Glycerol monolaurate improved intestinal barrier, antioxidant capacity, inflammatory response and microbiota dysbiosis in large yellow croaker (Larimichthys crocea) fed with high soybean oil diets.

Glycerol monolaurate (GML) is a potential candidate for regulating metabolic syndrome and inflammatory response. However, the role of GML in modulating intestinal health in fish has not been well determined. In this study, a 70-d feeding trial was conducted to evaluate the effect of GML on intestinal barrier, antioxidant capacity, inflammatory response and microbiota community of large yellow croaker (13.05 ± 0.09 g) fed with high level soybean oil (SO) diets. Two basic diets with fish oil (FO) or SO were formulated. Based on the SO group diet, three different levels of GML 0.02% (SO0.02), 0.04% (SO0.04) and 0.08% (SO0.08) were supplemented respectively. Results showed that intestinal villus height and perimeter ratio were increased in SO0.04 treatment compared with the SO group. The mRNA expressions of intestinal physical barrier-related gene odc and claudin-11 were significantly up-regulated in different addition of GML treatments compared with the SO group. Fish fed SO diet with 0.04% GML addition showed higher activities of acid phosphatase and lysozyme compared with the SO group. The content of malonaldehyde was significantly decreased and activities of catalase and superoxide dismutase were significantly increased in 0.02% and 0.04% GML groups compared with those in the SO group. The mRNA transcriptional levels of inflammatory response-related genes (il-1ß, il-6, tnf-α and cox-2) in 0.04% GML treatment were notably lower than those in the SO group. Meanwhile, sequencing analysis of bacterial 16S rRNA V4-V5 region showed that GML addition changed gut microbiota structure and increased alpha diversity of large yellow croaker fed diets with a high level of SO. The correlation analysis results indicated that the change of intestinal microbiota relative abundance strongly correlated with intestinal health indexes. In conclusion, these results demonstrated that 0.02%-0.04% GML addition could improve intestinal morphology, physical barrier, antioxidant capacity, inflammatory response and microbiota dysbiosis of large yellow croaker fed diets with a high percentage of SO.

The SWGEDWGEIW from Soybean Peptides Reduces Insulin Resistance in 3T3-L1 Adipocytes by Activating p-Akt/GLUT4 Signaling Pathway.

Diabetes mellitus, a group of metabolic disorders characterized by persistent hyperglycemia, affects millions of people worldwide and is on the rise. Dietary proteins, from a wide range of food sources, are rich in bioactive peptides with anti-diabetic properties. Notably, the protective mechanism of the single peptide SWGEDWGEIW (TSP) from soybean peptides (SBPs) on insulin resistance of adipocytes in an inflammatory state was investigated by detecting the lipolysis and glucose absorption and utilization of adipocytes. The results showed that different concentrations of TSP (5, 10, 20 µg/mL) intervention can reduce 3T3-L1 adipocytes' insulin resistance induced by inflammatory factors in a dose-dependent manner and increase glucose utilization by 34.2 ± 4.6%, 74.5 ± 5.2%, and 86.7 ± 6.1%, respectively. Thus, TSP can significantly alleviate the lipolysis of adipocytes caused by inflammatory factors. Further mechanism analysis found that inflammatory factors significantly reduced the phosphorylation (p-Akt) of Akt, two critical proteins of glucose metabolism in adipocytes, and the expression of GLUT4 protein downstream, resulting in impaired glucose utilization, while TSP intervention significantly increased the expression of these two proteins. After pretreatment of adipocytes with PI3K inhibitor (LY294002), TSP failed to reduce the inhibition of p-Akt and GLUT4 expression in adipocytes. Meanwhile, the corresponding significant decrease in glucose absorption and the increase in the fat decomposition of adipocytes indicated that TSP reduced 3T3-L1 adipocytes' insulin resistance by specifically activating the p-Akt/GLUT4 signal pathway. Therefore, TSP has the potential to prevent obesity-induced adipose inflammation and insulin resistance.

Effects of Tributyrin Supplementation on Growth Performance, Intestinal Digestive Enzyme Activity, Antioxidant Capacity, and Inflammation-Related Gene Expression of Large Yellow Croaker (Larimichthys crocea) Fed with a High Level of Clostridium autoethanogenum Protein.

An 8-week growth experiment was conducted to investigate effects of tributyrin (TB) supplementation on growth performance, intestinal digestive enzyme activity, antioxidant capacity, and inflammation-related gene expression of juvenile large yellow croaker (Larimichthys crocea) (initial weight of 12.90 ± 0.02 g) fed diets with high level of Clostridium autoethanogenum protein (CAP). In the negative control diet, 40% fish meal was used as the major source of protein (named as FM), while 45% fish meal protein of FM was substituted with CAP (named as FC) to form a positive control diet. Based on the FC diet, grade levels of 0.05%, 0.1%, 0.2%, 0.4%, and 0.8% tributyrin were added to formulate other five experimental diets. Results showed that fish fed diets with high levels of CAP significantly decreased the weight gain rate (WGR) and specific growth rate (SGR) compared with fish fed the FM diet (P < 0.05). WGR and SGR were significantly higher than in fish fed diets with 0.05% and 0.1% tributyrin that fed the FC diet (P < 0.05). Supplementation of 0.1% tributyrin significantly elevated fish intestinal lipase and protease activities compared to FM and FC diets (P < 0.05). Meanwhile, compared to fish fed the FC diet, fish fed diets with 0.05% and 0.1% tributyrin showed remarkably higher intestinal total antioxidant capacity (T-AOC). Malondialdehyde (MDA) content in the intestine of fish fed diets with 0.05%-0.4% tributyrin was remarkably lower than those in the fish fed the FC diet (P < 0.05). The mRNA expressions of tumor necrosis factor α (tnfα), interleukin-1ß (il-1ß), interleukin-6 (il-6), and interferon γ (ifnγ) were significantly downregulated in fish fed diets with 0.05%-0.2% tributyrin, and the mRNA expression of il-10 was significantly upregulated in fish fed the 0.2% tributyrin diet (P < 0.05). In regard to antioxidant genes, as the supplementation of tributyrin increased from 0.05% to 0.8%, the mRNA expression of nuclear factor erythroid 2-related factor 2 (nrf2) demonstrated a trend of first rising and then decreasing. However, the mRNA expression of Kelch-like ECH-associated protein 1 (keap1) was remarkably lower in fish fed the FC diet than that fed diets with tributyrin supplementation (P < 0.05). Overall, fish fed tributyrin supplementation diets can ameliorate the negative effects induced by high proportion of CAP in diets, with an appropriate supplementation of 0.1%.

Myo-inositol improves growth performance and regulates lipid metabolism of juvenile Chinese mitten crab (Eriocheir sinensis) fed different percentage of lipid.

This study evaluated the effects of dietary myo-inositol (MI) on growth performance, antioxidant status and lipid metabolism of juvenile Chinese mitten crab (Eriocheir sinensis) fed different percentage of lipid. Crabs (4·58 (sem 0·05) g) were fed four diets including a normal lipid diet (N, containing 7 % lipid and 0 mg/kg MI), N with MI supplementation (N + MI, containing 7 % lipid and 1600 mg/kg MI), a high lipid diet (H, containing 13 % lipid and 0 mg/kg MI) and H with MI supplementation (H + MI, containing 13 % lipid and 1600 mg/kg MI) for 8 weeks. The H + MI group showed higher weight gain and specific growth rate than those in the H group. The dietary MI could improve the lipid accumulations in the whole body, hepatopancreas and muscle as a result of feeding on the high dietary lipid (13 %) in crabs. Besides, the crabs fed the H + MI diets increased the activities of antioxidant enzymes but reduced the malondialdehyde content in hepatopancreas compared with those fed the H diets. Moreover, dietary MI enhanced the expression of genes involved in lipid oxidation and exportation, yet reduced lipid absorption and synthesis genes expression in the hepatopancreas of crabs fed the H diet, which might be related to the activation of inositol 1,4,5-trisphosphate receptor (IP3R)/calmodulin-dependent protein kinase kinase-ß (CaMKKß)/adenosine 5'-monophosphate-activated protein kinase (AMPK) signalling pathway. This study demonstrates that MI could increase lipid utilisation and reduce lipid deposition in the hepatopancreas of E. sinensis fed a high lipid diet through IP3R/CaMKKß/AMPK activation. This work provides new insights into the function of MI in the diet of crustaceans.

Impact of Dietary Vitamin D3 Supplementation on Growth, Molting, Antioxidant Capability, and Immunity of Juvenile Chinese Mitten Crabs (Eriocheir sinensis) by Metabolites and Vitamin D Receptor.

Vitamin D3 (vit-D3), as an indispensable and fat-soluble nutrient, is associated with skeletal mineralization and health in mammals. However, such associations have not been well studied in economically important crustaceans. Six levels of vit-D3 with isonitrogenous and isolipidic diets were used to feed Eriocheir sinensis. The range of optimal vit-D3 requirements is 5685.43-10,000 IU/kg based on growth. The crabs fed 9000 IU/kg vit-D3 showed the best growth performance. This vit-D3 dose significantly increased antioxidant capacity in the hepatopancreas and intestine and was optimal for molting and innate immunity via quantitative polymerase chain reaction analysis. Transcriptomics analyses indicate that vit-D3 could alter protein processing in the endoplasmic reticulum, steroid biosynthesis, and antigen processing and presentation. As shown by the enzyme-linked immunosorbent assay, vit-D3 could improve vitamin D receptor, retinoic acid receptor, and C-type lectins concentrations. The 1α,25-dihydroxy vit-D3 content in serum was significantly higher in 3000-9000 IU/kg vit-D3. The study suggests that dietary vit-D3 and its metabolites can regulate molting and innate immunity in crabs.

Influences of dietary vitamin D3 on growth, antioxidant capacity, immunity and molting of Chinese mitten crab (Eriocheir sinensis) larvae.

This study investigates the effects of vitamin D3 (VD3) on growth performance, antioxidant capacity, immunity and molting of larval Chinese mitten crab Eriocheir sinensis. A total of 6,000 larvae (7.52 ± 0.10 mg) were fed with six isonitrogenous and isolipidic experimental diets with different levels of dietary VD3 (0, 3000, 6000, 9000, 12000 and 36000 IU/kg) respectively for 23 days. The highest survival and molting frequency were found in crabs fed 6000 IU/kg VD3. Weight gain, specific growth rate, and carapace growth significantly increased in crabs fed 3000 and 6000 IU/kg VD3 compared to the control. Broken-line analysis of molting frequency, weight gain and specific growth rate against dietary VD3 levels indicates that the optimal VD3 requirement for larval crabs is 4825-5918 IU/kg. The highest whole-body VD3 content occurred in the 12000 IU/kg VD3 group, and the 25-dihydroxy VD3 content decreased with the increase of dietary VD3. The malonaldehyde content was lower than the control. Moreover, the superoxide dismutase activity, glutathione peroxidase and total antioxidant capacity of crab fed 6000 IU/kg VD3 were significantly higher than in control. Crabs fed 9000 IU/kg showed the highest survival after 120 h of salinity stress, and the relative mRNA expressions indicate vitamin D receptor (VDR) is the important regulatory element in molting and innate immunity. The molting-related gene expressions showed that the response of crab to salinity was self-protective. This study would contribute to a new understanding of the molecular basis underlying molting and innate immunity regulation by vitamin D3 in E. sinensis.

Dietary yeast culture modulates immune response related to TLR2-MyD88-NF-kß signaling pathway, antioxidant capability and disease resistance against Aeromonas hydrophila for Ussuri catfish (Pseudobagrus ussuriensis).

The aim of the present study was to investigate effects of dietary yeast culture on immune response related to TLR2-MyD88-NF-kß signaling pathway, antioxidant capability and disease resistance against Aeromonas hydrophila for Ussuri catfish (Pseudobagrus ussuriensis). A total of 240 Ussuri catfish (mean weight of 7.39 ±â€¯0.32 g) were randomly distributed into four groups that fed diets containing 0 (Y0), 10 (Y1), 20 (Y2) and 30 (Y3) g kg-1 yeast culture for 8 weeks. The results indicated that dietary 10 g kg-1 yeast culture supplementation significantly down-regulated mRNA levels of TLR2, MyD88, NF-kß p65, IL-1ß and IL-8 in the liver tissue compared with the control group (P < 0.05). Simultaneously, serum lysozyme (LZM) activity, respiratory burst activity (RBA) of phagocytes, plasma alkaline phosphatase (AKP) activity and immunoglobulin M (IgM) content were significantly improved in fish fed Y1 diet (P < 0.05). Fish fed Y1 diet had significantly higher serum alternative complement pathway activity (ACH50) and plasma complement 3 (C3) content than the Y3 group (P < 0.05). However, no significant differences were observed in plasma acid phosphatase (ACP) activity and complement 4 (C4) content among the groups (P > 0.05). Fish cumulative mortality rate (CMR) in the Y1 and Y2 groups were significantly lower than that in Y0 and Y3 groups (P < 0.05), and the lowest CMR was observed in the Y1 group after challenge by A. hydrophila. The highest hepatic superoxide dismutase and glutathione peroxidase activities, total antioxidant capacity and the lowest malondialdehyde content were found in Y1 group, but no significant difference was found in hepatic catalase activity among the groups (P > 0.05). These results demonstrate that dietary 10 g kg-1 yeast culture could effectively improve the immunity, antioxidant capability and disease resistance against A. hydrophila for Ussuri catfish and could down-regulate the mRNA expression levels of pro-inflammatory cytokines modulated by TLR2-MyD88-NF-kß signaling pathway.

Phenylaminopyrimidines as inhibitors of Janus kinases (JAKs).

A series of phenylaminopyrimidines has been identified as inhibitors of Janus kinases (JAKs). Development of this initial series led to the potent JAK2/JAK1 inhibitor CYT387 (N-(cyanomethyl)-4-[2-[[4-(4-morpholinyl)phenyl]amino]-4-pyrimidinyl]-benzamide). Details of synthesis and SAR studies of these compounds are reported.

Discovery of CYT997: a structurally novel orally active microtubule targeting agent.

CYT997 was discovered as a potent tubulin polymerization inhibitor possessing potent cytotoxic activity against a range of cancer cells. Details of SAR studies, pharmacokinetic investigations and synthesis of compounds leading to the discovery of CYT997 are reported.

Discovery of 2-(alpha-methylbenzylamino) pyrazines as potent Type II inhibitors of FMS.

A series of 2-(alpha-methylbenzylamino) pyrazines have shown to be potent inhibitors of the FMS tyrosine receptor kinase. Details of SAR studies, modeling and synthesis of compounds within this series are reported.

DNA threading bis(9-aminoacridine-4-carboxamides): effects of piperidine sidechains on DNA binding, cytotoxicity and cell cycle arrest.

We describe the synthesis of a series of DNA-threading bis(9-aminoacridine-4-carboxamides) comprising ethylpiperidino and N-methylpiperidin-4-yl sidechains, joined via neutral flexible alkyl chains, charged flexible polyamine chains and a semi-rigid charged piperazine linker. Their cytotoxicity towards human leukaemic cells gives IC(50) values ranging from 99 to 1100 nM, with the ethylpiperidino series generally being more cytotoxic than the N-methylpiperidin-4-yl series. Measurements with supercoiled DNA indicate that they bisintercalate.

Synthesis and cytotoxic activity of N-[(alkylamino)alkyl]carboxamide derivatives of 7-oxo-7H-benz[de]anthracene, 7-oxo-7H-naphtho[1,2,3-de]quinoline, and 7-oxo-7H-benzo[e]perimidine.

7-Oxo-7H-naphtho[1,2,3-de]quinoline-11-carboxamides and analogues were prepared and evaluated for in vitro and in vivo antitumor activity. Chromophore variations included 'deaza' (7-oxo-7H-benz[de]anthracene) and 'diaza' (7-oxo-7H-benzo[e]perimidine) analogues, and side chain variations included chiral alpha-methyl compounds. The naphthoquinolines were the most cytotoxic, with IC(50) values of 5-20 nM, and showed the strongest DNA binding, with high selectivity for G-C rich DNA. The chiral alpha-methyl analogues were 10-20-fold more cytotoxic than the parent des-methyl compound. Both enantiomers provided substantial growth delays against s.c. colon 38 tumors in mice, with the R-enantiomer more active than the S (tumor growth delays of >35 and 12 days, respectively).

Bisintercalating threading diacridines: relationships between DNA binding, cytotoxicity, and cell cycle arrest.

We have synthesized a series of bis(9-aminoacridine-4-carboxamides) linked via the 9-position with neutral flexible alkyl chains, charged flexible polyamine chains, and a semirigid charged piperazine-containing chain. The carboxamide side chains comprise N,N-dimethylaminoethyl and ethylmorpholino groups. The compounds are designed to bisintercalate into DNA by a threading mode, in which the side chains are intended to form hydrogen-bonding contacts with the O6/N7 atoms of guanine in the major groove, and the linkers are intended to lie in the minor groove. By this means, we anticipate that they will dissociate slowly from DNA, and be cytotoxic as a consequence of template inhibition of transcription. The dimers remove and reverse the supercoiling of closed circular DNA with helix unwinding angles ranging from 26 degrees to 46 degrees, confirming bifunctional intercalation in all cases, and the DNA complexes of representative members dissociate many orders of magnitude more slowly than simple aminoacridines. Cytotoxicity for human leukemic CCRF-CEM cells was determined, the most active agents having IC(50) values of 35-50 nM in a range extending over 20-fold, with neither the dimethylaminoethyl nor the ethylmorpholino series being intrinsically more toxic. In common with established transcription inhibitors, the morpholino series, with one exception, have no effect on cell cycle distribution in randomly dividing CCRF-CEM populations. By contrast, the dimethylaminoethyl series, with two exceptions, cause G2/M arrest in the manner of topoisomerase poisons, consistent with possible involvement of topoisomerases in their mode of action. Thus, the cellular response to these bisintercalating threading agents is complex and appears to be determined by both their side chain and linker structures. There are no simple relationships between structure, cytotoxicity, and cell cycle arrest, and the origins of this complexity are unclear given that the compounds bind to DNA by a common mechanism.