Effects of parental acclimation and energy limitation in response to high CO2 exposure in Atlantic cod.

Ocean acidification (OA), the dissolution of excess anthropogenic carbon dioxide in ocean waters, is a potential stressor to many marine fish species. Whether species have the potential to acclimate and adapt to changes in the seawater carbonate chemistry is still largely unanswered. Simulation experiments across several generations are challenging for large commercially exploited species because of their long generation times. For Atlantic cod (Gadus morhua), we present first data on the effects of parental acclimation to elevated aquatic CO2 on larval survival, a fundamental parameter determining population recruitment. The parental generation in this study was exposed to either ambient or elevated aquatic CO2 levels simulating end-of-century OA levels (~1100 µatm CO2) for six weeks prior to spawning. Upon fully reciprocal exposure of the F1 generation, we quantified larval survival, combined with two larval feeding regimes in order to investigate the potential effect of energy limitation. We found a significant reduction in larval survival at elevated CO2 that was partly compensated by parental acclimation to the same CO2 exposure. Such compensation was only observed in the treatment with high food availability. This complex 3-way interaction indicates that surplus metabolic resources need to be available to allow a transgenerational alleviation response to ocean acidification.

Characterization of Ras k 1 a novel major allergen in Indian mackerel and identification of parvalbumin as the major fish allergen in 33 Asia-Pacific fish species.

BACKGROUND: Fish is a well-recognized cause of food allergy and anaphylaxis. The evolutionary and taxonomic diversity of the various consumed fish species pose a challenge in the identification and characterization of the major fish allergens critical for reliable diagnostics. Globally, fish is a rising cause of food allergy complicated by a large under-investigated variety of species as well as increasing global tourism and trade. This is the first comprehensive study on allergen profiles of heat-processed fish from Vietnam. OBJECTIVE: The aim of this study was to identify the major heat-stable allergens from frequently exported Asia-Pacific freshwater and marine fish and to characterize the major allergen parvalbumin (PV) from one of the most consumed and exported fish species from Asia, the Indian mackerel (Rastrelliger kanagurta). METHODS: Heated protein extracts from 33 fish species were separated by gel electrophoresis. PV isoforms were identified by immunoblotting utilizing 3 different PV-specific monoclonal and polyclonal antibodies and further characterized by mass spectrometry. IgE reactivity was investigated using sera from 21 patients with confirmed fish allergy. RESULTS: Heat-stable IgE-reactive PVs, with up to 5 isoforms per species, were identified in all 33 analysed fish species. In the Indian mackerel, 7 PV isoforms were identified by 2D-gel electrophoresis combined with mass spectrometric analyses. The amino acid sequence deduced from cDNA of the most expressed isoform showed a high identity (>90%) to PVs from 2 other mackerel species. CONCLUSIONS AND CLINICAL RELEVANCE: Different PVs were identified as the major heat-stable allergens in all 33 analysed freshwater and marine fish species from Vietnam, many of which are exported world-wide and 21 species that have never been investigated before. The Indian mackerel PV represents a novel fish allergen, now officially registered as Ras k 1. Improved diagnostics for fish allergy against Asia-Pacific species should be developed with focus on PV.

Bochdalek Congenital Diaphragmatic Hernia in an Adult Sheep.

Congenital diaphragmatic hernia (CDH) is a rare condition. The aetiology of CDH is often unclear. In our case, a hollow mass was noted on MRI. Cardiac ejection fraction was diminished (47.0%) compared to 60.5% (average of 10 other normal animals, P < 0.05). The final diagnosis of congenital diaphragmatic hernia (Bochdalek type) was made when the sheep underwent surgery. The hernia was right-sided and contained the abomasum. Lung biopsy demonstrated incomplete development with a low number of bronchopulmonary segments and vessels. The likely cause of this hernia was genetic malformation.

Liquid jet delivery method featuring S100A1 gene therapy in the rodent model following acute myocardial infarction.

The S100A1 gene is a promising target enhancing contractility and survival post myocardial infarction (MI). Achieving sufficient gene delivery within safety limits is a major translational problem. This proof of concept study evaluates viral mediated S100A1 overexpression featuring a novel liquid jet delivery (LJ) method. Twenty-four rats after successful MI were divided into three groups (n = 8 ea.): saline control (SA); ssAAV9.S100A1 (SS) delivery; and scAAV9.S100A1 (SC) delivery (both 1.2 × 10¹¹ viral particles). For each post MI rat, the LJ device fired three separate 100 µl injections into the myocardium. Following 10 weeks, all rats were evaluated with echocardiography, quantitative PCR (qPCR) and overall S100A1 and CD38 immune protein. At 10 weeks all groups demonstrated a functional decline from baseline, but the S100A1 therapy groups displayed preserved left ventricular function with significantly higher ejection fraction %; SS group (60 ± 3) and SC group (57 ± 4) versus saline (46 ± 3), P < 0.05. Heart qPCR testing showed robust S100A1 in the SS (10,147 ± 3993) and SC (35,155 ± 5808) copies per 100 ng DNA, while off-target liver detection was lower in both SS (40 ± 40), SC (34,841 ± 3164), respectively. Cardiac S100A1 protein expression was (4.3 ± 0.2) and (6.1 ± 0.3) fold higher than controls in the SS and SC groups, respectively, P < 0.05.

Expression of vitellogenin receptor gene in the ovary of wild and captive Atlantic bluefin tuna (Thunnus thynnus).

The cDNA sequences of vitellogenin receptor proteins (VgR(+) and VgR(-)), containing or lacking the O-linked sugar domain, were determined in Atlantic bluefin tuna (Thunnus thynnus L.). VgR(-) gene expression in the ovary was compared in captive-reared and wild Atlantic bluefin tuna during the reproductive cycle. Gonad samples from adult fish were sampled from 2008 to 2010 from stocks reared in captivity at different commercial fattening operations in the Mediterranean Sea and from wild individuals caught either by traditional tuna traps during their migration towards the spawning grounds in the Mediterranean Sea or by the long-line artisanal fishery. In addition, juvenile male and female Atlantic bluefin tuna were sampled from a farming facility, to obtain baseline information and pre-adulthood amounts of VgR(-). The total length of VgR(+) cDNA was 4006 nucleotides (nt) and that of VgR(-) was 3946 nt. Relative amounts of VgR(-) were greater in juvenile females and in those adults having only previtellogenic oocytes (119 ± 55 and 146 ± 26 folds more than juvenile males, respectively). Amounts of VgR(-) were less in individuals with yolked oocytes (ripening stage, May-June) and increased after spawning in July (92 ± 20 and 113 ± 13 folds more than juvenile males in ripening and post-spawning fish, respectively). These data suggest that regulation of VgR(-) is not under oestrogen control. During the ripening period, greater VgR(-) gene expression was observed in wild fish than in fish reared in captivity, possibly because of (a) differences in water temperature exposure and/or energy storage, and/or (b) an inadequate diet in reared Atlantic bluefin tuna.

Gene delivery technologies for cardiac applications.

Ischemic heart disease (IHD) and heart failure (HF) are major causes of morbidity and mortality in the Western society. Advances in understanding the molecular pathology of these diseases, the evolution of vector technology, as well as defining the targets for therapeutic interventions has placed these conditions within the reach of gene-based therapy. One of the cornerstones of limiting the effectiveness of gene therapy is the establishment of clinically relevant methods of genetic transfer. Recently there have been advances in direct and transvascular gene delivery methods with the use of new technologies. Current research efforts in IHD are focused primarily on the stimulation of angiogenesis, modify the coronary vascular environment and improve endothelial function with localized gene-eluting catheters and stents. In contrast to standard IHD treatments, gene therapy in HF primarily targets inhibition of apoptosis, reduction in adverse remodeling and increase in contractility through global cardiomyocyte transduction for maximal efficacy. This article will review a variety of gene-transfer strategies in models of coronary artery disease and HF and discuss the relative success of these strategies in improving the efficiency of vector-mediated cardiac gene delivery.

GnRHa-mediated stimulation of the reproductive endocrine axis in captive Atlantic bluefin tuna, Thunnus thynnus.

A controlled-release implant loaded with GnRH agonist (GnRHa) was used to induce spawning in Atlantic bluefin tuna (Thunnus thynnus) during two consecutive reproductive seasons. The fish were implanted underwater and sampled between days 2 and 8 after treatment. At the time of GnRHa treatment, females were in full vitellogenesis and males in spermiation. There was a rapid burst of pituitary luteinizing hormone (LH) release at day 2 after treatment in GnRHa-treated fish, and circulating LH remained elevated up to day 8 after treatment. In contrast, control fish had significantly lower levels in the plasma, but higher LH content in the pituitary, as observed in many other cultured fishes that fail to undergo oocyte maturation, ovulation and spawning unless induced by an exogenous GnRHa. Plasma testosterone (T) and 17ß-estradiol (E(2)) were elevated in response to the GnRHa treatment in females, while 11-ketotestosterone (11-KT) but not T was elevated in males. Even though oocyte maturation and ovulation did occur in GnRHa-induced fish, no significant elevations in 17,20ß-dihydroxy-4-pregnen-3-one (17,20ß-P) or 17,20ß,21-trihydroxy-4-pregnen-3-one (20ß-S), in either the free, conjugated or 5ß-reduced,3α-hydroxylated forms was observed in fish sampled within 6 days after treatment. Interestingly, a significant peak in plasma free 17,20ß-P levels occurred in both males and females at day 8 after treatment. Histological sections of the ovaries in these females contained oocytes at the migrating germinal vesicle stage, suggesting the role of this hormone as a maturation-inducing steroid in Atlantic bluefin tuna. In conclusion, the GnRHa implants activated effectively the reproductive endocrine axis in captive Atlantic bluefin tuna broodstocks, through stimulation of sustained elevations in plasma LH, which in turn evoked the synthesis and secretion of the relevant sex steroids leading to gamete maturation and release.

Myocardial gene delivery using molecular cardiac surgery with recombinant adeno-associated virus vectors in vivo.

We use a novel technique that allows for closed recirculation of vector genomes in the cardiac circulation using cardiopulmonary bypass, referred to here as molecular cardiac surgery with recirculating delivery (MCARD). We demonstrate that this platform technology is highly efficient in isolating the heart from the systemic circulation in vivo. Using MCARD, we compare the relative efficacy of single-stranded (ss) adeno-associated virus (AAV)6, ssAAV9 and self-complimentary (sc)AAV6-encoding enhanced green fluorescent protein, driven by the constitutive cytomegalovirus promoter to transduce the ovine myocardium in situ. MCARD allows for the unprecedented delivery of up to 48 green fluorescent protein genome copies per cell globally in the sheep left ventricular (LV) myocardium. We demonstrate that scAAV6-mediated MCARD delivery results in global, cardiac-specific LV gene expression in the ovine heart and provides for considerably more robust and cardiac-specific gene delivery than other available delivery techniques such as intramuscular injection or intracoronary injection; thus, representing a potential, clinically translatable platform for heart failure gene therapy.

Comparative study of liver vitellogenin gene expression and oocyte yolk accumulation in wild and captive Atlantic bluefin tuna (Thunnus thynnus L.).

The sequence of vitellogenin A (VgA) and vitellogenin B (VgB) cDNAs in Atlantic bluefin tuna (Thunnus thynnus L.) were determined, and vitellogenin expression levels in the liver and oocyte yolk accumulation were compared in wild and captive-reared individuals. Liver and ovary samples were taken from 31 individuals reared experimentally in three commercial Atlantic bluefin tuna fattening sites in the Mediterranean Sea and from 33 wild individuals caught by commercial traps during the fish's migration towards their Mediterranean spawning grounds. The total length of VgA cDNA was 5585 nucleotides and that of VgB was 5267 nucleotides. The identity and similarity between deduced amino acid sequences of VgA and VgB were 60% and 78%, respectively. The Atlantic bluefin tuna VgA and VgB amino acid sequences have high similarities with those of other teleost fishes. Relative levels of VgA and VgB mRNAs were low in April, increased significantly during the reproductive period in May and June, and declined in July. There was a trend towards higher relative levels of VgA and VgB mRNAs in captive fish compared to wild individuals during the reproductive period. The surface occupied by eosinophilic yolk granules in fully vitellogenic oocytes, as well as the frequency of oocytes in late vitellogenesis, was significantly higher in captive compared to wild individuals. The study suggests that the experimental conditions under which Atlantic bluefin tuna individuals were reared allowed the occurrence of normal vitellogenesis, based on gene expression of VgA and VgB in the liver and yolk accumulation in the oocytes. The higher yolk accumulation and frequency of vitellogenic oocytes observed in the ovaries of captive fish suggest that improvements in feeding practices may result in an improved vitellogenic process.

Proliferation and apoptosis of male germ cells in captive Atlantic bluefin tuna (Thunnus thynnus L.) treated with gonadotropin-releasing hormone agonist (GnRHa).

The effects of administration of gonadotropin-releasing hormone agonist (GnRHa) on proliferation and apoptosis of male germ cells were evaluated on Atlantic bluefin tuna (Thunnus thynnus L.) reared in captivity. Fish (n=19) were treated with a sustained-release delivery system loaded with GnRHa during the natural spawning season of 2004 and 2005 (June-July). Untreated Control fish (n=17) and adult wild spawners were used for comparison. Fish were sacrificed 2-8 d after GnRHa implantation and body weight and gonad weight were recorded, and gonads and blood were taken. Germ cell proliferation and apoptosis were evaluated through the immunohistochemical detection of proliferating cell nuclear antigen (PCNA) and the terminal deoxynucleotidyl transferase-mediated d'UTP nick end labelling (TUNEL) method, respectively. Plasma 11 ketotestosterone (11-KT) levels were measured using an ELISA method. Mean gonado-somatic index and seminiferous lobule diameter did not differ between GnRHa-treated and Control fish, and were significantly lower in captive-reared individuals than in wild spawners. Significant increases in 11-KT plasma levels and spermatogonial mitosis, along with a reduction of germ cell apoptosis were demonstrated in GnRHa-treated fish compared to Controls. The results suggest that GnRHa administration was effective in enhancing germ cell proliferation and reducing apoptosis in captive males through the stimulation of luteinizing hormone (LH) release and testicular 11-KT production.

A histological investigation of the occurrence of non-reproductive female bluefin tuna Thunnus thynnus in the Mediterranean Sea.

The presence of non-reproductive Atlantic bluefin tuna Thunnus thynnus females in the Mediterranean Sea was investigated through histological analysis of the gonads. Three hundred and twenty-six ovary samples were collected from adults captured at different locations in the Mediterranean Sea during the reproductive seasons between 1998 and 2008. Only three specimens were considered to be in a non-reproductive state: two of them were in a reabsorbing state showing ovaries with early vitellogenic oocytes and extensive alpha and beta atresia of vitellogenic follicles; the third showed gonads with perinucleolar oocytes and was considered to be in a resting state. The low occurrence of non-reproductive individuals found in this study makes it unlikely that non-reproductive individuals aggregate with reproductive ones during their migration towards spawning grounds. Further research is suggested in order to investigate the potential presence of non-reproductive individuals on non-spawning grounds during the reproductive season.

Effects of hypoxia on the energy status and nitrogen metabolism of African lungfish during aestivation in a mucus cocoon.

We examined the energy status, nitrogen metabolism and hepatic glutamate dehydrogenase activity in the African lungfish Protopterus annectens during aestivation in normoxia (air) or hypoxia (2% O(2) in N(2)), with tissues sampled on day 3 (aerial exposure with preparation for aestivation), day 6 (entering into aestivation) or day 12 (undergoing aestivation). There was no accumulation of ammonia in tissues of fish exposed to normoxia or hypoxia throughout the 12-day period. Ammonia toxicity was avoided by increased urea synthesis and/or decreased endogenous N production (as ammonia), but the dependency on these two mechanisms differed between the normoxic and the hypoxic fish. The rate of urea synthesis increased 2.4-fold, with only a 12% decrease in the rate of N production in the normoxic fish. By contrast, the rate of N production in the hypoxic fish decreased by 58%, with no increase in the rate of urea synthesis. Using in vivo (31)P NMR spectroscopy, it was demonstrated that hypoxia led to significantly lower ATP concentration on day 12 and significantly lower creatine phosphate concentration on days 1, 6, 9 and 12 in the anterior region of the fish as compared with normoxia. Additionally, the hypoxic fish had lower creatine phosphate concentration in the middle region than the normoxic fish on day 9. Hence, lowering the dependency on increased urea synthesis to detoxify ammonia, which is energy intensive by reducing N production, would conserve cellular energy during aestivation in hypoxia. Indeed, there were significant increases in glutamate concentrations in tissues of fish aestivating in hypoxia, which indicates decreases in its degradation and/or transamination. Furthermore, there were significant increases in the hepatic glutamate dehydrogenase (GDH) amination activity, the amination/deamination ratio and the dependency of the amination activity on ADP activation in fish on days 6 and 12 in hypoxia, but similar changes occurred only in the normoxic fish on day 12. Therefore, our results indicate for the first time that P. annectens exhibited different adaptive responses during aestivation in normoxia and in hypoxia. They also indicate that reduction in nitrogen metabolism, and probably metabolic rate, did not occur simply in association with aestivation (in normoxia) but responded more effectively to a combined effect of aestivation and hypoxia.

The canine right caudal and accessory lobe pulmonary veins: revised anatomical description, clinical relevance, and embryological implications.

Necropsy dissections were performed on nine dogs to provide an anatomical description of the right caudal and accessory lobe pulmonary veins. In all dogs, the pulmonary vein from the right caudal lung lobe initially paralleled the right caudal lung lobe bronchus, running cranially, medially, and ventrally. It diverged from the bronchus at the level of the pulmonary artery and bronchus of the accessory lung lobe. At this point, the pulmonary vein from the right caudal lung lobe coursed dorsal to the pulmonary artery and bronchus of the accessory lung lobe. Medial to the bronchus of the accessory lung lobe, it received the pulmonary vein from the accessory lung lobe on its ventral surface. Within the pericardium, this common venous trunk merged with the caudal aspect of the left atrium either with or immediately adjacent to the left caudal lobe pulmonary vein. These findings were corroborated during surgical dissection to achieve isolation of the heart in five dogs as part of an experimental study on intravascular gene delivery to the heart. These anatomical findings are relevant to clinical and experimental surgery and raise interesting questions about the embryological development of pulmonary veins in the dog.

Immunohistochemical localization of CYP1A, vitellogenin and Zona radiata proteins in the liver of swordfish (Xiphias gladius L.) taken from the Mediterranean Sea, South Atlantic, South Western Indian and Central North Pacific Oceans.

Cytochrome P4501A (CYP1A) monoxygenase, vitellogenin (Vtg) and Zona radiata proteins (Zrp) are frequently used as biomarkers of fish exposure to organic contaminants. In this work, swordfish liver sections obtained from the Mediterranean Sea, the South African coasts (South Atlantic and South Western Indian Oceans) and the Central North Pacific Ocean were immunostained with antisera against CYP1A, Zrp, and Vtg. CYP1A induction was found in hepatocytes, epithelium of the biliary ductus and the endothelium of large blood vessels of fish from the Mediterranean Sea and South African waters, but not from the Pacific Ocean. Zrp and Vtg were immunolocalized in hepatocytes of male swordfish from the Mediterranean Sea and from South African waters. Plasma Dot-Blot analysis, performed in Mediterranean and Pacific specimens, revealed the presence of Zrp and Vtg in males from Mediterranean but not from Pacific. These results confirm previous findings about the potential exposure of Mediterranean swordfish to endocrine, disrupting chemicals and raise questions concerning the possible presence of xenobiotic contaminants off the Southern coasts of South Africa in both the South Atlantic and South Western Indian Oceans.

Histological and immunohistochemical investigation on ovarian development and plasma estradiol levels in the swordfish (Xiphias gladius L.).

The paper reports a histological and immunohistochemical description of oocyte growth and ultrastructural aspects of zona radiata (ZR) formation as well as the relationship between plasma estradiol-17beta, (E2) levels and ovarian development in swordfish (Xiphias gladius L.) from the Mediterranean Sea. Ovaries were inactive during March to mid April; maturation occurred during late April to June and spawning in June and July. Zona radiata formation starts, as Pas positive material, in oocytes at the lipid stage. In this stage a deposit of electrondense material between oolemma and follicular cells appears. In the cortical alveoli stage and through the early vitellogenic stage, the deposition of a moderately electrondense material occurred on the inner side of the ZR. Finally, in late vitellogenic oocytes a third layer, made of microfibrillar material, appeared. The immunohistochemical analyses revealed that the initial internalisation of hepatic zona radiata proteins (Zrp) in the swordfish oocyte starts before the uptake of vitellogenin (Vtg) and that it is associated with the low previtellogenic E2 plasma levels, while a significant E2 increase in plasma is associated with the beginning of Vtg uptake. This would appear to confirm the hypothesis that the differential and sequential induction of zonagenesis and vitellogenesis may reflect a general feature of teleost oogenesis.

Evidence of a high percentage of intersex in the Mediterranean swordfish (Xiphias gladius L.).

The first evidence of the presence of intersexuality in a wild population of Mediterranean swordfish (Xiphias gladius L.) is reported. Forty of 162 specimens (25%) macroscopically classified as males, showed the presence of female germ cells within the testes. In two specimens grouped previtellogenic oocytes were present; all the other specimens possessed single scattered previtellogenic oocytes. The presence of vitellogenin was demonstrated immunohistochemically in the liver of both intersex and normal males. These findings could be due to the exposure to oestrogen-mimicking substances.

Structural and functional properties of hemocyanin from Cyanagraea praedator, a deep-sea hydrothermal vent crab.

Cyanagraea praedator (Crustacea: Decapoda: Brachyura) is an endemic species of the East Pacific Rise hydrothermal vents, living in the upper part of black smoker chimneys. Because we were seeking species that have made respiratory adaptations to the hydrothermal environment, we looked at Cyanograea hemocyanin (Hc) and determined its quaternary structure and the oxygen-binding properties in relation to temperature, pH, and lactate. C. praedator Hc is composed of dodecamers and hexamers, with dodecamers formed by the perpendicular association of two hexamers. The composition of these polymers was determined by electrophoresis and, for the first time, by electrospray mass spectrometry. Dodecamers and hexamers are composed of six subunits common to the two forms, with molecular mass ranging from 75,008 Da to 75,534 Da. In addition, we found two dodecamer-specific subunits, at 75,419 Da and 75,629 Da. The native hemocyanin possesses a high oxygen affinity (P(50) varies between 4 and 10 Torr at pH 7.5, 15 degrees C) and a large Bohr coefficient (Delta log P(50)/DeltapH approximately -1.8). Oxygen affinity is not affected by lactate or, surprisingly, temperature between 5 degrees C and 35 degrees C (DeltaH = 1.16 kJ/mol(1) 5-35 degrees C). Dialysis of native hemolymph elicited a significant increase in Hc-O(2) affinity (DeltaP(50) = 2.5 Torr at pH 7.5), an effect opposite the usual trend observed for crustacean hemocyanins. In this article these functional properties are interpreted in relation to characteristics of the environment.

The Batista procedure. Theoretical analysis and clinical implications.

BACKGROUND: The Batista procedure leads to dramatic early improvement in left ventricular function in some patients and a worsening in function in others. The theoretical and actual clinical effects of the procedure on early postoperative left ventricular function remain controversial. The purpose of this study is to utilize an appropriate mathematical model to determine the effects of the Batista procedure on stroke volume and myocardial wall stress. Our hypothesis is that the preoperative end-systolic stress (ses) is an important predictor of early postoperative myocardial function after this procedure. A corollary is that an index related to ses may be useful in selecting patients for this procedure. METHODS: An analysis of the Batista procedure is developed, based upon a spherical membrane model of the ventricle. This model shows how ventricular dilatation distorts the systolic and diastolic pressure-volume relations. RESULTS: Dilatation initially improves ventricular performance; but further dilatation, beyond a critical value, produces an unstable state with sharply falling performance. For a ventricle operating significantly beyond the point of critical dilatation, our theoretical results suggest that the Batista procedure not only reduces myocardial stress but may improve stroke volume. The end-systolic stress, ses is an indicator of how close a ventricle is to the critical dilatation point. CONCLUSIONS: There is a theoretical basis for the Batista procedure. Resection of myocardium not only decreases wall stress but may improve stroke volume for sufficiently dilated and depressed ventricles. Patients with markedly elevated end-systolic stress may benefit most from the Batista procedure.