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Despite teleost fish being the first animal group in which all elements of adaptive immunity are present, the lack of follicular structures, as well as the fact that systemic Ab responses rely exclusively on unswitched low-affinity IgM responses, strongly suggests that fish B cell responses resemble mammalian B1 cell responses rather than those of B2 cells. In line with this hypothesis, in the current study, we have identified a homolog of CD5 in teleost fish. This pan-T marker belonging to the scavenger receptor cysteine-rich family of receptors is commonly used in mammals to distinguish a subset of B1 cells. Subsequently, we have demonstrated that a very high percentage of teleost IgM+ B cells express this marker, in contrast to the limited population of CD5-expressing B1 cells found in most mammals. Furthermore, we demonstrate that fish IgM+ B cells share classical phenotypic features of mammalian B1 cells such as large size, high complexity, high surface IgM, and low surface IgD expression, regardless of CD5 expression. Additionally, fish IgM+ B cells, unlike murine B2 cells, also displayed extended survival in cell culture and did not proliferate after BCR engagement. Altogether, our results demonstrate that although fish are evolutionarily the first group in which all the elements of acquired immunity are present, in the absence of follicular structures, most teleost IgM+ B cells have retained phenotypical and functional characteristics of mammalian B1 cells. [ABSTRACT FROM AUTHOR]

Stress hormone and sleep differences in a competition versus training setting are yet to be evaluated in elite female team-sport athletes. The aim of the current study was to evaluate salivary cortisol and perceptual stress markers during competition and training and to determine the subsequent effects on sleep indices in elite female athletes. Ten elite female netball athletes (mean ± SD; age: 23 ± 6 years) had their sleep monitored on three occasions; following one netball competition match (MATCH), one netball match simulation session (TRAIN), and one rest day (CONTROL). Perceived stress values and salivary cortisol were collected immediately pre- (17:15 pm) and post-session (19:30 pm), and at 22:00 pm. Sleep monitoring was performed using wrist actigraphy assessing total time in bed, total sleep time (TST), efficiency (SE%), latency, sleep onset time and wake time. Cortisol levels were significantly higher (p < .01) immediately post MATCH compared with TRAIN and CONTROL (mean ± SD; 0.700 ± 0.165, 0.178 ± 0.127 and 0.157 ± 0.178 μg/dL, respectively) and at 22:00 pm (0.155 ± 0.062, 0.077 ± 0.063, and 0.089 ± 0.083 μg/dL, respectively). There was a significant reduction in TST (−118 ± 112 min, p < .01) and SE (−7.7 ± 8.5%, p < .05) following MATCH vs. TRAIN. Salivary cortisol levels were significantly higher, and sleep quantity and quality were significantly reduced, following competition when compared to training and rest days. [ABSTRACT FROM AUTHOR]

Chemokines are a superfamily of cytokines that appeared about 650 million years ago, at the emergence of vertebrates, and are responsible for regulating cell migration under both inflammatory and physiological conditions. The first teleost chemokine gene was reported in rainbow trout in 1998. Since then, numerous chemokine genes have been identified in diverse fish species evidencing the great differences that exist among fish and mammalian chemokines, and within the different fish species, as a consequence of extensive intrachromosomal gene duplications and different infectious experiences. Subsequently, it has only been possible to establish clear homologies with mammalian chemokines in the case of some chemokines with well-conserved homeostatic roles, whereas the functionality of other chemokine genes will have to be independently addressed in each species. Despite this, functional studies have only been undertaken for a few of these chemokine genes. In this review, we describe the current state of knowledge of chemokine biology in teleost fish. We have mainly focused on those species for which more research efforts have been made in this subject, specifically zebrafish (Danio rerio), rainbow trout (Oncorhynchus mykiss) and catfish (Ictalurus punctatus), outlining which genes have been identified thus far, highlighting the most important aspects of their expression regulation and addressing any known aspects of their biological role in immunity. Finally, we summarise what is known about the chemokine receptors in teleosts and provide some analysis using recently available data to help characterise them more clearly. [ABSTRACT FROM AUTHOR]

This study builds upon previous work studying antimicrobial peptide (AMP) gene expression in rainbow trout ( Oncorhynchus mykiss ) fed a peptidoglycan (PG) enriched diet. The aims here were 1) to evaluate how long AMP expression is elevated in skin with continuous feeding of fish with the PG enriched diet for 21 or 28 days, and 2) to assess the impact of stopping PG feeding at day 14 when sampled at day 21 or 28. The rainbow trout were divided into 6 groups, with two fed a control commercial diet for the duration of the experiment and the other four given the same diet enriched with 10 mg PG/Kg for 14 days (PG 1–14) or continuously (PG continuous), the former reverting back to the commercial diet at day 14. No mortalities occurred during the study and there were no significant differences in growth among the fish in the different diet groups. The expression of six AMP genes was studied by real-time PCR in the skin, since these genes were shown to be induced in response to the PG enriched diets in a previous experiment. We show that continuous PG treatment for 21 or 28 days maintained high levels of AMP expression, although in general the levels decreased with time on the diets. Withdrawal of the PG diets at day 14 resulted in a fall in expression level especially apparent with omCATH-1, omCATH-2 and omLEAP-2a, but with omDB-3 and omDB-4 remaining at elevated levels (x10) in comparison to fish given a control diet. These results confirm that orally administered PG clearly enhances the trout innate immune system and could be used as a means to boost fish defences. Future studies should be conducted to verify the impact on survival after pathogen challenge in trout fed PG enriched diets under these regimes. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study was to investigate the effect of feeding rainbow trout (Oncorhynchus mykiss) peptidoglycan (PG) enriched diets on antimicrobial peptide (AMP) gene expression. Fish were divided into 5 groups and fed diets containing 0, 5, 10, 50 and 100 mg PG/Kg, and sampled 1, 7 and 14 days later. The expression of eight AMP genes (four defensins, two cathelicidins and two liver expressed AMPs) was determined in skin, gill, gut and liver, tissues important for first lines of defence or production of acute phase proteins. Up-regulation of many AMPs was found after feeding the PG enriched diets, with sequential expression seen over the time course studied, where defensins were typically expressed early and cathelicidins and LEAPs later on. A number of clear differences in AMP responsiveness between the tissues examined were also apparent. Of the four PG concentrations used, 5 mg PG/Kg did not always elicit AMP gene induction or to the same degree as seen with the other diets. The three higher dose groups generally showed similar trends although differences in fold change were more pronounced in the 50 and 100 mg PG/Kg groups. Curiously several AMPs were down-regulated after 14 days of feeding in gills, gut and liver. Nevertheless, overall the PG enriched diets had a positive effect on AMP expression. Further investigations now need to be undertaken to confirm whether this higher AMP gene expression correlates with protection against common bacterial diseases and if PG enriched diets have value as a means to temporarily boost the piscine immune system. [Copyright &y& Elsevier]

Abstract: To date two closely related interleukin-1β genes (IL-1β1 and IL-β2) have been found in salmonids. The cloning of trout and salmon IL-1β3, and a salmon IL-1β4 pseudogene reveals that two types of IL-1β genes exist in teleost species. Type I teleost IL-1β genes, including salmonid IL-1β3, share a similar 6 coding exon structure as in tetrapods. Type II teleost IL-1β genes, e.g. salmonid IL-1β1–2, lack one or two coding exons at their 5′-end, and share higher identities within this subgroup than within the type I subgroup. Both types of IL-1β genes have been found in species of Salmoniformes, Perciformes and Beloniformes, suggesting the divergence occurred early in teleost evolution. Trout IL-1β3 is highly expressed in ovary suggesting a role in reproduction. A relatively high constitutive expression in gills, spleen and kidney and the up-regulation by PAMPs, proinflammatory cytokines and viral infection suggests IL-1β3 also has a role in inflammation and host defence. [Copyright &y& Elsevier]

The foreign language vocabulary learning research literature often attributes strong mnemonic potency to the cognitive processing of meaning when learning words. Routinely cited as support for this idea are experiments by Craik and Tulving (C&T) demonstrating superior recognition and recall of studied words following semantic tasks ("deep" encoding) compared to structure-related tasks ("shallow" encoding). However, participants in C&T were not language learners but native speakers of English studying known English nouns. These experiments have never been directly replicated using nonnatives to establish the relevance of the findings to nonnatives and learners. The present study replicated C&T Experiment 5, comparing effects of shallow and deep encoding tasks on subsequent recognition of target words by native and nonnative speakers of English with equivalent short-term memory function. The results showed depth effects similar to C&T for all participants, indicating that C&T's results do generalize to less proficient speakers of the target language. It is crucial, however, that nonnative speakers of English benefited less from semantic encoding than native speakers, suggesting an effect of preexisting knowledge representations on mnemonic effects derived from semantic processing, and hence, a limit to the relevance of C&T for learners. Results are discussed in terms of the constructs of the mental lexicon, expert knowledge, distinctiveness and levels of processing in memory research and language learning. [ABSTRACT FROM AUTHOR]

There were eight occurrences of an incorrect spelling of the adjectival term language-learning as language-leaning in the original article (Bird, 2010). These typographical errors occurred in the first few pages only, which are reprinted herein. We regret these errors and any problems they may have caused. [ABSTRACT FROM AUTHOR]

Background: The innate immune system relies upon a wide range of germ-line encoded receptors including a large number of immunoglobulin superfamily (IgSF) receptors. Different Ig-like immune receptor families have been reported in mammals, birds, amphibians and fish. Most innate immune receptors of the IgSF are type I transmembrane proteins containing one or more extracellular Ig-like domains and their regulation of effector functions is mediated intracellularly by distinct stimulatory or inhibitory pathways. Methodology/Principal Findings: Carp SITR was found in a substracted cDNA repertoire from carp macrophages, enriched for genes up-regulated in response to the protozoan parasite Trypanoplasma borreli. Carp SITR is a type I protein with two extracellular Ig domains in a unique organisation of a N-proximal V/C2 (or I-) type and a C-proximal V-type Ig domain, devoid of a transmembrane domain or any intracytoplasmic signalling motif. The carp SITR C-proximal V-type Ig domain, in particular, has a close sequence similarity and conserved structural characteristics to the mammalian CD300 molecules. By generating an anti-SITR antibody we could show that SITR protein expression was restricted to cells of the myeloid lineage. Carp SITR is abundantly expressed in macrophages and is secreted upon in vitro stimulation with the protozoan parasite T. borreli. Secretion of SITR protein during in vivo T. borreli infection suggests a role for this IgSF receptor in the host response to this protozoan parasite. Overexpression of carp SITR in mouse macrophages and knock-down of SITR protein expression in carp macrophages, using morpholino antisense technology, provided evidence for the involvement of carp SITR in the parasite-induced NO production. Conclusion/Significance: We report the structural and functional characterization of a novel soluble immune-type receptor (SITR) in a teleost fish and propose a role for carp SITR in the NO-mediated response to a protozoan parasite. [ABSTRACT FROM AUTHOR]

A longitudinal study compared the effects of distributed and massed practice schedules on the learning of second language English syntax. Participants were taught distinctions in the tense and aspect systems of English at short and long practice intervals. They were then tested at short and long intervals. The results showed that distributed practice led to superior test scores on the long-term tests, indicating that the learning of second language syntax can benefit from distributed practice in a manner very similar to that reported for other skills and information types in the experimental psychology literature. Implications for intensive language-leaning programs and syllabus design in general are discussed. [ABSTRACT FROM AUTHOR]

An interleukin-7 ( IL-7) gene has been cloned and sequenced within the Japanese pufferfish, Takifugu rubripes ( Fugu), providing the first conclusive evidence for the existence of IL-7 in teleosts. The IL-7 cDNA was composed of 1438 bp, with a 117 bp 5′-UTR, an 826 bp 3′-UTR containing two mRNA instability motifs, and a 498 bp ORF which translates into a peptide of 166 amino acid residues. The Fugu IL-7 peptide contained a predicted signal peptide of 19 amino acid residues and the IL-7/9 family signature (Arg140–Val149). Homology analysis of Fugu IL-7 with other known IL-2 family members showed some similarity to the known mammalian IL-7 and Fugu IL-21 molecules, and phylogenetic tree analysis showed that the Fugu IL-7 clustered with the chicken and mammalian IL-7 and mammalian IL-9, away from other IL-2 family members (IL-2, IL-4, IL-15 and IL-21). The organization of the Fugu IL-7 gene was the same as in mouse, and consisted of five exons and four introns, but differed from the human gene, which is composed of six exons and five introns. Comparison of the Fugu and human genomes showed that some synteny existed around the IL-7 gene, with the presence of both the protein kinase inhibitor-α and chromosome 8 ORF 70 ( C8orf70) genes, with IL-7 and C8orf70 having the same transcriptional orientation. The Fugu IL-7 gene was expressed within unstimulated tissues, such as the head kidney, spleen, liver, intestine, gill and muscle. Stimulation of head kidney cells with lipopolysaccharide, polyinosinic polycytidylic acid or phytohaemagglutinin increased this expression. This discovery of IL-7 in Fugu will allow a more complete analysis of fish immune responses in the future. [ABSTRACT FROM AUTHOR]

In this study, we assessed the performance of trained senior (n = 6) and veteran (n = 6) cyclists (mean age 28 years, s = 3 and 57 years, s = 4 respectively). Each competitor completed two cycling tests, a ramped peak aerobic test and an indoor 16.1-km time-trial. The tests were performed using a Kingcycle™ ergometer with the cyclists riding their own bicycle fitted with an SRM™ powermeter. Power output, heart rate, and gas exchange variables were recorded continuously and blood lactate concentration [HLa] was assessed 3 min after the peak ramped test and at 2.5-min intervals during the time-trial. Peak values for power output (RMPmax), heart rate (HRpeak), oxygen uptake ([Vdot]O2peak), and ventilation ([Vdot]Epeak) attained during the ramped test were higher in the senior group (P < 0.05), whereas [HLa]peak, RERpeak, [Vdot]E:[Vdot]O2peak, and economypeak were similar between groups (P > 0.05). Time-trial values (mean for duration of race) for power output (WTT), heart rate (HRTT), [Vdot]O2 ([Vdot]O2TT), and [Vdot]E ([Vdot]ETT) were higher in the seniors (P < 0.05), but [HLa]TT, RERTT, [Vdot]ETT:[Vdot]O2TT, and economyTT were similar between the groups (P > 0.05). Time-trial exercise intensity, expressed as %RMPmax, %HRpeak, %[Vdot]O2peak, and %[Vdot]Epeak, was similar (P > 0.05) for seniors and veterans (WTT: 81%, s = 2 vs. 78%, s = 8; HRTT: 96%, s = 4 vs. 94%, s = 4; [Vdot]O2TT: 92%, s = 4 vs. 95%, s = 10; [Vdot]ETT: 89%, s = 8 vs. 85%, s = 8, respectively). Overall, seniors attained higher absolute values for power output, heart rate, [Vdot]O2, and [Vdot]E but not blood lactate concentration, respiratory exchange ratio (RER), [Vdot]E:[Vdot]O2, and economy. Veterans did not accommodate age-related declines in time trial performance by maintaining higher relative exercise intensity. [ABSTRACT FROM AUTHOR]

In this study, we assessed age-related changes in indoor 16.1-km cycling time-trial performance in 40 competitive male cyclists aged 25 - 63 years. Participants completed two tests: (1) a maximal ramped Kingcycle™ ergometer test, with maximal ramped minute power (RMPmax, W) recorded as the highest mean external power during any 60 s and maximal heart rate (HRmax, beats · min-1) as the highest value during the test; and (2) an indoor Kingcycle 16.1-km time-trial with mean external power output (W), heart rate (beats · min-1), and pedal cadence (rev · min-1) recorded throughout the event. Results revealed age-related declines (P < 0.05) in absolute and relative time-trial external power output [(24 W (7.0%) per decade], heart rate [7 beats · min-1 (3.87%) per decade], and cadence [3 rev · min-1 (3.1%) per decade]. No relationships (P > 0.05) were observed for mean power output and heart rate recorded during the time-trial versus age when expressed relative to maximal ramped minute power and maximal heart rate respectively. Strong relationships (P < 0.05) were observed for maximal ramped minute power and time-trial power (r = 0.95) and for maximal heart rate and time-trial heart rate (r = 0.95). Our results show that indoor 16.1-km time-trial performance declines with age but relative exercise intensity (%RMPmax and %HRmax) does not change. [ABSTRACT FROM AUTHOR]

Abstract: The first IL-6 sequence in fish has been determined in Fugu rubripes by exploiting the synteny that is found between some regions of the human and Fugu genomes. The predicted 227 aa IL-6 homologue contains the IL-6/G-CSF/MGF motif, has a predicted secondary structure of four α-helixes but only contains two of the four cysteines important in disulphide bond formation. It shows low amino acid identities (20–29%) with known IL-6 sequences, although phylogenetic analysis groups the Fugu molecule with the other IL-6 molecules. The gene organisation of Fugu IL-6 and the level of synteny between the human and Fugu genomes has been well conserved during evolution with the order and orientation of the genes matching exactly to human chromosome 7. PHA stimulation of Fugu kidney cells resulted in a large increase in the Fugu IL-6 transcript, whereas LPS and Poly I:C resulted in a significant increase within spleen cells. The discovery of IL-6 in fish will now allow more detailed investigations of local inflammatory responses. [Copyright &y& Elsevier]

This investigation provides the first conclusive evidence for the existence of the interleukin 2 (IL-2) andIL-21genes in bony fish. The IL-2 and IL-21 sequences have been determined inFugu rubripesby exploiting the conservation of synteny that is found between regions of the human andFugugenomes. The predicted 149-amino acid IL-2 homologue contains the IL-2 family signature, has a predicted secondary structure of three a helixes and has the two cysteines important in disulphide-bond formation. It shows low amino acid identities (24-34%) with other known IL-2 sequences. The predicted 155-amino acid IL-21 homologue has a predicted secondary structure of four a helixes and has the four cysteines important in disulphide-bond formation. It shows low amino acid identities (29-31%) with other known IL-21 sequences. The gene organisation ofFuguIL-2andIL-21and the level of synteny between the human andFugugenomes has been well conserved during evolution, with the order and orientation of the genes matching exactly to human Chromosome 4. Phytohaemagglutinin stimulation ofFugukidney cells resulted in a large increase in theFuguIL-2 and IL-21 transcripts. In vivo stimulation ofFuguwith LPS and poly I:C showedIL-21expression to be localised within mucosal tissues. The discovery ofIL-2andIL-21in fish will now allow more detailed investigations into T-helper cell responses. [ABSTRACT FROM AUTHOR]

In this study, we assessed the agreement between the powers recorded during a 30 s upper-body Wingate test using three different methods. Fifty-six men completed a single test on a Monark 814E mechanically braked ergometer fitted with a Schoberer Rad Messtechnik (SRM) powermeter. A commercial software package (Wingate test kit version 2.21, Cranlea, UK) was used to calculate conventional and corrected (with accelerative forces) values of power based on a resistive load (5% body mass) and flywheel velocity. The SRM calculated powers based on torque (measured at the crank arm) and crank rate. Values for peak 1 and 5 s power and mean 30 s power were measured. No significant differences ( P  >0.05) were found between the three methods for 30 s power values. However, the corrected values for peak 1 and 5 s power were 36 and 23% higher ( P  <0.05) respectively than those for the conventional method, and 27 and 16% higher ( P  <0.05) respectively than those for the SRM method. The conventional and SRM values for peak 1 and 5 s power were similar ( P  >0.05). Power values recorded using each method were influenced by sample time ( P  <0.05). Our results suggest that these three measures of power are similar when sampled over 30 s, but discrepancies occur when the sample time is reduced to either 1 or 5 s. [ABSTRACT FROM AUTHOR]

A homologue of interleukin 18 has been identified from rainbow trout, Oncorhynchus mykiss. The trout IL-18 gene spans 3.7 kb and consists of six exons and five introns, sharing the same gene organization with its human counterpart. The putative translated protein is 199 amino acids in length with no predicted signal peptide. Analysis of the multiple sequence alignment reveals a conserved ICE cut site, resulting in a mature peptide of 162 amino acids. The trout IL-18 shares 41­45% similarity with known IL-18 molecules and contains an IL-1 family signature motif. It is constitutively expressed in a wide range of tissues including brain, gill, gut, heart, kidney, liver, muscle, skin and spleen. Transcription is not modulated by lipopolysaccharide, poly(I:C) or trout recombinant IL-1β in primary head kidney leucocyte cultures and RTS-11 cells, a macrophage cell line. However, expression is downregulated by lipopolysaccharide and rIL-1β in RTG-2 cells, a fibroblast-like cell line. An alternatively spliced form of IL-18 mRNA has also been found and translates into a 182 amino acid protein with a 17 amino acid deletion in the precursor region of the authentic form. This alternatively spliced form is also widely expressed although much lower than the authentic form. Interestingly, its expression is upregulated by lipopolysaccharide and poly(I:C), but is not affected by rIL-1β in RTG-2 cells. The present study suggests that alternative splicing may play an important role in regulating IL-18 activities in rainbow trout. [ABSTRACT FROM AUTHOR]

Presents a heterosexual couple's attempts to challenge the traditional model of marriage. Views towards the institution and practice of marriage; Woman's rejection of marriage and the traditional socio-cultural expectations attached to it; Man's account of how being married to a feminist has rasied his awareness of its social construction.

All jawed vertebrates possess a complex immune system, which is capable of anticipatory and innate immune responses. Jawless vertebrates posses an equally complex immune system but with no evidence of an anticipatory immune response. From these findings it has been speculated that the initiation and regulation of the immune system within vertebrates will be equally complex, although very little has been done to look at the evolution of cytokine genes, despite well-known biological activities within vertebrates. In recent years, cytokines, which have been well characterised within mammals, have begun to be cloned and sequenced within non-mammalian vertebrates, with the number of cytokine sequences available from primitive vertebrates growing rapidly. The identification of cytokines, which are mammalian homologues, will give a better insight into where immune system communicators arose and may also reveal molecules, which are unique to certain organisms. Work has focussed on interleukin-1 (IL-1), a major mediator of inflammation which initiates and/or increases a wide variety of non-structural, function associated genes that are characteristically expressed during inflammation. Other than mammalian IL-1β sequences there are now full cDNA sequences and genomic organisations available from bird, amphibian, bony fish and cartilaginous fish, with many of these genes having been obtained using an homology cloning approach. This review considers how the IL-1β gene has changed through vertebrate evolution and whether its role and regulation are conserved within selected non-mammalian vertebrates. [ABSTRACT FROM AUTHOR]

The stat gene family diversified during early vertebrate evolution thanks to two rounds of whole genome duplication (WGD) to produce a typical repertoire composed of 6 STAT factors (named 1–6). In contrast, only one or two stat genes have been reported in C. elegans and in D. melanogaster. The main types of STAT found from bony fish to mammals are present in Agnathan genomes, but a typical STAT1-6 repertoire is only observed in jawed vertebrates. Comparative syntenies showed that STAT6 was the closest to the ancestor of the family. An extensive survey of stat genes across fish including polyploid species showed that whole genome duplications did not lead to a uniform expansion of stat genes. While 2 to 5 stat 1 are present in salmonids, whose genome duplicated about 35My ago, only one copy of stat 2 and stat 6 is retained. In contrast, common carp, with a recent whole genome duplication (5–10My), possesses a doubled stat repertoire indicating that the elimination of stat 2 and stat 6 additional copies is not immediate. Altogether our data shed light on the multiplicity of evolutionary pathways followed by key components of the canonical cytokine receptor signalling pathway, and point to differential selective constraints exerted on these factors. • Genome duplications events did not lead to a uniform expansion of the stat genes. • stat6 is the closest to the ancestral gene of the family. • Repertoire of stat genes was resolved for salmonid fish species. [ABSTRACT FROM AUTHOR]

Simple Summary: It is widely accepted that climate change is a rapidly increasing threat to the aquaculture industry, with the biggest issue being a slow but continuous rise in water temperature and an associated decrease in dissolved oxygen in some areas. In New Zealand fish farms, during the hottest period of the year (January/February), increased water temperatures have been shown to be detrimental to Chinook salmon performance and are associated with mortalities. We report on the responses of Chinook salmon to a chronic, long-term increase in temperature and decrease in oxygen over a three-month period. We also identified, characterised, and developed relevant biomarkers for Chinook salmon and applied them to monitor responses to changes in the fish's environment. This paper provides evidence on how the use of molecular tools, alongside general health and haematological parameters, have the potential to better inform the aquaculture industry of stock health status, thereby enhancing resilience, efficiency, and productivity. In New Zealand, during the hottest periods of the year, some salmon farms in the Marlborough Sounds reach water temperatures above the optimal range for Chinook salmon. High levels of mortality are recorded during these periods, emphasising the importance of understanding thermal stress in this species. In this study, the responses of Chinook salmon (Oncorhynchus tshawytscha) to chronic, long-term changes in temperature and dissolved oxygen were investigated. This is a unique investigation due to the duration of the stress events the fish were exposed to. Health and haematological parameters were analysed alongside gene expression results to determine the effects of thermal stress on Chinook salmon. Six copies of heat shock protein 90 (HSP90) were discovered and characterised: HSP90AA1.1a, HSP90AA1.2a, HSP90AA1.1b, HSP90AA1.2b, HSP90AB1a and HSP90AB1b, as well as two copies of SOD1, named SOD1a and SOD1b. The amino acid sequences contained features similar to those found in other vertebrate HSP90 and SOD1 sequences, and the phylogenetic tree and synteny analysis provided conclusive evidence of their relationship to other vertebrate HSP90 and SOD1 genes. Primers were designed for qPCR to enable the expression of all copies of HSP90 and SOD1 to be analysed. The expression studies showed that HSP90 and SOD1 were downregulated in the liver and spleen in response to longer term exposure to high temperatures and lower dissolved oxygen. HSP90 was also downregulated in the gill; however, the results for SOD1 expression in the gill were not conclusive. This study provides important insights into the physiological and genetic responses of Chinook salmon to temperature and oxygen stress, which are critical for developing sustainable fish aquaculture in an era of changing global climates. [ABSTRACT FROM AUTHOR]

Abstract: Atlantic bluefin tuna (BFT) (Thunnus thynnus) is of great economic significance for world aquaculture and therefore it is necessary to ensure optimal and sustainable conditions for the farming of this species. Intensive culture of fish may be limited by infectious diseases that can impact on growth performance and cause heavy losses. However, to date there are no reports of cloning and expression analysis of any major immune genes of Atlantic BFT although some immune genes are known in other BFT species. Therefore the aim of this study was to characterize the first cytokine molecules in Atlantic BFT, through: 1) Isolation of full-length cDNA and gene sequences of TNFα1, TNFα2 and IL-1β, 2) comparison of these molecules to known sequences in other vertebrates, especially teleost fish, by multiple sequence alignment, phylogenetic tree analysis and homology modeling; 3) Quantification of in vivo expression of these cytokines in selected tissues in reared BFT over the duration of the farming process. The results indicated that these three cytokines could have value for monitoring Atlantic BFT health status. Curiously, the liver seemed to be an important site of cytokine production during poor health conditions in this species, perhaps reflecting its role as an important organ involved in fish defenses. [Copyright &y& Elsevier]

Abstract: CD79α (also known as Igα) is a component of the B cell antigen receptor complex and plays an important role in B cell signalling. The CD79α protein is present on the surface of B cells throughout their life cycle, and is absent on all other healthy cells, making it a highly reliable marker for B cells in mammals. In this study the spiny dogfish (Squalus acanthias) CD79α (SaCD79α) is described and its expression studied under constitutive and stimulated conditions. The spiny dogfish CD79α cDNA contains an open reading frame of 618 bp, encoding a protein of 205 amino acids. Comparison of the SaCD79α gene with that of other species shows that the gross structure (number of exons, exon/intron boundaries, etc.) is highly conserved across phylogeny. Additionally, analysis of the 5′ flanking region shows SaCD79α lacks a TATA box and possesses binding sites for multiple transcription factors implicated in its B cell-specific gene transcription in other species. Spiny dogfish CD79α is most highly expressed in immune tissues, such as spleen, epigonal and Leydig organ, and its transcript level significantly correlates with those of spiny dogfish immunoglobulin heavy chains. Additionally, CD79α transcription is up-regulated, to a small but significant degree, in peripheral blood cells following stimulation with pokeweed mitogen. These results strongly indicate that, as in mammals, spiny dogfish CD79α is expressed by shark B cells where it associates with surface-bound immunoglobulin to form a fully functional BCR, and thus may serve as a pan-B cell marker in future shark immunological studies. [Copyright &y& Elsevier]

The human IL-1 family contains 11 genes encoded at three separate loci. Nine, including IL-IR antagonist (IL-1RN), are present at a single locus on chromosome 2, whereas IL-18 and IL-33 lie on chromosomes 11 and 9, respectively. There are currently only two known orthologs in the chicken, IL-1&bgr; and IL-18, which are encoded on chromosomes 22 and 24, respectively. Two novel chicken IL-1 family sequences were identified from expressed sequence tag libraries, representing secretory and intracellular (icIL-1RN) structural variants of the IL-1RN gene, as seen in mammals. Two further putative splice variants (SVs) of both chicken IL-1RN (chlL-1RN) structural variants were also isolated. Alternative splicing of human icIL-1RN gives three different transcripts; there are no known SVs for human secretory IL-1RN. The chicken icIL-1RN SVs differ from those found in human icIL-1RN in terms of the rearrangements involved. In mammals, IL-1RN inhibits IL-1 activity by physically occupying the IL-1 type I receptor. Both full-length structural variants of chlL-1RN exhibited biological activity similar to their mammalian orthologs in a macrophage cell line bioassay. The four SVs, however, were not biologically active. The chicken IL-1 family is more fragmented in the genome than those of mammals, particularly in that the large multigene locus seen in mammals is absent. This suggests differential evolution of the family since the divergence of birds and mammals from a common ancestor, and makes determination of the full repertoire of chicken IL-1 family members more challenging. [ABSTRACT FROM AUTHOR]

Background: In mammalian vertebrates, the cytokine interleukin (IL)-12 consists of a heterodimer between p35 and p40 subunits whereas interleukin-23 is formed by a heterodimer between p19 and p40 subunits. During an immune response, the balance between IL-12 and IL-23 can depend on the nature of the pathogen associated molecular pattern (PAMP) recognized by, for example TLR2, leading to a preferential production of IL-23. IL-23 production promotes a Th17-mediated immune response characterized by the production of IL-17A/F and several chemokines, important for neutrophil recruitment and activation. For the cold blooded vertebrate common carp, only the IL-12 subunits have been described so far. Methodology/Principal Findings: Common carp is the natural host of two protozoan parasites: Trypanoplasma borreli and Trypanosoma carassii. We found that these parasites negatively affect p35 and p40a gene expression in carp. Transfection studies of HEK293 and carp macrophages show that T. carassii-derived PAMPs are agonists of carp TLR2, promoting p19 and p40c gene expression. The two protozoan parasites induce different immune responses as assessed by gene expression and histological studies. During T. carassii infections, in particular, we observed a propensity to induce p19 and p40c gene expression, suggestive of the formation of IL-23. Infections with T. borreli and T. carassii lead to an increase of IFN-γ2 gene expression whereas IL-17A/F2 gene expression was only observed during T. carasssii infections. The moderate increase in the number of splenic macrophages during T. borreli infection contrasts the marked increase in the number of splenic neutrophilic granulocytes during T. carassii infection, along with an increased gene expression of metalloproteinase-9 and chemokines. Conclusion/Significance: This is the first study that provides evidence for a Th17-like immune response in fish in response to infection with a protozoan parasite. [ABSTRACT FROM AUTHOR]

Abstract: Signal Transducer and Activator of Transcription (STAT)-2 is a molecule involved in the type I interferon (IFN) signalling pathway. The full length cDNA sequence of Atlantic salmon (Salmo salar) ssSTAT2 was determined and phylogenetic analysis of the amino acid sequence grouped this novel salmon gene to the STAT2 clade. This represents the first fish STAT2 report. The gene encodes for a 802 aa polypeptide that has 38% identity to the human or murine STAT2. The expression was monitored by qPCR in the kidney of animals over the time of infection with the Infectious Salmon Anaemia Virus (ISAV) and in TO cells infected with Infectious Pancreatic Necrosis Virus (IPNV) or with the Salmon Alphavirus (SAV). SAV and ISAV induced an approximate 10-fold increase in the level of expression of ssSTAT2 gene whilst IPNV only induced a 1.5-fold increase. [Copyright &y& Elsevier]

Abstract: Reporter constructs of three interferon (IFN)-γ-induced rainbow trout genes were generated to examine specificity to type I or type II IFN. Constructs included γIP-10, LMP2 and TAP2 and were used to transfect trout fibroblast cells (RTG-2) which were then exposed to rainbow trout rIFNs. The γIP-10 construct showed high reporter activity even in the absence of rIFNs. The LMP2 promoter contained one GAS element and two double ISRE elements, of four constructs made, only those with ISRE elements showed significant reporter activity following rIFN-γ stimulation. The TAP2 regulatory region contained two GAS, two ISRE and one C/EBP element from which four constructs were made. Reporter expression for the construct containing all five elements showed an 11- and 2-fold increase in response to rIFN-γ and type I rIFN, respectively. Constructs containing only the GAS elements did not respond to rIFNs. The TAP2 construct with two ISRE and the C/EBP gave the greatest dose-dependent reporter response to rIFN-γ, with no significant response to type I rIFN. These data suggest that the ISRE elements, or elements nearby, are essential for the induction of type II IFN responsive genes in trout. The TAP2 construct is a candidate to develop a IFN-γ reporter stable cell line. [Copyright &y& Elsevier]

Abstract: In this paper we investigate the interleukin-10 (IL-10) expression and 3D modelling of the European sea bass (Dicentrarchus labrax L.). IL-10 is a regulatory cytokine that has been intensively studied in mammals and has been found mainly involved in the suppression or deactivation of activated immune responses. The full-length sea bass IL-10 cDNA consists of 1015 bp and is translated in a predicted molecule of 187 amino acids. A multiple alignment of this peptide with other known sequences showed the conservation of fundamental IL-10 family characteristics. Expression analysis by real-time PCR in control fish showed a high basal expression in the head kidney (HK), followed by gut and brain. In vitro treatment of HK leucocytes with LPS showed an intense increase of IL-10 expression at 4 h and a significant decrease at 24 h, with PHA-L no differences were evidenced. A 3D model for sea bass IL-10 was obtained by accurate homology procedures and putative interaction residues with the IL-10 receptor described. The results suggested that sea bass IL-10 could be involved in anti-inflammatory responses, but further experiments are needed to assess its importance in response to pathogenic agents, vaccinations and immunostimulants. [Copyright &y& Elsevier]

Abstract: In this paper we describe the cloning, expression and structural study by modelling techniques of the CD8α from sea bass (Dicentrarchus labrax L.). The sea bass CD8α cDNA is comprised of 1490bp and is translated in one reading frame to give a protein of 217 amino acids, with a predicted 26 amino acids signal peptide, a 88bp 5′-UTR and a 748bp 3′-UTR. A multiple alignment of CD8α from sea bass with other known CD8α sequences shows the conservation of most amino acid residues involved in the peculiar structural domains found within CD8α''s. Cysteine residues that are involved in disulfide bonding to form the V domain are conserved. In contrast, an extra cysteine residue found in most mammals in this region is not present in sea bass. The transmembrane and cytoplasmic regions are the most conserved regions within the molecule in the alignment analysis. However, the motif (CXCP) that is thought to be responsible for binding p56lck is missing in the sea bass sequence. Phylogenetic analysis conducted using amino acid sequences showed that sea bass CD8α grouped with other known teleost sequences and that three different clusters were formed by the mammalian, avian and fish CD8α sequences. The thymus was the tissue with the highest CD8α expression, followed by gut, gills, peripheral blood leukocytes and spleen. Lower CD8α mRNA levels were found in head kidney, liver and brain. It was possible to create a partial 3D model using the human and mouse structures as template. The CD8α 11–120 amino acid region was taken into consideration and the best obtained 3D model shows the presence of ten β-strands, involving about 50% of the sequence. The global structure was defined as an immunoglobulin-like β-sandwich made of two anti-parallel sheets. Two cysteines were present in this region and they were at a suitable distance to form an S seen in the template human and mouse structures. [Copyright &y& Elsevier]

Abstract: A lymphotoxin-beta (LT-β) gene has been cloned and sequenced in rainbow trout and provides the first conclusive evidence for the existence of LT-β in teleost. Two isoforms of LT-β were isolated. LT-β1 cDNA was composed of 952bp (with a 139bp 5′-UTR and a 201bp 3′-UTR) and LT-β2 cDNA was 836bp (with a 237bp 5′-UTR and a 197bp 3′-UTR) both of which translated into a protein of 203 amino acid residues. Both isoforms contained a predicted transmembrane domain of 21 amino acid residues (Leu 11-Val 31) and the TNF family signature (Val 104-Phe 120). Homology and phylogenetic analysis of trout LT-β''s with other known TNF family member showed good similarity to TNF-N (teleost) and other LT-β (mammals and frog). LT-β1 and TNF-α (1 and 2) genes were highly expressed in unstimulated trout head kidney, spleen, gill and intestine, whereas LT-β2 was weakly expressed only in the gill. The expression of LT-β1 and -β2 genes was not found in macrophage (RTS-11) and fibroblast (RTG-2) like cell lines, although the TNF-α2 gene was expressed in both cell lines with the TNF-α1 gene only expressed in RTS-11 cells. In head kidney cells, expression of LT-β1 and TNF-α (1, 2) genes was increased by stimulation with PHA or LPS. The discovery of trout LT-β will allow a more complete analysis of fish inflammatory responses. [Copyright &y& Elsevier]

Background: Ketamine ester analogs, SN 35210 and SN 35563, demonstrate different pharmacological profiles to ketamine in animal models. Both confer hypnosis with predictably rapid offset yet, paradoxically, SN35563 induces a prolonged anti-nociceptive state. To explore underlying mechanisms, broad transcriptome changes were measured and compared across four relevant target regions of the rat brain. Results: SN 35563 produced large-scale alteration of gene expression in the Basolateral Amygdala (BLA) and Paraventricular Nucleus of the Thalamus (PVT), in excess of 10x that induced by ketamine and SN 35210. A smaller and quantitatively similar number of gene changes were observed in the Insula (INS) and Nucleus Accumbens (ACB) for all three agents. In the BLA and PVT, SN 35563 caused enrichment for gene pathways related to the function and structure of glutamatergic synapses in respect to: release of neurotransmitter, configuration of postsynaptic AMPA receptors, and the underlying cytoskeletal scaffolding and alignment. Conclusion: The analgesic ketamine ester analog SN 35563 induces profound large-scale changes in gene expression in key pain-related brain regions reflecting its unique prolonged pharmacodynamic profile. [ABSTRACT FROM AUTHOR]

A number of Seriola species are currently farmed or being investigated as future aquaculture species in countries around the world. However they face a number of issues and limitations which will need to be overcome to ensure future stability and growth, one of which are disease outbreaks. Despite this, very little has been done to understand the immune system of Seriola species and very few immune genes have been characterised. Antimicrobial peptides (AMP) are naturally occurring low molecular weight polypeptides that play a major role in an organism's immune system and act effectively as a first line of defence. This investigation isolates the full length cDNA sequences of two AMP's, piscidin and hepcidin from the yellowtail kingfish ( Seriola lalandi ). The full-length cDNA of the piscidin gene encodes a 65 amino acid prepropeptide, containing a 25-residue peptide, predicted to form an amphipathic helix-loop-helix structure. Phylogenetic analysis using fish piscidin sequences, showed that this AMP is only found in bony fish within the Acanthomorpha clade and that a possible three groups within the piscidin family exists, with S. lalandi belonging to a particular group. The full-length cDNA of the hepcidin gene encodes a 90 amino acid preprohepcidin, which contains a typical RX(R/K)R motif for cleavage of the mature peptide which comprises of eight conserved cysteine residues. Phylogenetic analysis of known vertebrate hepcidin antimicrobial peptide (HAMP) sequences, shows sequences from the Neoteleostei clade of bony fish form two very separate groups, HAMP1 and HAMP2, with the S. lalandi hepcidin gene grouped with the HAMP1 sequences. HAMP2 sequences are found to have multiple copies within fish and genome analysis showed very clearly that these two groups of genes are located on separate regions on the genome, with the multiple HAMP2 copies formed from tandem gene duplications. Lastly, using qPCR the expression of the S. lalandi piscidin gene within healthy fish was highest within, spleen and gills and lowest in liver, whereas hepcidin was highest in the liver with little or no expression in the spleen and gills. [ABSTRACT FROM AUTHOR]

A continued programme of research is essential to overcome production bottlenecks in any aquacultured fish species. Since the introduction of genetic and molecular techniques, the quality of immune research undertaken in fish has greatly improved. Thousands of species specific cytokine genes have been discovered, which can be used to conduct more sensitive studies to understand how fish physiology is affected by aquaculture environments or disease. Newly available transcriptomic technologies, make it increasingly easier to study the immunogenetics of farmed species for which little data exists. This paper reviews how the application of transcriptomic procedures such as RNA Sequencing (RNA-Seq) can advance fish research. As a case study, we present some preliminary findings using RNA-Seq to identify cytokine related genes in Seriola lalandi. These will allow in-depth investigations to understand the immune responses of these fish in response to environmental change or disease and help in the development of therapeutic approaches. [ABSTRACT FROM AUTHOR]

DNA damage quantitation assays such as the comet assay have focused on the measurement of total nuclear damage per cell. The adoption of PCR-based techniques to quantify DNA damage has enabled sequence- and organelle-specific assessment of DNA lesions. Here we report on an adaptation of a qPCR technique to assess DNA damage in nuclear and mitochondrial targets relative to control. Novel aspects of this assay include application of the assay to the Rotor-Gene platform with optimized DNA polymerase/fluorophore/primer set combination in a touchdown PCR protocol. Assay validation was performed using ultraviolet C radiation in A549 and THP1 cancer cell lines. A comparison was made to the comet assay applied to peripheral blood mononuclear cells, and an estimation of the effects of cryopreservation on ultraviolet C-induced DNA damage was carried out. Finally, dose responses for DNA damage were measured in peripheral blood mononuclear cells following exposure to the cytotoxic agents bleomycin and cisplatin. We show reproducible experimental outputs across the tested conditions and concordance with published findings with respect to mitochondrial and nuclear genotoxic susceptibilities. The application of this DNA damage assay to a wide range of clinical and laboratory-derived samples is both feasible and resource-efficient. [ABSTRACT FROM AUTHOR]

IFN-γ is a major effector cytokine, produced to induce type I immune responses. It has been cloned in several fish species including zebrafish, however to date few studies have looked at IFN-γ protein expression and bioactivity in fish. Hence, the current study focused on developing a monoclonal antibody (moAb) against zfIFN-γ. We show that the zfIFN-γ moAb specifically recognises E. coli produced recombinant IFN-γ protein and zfIFN-γ produced in transfected HEK293 cells, by Western blot analysis. Next we analysed the production of the native protein following expression induced by PHA stimulation of leukocytes in vitro or antigen re-stimulation in vivo . We show the IFN-γ protein is produced as a dimer, and that a good correlation exists between transcript expression levels and protein levels. [ABSTRACT FROM AUTHOR]

The tumour necrosis factor superfamily (TNFSF) members CD40L and BAFF play critical roles in mammalian B cell survival, proliferation and maturation, however little is known about these key cytokines in the oldest jawed vertebrates, the cartilaginous fishes. Here we report the cloning of CD40L and BAFF orthologues (designated ScCD40L and ScBAFF) in the small-spotted catshark ( Scyliorhinus canicula ). As predicted both proteins are type II membrane-bound proteins with a TNF homology domain in their extracellular region and both are highly expressed in shark immune tissues. ScCD40L transcript levels correlate with those of TCRα and transcription of both genes is modulated in peripheral blood leukocytes following in vitro stimulation. Although a putative CD40L orthologue was identified in the elephant shark genome the work herein is the first molecular characterisation and transcriptional analysis of CD40L in a cartilaginous fish. ScBAFF was also cloned and its transcription characterised in an attempt to resolve the discrepancies observed between spiny dogfish BAFF and bamboo shark BAFF in previously published studies. [ABSTRACT FROM AUTHOR]

Interleukin (IL)-23 is a heterodimeric IL-12 family cytokine composed of a p19 α-chain, linked to a p40 β-chain that is shared with IL-12. IL-23 is distinguished functionally from IL-12 by its ability to induce the production of IL-17, and differentiation of Th17 cells in mammals. Three isoforms of p40 (p40a, p40b and p40c) have been found in some 3R teleosts. Salmonids also possess three p40 isoforms (p40b1, p40b2 and p40c) although p40a is missing, and two copies (paralogues) of p40b are present that have presumably been retained following the 4R duplication in this fish lineage. Teleost p19 has been discovered recently in zebrafish, but to date there is limited information on expression and modulation of this molecule. In this report we have cloned two p19 paralogues (p19a and p19b) in salmonids, suggesting that a salmonid can possess six potential IL-23 isoforms. Whilst Atlantic salmon has two active p19 genes, the rainbow trout p19b gene may have been pseudogenized. The salmonid p19 translations share moderate identities (22.8–29.9%) to zebrafish and mammalian p19 molecules, but their identity was supported by structural features, a conserved 4 exon/3 intron gene organisation, and phylogenetic tree analysis. The active salmonid p19 genes are highly expressed in blood and gonad. Bacterial ( Yersinia ruckeri ) and viral infection in rainbow trout induces the expression of p19a, suggesting pathogen-specific induction of IL-23 isoforms. Trout p19a expression was also induced by PAMPs (poly IC and peptidoglycan) and the proinflammatory cytokine IL-1β in primary head kidney macrophages. These data may indicate diverse functional roles of trout IL-23 isoforms in regulating the immune response in fish. [ABSTRACT FROM AUTHOR]

Adaptive immunity in homeotherms depends greatly on CD4+ Th cells which release cytokines in response to specific antigen stimulation. Whilst bony fish and poikilothermic tetrapods possess cells that express TcR and CD4-related genes (that exist in two forms in teleost fish; termed CD4-1 and CD4-2), to date there is no unequivocal demonstration that cells equivalent to Th exist. Thus, in this study we determined whether CD4-1+ lymphocytes can express cytokines typical of Th cells following antigen specific stimulation, using the zebrafish (Danio rerio). Initially, we analyzed the CD4 locus in zebrafish and found three CD4 homologues, a CD4-1 molecule and two CD4-2 molecules. The zfCD4-1 and zfCD4-2 transcripts were detected in immune organs and were most highly expressed in lymphocytes. A polyclonal antibody to zfCD4-1 was developed and used with an antibody to ZAP70 and revealed double positive cells by immunohistochemistry, and in the Mycobacterium marinum disease model CD4-1+ cells were apparent surrounding the granulomas typical of the infection. Next a prime-boost experiment, using human gamma globulin as antigen, was performed and revealed for the first time in fish that zfCD4-1+ lymphocytes increase the expression of cytokines and master transcription factors relevant to Th1/Th2-type responses as a consequence of boosting with specific antigen. [ABSTRACT FROM AUTHOR]

The IL-1 family consists of 11 members that play an important role as key mediators in inflammation and immunity. Here, we report the identification of a new member of the IL-1 family (IL-1Fm2) that is present in species belonging to the most evolutionarily advanced group of teleost fish (Series Percomorpha), including Perciformes, Beloniformes, Gasterosteiformes, Cyprinodontiformes and Pleuronectiformes. However, IL-1Fm2 seems to be absent in Tetraodontiformes, which also belong to the Percomorpha. The expression pattern of gilthead seabream IL-1Fm2 revealed that although it was hardly induced by PAMPs, the combination of PAMPs and recombinant IL-1Fm2 synergistically induced its expression in macrophages and granulocytes. In addition, recombinant IL-1Fm2 was able to activate the respiratory burst of seabream phagocytes and to synergistically induce the expression of IL-1β, TNF-α, IL-8 and IL-10 when combined with PAMPs. Finally, although gilthead seabream IL-1Fm2 did not show a conserved caspase-1 processing site, macrophages processed IL-1Fm2 before being released. However, both pan-caspase and caspase-1 inhibitors failed to inhibit the processing and release of IL-1Fm2. These results demonstrate an important role of IL-1Fm2 in the regulation of fish immune responses, shed light on the evolution of the IL-1 family in vertebrates and point to the complexity of this cytokine family. [ABSTRACT FROM AUTHOR]

Highlights: [•] An in depth analysis of the phylogeny of vertebrate CXC chemokines was undertaken. [•] A nomenclature for fish specific CXC chemokines have been proposed. [•] IL-1β, type I and II IFN differentially modulate trout CXC chemokine expression. [•] The chemoattractant activity of three of the trout CXC chemokines was studied. [ABSTRACT FROM AUTHOR]

ESX-5 is a mycobacterial type VII protein secretion system responsible for transport of numerous PE and PPE proteins. It is involved in the induction of host cell death and modulation of the cytokine response in vitro. In this work, we studied the effects of ESX-5 in embryonic and adult zebrafish using Mycobacterium marinum. We found that ESX-5-deficient M. marinum was slightly attenuated in zebrafish embryos. Surprisingly, the same mutant showed highly increased virulence in adult zebrafish, characterized by increased bacterial loads and early onset of granuloma formation with rapid development of necrotic centres. This early onset of granuloma formation was accompanied by an increased expression of pro-inflammatory cytokines and tissue remodelling genes in zebrafish infected with the ESX-5 mutant. Experiments using RAG-1-deficient zebrafish showed that the increased virulence of the ESX-5 mutant was not dependent on the adaptive immune system. Mixed infection experiments with wild-type and ESX-5 mutant bacteria showed that the latter had a specific advantage in adult zebrafish and outcompeted wild-type bacteria. Together our experiments indicate that ESX-5-mediated protein secretion is used by M. marinum to establish a moderate and persistent infection. [ABSTRACT FROM AUTHOR]

Abstract: B-cell activating factor (BAFF), also known as tumour necrosis factor (TNF) ligand superfamily member 13B, is an important immune regulator with critical roles in B-cell survival, proliferation, differentiation and immunoglobulin secretion. A BAFF gene has been cloned from spiny dogfish (Squalus acanthias) and its expression studied. The dogfish BAFF encodes for an anchored type-II transmembrane protein of 288 aa with a putative furin protease cleavage site and TNF family signature as seen in BAFFs from other species. The identity of dogfish BAFF has also been confirmed by conserved cysteine residues, and phylogenetic tree analysis. The dogfish BAFF gene has an extra exon not seen in teleost fish, birds and mammals that encodes for 29 aa and may impact on receptor binding. The dogfish BAFF is highly expressed in immune tissues, such as spleen, and is up-regulated by PWM in peripheral blood leucocytes, suggesting a potentially important role in the immune system. [Copyright &y& Elsevier]

Abstract: This paper reports the cloning and sequencing of interleukin (IL)-23 p19 subunit for the first time within a non-mammalian species, the zebrafish (Danio rerio), which was discovered using a synteny approach. In addition, amino acid sequences were for IL-23 p19 subunits were also predicted from the stickleback, Fugu and Tetraodon genomes and included in this investigation. The zebrafish IL-23 p19 cDNA consisted of a 66bp 5′ UTR, a 249bp 3′ UTR and a single open reading frame of 567bp giving a predicted 188 aa IL-23 p19 molecule. Multiple alignment of zebrafish IL-23 p19, with other known IL-23 p19 and IL-12 p35 amino acid sequences revealed areas of amino acid conservation, such as the presence of four predicted α-helixes, cysteines important for disulphide bond formation and the conservation of a tryptophan known to interact with the receptor. Amino acid homologies and phylogenetic analysis confirmed the relationship of the fish IL-23 p19 subunits with their mammalian homologues. All the teleost fish IL-23 p19 subunits were found to have 4 exons and 3 introns similar to that of human and mouse IL-23 p19 and a limited degree of synteny was found between the organisms for the regions containing the IL-23 p19 genes with only PAB-dependent poly(A)-specific ribonuclease subunit 2 (PAN2) and IL-23 p19 found in the same order on human chromosome 12 and all the fish genomes looked at. Lastly using real-time PCR, constitutive expression of IL-12 p40 and IL-23 p19 was observed in the kidney, liver, gut and muscle with IL-12 p40 expression higher than IL-23 p19. As soon as an hour after stimulation with LPS, there was an increase of IL-23 p19 in zebrafish leukocytes and an increase of IL-1β, IL-12 p40 and IL-23 p19 expression was found after infection of zebrafish for 1 or 6 days with Mycobaterium marinum strain E11. [Copyright &y& Elsevier]