NKHs27, a sevenband grouper NK-Lysin peptide that possesses immunoregulatory and antimicrobial activity.

As an effective and broad-spectrum antimicrobial peptide, NK-Lysin is attracted more and more attention at present. However, the functions and action mechanism of NK-Lysin peptides are still not comprehensive enough at present. In this study, a sevenband grouper (Hyporthodus septemfasciatus) NK-Lysin peptide, NKHs27, was identified and synthesized, and its biological functions were studied. The results indicated that NKHs27 shares 44.44%∼88.89% overall sequence identities with other teleost NK-Lysin peptides. The following antibacterial activity assay exhibited that NKHs27 was active against both Gram-negative and Gram-positive bacteria, including Staphylococcus aureus, Listonella anguillarum, Vibrio parahaemolyticus and Vibrio vulnificus. Additionally, NKHs27 showed a synergistic effect when it was combined with rifampicin or erythromycin. In the process of interaction with the L. anguillarum cells, NKHs27 changed the cell membrane permeability and retained its morphological integrity, then penetrated into the cytoplasm to act on genomic DNA or total RNA. Then, in vitro studies showed that NKHs27 could enhance the respiratory burst ability of macrophages and upregulate immune-related genes expression in it. Moreover, NKHs27 incubation improved the proliferation of peripheral blood leukocytes significantly. Finally, in vivo studies showed that administration of NKHs27 prior to bacterial infection significantly reduced pathogen dissemination and replication in tissues. In summary, these results provide new insights into the function of NK-Lysin peptides in teleost and support that NKHs27, as a novel broad-spectrum antibacterial peptide, has potential applications in aquaculture against pathogenic infections.

Antimicrobial and immunoregulatory activities of the derived peptide of a natural killer lysin from black rockfish (Sebastes schlegelii).

Natural killer lysin (NK-lysin) is a small molecule antimicrobial peptide secreted by natural killer cells and T lymphocytes. In this study, we characterized a cDNA sequence encoding an NK-lysin homologue (SsNKL1) from black rockfish, Sebastes schlegelii. The open reading frame (ORF) of SsNKL1 encodes a putative protein of 149 amino acids and shares 44%-87% overall sequence identities with other teleost NK-lysins. SsNKL1 possesses conserved NK-lysin family features, including a signal sequence and a surfactant-associated protein B (SapB) domain, sequence analysis revealed that SsNKL1 is most closely related to false kelpfish (Sebastiscus marmoratus) NK-lysin (with 87% sequence identity). SsNKL1 transcripts were detected in all the tested tissues, with the highest level in the kidney, followed by the spleen and gills. Upon Listonella anguillarum infection, the mRNA expression of SsNKL1 in the black rockfish was significantly up-regulated in the liver and kidney. The derived peptide SsNKLP27 from SsNKL1 was synthesized, and its biological function was studied. SsNKLP27 showed direct antibacterial activity against Gram-negative and Gram-positive bacteria, including Staphylococcus aureus, Bacillus subtilis, L. anguillarum, Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio vulnificus. SsNKLP27 treatment facilitated the bactericidal process of erythromycin by enhancing the permeability of the outer membrane. In the process of interaction with the target bacterial cells, SsNKLP27 changed the permeability and retained the morphological integrity of the cell membrane, then penetrated into the cytoplasm, and induced the degradation of genomic DNA and total RNA. In vivo studies showed that administration of SsNKLP27 before bacterial and viral infection significantly reduced the transmission and replication of pathogens in tissues. In vitro analysis showed that SsNKLP27 could enhance the respiratory burst ability and regulate the expression of some immune-related genes of macrophages. In summary, these results provided new insights into the function of NK-lysins in teleost fish and support that SsNKLP27 is a new broad-spectrum antimicrobial peptide that has a potential application prospect in aquaculture against pathogenic infection.

[Development and Evaluation of Prognostic Nomogram Model for Adult Ventricle Glioma Patients].

Objective: To explore the prognostic factors of adult ventricle glioma (AVG) and to construct and evaluate a survival-related prognostic nomogram model, which could provide further reference for the clinical management of AVG patients. Methods: The patients covered in the study were selected from the Surveillance Epidemiology and End Results (SEER) database (1973-2016). They all had definite histological diagnosis of AVG. They were assigned randomly to the training cohort and the validation cohort by random number table at a 2/1 ratio. Survival analysis was performed by Kaplan-Meier analysis. Cox regression analysis was employed to determine the independent prognostic factors for overall survival (OS) and cancer-specific survival (CSS). Then, integrating the basic characteristics of patients, the survival-related nomogram predictive model for OS and CSS in the training cohort was constructed, respectively. After that, internal cross validation and external validation of the model were carried out with the training cohort and the validation cohort in succession. The authenticity and reliability of the nomogram model were evaluated by calculating the concordance index (C-index). Calibration plots were constructed to assess the agreement between the predicted values and the observed values in the training cohort and the validation cohort. Results: A total of 369 AVG patients, including 218 males and 151 females, were included. The median age of the patients was 53. According to the WHO classification of gliomas, 66 (17.9%) patients had grade Ⅱ gliomas, 73 (19.8%) had grade Ⅲ gliomas, and 230 (62.3%) had grade Ⅳ gliomas. Regarding the extent of resection (EOR), 59 (16.0%) had gross total resection (GTR) and 145 (39.3%) had subtotal resection (STR) or partial resection (PR). Of all the patients, 167 (45.3%) received postoperative radiotherapy and 143 (38.8%) received postoperative chemotherapy. Patients were randomized into the training cohort ( n=246) and the validation cohort ( n=123), and there was no significant difference ( P>0.05) in the basic clinical characteristics between the training cohort and the validation cohort. In the training cohort, Cox regression analysis revealed that the independent prognostic factors for OS and CSS included age≥65, grades Ⅲ and Ⅳ according to the WHO classification of gliomas, and not receiving radiotherapy. Furthermore, 5 variables, including age, gender, WHO grades, surgery, and radiotherapy, were used to construct the nomogram model for predicting 6-month, 1-year, and 2-year OS and CSS. The results of internal cross validation in the training cohort showed that the C-indexes of OS and CSS were 0.758 and 0.765, respectively. The external validation results of the validation cohort showed that the C-indexes of OS and CSS were 0.733 and 0.719, respectively. Calibration plots for 6-month, 1-year, and 2-year OS in the training cohort showed relatively good agreement, while in the validation cohort the agreement was relatively low. The 6-month, 1-year, and 2-year CSS calibration plots had results similar to the calibration plots of OS. Conclusion: This nomogram predictive model of OS and CSS showed moderately reliable predictive performance, providing helpful reference information for clinicians to make quick and simple assessment of the survival probability of AVG patients.

Evaluation of the efficacy of a novel Vibrio vulnificus vaccine based on antibacterial peptide inactivation in turbot, Scophthalmus maximus.

Tongue sole tissue factor pathway inhibitor 2 (TFPI-2) C-terminus derived peptide, TC38, has previously been shown to kill Vibrio vulnificus cells without lysing the cell membrane; thus, the remaining bacterial shell has potential application as an inactivated vaccine. Therefore, this study aimed to evaluate the immune response induced by the novel V. vulnificus vaccine. The protective potential of TC38-killed V. vulnificus cells (TKC) was examined in a turbot model. Fish were intramuscularly vaccinated with TKC or FKC (formalin-killed V. vulnificus cells) and challenged with a lethal-dose of V. vulnificus. The results showed that compared with FKC, TKC was effective in protecting fish against V. vulnificus infection, with relative percent of survival (RPS) rates of 53.29% and 63.64%, respectively. The immunological analysis revealed that compared with the FKC and control groups, the TKC group exhibited: 1) significantly higher respiratory burst ability and bactericidal activity of macrophages at 7 d post-vaccination; 2) increased alkaline phosphatase, acid phosphatase, lysozyme, and total superoxide dismutase levels post-vaccination; 3) higher serum agglutinating antibody titer with corresponding higher serum bactericidal ability, and a more potent serum agglutination effect, as well as an increased IgM expression level; 4) higher expression of immune relevant genes, which were involved in both innate and adaptive immunity. Taken together, this is the first study to develop a novel V. vulnificus inactivated vaccine based on AMP inactivation, and TKC is an effective vaccine against V. vulnificus infection for aquaculture.

TC26, a teleost TFPI-1 derived antibacterial peptide that induces degradation of bacterial nucleic acids and inhibits bacterial infection in vivo.

At present, several reports have indicated that the C-terminal peptides of tissue factor pathway inhibitor 1 (TFPI-1) were active antibacterial peptides. However, the functions of TFPI-1 C-terminal peptides in teleost are still very limited. In this study, a C-terminal peptide, TC26 (with 26 amino acids), derived from common carp (Cyprinus carpio) TFPI-1, was synthesized and investigated for its antibacterial spectrum, action mechanism, as well as the in vivo effects on bacterial invasion. Our results showed that TC26 was active against Gram-positive bacteria Micrococcus luteus and Staphylococcus aureus, as well as Gram-negative bacterium Vibrio vulnificus. TC26 treatment facilitated the bactericidal process of erythromycin by enhancing the out-membrane permeability of V. vulnificus. During the bactericidal process, TC26 killed the target bacterial cells Vibrio vulnificus, by destroying cell membrane integrity, penetrating into the cytoplasm and inducing degradation of genomic DNA and total RNA. In vivo study showed that administration of turbot with TC26 before bacterial infection significantly reduced pathogen dissemination and replication in tissues. These results indicated that TC26 is a novel and active antibacterial peptide and may play a vital role in fighting pathogenic infection in aquaculture.

Metabolomic analysis of the toxic effect of chronic low-dose exposure to acephate on rats using ultra-performance liquid chromatography/mass spectrometry.

To study the toxic effect of chronic exposure to acephate at low-dose levels, a metabolomics approach based on ultra-performance liquid chromatography/mass spectrometry (UPLC-MS) was applied. Three different doses of 0.5 mg/kg/day, 1.5 mg/kg/day, and 4.5 mg/kg/day acephate were administered to Wistar rats for 24 weeks. Endogenous metabolite profiles were obtained with UPLC-MS for all rats at six time points after treatment. Some metabolites like dimethylthiophosphate and uric acid in urine were detected at week 4. Dimethylthiophosphate, which had the most significant elevations compared with other biomarkers, was considered as an early, sensitive biomarker of exposure to acephate. Moreover, there were some endogenous metabolite changes, which demonstrated that the doses of 1.5 mg/kg/day and 4.5 mg/kg/day of acephate led to renal injury and perturbed the normal metabolic processes of rats, including glucose, nucleic acid, and protein metabolism. A connection between exposure to acephate and the metabolic disturbance has been found and interpreted. Our study indicates that the metabolomics approach based on UPLC-MS of urine provides more information on toxicity than the conventional toxicological evaluation methods in measuring changes and can be considered as a promising technique for the study of the toxic effect of acephate.

A teleost bactericidal permeability-increasing protein-derived peptide that possesses a broad antibacterial spectrum and inhibits bacterial infection as well as human colon cancer cells growth.

The bactericidal permeability-increasing protein (BPI) is a multifunctional cationic protein produced by neutrophils with antibacterial, antitumor, and LPS-neutralizing properties. In teleost, a number of BPIs have been reported, but their functions are very limited. In this study, an N-terminal peptide, BO18 (with 18 amino acids), derived from rock bream (Oplegnathus fasciatus) BPI, was synthesized and investigated for its antibacterial spectrum, action mechanism, immunoregulatory property as well as the inhibition effects on bacterial invasion and human colon cancer cells growth. The results showed that BO18 was active against Gram-positive bacteria Bscillus subiilis, Micrococcus luteus, and Staphylococcus aureus, as well as Gram-negative bacteria Vibrio alginolyticus, Vibrio litoralis, Vibrio parahaemolyticus and Vibrio vulnificus. BO18 treatment facilitated the bactericidal process of erythromycin and rifampicin by enhancing the permeability of the outer membrane. During its interaction with V. alginolyticus, BO18 exerted its antibacterial activity by destroying cell membrane integrity, penetrating into the cytoplasm and binding to genomic DNA and total RNA. In vitro analysis indicated BO18 could enhance the respiratory burst ability and regulate the expression of immune related genes of macrophages. In vivo detection showed the administration of fish with BO18 before bacterial infection significantly reduced pathogen dissemination and replication in tissues. In addition, BO18 exerted a cytotoxic effect on the growth of human colon cancer cells HT-29. Together, these results add new insights into the function of teleost BPIs, and support that BO18 is a novel and broad-spectrum antibacterial peptide with potential to apply in fighting pathogenic infection in aquaculture.