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The role of bile acids (BA) extends far beyond lipid digestion and cholesterol metabolism. The transcriptional regulation of multiple genes within the liver and intestine are under their influence. BA exert these effects through binding and activating receptors in much the same way as endocrine hormones. The farnesoid X receptor (FXR) is the intracellular transcription factor for BA; TGR5 is the cell-surface receptor. The main target genes of FXR are those involved in BA and cholesterol metabolism. Yet more recently, FXR has also been shown to influence and promote certain protective pathways within the liver. These pathways are being harnessed by semisynthetic BAs in Phase II and III clinical trials. FXR activation within the intestine is also associated with similar protective pathways. This article examines the consequences of altered FXR activation in the context of BA malabsorption in Crohn's disease and the potential benefits of FXR agonists in Crohn's disease.

This research was funded by the European Community's Seventh Framework program (FP7/2007–2013) under grant agreement No 222,719- LIFECYCLE and by an EU Research Infrastructure Action under the FP7 “Capacities” Programme, ASSEMBLE (no. 227,799, CCMAR Ref 00,215/2011) and by the Marine Science & Technology pooling initiative and Scottish Funding Council grant HR09011 and contributing institutions. Skeletal muscle, cartilage and bone must function in a co-ordinated fashion during locomotion and growth. In the present study on the gilthead sea bream (Sparus aurata) we tested the hypothesis that muscle and bone differ in their responsiveness to stimuli eliciting fast growth, providing a potential mechanism for generating the skeletal deformities observed in aquaculture. To investigate transcription regulation in skeletal muscle and bone we stimulated protein synthesis using a flooding dose of the branched chain amino acid leucine and compared the results with saline-injected controls. To increase the amount of available sequence information for gene expression analysis a de novo transcriptome was assembled using publicly available Next Generation Sequencing libraries from embryo, fast skeletal muscle, bone and cartilage. The resulting 5 million reads were assembled into 125,646 isotigs representing around 16,000 unique genes, including most components of the Pi3k/Akt/mTor signalling pathway. Principal Components Analysis was able to distinguish the transcriptional responses between leucine and saline injected controls in skeletal muscle, but not in bone. General Linear Modelling revealed significant temporal changes in gene expression following leucine injection including the tissue-specific markers sparc, bglap (bone), mlc2 and myod2 (muscle) and gene transcripts associated with Pi3k/Akt/mTor signalling, p70sk6, akt2, ampka and mtor. Skeletal muscle showed more pronounced and rapid changes in transcript abundance than bone to the same pro-growth signal. The observed differences in transcriptional response are consistent with the idea that fast growth results in a miss-match between muscle and bone development and may contribute to a higher incidence of skeletal deformities. Postprint

© ASCE. Due to the prevalent use of the thermal response test (TRT) for estimating the key design parameters of ground source heat pump systems, it is important to understand how external factors may impact on their accuracy. The infinite line source model (ILSM) is typically used to interpret TRT data, relying on many simplifying assumptions, which when violated may lead to errors in the results. Seasonal variation causes the ground temperature profile to vary significantly at the surface, with decreasing effect at increasing depth, while the ILSM assumes this profile is constant. A numerical study of ILSM performance under different seasonal conditions using a state-of-the-art FE model is presented. The study aims to determine if TRT interpretation may become misleading when tests are conducted at different times of the year. Results suggest there is little variability in effective thermal conductivity, but up to ~50% in GHE thermal resistance, and as much as ~9°C in mean fluid temperature.

Stac3 was identified as a nutritionally regulated gene from an Atlantic salmon subtractive hybridization library with highest expression in skeletal muscle. Salmon Stac3 mRNA was highly correlated with myogenin and myoD1a expression during differentiation of a salmon primary myogenic culture and was regulated by amino acid availability. In zebrafish embryos, stac3 was initially expressed in myotomal adaxial cells and in fast muscle fibers post-segmentation. Morpholino knockdown resulted in defects in myofibrillar protein assembly, particularly in slow muscle fibers, and decreased levels of the hedgehog receptor patched. The function of Stac3 was further characterized in vitro using the mammalian C2C12 myogenic cell line. Stac3 mRNA expression increased during the differentiation of the C2C12 myogenic cell line. Knockdown of Stac3 by RNAi inhibited myotube formation, and microarray analysis revealed that transcripts involved in cell cycle, focal adhesion, cytoskeleton, and the pro-myogenic factors Igfbp-5 and Igf2 were down-regulated. RNAi-treated cells had suppressed Akt signaling and exogenous insulin-like growth factor (Igf) 2 was unable to rescue the phenotype, however, Igf/Akt signaling was not blocked. Overexpression of Stac3, which results in increased levels of Igfbp-5 mRNA, did not lead to increased differentiation. In synchronized cells, Stac3 mRNA was most abundant during the G(1) phase of the cell cycle. RNAi-treated cells were smaller, had higher proliferation rates and a decreased proportion of cells in G(1) phase when compared with controls, suggesting a role in the G(1) phase checkpoint. These results identify Stac3 as a new gene required for myogenic differentiation and myofibrillar protein assembly in vertebrates.

Malabsorption occurs when the function of the gastrointestinal tract is suboptimal and nutrient absorption is reduced. Malnutrition, weight loss, diarrhoea, steatorrhoea, anaemia and other specific nutrient deficiencies can be produced. This article will review the principles of normal nutrient absorption and the pathophysiology in disorders which result in malabsorption. Normal absorption needs coordinated processes of motility, hormone release, digestive secretion from the salivary glands, stomach, pancreas, liver and intestine, and the expression of specific enzymes and transporter molecules. Gastric, pancreatic and intestinal disorders can all produce malabsorption. These can be complications of surgical procedures, or be due to inflammatory and autoimmune disorders such as coeliac disease, Crohn's disease, small intestinal bacterial overgrowth, chronic pancreatitis, or autoimmune gastritis. Understanding the mechanisms involved and how these are affected by surgical procedures and disease will enable malabsorption to be recognized, investigated and treated appropriately.

We investigated postprandial changes in transcript abundance following a single satiating meal in juvenile Atlantic salmon (Salmo salar L.) (about 70 g body mass) following fasting for 1 week at 12°C. The expression of twenty-three growth-related genes was determined in fast myotomal muscle using quantitative real-time PCR at the following postprandial time points: − 12, 0, 1, 3, 6, 12, 24, 48 and 96 h. The gut was fullest 1–6 h after feeding and emptied within 48–96 h. IGF-I, MyoD1c, MRF4 and myf5 transcripts were sharply up-regulated within 1 h of refeeding and are promising candidate genes involved in a fast-response signalling system that regulates fish myotomal muscle growth. These genes clustered together with MyoD1b and suggest a coordinated regulation to favour resumption of myogenesis as an early response to feeding. Insulin-like growth factor (IGF)-II and the ubiquitin ligase MAFbx/atrogin-1 were initially down-regulated but restored to initial values after 12 h. It is also suggested that local production of IGF-I within the muscle might suppress catabolic pathways depressing MAFbx/atrogin-1.

Intraspecific phenotypic variation is ubiquitous and often associated with resource exploitation in emerging habitats. For example, reduced body size has evolved repeatedly in Arctic charr ( Salvelinus alpinus L.) and threespine stickleback ( Gasterosteus aculeatus L.) across post-glacial habitats of the Northern Hemisphere. Exploiting these models, we examined how body size and myogenesis evolve with respect to the ‘optimum fibre size hypothesis’, which predicts that selection acts to minimize energetic costs associated with ionic homeostasis by optimizing muscle fibre production during development. In eight dwarf Icelandic Arctic charr populations, the ultimate production of fast-twitch muscle fibres (FN max ) was only 39.5 and 15.5 per cent of that in large-bodied natural and aquaculture populations, respectively. Consequently, average fibre diameter (FD) scaled with a mass exponent of 0.19, paralleling the relaxation of diffusional constraints associated with mass-specific metabolic rate scaling. Similar reductions in FN max were observed for stickleback, including a small-bodied Alaskan population derived from a larger-bodied oceanic stock over a decadal timescale. The results suggest that in species showing indeterminate growth, body size evolution is accompanied by strong selection for fibre size optimization, theoretically allowing resources saved from ionic homeostasis to be allocated to other traits affecting fitness, including reproduction. Gene flow between small- and large-bodied populations residing in sympatry may counteract the evolution of this trait.

Global warming is intensifying interest in the mechanisms enabling ectothermic animals to adjust physiological performance and cope with temperature change. Here we show that embryonic temperature can have dramatic and persistent effects on thermal acclimation capacity at multiple levels of biological organization. Zebrafish embryos were incubated until hatching at control temperature ( T E = 27 °C) or near the extremes for normal development ( T E = 22 °C or 32 °C) and were then raised to adulthood under common conditions at 27 °C. Short-term temperature challenge affected aerobic exercise performance ( U crit ), but each T E group had reduced thermal sensitivity at its respective T E . In contrast, unexpected differences arose after long-term acclimation to 16 °C, when performance in the cold was ∼20% higher in both 32 °C and 22 °C T E groups compared with 27 °C T E controls. Differences in performance after acclimation to cold or warm (34 °C) temperatures were partially explained by variation in fiber type composition in the swimming muscle. Cold acclimation changed the abundance of 3,452 of 19,712 unique and unambiguously identified transcripts detected in the fast muscle using RNA-Seq. Principal components analysis differentiated the general transcriptional responses to cold of the 27 °C and 32 °C T E groups. Differences in expression were observed for individual genes involved in energy metabolism, angiogenesis, cell stress, muscle contraction and remodeling, and apoptosis. Therefore, thermal acclimation capacity is not fixed and can be modified by temperature during early development. Developmental plasticity may thus help some ectothermic organisms cope with the more variable temperatures that are expected under future climate-change scenarios.

Ecological factors have a major role in shaping natural variation in body size, although the underlying mechanisms are poorly understood. Icelandic Arctic charr (Salvelinus alpinus L.) populations represent an ideal model to understand body-size evolution, because adult dwarfism has arisen independently on multiple occasions in response to parallel environmental pressures. The mechanistic target of rapamycin (mTOR) pathway transmits signals from the environment to control cellular growth and is a primary candidate to be under selection for the dwarf phenotype. To test this hypothesis, we modified ‘inputs’ to this pathway in five dwarf and two generalist populations (with ancestral life history and body-size traits), using a standardized manipulation of food intake in a common environment. The skeletal muscle transcript levels of 21 mTOR-pathway genes were quantified in 274 individuals (∼6000 datapoints), and statistical modelling was used to elucidate sources of variation. Constitutive expression differences between populations were the main component of variation for around three-quarters of the studied genes, irrespective of nutritional-state and body-size phenotype. There was evidence for stabilizing selection acting among populations, conserving the nutritionally dependent regulation of pathway genes controlling muscle atrophy. There were three genes (mTOR, 4E-BP-1 and IGFBP4), where the expression variation between dwarf and generalist populations exceeded the between-population variation. Divergence in the expression of these candidate adaptive genes was most evident during a period of rapid growth following sustained fasting and was directionally consistent with their functions regulating growth and protein synthesis. We concluded that selection has operated efficiently to shape gene expression evolution in Icelandic charr populations and that the regulation of certain mTOR-pathway genes evolved adaptively in locations favouring dwarfism, resulting in reduced muscle protein accretion under growth-favouring conditions.

There is currently a great deal of interest in the problem of driver distraction. Most research focuses on distractions from inside the vehicle, but drivers can also be distracted by objects outside the vehicle. Major roads are increasingly becoming sites for advertising billboards, and there is little research on the potential effects of this advertising on driving performance. The driving simulator experiment presented here examines the effects of billboards on drivers, including older and inexperienced drivers who may be more vulnerable to distractions. The presence of billboards changed drivers' patterns of visual attention, increased the amount of time needed for drivers to respond to road signs, and increased the number of errors in this driving task.

Geothermal energy, whether as a source of electricity or to heat or cool buildings, has an enormous potential as a renewable energy source. This paper presents a broad overview of geothermal energy, with a focus on the emerging technologies of Enhanced Geothermal Systems (EGS) and Ground Source Heat Pumps (GSHPs). EGS and GSHP systems are important because of their potential for widespread use, as opposed to traditional geothermal energy systems which are generally limited to tectonically active regions. Commercial application of EGS technology is some years off but its enormous potential demands investment for further research. GSHP heat pump technology is available now — policy makers need to be educated and the potential of GSHPs demonstrated. Research should focus on optimising the design of the in-ground component of GSHP systems to maximise their economic, as well as environmental, benefits.

Research applications and cell therapies involving genetically modified cells require reliable, standardized, and cost-effective methods for cell manipulation. We report a novel nanomagnetic method for integrated cell separation and gene delivery. Gene vectors associated with magnetic nanoparticles are used to transfect/transduce target cells while being passaged and separated through a high gradient magnetic field cell separation column. The integrated method yields excellent target cell purity and recovery. Nonviral and lentiviral magselectofection is efficient and highly specific for the target cell population as demonstrated with a K562/Jurkat T-cell mixture. Both mouse and human enriched hematopoietic stem cell pools were effectively transduced by lentiviral magselectofection, which did not affect the hematopoietic progenitor cell number determined by in vitro colony assays. Highly effective reconstitution of T and B lymphocytes was achieved by magselectofected murine wild-type lineage-negative Sca-1+ cells transplanted into Il2rg−/− mice, stably expressing GFP in erythroid, myeloid, T-, and B-cell lineages. Furthermore, nonviral, lentiviral, and adenoviral magselectofection yielded high transfection/transduction efficiency in human umbilical cord mesenchymal stem cells and was fully compatible with their differentiation potential. Upscaling to a clinically approved automated cell separation device was feasible. Hence, once optimized, validated, and approved, the method may greatly facilitate the generation of genetically engineered cells for cell therapies.

Forecasts of an "explosion" in the level of road trauma as many of the world's most populous nations rapidly motorise have contributed to the urgency of documenting best practice thinking and management systems to improve road safety performance. By examining the success stories of nations with either the lowest fatailty rates per head of population or with the largest recent improvements a number of "lessons" have emerged. Data-driven problem identification and the development of evidence-driven countermeasure packages formalised in a strategy for effective implementation, with ambitious, quantitative targets and transparent lines of institutional accountability are the critical success factors. Basing this planning upon the cornerstones of the safe system conceptual model constitutes best practice thinking. This paper argues that critical elements of the safe system model are in discord with behavioural mores in the cultures of many western motorised nations and that this hampers the adoption of the most effective safety programs within key institutions and within political systems. A case is made for a systematic examination of car use and safety cultures in order to add a further dimension to best practice learnings.

A genomics approach was used to identify nutritionally regulated genes involved in growth of fast skeletal muscle in Atlantic salmon ( Salmo salar L. ). Forward and reverse subtractive cDNA libraries were prepared comparing fish with zero growth rates to fish growing rapidly. We produced 7,420 ESTs and assembled them into nonredundant clusters prior to annotation. Contigs representing 40 potentially unrecognized nutritionally responsive candidate genes were identified. Twenty-three of the subtractive library candidates were also differentially regulated by nutritional state in an independent fasting-refeeding experiment and their expression placed in the context of 26 genes with established roles in muscle growth regulation. The expression of these genes was also determined during the maturation of a primary myocyte culture, identifying 13 candidates from the subtractive cDNA libraries with putative roles in the myogenic program. During early stages of refeeding DNAJA4, HSPA1B, HSP90A, and CHAC1 expression increased, indicating activation of unfolded protein response pathways. Four genes were considered inhibitory to myogenesis based on their in vivo and in vitro expression profiles ( CEBPD, ASB2, HSP30, novel transcript GE623928). Other genes showed increased expression with feeding and highest in vitro expression during the proliferative phase of the culture ( FOXD1, DRG1) or as cells differentiated ( SMYD1, RTN1, MID1IP1, HSP90A, novel transcript GE617747). The genes identified were associated with chromatin modification ( SMYD1, RTN1), microtubule stabilization ( MID1IP1), cell cycle regulation ( FOXD1, CEBPD, DRG1), and negative regulation of signaling ( ASB2) and may play a role in the stimulation of myogenesis during the transition from a catabolic to anabolic state in skeletal muscle.

Vertebrate akirin genes usually form a family with one-to-three members that regulate gene expression during the innate immune response, carcinogenesis and myogenesis. We recently established that an expanded family of eight akirin genes is conserved across salmonid fish. Here, we measured mRNA levels of the akirin family of Atlantic salmon (Salmo salar L.) during the differentiation of primary myoblasts cultured from fast-skeletal muscle. Using hierarchical clustering and correlation, the data was positioned into a network of expression profiles including twenty further genes that regulate myogenesis. akirin1(2b) was not significantly regulated during the maturation of the cell culture. akirin2(1a) and 2(1b), along with IGF-II and several igfbps, were most highly expressed in mononuclear cells, then significantly and constitutively downregulated as differentiation proceeded and myotubes formed/matured. Conversely, akirin1(1a), 1(1b), 1(2a), 2(2a) and 2(2b) were expressed at lowest levels when mononuclear cells dominated the culture and highest levels when confluent layers of myotubes were evident. However, akirin1(2a) and 2(2a) were first upregulated earlier than akirin1(1a), 1(1b) and 2(2b), when rates of myoblast proliferation were highest. Interestingly, akirin1(1b), 1(2a), 2(2a) and 2(2b) formed part of a module of co-expressed genes involved in muscle differentiation, including myod1a, myog, mef2a, 14-3-3β and 14-3-3γ. All akirin paralogues were expressed ubiquitously across ten tissues, although mRNA levels were regulated between cell-types and family members. Gene expression patterns were often highly correlated between akirin paralogues, suggesting that natural selection has maintained an intricate network of co-regulation among family members. We concluded that the Atlantic salmon akirin family performs a multifaceted role during myogenesis and has physiological functions spanning many cell-types.

MAFbx is an E3 ubiquitin ligase which plays important roles in myogenesis and muscle atrophy. We characterised the Atlantic salmon MAFbx gene, identifying two alternatively spliced MAFbx isoforms. The mRNA sequence of Atlantic salmon MAFbx-alpha is 1698 nucleotides long including a 134 bp 5' UTR and 1065 bp coding sequence which encodes a 355 amino acid protein with a predicted mass of 41,657Da and pI 8.74. Two different 3' UTRs were identified of 495 and 314 bp in length. MAFbx-beta is produced by the removal of the 116 bp exon 2 from MAFbx-alpha, resulting in a frame shift mutation and introduction of a premature stop codon. In contrast to mammals, MAFbx-alpha and beta were ubiquitously expressed in all salmon tissues examined. In vivo, expression was 600-fold (MAFbx-alpha) and 200-fold (MAFbx-beta) higher in fasted individuals than following 21 days refeeding to satiation. In primary myogenic cell cultures, MAFbx-alpha mRNA was highest in differentiated myotubes while MAFbx-beta mRNA had peak expression in mono-nucleated cells. Starving cells of serum and amino acids resulted in a 6-fold increase in MAFbx-alpha, whereas MAFbx-beta remained similar to control levels. In starved cells, MAFbx-alpha mRNA levels declined in response to amino acid, IGF-I and IGF-II treatments whereas MAFbx-beta levels only decreased in response to IGF-I. Addition of amino acids and IGF or insulin to starved cells increased MAFbx-beta levels after 12 and 24h. These results indicate that regulation of MAFbx in Atlantic salmon occurs at both the transcriptional and post-transcriptional level through production of the alternatively spliced MAFbx-beta, which is a likely target for non-sense mediated decay.

A new tensile strength method was developed to quantify the force required to tear a standardized block of Atlantic salmon muscle with the aim of identifying those samples more prone to factory downgrading as a result of softness and fillet gaping. The new method effectively overcomes problems of sample attachment encountered with previous tensile strength tests. The repeatability and sensitivity and predictability of the new technique were evaluated against other common instrumental texture measurement methods. The relationship between sensory assessments of firmness and parameters from the instrumental texture methods was also determined. Data from the new method were shown to have the strongest correlations with gaping severity (r =−0.514, P < 0.001) and the highest level of repeatability of data when analyzing cold-smoked samples. The Warner Bratzler shear method gave the most repeatable data from fresh samples and had the highest correlations between fresh and smoked product from the same fish (r = 0.811, P < 0.001). A hierarchical cluster analysis placed the tensile test in the top cluster, alongside the Warner Bratzler method, demonstrating that it also yields adequate data with respect to these tests. None of the tested sensory analysis attributes showed significant relationships to mechanical tests except fillet firmness, with correlations (r) of 0.42 for cylinder probe maximum force (P = 0.005) and 0.31 for tensile work (P = 0.04). It was concluded that the tensile test method developed provides an important addition to the available tools for mechanical analysis of salmon quality, particularly with respect to the prediction of gaping during factory processing, which is a serious commercial problem. Practical Application: A novel, reliable method of measuring flesh tensile strength in salmon, provides data of relevance to gaping.

The most studied members of the calpain protease superfamily are CAPN1 and 2, which are conserved across vertebrates. Another similar family member called mu/m-CAPN has been identified in birds alone. Here, we establish that mu/m-CAPN shares one-to-one orthology with CAPN11, previously described only in eutherians (placental mammals). We use the name CAPN11 for this family member and identify orthologues across vertebrate lineages, which form a monophyletic phylogenetic clade directly ancestral to CAPN1 and 2. In lineages branching before therians (live-bearing mammals), the CAPN11 coding region has evolved under strong purifying selection, with low nonsynonymous (d(N)) versus synonymous (d(S)) substitution rates (d(N)/d(S) = 0.076 across pretherians), and its transcripts were detected widely across different tissues. These characteristics are present in CAPN1 and 2 across vertebrate lineages and indicate that pretherian CAPN11 likewise has conserved a wide physiological function. However, an approximately 7-fold elevation in d(N)/d(S) is evident along the CAPN11 branch splitting eutherians from platypus, paralleled by a shift to "testis-specific" gene regulation. Estimates of d(N)/d(S) in eutherians were approximately 3-fold elevated compared with pretherians and coding and transcriptional-level evidence suggests that CAPN11 is functionally absent in marsupials. Many CAPN11 sites are functionally constrained in eutherians to conserve a residue with radically different biochemical properties to a fixed state shared between pretherian CAPN11 and CAPN1 and 2. Protein homology modeling demonstrated that many such eutherian-specific residue replacements modify or ablate interactions with the calpain inhibitor calpastatin that are observed in both pretherian orthologues and CAPN1/2. We propose a model akin to the Dykhuizen-Hartl effect, where inefficient purifying selection and increased genetic drift associated with a reduction in effective population size, drove the fixation of mutations in regulatory and coding regions of CAPN11 of a common marsupial-eutherian ancestor. A subset of these changes had a cumulative adaptive advantage in a eutherian ancestor because of lineage-specific aspects of sperm physiology, whereas in marsupials, no advantage was realized and the gene was disabled. This work supports that functional divergence among gene family member orthologues is possible in the absence of widespread positive selection.

The scallop adductor muscle consists of striated fibres responsible for the fast closure of the shells, and smooth fibres able to maintain tension in a prolonged state of contraction called catch. Formation of the force-bearing catch linkages has been demonstrated to be initiated by dephosphorylation of the key catch-regulating factor twitchin by a calcineurin-like phosphatase, while the involvement of other thick filament proteins is uncertain. Here we report on the development of catchability of the adductor smooth muscle in the great scallop (Pecten maximus) by analysing the spatio-temporal gene expression patterns of the myosin regulatory light chain (MLCr), twitchin, myorod and calcineurin using whole mount in situ hybridization and real-time quantitative PCR. The MLCr signal was identified in the retractor and adductor muscles of the pediveliger larvae, and the juvenile and adult scallop displayed abundant mRNA levels of MLCr in the smooth and striated adductor muscles. Twitchin was mainly expressed in the smooth adductor muscle during metamorphosis, whereas the adult striated adductor muscle contained seven-folds higher twitchin mRNA levels compared to the smooth portion. Calcineurin expression predominated in the gonads and in the smooth adductor, and five-folds higher mRNA levels were measured in the smooth than in the striated fibres at the adult stage. In contrast to the other genes examined, the expression of myorod was confined to the smooth adductor muscle suggesting that myorod plays a permissive role in the molluscan catch muscles, which are first required at the vulnerable settlement stage as a component of the predator defence system.

We investigated the effects of embryonic temperature (ET) treatments (22, 26 and 31 degrees C) on the life-time recruitment of fast myotomal muscle fibres in zebrafish Danio rerio L. reared at 26/27 degrees C from hatching. Fast muscle fibres were produced until 25 mm total length (TL) at 22 degrees C ET, 28 mm TL at 26 degrees C ET and 23 mm TL at 31 degrees C ET. The final fibre number (FFN) showed an optimum at 26 degrees C ET (3600) and was 19% and 14% higher than for the 22 degrees C ET (3000) and 31 degrees C ET (3100) treatments, respectively. Further growth to the maximum TL of approximately 48 mm only involved fibre hypertrophy. Microarray experiments were used to determine global changes in microRNA (miRNA) and mRNA expression associated with the transition from the hyperplasic myotube-producing phenotype (M(+), 10-12 mm TL) to the hypertrophic growth phenotype (M(-), 28-31 mm TL) in fish reared at 26-27 degrees C over the whole life-cycle. The expression of miRNAs and mRNAs obtained from microarray experiments was validated by northern blotting and real-time qPCR in independent samples of fish with the M(+) and M(-) phenotype. Fourteen down-regulated and 15 up-regulated miRNAs were identified in the M(-) phenotype together with 34 down-regulated and 30 up-regulated mRNAs (>2-fold; P

Super-paramagnetic CD44 MicroBeads (Miltenyi) designed for the isolation of infectious HIV-1 from dilute or difficult biological samples dramatically enhance the infectivity of bound HIV virions, even if the original viral suspension is merely incubated with beads. Infection of the CEM T cell line with the NL4-3 virus clone or primary human CD4 T cells with X4- and R5-tropic clones and a clade C primary virus isolate all showed accelerated p24 production and larger fractions of infected target cells. Effects could be detected very early; incubation of virus with the CD44 MicroBeads promoted higher levels of viral integration within the first infection cycle. In summary, CD44 MicroBeads provide the means not only to concentrate dilute viral samples, but also to directly facilitate within days rather than weeks the in vitro expansion of patient isolates independent of coreceptor usage and the performance of HIV replication assays that require a large fraction of infected primary T cells.

SUMMARY In this study we describe the complete coding sequence for insulin-like growth factor I (IGF-I), insulin-like growth factor II (IGF-II), insulin-like growth factor binding protein (IGFBP) 1, 2, 4, 5 and 6 and IGFBP-related protein 1 (IGFBP-rP1) of Atlantic salmon (Salmo salar L.). We also report the characterisation of two gene paralogues of IGFBP-2 and IGFBP-5. Following 22 days restricted feeding (0 d) to achieve zero growth, fish were fed to satiation and sampled at 3, 5, 7, 14, 30 and 60 days. Expression profiles for genes involved in the IGF signalling pathway in fast myotomal muscle were determined using real-time quantitative RT-PCR. The transition from zero to fast growth is characterised by constitutive upregulation of IGF-I and IGFBP-4, a transient increase in IGFBP-5.2, and downregulation of IGFBP-2.1, IGF-II, IGF2R (IGF-II receptor) and IGFR1a (IGF-I receptor a). Expression of IGFBP-2.2, IGFBP-5.1, IGFBP-6, IGFBP-rP1 and IGFR1b showed little or no response to feeding. Expression of the myogenic marker genes myogenin, MHC and MLC2 were higher with feed restriction, and decreased as an early response to feeding, before increasing to a peak at 14 days,corresponding with a peak in IGF-I expression. IGFBP-4, which contains a putative connective tissue localisation signal, was the only IGFBP constitutively upregulated following feeding, and was positively correlated with IGF-I expression. Together, these data show that switching to fast growth in Atlantic salmon skeletal muscle involves the local upregulation of IGF-I,IGFBP-5.2 and IGFBP-4, with downregulation of IGFBP-2.1.

We investigated the effects of development temperature and pantophysin (PanI) geno- type on muscle fibre recruitment in Atlantic cod Gadus morhua L. reared under controlled laboratory conditions. Fertilized eggs of farmed cod originating from the Norwegian coastal (NC) and northeast Arctic stock (NEA) were incubated at 4, 6 or 8°C until hatching. At hatching, the number of fast myotomal muscle fibres (determined at 0.7 standard length) was generally greater at higher rearing temperature (10.4% greater at 8 than at 4°C in NEA cod) and was greater for NC than for NEA pop- ulations (32.7% greater at 4°C). After first feeding, the temperature groups were reared under identi- cal feeding and environmental conditions until the final number of fast myotomal muscle fibres was reached by adult fish. Development temperature and PanI genotype affected the final number of myotomal muscle fibres (FFN) in adult cod. In the NEA population, FFN was 11% higher at 4°C (92 000 ± 4000 SE, n = 13) than at 8°C (83 000 ± 3000 SE, n = 20), reversing the trend seen at hatching. Analysis of the NEA population revealed that FFN was 16% higher in PanI BB (n = 19) than in PanI AB (n = 27) genotypes (Tukey's test; difference of means ± SE: 12 900 ± 3400; p = 0.001). Results indicate association between the PanI locus and the gene(s) regulating the final muscle fibre number, and also demonstrate persistent effects of embryonic environment on adult muscle phenotype.

A sampling method is described to determine accurately the number of fast myotomal muscle fibres (NF) in a large flatfish species, the Atlantic halibut Hippoglossus hippoglossus. An unusual feature of the fast myotomal muscle is the presence of internalized strips of slow muscle fibres. In fish of 1·5–3·5 kg (n = 24), the total cross-sectional area (ATC) of fast muscle was 18% greater in the dorsal than ventral myotomal compartments (P < 0·05), whereas there was no significant difference between left- and right-hand sides of the body. Due the bilateral asymmetry, muscle blocks (5 × 5 × 5 mm) were prepared to systematically sample each myotomal quadrant (dorsal, ventral, left- and right-side) and the diameters of 150 fast fibres measured per block. Smooth non-parametric probability functions were fitted to a minimum of 800 measurements of fibre diameter per quadrant (n = 5). There were no significant differences in the distribution of muscle fibre diameters between myotomal compartments and therefore NF could be estimated from a single quadrant. The number of blocks required to estimate NF with a repeatability of ±2·5% increased from six at 300 g body mass to 17 at 96·5 kg, caused by variation within and between blocks. Gompertz curves were fitted to measurements of fibre number and fork length (LF). The estimated final fibre number was 8·96 × 105 (7·99–9·94 × 105, 95% CI) for males and 1·73 × 106 (1·56–1·90 × 106, 95% CI) for female fish. The estimated LF for cessation of fibre recruitment in the fast muscle of female fish (1775 mm) was almost twice that in males (810 mm), reflecting their greater ultimate body size.

Atlantic halibut (Hippoglossus hippoglossus L.) were commercially farmed in Helgeland, Norway (May 2004-May 2005). The average weight (Mb) of fish increased over the 12 month production cycle by approximately 73% for females and approximately 50% for males, although during the winter months (November-early May) Mb was unchanged in females and declined by 18% in males because of sexual maturation and sperm release. Periods of zero or negative growth were associated with up to 5.7% (females) and 17.9% (males) decline in fast muscle protein content. The activities of cathepsins B, B + L, H, and D showed a reciprocal relationship and were highly correlated with the changes in protein content. Water-holding capacity was measured as the liquid loss increased from 3-5% in November to 11-13% in May. Two general additive models (GAMs) showed that cathepsin B + L, cathepsin D, and collagenase explained 73.1% of the total variance in protein content, while cathepsin H was the largest contributor to liquid loss, explaining approximately 48.8% of the total variance. The results indicate that to obtain the best flesh quality Atlantic halibut should be harvested in the fall or early winter when the liquid loss and cathepsin activities are low and less likely to cause problems during secondary processing and storage.

We investigated how adult growth in Atlantic salmon (Salmo salarL.) was affected by changing embryonic temperature from fertilization until the completion of eye pigmentation. Fertilized eggs from several hundred families were divided between four temperature treatments (2, 5, 8 or 10°C) and subsequently reared in identical conditions in replicated tanks. Fish exposed to 2 and 5°C treatments were significantly smaller at smoltification than groups at higher temperatures, but showed substantial compensatory catch-up growth. Remarkably, temperature during this short window of embryogenesis dictated adult myogenic phenotype three years later with significant treatment effects on the muscle fibre final number (FFN), maximum diameter, nuclear density and size distribution. FFN was highest for the 5°C treatment and was reduced at higher and lower treatment temperatures. Our results require direct temperature effects on embryonic tissues, such as the stem cell-containing external cell layer, in order to produce persistent effects on juvenile and adult growth.

We examined the polymorphism of the lysyl oxidase (LOX) locus, involved in the initiation of muscle collagen cross-linking, in three populations of Atlantic salmon with different life histories and growth rates and compared it with a closely related species (rainbow trout). Up to four alleles were observed per individual, probably as a consequence of the tetraploid origin of the salmonid genome. We found high polymorphism in the LOX locus (16 alleles expressed in total and several low frequency private alleles) in two natural Atlantic salmon populations and extremely reduced diversity in a farmed population (3 alleles) with low density of collagen crosslinks. We also assessed the relative role of selection in maintaining LOX genetic variability in Atlantic salmon. Results from several neutrality tests suggest that selection is playing a role in shaping diversity at the LOX locus. Positive selection was inferred by three different likelihood phylogeny-based methods and one selected site, identified by all three different methods (PAML, FEL and REL) was located within the "copper-talon" characteristic of LOX proteins. We suggest that the retention of four alleles in the salmon LOX locus could be related to its multiple functions.

The surface protein composition of extracellular vesicles (EVs) is related to the originating cell and may play a role in vesicle function. Knowledge of the protein content of individual EVs is still limited because of the technical challenges to analyse small vesicles. Here, we introduce a novel multiplex bead-based platform to investigate up to 39 different surface markers in one sample. The combination of capture antibody beads with fluorescently labelled detection antibodies allows the analysis of EVs that carry surface markers recognized by both antibodies. This new method enables an easy screening of surface markers on populations of EVs. By combining different capture and detection antibodies, additional information on relative expression levels and potential vesicle subpopulations is gained. We also established a protocol to visualize individual EVs by stimulated emission depletion (STED) microscopy. Thereby, markers on single EVs can be detected by fluorophore-conjugated antibodies. We used the multiplex platform and STED microscopy to show for the first time that NK cell–derived EVs and platelet-derived EVs are devoid of CD9 or CD81, respectively, and that EVs isolated from activated B cells comprise different EV subpopulations. We speculate that, according to our STED data, tetraspanins might not be homogenously distributed but may mostly appear as clusters on EV subpopulations. Finally, we demonstrate that EV mixtures can be separated by magnetic beads and analysed subsequently with the multiplex platform. Both the multiplex bead-based platform and STED microscopy revealed subpopulations of EVs that have been indistinguishable by most analysis tools used so far. We expect that an in-depth view on EV heterogeneity will contribute to our understanding of different EVs and functions.Keywords: exosome; flow cytometry; STED; magnetic isolation; B cell; platelet; NK cell(Published: 19 February 2016)Citation: Journal of Extracellular Vesicles 2016, 5: 29975 - http://dx.doi.org/10.3402/jev.v5.29975

Factors contributing to the texture of fish flesh, including pH, water content, density of fast muscle fibers, and the concentration of collagen and hydroxylysyl pyridinoline (PYD) cross-links, were investigated post-rigor in commercially farmed Atlantic halibut (Hippoglossus hippoglossus L.). The fish was sampled every quarter for a 12 month period from May 2004 to May 2005. Hydroxyproline (HYP) as a measure of collagen and PYD were determined using a high-performance liquid chromatography (HPLC) method. An ANCOVA model with fork length and season as covariates were used to explore the seasonal effects on texture, pH, muscle fiber density, alkaline-insoluble collagen (a-i HYP), alkaline-soluble collagen (a-s HYP), and PYD cross-links. A multiple linear regression (MLR) showed that the most important factors contributing to texture were PYD > water (%) > a-i HYP > fiber density, while pH and a-s HYP did not show any correlation to texture. The contribution of fast muscle fiber density to texture was found to vary between sexes and with the season, contributing more in males and in the spring. The most important parameter affecting texture was PYD, explaining 64% (p < 0.001) of the total variation in a linear regression analysis. It is concluded that cross-linking processes are of great importance for the rigidity and strength of the collagen in Atlantic halibut flesh. Farmed halibut should be harvested in the fall or early winter when texture and nutrition are good to obtain optimal quality.

We tested the hypothesis that fast growth prior to harvest increased the incidence of soft flesh and gaping in Atlantic salmon ( Salmo salar L.) fillets. Growth trials were conducted at Salar, South Uist Western Isles, Scotland and at the Gildeskal Research Station-Gifas, Northern Norway in duplicate 5 m × 5 m × 5 m net pen sea cages, and feeding commercial diets. Individual growth rate was measured as the thermal growth coefficient (TGC) and a range of flesh quality attributes were measured including fillet firmness using an instrumental texture analyser. The Uist trial comprised the offspring of seven families from the Stofnfiskur breeding programme (Stofnfiskur A/S, Iceland). Salmon from the Uist trial harvested in October 2003 had an average TGC of 2.1 (range 1.4 to 3.6) and showed a very minor but significant positive relationship between the work done (WD) (mJ) to shear a standardised fillet slab and TGC ( R 2 = 0.041; degrees of freedom 160; P = 0.01). In the Gifas trial 1200 PIT-tagged 03 input S1 smolt of the NLA strain were tightly graded and stocked into 4 net pens in June 2004 at a mean weight of 1447 g (16% coefficient of variation). In order to gain salmon with a wide spectrum of growth rates of comparable harvest weight the fish were fed in duplicate cages either to satiation or restricted ration to target a lower growth rate whilst maintaining the same feed conversion rate. In September 2004 104 fish were harvested, selected for high growth rates (TGC = 3.7 ± 0.06, mean ± SE; range 2.6 to 5.0) and 106 lower growth rate fish (TGC = 2.7 ± 0.04, mean ± SE; range 1.7 to 3.6) were harvested in November 2004. For the September harvest there was no relationship between TGC and WD whereas for the November harvest there was a very minor but significant negative correlation between TGC and WD ( R 2 = 0.046; degrees of freedom 102; P = 0.04). There was no relationship between TGC and the incidence of gaping 3d post- rigor as measured by the length (cm) gapes m − 2 fillet cross-sectional area (Uist trial) or gaping score (0 = no gaping to 4 = severe gaping) (Gifas trial). In practical terms it was concluded that there was no evidence that fast growing fish had a materially higher incidence of soft flesh and gaping than slow growing fish for the stocks and sites studied.

Physicians managing children and adolescents with mental health issues are often called on to prescribe medications, which in themselves, represent significant concerns. Warnings about potentially serious side effects relating specifically to the paediatric population have been placed on antidepressants since May 2004 (1,2). More recently, there have been concerns expressed about the safety of stimulant medications. Little has been published about the perception of patients and their families when faced with these issues. In February 2005, the proprietary medication Adderall XR (Shire BioChem Inc, Canada) (mixed amphetamine/dextroamphetamine salts) was withdrawn from the Canadian market by Health Canada (3). The concerns were based on 20 international case reports of sudden deaths related to cardiotoxic and cerebral vascular events in patients taking Adderall XR or Adderall immediate-release formula. Fourteen of the 20 cases occurred in the paediatric population. Health Canada later reconsidered its decision, and the medication was returned to the market in November 2005 (4). The present study was undertaken primarily due to concern that this medication withdrawal could adversely affect the clinical relationship between the physician, the patient and their caregivers. In particular, the study was interested in determining how much anxiety these events provoked in families, and whether these events diminished the trust between physicians and the families. To this end, a questionnaire was developed and distributed to caregivers of patients affected by this withdrawal to explore the effect of the drug recall on patients and their families. The study was completed during the period between the recall and return of Adderall XR to the Canadian market. The authors hoped to gain some insight into how these events changed families’ views of the health care system, health care providers and pharmacotherapy for attention deficit hyperactivity disorder (ADHD).

Muscleblind-like (Mbnl) proteins are required for terminal muscle differentiation in mammals. In this study we have identified two mbnl paralogues from the tiger pufferfish, tmbnl2a and tmbnl3, which are the first examples of non-mammalian mbnl genes. Tmbnl2a and tmbnl3 were found in regions of conserved synteny and had a high degree of global conservation with their mammalian homologues. Phylogenetic analysis showed that the T. rubripes genome contains one mbnl3 gene and two copies of mbnl1 and mbnl2. Moreover, the mbnl1 and mbnl3 paralogues are derived from duplication of a common ancestral gene. The average rates of synonymous substitutions between T. rubripes, mouse and human mbnl2 and mbnl3 genes were much higher than the corresponding rates of non-synonymous mutations, suggesting that Mbnl2 and Mbnl3 are subjected to strong purifying selection. Quantitation of tmbnl2a and tmbnl3 transcripts by real-time PCR revealed that these two paralogues are differentially expressed in fast and slow myotomal muscle, heart, liver, skin, brain and testes. Tmbnl2a was expressed at similar levels in all tissues examined, as was the mouse orthologue. Tmbnl3 was expressed at higher levels than tmbnl2a, with a ubiquitous tissue distribution. Expression of tmbnl3 remained high in adult pufferfish muscle whereas the mouse orthologue was down-regulated in adults, perhaps reflecting the indeterminate and determinate growth patterns of these taxa, respectively.

Two year classes of Atlantic salmon (Salmo salar L.) (Fanad-Mowi strain) were studied from the Marine Harvest family selection program. Fish were harvested at ∼3 years of age in 2004 and 2005 and comprised 149 and 157 full-sib families. All the fish in each year class were reared under the same conditions. Following paternity analysis using DNA microsatellites a sub-set of 142 fish in 2004 and 70 fish in 2005 were selected from 28 families and the number and size of fast myotomal muscle fibres determined at 0.7 fork length (FL). Muscle fat content, flesh colour and pigment concentration were also measured. There was a tendency for fish sampled in 2005 to be heavier for a given fork length than in 2004, and for males to be longer than females reflecting sexual maturation. It was established that muscle fibre recruitment had ceased in all the fish sampled and the final fibre number had been reached. The average muscle fibre number per family ranged from 3.48 x 10 5 ±1.7×10 4 to 4.65 ×10 5 ±1.9 × 10 4 (Mean±SE, n=5 and 8 fish respectively). Fibre number showed no significant differences between male and female fish or with year class. In contrast, muscle fibre density (fibres per mm 2 muscle) was inversely related to FL, and higher in 2004 than 2005 reflecting a lower average fibre size. Myogenic progenitor cells were quantified using a specific marker, an antibody to paired box protein 7 (Pax-7). There were significant positive correlations between the density of Pax-7 positive cells and fibre number (R 2 =0.20; F 1,59 =15.0; P=0.001) and muscle fibre density (R 2 =0.30; F 1,59 =25.8; P

A microfluidic platform is presented which fully automates all incubation steps of a three-stage, multiplexed magnetic bead immunoassay, such as the Luminex® xMAP technology. Magnetic actuation is used to transfer the microbeads between co-infused adjacent laminar flow streams to transport the beads into and out of incubation and wash solutions, with extended incubation channels to allow sufficient bead incubation times (1–30 min, commonly 5 min per stage) to enable high-sensitivity. The serial incubation steps of the immunoassay are completed in succession within the device with no operator interaction, and the continuous flow operation with magnetic bead transfer defines the incubation sequencing requiring no external fluidic controls beyond syringe pump infusion. The binding kinetics of the assay is empirically characterized to determine the required incubation times for specific assay sensitivities in the range 1 pg/ml to 100 ng/ml. By using a Luminex® xMAP duplex assay, concurrent detection of IL-6 and TNF-α was demonstrated on-chip with a detection range 10 pg/ml to 1 ng/ml. This technology enables rapid automation of magnetic microbead assays, and has the potential to perform continuous concentration monitoring.

Objectives: Chronic diarrhea caused by primary bile acid diarrhea (PBAD) is a common condition. We have previously shown PBAD is associated with low fasting serum levels of the ileal hormone, fibroblast growth factor 19 (FGF19). FGF19 is a negative regulator of hepatic bile acid synthesis and is stimulated by farnesoid X receptor agonists, which produce symptomatic improvement in PBAD. We aimed to assess possible causes for low serum FGF19 in patients with PBAD.Methods: Patients with PBAD, defined by reduced (75)Se-labelled homocholic acid taurine (SeHCAT) retention, and idiopathic diarrhea controls had measurements of fasting lipids and fasting/post-prandial FGF19 serum profiles. Specific functional variants in candidate genes were investigated in exploratory studies. In further groups, basal and bile acid-stimulated transcript expression was determined in ileal biopsies and explant cultures by quantitative PCR.Results: FGF19 profiles in PBAD patients included low fasting and meal-stimulated responses, which were both strongly correlated with SeHCAT. A subgroup of 30% of PBAD patients had fasting hypertriglyceridemia and higher FGF19. No clear significant differences were found for any genetic variant but there were borderline associations with FGFR4 and KLB. SeHCAT retention significantly correlated with the basal ileal transcript expression of FGF19 (rs=0.59, P=0.03) and apical sodium-dependent bile acid transporter (ASBT) (rs=0.49, P=0.04), and also with the degree of stimulation by chenodeoxycholic acid at 6 h for transcripts of FGF19 (median 184-fold, rs=0.50, P=0.02) and ileal bile acid binding protein (IBABP) (median 2.2-fold, rs=0.47, P=0.04). Median stimulation of FGF19 was lower in patients with SeHCAT retention Conclusions: These studies demonstrate a complex, multifactorial etiology of PBAD, including impairments in ileal FGF19 expression and responsiveness.

Background Coho salmon (Oncorhynchus kisutch) transgenic for growth hormone (Gh) express Gh in multiple tissues which results in increased appetite and continuous high growth with satiation feeding. Restricting Gh-transgenics to the same lower ration (TR) as wild-type fish (WT) results in similar growth, but with the recruitment of fewer, larger diameter, muscle skeletal fibres to reach a given body size. In order to better understand the genetic mechanisms behind these different patterns of muscle growth and to investigate how the decoupling of Gh and nutritional signals affects gene regulation we used RNA-seq to compare the fast skeletal muscle transcriptome in TR and WT coho salmon. Results Illumina sequencing of individually barcoded libraries from 6 WT and 6 TR coho salmon yielded 704,550,985 paired end reads which were used to construct 323,115 contigs containing 19,093 unique genes of which >10,000 contained >90 % of the coding sequence. Transcripts coding for 31 genes required for myoblast fusion were identified with 22 significantly downregulated in TR relative to WT fish, including 10 (vaspa, cdh15, graf1, crk, crkl, dock1, trio, plekho1a, cdc42a and dock5) associated with signaling through the cell surface protein cadherin. Nineteen out of 44 (43 %) translation initiation factors and 14 of 47 (30 %) protein chaperones were upregulated in TR relative to WT fish. Conclusions TR coho salmon showed increased growth hormone transcripts and gene expression associated with protein synthesis and folding than WT fish even though net rates of protein accretion were similar. The uncoupling of Gh and amino acid signals likely results in additional costs of transcription associated with protein turnover in TR fish. The predicted reduction in the ionic costs of homeostasis in TR fish associated with increased fibre size were shown to involve multiple pathways regulating myotube fusion, particularly cadherin signaling. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1782-z) contains supplementary material, which is available to authorized users.

Hereditary genetic disorders have been shown to be effectively treated by re-introduction of the wild-type gene product via viral modification of autologous CD34+ progenitor cells. However, current manufacturing processes are performed manually or at best by using semi-automated procedures. A standardized production of cellular therapeutic agents and their genetic modification is a major requirement to move cellular and gene therapies from their current translational setting into routine clinical use. A closed and highly automated manufacturing procedure would lead to large improvements in product performance, product safety and operator safety. We have recently developed a functionally closed and fully automated cell processing device, the CliniMACS Prodigy®, which enables complex cellular products to be manufactured.In this functionally closed system, the following steps have been automated: concentration and washing of blood products, magnetic labeling and enrichment of CD34+ cells, cultivation and pre-activation of the enriched CD34+ cells, transduction with lentiviral vectors and resuspension of the final cell product in buffer for infusion.Automated cell preparation, cell labeling and magnetic CD34+ cell separation was achieved from both mobilized and non-mobilized apheresis samples as well as bone marrow aspirates. CD34+ cells were enriched to purities exceeding 90%. Small scale experiments (10e5-10e7 purified CD34+ cells) performed with a newly developed automated transduction process, incorporating a single transduction cycle, resulted in high transduction efficiencies (up to 90%). A scale-up of the procedure was performed using larger numbers of cells (>10e7) enriched from mobilized apheresis material. CD34+ cells were transduced with a clinical-grade lentiviral vector using a two hit protocol already described in a clinical setting and used as standard for a control experiment run in parallel (Scaramuzza et al. 2013, Mol. Ther). Equivalent gene marking efficiencies were obtained. Moreover, gene modified cells maintained full engraftment potential as demonstrated by biodistribution studies in NSG mice.These preliminary data demonstrate that automated cell processing and genetic manipulation of haematopoetic progenitor cells can be efficiently performed in a closed system. Work in progress aims to further optimize this procedure and improve the efficiency of CD34+ cell transduction to levels superior to those currently achieved by manual protocols.

We have investigated the interactions between dietary vitamin C levels (at 33, 79, 135, and 424 mg kg-1 of wet mass feed) and growth rate on the collagen and cross-link contents of fast muscle in farmed juvenile Atlantic salmon (Salmo salar L.). The growth rate was measured over an 11 week period using the thermal growth coefficient (TGC). Alkaline-soluble (0.1 M NaOH) (a-s) hydroxyproline (HYP) and alkaline-insoluble (i-s) HYP were determined as a measure of collagen content and hydroxylysyl pyridinoline (PYD) as a measure of mature collagen cross-link concentration. There was a approximately 5-fold increase in muscle vitamin C concentration at similar feed conversion ratios ( approximately 0.82) as dietary vitamin C levels increased from 39 to 424 mg kg-1 of wet mass feed. However, even the lowest dietary vitamin C was sufficient for normal skeletal development and growth. The lowest dietary vitamin C level tested resulted in a approximately 27% decrease in the a-sHYP concentration relative to the other diets, whereas there was no significant effect of vitamin C on the i-sHYP and PYD concentrations. ANOVA revealed no significant interaction between vitamin C and growth rate, whereas the covariate TGC was significant for i-sHYP and PYD but not for a-sHYP. Pyridinoline cross-link and i-s HYP concentrations were 11.1 and 7.7% lower, respectively, in high (TGC > 3.9) mass than low (TGC < 3.9) growth rate fish. These small differences in collagen cross-linking were associated with a 15.6% decrease in fillet firmness measured with an instrumental texture analyzer. It was concluded that for healthy juvenile salmon reared under controlled growth conditions, the dietary vitamin C inclusion of 79 mg kg-1 of wet mass feed was sufficient to produce the required synthesis of soluble muscle collagen. Furthermore, post-translational modifications of the collagen leading to cross-linking showed a small decrease with increasing growth rate but was independent of vitamin C concentration in the diet at these levels.

Atlantic halibut (Hippoglossus hippoglossus L.) has for more than a decade been considered as a promising species for aquaculture, but are still to be considered as in the initiating stage. Despite its large growth potential (females of 300 kg are reported in the wild, males seldom reach 50 kg), Atlantic halibut displays a relatively poor growth in aquaculture. A well known and unwanted situation in aquaculture is the early maturation of males (NORBERG et al., 2001). The first objective of the present study is to investigate muscle fibre recruitment in Atlantic halibut farmed commercially under ambient photoperiod condition in the grow-out phase. The second objective is to compare muscle fibre recruitment and the distribution of muscle fibre diameters in male and female halibut to see if the sexual dimorphism observed in size are reflected in muscle fibre distribution (CALVO, 1989). The third objective is to investigate the effects of precocious sexual maturation on muscle growth in males. A group of commercially farmed Atlantic halibut reared under ambient conditions from Aga Marin’s (Donna, Norway) production were followed and sampled every third month during a 12 month production period (May 2004 – May 2005, n=100). The fish grew in average from 1.2 kg to 2.1 kg and all males matured, no maturation was observed amongst females. From the point of maturation females displayed a superior growth compared to males (P

Arctic charr (Salvelinus alpinus L.) from Lake Thingvallavatn, Iceland occur as four distinct morphs: large benthivorous (LB), dwarf benthivorous (DB), piscivorous (PI) and planktonivorous (PL). The morphs differ with respect to body size, head morphology, growth rate, and life history. The aim of this study was to investigate the paired box protein 7 (Pax7) gene as a candidate for such polymorphisms due to its importance in cranio-facial, skeletal muscle, and central nervous system development. No variation in coding and intronic sequences was found between morphs. We identified 10 alternate Pax7 isoforms with insertions/deletions: a four-residue (GNRT) deletion, a GEASS insertion truncated by the first serine residue (GEAS), and a thirteen-residue insertion (GQYA/TGPEYVYCGT). The latter insertion with a threonine (T) contains a putative casein kinase II (CK-2) phosphorylation site. Pax7 spatial expression patterns were identical in embryos of DB-, LB-, and PL-morphs, and were similar to those described for zebrafish Pax7c, but a difference in temporal expression for segmentation was observed between DB and LB morphs. At the end of segmentation, novel expression was observed in the mandibular region as two bilateral domains. The potential role of multiple alternative splicing of the Pax7 gene for the generation of different Arctic charr morphs is briefly discussed.

Endurance exercise training (2·4-2·6 body lengths s -1 , 16 h day -1 for 28 days) resulted in an increased density of myonuclei in fast muscle fibres relative to tank rested controls and induced slow muscle fibre hypertrophy. The results indicate that exercise is a powerful stimulus for the proliferation of myogenic cells and nuclear accretion.

Muscle characteristics that are important for flesh quality traits were assessed in populations of wild and farmed Atlantic salmon ( Salmo salar L.). The wild fish were obtained from a commercial fishery on the North Esk, Scotland in 2001 and 2003. The farmed fish comprised 7 families from the Stofnfiskur breeding programme (Stofnfiskur A/S, Iceland) grown in 5 m × 5 m × 5 m net pen sea cages in a dedicated trial unit on South Uist, Scotland. Total carotenoid pigment (mg kg − 1 wet mass flesh) concentrations were higher in farmed (7.68 and 8.43 for grilse and salmon, respectively) than wild fish (5.53 and 6.44 for grilse and salmon, respectively). In contrast, the average flesh colour, measured with a SalmonFan ™, was similar for all groups (27.4 to 28.0), indicating a superior colour visualisation per unit pigment concentration in wild fish. The flesh lipid content was 46%-higher (grilse) and 84%-higher (salmon) in farmed than wild fish. Vitamin E concentrations per unit wet mass lipid were similar in wild and farmed salmon and in the range (208–259 mg kg − 1 ). Fillet firmness was measured using a shear test and instrumental texture analyser. The work done (WD) to shear standardised post-rigor muscle samples was higher in grilse than salmon and was significantly greater for the wild (710 and 811 mJ, respectively) than farmed (415 and 602 mJ, respectively) fish, indicating a firmer texture in the wild fish. The grilse and salmon sampled had stopped recruiting fast muscle fibres and both wild and farmed fish had around 650,000 fibres per trunk cross-section. Thus differences in fillet texture and colour between these populations could not be explained by the muscle cellularity which was similar between groups. Hydroxyproline (HYP) was used to estimate collagen concentration. Alkaline-insoluble HYP was 72.5% higher in wild than farmed salmon (14.0 vs. 8.1 μmol g − 1 dry mass, respectively) whereas alkaline-soluble HYP was 5.2-fold higher in farmed than wild salmon (31.0 vs. 6.0 μmol g − 1 dry mass, respectively). The alkaline-insoluble collagen fraction is thought to be enriched with reducible and non-reducible cross-linked collagen. There was a significant positive correlation between pyridinoline (PYD) crosslink concentration and WD, explaining 25% of the within population variation in firmness. However, PYD concentration (pmol g − 1 dry mass) was similar between wild (476) and farmed (445) salmon and cannot explain the differences in flesh firmness observed between populations. It was hypothesised that the higher average values of firmness in the wild than farmed salmon flesh can in part be attributed to higher concentrations of reducible immature collagen crosslinks.

Paired box transcription factor 7 (Pax-7) cDNA was isolated from the skeletal muscle and brain of alevin and adult stages of Atlantic salmon, identifying 10 variants categorised as novel or established insertions (ins) or deletions (del). Two putative Pax-7 paralogs were identified (denoted Pax-7alpha and Pax-7beta) on the basis of the length and sequences of intron 3 (218 and 248 bp) and versions of ins1 and ins2. Pax-7beta contained a threonine variant of ins1 (GQY[T]GPEYVYCGT), and a shortened variant of ins2 (GEAS). Pattern identification revealed the threonine variant of ins1 includes a potential phosphorylation site (casein kinase II). Thus, the tetraploid Atlantic salmon genome appears to contain at least two putative copies and multiple splice variants of Pax-7. In situ hybridisation localised Pax-7 to mononuclear cells in the fast muscle of adult Atlantic salmon, while quantitative real-time PCR showed Pax-7alpha to be more highly expressed in brain than in skeletal muscle.

With the development of genomic science and its battery of technologies, polar biology stands on the threshold of a revolution, one that will enable the investigation of important questions of unprecedented scope and with extraordinary depth and precision. The exotic organisms of polar ecosystems are ideal candidates for genomic analysis. Through such analyses, it will be possible to learn not only the novel features that enable polar organisms to survive, and indeed thrive, in their extreme environments, but also fundamental biological principles that are common to most, if not all, organisms. This article aims to review recent developments in Antarctic genomics and to demonstrate the global context of such studies. Copyright © 2004 John Wiley and Sons, Ltd.

Myogenin (Myog) is a muscle-specific basic helix-loop-helix transcription factor that plays an essential role in the specification and differentiation of myoblasts. The myogenin genes from the tiger pufferfish, Takifugu rubripes, and green-spotted pufferfish, Tetraodon nigroviridis, were cloned and a comparative genomic analysis performed. The gene encoding myogenin is composed of three exons and has a relatively similar genomic structure in T. rubripes, T. nigroviridis and human. Introns 1 and 2 were approximately 2-fold and 8-fold longer respectively in human than pufferfish. Myogenin is located in a 100 kb region of conserved synteny between these organisms, corresponding to chromosome 1 in human, chromosome 11 in T. nigroviridis and scaffold 208 in T. rubripes. Pufferfish myogenin contained a serine-rich region at the carboxyl terminus that is highly conserved amongst teleosts. During embryonic development of T. rubripes, myogenin was expressed in a rostral-caudal gradient in the developing somites and subsequently during the pharyngula period in the pectoral fin bud primordia, jaw muscles and extraocular muscle precursors. In T. rubripes, the time required to form a somite pair during the linear phase of somitogenesis ( identical withsomite-interval) was 122 min, 97 min and 50 min in embryos incubated at 15, 18 and 21 degrees C, respectively. Myogenin mRNA transcripts were quantified using qPCR and normalised to the highest level of expression. Peak myogenin expression occurred later with respect to developmental stage (standardised using somite-intervals) and was over 2-fold higher at 21 degrees C than at either 18 or 15 degrees C. Changes in the relative timing and intensity of myogenin expression are a potential mechanism for explaining thermal plasticity of muscle phenotype in larvae via effects on the differentiation programme.

Three embryonic class II myosin heavy chains (MYHs) were cloned from the common carp (Cyprinus carpio L.), MYH e m b 1 , MYH e m b 2 and MYH e m b 3 . MYH DNA clones were also isolated from the slow muscle of adult carp acclimated to 10°C (MYH S 1 0 ) and 30°C (MYH S 3 0 ). Phylogenetic analysis demonstrated that MYH e m b 1 and MYH e m b 2 belonged to the fast skeletal muscle MYH clade. By contrast, the sequence of MYH e m b 3 was similar to the adult slow muscle isoforms, MYH S 1 0 and MYH S 3 0 . MYH e m b 1 and MYH e m b 2 transcripts were first detected by northern blot analysis in embryos 61 h post-fertilization (h.p.f.) at the heartbeat stage, with peak expression occurring in 1-month-old juveniles. MYH e m b 1 continued to be expressed at low levels in 7-month-old juveniles when MYH e m b 2 was not detectable. MYH e m b 3 transcripts appeared at almost the same stage as MYH e m b 1 transcripts did (61 h.p.f.), and these genes showed a similar pattern of expression. Whole mount in situ hybridization analysis revealed that the transcripts of MYH e m b 1 and MYH e m b 2 were expressed in the inner part of myotome, whereas MYH e m b 3 was expressed in the superficial compartment. MYH S 1 0 and MYH S 3 0 mRNAs were first detected at hatching. In adult stages, the expression of slow muscle MYH mRNAs was dependent on acclimation temperature. MYH S 1 0 mRNA was expressed at an acclimation temperature of 10 and 20°C, but not at 30°C. In contrast, MYH S 3 0 mRNA was strongly expressed at all acclimation temperatures. The predominant MYH transcripts found in adult slow muscle and in embryos at hatching were expressed in adult fast muscle at some acclimation temperatures but not others. A MYH DNA clone was isolated from the cardiac muscle of 10°C-acclimated adult fish (MYH c a r d ). MYH c a r d mRNA was first detected at 61 h.p.f., but strong signals were only observed in the adult myocardium. The present study has therefore revealed a complex pattern of expression of MYH genes in relation to developmental stage, muscle type and acclimation temperature. None of the skeletal muscle MYHs identified so far was strongly expressed during the late juvenile stage, indicating further developmentally regulated members of the MYH II gene family remain to be discovered.

The suborder Notothenioidei comprises 122 species divided into 8 families, with members of 6 of the families living outside Antarctic waters. The Antarctic species underwent an extensive radiation from a small demersal ancestor to occupy different ecological niches and levels in the water column. The axial muscle of Antarctic and some Subantarctic notothenioids is unusual in containing very large diameter muscle fibres and a low muscle fibre number. Maximum fibre diameters are greater than 500 mm in many species. There is no indication of systematic differences in fibre number, fibre type composition, ATPase activity, time of cessation of fibre recruitment (hyperplasia) and swimming performance between Antarctic and Subantarctic species. Instead, fibre number is significantly decreased in species belonging to the most derived families relative to the more basal families (a trend that also correlates with an increase in the diameter of the fibres). The length of the cell cycle of the muscle fibres shows cold compensation in the Antarctic species H. antarcticus relative to the closely related Subantarctic one (H. bispinis). Feeding after a starvation period results in a strong stimulation of the proliferation of muscle fiber progenitors in H. bispinis. Similar studies have not yet been performed on any Antarctic species. El suborden Notothenioidei comprende 122 especies divididas en 8 familias, incluyendo miembros de 6 de las familias que viven en aguas subantárticas. Las especies antárticas sufrieron una impresionante radiación a partir de un ancestro pequeño y demersal que les permitió ocupar diferentes nichos ecológicos y niveles en la columna de agua. El número total de fibras de la musculatura axial de nototénidos antárticos y algunos subantárticos es escaso y las fibras presentan un tamaño inusualmente grande, alcanzando diámetros máximos mayores a 500 ?m en muchas de las especies estudiadas. No existen diferencias sistemáticas entre la musculatura axial de nototénidos antárticos y subantárticos en el número de fibras musculares, los tipos de fibras musculares, la actividad ATPasa, el momento de cese de incorporación de fibras musculares (hiperplasia), ni en la capacidad (performance) natatoria a diferentes temperaturas. De hecho, el número de fibras musculares es significativamente menor en las especies pertenecientes a las familias más derivadas en comparación con especies de las familias más basales (una tendencia que también se correlaciona con un incremento en el diámetro de las fibras). La duración del ciclo celular muestra una compensación al frío en la especie antártica H. antarcticus en comparación con la especie subantártica más relacionada (H. bispinis). La alimentación luego de un período de ayuno produce una fuerte estimulación en la proliferación de progenitores de las fibras musculares en H. bispinis. No se han realizado aún estudios similares en especies antárticas.

Growth was investigated over 16 d in juvenile common carp (Cyprinus carpio L.) held in either static water (tank rested, TR16) or exercised in a flume at 2.5-3.2 body lengths s-1 for 18 h a day (exercised, E16). Relative to the start of the experiment (TR0), the TR16 group showed a 31% increase in body mass (specific growth rate, 1.57% d-1), whereas there was no net change in the E16 group. There was, however, a significant exercise-induced hypertrophy of slow muscle fibres with average fibre cross-sectional area (FCSA) increasing by 35% in the E16 group, compared with 11% in the TR16 group. In contrast, FCSA of fast muscle fibres increased by 34% in the TR16 group compared to just 18% in the E16 group. The relative concentrations and subcellular localisation of proteins hypothesised to play a role in the regulation of muscle growth were measured. MyoD concentration was similar in the TR0, TR16 and E16 groups in both slow and fast muscle. However, there was a small (5%-10%) but statistically significant increase in nuclear localisation of MyoD in those groups showing a significant increase in FCSA over the time course of the experiment. PCNA concentration was 31% and 12% higher in the TR16 than in either the TR0 or E16 groups for slow and fast muscle, respectively. Exercise resulted in a approximately 10% increase in nuclear factor of T-cells (NFAT2) concentration in slow muscle but no change in NFAT2 localisation. Calcineurin B concentration was similar in tank rested and exercised groups. The results do not support a major role for the calcineurin-signalling pathway in the regulation of muscle hypertrophy in the common carp.