculture. Bacteria that reside free and inside phagocytes cause acute and chronic forms of photobacteriosis. Infections of juveniles rapidly kill up to 90-100% fish. Factors underlying failure of the immune protection against bacteria remain largely unknown. The reported study used a transcriptomic approach to address this issue. juvenile sea breams (0.5 g) were challenged by immersion in salt water containing 2.89 x 10(8) CFU of a virulent Phdp and the head kidney was sampled after 24- and 48-h. Analyses were performed using the second version of a 44 k oligonucleotide DNA microarray that represents 19,734 sea bream unique transcripts and covers diverse immune pathways. Expression changes of selected immune genes were validated with qPCR. Results suggested rapid recognition of the pathogen, as testified by up-regulation of lectins and antibacterial proteins (bactericidal permeability-increasing protein lectins, lysozyme, intracellular and extracellular proteases), chemokines and chemokine receptors. Increased expression of proteins involved in iron and heme metabolism also could be a response against bacteria that are dependent on iron. However, negative regulators of immune/inflammatory response were preponderant among the up-regulated genes. A remarkable finding was the increased expression of IL-10 in concert with up-regulation of arginase I and II and proteins of the polyamine biosynthesis pathway that diverts the arginine flux from the production of reactive nitrogen species. Such expression changes are characteristic for alternatively activated macrophages that do not develop acute inflammatory responses. Immune suppression can be induced by the host to reduce tissue damages or by the pathogen to evade host response. (C) 2013 Elsevier Ltd. All rights reserved

Dynamic patterns in similarity-based cooperation: an agent-based investigation

Understanding what motivates and fosters collective actions has major implications in the governance and management of organizations, in the regulation and design of public policies, and has long attracted the interests of scholars and practitioners in business and economics. This paper deals with how groups of agents emerge in a dynamic contest characterized by lack of formal structure and uncertainty regarding the possible individual outcomes, focusing on the features of the cooperators and on the dynamics emerging among them. Through the development of a stylized agent-based model we start by showing how similarity in values can be a successful driver for cooperation but are also able to highlight the limits of such process, by looking at how and how much agents cooperate with similar others. A second-version of the model, where memory of past interactions has a role, introduces further dynamics and is able to create successful and relatively stable groups

Does sharing values lead to cooperation? A similarity-based investigation

Understanding what motivates and fosters collective actions has major implications in the regulation and design of public policies, in the governance and management of organizations and has long attracted the interests of scholars and practitioners in economics and business. If trust and reciprocity certainly qualify as possible drivers of collective actions in some specific environments, as the uncertainty regarding the interaction structure increases, they are not likely to be able to explain the emergence of stable interacting groups. This paper deals with how groups of agents emerge in a dynamic contest characterized by lack of formal structure and uncertainty regarding the possible individual outcomes. Through the development of a stylized agent-based model we aim to show how similarity in values can be a successful driver for cooperation. A second-version of the model, where memory of past interactions has a role, introduces further dynamics and is able to create successful and relatively stable groups. The model nicely tries some stylized facts and sheds some light on potential avenues for the resolution of social dilemmas, such as contribution to public goods, addressing the role of perceived similarity in nurturing the cooperative process

"Delirium Day": A nationwide point prevalence study of delirium in older hospitalized patients using an easy standardized diagnostic tool

Background: To date, delirium prevalence in adult acute hospital populations has been estimated generally from pooled findings of single-center studies and/or among specific patient populations. Furthermore, the number of participants in these studies has not exceeded a few hundred. To overcome these limitations, we have determined, in a multicenter study, the prevalence of delirium over a single day among a large population of patients admitted to acute and rehabilitation hospital wards in Italy. Methods: This is a point prevalence study (called "Delirium Day") including 1867 older patients (aged 65 years or more) across 108 acute and 12 rehabilitation wards in Italian hospitals. Delirium was assessed on the same day in all patients using the 4AT, a validated and briefly administered tool which does not require training. We also collected data regarding motoric subtypes of delirium, functional and nutritional status, dementia, comorbidity, medications, feeding tubes, peripheral venous and urinary catheters, and physical restraints. Results: The mean sample age was 82.0 ± 7.5 years (58 % female). Overall, 429 patients (22.9 %) had delirium. Hypoactive was the commonest subtype (132/344 patients, 38.5 %), followed by mixed, hyperactive, and nonmotoric delirium. The prevalence was highest in Neurology (28.5 %) and Geriatrics (24.7 %), lowest in Rehabilitation (14.0 %), and intermediate in Orthopedic (20.6 %) and Internal Medicine wards (21.4 %). In a multivariable logistic regression, age (odds ratio [OR] 1.03, 95 % confidence interval [CI] 1.01-1.05), Activities of Daily Living dependence (OR 1.19, 95 % CI 1.12-1.27), dementia (OR 3.25, 95 % CI 2.41-4.38), malnutrition (OR 2.01, 95 % CI 1.29-3.14), and use of antipsychotics (OR 2.03, 95 % CI 1.45-2.82), feeding tubes (OR 2.51, 95 % CI 1.11-5.66), peripheral venous catheters (OR 1.41, 95 % CI 1.06-1.87), urinary catheters (OR 1.73, 95 % CI 1.30-2.29), and physical restraints (OR 1.84, 95 % CI 1.40-2.40) were associated with delirium. Admission to Neurology wards was also associated with delirium (OR 2.00, 95 % CI 1.29-3.14), while admission to other settings was not. Conclusions: Delirium occurred in more than one out of five patients in acute and rehabilitation hospital wards. Prevalence was highest in Neurology and lowest in Rehabilitation divisions. The "Delirium Day" project might become a useful method to assess delirium across hospital settings and a benchmarking platform for future surveys