Dietary vitamin C reduces mortality of pacific white shrimp (Penaeus vannamei) post-larvae by Vibrio parahaemolyticus challenge.

This study was conducted to investigate whether optimal vitamin C (VC) levels can enhance non-specific immune response and antioxidant capacity and reduce mortality of Pacific white shrimp (Penaeus vannamei) post-larvae when infected with Vibrio parahaemolyticus. Six experimental diets were formulated to contain six different VC levels of 0, 40, 80, 120, 160 and 320 mg/kg diet (designated as C0, C40, C80, C120, C160 and C320, respectively). Shrimp post-larvae (39.1 ± 0.47 mg) were randomly distributed to 24 tanks with 40 shrimp per tank. Four replicate groups of shrimp were fed one of the diets for 43 days. VC supplemented groups showed significantly higher growth performance than C0 group. Shrimp fed C120 diet had significantly improved feed utilization efficiency than shrimp fed C0 diet. VC concentrations in hepatopancreas and gills were significantly higher with the increase in dietary VC levels. Optimal dietary VC levels significantly upregulated the expressions of growth and digestive enzyme-related genes such as IGF-1, IGF-BP, amylase, trypsin and chymotrypsin, and also upregulated the expressions of innate immunity and antioxidant-related genes such as prophenoloxidase, crustin, penaiedin-3a, superoxide dismutase, glutathione peroxidase and catalase in hepatopancreas. Shrimp fed C80, C120 and C160 diets showed significantly increased resistance to V. parahaemolyticus than shrimp fed C0 diet. The optimum dietary VC level for the shrimp post-larvae was established to be 80.2 mg/kg diet by a broken-line regression analysis based on the growth. The findings from the challenge test indicated that VC levels over 83.0 mg/kg diet could enhance disease resistance of the shrimp against V. parahaemolyticus.

Dietary Riboflavin Requirement of Pacific White Shrimp (Litopenaeus vannamei).

This study was conducted to determine the dietary riboflavin requirement and its effects on growth performance, feed utilization, innate immunity, and diet digestibility of Litopenaeus vannamei. A riboflavin-free basal diet (R0) was formulated as a control, and six other diets were prepared by adding riboflavin of 10, 20, 30, 40, 50, and 60 mg/kg to the basal diet (designated as R10, R20, R30, R40, R50, and R60, respectively). Quadruplicate groups of shrimp (initial average weight 0.17 ± 0.00 g) were fed the diets six times a day for 8 weeks. Weight gain, specific growth rate, and protein efficiency ratio were significantly increased by riboflavin (p < 0.05). The maximum values were observed in shrimp fed R40 diet. The highest activities of phenoloxidase, nitro blue tetrazolium, superoxide dismutase, and glutathione peroxidase were observed in shrimp fed R40 diet. Lysozyme activity was significantly higher in shrimp fed R30 and R40 diets than that of shrimp fed R60 diet (p < 0.05). Intestinal villi were significantly longer in shrimp fed R50 and R60 diets compared to those of all other groups while the shortest villi were observed in R0 group (p < 0.05). Intestinal villi were clearly distinguished in shrimp fed higher levels of riboflavin compared to those of shrimp fed R0 and R10 diets. Apparent digestibility coefficients of dry matter and protein in diets were not significantly affected by riboflavin levels (p < 0.05). Whole-body proximate composition and hemolymph biochemical parameters were not significantly altered by dietary riboflavin (p < 0.05). Therefore, the results of this study indicate that riboflavin is essential to enhance growth performance, feed utilization, nonspecific immunity, and intestine morphology of shrimp. An optimal riboflavin requirement for the maximum growth of L. vannamei seems to be approximately 40.9 mg/kg diet.

Effects of dietary piperine supplementation on innate immunity, growth performance, feed utilization and intestinal morphology of olive flounder (Paralichthys olivaceus).

Piperine, the main bioactive component of black pepper (Piper nigrum) or long pepper (Piper longum), has anti-inflammatory, antifungal, and antibacterial properties. This study was carried out to evaluate the supplemental effects of piperine in olive flounder (Paralichthys olivaceus) diets. Six isonitrogenous and isolipidic diets were formulated to contain different levels of piperine at 0.00, 0.25, 0.50, 0.75, 1.00, and 2.00 g/kg (Con, P25, P50, P75, P100, and P200, respectively). Diets were randomly allocated to triplicate groups of fish (initial weight 27.6 ± 0.4 g, 30 fish/tank) and fed three times daily for 8 weeks. Results showed that dietary piperine significantly improved fish growth and feed utilization efficiency. The highest growth, including the highest Igf-1 mRNA expression, was observed in the P50 group, while P50 and P75 groups showed the highest protein efficiency ratio. Compared to the Con group piperine supplemented groups had significantly higher lysozyme activity, immunoglobulin level, and phagocytosis activities. Plasma cholesterol was significantly lower in fish fed P200 diet. Dry matter and protein digestibility were higher in P25, P50, and P75 groups than in Con group. Dietary piperine increased the intestinal villi length and goblet cell counts. In the challenge test against Edwardsiella tarda, all the groups supplemented with piperine showed higher cumulative survival compared to Con group. Therefore, these findings indicate that dietary piperine supplementation can improve growth performance, innate immunity, disease resistance, diet digestibility, and intestinal morphology of olive flounder. The optimum dietary piperine level seems to be approximately 0.5 g/kg for the fish.

Antioxidant and Neuroprotective Effects of N-((3,4-Dihydro-2H-benzo[h]chromen-2-yl)methyl)-4-methoxyaniline in Primary Cultured Rat Cortical Cells: Involvement of ERK-CREB Signaling.

Excitotoxicity and oxidative stress play vital roles in the development of neurodegenerative disorders including Alzheimer's disease (AD). In the present study, we investigated the effect of N-((3,4-dihydro-2H-benzo[h]chromen-2-yl)methyl)-4-methoxyaniline (BL-M) on excitotoxic neuronal cell damage in primary cultured rat cortical cells, and compared to that of memantine, a non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist clinically used to treat AD. We found that BL-M inhibited glutamate- or N-methyl-d-aspartate (NMDA)-induced excitotoxic cell damage. The IC50 value of BL-M against NMDA toxicity was comparable to that of memantine. BL-M potently inhibited intracellular reactive oxygen species generated by glutamate or NMDA. Additionally, it inhibited the formation of 1,1-diphenyl-2-picryl-hydrazyl radicals in vitro and lipid peroxidation in rat brain homogenates. In contrast, memantine showed minimal or negligible antioxidant activity. Western blotting and immunocytochemical analyses showed that BL-M, not memantine, increased the ERK1/2 phosphorylation and subsequent phosphorylation of cAMP response element-binding protein (CREB). The inhibition of NMDA toxicity by BL-M was dramatically reversed by U0126, a well-known MEK inhibitor, suggesting that ERK1/2-mediated CREB phosphorylation is required for the neuroprotective action. Collectively, in this study, we demonstrated the neuroprotective effect of a newly synthesized chromene derivative BL-M and its underlying action mechanism(s). In contrast to memantine, BL-M exhibited marked antioxidant activity. Furthermore, it enhanced the ERK-mediated phosphorylation of CREB, which plays a crucial neuroprotective role. Our findings suggest that BL-M may be beneficial for AD and other neurodegenerative disorders associated with excitotoxicity as well as oxidative stress.

Effects of protein hydrolysates supplementation in low fish meal diets on growth performance, innate immunity and disease resistance of red sea bream Pagrus major.

This study was conducted to evaluate the supplemental effects of three different types of protein hydrolysates in a low fish meal (FM) diet on growth performance, feed utilization, intestinal morphology, innate immunity and disease resistance of juvenile red sea bream. A FM-based diet was used as a high fish meal diet (HFM) and a low fish meal (LFM) diet was prepared by replacing 50% of FM by soy protein concentrate. Three other diets were prepared by supplementing shrimp, tilapia or krill hydrolysate to the LFM diet (designated as SH, TH and KH, respectively). Triplicate groups of fish (4.9 ± 0.1 g) were fed one of the test diets to apparent satiation twice daily for 13 weeks and then challenged by Edwardsiella tarda. At the end of the feeding trial, significantly (P < 0.05) higher growth performance was obtained in fish fed HFM and hydrolysate treated groups compared to those fed the LFM diet. Significant improvements in feed conversion and protein efficiency ratios were obtained in fish fed the hydrolysates compared to those fed the LFM diet. Significant enhancement in digestibility of protein was found in fish fed SH and KH diets and dry matter digestibility was increased in the group fed SH diet in comparison to LFM group. Fish fed the LFM diet showed significantly higher glucose level than all the other treatments. Whole-body and dorsal muscle compositions were not significantly influenced by dietary treatments. Histological analysis revealed significant reductions in goblet cell numbers and enterocyte length in the proximal intestine of fish fed the LFM diet. Superoxide dismutase activity and total immunoglobulin level were significantly increased in fish fed the diets containing protein hydrolysates compared to the LFM group. Also, significantly higher lysozyme and antiprotease activities were found in fish fed the hydrolysates and HFM diets compared to those offered LFM diet. Fish fed the LFM diet exhibited the lowest disease resistance against E. tarda and dietary inclusion of the hydrolysates resulted in significant enhancement of survival rate. The results of the current study indicated that the inclusion of the tested protein hydrolysates, particularly SH, in a LFM diet can improve growth performance, feed utilization, digestibility, innate immunity and disease resistance of juvenile red sea bream.

Choline Essentiality and Its Requirement in Diets for Juvenile Parrot Fish (Oplegnathus fasciatus).

A 12-wk feeding trial was conducted to evaluate the essentiality of choline supplementation in diets for parrot fish. Five isonitrogenous and isocaloric diets were supplemented with 0 (as control), 500, 1,000, and 2,000 mg choline per kg diet, and a positive control diet without choline contained 0.3% of 2-amino-2-methyl-1-propanol as choline biosynthesis inhibitor (designated as Con, C500, C1000, C2000 and Con(+), respectively). Triplicate groups of fish (body weight, 8.8±0.01 g) were fed one of the experimental diets at a rate of 4% body weight twice daily. The fish fed Con(+) diet revealed significantly lower growth performance and feed utilization efficiency than other fish groups. Supplementation of choline to the basal diet did not significantly influence fish growth. The highest liver lipid content was observed in fish fed the Con(+) diet and inversely correlated with liver choline concentration although the differences were not significant. Also, significantly higher liver linoleic, eicosapentaenoic and docosahexaenoic acid contents were found in fish fed the Con(+) diet. Innate immune parameters including respiratory burst and myeloperoxidase activities were not significantly affected by dietary choline levels. The findings in this study conclude that choline concentration of approximately 230 mg kg(-1) diet meets the requirement of parrot fish.

Effects of Dietary Supplementation of Barodon, an Anionic Alkali Mineral Complex, on Growth Performance, Feed Utilization, Innate Immunity, Goblet Cell and Digestibility in Olive Flounder (Paralichthys olivaceus).

A 15-wk feeding trial was conducted to examine the supplemental effects of Barodon on growth performance, gastrointestinal histology, feed digestibility and innate immunity in olive founder. A basal commercial diet was used as a control and two other diets were prepared by spraying 0.1% or 0.2% of Barodon. Triplicate groups of fish (BW, 145 g) were fed one of the test diets to apparent satiation twice daily. At the end of the feeding trial, fish growth performance was not significantly affected by dietary treatments; however, feed utilization was significantly improved (linear and quadratic, p<0.05) by Barodon supplementation. Significantly higher (p<0.05) survival rates were obtained in fish fed Barodon containing diets. Hepatosomatic index increased significantly in Barodon treated groups. Also, the use of Barodon resulted in significant increase (linear and quadratic, p<0.05) of intestine length and number of goblet cells. Significantly higher (Quadratic, p<0.05) apparent digestibility coefficient of DM was obtained by supplementation of Barodon. Lysozyme and myeloperoxidase activities increased quadratically and linearly, respectively, in Barodon treated fish. Also, significantly higher (linear and quadratic, p<0.05) superoxide dismutase activity was found in Barodon fed fish. The findings in this study show that inclusion of Barodon in diets for olive flounder improves feed utilization and digestibility, and positively affects digestive tract histology and innate immunity.

Dietary Poly-ß-Hydroxybutyrate Improved the Growth, Non-specific Immunity, Digestive Enzyme Activity, Intestinal Morphology, Phagocytic Activity, and Disease Resistance Against Vibrio parahaemolyticus of Pacific White Shrimp, Penaeus vannamei.

This study assessed the effects of dietary supplementation of poly-ß-hydroxybutyrate (PHB) on growth performance, feed efficiency, non-specific immunity, digestive enzyme capacity, phagocytic activity, hemocyte count, intestinal morphology, and disease resistance against Vibrio parahaemolyticus of Pacific white shrimp (Penaeus vannamei). Six diets were prepared by supplementing graded levels of PHB at 0.00, 0.25, 0.50, 1.00, 2.00, and 4.00% (Con, P0.25, P0.5, P1.0, P2.0, and P4.0, respectively). Triplicate groups of 90 shrimps (initial body weight 0.25 ± 0.01 g) per treatment were randomly assigned and fed an experimental diet for 56 days. The growth performance of shrimp was significantly improved by 1% dietary PHB supplementation. PHB-included diets fed shrimp showed significantly improved hepatopancreatic trypsin, chymotrypsin, and pepsin activities. Villus height was significantly increased with dietary PHB supplementation, and villus width was increased at a 1% inclusion level. P0.25, P0.5, and P4.0 groups significantly increased phenoloxidase activity, and the P2.0 group significantly increased anti-protease activity compared to the Con group. The survival of shrimp challenged against V. parahaemolyticus was higher in P0.5, P1.0, and P2.0 groups than in the Con diet. Dietary PHB supplementation improved weight gain, digestive enzyme activity, intestinal morphology, non-specific immunity, and disease resistance against V. parahaemolyticus of shrimp. According to the above observations, the optimal dietary PHB supplementation level for maximum weight gain would be 1% for Pacific white shrimp.

Effects of dietary supplementation of inosine monophosphate on growth performance, innate immunity and disease resistance of olive flounder (Paralichthys olivaceus).

This study was investigated to examine the effects of dietary inosine monophosphate (IMP) supplementation on growth performance, feed utilization, innate immunity, hematological parameters and disease resistance of juvenile olive flounder. Five experimental diets were formulated to contain IMP at levels of 0, 0.1, 0.2, 0.4 and 1.0%. All diets were maintained isonitrogenous (48% crude protein) and isocaloric (20.7 MJ/kg diet). Triplicate groups of olive flounder (initial body weight, 7.5 ± 0.02 g) were fed one of the experimental diets to apparent satiation (twice a day) for 14 weeks. Final body weight of fish fed 0.1-0.2% IMP were significantly higher than that of fish fed the 1.0% IMP. Groups of fish fed 0.2 or 0.4% IMP diet had significantly higher myeloperoxidase and lysozyme activities than fish fed the control diet. However, nitro-blue-tetrazolium and superoxide dismutase activities were not significantly different among all treatments. In the challenge test against Streptococcus iniae, cumulative mortality of fish fed IMP supplemented diets was significantly lower (15%, 4%, 4% and 9% for 0.1%, 0.2%, 0.4% and 1.0% IMP, respectively) than that of fish fed the control group (87%). The results suggest that IMP supplementation of 0.46-1.84 g into a kg of fish meal based diet (0.1-0.4% IMP product) can enhance innate immunity and disease resistance of olive flounder.

Comparison of Growth Performance and Whole-body Amino Acid Composition in Red Seabream (Pagrus major) Fed Free or Dipeptide Form of Phenylalanine.

This study was conducted to evaluate the efficacy of the dipeptide form of phenylalanine as a new source of amino acid in terms of growth performance and whole-body amino acid composition in comparison to the free form for red seabream (Pagrus major). Fish (1.46±0.001 g) were fed four isonitrogenous and isocaloric experimental diets containing 0.7 or 1.4% phenylalanine either in free or dipeptide form. A feeding trial was carried out in three replicates and the fish were fed to apparent satiation for six weeks. At the end of the feeding trial, feed intake of fish was influenced by both phenylalanine form and level and significantly higher values were obtained at an inclusion level of 0.7% and by the use of dipeptide form. However, the other growth parameters did not significantly differ among treatments. Whole-body amino acid compositions revealed no significant changes in concentrations of both essential and non-essential amino acids regardless of the increase in phenylalanine levels or the use of its different forms. The finding in this study indicates that juvenile red seabream can utilize dipeptide phenylalanine as efficiently as free form without any undesirable effects on growth performance or whole-body amino acid composition.

Effects of Decreasing Fishmeal as Main Source of Protein on Growth, Digestive Physiology, and Gut Microbiota of Olive Flounder (Paralichthys olivaceus).

In olive flounder (Paralichthys olivaceus), growth performance, expression of growth-related factors, digestive physiology, and gut microbiota were assessed under farm conditions in the fish fed diets with low levels of fishmeal. Four experimental diets were prepared, FM70 [control (CON), 70% fishmeal], FM45 (45% fishmeal), FM35A (35% fishmeal), and FM35B (35% fishmeal + insect meal), and fed to the fish for five months. The CON-fed fish had the highest plasma GH, but IGF-1 and hepatic IGF-1 mRNA expression of the olive flounder fed diets with low-fishmeal levels did not significantly differ among diets. The intestinal villus length, muscular thickness, and the number of goblet cells were statistically similar, and ocular examination of hepatopancreas showed no discernable difference in all experimental diets. The chymotrypsin content of FM35B-fed fish is significantly lower, but trypsin and lipase contents are similar. The diversity of gut microbiota did not differ among groups, although the FM35B group had a higher composition of Firmicutes. Thus, a diet with reduced fishmeal content and several alternative protein sources can be used as feed ingredients in feed formulation for olive flounder reared under typical aquaculture farm conditions.

Digestibility of insect meals for Pacific white shrimp (Litopenaeus vannamei) and their performance for growth, feed utilization and immune responses.

This study was conducted to examine digestibility of insect meals for Pacific white shrimp (Litopenaeus vannamei) and their utilization as fish meal substitutes. The tested insect meals were mealworm, silkworm, black soldier fly, rice grasshopper, two-spotted cricket, dynastid beetle and white-spotted flower chafer. Apparent digestibility coefficients of the tested insect meals were 83-89% for protein, 91-98% for lipid, 84-90% for energy, 77-81% for dry matter, 28-36% for chitin, 76-96% for amino acids and 89-93% for fatty acids. The amino acid availability of insect meals was high in taurine (93-96%), arginine (91-95%) and lysine (90-95%). Availability of fatty acids were 89-93% for saturated fatty acids, 90-93% for monounsaturated fatty acids and 88-93% for polyunsaturated fatty acids. For a feeding trial, a control diet was formulated using 27% tuna byproduct meal as a fish meal source and seven other diets were prepared replacing 10% tuna byproduct meal in the control diet with each insect meal. Triplicate groups of shrimp (initial body weight: 0.17 g) were fed the diets for 65 days. The growth performance was significantly improved when the shrimp were fed black soldier fly or dynastid beetle included diet. Dietary supplementation of insect meals significantly improved non-specific immune responses and antioxidant enzyme activity in the shrimp. These results indicate that the tested insect meals have high potentials to be used as a protein source that could replace fish meal in diets for the shrimp.

Effect of Fishmeal Content in the Diet on the Growth and Sexual Maturation of Olive Flounder (Paralichthysolivaceus) at a Typical Fish Farm.

Olive flounder (Paralichthys olivaceus) is a commercially important and valuable species for aquaculture in Korea. Due to the unstable supply of fishmeal for farmed fish, an optimum fish-feed formulation should be researched to ensure the sustainability of P. olivaceus aquaculture. This study investigated the effect of three experimental diets: Con (basal diet); FM20 (20% fishmeal replacement of CON); and FM30 (30% fishmeal replacement of CON) on P. olivaceus over 20 weeks at a typical farm by monitoring the growth and factors relating to sexual maturation. The results showed that no differences in growth were observed between the CON and diet-replacement groups. Gonadal oocyte development was similar between the CON and diet-replacement groups. Moreover, sbGnRH and GH expression did not differ between the CON and diet-replacement groups. The levels of Erß and Vtg expression were significantly higher in the FM20 group than in the CON and FM30 groups after the experimental period. The expression of PSS-I was significantly higher in the FM30 group than in the CON and FM20 groups. Therefore, although growth occurred when 30% of the fishmeal was replaced, such high dietary protein replacement may be ill-advised during the maturation of olive flounder at the commercial fish farm.

Beta-sitosterol-induced-apoptosis is mediated by the activation of ERK and the downregulation of Akt in MCA-102 murine fibrosarcoma cells.

Beta-sitosterol (SITO) is a potential candidate for cancer chemotherapy, however, little is known about the cellular and molecular mechanisms in cancer cells. We herein identified how SITO induces anti-proliferation and cell death in MCA-102 fibrosarcoma cells. SITO exposure induced-apoptosis and the cell death resulted from a significant loss of the Bcl-2 and the inhibitor of apoptosis protein (IAP) family (XIAP, cIAP-1 and cIAP-2), and increased Bax with an alteration of p53 and p21. SITO-induced cell death significantly also increased caspase activity and poly(ADP-ribose) polymerase (PARP) cleavage, and caspase-3 inhibitor z-DEVD-fmk significantly inhibited SITO-induced cell death. These data suggest that the activation of caspase-3 is associated with SITO-induced-apoptosis. Treatment with SITO also induced phosphorylation of extracellular-signal regulating kinase (ERK) and p38 mitogen-activated protein kinase (MARK), but not c-Jun N-terminal kinase (JNK). A specific ERK inhibitor PD98059 significantly blocks SITO-induced-apoptosis, whereas a JNK inhibitor SP600125 has no affect. A p38 MAPK inhibitor SB203580 very slightly suppressed cell death. The induction of apoptosis was also accompanied by an inactivation of phosphatidylinositol 3-kinase (PI3K)/Akt, and PI3K inhibitor LY29004 significantly increases SITO-induced cell death. These findings provide evidence demonstrating that the proapoptotic effect of SITO is mediated through the activation of ERK and the block of the PI3K/Akt signal pathway in MCA-102 cells. Therefore, SITO has a strong potential as a therapeutic agent for preventing cancers such as fibrosarcoma.

Bee venom and melittin reduce proinflammatory mediators in lipopolysaccharide-stimulated BV2 microglia.

Bee venom (BV), well known as a traditional Oriental medicine, has been shown to exhibit anti-arthritic and anti-carcinogenic effects. However, the molecular mechanisms responsible for the anti-inflammatory activity of BV have not been elucidated in microglia. In the present study, we investigated the anti-inflammatory effect of BV and its major component, melittin (MEL), on lipopolysaccharide (LPS)-stimulated BV2 microglia. Our results indicate that BV and MEL suppress LPS-induced nitric oxide (NO) and inducible NO synthase (iNOS) expression in a dose-dependent manner, without causing cytotoxicity in BV2 microglia. Moreover, BV and MEL suppressed LPS-induced activation of nuclear factor kappa B (NF-kappaB) by blocking degradation of IkappaBalpha and phosphorylation of c-Jun N-terminal kinase (JNK) and Akt, which resulted in inhibition of iNOS expression. Our data also indicate that BV and MEL exert anti-inflammatory effects by suppressing the transcription of cyclooxygenase (COX)-2 genes and proinflammatory cytokines, such as interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)-alpha. BV and MEL also attenuated the production of prostaglandin E(2) (PGE(2)). These results demonstrate that BV and MEL possess a potent suppressive effect on proinflammatory responses of BV2 microglia and suggest that these compounds may offer substantial therapeutic potential for treatment of neurodegenerative diseases that are accompanied by microglial activation.

Inhibitory effects of eicosapentaenoic acid on lipopolysaccharide-induced activation in BV2 microglia.

Upon activation, microglia release proinflammatory mediators that play important roles in eliciting neuroinflammatory responses associated with neurodegenerative diseases. The anti-inflammatory properties of eicosapentaenoic acid (EPA) have been known, however, the effects responsible for lipopolysaccharide (LPS)-induced activation remain poorly understood in microglia. In the present study, we investigated the effects of EPA on the expression of proinflammatory mediators in LPS-stimulated BV2 microglia. EPA significantly inhibited the release of nitric oxide (NO), prostaglandin E(2) (PGE(2)) and proinflammatory cytokines such as interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)-alpha in a dose-dependent manner. EPA also attenuated the production of cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS) and proinflammatory cytokines at mRNA and/or protein levels. Moreover, EPA suppressed NF-kappaB activation by blocking IkappaB degradation, and also blocked the mitogen-activated protein kinases (MAPKs) such as ERK, p38 and JNK, and the Akt pathway. The anti-inflammatory properties of EPA may be useful for ameliorating neurodegenerative diseases as well as suppressing LPS-induced shock.

Statistical selection strategy for risk and protective rare variants associated with complex traits.

In genetic association studies with deep sequencing data, it is a challenging statistical problem to precisely locate rare variants associated with complex diseases or traits due to the limited number of observed genetic mutations. In particular, both risk and protective rare variants can be present in the same gene or genetic region. There currently exist very few statistical methods to separate casual rare variants from noncausal variants within a disease/trait-related gene or a genetic region, while there are relatively many statistical tests to detect a phenotypic association of a group of rare variants such as a gene or a genetic region. In this article, we propose a new statistical selection strategy that is able to locate causal rare variants within the disease/trait-related gene or a genetic region. The proposed procedure is to linearly combine potential risk and protective variants in order to find the optimal combination of rare variants that can have the strongest association signal. It is also computationally very efficient since the procedure is based on forward selection. In simulation studies we demonstrate that the selection performance of the proposed procedure is more powerful than other existing methods when both risk and protective variants are present. We also applied it to the real sequencing data on the ANGPTL gene family from the Dallas Heart Study.

High-performance liquid chromatographic determination of gossypol and gossypolone enantiomers in fish tissues using simultaneous electrochemical and ultraviolet detectors.

There is a need to measure dietary transfer of gossypol and its metabolite, gossypolone in aquatic animals because of common use of cottonseed meal as a feed ingredient and fertilizer. The analytical method for gossypol and gossypolone enantiomers, therefore, becomes important. HPLC techniques have been developed by using mainly UV detection. We simultaneously used both UV and electrochemical (EC) detectors, and found that each individual detector has its own advantage which can increase accuracy and ease of identification. EC detection (2.5 and 50 ng/ml) exhibited a significantly lower detection level for both gossypol and gossypolone enantiomers than the UV detection (40 and 300 ng/ml) in the rainbow trout tissues, while UV detectors showed more stable detection than EC. We were able to detect a very low concentration of each gossypol enantiomer by EC but not UV detection especially in seminal plasma. For the first time gossypolone enantiomers were quantified in fish tissues by HPLC and its method was described. The technique, simultaneous adoption of both UV and EC detectors, could be helpful for a very low concentration of gossypol and/or gossypolone enantiomers in tissues of other animals and humans.

Gossypol and gossypolone enantiomers in tissues of rainbow trout fed low and high levels of dietary cottonseed meal.

Gossypol is an antifertilizing agent in males and females. However, gossypol and its metabolite, gossypolone, have also gained interest because of their anticarcinogenic activities. This paper examines for the first time both enantiomers of tissue gossypol and gossypolone in mature rainbow trout fed two diets containing low (15%) and high (60%) levels of cottonseed meal (CM) for 9 months. The gossypol concentration was highest in liver followed by kidney, intestine, testis, blood plasma, stomach, and muscle. Gossypol was detected in muscles of fish fed low- and high-CM diets (0.31 +/- 0.03 and 1.95 +/- 0.59 microg of total gossypol/g, wet basis, respectively). The (+)-gossypol enantiomer was predominantly retained in all tissues. The ratio of (-)- to total gossypol ranged from 30 to 44% in fish fed the high-CM diet and from 23 to 30% in fish fed the low-CM diet except for muscle tissue (44%). Higher gossypolone concentrations were found in intestine than in liver. Gossypolone, however, was not detected in blood plasma, muscle, and testis of fish fed the low-CM diet. The ratio of gossypolone to gossypol was highest in muscle (1.75), followed by intestine (1.59), stomach (1.50), kidney (0.43), liver (0.34), testis (0.28), and blood plasma (0.27). This study indicated that the retention of the (-)-gossypol enantiomer is dependent on dietary concentrations and that the oxidative conversion of gossypol to gossypolone occurs more actively in the digestive tract and muscle than in other tissues in rainbow trout.

Effect of dietary ascorbic acid on growth and non-specific immune responses of tiger puffer, Takifugu rubripes.

We report nutritional physiology and non-specific immune responses of ascorbic acid (AA) in puffer fish for the first time. This study aimed to examine the essentiality and requirements of AA in diets for the tiger puffer, Takifugu rubripes based on growth performance, liver AA and bone collagen concentration, and non-specific immune responses. Five casein-gelatin based semi-purified diets were formulated to contain five graded levels of l-ascorbyl-2-monophosphate at 0, 40, 80, 160 and 700mg/kg (designated as AMP0, AMP40, AMP80, AMP160 and AMP700, respectively) and fed to triplicate groups of fish. After 10weeks of feeding trial, growth performances of fish (initial body weight, 35g) fed the AMP0 were significantly lower compared to that of fish fed diets supplemented with AMP. The fish fed the AMP0 diet also exhibited significantly lower hematocrit, condition factor and hepatosomatic index compared to the fish fed diets supplemented with AMP. Phagocytic activity (NBT assay) was significantly lower in fish fed the AMP0 diet than in fish fed the AMP containing diets. Plasma lysozyme activity of fish fed the AMP80 and AMP160 was significantly higher than that of fish fed the AMP0. Dietary supplementation of AMP significantly increased the liver superoxide dismutase in the fish. Myeloperoxidase activity of fish fed the AMP0 was significantly lower compared to that of fish fed the AMP containing diets. Bone collagen level tended to increase numerically and total AA concentration in liver of fish was significantly increased in a dose dependent manner by the supplementation of AMP. Therefore, tiger puffer requires exogenous ascorbic acid and the optimum dietary level could be 29mg AA/kg diet for normal growth and physiology. Dietary AA concentration over 82mg/kg could be required to enhance non-specific immune responses of the fish. However, it does not seem that the fish needs an overdose of dietary AA (>160mg/kg) for better non-specific immune responses.