The Yin Yang 1 of Penaeus vannamei regulates transcription of the small subunit hemocyanin gene during Vibrio parahaemolyticus infection.

Hemocyanin is a respiratory protein, it is also a multifunctional immune molecule that plays a vital role against pathogen invasion in shrimp. However, the regulation of hemocyanin gene expression in shrimp hemocytes and the mechanisms involved during pathogen infection remains unclear. Here, we used DNA pull-down followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the Yin Yang 1 transcription factor homolog in Penaeus vannamei (PvYY1) as a key factor that modulates transcription of the small subunit hemocyanin gene of P. vannamei (PvHMCs) in hemocytes during Vibrio parahaemolyticus AHPND (VPAHPND) infection. Bioinformatics analysis revealed that the core promoter region of PvHMCs contains two YY1 motifs. Mutational and oligoprecipitation analyses confirmed that PvYY1 could bind to the YY1 motifs in the PvHMCs core promoter region, while truncation of PvYY1 revealed that the N-terminal domain of PvYY1 is essential for the transactivation of PvHMCs core promoter. Besides, the REPO domain of PvYY1 could repress the activity of the PvHMCs core promoter. Overexpression of PvYY1 significantly activates the promoter activity of PvHMCs core promoter, while PvYY1 knockdown significantly decreases the expression level of PvHMCs in shrimp hemocytes and survival rate of shrimp upon infection with VPAHPND. Our present study provides new insights into the transcriptional regulation of PvHMCs by PvYY1 in shrimp hemocytes during bacteria (VPAHPND) infection.

Effects of dietary supplementation of Gracilaria lemaneiformis-derived sulfated polysaccharides on the growth, antioxidant capacity, and innate immunity of rabbitfish (Siganus canaliculatus).

The dietary supplementation of red seaweed-derived polysaccharides has been shown to be beneficial to fish and shellfish aquaculture. However, the function of red seaweed (Gracilaria lemaneiformis)-extracted polysaccharide (GLP) on the health status of rabbitfish (Siganus canaliculatus) is still unknown. This study explored the influences of GLP on growth performance, antioxidant activity, and immunity of rabbitfish. Herein, the fish were fed commercial pelleted feed incorporated with the diverse amount of GLP: 0 (control), 0.10 (GLP0.10), and 0.15 g kg-1 (GLP0.15) for 60 days. The results demonstrated that dietary GLP0.15 significantly elevated FBW and WG, while feed utilization efficiency improved (reduced feed conversion ratio and increased protein efficiency ratio) upon GLP0.10 treatment, regarding the control (P < 0.05). Also, dietary administration of GLP0.15 suggestively improved the serum acid phosphatase and lysozyme activity as well as hepatic total antioxidant capacity, catalase, and superoxide dismutase activity. In contrast, GLP0.15decreased the serum alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, and malonaldehyde activity when compared to the control (P<0.05). Moreover, the lipase (36.08 and 16.46 U/mgprot in GLP0.10 and GLP0.15, respectively) and amylase (0.43 and 0.23 U/mgprot in GLP0.10 and GLP0.15, respectively) activity recorded the maximum values than the control (8.61 and 0.13 U/mgprot, respectively).Further, the intestinal morphometry was developed (such as increased villus length, width, and area) in the fish fed with a GLP-supplemented diet compared to the control. The KEGG pathway analysis unveiled that several differentially expressed genes (DEGs) in control vs. GLP0.10 and control vs. GLP0.15 were associated with metabolic or immune-associated pathways like antigen processing and presentation, phagosome, complement and coagulation cascades, and platelet activation. The DEGs, namely C3, f5, fgb, MHC1, and cfb, were evaluated in control vs. GLP0.10 and C3 and MHC1 in control vs. GLP0.15, suggesting their possible contributions to GLP-regulated immunity. Additionally, the cumulative mortality of rabbitfish after the Vibrio parahaemolyticus challenge was lower in both GLP0.10 (8.88%) and GLP0.15 (11.11%) than in control (33.33%) (P<0.05). Thus, these findings direct the potential use of GLP as an immunostimulant and growth promoter in rabbitfish aquaculture.

Taurine metabolism is modulated in Vibrio-infected Penaeus vannamei to shape shrimp antibacterial response and survival.

BACKGROUND: Numerous microorganisms are found in aquaculture ponds, including several pathogenic bacteria. Infection of cultured animals by these pathogens results in diseases and metabolic dysregulation. However, changes in the metabolic profiles that occur at different infection stages in the same ponds and how these metabolic changes can be modulated by exogenous metabolites in Penaeus vannamei remain unknown. RESULTS: Here, we collected gastrointestinal tract (GIT) samples from healthy, diseased, and moribund P. vannamei in the same aquaculture pond for histological, metabolic, and transcriptome profiling. We found that diseased and moribund shrimp with empty GITs and atrophied hepatopancreas were mainly infected with Vibrio parahaemolyticus and Vibrio harveyi. Although significant dysregulation of crucial metabolites and their enzymes were observed in diseased and moribund shrimps, diseased shrimp expressed high levels of taurine and taurine metabolism-related enzymes, while moribund shrimp expressed high levels of hypoxanthine and related metabolism enzymes. Moreover, a strong negative correlation was observed between taurine levels and the relative abundance of V. parahaemolyticus and V. harveyi. Besides, exogenous taurine enhanced shrimp survival against V. parahaemolyticus challenge by increasing the expression of key taurine metabolism enzymes, mainly, cysteine dioxygenase (CDO) and cysteine sulfinic acid decarboxylase (CSD). CONCLUSIONS: Our study revealed that taurine metabolism could be modulated by exogenous supplementation to improve crustacean immune response against pathogenic microbes. Video Abstract.

Identification of antibacterial metabolites produced by a marine bacterium Halobacillus marinus HMALI004.

AIMS: This study examined and characterized the extract for metabolites of Halobacillus marinus HMALI004 to understand their antibacterial activities against opportunistic marine pathogens, that is, Vibrio parahaemolyticus and Vibrio cholerae. METHODS AND RESULTS: The bacterial strain HMALI004 was characterized as H. marinus, and an antibacterial spectral test revealed its inhibition against two opportunistic marine pathogens (V. parahaemolyticus and V. cholera). Fermentation broth of strain HMALI004 was subjected to column chromatography and high-performance liquid chromatography to separate antibacterial substances. Two compounds were successfully isolated and identified as 1H-pyrrole-2-carboxylic acid and 4-chloro-1H-pyrrole-2-carboxylic acid by mass spectrometry (MS) and nuclear magnetic resonance. The minimal inhibition concentration (MIC) values of 1H-pyrrole-2-carboxylic acid and 4-chloro-1H-pyrrole-2-carboxylic acid for V. parahaemolyticus were 25 µg/ml, while their MIC values for V. cholerae were 50 and 100 µg/ml, respectively. The reactive oxygen species (ROS) production of two pathogen strains treated with 1H-pyrrole-2-carboxylic acid and 4-chloro-1H-pyrrole-2-carboxylic acid were detected to investigate the antimicrobial mechanism. The results suggested that 4-chloro-1H-pyrrole-2-carboxylic acid exerted enhanced ROS production in V. parahaemolyticus, whereas 1H-pyrrole-2-carboxylic acid had a weaker effect. Both compounds caused a significant rise in ROS production in V. cholerae, causing severe damage to the cell wall and cytoplasm, leading to cell death. CONCLUSIONS: The bacterium H. marinus HMALI004 was isolated from a shrimp pond and was found to produce antimicrobial compounds, which could inhibit the growth of opportunistic marine pathogens V. parahaemolyticus and V. cholerae by increasing ROS. SIGNIFICANCE AND IMPACT OF THE STUDY: Successfully isolated antibacterial-producing strain, H. marinus HMALI004, and its antimicrobial compounds could be used as biological control agents for marine pathogens.

Disruption of YY1-EZH2 Interaction Using Synthetic Peptides Inhibits Breast Cancer Development.

Enhancer of zeste homolog 2 (EZH2) is a methyltransferase to mediate lysine 27 trimethylation in histone H3 (i.e., H3K27me3) and repress gene expression. In solid tumors, EZH2 promotes oncogenesis and is considered a therapeutic target. As a transcription factor, Yin Yang 1 (YY1) recruits EZH2 through its oncoprotein binding (OPB) domain to establish gene repression. In this study, we mapped the YY1 protein binding (YPB) domain on EZH2 to a region of 27 amino acids. Both YPB and OPB domain synthetic peptides could disrupt YY1EZH2 interaction, markedly reduce breast cancer cell viability, and efficiently inhibit tumor growth in a xenograft mouse model. We analyzed MDA-MB-231 cells treated with YPB, OPB, and control peptides by chromatin immunoprecipitation DNA sequencing (ChIP-seq) using an antibody against H3K27me3. YPB and OPB treatments altered H3K27me3 on 465 and 1137 genes, respectively, compared to the control. Of these genes, 145 overlapped between the two peptides. Among them, PTENP1, the PTEN pseudogene, showed reduced H3K27me3 signal when treated by either YPB or OPB peptide. Consistently, the two peptides enhanced both PTENP1 and PTEN expression with concomitantly reduced AKT activation. Further studies validated PTENP1's contribution to the anticancer activity of YPB and OPB peptides.

Streptococcus suis sortase A is Ca2+ independent and is inhibited by acteoside, isoquercitrin and baicalin.

Sortase A (SrtA) has long been recognized as an ideal drug target for therapeutic agents against Gram-positive pathogens. However, the SrtA of Streptococcus suis (Ss-SrtA), an important zoonotic agent, has not been studied. In this study, the enzymatic properties of Ss-SrtA were investigated, and inhibition of Ss-SrtA by natural products was evaluated. Ss-SrtA was expressed and purified. The purified recombinant Ss-SrtA had maximal activity at pH 6.0-7.5, 45°C, and showed a Km of 6.7 µM for the hydrolysis of substrate abz-LPATG-dnp. Different from Staphylococcus aureus SrtA (Sa-SrtA) which is stimulated by Ca2+, Ss-SrtA was observed to be Ca2+ independent. Structural analysis showed that salt bridges formed between K111 and D180 in Ss-SrtA replaced the function of Ca2+ in Sa-SrtA to stabilize the substrate-binding cleft. Site-directed mutagenesis identified H126, C192 and R200 as the key residues of Ss-SrtA active site. To discover potential inhibitors, the percent inhibition of sortase activity by natural products was measured. Among these selected natural products, acteoside, isoquercitrin and baicalin were discovered as novel SrtA inhibitors, with IC50 values of 36.3 ± 1.3 µM, 100.0 ± 1.3 µM and 85.4 ± 1.5 µM, respectively. The inhibitory effects of these three natural products were further confirmed on endogenous Sa-SrtA. Using a previously established S. aureus model with a fluorescent-labeled Sa-SrtA substrate, acteoside, isoquercitrin, and baicalin showed 86%, 28% and 45% inhibition on endogenous Sa-SrtA activity, respectively. Overall, these findings shed new light on enzymatic properties, Ca2+-independent catalytic mechanism and potential inhibitors of Ss-SrtA.

Identification and characterization of the related immune-enhancing proteins in crab Scylla paramamosain stimulated with rhubarb polysaccharides.

Recently, considerable interest has been focused on immunostimulants to reduce diseases in crab aquaculture. However, information regarding to the related immune-enhancing proteins in crabs is not available yet. In this study, rhubarb polysaccharides were tested for enhancement of the immune activity in crab Scylla paramamosain. Compared with those in the control group, values of, phenoloxidase (PO), alkaline phosphatase (AKP) and alkaline phosphatasein (ACP) activity in the, experimental group were improved significantly 4 d after the treatment. Furthermore, 15 and 17 altered proteins from haemocytes and hepatopancreas, respectively, were found in rhubarb polysaccharide-treated crabs using 2-DE approach. Of these, hemocyanin, chymotrypsin, cryptocyanin, C-type lectin receptor, and ferritin protein were identified by mass spectrometry. In addition, RT-PCR, analysis showed that the mRNA levels of hemocyanin and chymotrypsin increased about 2.4- and 1.4-fold in the experiment group. Moreover, the hemocyanin gene in S. paramamosain (SpHMC) was, cloned and characterized. SpHMC contains one open reading frame of 2022 bp and encodes a polypeptide of 673 amino acids. It is clustered into one branch along with crab hemocyanin in a phylogenetic tree. The mRNA transcripts of SpHMC were detected mainly in the tissues of, hepatopancreas, hemocyte and intestines, and its levels were up-regulated significantly in hemocytes, of S. paramamosain treated with Vibrio parahemolyticus, Beta streptococcus or poly I:C for 6-48 h. Taken together, these studies found 5 related immune-enhancing proteins and a novel heomcyanin homologue with potential pathogen-resistant activities in crab.

Proteomic identification of the related immune-enhancing proteins in shrimp Litopenaeus vannamei stimulated with vitamin C and Chinese herbs.

Recently, strong interest has been focused on immunostimulants to reducing the diseases in shrimp aquaculture. However, information regarding to the related immune-enhancing proteins in shrimps is not available yet. In this study, vitamin C (Vc), Chinese herbs (CH), and the mixture of vitamin C and Chinese herbs (Mix) were tested for their enhancement on shrimp's immune activity. Compared with those in the control group, values of phenoloxidase (PO), superoxide dismutase (SOD) and antibacterial (Ua) activity in the Mix-treated group were improved significantly 12 or 24 days after the treatment. The cumulative mortality was also lower in the Mix-treated group after infection with Vibrio parahemolyticus. Furthermore, comparative proteomic approach was used to assess the protein expression profile in shrimps. Approximately 220-290 and 300-400 protein spots were observed in the 2-DE gels. Among them, 29 and 28 altered proteins from hemocytes and hepatopancreas, respectively, were subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) analysis. The results revealed that the main altered proteins showed high homologies with Litopenaeus vannamei hemocyanin, hemolymph clottable protein, hemoglobin beta, cytosolic MnSOD, trypsin, cathepsin I(L) and zinc proteinase Mpc1. Together, these studies found Vc and CH were suitable immunostimulants to shrimp L. vannamei, and 7 altered proteins could be involved in the enhanced immune activities.

Crystallization and preliminary X-ray study of native and selenomethionyl beta-1,4-mannanase AaManA from Alicyclobacillus acidocaldariusTc-12-31.

AaManA, a beta-1,4-mannanase from the thermoacidophile Alicyclobacillus acidocaldarius Tc-12-31, was expressed in Escherichia coli and purified in a form suitable for X-ray crystallographic analysis. Crystals were obtained using the hanging-drop vapour-diffusion method at 291 K using ammonium dihydrogen phosphate as a precipitant. Data were collected from native mannanase and from a selenomethionyl derivative to 1.90 and 1.99 A, respectively, at 100 K. The native crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 44.34, b = 75.55, c = 88.02 A. The derivative crystal belonged to the same space group as native AaManA, with unit-cell parameters a = 44.55, b = 75.70, c = 92.66 A.

[Effects of ginkgolides injection on experimental cerebral ischemia in mice and rats].

OBJECTIVE: To investigate the effects of ginkgolides injection on experimental cerebral ischemia and its related mechanism of action. METHOD: The middle cerebral artery occlusion (MACO) model was induced by the FeCl3-occluding method to explore the protective effects of ginkgolides injection on the score of neurological deficits, the rate of cerebral infarction and the histomorphology of cerbral ischemia in rats. Thrombosis formation in vivo was induced by adrenaline-collagen in mice to explore the antithrombotic effect. Platelet aggregation was induced by ADP and hemorrheological parameters with hyper-viscosity by dextran T-500 were used to explore the effects of antiplatelet aggregation and decreasing viscosity of blood. RESULT: Ginkgolides injection could markedly decrease the infarct size and behavior deficits score, inhibit the thrombus formation in mice, decrease blood viscosity and ameliorate hemorrheological parameters in rat. CONCLUSION: Ginkgolides injection has the protective effects on focal cerebral ischemia, and its mechanism may be relative to its inhibition of platelet-dependent thrombosis and amelioration of hemarheological partments.