The growth inhibitory effects and non-targeted metabolomic profiling of Microcystis aeruginosa treated by Scenedesmus sp.

The health of the aquatic ecosystem has recently been severely affected by cyanobacterial blooms brought on by eutrophication. Therefore, it is critical to develop efficient and secure methods to control dangerous cyanobacteria, such as Microcystis aeruginosa. In this research, we tested the inhibition of M. aeruginosa growth by a Scenedesmus sp. strain isolated from a culture pond. Scenedesmus sp. culture filtrate that had been lyophilized was added to M. aeruginosa, and cultivation for seven days, the cell density, chlorophyll a (Chl-a) concentration, maximum quantum yield of photosystem II (Fv/Fm), the activities of superoxide dismutase (SOD), catalase (CAT), and the concentration of malondialdehyde (MDA) and glutathione (GSH) were measured. Moreover, non-targeted metabolomics was carried out to provide light on the inhibitory mechanism in order to better understand the metabolic response. According to the results, M. aeruginosa is effectively inhibited by the lyophilized Scenedesmus sp. culture filtrate at a rate of 51.2%. Additionally, the lyophilized Scenedesmus sp. clearly inhibit the photosystem and damages the antioxidant defense system of M. aeruginosa cells, resulting in oxidative damage, which worsens membrane lipid peroxidation, according to changes in Chl-a, Fv/Fm, SOD, CAT enzyme activities and MDA, GSH. Metabolomics analysis revealed that the secondary metabolites of Scenedesmus sp. significantly interfere with the metabolism of M. aeruginosa involved in amino acid synthesis, membrane creation and oxidative stress, which is coherent with the morphology and physiology outcomes. These results demonstrate that the secondary metabolites of Scenedesmus sp. exert algal inhibition effect by breaked the membrane structure, destroyed the photosynthetic system of microalgae, inhibited amino acid synthesis, reduced antioxidant capacity, and eventually caused algal cell lysis and death. Our research provides a reliable basis for the biological control of cyanobacterial blooms on the one hand, and on other hand supply application of non-targeted metabolome on the study of microalgae allelochemicals.

Molecular characterization of peptidoglycan recognition proteins from Mytilus coruscus.

Mytilus shows great immune resistance to various bacteria from the living waters, indicating a complex immune recognition mechanism against various microbes. Peptidoglycan recognition proteins (PGRPs) play an important role in the defense against invading microbes via the recognition of the immunogenic substance peptidoglycan (PGN). Therefore, eight PGRPs were identified from the gill transcriptome of Mytilus coruscus. The sequence features, expression pattern in various organs and larval development stages, and microbes induced expression profiles of these Mytilus PGRPs were determined. Our data revealed the constitutive expression of PGRPs in various organs with relative higher expression level in immune-related organs. The expression of PGRPs is developmentally regulated, and most PGRPs are undetectable in larvae stages. The expression level of most PGRPs was significantly increased with in vivo microbial challenges, showing strong response to Gram-positive strain in gill and digestive gland, strong response to Gram-negative strain in hemocytes, and relative weaker response to fungus in the three tested organs. In addition, the function analysis of the representative recombinant expressed PGRP (rMcPGRP-2) confirmed the antimicrobial and agglutination activities, showing the immune-related importance of PGRP in Mytilus. Our work suggests that Mytilus PGRPs can act as pattern recognition receptors to recognize the invading microorganisms and the antimicrobial effectors during the innate immune response of Mytilus.

Nifuroxazide in combination with CpG ODN exerts greater efficacy against hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a common malignant tumour in China that remains a major challenge to the medical community, and effective treatment is urgently needed. Due to complex tumorigenesis, monotherapy shows poor therapeutic effects, and combined treatment becomes a necessary option. YW002, a CpG ODN-containing sequence, has been proven to enhance antitumor effects in tumour-bearing mouse models. Moreover, as a broad-spectrum antimicrobial drug, nifuroxazide exhibited an anti-HCC effect through activation of p-Stat3. Here, we tested the effect of nifuroxazide on HCC in vitro and then explored the therapeutic effect of combined nifuroxazide and CpG ODN on HCC in vivo. Nifuroxazide inhibited proliferation, induced apoptosis and suppressed migration and invasion in HepG2 cells in vitro. The combination therapy using nifuroxazide and CpG ODN significantly suppressed the growth of tumours in tumour-bearing mice with few side effects and achieved better therapeutic effects on HCC than monotherapy. Moreover, combined nifuroxazide and CpG ODN therapy significantly induced apoptosis, enhanced the infiltration of CD4+ and CD8+ T lymphocytes and macrophages in tumour tissue, and increased the ratio of CD4+ and CD8+ T lymphocytes in the spleens of tumour-bearing mice. The introduction of this combination therapy combining nifuroxazide and CpG ODN provided a new strategy for HCC treatment.

A novel polypeptide vaccine and adjuvant formulation of EV71.

Hand foot and mouth disease (HFMD) is an infectious disease mainly caused by Enterovirus 71 (EV 71). However, the effective treatment is limited currently. The aim of this study was to investigate the activity of the vaccine including the EV71 polypeptides mixed with a novel adjuvant containing CpG oligodeoxynucleotides (CpG ODNs). After collecting mouse sera, we determined the antibody concentration in serum by enzyme-linked immunosorbent assays (ELISA). Then, CD19+CD27+ B cells in the spleen were analysed by flow cytometry. The assay revealed that a substantial increase in antibody titers was achieved. This indicates a high level of immunogenicity for peptide vaccine and the good stability of adjuvant, also suggests that the combination of vaccine and adjuvant can stimulate the production of high-level antibodies and CD19+CD27+ B lymphocytes in mice. Furthermore, the antibody could effectively identify EV71 inactivated virus. The results demonstrated that the autonomous construction of EV71 polypeptide vaccine had a good immunogenicity. Moreover, the peptide vaccine injection with a novel adjuvant, which is easy to prepare, could cause a high antibody level of EV71 and shown a good application prospect.

Molecular Characterization of a Novel Shell Matrix Protein With PDZ Domain From Mytilus coruscus.

Mollusk shells are products of biomineralization and possess excellent mechanical properties, and shell matrix proteins (SMPs) have important functions in shell formation. A novel SMP with a PDZ domain (PDZ-domain-containing-protein-1, PDCP-1) was identified from the shell matrices of Mytilus coruscus. In this study, the gene expression, function, and location of PDCP-1 were analyzed. PDCP-1 was characterized as an ∼70 kDa protein with a PDZ (postsynaptic density/discs large/zonula occludes) domain and a ZM (ZASP-like motif) domain. The PDCP-1 gene has a high expression level and specific location in the foot, mantle and adductor muscle. Recombinantly expressed PDCP-1 (rPDCP-1) altered the morphology of calcite crystals, the polymorph of calcite crystals, binding with both calcite and aragonite crystals, and inhibition of the crystallization rate of calcite crystals. In addition, anti-rPDCP-1 antibody was prepared, and immunohistochemistry and immunofluorescence analyses revealed the specific location of PDCP-1 in the mantle, the adductor muscle, and the aragonite (nacre and myostracum) layer of the shell, suggesting multiple functions of PDCP-1 in biomineralization, muscle-shell attachment, and muscle attraction. Furthermore, pull-down analysis revealed 19 protein partners of PDCP-1 from the shell matrices, which accordingly provided a possible interaction network of PDCP-1 in the shell. These results expand the understanding of the functions of PDZ-domain-containing proteins (PDCPs) in biomineralization and the supramolecular chemistry that contributes to shell formation.

Recombinant transgelin-like protein 1 from Mytilus shell induces formation of CaCO3 polymorphic crystals in vitro.

Transgelin is an actin cross-linking/gelling protein of the calponin family, which is associated with actin stress fibres, cell motility, adhesion and the maintenance of cell morphology. Transgelin-like proteins (TLPs) have also been identified as shell matrix proteins (SMPs) in several mollusc species; however, the functions of TLPs in biomineralization remain unknown. Transgelin-like protein 1 (TLP-1) was previously identified from the shell of Mytilus coruscus as a novel 19 kDa SMP with a calponin homology (CH) domain. To understand the role of TLP-1 in shell formation, the expression level and localization of the TLP-1 gene in biomineralization-related tissues were determined in this study. Furthermore, recombinant TLP-1 was expressed in a prokaryotic expression system with codon optimization, and an anti-rTLP-1 antibody was prepared based on the expressed recombinant TLP-1 (rTLP-1) protein. In vitro, rTLP-1 induced the formation of CaCO3 polymorphic crystals with distinct morphologies and inhibited crystallization rate and crystal interactions. Immunohistochemical, immunofluorescence, and pull-down analyses using the anti-rTLP-1 antibody revealed the specific locations of TLP-1 in biomineralization-related tissues and shell myostracum layer, and suggested the existence of a possible TLP-1 interaction network in the shell matrix. Our results are beneficial for understanding the functions of TLP-1, particularly through its CH domain, during shell mineralization.

Characterization of a novel shell matrix protein with vWA domain from Mytilus coruscus.

Mollusk shell is a product of biomineralization with excellent mechanical properties, and the shell matrix proteins (SMPs) have important functions in shell formation. A vWA domain-containing protein (VDCP) was identified from the shell of Mytilus coruscus as a novel shell matrix protein. The VDCP gene is expressed at a high level in specific locations in the mantle and adductor muscle. Recombinant VDCP (rVDCP) showed abilities to alter the morphology of both calcite and aragonite, induce the polymorph change of calcite, bind calcite, and decrease the crystallization rate of calcite. In addition, immunohistochemistry analyses revealed the specific location of VDCP in the mantle, the adductor muscle, and the myostracum layer of the shell. Furthermore, a pull-down analysis revealed eight protein interaction partners of VDCP in shell matrices and provided a possible protein-protein interaction network of VDCP in the shell.

Molecular characterization of a whirlin-like protein with biomineralization-related functions from the shell of Mytilus coruscus.

Mollusc shells are produced from calcified skeletons and have excellent mechanical properties. Shell matrix proteins (SMPs) have important functions in shell formation. A 16.6 kDa whirlin-like protein (WLP) with a PDZ domain was identified in the shell of Mytilus coruscus as a novel SMP. In this study, the expression, function, and location of WLP were analysed. The WLP gene was highly expressed and specifically located in the adductor muscle and mantle. The expression of recombinant WLP (rWLP) was associated with morphological change, polymorphic change, binding ability, and crystallization rate inhibition of the calcium carbonate crystals in vitro. In addition, an anti-rWLP antibody was prepared, and the results from immunohistochemistry and immunofluorescence analyses revealed the specific location of the WLP in the mantle, adductor muscle, and myostracum layer of the shell, suggesting multiple functions for WLP in biomineralization, muscle-shell attachment, and muscle attraction. Furthermore, results from a pull-down analysis revealed 10 protein partners of WLP in the shell matrices and a possible network of interacting WLPs in the shell. In addition, in this study, one of the WLP partners, actin, was confirmed to have the ability to bind WLP. These results expand the understanding of the functions of PDZ-domain-containing proteins in biomineralization and provide clues for determining the mechanisms of myostracum formation and muscle-shell attachment.

Microstructure and in-depth proteomic analysis of Perna viridis shell.

For understanding the structural characteristics and the proteome of Perna shell, the microstructure, polymorph, and protein composition of the adult Perna viridis shell were investigated. The P. viridis shell have two distinct mineral layers, myostracum and nacre, with the same calcium carbonate polymorph of aragonite, determined by scanning electron microscope, Fourier transform infrared spectroscopy, and x-ray crystalline diffraction. Using Illumina sequencing, the mantle transcriptome of P. viridis was investigated and a total of 69,859 unigenes was generated. Using a combined proteomic/transcriptomic approach, a total of 378 shell proteins from P. viridis shell were identified, in which, 132 shell proteins identified with more than two matched unique peptides. Of the 132 shell proteins, 69 are exclusive to the nacre, 12 to the myostracum, and 51 are shared by both. The Myosin-tail domain containing proteins, Filament-like proteins, and Chitin-binding domain containing proteins represent the most abundant molecules. In addition, the shell matrix proteins (SMPs) containing biomineralization-related domains, such as Kunitz, A2M, WAP, EF-hand, PDZ, VWA, Collagen domain, and low complexity regions with abundant certain amino acids, were also identified from P. viridis shell. Collagenase and chitinase degradation can significantly change the morphology of the shell, indicating the important roles of collagen and chitin in the shell formation and the muscle-shell attachment. Our results present for the first time the proteome of P. viridis shell and increase the knowledge of SMPs in this genus.

In-depth proteomic analysis of Boleophthalmus pectinirostris skin mucus.

Fish skin mucus serves as the first line of defence against pathogens and external stressors. The mudskipper Boleophthalmus pectinirostris inhabits intertidal mudflats containing abundant and diverse microbial populations; thus, the skin and mucus of B. pectinirostris are very important for immune defence. However, the molecules involved in the immune response and mucus secretion in the skin of this fish are poorly understood. To explore the proteomic profile of the skin mucus and understand the molecular mechanisms underlying B. pectinirostris adaption to amphibious environments, the microstructure of B. pectinirostris skin was analysed, and a series of histochemical procedures were employed for mucous glycoprotein localization and characterization. In addition, the antibacterial activity of B. pectinirostris skin mucus was studied, and the transcriptome of the skin and in-depth proteome of the mucus were determined. These studies revealed the hierarchical structure of B. pectinirostris skin and different types of glycoproteins (GPs) in the dermal bulge (DB) of the B. pectinirostris skin epidermis. The mucus has a broad antimicrobial spectrum and significant effects on the bacterial morphology. Furthermore, 93,914 unigenes were sequenced from B. pectinirostris skin tissue, and a total of 559 proteins were identified from B. pectinirostris skin mucus. SIGNIFICANCE.

Production and Function of Different Regions from Mytichitin-1 of Mytilus coruscus.

Chitinase is an important enzyme for many physiological processes. Mytichitin-1 is a chitinase-like protein in Mytilus coruscus, and its C-terminal 55-AA fragment (mytichitin-CB) is a novel antimicrobial peptide, suggesting a new immune process in which chitinase is involved; mytichtin-1 may have various forms in the different biological processes of M. coruscus. Thus, the study of mytichitin-1 will be helpful for understanding the mechanism of mussel immune biology and the functional diversity of chitinase. In this study, mytichitin-1 was recombinantly expressed with different lengths, full-length mytichtin-1 (rMchi-F) and the N-terminal region (rMchi-N) in Escherichia coli BL21 with codon optimization. The results of SDS-PAGE, Western blotting, and mass spectrometry confirmed that the two forms of mytichitin-1 had been successfully recombinant expressed with a yield of 40 mg purified enzyme per L culture. In addition, the 55-AA fragment of mytichitin-CB was chemically synthesized (sMchi-CB). After purification and oxidation, the functions of the three protein products were analysed, including chitin degradation, chitin binding, and antimicrobial activities. Both rMchi-F and rMchi-N displayed enzymatic activity with the optimum pH of 4.0 and optimum temperature of 40 °C, and rMchi-N showed a stronger activity than rMchi-F. Enzymatic activities of rMchi-F and rMchi-N were stimulated by the metal ions Fe2+, Ba2+, and Na+ and partially inhibited by Cu2+, Ni2+ and Zn2+. rMchi-F, rMchi-N, and sMchi-CB had the ability to combine with colloid chitin. The antimicrobial activities of these proteins were tested against bacteria and fungi, and the results indicated the strongest activity for sMchi-CB and the weakest activity for rMchi-N. Using a prepared anti-rMchi-F polyclonal antibody, immunohistochemistry and immunoprecipitation were performed and the results revealed the location of mytichitin-1 in mantle, digestive gland and blood cells. In addition, two forms of mytichitin-1, mytichitin-CB (6 kD) and full-length mytichitin-1 (48 kD), were detected, and a 35 kD protein was identified as the third form of mytichitin-1, existing in various tissues of M. coruscus. These findings suggest that mytichitin-1 may play different roles, with at least three forms, in different M. coruscus tissues.

Comparative proteomic analysis of Ulva prolifera response to high temperature stress.

BACKGROUND: Ulva prolifera belongs to green macroalgae and is the dominant species of green tide. It is distributed worldwide and is therefore subject to high-temperature stress during the growth process. However, the adaptation mechanisms of the response of U. prolifera to high temperatures have not been clearly investigated yet. METHODS: In this study, isobaric tags for relative and absolute quantitation (iTRAQ) labelling was applied in combination with the liquid chromatography-tandem mass spectrometry (LC-MS/MS) to conduct comparative proteomic analysis of the response of U. prolifera to high-temperature stress and to elucidate the involvement of this response in adaptation mechanisms. Differentially expressed proteins (DEPs) of U. prolifera under high temperature (denote UpHT) compared with the control (UpC) were identified. Bioinformatic analyses including GO analysis, pathway analysis, and pathway enrichment analysis was performed to analyse the key metabolic pathways that underlie the thermal tolerance mechanism through protein networks. Quantitative real-time PCR and western blot were performed to validate selected proteins. RESULTS: In the present study, 1223 DEPs were identified under high temperature compared with the control, which included 790 up-regulated and 433 down-regulated proteins. The high-temperature stimulus mainly induced the expression of glutathione S-transferase, heat shock protein, ascorbate peroxidase, manganese superoxide dismutase, ubiquitin-related protein, lhcSR, rubisco activase, serine/threonine protein kinase 2, adenylate kinase, Ca2+-dependent protein kinase (CDPK), disease resistance protein EDS1, metacaspase type II, NDPK2a, 26S proteasome regulatory subunit, ubiquinone oxidoreductase, ATP synthase subunit, SnRK2s, and cytochrome P450. The down-regulated proteins were photosynthesis-related proteins, glutathione reductase, catalase-peroxidase, thioredoxin, thioredoxin peroxidase, PP2C, and carbon fixation-related proteins. Furthermore, biological index analysis indicated that protein content and SOD activity decreased; the value of Fv/Fm dropped to the lowest point after culture for 96 h. However, APX activity and MDA content increased under high temperature. CONCLUSION: The present study implied an increase in proteins that were associated with the stress response, oxidative phosphorylation, the cytokinin signal transduction pathway, the abscisic acid signal transduction pathway, and the glutathione metabolism pathway. Proteins that were associated with photosynthesis, carbon fixation in photosynthesis organisms, and the photosynthesis antenna protein pathway were decreased. These pathways played a pivotal role in high temperature regulation. These novel proteins provide a good starting point for further research into their functions using genetic or other approaches. These findings significantly improve the understanding of the molecular mechanisms involved in the tolerance of algae to high-temperature stress.

Integration of deep transcriptome and proteome analyses of salicylic acid regulation high temperature stress in Ulva prolifera.

To investigate changes in transcript and relative protein levels in response to salicylic acid regulation of the thermotolerance in U. prolifera, complementary transcriptome and proteome analyses were performed with U. prolifera grown at 35 °C (UpHT) and with the addition of SA at high temperature (UpSHT). At mRNA level,12,296 differentially expressed genes (DEGs) were obtained from the comparison of UpSHT with UpHT. iTRAQ-labeling proteome analysis showed that a total of 4,449 proteins were identified and reliably quantified. At mRNA level, the up-regulated genes involved in antioxidant activity were thioredoxin,peroxiredoxin,FeSOD, glutathione peroxidase, partion catalase and MnSOD. The down-regulated genes were ascorbate peroxidase, glutathione S-transferase, catalase and MnSOD. In addition, the DEGs involved in plant signal transduction pathway (such as auxin response factors, BRI1 and JAZ) were down-regulated. At protein level, the up-regulated proteins involved in carbon fixation and the down-regulated protein mainly were polyubiquitin, ascorbate peroxidase. The expression of Ca2+-binding protein, heat shock protein and photosynthesis-related proteins, EDS1 were also significantly regulated both at mRNA and protein level. The results indicated that SA alleviated the high-temperature stimulus through partion antioxidant related proteins up-regulated, JA signal pathway enchanced, Ca2+-binding proteins, photosynthesis-related proteins significantly changed, antioxidant enzyme activities increased and photosynthesis index changed.

In-depth proteomic analysis of the byssus from marine mussel Mytilus coruscus.

UNLABELLED: Mussels attach to various submerged surfaces by using the byssus, which contains different proteins and is a promising source of water-resistant bio-adhesives for potential use in biotechnological and medical applications. The protein composition of the byssus has not yet been fully understood although at least eleven byssal proteins were characterized previously. In order to increase genomic resources and identify new byssal proteins from mussel Mytilus coruscus, high-throughput Illumina sequencing was undertaken on the foot, and 79,997,776 paired-ends reads were generated, yielding a library containing 88,825ft unigenes. The M. coruscus byssus was divided into three parts, the proximal thread, the distal thread, and the plaque. Byssal proteins from each part of the byssus were analyzed by shotgun-LTQ analysis. The MS/MS spectra were searched against the foot unigenes dataset and 48 byssal proteins were identified from the M. coruscus byssus. From the whole set, 17, 5, and 11 proteins were exclusive to the proximal thread, the distal thread, and the plaque, respectively. These data can be used as a resource for further studies on the roles of byssal proteins in the deposition of different byssus parts (thread vs. plaque) or in the different mechanical properties (tenacity vs. adhesion). BIOLOGICAL SIGNIFICANCE: Byssal proteins are the major component that controls different aspects of the byssal formation process and thus a source of bioactive molecules that would offer interesting perspectives in biomaterials and bio-adhesive fields. In this paper, we characterized the protein set from different partsof Mytilus coruscus byssus by a combination of transcriptome/proteome technical. A whole set of 48 byssal proteins were described here, including proteins of collagen-like, C1q domain-containing, protease inhibitor-like, tyrosinase-like, SOD, and others. Thread (the distal portion and the proximal portion) and plaque showed distinct protein composition. Of the whole byssal protein set, 11 are exclusive to the plaque, 17 are exclusive to the proximal thread, and 5 are exclusive to the distal thread. Only four proteins are shared by all the three parts of the byssus. The new byssal proteins reported here represent a significant expansion of the knowledge base of Mytilus byssal proteins, and are important for further exploring the mechanism of adhesion in mussel.

Interaction between Vaccinium bracteatum Thunb. leaf pigment and rice proteins.

In this study, we investigated the interaction of Vaccinium bracteatum Thunb. leaf (VBTL) pigment and rice proteins. In the presence of rice protein, VBTL pigment antioxidant activity and free polyphenol content decreased by 67.19% and 68.11%, respectively, and L(∗) of the protein-pigment complex decreased significantly over time. L(∗) values of albumin, globulin and glutelin during 60-min pigment exposure decreased by 55.00, 57.14, and 54.30%, respectively, indicating that these proteins had bound to the pigment. A significant difference in protein surface hydrophobicity was observed between rice proteins and pigment-protein complexes, indicating that hydrophobic interaction is a major binding mechanism between VBTL pigment and rice proteins. A significant difference in secondary structures between proteins and protein-pigment complexes was also uncovered, indicating that hydrogen bonding may be another mode of interaction between VBTL pigment and rice proteins. Our results indicate that VBTL pigment can stain rice proteins with hydrophobic and hydrogen interactions.

Layer-by-Layer Proteomic Analysis of Mytilus galloprovincialis Shell.

Bivalve shell is a biomineralized tissue with various layers/microstructures and excellent mechanical properties. Shell matrix proteins (SMPs) pervade and envelop the mineral crystals and play essential roles in biomineralization. Despite that Mytilus is an economically important bivalve, only few proteomic studies have been performed for the shell, and current knowledge of the SMP set responsible for different shell layers of Mytilus remains largely patchy. In this study, we observed that Mytilus galloprovincialis shell contained three layers, including nacre, fibrous prism, and myostracum that is involved in shell-muscle attachment. A parallel proteomic analysis was performed for these three layers. By combining LC-MS/MS analysis with Mytilus EST database interrogations, a whole set of 113 proteins was identified, and the distribution of these proteins in different shell layers followed a mosaic pattern. For each layer, about a half of identified proteins are unique and the others are shared by two or all of three layers. This is the first description of the protein set exclusive to nacre, myostracum, and fibrous prism in Mytilus shell. Moreover, most of identified proteins in the present study are novel SMPs, which greatly extended biomineralization-related protein data of Mytilus. These results are useful, on one hand, for understanding the roles of SMPs in the deposition of different shell layers. On the other hand, the identified protein set of myostracum provides candidates for further exploring the mechanism of adductor muscle-shell attachment.

In-depth proteomic analysis of nacre, prism, and myostracum of Mytilus shell.

Mytilus is an economically important bivalve and its shell is a biomineralized tissue with various microstructures/layers. In the present study, the shell of marine mussel, Mytilus coruscus, was analyzed and three shell layers with different morphologies and polymorphs were observed, which includes nacre, fibrous prism, and myostracum strongly attached by adductor muscles to the interior of the shell surface. In order to understand whether these different shell layers contain different shell matrix proteins (SMPs), the transcriptome sequencing of M. coruscus mantle and a parallel proteomic analysis of SMPs in the three shell layers were performed. A combination of LC-MS/MS analysis with the mantle transcriptome dataset search resulted in the identification of a total of 63 proteins from M. coruscus shell. From this protein set, fifteen, fourteen, and eight proteins were found to be unique to nacre, fibrous prism, and myostracum layers, respectively. In addition, many novel shell proteins were also identified. The data in this study could be used as a background to explore the roles of SMPs in the deposition of different shell layers (nacre vs. fibrous prism vs. myostracum), the different polymorphisms of calcium carbonate (aragonite vs. calcite); and further, the identified proteins from the myostracum could provide candidates for studying the mechanism of adductor muscle-shell attachment. BIOLOGICAL SIGNIFICANCE: In this paper, we characterized for the first time the protein set from different shell layers in Mytilus. Shell matrix proteins are the major component that controls different aspects of the shell formation process and thus a source of bioactive molecules that would offer interesting perspectives in biomaterials and biomedical fields. Our data can be used as a resource for further exploring the roles of shell matrix proteins in the deposition of different shell layers (nacre vs. fibrous prism vs. myostracum) or different polymorphisms of calcium carbonate (aragonite vs. calcite), and the identified protein set of myostracum provided candidates for studying the mechanism of adductor muscle-shell attachment.

Characterization of a novel antimicrobial peptide with chitin-biding domain from Mytilus coruscus.

Using reverse phase high performance liquid chromatography (RP-HPLC), a novel antimicrobial peptide with 55 amino acid residues was isolated from the hemolymph of Mytilus coruscus. This new antimicrobial peptide displays predominant antimicrobial activity against fungi and Gram-positive bacteria. The molecular mass and the N-terminal sequence of this peptide were analyzed by Mass Spectrometry and Edman degradation, respectively. This antimicrobial peptide, with molecular mass of 6621.55 Da, is characterized by a chitin-biding domain and by 6 Cysteine residues engaged in three intra-molecular disulfide bridges. The full-length of cDNA sequence of this new peptide was obtained by rapid amplification of cDNA ends (RACE) and the encoded precursor was turn out to be a chitotriosidase-like protein. Therefore, we named the precursor with mytichitin-1 and the new antimicrobial peptide (designated as mytichitin-CB) is the carboxyl-terminal part of mytichitin-1. The mRNA transcripts of mytichitin-1 are mainly detected in gonad and the expression level of mytichitin-1 in gonad was up-regulated and reached the highest level at 12 h after bacterial challenge, which was 9-fold increase compared to that of the control group. These results indicated that mytichitin-1 was involved in the host immune response against bacterial infection and might contribute to the clearance of invading bacteria.

Keratocytes create stromal spaces to promote corneal neovascularization via MMP13 expression.

PURPOSE: To investigate the exact mechanism by which keratocytes promote corneal neovascularization. METHODS: The expression of matrix metalloproteinase 13 (MMP13), cluster of differentiation 146 (CD146), VEGFa, VEGFc, VEGF receptor (r)2, and VEGFr3 by normal and alkali-burned rat corneas was determined via quantitative (q)RT-PCR and/or Western blot analysis or in situ hybridization. Corneal neovascularization was observed under a slit lamp microscope and evaluated via immunohistochemistry. The cells that expressed MMP13 in the corneas were determined via sequential immunohistochemistry and in situ hybridization. The degradation of type I collagen was evaluated via the detection of hydroxyproline content and Western blot analysis. The effects of VEGFa and VEGFc on MMP13 expression were determined via luciferase reporter assay for the MMP13 promoter and primary keratocyte culture. RESULTS: Matrix metalloproteinase 13 was predominantly expressed by epithelial cells in normal rat corneas, but it was expressed by cells in corneal stromas after alkali burns. The formation of new blood vessels was consistent with MMP13 expression and attenuated by a selective MMP13 inhibitor in alkali-burned corneas. Keratocytes were the major cells expressing MMP13 in corneal stromas after alkali burns. Through MMP13 expression, keratocytes directly degraded collagen type I to create stromal spaces, which were convenient for newly formed blood vessels to grow into. Expression of MMP13 and collagen type I degradation via keratocytes were induced by VEGFc through VEGFr3 and inhibited by antibodies for VEGFc and VEGFr3. CONCLUSIONS: Keratocytes could directly degrade type I collagen and create stromal spaces, promoting corneal neovascularization through VEGFc/VEGFr3-induced MMP13 expression.