Antimicrobial Protein LECT2-b Helps Maintain Gut Microbiota Homeostasis via Selectively Targeting Certain Pathogenic Bacteria.

Antimicrobial peptides/proteins (AMPs) constitute a critical component of gut immunity in animals, protecting the gut from pathogenic bacteria. However, the interactions between AMPs and gut microbiota remain elusive. In this study, we show that leukocyte-derived chemotaxin-2 (LECT2)-b, a recently discovered AMP, helps maintain gut homeostasis in grass carp (Ctenopharyngodon idella), one of the major farmed fish species globally, by directly regulating the gut microbiota. Knockdown of LECT2-b resulted in dysregulation of the gut microbiota. Specifically, LECT2-b deficiency led to the dominance of Proteobacteria, consisting of proinflammatory bacterial species, over Firmicutes, which includes anti-inflammatory bacteria. In addition, the opportunistic pathogenic bacteria genus Aeromonas became the dominant genus replacing the probiotic bacteria Lactobacillus and Bacillus. Further analysis revealed that this effect was due to the direct and selective inhibition of certain pathogenic bacterial species by LECT2-b. Moreover, LECT2-b knockdown promoted biofilm formation by gut microbiota, resulting in tissue damage and inflammation. Importantly, LECT2-b treatment alleviated the negative effects induced by LECT2-b knockdown. These findings highlight the crucial role of LECT2-b in maintaining the gut microbiota homeostasis and mucosal health. Overall, our study provides important data for understanding the roles of AMPs in the regulation of gut homeostasis in animals.

Phagocytic Plasma Cells in Teleost Fish Provide Insights into the Origin and Evolution of B Cells in Vertebrates.

Teleost IgM+ B cells can phagocytose, like mammalian B1 cells, and secrete Ag-specific IgM, like mammalian B2 cells. Therefore, teleost IgM+ B cells may have the functions of both mammalian B1 and B2 cells. To support this view, we initially found that grass carp (Ctenopharyngodon idella) IgM+ plasma cells (PCs) exhibit robust phagocytic ability, akin to IgM+ naive B cells. Subsequently, we sorted grass carp IgM+ PCs into two subpopulations: nonphagocytic (Pha-IgM+ PCs) and phagocytic IgM+ PCs (Pha+IgM+ PCs), both of which demonstrated the capacity to secrete natural IgM with LPS and peptidoglycan binding capacity. Remarkably, following immunization of grass carp with an Ag, we observed that both Pha-IgM+ PCs and Pha+IgM+ PCs could secrete Ag-specific IgM. Furthermore, in vitro concatenated phagocytosis experiments in which Pha-IgM+ PCs from an initial phagocytosis experiment were sorted and exposed again to beads confirmed that these cells also have phagocytic capabilities, thereby suggesting that all teleost IgM+ B cells have phagocytic potential. Additionally, we found that grass carp IgM+ PCs display classical phenotypic features of macrophages, providing support for the hypothesis that vertebrate B cells evolved from ancient phagocytes. These findings together reveal that teleost B cells are a primitive B cell type with functions reminiscent of both mammalian B1 and B2 cells, providing insights into the origin and evolution of B cells in vertebrates.

An Atlas of Grass Carp IgM+ B Cells in Homeostasis and Bacterial Infection Helps to Reveal the Unique Heterogeneity of B Cells in Early Vertebrates.

Teleost B cells are primitive lymphocytes with both innate and adaptive immune functions. However, the heterogeneity and differentiation trajectory of teleost B cells remain largely unknown. In this study, the landscape of grass carp IgM+ (gcIgM+) B cells was revealed by single-cell RNA sequencing. The results showed that gcIgM+ B cells mainly comprise six populations: (im)mature B cells, innate B cells, proliferating B cells, plasma cells, CD22+ cells, and CD34+ cells, among which innate B cells and proliferating B cells were uncommon B cell subsets with, to our knowledge, new characteristics. Remarkably, three functional IgMs were discovered in grass carp, and a significant percentage of gcIgM+ B cells, especially plasma cells, expressed multiple Igµ genes (Igµ1, Igµ2, and/or Igµ3). More importantly, through single-cell sorting combined with Sanger sequencing, we found that distinct VHDJH recombination patterns of Igµ genes were present in single IgM+ B cells, indicating that individual teleost B cells might produce multiple Abs by coexpressing rearranged IgM subclass genes. Moreover, the percentage of IgM1highIgM2highIgM3high plasma cells increased significantly after bacterial infection, suggesting that individual plasma cells might tend to produce multiple IgMs to resist the infection in teleost fish. In summary, to our knowledge, this study not only helps to uncover the unique heterogeneity of B cells in early vertebrates but also provided significant new evidence supporting the recently proposed "one cell-multiple Abs" paradigm, challenging the classical rule of "one cell-one Ab."

Identification and functional analyses of CD4-1+ cells in grass carp (Ctenopharyngodon idella).

In mammals, CD4 is found to be expressed on T cells and innate immune cells, however, teleost cells bearing CD4 have not been well identified and characterized. In this study, we identified two different CD4-1+ cell subsets in grass carp (Ctenopharyngodon idella): CD4-1+ lymphocytes (Lym) and CD4-1+ myeloid cells (Mye), both of which had the highest proportions in the head kidney. The mRNA expression analysis showed that CD4-1, CD4-2, TCRß, CD3γ/δ, and LCK1 are highly expressed in CD4-1+ Lym and also expressed in CD4-1+ Mye. Furthermore, we found that CD4-1+ Lym have a Lym morphology and highly express T-cell cytokines, suggesting that they are CD4+ T cells equivalent to mammalian Th cells. On the other hand, CD4-1+ Mye were found to have a morphology of macrophage and highly express macrophage marker gene MCSFR, indicating that they are macrophages. In addition, functional analysis revealed that CD4-1+ Mye possess phagocytic ability and great antigen-processing ability. Taken together, our study sheds further light on the composition and function of CD4+ cells in teleost fish.

Insights into the Antibacterial Properties of Complement Peptides C3a, C4a, and C5a across Vertebrates.

Complement peptides C3a, C4a, and C5a are important components of innate immunity in vertebrates. Although they diverged from a common ancestor, only C3a and C4a can act as antibacterial peptides in Homo sapiens, suggesting that C5a has evolved into a purely chemotactic molecule; however, the antibacterial properties of C3a, C4a, and C5a across vertebrates still require elucidation. In this article, we show that, unlike those in H. sapiens, Mus musculus C3a, C4a, and C5a all possess antibacterial activities, implying that the antibacterial properties of C3a, C4a, and C5a have evolved divergently in vertebrates. The extremely different net charge, a key factor determining the antibacterial activities of cationic antimicrobial peptides, of vertebrate C3a, C4a, and C5a supports this speculation. Moreover, the antibacterial activity of overlapping peptides covering vertebrate C3a, C4a, and C5a further strongly supports the speculation, because their activity is positively correlated with the net charge of source molecules. Notably, the structures of C3a, C4a, and C5a are conserved in vertebrates, and the inactive overlapping peptides can become antibacterial peptides if mutated to possess enough net positive charges, indicating that net charge is the only factor determining the antibacterial properties of vertebrate C3a, C4a, and C5a. More importantly, many vertebrate C3a-, C4a-, and C5a-derived peptides possess high antibacterial activities yet exhibit no hemolytic activities, suggesting the application potential in anti-infective therapy. Taken together, our findings reveal that vertebrate C3a, C4a, and C5a are all sources of antibacterial peptides that will facilitate the design of excellent peptide antibiotics.

Insights into the Antibacterial Properties of Complement Peptides C3a, C4a, and C5a across Vertebrates.

Complement peptides C3a, C4a, and C5a are important components of innate immunity in vertebrates. Although they diverged from a common ancestor, only C3a and C4a can act as antibacterial peptides in Homo sapiens, suggesting that C5a has evolved into a purely chemotactic molecule; however, the antibacterial properties of C3a, C4a, and C5a across vertebrates still require elucidation. In this article, we show that, unlike those in H. sapiens, Mus musculus C3a, C4a, and C5a all possess antibacterial activities, implying that the antibacterial properties of C3a, C4a, and C5a have evolved divergently in vertebrates. The extremely different net charge, a key factor determining the antibacterial activities of cationic antimicrobial peptides, of vertebrate C3a, C4a, and C5a supports this speculation. Moreover, the antibacterial activity of overlapping peptides covering vertebrate C3a, C4a, and C5a further strongly supports the speculation, because their activity is positively correlated with the net charge of source molecules. Notably, the structures of C3a, C4a, and C5a are conserved in vertebrates, and the inactive overlapping peptides can become antibacterial peptides if mutated to possess enough net positive charges, indicating that net charge is the only factor determining the antibacterial properties of vertebrate C3a, C4a, and C5a. More importantly, many vertebrate C3a-, C4a-, and C5a-derived peptides possess high antibacterial activities yet exhibit no hemolytic activities, suggesting the application potential in anti-infective therapy. Taken together, our findings reveal that vertebrate C3a, C4a, and C5a are all sources of antibacterial peptides that will facilitate the design of excellent peptide antibiotics.

LECT2 Is a Novel Antibacterial Protein in Vertebrates.

In vertebrates, leukocyte-derived chemotaxin-2 (LECT2) is an important immunoregulator with conserved chemotactic and phagocytosis-stimulating activities to leukocytes during bacterial infection. However, whether LECT2 possesses direct antibacterial activity remains unknown. In this article, we show that, unlike tetrapods with a single LECT2 gene, two LECT2 genes exist in teleost fish, named LECT2-a and LECT2-b Using grass carp as a research model, we found that the expression pattern of grass carp LECT2-a (gcLECT2-a) is more similar to that of LECT2 in tetrapods, while gcLECT2-b has evolved to be highly expressed in mucosal immune organs, including the intestine and skin. Interestingly, we found that gcLECT2-b, with conserved chemotactic and phagocytosis-stimulating activities, can also kill Gram-negative and Gram-positive bacteria directly in a membrane-dependent and a non-membrane-dependent manner, respectively. Moreover, gcLECT2-b could prevent the adherence of bacteria to epithelial cells through agglutination by targeting peptidoglycan and lipoteichoic acid. Further study revealed that gcLECT2-b can protect grass carp from Aeromonas hydrophila infection in vivo, because it significantly reduces intestinal necrosis and tissue bacterial load. More importantly, we found that LECT2 from representative tetrapods, except human, also possesses direct antibacterial activities, indicating that the direct antibacterial property of LECT2 is generally conserved in vertebrates. Taken together, to our knowledge, our study discovered a novel function of LECT2 in the antibacterial immunity of vertebrates, especially teleost fish, greatly enhancing our knowledge of this important molecule.

Preparation of the monoclonal antibody against Nile tilapia Igλ and study on the Igλ+ B cell subset in Nile tilapia.

Immunoglobulins (Igs) are important effector molecules that mediate humoral immunity. A typical Ig consists of two heavy and two light chains. In teleosts, three Ig heavy chain isotypes (Igµ, Igδ and Igτ) and three Ig light chain isotypes (Igκ, Igλ and Igσ) have been identified. Compared to the heavy chains, teleost Ig light chains have been poorly studied due to the lack of antibodies. In this study, a mouse anti-Nile tilapia Igλ monoclonal antibody (mAb) was prepared, which could specifically recognize Igλ in serum and Igλ+ B cells in tissues. Further, the composition of IgM+ and Igλ+ B cell subsets was analyzed using this antibody and a mouse anti-tilapia IgM heavy chain mAb. The ratio of IgM+Igλ+ B cells to total IgM+ B cells in head kidney and peripheral blood was about 30%, while that in spleen was about 50%; the ratio of IgM-Igλ+ B cells to total Igλ+ B cells in head kidney and peripheral blood was about 45%, while that in spleen was about 25%. The IgM-Igλ+ B cells was speculated to be IgT+ B cells. Finally, we detected an increase in the level of specific antibodies against the surface antigen-Sip of Streptococcus agalactiae in serum after S. agalactiae infection, indicating that mouse anti-tilapia Igλ mAb can be used to detect the antibody level after immunization of Nile tilapia, which lays a foundation for the evaluation of immunization effect of tilapia vaccine.

Combination of canagliflozin and puerarin alleviates the lipotoxicity to diabetic kidney in mice.

Diabetic kidney disease is one of the most serious complications of diabetes. Although diabetic kidney disease can be effectively controlled through strict blood glucose management and corresponding symptomatic treatment, these therapies cannot reduce its incidence in diabetic patients. The sodium-glucose cotransporter 2 (SGLT2) inhibitors and the traditional Chinese herb "Gegen" have been widely used in diabetes-related therapy. However, it remains unclear whether the combined use of these two kinds of medicines contributes to an increased curative effect on diabetic kidney disease. In this study, we examined this issue by evaluating the efficacy of the combination of puerarin, an active ingredient of Gegen, and canagliflozin, an SGLT2 inhibitor for a 12-week intervention using a mouse model of diabetes. The results indicated that the combination of puerarin and canagliflozin was superior to canagliflozin alone in improving the metabolic and renal function parameters of diabetic mice. Our findings suggested that the renoprotective effect of combined puerarin and canagliflozin in diabetic mice was achieved by reducing renal lipid accumulation. This study provides a new strategy for the clinical prevention and treatment of diabetic kidney disease. The puerarin and SGLT2 inhibitor combination therapy at the initial stage of diabetes may effectively delay the occurrence of diabetic kidney injury, and significantly alleviate the burden of renal lipotoxicity.

Chromosome-level genome assembly of grass carp (Ctenopharyngodon idella) provides insights into its genome evolution.

BACKGROUND: The grass carp has great economic value and occupies an important evolutionary position. Genomic information regarding this species could help better understand its rapid growth rate as well as its unique body plan and environmental adaptation. RESULTS: We assembled the chromosome-level grass carp genome using the PacBio sequencing and chromosome structure capture technique. The final genome assembly has a total length of 893.2 Mb with a contig N50 of 19.3 Mb and a scaffold N50 of 35.7 Mb. About 99.85% of the assembled contigs were anchored into 24 chromosomes. Based on the prediction, this genome contained 30,342 protein-coding genes and 43.26% repetitive sequences. Furthermore, we determined that the large genome size can be attributed to the DNA-mediated transposable elements which accounted for 58.9% of the repetitive sequences in grass carp. We identified that the grass carp has only 24 pairs of chromosomes due to the fusion of two ancestral chromosomes. Enrichment analyses of significantly expanded and positively selected genes reflected evolutionary adaptation of grass carp to the feeding habits. We also detected the loss of conserved non-coding regulatory elements associated with the development of the immune system, nervous system, and digestive system, which may be critical for grass carp herbivorous traits. CONCLUSIONS: The high-quality reference genome reported here provides a valuable resource for the genetic improvement and molecular-guided breeding of the grass carp.

Supramolecular Polymerization in Liquid Crystalline Media: Toward Modular Synthesis of Multifunctional Core-Shell Columnar Liquid Crystals.

Recently, we discovered a modular synthetic approach for constructing core-shell columnar liquid crystals (LCs) by supramolecular polymerization in LC media. In the present work, we successfully confirmed that our modular synthetic approach has the potential to be widely extended to the development of multifunctional columnar LCs. Herein, we constructed the first core-shell columnar LC that was proved to be orientable by both electric and magnetic fields by the supramolecular polymerization of NODiskNH* in a nematic LC medium of 4-cyano-4'-pentyloxybiphenyl (5OCB). NODiskNH* is a chiral benzenetricarboxamide derivative bearing 2,2,6,6-tetramethylpiperidine 1-oxyl termini, which is known to form a helical supramolecular polymer via a triple hydrogen-bonding array. NODiskNH* alone formed a hydrogen-bonded liquid phase without any long-range structural ordering. However, a nematic LC medium of 5OCB, when mixed with NODiskNH* at a molar ratio of 1:3, underwent a "structural order-increasing" mesophase transition, affording an optically active single LC phase with a hexagonally arranged core-shell columnar geometry in a temperature range from 113 to 51 °C. Unprecedentedly, this core-shell columnar LC can orient its columns both electrically and magnetically, resulting in unidirectional columnar ordering.

Molecular and functional characterization of the indoleamine 2,3-dioxygenase in grass carp (Ctenopharyngodon idella).

Indoleamine 2,3-dioxygenase (IDO) is a kind of dioxygenase that can catalyze the degradation of levo-tryptophan (L-Trp) and plays key roles in immune tolerance. In this study, the IDO gene was cloned and functionally characterized from grass carp (gcIDO). The results showed that gcIDO overexpressed in GCO cells could catalyze the degradation of L-Trp through the L-Trp - kynurenine pathway, and this activity could be promoted by δ-aminolevulinic acid (ALA) while inhibited by levo-1-methyl tryptophan (L-1MT). Moreover, gcIDO was constitutively expressed in various tissues, and its expression could be significantly up-regulated by LPS and Poly (I:C) in peripheral blood leukocytes (PBLs). Furthermore, recombinant TGF-ß1 of grass carp could up-regulate the expression of IDO, TGF-ß1, CD25, and Foxp3 in PBLs, indicating that the TGF-ß1/IDO pathway is present in fish. In the soybean meal induced enteritis (SBMIE) model, the expression of gcIDO in the intestine was up-regulated significantly, demonstrating that gcIDO may play an immunoregulatory role in SBMIE. Taken together, these data suggest that the IDO plays multiple roles in the immunity of fish.

Spring Viremia of Carp Virus N Protein Suppresses Fish IFNφ1 Production by Targeting the Mitochondrial Antiviral Signaling Protein.

For a virus to replicate efficiently, it must try and inhibit host IFN expression because IFN is an important host defense at early stages after viral infection. For aquatic viruses, the mechanisms used to escape the hosts IFN system are still unclear. In this study, we show that the N protein of spring viremia of carp virus (SVCV) inhibits zebrafish IFNφ1 production by degrading the mitochondrial antiviral signaling protein (MAVS). First, the upregulation of IFNφ1 promoter activity stimulated by polyinosinic:polycytidylic acid, retinoic acid-inducible gene I (RIG-I) or MAVS was suppressed by the SVCV infection. However, the upregulation by the downstream factor of the RIG-I-like receptor signaling pathway, TANK-binding kinase 1, was not affected. Notably, at the protein level, MAVS decreased remarkably when cells were infected with SVCV. Second, consistent with the result of the SVCV infection, overexpression of the N protein of SVCV blocked the IFNφ1 transcription activated by MAVS and downregulated MAVS expression at the protein level but not at the mRNA level. Further analysis demonstrated that the N protein targeted MAVS for K48-linked ubiquitination, which promoted the degradation of MAVS. These data indicated that fish MAVS could be degraded by the N protein of SVCV through the ubiquitin-proteasome pathway. To our knowledge, this is the first article of a fish RIG-I-like receptor pathway interfered by an aquatic virus in an ubiquitin-proteasome manner, suggesting that immune evasion of a virus also exists in lower vertebrates.

Distinctive structural hallmarks and biological activities of the multiple cathelicidin antimicrobial peptides in a primitive teleost fish.

Cathelicidin antimicrobial peptides (CAMPs) represent a crucial component of the innate immune system in vertebrates. Although widely studied in mammals, little is known about the structure and function of fish CAMPs. Further to the previous findings, two more cathelicidin genes and multiple transcripts from rainbow trout were identified in the present study. Interestingly, we found that trout have evolved energy-saving forms of cathelicidins with the total deletion of the characteristic cathelin-like domain. Sequence analysis revealed that salmonid CAMPs have formed a special class of antimicrobial peptides in vertebrates with three distinctive hallmarks: the N terminus is intensified by positive charges, the central region consists of repetitive motifs based on RPGGGS, and the C terminus is lowly charged. Immunofluorescence localization of trout CAMPs demonstrated that these peptides expressed mainly at the mucosal layer of gut. Meanwhile, signals around sinusoids were also detected in head kidney. Moreover, the biological activities of trout CAMPs were proved to be mediated by the N terminus. Additionally, the repetitive motifs characteristically existing in Salmonidae increased the structural flexibilities of peptides and further increased the antibacterial and IL-8-stimulating activities. Unlike most α helical and cytotoxic mammalian CAMPs, trout CAMPs, mainly consisting of ß-sheet and random coil, exhibited no cytotoxic activities. The distinctive structural features of trout CAMPs provide new insights into the understanding of the evolution of CAMPs in vertebrates. Moreover, the high bacterial membrane selectivity of trout CAMPs will help to design excellent peptide antibiotics.

Identification and characterization of Bacillus subtilis from grass carp (Ctenopharynodon idellus) for use as probiotic additives in aquatic feed.

Bacillus subtilis is widely used as probiotic species in aquaculture for water quality control, growth promoting, or immunity enhancing. The aim of this study is to find novel B. subtilis strains from fish as potential probiotics for aquaculture. Eleven B. subtilis isolates derived from the intestinal tract of grass carp were identified by gene sequencing and biochemical tests. These isolates were classified into 4 groups, and the representatives (GC-5, GC-6, GC-21 and GC-22) of each group were further investigated for antibiotic susceptibility, sporulation rate, biofilm formation, activity against pathogenic bacteria, resistance to stress conditions of intestinal tract (high percentage of bile and low pH) and high temperature, which are important for probiotics to be used as feed additives. Additionally, the adhesion properties of the 4 characterized strains were assessed using Caco-2 cell and gut mucus models. The results showed that the 4 strains differed in their capacities to adhere to intestinal epithelial cells and mucus. Furthermore, the strains GC-21 and GC-22 up-regulated the expression levels of IL-10 and TGF-ß but down-regulated IL-1ß, suggesting their potential anti-inflammatory abilities. Based on physiological properties of the 4 characterized B. subtilis strains, one or more strains may have potential to be used as probiotics in aquaculture.

Investigating the potential immune role of fish NCAMs: Molecular cloning and expression analysis in mandarin fish.

The immune role of NCAMs has been revealed in mammals, yet there is no such report in fish. Hence, we analyzed the molecular characterizations and immune-associated expression patterns of NCAMs in mandarin fish. Three NCAM members, named mfNCAM1a, mfNCAM1b and mfNCAM2, were identified. Among the cDNA sequences of mfNCAMs, AU-rich elements in the 3' UTRs of mfNCAM1b and mfNCAM2 as well as VASE sequences in the fourth Ig-like domain-encoding regions of mfNCAM1a and mfNCAM1b were discovered. Moreover, the syntenic analysis suggested that the duplication of NCAM1 is fish-specific. At mRNA and protein levels, the expression analyses revealed that mfNCAMs existed in both systemic and mucosal immune tissues, and located within lymphoid cells. Upon stimulated either by LPS or poly I:C, the expression level of mfNCAM1a was significantly up-regulated in head kidney, spleen, liver, and gut, whereas mfNCAM1b only in head kidney and liver, and mfNCAM2 only in liver. Additionally, the cells coexpressed mfNCAM1 and mfNCCRP-1 might imply the equivalents to mammalian NK cells. Our finding firstly demonstrates the member-specific immune-related tissue expression pattern and immune activity for fish NCAMs. Current data indicate that mfNCAM2 has little immune activity, while the immune activity of mfNCAM1a exists in more tissues than mfNCAM1b, and mfNCAM1a may tend to respond more actively to viral while mfNCAM1b to bacterial stimulants. Additionally, NCAM1b should be a fish-specific member with unique immune function, judging from its different expression pattern, immune activity as well as phylogenetic relationship to mfNCAM1a.

Supramolecular copolymerization of hydrophobic and hydrophilic monomers in liquid crystalline media.

In the case of covalent polymers, immiscible polymers can be integrated by covalently linking them together, but such a strategy is not possible in supramolecular polymers. Here we report the supramolecular copolymerization of two porphyrin-based monomers, C10P2H and TEGPCu with side chains bearing cyanobiphenyl (CB) groups at the ends of hydrophobic alkyl or hydrophilic tetraethylene glycol chains, respectively. These monomers undergo self-sorting supramolecular polymerization in highly diluted solutions ([monomer] = 3.4 × 10-9 mol% (2.0 × 10-8 mol L-1)) in nonpolar media due to the incompatibility of the side chains. Surprisingly, these monomers undergo supramolecular copolymerization under high concentration conditions ([monomer] = 7.7 mol%) in the medium of 4-cyano-4'-pentyloxybiphenyl (5OCB) to form a columnar liquid crystalline phase under thermodynamic conditions, where the individual columns are composed of supramolecular block copolymers. The combination of CB ends of both monomers and the 5OCB medium is essential for the two monomers to form an integrated structure in a condensed system without phase separation.

Comparative study on antibacterial characteristics of the multiple liver expressed antimicrobial peptides (LEAPs) in teleost fish.

Antimicrobial peptides are important components of the host innate immune system, forming the first line of defense against infectious microorganisms. Among them, liver-expressed antimicrobial peptides (LEAPs) are a family of antimicrobial peptides that widely exist in vertebrates. LEAPs include two types, named LEAP-1 and LEAP-2, and many teleost fish have two or more LEAP-2s. In this study, LEAP-2C from rainbow trout and grass carp were discovered, both of which are composed of 3 exons and 2 introns. The antibacterial functions of the multiple LEAPs were systematically compared in rainbow trout and grass carp. The gene expression pattern revealed that rainbow trout and grass carp LEAP-1, LEAP-2A, LEAP-2B and/or LEAP-2C were differentially expressed in various tissues/organs, mainly in liver. After bacterial infection, the expression levels of LEAP-1, LEAP-2A, LEAP-2B and/or LEAP-2C in the liver and gut of rainbow trout and grass carp increased to varying degrees. Moreover, the antibacterial assay and bacterial membrane permeability assay showed that rainbow trout and grass carp LEAP-1, LEAP-2A, LEAP-2B and LEAP-2C all have antibacterial activities against a variety of Gram-positive and Gram-negative bacteria with varying levels through membrane rupture. Furthermore, cell transfection assay showed that only rainbow trout LEAP-1, but not LEAP-2, can lead to the internalization of ferroportin, the only iron exporter on cell surface, indicating that only LEAP-1 possess iron metabolism regulation activity in teleost fish. Taken together, this study systematically compared the antibacterial function of LEAPs in teleost fish and the results suggest that multiple LEAPs can enhance the immunity of teleost fish through different expression patterns and different antibacterial activities to various bacteria.

Plasmablasts induced by chitosan oligosaccharide secrete natural IgM to enhance the humoral immunity in grass carp.

Chitosan oligosaccharide (COS) is an attractive immunopotentiator capable of driving humoral immunity in vertebrates, but its cellular and molecular mechanisms still require elucidation. In this study, COS induced the proliferation and differentiation of splenic IgM+ B cells into IgMlo and IgMhi B cell subsets in grass carp (Ctenopharyngodon idella). The IgMlo B cells were further identified as short-lived plasmablasts that secreted natural IgM with binding-abilities to lipopolysaccharide (LPS) and peptidoglycan (PGN). Moreover, the mannose receptor (MR) and integrins were discovered and identified as the binding-receptors of COS on IgMlo plasmablasts. The MR synergized with integrins to trigger intracellular signal transduction to boost plasmablast generation and expansion. Notably, IgMlo plasmablasts originally generated in spleen but they migrated into blood to secrete natural IgM, which augmented the serum bactericidal activity. Taken together, this study revealed the cellular and molecular mechanisms of COS-triggered humoral immunity in fish.

Anomalous Chiral Transfer: Supramolecular Polymerization in a Chiral Medium of a Mesogenic Molecule.

Here, we report a medium-to-polymer anomalous chiral transfer in the supramolecular polymerization of a tetraphenylporphyrin-based achiral hydrogen-bonding monomer (TPP) in a chiral medium of 5-cyanobiphenyl CB*. A mixture of TPP in (R)-CB* ([TPP]=7.7 mol %) at 40 °C gave a columnar oblique LC mesophase, where the individual columns were composed of an optically active helical supramolecular polymer of TPP as a consequence of a successful medium-to-polymer chiral transfer. Meanwhile, upon dilution of CB* with achiral 5-cyanobiphenyl CB, the optical activity of the system showed an anomalous bell-shaped dependency on the composition of CB*/CB, where the gabs value of 0.049 at CB*/CB=50/50 was 6.0 times larger than the gabs value of CB* alone. Such anomalous chiroptical amplification in CD is most likely due to a change in the stacking geometry of TPP in the oblique columnar LC upon lateral compression.