Molecular characterization, antibacterial and immunoregulatory activities of liver-expressed antimicrobial peptide 2 in black rockfish, Sebastes schlegelii.

LEAP2 (liver expression antimicrobial peptide 2), is an antimicrobial peptide widely found in vertebrates and mainly expressed in liver. LEAP2 plays a vital role in host innate immunity. In teleosts, a number of LEAP2 homologs have been reported, but their in vivo effects on host defense are still limited. In this study, a LEAP2 homolog (SsLEAP2) was identified from black rockfish, Sebastes schlegelii, and its structure, expression as well as biological functions were analyzed. The results showed that the open reading frame of SsLEAP2 is 300 bp, with a 5'- untranslated region (UTR) of 375 bp and a 3' - UTR of 238 bp. The deduced amino acid sequence of SsLEAP2 shares the highest overall identity (96.97%) with LEAP2 of Sebastes umbrosus. SsLEAP2 possesses conserved LEAP2 features, including a signal peptide sequence, a prodomain and a mature peptide, in which four well-conserved cysteines formed two intrachain disulphide domain. The expression of SsLEAP2 was highest in liver and could be induced by experimental infection with Listonella anguillarum, Edwardsiealla piscicida and Rock bream iridovirus C1 (RBIV-C1). Recombinant SsLEAP2 (rSsLEAP2) purified from Escherichia coli was able to bind with various Gram-positive and Gram-negative bacteria. Further analysis showed that rSsLEAP2 could enhance the respiratory burst activity, and induce the expression of immune genes including interleukin 1-ß (IL-1ß) and serum amyloid A (SAA) in macrophages; additionally, rSsLEAP2 could also promote the proliferation and chemotactic of peripheral blood lymphocytes (PBLs). In vivo experiments indicated that overexpression of SsLEAP2 could inhibit bacterial infection, and increase the expression level of immune genes including IL-1ß, tumor necrosis factor ligand superfamily member 13B (TNF13B) and haptoglobin (HP); conversely, knock down of SsLEAP2 promoted bacterial infection and decreased the expression level of above genes. Taken together, these results suggest that SsLEAP2 is a novel LEAP2 homolog that possesses apparent antibacterial activity and immunoregulatory property, thus plays a critical role in host defense against pathogens invasion.

Identification, expression pattern and functional characterization of IFN-γ involved in activating JAK-STAT pathway in Sebastes schlegeli.

IFN-γ (interferon gamma) is a critical cytokine in the immune system involved both directly and indirectly in antiviral activity, stimulation of bactericidal activity, antigen presentation and activation of macrophages via the Janus kinase/signal transducer and activator of transcription (JAK-STAT) pathway. The IFN-γ function is best described in cell defense against intracellular pathogens in mammals, but IFN-γ cytokine-induced metabolic change and its role in anti-infection remain unknown in teleost fish. In this study, a novel IFN-γ (SsIFN-γ) was identified from black rockfish (Sebastes schlegeli) by rapid amplification of cDNA ends (RACE). The open reading frame (ORF) of SsIFN-γ encoded a putative protein of 215 amino acids and shares 60.2%-93.5% overall sequence identities with other teleost IFN-γ. SsIFN-γ was distributed ubiquitously in all the detected tissues and immune cells, which was highly expressed in the spleen, gills, head kidney by quantitative real-time PCR. The mRNA expression of SsIFN-γ was significantly upregulated in the spleen, head kidney, head kidney (HK) macrophages and peripheral blood lymphocytes (PBLs) during pathogen infection. Meanwhile, the recombinant protein (rSsIFN-γ) exhibited an immunomodulatory function to enhance respiratory burst activity and nitric oxide response of HK macrophages. Furthermore, rSsIFN-γ could effectively upregulate the expression of macrophage proinflammatory cytokine, the expression of JAK-STAT signaling pathway related genes and interferon-related downstream genes in the head kidney and spleen. Luciferase assays showed ISRE and GAS activity were obviously enhanced after rSsIFN-γ treatment. These results indicated that SsIFN-γ possessed apparent immunoregulatory properties and played a role in fighting pathogen infection which will be helpful to further understanding of the immunologic mechanism of teleosts IFN-γ in innate immunity.

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.

Microbial composition and nitrogen removal pathways in a novel sequencing batch reactor integrated with semi-fixed biofilm carrier: evidence from a pilot study for low- and high-strength sewage treatment.

The sequencing batch reactor (SBR) activated sludge process is a well-established technology for sewage treatment. One of the drawbacks of SBRs, however, total nitrogen (TN) removals is insufficient. By means of introducing four improvements, including semi-fixed biofilm carrier, sludge elevation mixing and change for the mode of influent and effluent, compliant standard for TN discharge was obtained in this novel SBR configuration during low- and high-strength sewage load. To illustrate the microbial compositions and functions of the attached biofilm on semi-fixed carrier and the suspended aggregates, as well as the nitrogen removal pathway, high throughput 16S rRNA gene amplicon sequencing, PICRUSt2 algorithm, and KEGG database were applied. The results revealed that (i) the microbial communities from suspended aggregates and biofilm samples were significantly different from each other; (ii) during low-strength sewage loads, TN removal was mainly by nitrification-denitrification. The suspended aggregates was responsible for denitrification, while the biofilm was focused on ammonium oxidation; (iii) during high-strength sewage loads, function of nitrate reductase from suspended aggregates was faded, and anammox and N assimilation by biofilm became dominant. Meanwhile, TN removal referring to the formation of L-glutamine via assimilation was the main pathway.

Biopharmaceutical and Pharmacokinetic Activities of Oxymatrine Determined by a Sensitive UHPLC-MS/MS Method.

BACKGROUND: Oxymatrine is known as one of the most promising alkaloids from Sophora flavescens for its excellent pharmacological effects. OBJECTIVE: The aim of this research is to assess the biopharmaceutical and pharmacokinetic activities of oxymatrine and clarify its mechanisms of absorption and metabolism. METHODS: The biological characteristics of oxymatrine were systematically investigated by UHPLC-MS/MS. The mechanisms of absorption and metabolism of oxymatrine were further clarified through incubation in rat liver microsomes and transport across the Caco-2 monolayer cell absorption model. RESULTS: It was found that the absolute oral bioavailability of oxymatrine was 26.43%, and the pharmacokinetic parameters Cmax, Tmax, and t1/2 were 605.5 ng/mL, 0.75 h, and 4.181 h after oral administration, indicating that oxymatrine can be absorbed quickly. The tissue distribution tests showed that oxymatrine distributed throughout all the organs, with the small intestine accumulating the highest level, followed by the kidney, stomach, and spleen. The Papp in Caco-2 cell line absorption model was over 1 × 10-5 and PDR 1.064, and t1/2 of oxymatrine in rat liver microsome in vitro was 1.042 h, indicating that oxymatrine can be absorbed easily through passive diffusion and CYP450 enzymes could be involved in its metabolism. The plasma protein binding rate of oxymatrine was 2.78 ± 0.85%. CONCLUSION: Oxymatrine can be absorbed into blood easily through passive diffusion, mainly distributed in the intestine, stomach, liver, and spleen in vivo, and CYP450 enzymes in the liver could be involved in its metabolism.

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.

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.

Identification and characterization of a cathepsin K homologue that interacts with pathogen bacteria in black rockfish, Sebastes schlegelii.

Cathepsin K belongs to the family of cysteine cathepsins. It is well known that the cysteine cathepsins participate in various physiological processes and host immune defense in mammals. However, in teleost fish, the function of cathepsin K is very limited. In the present study, a cathepsin K homologue (SsCTSK) from the teleost black rockfish (Sebastes schlegelii) was identified and examined at expression and functional levels. In silico analysis showed that three domains, including signal peptide, cathepsin propeptide inhibitor I29 domain, and functional domain Pept_C1, are existed in SsCTSK. SsCTSK also possesses a peptidase domain with three catalytically essential residues (Cys25, His162 and Asn183). Phylogenetic profiling indicated that SsCTSK was evolutionally close to the cathepsin K of other teleost fish. Expression of SsCTSK occurred in multiple tissues and was induced by bacterial infection. Purified recombinant SsCTSK (rSsCTSK) exhibited apparent maximal peptidase activity at 45 °C, and its enzymatic activity was remarkably declined in the presence of the cathepsin inhibitor E-64. Moreover, rSsCTSK possesses the ability to bind with PAMPs and bacteria. Finally, knockdown of SsCTSK expression facilitated bacterial invasion in black rockfish. Collectively, these results indicated that SsCTSK functions as a cysteine protease and may serves as a target for pathogen manipulation of host defense system.

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.

Characterization, expression, enzymatic activity, and functional identification of cathepsin S from black rockfish Sebastes schlegelii.

Cathepsin S belong to the cathepsin L-like family of cysteine cathepsins. It is well known that Cathepsin S participate in various physiological processes and host immune defense in mammals. However, in teleost fish, the function of cathepsin S is less investigated. In the present study, a cathepsin S homologue (SsCTSS) from the teleost fish black rockfish (Sebastes schlegelii) were identified and examined at expression and functional levels. In silico analysis showed that three domains, including signal peptide, cathepsin propeptide inhibitor I29 domain, and functional domain Pept_C1, were existed in the cathepsin. SsCTSS possesses a peptidase domain with three catalytically essential residues (Cys25, His162, and Asn183). Phylogenetic profiling indicated that SsCTSS are evolutionally close to the cathepsin S of other teleost fish. The expression of SsCTSS in immune-related tissues was upregulated in a time-dependent manner upon bacterial pathogen infection. Purified recombinant SsCTSS (rSsCTSS) exhibited apparent peptidase activity, which was remarkably declined in the presence of the cathepsin inhibitor E-64. rSsCTSS showed strong binding ability to LPS and PGN, the major constituents of the outer membranes of Gram-negative and Gram-positive bacteria, respectively. rSsCTSS also exhibited the capability of agglutination to different bacteria. The knockdown of SsCTSS attenuated the ability of host to eliminate pathogenic bacteria. Taken together, our results suggested that SsCTSS functions as cysteine protease which might be involved in the antibacterial immunity of black rockfish.

[Biological characteristics of bacteriophages infecting three typic rhizobia of legume]. / 侵染3æ ªæ¨¡å¼è±†ç§‘æ ¹ç˜¤èŒçš„å™¬èŒä½“ç”Ÿç‰©å­¦ç‰¹æ€§.

Bacteriophages infecting rhizobia of legume leads to a significant decrease in the number of rhizobia in soil and nodulation in legume, which finally affects nitrogen fixation and remarkably reduces crop yield. However, limited studies have focused on bacteriophages of rhizobia. In this study, three typic rhizobium strains of Bradyrhizobium diazoefficiens USDA110T, Sinorhizobium sojae CCBAU05684T and Sinorhizobium meliloti USDA1002T were used as the host bacteria. From each host, three rhizobiophages were isolated from an agricultural black soil with double-layer plate method. We isolated nine rhizobiophages and investigated their morphological feature and biological characteristics. The results showed that the phages of SMM (infecting Sinorhizobium meliloti USDA1002T) and BDM (infecting Bradyrhizobium diazoefficiens USDA110T) belonged to Myoviridae family, while phages of SSS (infecting Sinorhizobium sojae CCBAU05684T) belonged to Siphoviridae family. The optimal multiplicity of infection for nine phages ranged from 0.001 to 1.0. The one-step growth curve showed that the latent and rising periods of BDM were remarkably longer than that of SMM and SSS, but the bust size was minimal. Nine phages had the strongest infecting activity at 30-40 ℃ and at neutral pH condition. Comparative analysis showed that the biological characteristics of phages infected with the same host were different, with the differentiation being smaller than that of phages infected with different hosts.

A novel calreticulin-related molecule that interacts with bacteria and enhances host resistance against bacterial infection in black rockfish, Sebastes schlegeli.

Calreticulin (CRT) is a highly conserved and multi-functional protein with diverse localizations. CRT has lectin-like properties and possesses important immunological activities in mammalian. In teleost, very limited studies on CRT immunologic function have been documented. In the present study, a CRT homologue (SsCRT) was cloned, identified and characterized from black rockfish, Sebastes schlegeli, an important aquaculture species in East Asia. The full length of SsCRT cDNA is 2180 bp and encoded a polypeptide of 425 amino acids. SsCRT contains a signal peptide, three distinct structural and functional domains (N-, P- and C-domains), and an endoplasmic reticulum (ER) retrieval signal sequence (KDEL). The deduced amino acid sequence of SsCRT shares 89-92% overall sequence identities with the CRT proteins of several fish species. SsCRT was distributed ubiquitously in all the detected tissues and was highly expressed in the spleen, muscle and liver. After the infection of fish extracellular bacterial pathogen Vibrio anguillarum and intracellular bacterial pathogen Edwardsiella tarda, the mRNA transcripts of SsCRT in spleen, liver, and head kidney were significantly up-regulated. The expression patterns were time-dependent and tissue-dependent. Recombinant SsCRT (rSsCRT) exhibited apparent binding activities against different bacteria and PAMPs. In vivo studies showed that the expressions of multiple immune-related genes such as TNF13B, IL-1ß, IL-8, SAA, Hsp70, and ISG15 in head kidney were significantly enhanced when black rockfish were treated with rSsCRT. Furthermore, rSsCRT reduced pathogen dissemination and replication in fish kidney and spleen. These results indicated that SsCRT served as an immune receptor to recognize and eliminate the invading pathogens, which played a vital role in the immune response of Sebastes schlegeli. These findings provide new insights into understanding the roles of CRT proteins in immune response and pathogen infection in teleost.

A novel C1q domain containing protein in black rockfish (Sebastes schlegelii) serves as a pattern recognition receptor with immunoregulatory properties and possesses binding activity to heat-aggregated IgG.

C1q-domain-containing (C1qDC) proteins, which are involved in a series of immune responses, are important pattern recognition receptors in innate immunity in vertebrates and invertebrates. Functional studies of C1qDC proteins in vertebrates are scarce. In the present study, a C1qDC protein (SsC1qDC) from the teleost black rockfish (Sebastes schlegelii) was identified and examined at expression and functional levels. The open reading frame of SsC1qDC is 636 bp, and the predicted amino acid sequence of SsC1qDC shares 62%-69% overall identity with the C1qDC proteins of several fish species. SsC1qDC possesses conserved C1qDC features, including a signal sequence and a C1q domain. SsC1qDC was expressed in different tissues and its expression was up-regulated by bacterial and viral infection. Recombinant SsC1qDC (rSsC1qDC) exhibited apparent binding activities against PAMPs including LPS and PGN. rSsC1qDC had antibacterial activity against Vibrio parahaemolyticus, and was able to enhance the phagocytic activity of macrophages towards Vibrio anguillarum. rSsC1qDC interacted with human heat-aggregated IgG. Furthermore, in the presence of rSsC1qDC, fish exhibited enhanced resistance against bacterial infection. Collectively, these results indicated that SsC1qDC serves as a pattern recognition receptor and plays a vital role in the defense system of black rockfish.

High mobility group box 2 of black rockfish Sebastes schlegelii: Gene cloning, immunoregulatory properties and antibacterial effect.

High-mobility group box 2 (HMGB2) is a non-histone chromosomal protein that involved diverse functions such as transcriptional regulation and innate immune responses in mammalian. In teleost, very limited studies on HMGB2 proteins have been documented. Black rockfish (Sebastes schlegelii) is an economic fish species and cultured worldwide. However, the study of black rockfish about immunology is very scarce. In the present study, a HMGB2 homologue gene (SsHMGB2) was identified and characterized in black rockfish. The open reading frame of SsHMGB2 is 648 bp, and the deduced amino acid sequence of SsHMGB2 shares 74.4%-91.2% overall sequence identities with the HMGB2 proteins of several fish species. In silico analysis identified several conserved features, including two basic HMG boxes and an acidic C-terminal tail composed of 24 Asp/Glu residues. Expression of SsHMGB2 occurred in multiple tissues and was upregulated during pathogens infection. Recombinant SsHMGB2 (rSsHMGB2) exhibited apparent binding activities against DNA. In vivo studies showed that the expressions of multiple immune-related genes in head kidney were significantly enhanced when black rockfish were treated with rSsHMGB2. Furthermore, rSsHMGB2 reduced pathogen dissemination and replication in fish kidney and spleen. Taken together, these results suggest that SsHMGB2 possesses apparent immunoregulatory properties and played a role in fighting bacterial infection.

[Comparison on fungal molecular ecological networks of agricultural soils with different latitudes in the black soil region of Northeast China]. / 东北黑土区不同纬度农田土壤真菌分子生态网络比较.

To investigate the differences of fungal network structures and interaction among fungal species of in black soil region of Northeast China, Illumina MiSeq sequencing was used to reveal the fungal communities in the three long-term fertilization experimental fields. Fungal molecular ecological networks were constructed based on random matrix theory (RMT). The results demonstrated that Ascomycota, Basidiomycota and Zygomycota were the dominant phyla and Hypocreales, Pleosporales and Sordariales were the dominant order, but the relative abundance of some dominant taxa significantly varied in different locations. Fungal molecular ecological network structures in three locations showed significant difference, with more complex fungal network being observed in north location with more competitive relations among species. The fungal network in south location was more easily disturbed by environmental perturbations with less stability. Only seven shared nodes were detected among three fungal molecular ecological networks. There were large differences in connectivity of shared nodes within individual fungal network. The subnetwork of Hypocreales was gradual complex from south to north location while subnetwork of Pleosporales presented reversed trend. The key species of south, middle and north locations were Chaetomiaceae, Pleosporales and Penicillium coralligerum, respectively. Soil pH and total N content were the main soil properties simultaneously influencing three fungal networks.

A teleost TFPI-2 peptide that possesses a broad antibacterial spectrum and immune-stimulatory properties.

Tissue factor pathway inhibitor 2 (TFPI-2) is an analogue of TFPI-1 and a potent endogenous inhibitor of tissue factor (TF)-mediated blood coagulation. Previous reports have shown that several peptides derived from human and vertebrates TFPI-2 possess antibacterial activity against diverse bacteria. In this study, a C-terminal peptide, TO24 (with 24 amino acids), derived from red drum (Sciaenops ocellatus) TFPI-2, was synthesized and investigated for its antimicrobial spectrum, action mode, as well as the immune-stimulatory property. Our results indicated that TO24 was active against Gram-positive bacteria Micrococcus luteus and Staphylococcus aureus; Gram-negative bacteria Vibrio litoralis, Vibrio ichthyoenteri, Vibrio vulnificus and Vibrio scophthalmi, as well as fish megalocytivirus, infectious spleen and kidney necrosis virus (ISKNV). During its interaction with V. vulnificus, TO24 exerted its antibacterial activity by destroying cell membrane integrity, penetrating the cytoplasm and inducing degradation of genomic DNA and total RNA. In addition, TO24 had no hemolytic activity against red drum blood cells. In vitro, TO24 enhanced bactericidal activity of red drum macrophages. In vivo, administration of red drum with TO24 before bacterial infection significantly reduced pathogen dissemination and replication in tissues. These results indicate that TO24 is a broad-spectrum antimicrobial peptide with immune-stimulatory properties and it has the potential to be used as an antimicrobial agent in aquaculture.

Constitution of traditional chinese medicine and related factors in women of childbearing age.

BACKGROUND: This study investigates the constitution of traditional Chinese medicine (TCM) among women who want to be pregnant in one year and explores factors related to TCM constitution. METHODS: This study was conducted on women who participated in free preconception check-ups provided by the Zhabei District Maternity and Child Care Center in Shanghai, China. The information regarding the female demographic characteristics, physical condition, history of pregnancy and childbearing, diet and behavior, and social psychological factors was collected, and TCM constitution assessment was performed. The Chi-square test, t-test, logistic regression analysis, and multinomial logistic regression analysis were used to explore the related factors of TCM constitution. RESULTS: The participants in this study were aged 28.3 ± 3.0 years. Approximately fifty-five women in this study had Unbalanced Constitution. Logistic regression analysis showed that Shanghai residence, dysmenorrhea, gum bleeding, aversion to vegetables, preference for raw meat, job stress, and economic stress were significantly and negatively associated with Balanced Constitution. Multinomial logistic analysis showed that Shanghai residence was significantly associated with Yang-deficiency, Yin-deficiency, and Stagnant Qi Constitutions; gum bleeding was significantly associated with Yin-deficiency, Stagnant Blood, Stagnant Qi, and Inherited Special Constitutions; aversion to vegetables was significantly associated with Damp-heat Constitution; job stress was significantly associated with Yang-deficiency, Phlegm-dampness, Damp-heat, Stagnant Blood, and Stagnant Qi Constitutions; and economic stress was significantly associated with Yang-deficiency, and Stagnant Qi Constitutions. CONCLUSION: The application of TCM constitution to preconception care would be beneficial for early identification of potential TCM constitution risks and be beneficial for early intervention (e.g., health education, and dietary education), especially during the women who do not have a medical condition and those who have related factors found in this study.

TO17: A teleost antimicrobial peptide that induces degradation of bacterial nucleic acids and inhibits bacterial infection in red drum, Sciaenops ocellatus.

Tissue factor pathway inhibitor (TFPI)-1 is well known for its role as an inhibitor of blood coagulation. Several studies have demonstrated that the C-terminal peptides of TFPI-1 are active against a broad spectrum of microorganisms. In a previous study, we found that TO17 (with 17 amino acids), a TFPI-1 C-terminal peptide from red drum (Sciaenops ocellatus), was active against Edwardsiella tarda. In the present study, we investigated further the antimicrobial spectrum, action mode, as well as the immunostimulatory property of TO17. Our results showed that TO17 displayed antimicrobial activity against Staphylococcus aureus, Micrococcus luteus, Vibrio vulnificus, and infectious spleen and kidney necrosis virus, independent of host serum. Furthermore, the activity of TO17 was influenced by the length or type of amino acids at the N and C termini. During its interaction with V. vulnificus, TO17 exerted its antibacterial activity by destroying cell membrane integrity, penetrating the cytoplasm and inducing degradation of genomic DNA and total RNA. In addition, TO17 had no hemolytic activity against red drum blood cells. In vitro, TO17 enhanced production of nitric oxide and bactericidal activity of red drum macrophages. In vivo, administration of red drum with TO17 before bacterial infection significantly reduced pathogen dissemination and replication in tissues. These results indicate that TO17 is a broad-spectrum antimicrobial peptide with immunostimulatory properties and it has the potential to be used as an antimicrobial agent in aquaculture.

TC38, a teleost TFPI-2 peptide that kills bacteria via penetration of the cell membrane and interaction with nucleic acids.

Tissue factor pathway inhibitor 2 (TFPI-2) is an analog of TFPI-1 and a potent endogenous inhibitor of tissue factor (TF)-mediated blood coagulation. Recent reports have proven that the C-terminal of TFPI-2 peptides in humans and several other vertebrates possesses antibacterial activity against Gram-positive and Gram-negative bacteria. In our previous study, we reported that the TFPI-2 peptide, TC38 in tongue sole (Cynoglossus semilaevis) was active against Micrococcus luteus. In this study, we further examine the antimicrobial spectrum, mechanism of action, and function of TC38 in tongue sole. Our results indicate that TC38 is active against the Gram-negative bacteria Vibrio ichthyoenteri, Vibrio litoralis, Vibrio parahaemolyticus, and Vibrio vulnificus, as well as the fish Megalocytivirus, infectious spleen and kidney necrosis virus (ISKNV). The mechanism of action of TC38 against V. vulnificus was explored. The results showed that TC38 killed V. vulnificus cells without lysis of the cell membrane. FITC-labeled TC38 was able to penetrate the cell membrane and bind to DNA and RNA, then disrupt cellular function, eventually leading to cell death. Administration of TC38 to tongue sole significantly improved its defense against V. vulnificus infection. Overall, these results indicate that TC38 is a novel peptide with a broad antimicrobial spectrum. Furthermore, the unique action of TC38 against V. vulnificus adds new insights to the mechanism of action of vertebrate TFPI peptides. Moreover, TC38 is an interesting antimicrobial agent that could be useful in the fight against pathogenic invasion in aquaculture.