QseBC regulates in vitro and in vivo virulence of Aeromonas hydrophila in response to norepinephrine.

The inter-kingdom communication between host and pathogenic bacteria mediated by the host hormones epinephrine (Epi)/norepinephrine (NE)/autoinducer-3 (AI-3) and transduced by the bacterial two-component signal transduction system QseBC has been well demonstrated in mammalian pathogens. Aeromonas hydrophila, a common opportunistic pathogen in freshwater aquaculture, responds to NE by increased bacterial growth and enhanced virulence. However, the underlying mechanisms remain poorly understood. Our study demonstrated that deletion of qseB and qseC significantly inhibited NE-promoted growth, biofilm formation, and hemolytic activity of A. hydrophila. The adhesion ability of ΔqseB and ΔqseC to J774a.1 cells was significantly decreased compared with the wild-type strain in the presence and absence of NE, whereas NE still enhanced the adhesion ability of the mutant and wild-type strains with a similar effect, suggesting that NE-enhanced cell adhesion was independent of QseBC. Moreover, QseBC did not affect the swimming and swarming motility of A. hydrophila with or without NE. Quantitative real-time PCR analyses revealed the down-regulated expression of some virulence-related genes (hly, ast, act, aerA) in each mutant compared with the wild-type strain in the presence of NE. Tilapia infection experiments indicated that deletion of qseB or qseC weakened NE-promoted virulence of A. hydrophila. In conclusion, our study suggests that NE stimulates the growth, biofilm formation, and hemolytic activity of A. hydrophila and enhances the virulence of the pathogen in fish via the QseBC system.

The role of dietary chromium supplementation in relieving heat stress of juvenile blunt snout bream Megalobrama amblycephala.

The present study assessed the role of dietary chromium (Cr) supplementation in relieving heat stress (HS) of juvenile blunt snout bream Megalobrama amblycephala. The supplemented Cr contents by chromium picolinate (Cr-Pic) was 0 mg/kg (control group), 0.4 mg/kg, 1.6 mg/kg and 12.0 mg/kg, respectively. The fish continued to be fed four diets at suitable temperatures (26 °C) for 2 weeks, and then the temperature was then heated up to 33 °C through thermo-regulated system. The results showed that Cr supplementation had no significant effect on the immune indices and antioxidant indices before HS (P > 0.05). However, Cr supplementation played an important role in relieving HS. After HS, compared with the control group, 1.6 mg/kg and 12.0 mg/kg Cr supplementation groups significantly lowered the plasma glucose level and aspartate transaminase (AST) activity (P < 0.05), and 0.4 mg/kg and 1.6 mg/kg Cr supplementation groups significantly lowered alanine aminotransferase (ALT) activity (P < 0.05). 0.4 mg/kg and 1.6 mg/kg supplementation groups significantly improved hepatic total superoxide dismutase (T-SOD) activity (P < 0.05). Furthermore, 0.4mg/kg-12.0 mg/kg Cr supplementation groups significantly improved the activities of hepatic glutathione peroxidase (GPx) and catalase (CAT) and lowered hepatic malondialdehyde (MDA) level in liver (P < 0.05). The mRNA levels of hepatic copper zinc superoxide dismutase (Cu/Zn-SOD), CAT and GPx were significantly improved in 0.4mg/kg-12.0 mg/kg supplementation Cr groups (P < 0.05), however, there was no significant variation of hepatic manganese superoxide dismutase (Mn-SOD) mRNA levels under different levels of supplementation (P > 0.05). Significantly lower mRNA levels of hepatic pro-inflammatory cytokines observed in 0.4mg/kg-12.0 mg/kg Cr supplementation groups including tumour necrosis factor-α (TNF-α), interleukin 1ß (IL-1ß) and interleukin 8 (IL-8) (P < 0.05), and 0.4mg/kg-12.0 mg/kg Cr supplementation significantly improved the relative expressions of hepatic heat shock protein 70 (HSP70) and heat shock protein 90 (HSP90) (P < 0.05). The present study indicated that dietary Cr supplementation might have no significant effect on immune capacity and antioxidant capacity under normal physiological conditions, whereas it played an important role in relieving HS.

A study of the potential effect of yellow mealworm (Tenebrio molitor) substitution for fish meal on growth, immune and antioxidant capacity in juvenile largemouth bass (Micropterus salmoides).

This study aimed to evaluate the effects of partial replacement of fish meal (FM) with yellow mealworm (Tenebrio molitor, TM) on the growth performance, food utilization and intestinal immune response of juvenile largemouth bass (Micropterus salmoides). Seven diets containing increasing levels of TM (FM substitution) were designed (approximately 0% (0%), 4% (11.1%), 8.1% (22.2%), 12.2% (33.3%), 16.3% (44.4%), 20.4% (55.5%), and 24.5% (66.6%), designated TM0, TM11, TM22, TM33, TM44, TM55, and TM66, respectively). 420 fish were randomly selected and placed in 21 cages (1 m*1 m*1 m, 7 treatments for triplicate, 20 fish per cage). Fish (initial weight 6.25 ± 0.03 g) were fed seven isonitrogenous (47%) and isocaloric (19 MJ kg-1) diets to satiety twice daily for 8 weeks. Compared to the control group (TM0), TM11 showed no significant difference in the weight gain rate (WGR), specific growth rate (SGR) or feed conversion ratio (FCR), while all other TM inclusion groups presented different degrees of decline. There was no significant difference in the whole-body composition among all groups (P > 0.05). Plasma total protein (TP), triglyceride (TG) and albumin (ALB) contents were significantly decreased in TM55 and TM66 (P < 0.05). The highest plasma aspartate transaminase (AST) activity was observed in TM66 (P < 0.05). TM33, TM44 and TM55 showed the lowest activities of plasma alanine amiotransferase (ALT) and alkaline phosphatase (ALP) (P < 0.05). Moreover, increased mRNA levels of superoxide dismutase (SOD) and catalase (CAT) were measured in the TM11 to TM55 groups, while intestinal SOD activity peaked in TM11 (P < 0.05). With the exception of TM11, the other TM inclusion groups showed significant inhibition of the relative expression of RelA, C3 and TNF-α (P < 0.05). All experimental groups exhibited lower expression of IL-10 than TM0 (P < 0.05). The TM11 group showed significantly upregulated expression of IL-1ß and TGF-ß (P < 0.05). In addition, TLR2 expression was increased in TM11 and TM22 (P < 0.05). Considering enzyme activities and immune-related gene expression, TM supplementation levels should not exceed 4% (TM11).

A Mechanochemical, Catalyst-Free Cascade Synthesis of 1,3-Diols and 1,4-Iodoalcohols Using Styrenes and Hypervalent Iodine Reagents.

A mechanochemical and solvent/catalyst-free functionalization of olefins with hypervalent iodine reagents has been developed, enabling the synthesis of 1,3-dioxygenated compounds. Under similar reaction conditions with the addition of molecular iodine, 1,4-iodoalcohols can be synthesized. These valuable products are non-trivial to achieve via standard solution-phase methods. Mechanistic study reveals that the hypervalent iodine reagent might dimerize at solid state with the help of mechanical force. The active monomeric form of hypervalent iodine reagent might trigger the 1,3- and 1,4-difunctionalization reactions in an intermolecular cascade manner.

Culture salinity modulates Nrf2 antioxidant signaling pathway and immune response of juvenile Genetically Improved Farmed Tilapia (GIFT) (Oreochromis niloticus) under different dietary protein levels.

This study aimed to evaluate that dietary protein levels and culture salinity levels affect the health status of juvenile genetically improved farmed tilapia (GIFT, Oreochromis niloticus). Graded protein levels of six diets were prepared, ranging from 18.20% to 49.49% (dry basis), and were used in cultured GIFT at two salinity levels (0‰ and 8‰) for 8 weeks. The results suggested that appropriate protein levels reduced pro-inflammatory gene expressions in the intestine including interleukin 1ß (IL-1ß), interleukin 8 (IL-8) and tumour necrosis factor-α (TNF-α) mRNA levels at two salinity levels (P < 0.05). 8‰ salinity significantly decreased the expression levels of IL-1ß, TNF-α and nuclear factor-kappa B (NF-κB) (P < 0.05). The anti-inflammatory factor interleukin 10 (IL-10) was significantly increased by 36.42% protein level (P < 0.05). Regarding antioxidant capacity, appropriate protein levels and 8‰ salinity significantly improved the antioxidant capacity of fish by regulating the activities of intestinal total superoxide dismutase (T-SOD), glutathione peroxidase (GPx), and the levels of glutathione (GSH) and malondialdehyde (MDA). Furthermore, appropriate protein levels and 8‰ salinity also significantly enhanced the antioxidant gene expressions associated with the Nrf2/keap1 signaling pathway by regulating the expression levels of heme oxygenase-1 (HO-1), GPx, catalase (CAT) and superoxide dismutase (SOD). According to GPx activities and the mRNA levels of IL-10, the optimum dietary protein levels for GIFT juveniles were 31.12%-32.18% (0‰) and 34.25-35.38% (8‰) based on second-degree polynomial regression analysis. The present study found that appropriate protein levels and 8‰ culture salinity are critical in maintaining the health of GIFT juveniles by improving antioxidant and immune capacity.

Molecular cloning and expression mechanism of Mnp65 in Megalobrama amblycephala response to Aeromonas hydrophilia challenge.

p65 is one of the important subunits of the inflammation-related transcription factor NF-κB. In the present study, we cloned and identified the p65 from Megalobrama amblycephala (Mnp65) by homologous cloning and RACE technique. The full-length Mnp65 cDNA consisted of 2331 bp, and included one open reading frame encoding a 604-amino acid putative protein. The protein sequence included a DNA binding motif, a well conserved N-terminal Rel-homology domain (RHD), and a C-terminal IG-like plexins transcription (IPT). Mnp65 was closely related with the other p65 proteins of Cypriniformes and clearly distinct from that of Perciformes and Salmoniformes in terms of sequence homology. Mnp65 homodimer may interact with IκBα in the IPT domain based on the predicted 3D structure of IκBα/Mnp65 complex. Mnp65 was ubiquitously expressed in M. amblycephala tissues, and the highest levels were detected in muscle and liver. Intragastric infection with Aeromonas hydrophila caused respiratory burst and cytokine storm from 8 h to 48 h, showing significantly higher level of respiratory burst activities and significantly high cytokines levels, such as TNF-α, IL-1ß, IL-6, IL-8 etc., compared to 0 h. In addition, the bacterial challenge downregulated the IkBα, and upregulated Mnp65 and TNF-α in the liver. IkBα-Mnp65 was regulated by the negative feedback of cytokine storm, to increase IkBα and decrease Mnp65. Then cytokine storm was relieved at 96 h. Finally, severe intestinal inflammation was observed from 24 h to 48 h after infection, characterized by extensive villous necrosis, epithelial hyperplasia and lymphocyte infiltration, all of which were relieved at 96 h. Taken together, Mnp65 plays a crucial role in the physiological response of teleost fish to bacterial infection.

Molecular cloning and functional characterization of the hypoxia-inducible factor-1α in bighead carp (Aristichthys nobilis).

HIF-l is the earliest documented and most widely studied hypoxia-inducible factor (HIF) and plays a key role in the cell hypoxia signal transduction pathway. Particularly, the HIF-1α protein is sensitive to oxygen and plays a critical role in hypoxia regulation. This study is the first to report on the molecular cloning and characterization of HIF-1α in bighead carp (Aristichthys nobilis; anHIF-1α). The full-length cDNA of anHIF-1α was 2361 bp, and encodes an estimated 674 amino acids with a predicted molecular mass of 76.10 kDa and a theoretical isoelectric point of 7.72. Moreover, the conserved basic Helix-Loop-Helix domain along with two Per-ARNT-Sim domains (A/B), and C-TAD were identified in this protein. Interestingly, the tertiary structure of the anHIF-1α protein was found to be extremely similar to that of mice. Multiple comparison and phylogenetic tree results demonstrated that anHIF-1α was highly conserved. Under normoxic conditions, anHIF-1α mRNA transcripts could be detected in all tissues examined with the highest expression level in the heart. With gradually decreasing oxygen concentrations, anHIF-1α mRNA level was upregulated significantly in the gill, liver, kidney, spleen, intestine, brain, and muscle tissues (P < 0.05). Similarly, anHIF-1α was expressed in all examined bighead carp tissues, and the results suggested that the upregulation of anHIF-1α at the transcriptional level may be an important stress response adaptation to hypoxia in bighead carp. Finally, based on the tertiary structure comparative analyses between anHIF-1α with mouse HIF-1α, we think the physiological function, and protein structure of HIF-1α could be compared between fish and mammal in the future.

The immunoreaction and antioxidant capacity of juvenile blunt snout bream (Megalobrama amblycephala) involves the PI3K/Akt/Nrf2 and NF-κB signal pathways in response to dietary methionine levels.

A 75-day rearing trail was designed to evaluate the immunoreaction and antioxidant capacity of juvenile blunt snout bream in response to dietary methionine levels. Three practical diets were extruded to feed juveniles with graded methionine levels (0.40%, 0.84% and 1.28% dry matter). The data indicated that the plasma concentrations of immunoglobulin M (IgM), complement component 3 (C3) and glutathione (GSH) in the 0.84% methionine diet were markedly upper than those in the 0.40% group (P < 0.05). The activities of plasma antioxidant parameters involving catalase (CAT), total superoxide dismutase (T-SOD), total antioxidant capacity (T-AOC) and glutathione peroxidase (GPx) were significantly increased by the 0.84% diet compared with the 0.40% diet, whereas plasma alanine aminotransferase (ALT) and malondialdehyde (MDA) levels were significantly induced by 0.40% methionine (P < 0.05). Compared with the 0.40% group, 0.84% dietary methionine dramatically upregulated the mRNA expression levels of protein kinase B (Akt), phosphoinositide 3-kinase (PI3K) and nuclear factor erythroid 2-related factor 2 (Nrf2) pathway related genes including CAT, manganese superoxide dismutase (Mn-SOD), heme oxygenase 1 (HO-1) and glutathione peroxidase-1 (GPx-1) in the kidney and liver, and downregulated Kelch-like ECH-associated protein 1 (Keap1) mRNA levels (P < 0.05). Compared with the 0.40% group, the 0.84% dietary methionine strikingly suppressed the mRNA levels of renal and hepatic nuclear factor-kappa B (NF-κB) and pro-inflammatory cytokines (interleukin 1ß (IL-1ß), tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6)), however, improved the mRNA expression levels of anti-inflammatory cytokines involved renal and hepatic transforming growth factor-ß (TGF-ß) and hepatic interleukin 10 (IL-10) (P < 0.05). Renal IL-10 and interleukin 8 (IL-8) mRNA expression levels were not markedly influenced by experimental diets (P > 0.05). Dietary methionine (0.84%) significantly upregulated renal and hepatic heat stress protein 70 (Hsp70), renal B-cell lymphoma-2 (Bcl-2) gene expression levels compared with the 0.40% diet (P < 0.05). In a word, the data represented that 0.84% dietary methionine could enhance the immune and antioxidant capacity of this fish species by inducing PI3K/Akt/Nrf2 pathway and inhibiting NF-κB pathway.

Effects of dietary fenugreek seed extracts on growth performance, plasma biochemical parameters, lipid metabolism, Nrf2 antioxidant capacity and immune response of juvenile blunt snout bream (Megalobrama amblycephala).

Dietary administration of some plant-derived substances have been proved of great economic value in aquaculture. In order to investigate the effects of dietary fenugreek seed extracts (FSE) on juvenile blunt snout bream (Megalobrama amblycephala), a feeding trial was conducted for 8 weeks. The results showed that final weight (FW), weight gain (WG), feed conversion ratio (FCR) and specific growth rate (SGR) were not significantly affected by dietary FSE levels. The whole body lipid contents of fish fed with 0.04%, 0.08% and 0.16% FSE diets were significantly lowered compared to the control group. Dietary FSE diets significantly affected plasma complement component 3 (C3), immunoglobulin M (IgM), albumin (ALB) and total protein (TP). The relative expressions of acetyl CoA carboxylase (ACC), fatty acid synthase (FAS) and sterol regulatory element binding protein-1 (SREBP1) mRNA in the liver of fish decreased significantly with increasing dietary FSE levels from 0% up to 0.04%. FSE supplementation diets lowered the liver pro-inflammatory genes expressions by regulating tumor necrosis factor-α (TNF-α) and interleukin 8 (IL-8) mRNA levels and increased anti-inflammatory genes expression by regulating transforming growth factor (TGF-ß) and interleukin 10 (IL-10). FSE diets increased growth factor-1 (IGF-1) and target of rapamycin (TOR) mRNA levels from 0% up to 0.04%, 0.04% FSE diets significantly increased growth factor-1 (IGF-1) mRNA levels and S6 kinase-polypeptide 1 (S6K1) mRNA levels compared to the control group. 0.04% FSE diets significantly increased superoxide dismutase (SOD) activities and 0.08% FSE diets significantly increased catalase (CAT) and glutathione peroxidase (GPx) activities, 0.16% FSE diets significantly increased total antioxidant capacity (T-AOC) activities compared to the control group. Additionally, compared to the control group, 0.04% dietary FSE significantly up-regulated nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA levels and glutathione peroxidase-1 (GPx1) mRNA levels, at the same time, 0.02%, 0.04%, 0.08%, 0.16% FSE diets significantly down-regulated kelch-like ECH-associated protein 1 (Keap1) mRNA levels. However, no significant effects were observed on copper zinc superoxide dismutase (Cu/Zn-SOD) and manganese superoxide dismutase (Mn-SOD). Our study indicated that dietary FSE could improve plasma biochemical parameters, regulate lipid metabolism related genes, promote Nrf2 antioxidant capacity and enhance immune response of juvenile blunt snout bream.

Effects of dietary tryptophan levels on antioxidant status and immunity for juvenile blunt snout bream (Megalobrama amblycephala) involved in Nrf2 and TOR signaling pathway.

Dietary administration of tryptophan has been proved improving growth performance of fish. An 8-week feeding trial was conducted to investigate the effects of dietary tryptophan level on antioxidant capacity and immune response through Nrf2 and TOR signaling pathway. The results showed that, 0.08% tryptophan level significantly increased plasma aspartate aminotransferase (AST), while immunoglobulin M (IgM) and alkaline phosphatase (ALP) were strikingly increased by 0.40% level. The level of plasma complement component 3 (C3), alanine aminotransferase (ALT) and albumin (ALB) were independent of tryptophan supplementation. Total superoxide dismutase (T-SOD), catalase (CAT), total antioxidant capacity (T-AOC) and glutathione (GSH) activity were increased with increasing dietary tryptophan level until 0.40% and then decreased, while the level of malondialdehyde (MDA) showed a reverse trend. 0.19% and 0.28% tryptophan level significantly improved the glutathione peroxidase 1 (GPx-1) activity. Compared with 0.08% dietary tryptophan level, 0.40% level significantly improved nuclear factor erythroid 2-related factor 2 (Nrf2), GPx, manganese superoxide dismutase (Mn-SOD), CAT and transforming growth factor-ß (TGF-ß) mRNA level, while Kelch-like ECH-associated protein 1 (Keap1) and interleukin 1ß (IL-1ß) mRNA level were significantly decreased. The relative expression of copper zinc superoxide dismutase (Cu/Zn-SOD), heme oxygenase-1 (HO-1), target of rapamycin (TOR), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), protein kinase B (Akt) and interleukin 10 (IL-10) were significantly improved by 0.28% diet, while the mRNA level of tumor necrosis factor-α (TNF-α) and nuclear factor-kappa B (NF-κB) were increased by 0.08% diet. Interleukin 8 (IL-8) mRNA level was not significantly affected by dietary tryptophan. Based on MDA and T-SOD value, the optimal dietary tryptophan level of juvenile blunt snout bream was determined to be 0.33% (1.03% of dietary protein) and 0.36% (1.13% of dietary protein), respectively, using quadratic regression analysis.

Comparative transcriptomic and proteomic analyses reveal upregulated expression of virulence and iron transport factors of Aeromonas hydrophila under iron limitation.

BACKGROUND: Iron plays important roles in the growth, reproduction and pathogenicity of Aeromonas hydrophila. In this study, we detected and compared the mRNA and protein expression profiles of A. hydrophila under normal and iron restricted medium with 200 µM 2,2-Dipyridyl using RNA Sequencing (RNA-seq) and isobaric tags for relative and absolute quantification (iTRAQ) analyses. RESULTS: There were 1204 genes (601 up- and 603 down-regulated) and 236 proteins (90 up- and 146 down-regulated) shown to be differentially expressed, and 167 genes and proteins that showed consistent expression. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the differentially expressed genes and proteins were mainly involved in iron ion transport, protein activity, energy metabolism and virulence processes. Further validation of the RNA-seq and iTRAQ results by quantitative real-time PCR (qPCR) revealed that 18 of the 20 selected genes were consistently expressed. The iron-ion absorption and concentration of A. hydrophila under iron-limited conditions were enhanced, and most virulence factors (protease activity, hemolytic activity, lipase activity, and swimming ability) were also increased. Artificial A. hydrophila infection caused higher mortality in cyprinid Megalobrama amblycephala under iron-limited conditions. CONCLUSION: Understanding the responses of pathogenic Aeromonas hydrophila within the hostile environment of the fish host, devoid of free iron, is important to reveal bacterial infection and pathogenesis. This study further confirmed the previous finding that iron-limitation efficiently enhanced the virulence of A. hydrophila using multi-omics analyses. We identified differentially expressed genes and proteins, related to enterobactin synthesis and virulence establishment, that play important roles in addressing iron scarcity.

Effects of dietary arginine on intestinal antioxidant status and immunity involved in Nrf2 and NF-κB signaling pathway in juvenile blunt snout bream, Megalobrama amblycephala.

The present study assessed the effects of dietary arginine on intestinal antioxidant status and immunity involved in Nrf2 and NF-κB signaling pathway in juvenile blunt snout bream. Fish were fed three practical diets with graded arginine levels (0.87%, 1.62% and 2.70%) for 8 weeks. Compared with the control group (0.87%), the counts of white blood cell (WBC), red blood cell (RBC) and hemoglobin (HGB) content were significantly improved at dietary arginine levels of 1.62% (P<0.05). Plasma albumin (ALB) levels and alkaline phosphatase (ALP) activities were significantly improved at dietary arginine levels of 1.62% and 2.70% (P < 0.05). Alanine transaminase (ALT) activity was decreased in fish fed with 1.62% dietary arginine level (P<0.05). Plasma glutathione peroxidase (GPx) activities, copper-zinc superoxide dismutase (Cu/Zn-SOD) activities, total antioxidant capacity (T-AOC) activities and glutathione (GSH) levels were significantly increased at dietary arginine levels of 1.62% and 2.70% (P<0.05). Plasma total superoxide dismutase (T-SOD) activities and catalase (CAT) activities were significantly improved in fish fed with 1.62% dietary arginine level. Significantly higher manganese superoxide dismutase (Mn-SOD) activity was observed in fish fed with 1.62% dietary arginine level compared with 2.70% dietary arginine level (P<0.05). 1.62% and 2.70% dietary arginine levels significantly lowered malondialdehyde (MDA) levels. The relative expression of nuclear factor erythroid 2-related factor 2 (Nrf2) was significantly increased in fish fed with 1.62% dietary arginine level, inversely, the relative expression of Kelch-like ECH-associated protein 1 (Keap1) showed a converse trend. 1.62% and 2.70% dietary arginine levels significantly improved the relative expressions of Cu/Zn-SOD, GPx and CAT. Furthermore, 2.70% dietary arginine level significantly lowered the relative expression of Mn-SOD compared with the control group and 1.62% dietary arginine levels. The relative expressions of Interleukin 1ß (IL-1ß), tumour necrosis factor-α (TNF-α) and nuclear factor-kappa B (NF-κB) were lowered in fish fed with 1.62% dietary arginine level. 1.62% and 2.70% dietary arginine levels significantly improved the relative expressions of transforming growth factor-ß (TGF-ß). Hematocrit (HCT), aspartate aminotransferase (AST) activities, interleukin 8 (IL-8) and interleukin 10 (IL-10) expressions were not significantly affected by the graded dietary arginine levels. These results suggest that the optimal dietary arginine level plays an important role in enhancing antioxidant and immune status to maintain the intestinal health of juvenile blunt snout bream.

Effects of dietary arginine on antioxidant status and immunity involved in AMPK-NO signaling pathway in juvenile blunt snout bream.

The present study assessed the effects of dietary arginine on antioxidant status and immunity involved in AMPK-NO signaling pathway in juvenile blunt snout bream. Fish were fed six practical diets with graded arginine levels ranging from 0.87% to 2.70% for 8 weeks. The results showed that compared with the control group (0.87% dietary arginine level), significantly higher mRNA levels of adenosine monophosphate activated protein kinase (AMPK) and nitric oxide synthetase (NOS), activities of total nitric oxide synthetase (T-NOS) and nitric oxide synthetase (iNOS), and plasma nitric oxide (NO) contents were observed in fish fed with 1.62%-2.70% dietary arginine levels. Significantly higher levels of NOS and iNOS were observed in fish fed with 1.62%-2.70% dietary arginine levels in enzyme-linked immune sorbent assay. At dietary arginine levels of 1.22%-2.70%, the mRNA levels of iNOS were significantly improved. Dietary arginine also significantly influenced plasma interleukin 8 (IL-8) and tumour necrosis factor-α (TNF-α) contents. Furthermore, dietary arginine significantly affected the activity and mRNA level of glutathione peroxidase (GPx), the mRNA levels of pro-inflammatory factor including IL-8 and TNF-α and plasma malondialdehyde (MDA) content. However, total superoxide dismutase (T-SOD) activity, plasma complement component 3 (C3) content, plasma immunoglobulin M (IgM) content, plasma interleukin 1ß (IL-1ß) content and the mRNA levels of copperzinc superoxide dismutase (Cu/Zn-SOD), manganese superoxide dismutase (Mn-SOD) and IL-1ß were not significantly affected by dietary arginine. After Aeromonas hydrophila challenge, the death rate was significantly lowered in fish fed with 1.62%-1.96% dietary arginine levels. Furthermore, the mRNA levels of AMPK, NOS and iNOS, plasma NO content and the activities of T-NOS and iNOS showed an upward trend with increasing dietary arginine levels. Significantly higher levels of NOS and iNOS were observed in fish fed with 1.62%-2.70% dietary arginine levels in enzyme-linked immune sorbent assay. At dietary arginine levels of 1.96%-2.31%, T-SOD activities were significantly improved. Significantly higher GPx activities were observed in fish fed with 1.22%-2.70% dietary arginine levels. At dietary arginine levels of 1.22%-2.31%, the plasma TNF-α and IL-8 contents were significantly decreased. Significantly lower plasma IL-1ß contents were observed in fish fed 1.62%-1.96% dietary arginine levels. Dietary arginine significantly influenced the mRNA levels of antioxidant and pro-inflammatory genes including Cu/Zn-SOD, Mn-SOD, GPx, IL-8, TNF-α and IL-1ß. Significantly higher plasma C3 contents and significantly lower plasma MDA contents were observed in fish fed with 1.62%-1.96% arginine levels. Furthermore, plasma IgM contents were significantly improved at dietary arginine levels of 1.62%-2.31%. However, high dietary arginine group (2.70%) significantly improved the mRNA levels of pro-inflammatory genes including IL-8, TNF-α and IL-1ß and plasma MDA, IL-8, TNF-α and IL-1ß contents as compared with optimal dietary arginine levels (1.62% and 1.96%). The present results indicate that optimal arginine level (1.62% and 1.96%) could improve antioxidant capacity, immune response and weaken tissues inflammatory involved in arginine-AMPK-NO signaling pathway, while high arginine level resulted in excessive NO production, leading to increase oxidative stress damage and inflammatory response in juvenile blunt snout bream.

Effect of nitrite exposure on the antioxidant enzymes and glutathione system in the liver of bighead carp, Aristichthys nobilis.

Nitrite (NO2-) can cause oxidative stress in aquatic animal when it accumulates in the organism, resulting in different toxic effects on fish. In the present study, we investigated the effects of nitrite exposure on the antioxidant enzymes and glutathione system in the liver of Bighead carp (Aristichthys nobilis). Fish [Initial average weight: (180.05 ±â€¯0.092) g] were exposed to 48.634 mg/L nitrite for 96 h, and a subsequent 96 h for the recovery test. Fish livers were collected to assay antioxidant enzymes activity, hepatic structure and expression of genes after 0 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h of exposure and12 h, 24 h, 48 h, 72 h, 96 h of recovery. The results showed that the activity of glutathione peroxidase (GSH-Px), glutathione S-transferase (GST), and glutathione reductase (GR) increased significantly in the early stages of nitrite exposure. The study also showed that nitrite significantly up-regulated the mRNA levels of glutathione peroxidase (GSH-Px), glutathione S-transferase (GST), and glutathione reductase (GR) after 6, 48, and 72 h of exposure respectively. Nitrite also increased the formation of malondialdehyde (MDA), oxidized glutathione (GSSG), and the activity of catalase (CAT). Nitrite was observed to reduce the activity of superoxide dismutase (SOD) and the level of glutathione (GSH). In the recovery test, GSH and the GSSG recovered but did not return to pre-stress levels. The results suggested that the glutathione system played important roles in nitrite-induced oxidative stress in fish. The bighead carp responds to oxidative stress by enhancing the activity of GSH-Px, GST, GR and up-regulating the expression level of GSH-Px, GST, GR, a whilst simultaneously maintaining the dynamic balance of GSH/GSSG. CAT was also indispensable. They could reduce the degree of lipid peroxidation, and ultimately protect the body from oxidative damage.

Design, synthesis and structure-activity relationship study of novel naphthoindolizine and indolizinoquinoline-5,12-dione derivatives as IDO1 inhibitors.

Indoleamine 2,3-dioxygenase 1 (IDO1) is regarded as a promising target for cancer immunotherapy. Many naphthoquinone derivatives have been reported as IDO1 inhibitors so far. Herein, two series of naphthoquinone derivatives, naphthoindolizine and indolizinoquinoline-5,12-dione derivatives, were synthesized and evaluated for their IDO1 inhibitory activity. Most of the target compounds showed significant inhibition potency and high selectivity for IDO1 over tryptophan 2,3-dioxygenase (TDO). The structure-activity relationship was also summarized. The most potent compounds 5c (IC50 23 nM, IDO1 enzyme), and 5b' (IC50 372 nM, HeLa cell) were identified as promising lead compounds.

In Vivo Analysis of miR-34a Regulated Glucose Metabolism Related Genes in Megalobrama amblycephala.

The Megalobrama amblycephala (M. amblycephala) is one of the most important economic freshwater fish in China. The molecular mechanism under the glucose intolerance responses which affects the growth performance and feed utilization is still confused. miR-34a was reported as a key regulator in the glucose metabolism, but how did the miR-34a exert its function in the metabolism of glucose/insulin in M. amblycephala was still unclear. In this study, we intraperitoneally injected the miR-34a inhibitor (80 nmol/100 g body weight) into M. amblycephala (fed with high starch diet, 45% starch) for 12 h, and then analyzed the gene expression profiling in livers by RNA-seq. The results showed that miR-34a expression in M. amblycephala livers was inhibited by injection of miR-34a inhibitor, and a total of 2212 differentially expressed genes (DEGs) were dysregulated (including 1183 up- and 1029 downregulated DEGs). Function enrichment analysis of DEGs showed that most of them were enriched in the peroxisome proliferator-activated receptor (PPAR), insulin, AMP-activated protein kinase (AMPK) and janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathways, which were all associated with the glucose/lipid metabolic and biosynthetic processes. In addition, we examined and verified the differential expression levels of some genes involved in AMPK signaling pathway by qRT-PCR. These results demonstrated that the inhibition of miR-34a might regulate glucose metabolism in M. amblycephala through downstream target genes.

Regulation mechanism of oxidative stress induced by high glucose through PI3K/Akt/Nrf2 pathway in juvenile blunt snout bream (Megalobrama amblycephala).

This study was conducted to investigate the effects of oral administration of a high concentration of glucose on the respiratory burst, antioxidant status, and hepatic gene expression of heme oxygenase-1 (ho1) and PI3K/Akt/Nrf2-related signaling molecules in juvenile blunt snout bream (Megalobrama amblycephala). Blunt snout bream juveniles with an initial body weight of 19.94 ± 0.58 g were orally fed with a high concentration of glucose (3 g/kg body weight). The results indicated that plasma glucose exhibited a biphasic response. Acute and persistent hyperglycemia due to the oral glucose administration significantly reduced (P < 0.05) the white blood cell count, red blood cell count, and hemoglobin content and caused oxidative stress (significantly increased alanine aminotransferase, aspartate transaminase, alkaline phosphatase, and glucose levels) and early apoptosis of hepatocytes in the fish. Hepatic superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activities increased rapidly (P < 0.05) as protection from oxidative stress and were downregulated (P < 0.05) because of persistent hyperglycemia. Blood respiratory burst was significantly reduced (P < 0.05) because of hyperglycemia and showed a trend that was opposite to that of plasma glucose. Slight upregulation of nrf2 mRNA and antioxidants acts as a compensative protection mechanism, and the downregulated PI3K/Akt pathway blocked this function of Nrf2. In conclusion, the PI3K/Akt pathway and Nrf2 mediated the antioxidative mechanism independently in the blunt snout bream juveniles subjected to the oral administration of a high glucose concentration.

Synthesis and Biological Evaluation of Novel Benzimidazole Derivatives and Analogs Targeting the NLRP3 Inflammasome.

A series of benzo[d]imidazole analogues of thiabenzole were synthesized and their antiinflammatory activities toward NLRP3 (nucleotide-binding domain leucine-rich repeat containing protein family，pyrin domain-containing 3，also known as cryopyrin or NALP3) inflammasome were evaluated in vitro. Two lead compounds, TBZ-09 and TBZ-21, were identified by antiproduction of IL-1ß. In the second round of biological evaluation, based on the lead, 34 more compounds were synthesized and their in vitro anti-inflammatory activities were investigated. Several compounds were identified as anti-inflammatory agents that can reduce IL-1ß expression in a dosedependent manner. A preliminary structure-activity relationship is also summarized here.

Molecular cloning and expression analysis of Megalobrama amblycephala transferrin gene and effects of exposure to iron and infection with Aeromonas hydrophila.

Transferrin (Tf) plays an important function in iron homeostasis and metabolism of organisms. In this study, we identified and characterized the Tf gene in Megalobrama amblycephala and evaluated its expression in basal conditions as well as after iron overload and experimental infection with Aeromonas hydrophila. Furthermore, we studied the iron binding properties of recombinant Tf. The full-length M. amblycephala Tf complementary DNA (cDNA) (GenBank accession no.: KX698308) of 2245 bp was cloned and contained a 1953 bp open reading frame (ORF) encoding 650 amino acid residues and flanked by a 68 bp 5' and a 204 bp 3' untranslated regions (UTR). Predicted conservative structure illustrated that M. amblycephala Tf consisted of two conservative Tf domains. Amino acid sequence alignment revealed that M. amblycephala Tf had high similarity with that of cyprinids deposited in Genbank, and phylogenetic analysis showed that M. amblycephala Tf clustered with Ctenopharyngodon idella and Hypophthalmichthys molitrix. Tissue expression pattern analyses demonstrated that the liver was the main Tf mRNA expressing organ, being significantly higher than other tissues (p < 0.05). In the liver, Tf mRNA expression in fish artificially injected with the pathogenic bacteria A. hydrophila was significantly upregulated, reaching a peak at 12 h post injection (hpi) and then decreasing afterward. The expression in FeCl3-injected fish showed a similar tendency, but reached a peak at 8 hpi. Meanwhile, fish serum iron significantly decreased following A. hydrophila injection, but increased to peak at 4 hpi and then decreased in FeCl3-injected fish. The recombinant M. amblycephala Tf showed iron binding capacity using CAS analysis. These results are helpful to understand the structure and regulation of expression of Tf, as well as the specific function of Tf for both immune responses and iron homeostasis.

The effect of dietary folic acid on biochemical parameters and gene expression of three heat shock proteins (HSPs) of blunt snout bream (Megalobrama amblycephala) fingerling under acute high temperature stress.

The effects of dietary folic acid on biochemical parameters and gene expression of three heat shock proteins (HSPs) of blunt snout bream (Megalobrama amblycephala) fingerling under acute high temperature stress. Six dietary folic acid groups (0.0, 0.5, 1.0, 2.0, 5.0, and 10.0) mg/kg diets were designed and assigned into 18 tanks in three replicates each (300 l/tank) and were administered for 10 weeks in a re-circulated water system. The fingerlings with an initial weight of 27.0 ± 0.03 g were fed with their respective diets four times daily. At the end of the experiment, samples were collected before challenge, 0, 24, 72 h, and 7 days. Serum total protein (TP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), cortisol, glucose, complement C3 (C3), complement C4 (C4, immunoglobulin M (IgM) hepatic superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and the expression of heat shock protein 60 (HSP60), 70 (HSP70), and 90 (HSP90) were studied. The results showed that fish fed with dietary folic acid between 1.0, 2.0, and 5.0 mg/kg significantly (P < 0.05) increased serum TP, C3, C4 hepatic SOD, CAT, and the expression of HSP60, HSP70, and HSP90 before and after temperature challenge of 32 °C. Also, serum ALP, cortisol, glucose, and hepatic MDA were significantly (P < 0.05) reduced by supplementation of dietary folic acid level 1.0, 2.0, and 5.0 mg/kg before and after the same temperature challenge of 32 °C. Before stress, 0, 24, 72 h, and 7 days significantly (P < 0.05) affects serum biochemical parameters, immune and antioxidant capacities, and expression level of three HSPs. Furthermore, there was no statistical evidence to show that dietary folic acid inclusion level and temperature duration have significant interactive effect on serum biochemical parameters, antioxidant parameters, and gene expression level (P > 0.05) of the three HSPs. However, there were statistical significant interactive effect between dietary folic acid inclusion level and temperature duration on serum C3 and C4 (P < 0.05) except IgM (P > 0.05). The present results indicate that supplementation of basal diet from 1.0 mg/kg; 2.0 and 5.0 mg/kg can enhance acute high temperature resistance ability in M. amblycephala fingerling to some degree and improve physiological response, immune and antioxidant capacities, and expression level of three HSPs.