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Ladderlectin is a member of C-type lectins (CTLs) in teleost fish and involved in innate immune defense. In this study, ayu (Plecoglossus altivelis) ladderlecin-like (PaLL-like) sequence was cloned, which encodes a polypeptide of 172 amino acids that includes a signal peptide and characteristic C-type lectin-like domains (CTLDs). Phylogenetically, PaLL-like was most closely related to its teleost counterpart from shishamo smelt (Spirinchus lanceolatus). Expression analysis revealed a ubiquitous expression profile, with highest expression detected in liver and its expression was up-regulated following Vibiro anguillarum infection. Similar to canonical CTLs, PaLL-like exhibited carbohydrate-binidng capacities to a wide range of well-defined mono-/di-saccharides and likely confer PaLL-like the ability to agglutinate all tested bacterial, including three Gram-positive species (i.e., Listeria monocytogenes, Staphylococcus aureus and Streptococcus iniae) and eight Gram-negative species (i.e., Edwardsiella tarda, Aeromonas (A.) hydrophila, Escherichia coli, Vibrio (V.) harveyi, V. anguillarum, V. parahemolyticus, A. versoni and V. vulnificus), in a calcium-dependent manner. Further functional studies revealed that PaLL-like displayed immunomodulatory activities leading to enhanced bactericidal activity of serum, pathogen opsonization and macrophage activation with increased expression of pro-inflammatory cytokines (i.e., PaIL-1β and PaTNF-α). Collectively, these immunomodulatory activities of PaLL-like suppressed proliferations of V. anguillarum in targeted tissued in vivo and likely contributed to the increased survival rate of infected-fish. Overall, our results demonstrated PaLL-like is a critical component of innate immunity and provides protective effects against bacterial infection.

Liver-expressed antimicrobial peptide 2 (LEAP-2) is a cationic peptide that plays an important role in a host’s innate immune system. We previously demonstrated that mudskipper (Boleophthalmus pectinirostris) LEAP-2 (BpLEAP-2) induces chemotaxis and activation of monocytes/ macrophages (MO/MФ). However, the molecular mechanism by which BpLEAP-2 regulates MO/MΦ remains unclear. In this study, we used yeast two-hybrid cDNA library screening to identify mudskipper protein(s) that interacted with BpLEAP-2, and characterized a sequence encoding motile sperm domain-containing protein 2 (BpMOSPD2). The interaction between BpLEAP-2 and BpMOSPD2 was subsequently confirmed by co-immunoprecipitation (Co-IP). Sequence analyses revealed that the predicted BpMOSPD2 contained an N-terminal extracellular portion composed of a CRAL-TRIO domain and a motile sperm domain, a C-terminal transmembrane domain, and a short cytoplasmic tail. Phylogenetic tree analysis indicated that BpMOSPD2 grouped tightly with fish MOSPD2 homologs and was most closely related to that of the Nile tilapia (Oreochromis niloticus). The recombinant BpMOSPD2 was produced by prokaryotic expression and the corresponding antibody was prepared for protein concentration determination. RNA interference was used to knockdown BpMOSPD2 expression in the mudskipper MO/MФ, and the knockdown efficiency was confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Knockdown of BpMOSPD2 significantly inhibited BpLEAP-2-induced chemotaxis of mudskipper MO/MФ and BpLEAP-2-induced bacterial killing activity. Furthermore, knockdown of BpMOSPD2 inhibited the effect of BpLEAP-2 on mRNA expression levels of BpIL-10, BpTNFα, BpIL-1β, and BpTGFβ in MO/MФ. In general, BpMOSPD2 directly interacted with BpLEAP-2, and mediated the effects of BpLEAP-2 on chemotaxis and activation of mudskipper MO/MФ. This is the first identification of MOSPD2 as a receptor for LEAP-2.

Due to continuous improvements in the beneficiation process, the size of tailings has decreased. In many mines, more than 50% of the total tailings are finer than 74 μm. Ultrafine tailings exhibit a slow settling velocity and uneven settling, which pose new challenges to the safety and stability of tailings dams. In this paper, ultrafine iron tailings from the Makeng Iron Mine in Longyan City, Fujian Province, were used as the research object. A four-factor, three-level orthogonal test method was used to study the sedimentation characteristics of ultrafine tailings with four common curing agent materials, including polyacrylamide, polyaluminum chloride, polyaluminum sulfate, and polyferric sulfate. The results show that when the pulp concentration is 30%, the polyacrylamide is cationic and the molecular weight is 10 million, the optimal content of the flocculant is 3‰, the optimum dose of the polyaluminum chloride with a content of 28 is 0.1‰, the optimum dose of polyaluminum sulfate is 1‰, and the optimum dose of polymeric ferric sulfate is 1‰. When the flocculant is mixed according to the proportion of 2‰ polyacrylamide, 0.05‰ polyaluminum chloride, 1‰ polyaluminum sulfate, and 1.0‰ polyferric sulfate, the sedimentation speed of the ultrafine tailings is fast, and the supernatant liquid is clear. The results of multivariate nonlinear regression analysis of the sedimentation curve show that the primary and secondary factors affecting sedimentation are polyacrylamide > polyaluminum sulfate > polyaluminum chloride > polyferric sulfate. When the optimal ratio is applied, the cohesion (c) of ultrafine tailings increases from 27 kPa to 68.75 kPa and the internal friction angle (φ) increases from 25.53° to 27.53°, which shows that the shear strength improves and the stability of the tailings dam increases. The economic analysis of the composite flocculant with the optimal ratio shows that the flocculant with the optimal proportion has an obvious economic advantage over polyacrylamide alone.

NK-lysin (NKL) is a cationic host defense peptide that plays an important role in host immune responses against various pathogens. However, the immunomodulatory activity of NKL in fishes is rarely investigated. In this study, we characterized a cDNA sequence encoding an NK-lysin homolog (BpNKL) from the fish, mudskipper (Boleophthalmus pectinirostris). Sequence analysis revealed that BpNKL is most closely related to tiger puffer (Takifugu rubripes) NKL. BpNKL transcript was detected in all the tested tissues, with the highest level in the gill, followed by the spleen and kidney. Upon Edwardsiella tarda infection, the mRNA expression of BpNKL in the mudskipper was significantly upregulated in the spleen, kidney, and gill. A shortened peptide derived from BpNKL, BpNKLP40, was then chemically synthesized and its biological functions were investigated. BpNKLP40 exhibited a direct antibacterial activity against some Gram-negative bacteria, including E. tarda, Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio harveyi, and induced hydrolysis of E. tarda genomic DNA. Intraperitoneal injection of 1.0 μg/g BpNKLP40 significantly improved the survival of mudskipper following E. tarda infection and reduced the bacterial burden in tissues and blood. Moreover, 1.0 μg/ml BpNKLP40 treatment had an enhanced effect on the intracellular killing of E. tarda by monocytes/macrophages (MO/MФ) as well as on the mRNA expression of pro-inflammatory cytokines in MO/MФ. In conclusion, our study reveals that BpNKL plays a role against E. tarda infection in the mudskipper by not only directly killing bacteria but also through an immunomodulatory activity on MO/MФ.

CC motif chemokine ligand 25 (CCL25) is a key chemokine that attracts various types of leukocytes, such as activated peritoneal macrophages. However, information on CCL25 in fish is limited. Here, a CCL25 gene (LjCCL25) was identified from Japanese sea bass (Lateolabrax japonicus), showing upregulation in multiple tissues against Vibrio harveyi infection. The recombinant LjCCL25 (rLjCCL25) only significantly induced the migration of monocytes/macrophages (MO/MΦ) both in vitro and in vivo, but didn't induce that of neutrophils or lymphocytes. Additionally, rLjCCL25 only induced migration of the lipopolysaccharide-stimulated MO/MΦ (M1 type). Knockdown of Japanese sea bass CC chemokine receptor 9 (LjCCR9) expression in MO/MФ by RNA interference inhibited the LjCCL25-induced chemotaxis of resting and M1 type MO/MФ. Moreover, administration of 300 ng/g rLjCCL25 effectively increased the survival of V. harveyi-infected fish and decreased bacterial load. Our study demonstrates that LjCCL25 functions as an MO/MФ chemoattractant via LjCCR9 in Japanese sea bass against V. harveyi.

Dalbergia hupeana Hance (D. hupeana) is a precious hardwood tree of the genus Dalbergia. It is one of the few species widely distributed within subtropical areas and is important for timber production and forest restoration. At present, there is little published genetic information on D. hupeana. Therefore, we performed a genome survey using next generation sequencing (NGS) and developed a set of novel genomic SSR (gSSR) markers from the assembled data, and assessed the transferability of these markers to other Dalbergia species in Asia. The results of the genome survey show the genome size of D. hupeana to be about 664 Mb and highly heterozygous. The assembly of sequencing data produced 2,431,997 contigs, and the initial assembly of the NGS data alone resulted in contig N50 of 393 kb with a total of 720 Mb. A total of 127,742 perfect SSR markers were found in the assembled contigs. A total of 37 highly polymorphic and easily genotyped gSSR markers were developed in D. hupeana, while the majority of gSSR markers could be successfully transferred to nine other Dalbergia species in Asia. The transferability rate of gSSR markers was highest in D. balansae, which is more closely related to D. hupeana. Seven gSSR markers were able to be amplified in all tested species. In addition, a preliminary assessment of the genetic diversity of three tree species in the Dalbergia genus suggested a high level of genetic diversity within populations distributed in the subtropical area in China. However, the determination of the global status of their genetic variation still requires further and more comprehensive assessment. Our findings will enable further studies on the genetic diversity, phylogenetics, germplasm characterization, and taxonomy of various Dalbergia species.

MPTA is a novel extract product derived from Macleaya cordata (Willd.) R. Br., which has good anti-inflammatory and antioxidant activity. The aim of this study was to investigate the acute oral toxicity and 90-day sub-chronic oral toxicity of MPTA. In the acute toxicity study, 50 SD rats of both sexes were randomly divided into 5 groups and dosed in a gradient from 197.53 mg/kg to 1000.00 mg/kg bw. Toxic effects were observed up to 14 days and LD

Eight new organometallic complexes were obtained by the reaction of trialkyl rare earth complexes Ln(CH2SiMe3)3(THF)2 (Ln = Y, Lu, Ho, Dy) with tetradentate phenol ether ligands. Among them the structure of complex 7 with Lu was confirmed by single crystal X-ray diffraction. These complexes exhibited moderate to high activity for 2-VP polymerization at ambient temperature in toluene. Complex 5 showed the highest catalytic activity, with a turnover frequency of 1714 h−1. Significantly, the 2-VP polymerization is also stereospecific, producing isotactic poly(2-VP) with an isotacticity of mm = 0.44–0.99. The catalytic experimental results indicate that the size of metal atom obviously influences the stereoregularity of the growing polymer chain, and catalysts with Ho atom exhibit better isotactic selectivity than other metal catalysts.

Chemokine (CC motif) ligand 2 (CCL2), also known as monocyte chemoattractant protein 1 (MCP-1), is one of the key chemokines that regulate migration and infiltration of monocytes/macrophages (MO/MФ) in mammals. However, the functional repertoire of fish CCL2 remains unclear. Here, we identified a cDNA sequence encoding a novel CCL2-like protein (PaCCL2L) in ayu, Plecoglossus altivelis. Sequence analysis revealed that PaCCL2L grouped with CCL2 homologs, and is most closely related to Mexican tetra (Astyanax mexicanus) and zebrafish (Danio rerio) homologs. PaCCL2 transcripts were expressed in all tested tissues from healthy ayu, with the highest level in the spleen. Upon Vibrio anguillarum infection, PaCCL2L transcripts increased significantly in tested tissues, including the liver, spleen, and head kidney. We then produced the recombinant PaCCL2L mature peptide (rPaCCL2L) by prokaryotic expression and generated the corresponding antibodies (anti-PaCCL2L). A significant increase in PaCCL2L protein and mRNA expression was observed in ayu MO/MФ following V. anguillarum challenge. Intraperitoneal injection of rPaCCL2L resulted in significantly improved survival and reduced tissue bacterial load in V. anguillarum-infected ayu. rPaCCL2L had a positive effect on the chemotaxis of MO/MΦ and neutrophils both in vitro and in vivo. Meanwhile, rPaCCL2L exhibited a positive effect on the chemotaxis of LPS-stimulated MO/MΦ (M1 type) in vitro, whereas it exhibited no chemotaxis effect on cAMP-stimulated MO/MΦ (M2 type). In addition, rPaCCL2L treatment exhibited an enhanced effect on MO/MΦ phagocytosis, bacterial killing, respiratory burst, and mRNA expression of proinflammatory cytokines, whereas anti-PaCCL2L treatment had an inhibitory effect. Our study demonstrates that PaCCL2L might play a role in the immune response of ayu against V. anguillarum infection through chemotactic recruitment and activation of MO/MΦ.

Dalbergia cultrata is a Near Threatened species with high ecological and economic values. In this study, its chloroplast genome was assembled using Illumina pair-end sequencing dataset. The chloroplast genome has a quadripartite structure with 156,385 bp in length and contains a pair of 16,392 bp inverted repeat (IR) regions, which were separated by large single copy (LSC: 86,040 bp) region and small single copy (SSC: 37,561 bp) region. A total of 121 genes were annotated, including 77 protein-coding genes (PCGs), 36 tRNAs, and 8 rRNAs. The overall GC content was 36.1%. The phylogenetic analysis revealed that D. cultrata has close relationship to D. hainanensis and D. odorifera. This complete chloroplast genome can be readily used for population genetic studies of D. cultrata.

NLRP3 inflammasome plays a crucial role in the innate immune system. Our group previously reported that the microglial adenosine 2A receptor (A

The rose is one of the most important ornamental woody plants because of its extensive use and high economic value. Herein, we sequenced a complete chloroplast genome of the miniature rose variety Rosa ‘Margo Koster’ and performed comparative analyses with sequences previously published for other species in the Rosaceae family. The chloroplast genome of Rosa ‘Margo Koster’, with a size of 157,395 bp, has a circular quadripartite structure typical of angiosperm chloroplast genomes and contains a total of 81 protein-coding genes, 30 tRNA genes and 4 rRNA genes. Conjunction regions in the chloroplast genome of Rosa ‘Margo Koster’ were verified and manually corrected by Sanger sequencing. Comparative genome analysis showed that the IR contraction and expansion events resulted in rps19 and ycf1 pseudogenes. The phylogenetic analysis within the Rosa genus showed that Rosa ‘Margo Koster’ is closer to Rosa odorata than to other Rosa species. Additionally, we identified and screened highly divergent sequences and cpSSRs and compared their power to discriminate rose varieties by Sanger sequencing and capillary electrophoresis. The results showed that 15 cpSSRs are polymorphic, but their discriminating power is only moderate among a set of rose varieties. However, more than 150 single nucleotide variations (SNVs) were discovered in the flanking region of cpSSRs, and the results indicated that these SNVs have a higher divergence and stronger power for profiling rose varieties. These findings suggest that nucleotide mutations in the chloroplast genome may be an effective and powerful tool for rose variety discrimination and DNA profiling. These molecular markers in the chloroplast genome sequence of Rosa spp. will facilitate population and phylogenetic studies and other related studies of this species.

Large yellow croaker (Larimichthys crocea, LYC) aquaculture is being threatened by intensive infectious diseases. Relevant studies have focused on LYC immune responses to infection. By contrast, little is known how and to what extent the gut microbiota responds to infection. Here, we explored the interactions between LYC immune responses and gut bacterial communities during Pseudomonas plecoglossicida infection. P. plecoglossicida successfully colonized into LYC gut microbiota, resulting in an increasing mortality rate. Relative gene expressions of pro-inflammatory cytokines (TNF-α1, TNF-α2 and IL-1β) and anti-inflammatory cytokine (IL-10) were consistently and significantly induced by P. plecoglossicida infection, whereas non-specific immune enzymes activities were only enhanced at the early infection stages. P. plecoglossicida infection caused an irreversible disruption in the gut microbiota, of which infection and hours post infection constrained 16.2% and 5.6% variations, respectively. In addition, top 18 discriminatory taxa that were responsible for the difference between treatments were identified, whose abundances were significantly associated with the immune activities of LYC. Using a structural equation modeling (SEM), we found that gut bacterial communities were primarily governed by the conjointly direct (-0.33) and indirect (0) effects of infection, which subsequently affect host immune responses. Our results suggest that an irreversible dysbiosis in gut microbiota could be the causality of increasing mortality. To our knowledge, this is the first study to provide an integrated overview among pathogen infection, immune response and gut microbiota of LYC.

To observe the effect of norcantharidin (NCTD) on collagen-induced arthritis (CIA) rats. Sixty Sprague-Dawley(SD) rats were randomly divided into 6 groups (n=10): normal group, CIA model group(model group), NCTD low-dose group [1.35 mg/(kg•d)], NCTD middle-dose group [2.7 mg/(kg•d)], NCTD high-dose group [5.4 mg/(kg•d)] and methotrexate (MTX) group [1.8 mg/(kg/w)]. Anesthetized rats were sacrificed by luxation of cervical vertebra after 4 weeks of administration. The arthritis scores were evaluated twice a week. The pathological changes in the ankle joints of rats were observed by hematoxylin-eosin (H&E) staining. The serum levels of interleukin (IL) 1β, IL-6, tumor necrosis factor (TNF)-α, vascular endothelial growth factor (VEGF), IL-17 and transform growth factor (TGF) β were detected by enzyme linked immunosorbent assay (ELISA). The mRNA expression of retinoid-related orphan nuclear receptorγt (RORγt) and forkhead box P3 (Foxp3) in peripheral blood lymphocytes were confirmed by real-time polymerase chain reaction. MTX and high-dose NCTD not only decreased the arthritis scores but also alleviated the pathological changes in CIA rats’ ankle joints compared with the model group (P

Many factors, such as temperature, solvent, the central metal atom and the type of coligands, may affect the nature of metal–organic frameworks (MOFs) and the framework formation in the self-assembly process, which results in the complexity of these compounds and the uncertainty of their structures. Two new isomeric ZnIImetal–organic frameworks (MOFs) based on mixed ligands, namely, poly[[μ-1,5-bis(2-methyl-1H-imidazol-1-yl)pentane-κ2N3:N3′](μ-5-methylisophthalato-κ2O1:O3)zinc(II)], [Zn(C9H6O4)(C13H20N4)]n, (I), and poly[[μ-1,5-bis(2-methyl-1H-imidazol-1-yl)pentane-κ2N3:N3′](μ3-5-methylisophthalato-κ3O1:O1′:O3)(μ3-5-methylisophthalato-κ4O1:O1′:O3,O3′)dizinc(II)], [Zn2(C9H6O4)2(C13H20N4)]n, (II), have been synthesized under hydrothermal conditions and characterized by single-crystal X-ray diffraction, IR spectroscopy, elemental analysis and thermogravimetric analysis. Complex (I) displays a two-dimensional layer net, while complex (II) exhibits a twofold interpenetrating three-dimensional framework. Both complexes show high stability and good fluorescence in the solid state at room temperature.

Liquidambar acalycina is a popular tree species with high ornamental and economic values because of the leaf shape and coloration. In this study, chloroplast genome of L.acalycina was assembled usi...

Objective: To study the effect of oxidative stress injury on miR-210 expression in spinal neurons and the neuroprotective effect of miR-210 inhibitor. Methods: Spinal neurons of rats were cultured and divided into control group (group A), H2O2 group (group B), NC group (group C), H2O2+NC group (group D) and H2O2+miR-210 inhibitor group (group E), miR-210 expression levels in group A and group B as well as the cell vitality and expression levels of oxidative stress molecules, apoptotic molecules and function-related molecules in group C, group D and group E were determined. Results: miR-210 expression level in group B was significantly higher than that in group A; after 12 h, 18 h and 24 h of treatment, OD values of group D were significantly lower than those of group C, and OD values of group E were significantly higher than those of group D; after 24 h of treatment, HO-1, NOX-2, CAT, GAP-43 and synapsin-I levels in group D were significantly lower than those in group C while ROS, 8-OHdG, c-fos, c-jun, AP1, caspase-3, PSD-93 and PSD-95 levels were significantly higher than those in group C; HO-1, NOX-2, CAT, GAP-43 and synapsin-I levels in group E were significantly higher than those in group D while ROS, 8-OHdG, c-fos, c-jun, AP1, caspase-3,PSD-93 and PSD-95 levels were significantly lower than those in group D. Conclusion: Oxidative stress injury can cause high miR-210 expression in spinal neurons, and miR-210 inhibitor can reduce the neuron injury caused by oxidative stress.

Mammalian interleukin 4 (IL-4) and interleukin 13 (IL-13) molecules are anti-inflammatory cytokines mediating the alternative activation of macrophages. However, the role of fish IL-4/13 homologs in monocytes/macrophages (MO/MФ) polarization remains unclear. In this study, we have functionally identified an IL-4/13B homolog in grass carp (Ctenopharyngodon idella), which is termed as CiIL-4/13B. Multiple alignment showed that CiIL-4/13B shared the typical characteristics and structure with other known fish IL-4/13. Phylogenetic analysis showed that CiIL-4/13B is evolutionarily closely related to zebrafish (Danio rerio) and common carp (Cyprinus carpio) IL-4/13B. CiIL-4/13B mRNA was constitutively expressed in tissues and peripheral blood lymphocytes (PBLs) examined, with its highest expression seen in PBLs. Following Aeromonas hydrophila infection, CiIL-4/13B mRNA expression was upregulated. Recombinant CiIL-4/13B (rCiIL-4/13B) was overexpressed in Escherichia coli and purified for a functional study. Using prepared anti-rCiIL-4/13B antiserum, Western blot analysis showed that native CiIL-4/13B in grass carp plasma is N-glycosylated. Intraperitoneal injection of bioactive rCiIL-4/13B significantly increased the survival rate of grass carp against A. hydrophila, and decreased the tissue bacterial load, with a higher dose having better effects. Bioactive rCiIL-4/13B treatment decreased nitrite production and mRNA expression of proinflammatory cytokines (IL-1β and TNF-α), while it increased arginase activity and mRNA expression of anti-inflammatory cytokines (TGF-β and IL-10). The phagocytosis by grass carp MO/MФ had no significant changes by the 8 h treatment of bioactive rCiIL-4/13B compared to that of the negative control, while it was significantly inhibited by the 24 h treatment of bioactive rCiIL-4/13B. The inhibitory effect of rCiIL-4/13B on MO/MФ phagocytosis may be a consequence of MO/MФ proliferation. In summary, our results suggest that CiIL-4/13B plays a protective effect in grass carp against A. hydrophila by inducing alternatively activated MO/MФ.

Cathelicidins (CATHs) are a family of endogenous antimicrobial peptides that are capable of both direct bacteria-killing and immunomodulatory effects. P2X7 receptor (P2X7R) is a mediator of CATH in mammalian immune cells. Here, we studied the function and regulation of CATH in head kidney-derived monocytes/macrophages (MO/MФ) from ayu, Plecoglossus altivelis. We investigated the chemotaxis of MO/MФ in response to ayu CATH (PaCATH), and found that PaCATH had a dose-dependent effect on MO/MФ chemotaxis with the optimal concentration of 10.0 μg/ml. The qPCR and Western blot analysis revealed that PaCATH inhibited the expression of ayu P2X7R (PaP2X7R) at both mRNA and protein levels. Knockdown of the PaP2X7R expression in ayu MO/MФ by RNA interference not only significantly inhibited the chemotactic effect of PaCATH on MO/MФ, but also obviously reduced the effect of PaCATH on the phagocytosis, bacteria-killing, respiratory burst, and cytokine expression of ayu MO/MФ. Our study revealed that the immunomodulatory effect of fish CATH is mediated by P2X7R.

Dalbergia cultrata is a Near Threatened species with high ecological and economic values. In this study, its chloroplast genome was assembled using Illumina pair-end sequencing dataset. The chlorop...

Liver-expressed antimicrobial peptide 2 (LEAP-2) is widespread in fish and plays an important role in the host's innate immune system. In this study, full-length cDNA for LEAP-2 (PaLEAP-2) gene was cloned and sequenced from ayu, Plecoglossus altivelis. PaLEAP-2 mRNA was detected in a wide range of tissues, with the highest level of transcripts found in the liver. Upon induction by Vibrio anguillarum, its expression significantly increased in the liver, kidney, spleen, gill, and heart, but decreased in the intestine. The PaLEAP-2 mature peptide was chemically synthesized; it exhibited selective antimicrobial activity against various bacteria in vitro. PaLEAP-2 at high concentration reduced the bacterial load and improved the survival rate of V. anguillarum-infected ayu. Moreover, it inhibited the expression of mRNAs for TNF-α and IL-1β in V. anguillarum-infected ayu, both at high and low concentrations. PaLEAP-2 induced hydrolysis of the pET-22b plasmid DNA and bacterium genomic DNA. These results suggest that PaLEAP-2 plays a role in ayu immune responses against bacterial infection.

Interleukin 1β (IL-1β), the first interleukin to be characterized, plays a key role in regulating the immune response. In this study, we determined the cDNA and genomic DNA sequences of the IL-1β gene from the large yellow croaker, Larimichthys crocea. Phylogenetic analysis indicated that the IL-1β (LcIL-1β) gene was most closely related to that of European seabass (Dicentrarchus labrax), sharing 67.8% amino acid identity. In healthy large yellow croaker, LcIL-1β transcription was detected in all tested tissues, with the highest level found in the head kidney. Upon Vibrio alginolyticus infection, LcIL-1β transcription in all tested tissues was significantly upregulated. Intraperitoneal injection of recombinant LcIL-1β (rLcIL-1β) improved the survival rate and reduced the tissue bacterial load after V. alginolyticus infection. In addition, rLcIL-1β induced monocytes/macrophages (MO/MΦ) chemotaxis and increased phagocytosis and bactericidal activity in vitro. These results suggest that LcIL-1β plays an important role in the large yellow croaker immune response against V. alginolyticus.

A new copper(II) coordination polymer [Cu2(C5H4NCOO)2(C7H5N4)2] n (1) has been synthesized with 4-pyridinecarboxylate and 3-(pyridin-2-yl)-1,2,4-triazolyl ligands. The crystal structure shows that two neighboring copper(II) ions are coordinated with two deprotonated (anionic) 3-(pyridin-2-yl)-1,2,4-triazole ligands to form a binuclear structure. Adjacent binuclear units are linked by 4-pyridinecarboxylate anions to form a three-dimensional network structure. The magnetic, fluorescent, and electrochemical properties and thermal stability of 1 were studied. The results show that 1 exhibits antiferromagnetic interaction, and upon excitation at 318 nm, it has an intense fluorescent emission at approximately 430 nm. The electron transfer of 1 is irreversible in electrode reactions. The thermogravimetric analysis shows that 1 is stable below 240 °C.

Rapid, low-cost, and user-friendly strategies are urgently needed for early disease diagnosis and timely treatment, particularly for on-site screening of pathogens in aquaculture. In this study, we successfully developed a real-time fluorogenic loop-mediated isothermal amplification assay integrated on a microfluidic disc chip (on-chip LAMP), which was capable of simultaneously detecting 10 pathogenic bacteria in aquatic animals, i.e., Nocardia seriolae, Pseudomonas putida, Streptococcus iniae, Vibrio alginolyticus, Vibrio anguillarum, Vibrio fluvialis, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio rotiferianus, and Vibrio vulnificus. The assay provided a nearly-automated approach, with only a single pipetting step per chip for sample dispensing. This technique could achieve limits of detection (LOD) ranging from 0.40 to 6.42pg per 1.414μL reaction in less than 30 min. The robust reproducibility was demonstrated by a little variation among duplications for each bacterium with the coefficient of variation (CV) for time to positive (Tp) value less than 0.10. The clinical sensitivity and specificity of this on-chip LAMP assay in detecting field samples were 96.2% and 93.8% by comparison with conventional microbiological methods. Compared with other well-known techniques, on-chip LAMP assay provides low sample and reagent consumption, ease-of-use, accelerated analysis, multiple bacteria and on-site detection, and high reproducibility, indicating that such a technique would be applicable for on-site detection and routine monitoring of multiple pathogens in aquaculture.

Haptoglobin (Hp) molecule has been cloned and characterized in two marine teleosts (gilthead seabream and European sea bass), obtaining putative proteins of 319 residues encoded by an ORF of 960 bp in both species. However, the matrix of similarity revealed low identities among bony fish species 78.9% (seabream-sea bass), 43% (seabream/seabass-zebrafish) and lower than 20% with sharks and human. The protein sequences showed a signal peptide from the position 1 to 23, a trypsin domain from 47 to 297, and several predicted disulfide bridges and glycosylation sites. The expression of hp transcript levels during ontogeny showed a progressive increase of expression in seabream whilst remained almost unaltered in sea bass. By tissues, this gene was found constitutively expressed with the highest levels on liver in both species. The main results on hp transcript levels showed the up-regulation in gilthead seabream suffering from naturally occurring lymphocystis disease; and the down-regulation and up-regulation after nodavirus infection in the resistant gilthead seabream and the susceptible European sea bass, respectively. These findings demonstrate for the first time an important role of haptoglobin against viral infections, operating differently in two of the most important marine farmed fish species., SI

Liver-expressed antimicrobial peptide 2 (LEAP-2) plays a vital role in the host innate immune system. In the present study, two LEAP-2 genes (LcLEAP-2A and LcLEAP-2C) from large yellow croaker (Larimichthys crocea) were cloned, both of which consist of 3 exons and 2 introns. The LcLEAP-2A transcripts were expressed in a wide range of tissues, with the highest mRNA levels found in the liver and intestine, while LcLEAP-2C transcripts showed obvious lower mRNA levels in all tested tissues compared to LcLEAP-2A. Upon infection by Vibrio alginolyticus, LcLEAP-2A transcripts were significantly up-regulated in liver, trunk kidney, spleen, head kidney, and gill, but down-regulated in intestine. In addition, significant up-regulation of LcLEAP-2C transcripts were also detected in all tissues tested, including intestine. The LcLEAP-2A and LcLEAP-2C mature peptides were chemically synthesized and found to exhibit selective antimicrobial activity in vitro against various species of bacteria. LcLEAP-2C, but not LcLEAP-2A, had antimicrobial activity against V. alginolyticus. Moreover, LcLEAP-2C treatment at low concentrations was evaluated and found to improve survival rate in V. alginolyticus-infected large yellow croaker, resulting in a decrease in bacterial load and expression of inflammatory cytokines. These results suggest that LcLEAP-2 isoforms play an important role in innate immunity by killing bacteria and inhibiting early inflammatory response in large yellow croaker.