B cell characteristics define HCV reinfection outcome.

BACKGROUND AND AIMS: In individuals highly exposed to hepatitis C virus (HCV), reinfection is common, suggesting that natural development of sterilising immunity is difficult. In those that are reinfected, some will develop a persistent infection, while a small proportion repeatedly clear the virus, suggesting natural protection is possible. The aim of this study was to characterise immune responses associated with rapid natural clearance of HCV reinfection. METHODS: Broad neutralising antibodies (BnAbs) and Envelope 2 (E2)-specific memory B cell (MBCs) responses were examined longitudinally in 15 subjects with varied reinfection outcomes. RESULTS: BnAb responses were associated with MBC recall, but not with reinfection clearance. Strong evidence of antigen imprinting was found, and the B cell receptor repertoire showed a high level of clonality with ongoing somatic hypermutation of many clones over subsequent reinfection events. Single cell transcriptomic analyses showed that cleared reinfections featured an activated transcriptomic profile in HCV-specific B cells that rapidly expanded upon reinfection. CONCLUSIONS: MBC quality, but not necessarily breadth of nAb responses, is important for protection against antigenically diverse variants, which is encouraging for HCV vaccine development.

Proinflammatory activation of microglia in the cerebellum hyperexcites Purkinje cells to trigger ataxia.

Specific medications to combat cerebellar ataxias, a group of debilitating movement disorders characterized by difficulty with walking, balance and coordination, are still lacking. Notably, cerebellar microglial activation appears to be a common feature in different types of ataxic patients and rodent models. However, direct evidence that cerebellar microglial activation in vivo is sufficient to induce ataxia is still lacking. Here, by employing chemogenetic approaches to manipulate cerebellar microglia selectively and directly, we found that specific chemogenetic activation of microglia in the cerebellar vermis directly leads to ataxia symptoms in wild-type mice and aggravated ataxic motor deficits in 3-acetylpyridine (3-AP) mice, a classic mouse model of cerebellar ataxia. Mechanistically, cerebellar microglial proinflammatory activation induced by either chemogenetic M3D(Gq) stimulation or 3-AP modeling hyperexcites Purkinje cells (PCs), which consequently triggers ataxia. Blockade of microglia-derived TNF-α, one of the most important proinflammatory cytokines, attenuates the hyperactivity of PCs driven by microglia. Moreover, chemogenetic inhibition of cerebellar microglial activation or suppression of cerebellar microglial activation by PLX3397 and minocycline reduces the production of proinflammatory cytokines, including TNF-α, to effectively restore the overactivation of PCs and alleviate motor deficits in 3-AP mice. These results suggest that cerebellar microglial activation may aggravate the neuroinflammatory response and subsequently induce dysfunction of PCs, which in turn triggers ataxic motor deficits. Our findings thus reveal a causal relationship between proinflammatory activation of cerebellar microglia and ataxic motor symptoms, which may offer novel evidence for therapeutic intervention for cerebellar ataxias by targeting microglia and microglia-derived inflammatory mediators.

Construction of Processable Ultrastiff Hydrogel for Periarticular Fracture Strutting and Healing.

Development of bioactive bone and joint implants that offer superior mechanical properties to facilitate personalized surgical procedures remains challenging in the field of biomedical materials. As for the hydrogel, mechanical property and processability are major obstructions hampering its application as load-bearing scaffolds in orthopedics. Herein, we constructed implantable composite hydrogels with appealing processability and ultrahigh stiffness. Central to our design is the incorporation of a thixotropic composite network into an elastic polymer network via dynamic interactions to synthesize a percolation-structured double-network (DN) hydrogel with plasticity, followed by in situ strengthening and self-strengthening mechanisms for fostering the DN structure to the cojoined-network structure and subsequently mineralized-composite-network structure to harvest excellent stiffness. The ultrastiff hydrogel is shapeable and can reach a compressive modulus of 80-200 MPa together with a fracture energy of 6-10 MJ/m3, comparable to the mechanical performance of cancellous bone. Moreover, the hydrogel is cytocompatible, osteogenic, and showed almost no volume shrinkage within 28 days in simulated body fluid or culture medium. Such characteristics enabled the utility of a hydrogel in the reduction and stabilization of periarticular fracture treatment on a distal femoral AO/OTA B1 fracture rabbit model and successfully avoided the recollapse of the articular surface.

Injectable Double-Network Hydrogel-Based Three-Dimensional Cell Culture Systems for Regenerating Dental Pulp.

The regeneration of dental pulp tissue is very important, but difficult, in dentistry. The biocompatibility, water content, and viscoelastic properties of pulp-like tissue must be optimized to achieve the efficient transfer of metabolites and nutrients, a suitable degradation rate, distribution of encapsulated cells, injectability, and gelation in situ under physiological conditions. As promising materials for pulp regeneration, hydrogel scaffolds have been produced to simulate the extracellular matrix and transmit signaling molecules. It is imperative to develop hydrogels to effectively regenerate pulp tissue for clinical application. Here, two injectable double-network (DN) hydrogel-based three-dimensional (3D) cell culture systems were developed for regenerating dental pulp. The microstructure, mechanical property, rheology property, and degradation behavior of the injectable DN glycol chitosan-based hydrogels in a simulated root canal model were characterized and compared to a single-network (SN) glycol chitosan-based hydrogel. Human dental pulp stem cells (hDPSCs) were then encapsulated into the GC-based hydrogels for the regeneration of pulp tissue, and the biological performance was investigated both in vitro and in vivo. The results showed that the DN hydrogels had ideal injectability under physiological conditions due to the dynamic nature of the crosslinks. Besides, the DN hydrogels exhibited better mechanical properties and longer degradation duration than the corresponding SN hydrogel. As a 3D cell culture system, the characteristics of the DN hydrogel facilitated odontogenic differentiation and mineralization of hDPSCs in vitro. Further in vivo analysis confirmed that the chemical composition, matrix stiffness, and degradation rate of the DN hydrogel matched those of pulp-like fibrous connective tissue, which might be related to Smad3 activation. These findings demonstrate that DN glycol chitosan-based hydrogels are suitable for the regeneration of pulp tissue.

[Progress in the application of evaluation criteria for clinical efficacy of acupuncture in the treatment of knee osteoarthritis].

Knee osteoarthritis is a chronic joint disease characterized by degeneration of knee cartilage and secondary osteoproliferation, pain and dysfunction, with a high morbidity.Clinical evaluation of efficacy was mainly based on scales, including pain scales, knee function scales, quality of life scales. In order to fully present comprehensive evaluation criteria of acupuncture effect of knee osteoarthritis, this paper reviewed the scales, contrasted their characteristics and scope of application, analyzed the existing problems, and offered proposals to develop and choose efficacy evaluation criteria. Expecting to provide refe-rence frame to evaluate the clinical efficacy of acupuncture in treating knee osteoarthritis in the future.

Janus Nanoparticle Coupled Double-Network Hydrogel.

For double network (DN) hydrogels, their performance can be tuned by adjusting the interaction between their two networks. A novel DN hydrogel toughening approach is proposed by employing Janus nanoparticles (JNs) as crosslinkers to gain a conjoined-network hydrogel. First, a kind of JNs modified by amino groups and quaternary ammonium salt is synthesized, named R3 N+ -JN-NH2 . The DN hydrogel is fabricated based on ionic coordination between calcium chloride (CaCl2 ) and sodium alginate (Alg), as well as covalent (benzoic imine) between glycol chitosan (GC) and benzaldehyde-capped poly(ethylene oxide) (BzCHO-PEO-BzCHO). Based on the same covalent and ionic dynamic crosslinking mechanism, the added R3 N+ -JN-NH2 interacts with two networks to promote crosslinking to form a dually crosslinked structure. The R3 N+ -JN-NH2 effectively provides more energy dissipation, and the hydrogel with conjoined networks shows better compression resistance.

Human CD8 T-stem cell memory subsets phenotypic and functional characterization are defined by expression of CD122 or CXCR3.

Long-lived T-memory stem cells (TSCM ) are key to both naturally occurring and vaccine-conferred protection against infection. These cells are characterized by the CD45RA+ CCR7+ CD95+ phenotype. Significant heterogeneity within the TSCM population is recognized, but distinguishing surface markers and functional characterization of potential subsets are lacking. Human CD8 TSCM subsets were identified in healthy subjects who had been previously exposed to CMV or Influenza (Flu) virus in flow cytometry by expression of CD122 or CXCR3, and then characterized in proliferation, multipotency, self-renewal, and intracellular cytokine production (TNF-α, IL-2, IFN-γ), together with transcriptomic profiles. The TSCM CD122hi -expressing subset (versus CD122lo ) demonstrated greater proliferation, greater multipotency, and enhanced polyfunctionality with higher frequencies of triple positive (TNF-α, IL-2, IFN-γ) cytokine-producing cells upon exposure to recall antigen. The TSCM CXCR3lo subpopulation also had increased proliferation and polyfunctional cytokine production. Transcriptomic analysis further showed that the TSCM CD122hi population had increased expression of activation and homing molecules, such as Ccr6, Cxcr6, Il12rb, and Il18rap, and downregulated cell proliferation inhibitors, S100A8 and S100A9. These data reveal that the TSCM CD122hi phenotype is associated with increased proliferation, enhanced multipotency and polyfunctionality with an activated memory-cell like transcriptional profile, and hence, may be favored for induction by immunization and for adoptive immunotherapy.

Biphasic Double-Network Hydrogel With Compartmentalized Loading of Bioactive Glass for Osteochondral Defect Repair.

Periarticular injury usually causes the defects of superficial cartilage and the underlying subchondral bone. Although some efficacious outcomes have been achieved by the existing therapeutic methods both in clinics and research, like symptomatic treatment, microfracture surgery, and tissue engineering technology, they still present specific disadvantages and complications. To improve this situation, we designed a biphasic (bi-) scaffold aiming to repair the structure of cartilage and subchondral bone synchronously. The scaffold consisted of a superior double-network (DN) hydrogel layer and a lower bioactive glass (BG) reinforced hydrogel layer, and the DN hydrogel included glycol chitosan (GC) and dibenzaldhyde functionalized poly(ethylene oxide) network, and sodium alginate (Alg) and calcium chloride (CaCl2) network. To investigate its effectiveness, we applied this biphasic scaffold to repair osteochondral full-thickness defects in rabbit models. We set up six observation groups in total, including Untreated group, Microfracture group, BG only group, DN gel group, bi-DN gel group, and bi-DN/TGF-ß gel group. With a follow-up period of 24 weeks, we evaluated the treatment effects by gross observation, micro-CT scan and histological staining. Besides, we further fulfilled the quantitative analysis of the data from ICRS score, O'Driscoll score and micro-CT parameters. The results revealed that neat GC/Alg DN hydrogel scaffold was only conductive to promoting cartilage regeneration and neat BG scaffold merely showed the excellent ability to reconstruct subchondral bone. While the biphasic scaffold performed better in repairing osteochondral defect synchronously, exhibiting more well-integrated cartilage-like tissue with positive staining of toluidine blue and col II immunohistochemistry, and more dense trabecular bone connecting closely with the surrounding host bone. Therefore, this method possessed the clinical application potential in treating articular injury, osteochondral degeneration, osteochondral necrosis, and sclerosis.

LDPE microplastics significantly alter the temporal turnover of soil microbial communities.

Although the ubiquitous presence of microplastics in various environments is increasingly well studied, knowledge of the effects of microplastics on ambient microbial communities is still insufficient. To estimate the response of soil bacterial community succession and temporal turnover to microplastic amendment, a soil microcosm experiment was carried out with polyethylene microplastics. The soil samples under control and microplastic amendment conditions were collected for sequencing analysis using Illumina MiSeq technology. Microplastic amendment was found to significantly alter soil bacterial community structure, and the community differences were increased linearly with the incubation time. Compared with the turnover rate of bacterial community in the control samples (0.0103, p < .05, based on Bray-Curtis similarity), the succession rate was significantly (p < .001) higher in the soil with microplastic amendment (0.0309, p < .001). In addition, the effects of microplastic amendment on the time-decay relationships (TDRs) on taxonomic divisions revealed considerable variations of TDRs values, indicating the effects were lineage dependent. Our results propose that the presence of microbial in soil ecosystem may lead to a faster succession rate of soil bacterial community, which provides new insights into the evolutionary consequences of microplastics in terrestrial environment.

LDPE microplastic films alter microbial community composition and enzymatic activities in soil.

Concerns regarding microplastic contamination have spread from aquatic environments to terrestrial systems with a growing number of studies have been reported. Notwithstanding, the potential effects on soil ecosystems remain largely unexplored. In this study, the effects of polyethylene microplastics on soil enzymatic activities and the bacterial community were evaluated, and the microbiota colonizing on microplastics were also investigated. Microplastic amendment (2000 fragments per kg soil) significantly increased the urease and catalase activities in soil after 15 days, and no discernible alteration of invertase activities was detected. Results from high-throughput sequencing of 16S rRNA revealed that the alpha diversities (richness, evenness, and diversity) of the microbiota in soil were not obviously changed by the PE amendment, whereas the diversity indexes of microbiota on plastic fragments were significantly lower than those in the control and amended soils. Different taxonomic composition was observed in between the control and amended soils after 90 days of incubation. Bacterial assemblages with distinct community structure colonized the PE microplastics. Additionally, several taxa including plastic-degrading bacteria and pathogens were more abundant on microplastics. Simultaneously, the predicted functional profiles showed that the pathways of amino acid metabolism and xenobiotics biodegradation and metabolism were higher on the microplastics. These results indicated that microplastics in soil, compared with those in aquatic environments, can also act as a distinct microbial habitat, potentially altering the ecological functions of soil ecosystems.

An Injectable Strong Hydrogel for Bone Reconstruction.

For treating bone defects in periarticular fractures, there is a lack of biomaterial with injectable characteristics, tough structure, and osteogenic capacity for providing a whole-structure support and osteogenesis in the defect area. An injectable hydrogel is an ideal implant, however is weak as load-bearing scaffolds. Herein, a new strategy, i.e., an in situ formation of "active" composite double network (DN), is raised for the preparation of an injectable strong hydrogel particularly against compression. As a demonstration, 4-carboxyphenylboronic acid grafted poly(vinyl alcohol) (PVA) is crosslinked using calcium ions to provide a tough frame while bioactive glass (BG) microspheres are associated by poly(ethylene glycol) to obtain an interpenetrated inorganic network for reinforcement. The injected PVA/BG DN hydrogel gains compressive strength, modulus, and fracture energy of 34 MPa, 0.8 MPa, and 40 kJ m-2 , respectively. Then, the properties can be "autostrengthened" to 57 MPa, 2 MPa, and 65 kJ m-2 by mineralization in 14 days. In vivo experiments prove that the injected DN hydrogel is more efficient to treat femoral supracondylar bone defects than the implanted bulk DN gel. The work suggests a facile way to obtain a strong hydrogel with injectability, cytocompatibility, and tailorable functionality.

Microplastics from mulching film is a distinct habitat for bacteria in farmland soil.

Microplastics, as an emerging pollutant of global importance, have been well documented in aquatic ecosystems. However, little is known about the effects of microplastics on agroecosystems, particularly for soil microbial communities. Herein, microplastics collected from cotton fields in Xinjiang, China, were analysed with a scanning electron microscope (SEM) and high-throughput sequencing to investigate the attached bacterial communities. Microplastic surfaces, especially pits and flakes, were colonized by various microorganisms, suggesting active hydrolysis of plastic debris. The bacterial communities colonizing microplastics were significantly different in structure from those in the surrounding soil, plant litter and macroplastics. In addition, statistical analysis of differentially abundant OTUs showed that microplastics serve as a "special microbial accumulator" in farmland soil, enriching some taxa that degrade polyethylene, such as Actinobacteria, Bacteroidetes and Proteobacteria. Co-occurrence network analysis revealed that the biotic interactions between microorganisms on microplastics are as complex as those in soil, and Acidobacteria, Chloroflexi, Gemmatimonadetes, and Bacteroidetes are considered keystone species in bacterial communities. Collectively, the findings imply that microplastics acted as a distinct habitat for bacteria in farmland soil, which increases our understanding of microplastic pollution.

Increasing aridity affects soil archaeal communities by mediating soil niches in semi-arid regions.

Soil archaea plays a vital role in the functioning of dryland ecosystems, which are expected to expand and get drier in the future as a result of climate change. However, compared with bacteria and fungi, the impacts of increasing aridity on archaea in these ecosystems remain largely unknown. Here, soil samples were collected along a typical aridity gradient in semi-arid regions in Inner Mongolia, China, to investigate whether and how the increasing aridity affects archaeal communities. The results showed that archaeal richness linearly decreased with increasing aridity. After partialling out the effects of soil properties based on partial least squares regression, the significant aridity-richness relationship vanished. The composition of archaeal communities was distributed according to the aridity gradient. These variations were largely driven by the changes in the relative abundance of Thaumarchaeota, Euryarchaeota and unclassified phyla. Niche-based processes were predominant in structuring the observed archaeal aridity-related pattern. The structural equation models further showed that aridity indirectly reduced archaeal richness through improving soil electrical conductivity (EC) and structured community composition by changing soil total nitrogen (TN). These results suggested that soil salinization and N-losses might be important mechanisms underlying the increasing aridity-induced alterations in archaeal communities, and highlighted the importance of soil niches in mediating the indirect impacts of increasing aridity on archaea.

Pedunculoside, a novel triterpene saponin extracted from Ilex rotunda, ameliorates high-fat diet induced hyperlipidemia in rats.

Pedunculoside (PE) is a novel triterpene saponin extracted from the dried barks of Ilex rotunda Thunb. The present study aims to explore lipid-lowering effects of PE on hyperlipidemia rat induced by high-fat diet. The rats were fed with the high-fat diet and subjected to intragastric administration of PE at doses of 30, 15, or 5 mg/kg daily for 7 weeks. The results demonstrated that treatment with PE for 7-week dramatically decreased serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) and reduced liver TC in hyperlipidemia rat induced by high-fat diet. Furthermore, the results also showed that PE modulated the expression of enzymes involved in lipid metabolism including peroxisome proliferator-activated receptor α (PPAR-α), sterol regulatory element-binding protein 1 (SREBP-1), fatty acid synthase (FAS) and stearoyl CoA desaturase-1 (SCD-1) mRNA in liver. Besides, PE-treated group decreased weights and diameters of epididymal adipose hyperlipidemia rat. Mechanism study demonstrated that PE regulated PPAR-γ, CCAAT/Enhancer-binding Protein α (C/EBPα)、and SREBP-1 expression as well as inhibited phosphorylation of AMPK in MDI (methylisobutylxanthine, dexamethasone, insulin) induced-3T3L1 cells. Molecular Docking confirmed interaction between PE with proteins involving PPAR-γ, C/EBPα and SREBP-1. In summary, these findings may support that PE is a novel lipid-lowering drug candidate.

Ultra-tough injectable cytocompatible hydrogel for 3D cell culture and cartilage repair.

In this work, we developed a very facile strategy, i.e. dual dynamic crosslinking, to prepare a high performance injectable hydrogel. Poly(vinyl alcohol) (PVA) was crosslinked by 4-carboxyphenylboronic acid (CPBA) through the generation of borate bonding and ionic interaction to bridge the polymer chains in the presence of calcium ions. The dynamic gathering of CPBA could induce a self-reinforcing effect inside the hydrogel matrix, leading to high tensile and compressive moduli of the hydrogel over 1.0 MPa including the highest compressive modulus up to 5.6 MPa. Meanwhile, the mechanical properties of the hydrogel can be broadly and accurately tuned. And owing to the flexible PVA network, the hydrogel is ultra-tough, showing maximum tensile strain, tensile and compressive fracture energies up to 1600%, 600 kJ m-2 and 25 kJ m-2, respectively. Besides, the dynamic bonding overcomes the barriers to forming an injected strong hydrogel, e.g. to obtain a modulus and a fracture energy exceeding 1.0 MPa and 40 kJ m-2, by using a commercial dual-syringe kit under physiological conditions. Such a mild gelation procedure benefits the administration, 3D encapsulation and proliferation of cells of the hydrogels. The application of the PVA hydrogel was demonstrated by effective cartilage repair.

[Degradation Process of Exogenous Naphthenic Acids and Their Effects on Microbial Community Structure in Soil].

Naphthenic acids (NAs) are a natural component of petroleum, which account for about 2% of severe ecological toxicity in addition to polycyclic aromatic hydrocarbons. With the growth in demand for energy, a large number of NAs have leaked into soil environments through oil industry processes, which have caused enormous potential threats to human health and ecosystems. However, there are few studies about the degradation process of exogenous NAs and their effects on microbial community structures in soil. This research explores the degradation process of NAs and their dynamics in microbial communities in soil by adding a high concentration of 180 mg·kg-1 of NAs to natural, clean soil with the aid of liquid chromatography and high-throughput sequencing technologies. This study found that:① Natural clean soil has a strong capability to degrade high concentration of NAs with about 50% of the NAs degraded within 5 days, which stabilized at 80% after 30 days of the experiment; ② Pollution with NAs obviously alters the microbial community structure as the number of specific OTU increased and were mainly distributed in phylum of unidentified Proteobacteria and Bacteroidetes; ③ Under high concentrations of NAs, the content of Bacteroidetes and Acidobacteria phylum and the γ-Proteobacteria of Proteobacteria phylum all increased swiftly and were speculated to be a potential agents for NA degradation, with the relative abundance ratio of Bacteroidetes and Acidobacteria increasing from 4.2% and 2% to 20.3% and 5.5%, respectively, while a 24.8% decrease was found in Actinobacteria phylum; ④ This study revealed the degradation process of exogenous NAs and their effects on microbial community structure in soil, which provided scientific support for the ecological restoration of petroleum pollution and further study in this area.

ß-Thymosins participate in antiviral immunity of red swamp crayfish (Procambarus clarkii).

ß-Thymosins participate in numerous biological activities, including cell proliferation and differentiation, wound healing, and anti-inflammatory and antimicrobial activities. Many studies have investigated vertebrate ß-thymosins, whereas few reports have focused on invertebrate ß-thymosins. In this study, nine isoforms of ß-thymosins (PcThy-1 to PcThy-8) were identified from the red swamp crayfish Procambarus clarkii. The isoforms contained different numbers of the thymosin ß actin-binding motif. PcThy-1 contained one thymosin ß actin-binding motif, whereas PcThy-8 contained eight motifs. Western blot analysis with anti-PcThy-4 antibody showed that three to six isoforms were present in one tissue, and PcThy-4, PcThy-5, PcThy-6, and PcThy-7 were the main isoforms in several tissues. Time course expression analysis of PcThys at the protein level showed that PcThy-4 was upregulated in hemocytes and gills after white spot syndrome virus (WSSV) challenge. PcThy-4, which contained four thymosin ß actin-binding motifs, was selected for further research. Tissue distribution analysis by quantitative real-time PCR showed that PcThy-4 was present in tissues of the hemocytes, heart, hepatopancreas, gills, stomach, and intestine at the transcriptional level. Transcriptional expression profiles showed that PcThy-4 was upregulated after WSSV challenge. In vivo RNAi and protein injection assay results showed that PcThy-4 inhibited the replication of WSSV in crayfish and enhanced the survival rate after WSSV infection. Furthermore, PcThy-4 promoted hemocyte phagocytosis of WSSV. Overall, results suggested that PcThys protected crayfish from WSSV infection and played an important role in antiviral immune response.

A new group of anti-lipopolysaccharide factors from Marsupenaeus japonicus functions in antibacterial response.

Anti-lipopolysaccharide factors (ALFs) are a group of critical effector molecules with a broad spectrum of antimicrobial activities in crustaceans. Four groups of ALFs (A, B, C, and D) have been identified in peneaid shrimp. In the study, we identified a new group of ALFs (designated as MjALF-E) from Marsupenaeus japonicus. This new group (group E) included MjALF-E1 and E2. MjALF-E1 was highly expressed in hemocytes, heart, and intestine, whereas E2 was highly expressed in gills, stomach, and intestine. Expressions of both MjALF-E1 and E2 were upregulated by bacterial challenge. Synthesized LPS-binding domain peptides of MjALF-E1 and E2 strongly bind to bacterial cell wall components lipopolysaccharide (LPS) and peptidoglycan (PGN). The recombinant rMjALF-E2 showed relatively weak binding activity to LPS and PGN. Both synthesized peptides and rMjALF-E2 exhibited antimicrobial activity against Gram-negative bacteria, whereas rMjALF-E2 could promote the clearance of bacteria in vivo. After knockdown of MjALF-E2 and infection with Vibrio anguillarum, shrimp showed high and rapid mortality compared with GFPi shrimp. These results suggest that MjALF-Es serves a protective function against bacterial infection in shrimp.

L-Type lectin from the kuruma shrimp Marsupenaeus japonicus promotes hemocyte phagocytosis.

L-Type lectins (LTLs) contain a luminal carbohydrate recognition domain, which exhibits homology to leguminous lectins. These type I membrane proteins are involved in the early secretory pathway of animals, and have functions in glycoprotein sorting, trafficking and targeting. Recent studies suggest that LTLs may be involved in immune responses in vertebrates, but no functional studies have been reported. This study reports an LTL, designated as MjLTL1, from the kuruma shrimp Marsupenaeus japonicus. MjLTL consists of a signal peptide, leguminous lectin domain, and transmembrane region. It was upregulated following challenge of shrimp with Vibrio anguillarum. MjLTL1 could agglutinate several bacteria with the presence of calcium, and bind to several Gram-positive and Gram-negative bacteria through lipopolysaccharide and peptidoglycan binding. MjLTL1 could enhance the clearance of V. anguillarum in vivo. MjLTL1 silencing by RNA interference could impair bacterial clearance ability. Further study suggested that MjLTL1 promoted hemocyte phagocytosis. To analyze the possible mechanism, a disintegrin and metalloprotease-like protein (MjADAM) mediating the proteolytic release of extracellular domains from the membrane-bound precursors was also studied in the shrimp. MjADAM exhibited similar tissue location and expression profiles to MjLTL1. After knockdown of MjADAM, the hemocyte phagocytosis rate also declined significantly. ADAM was reported to have an ectodomain shedding function to LTL and release the ectodomain of the lectin from cell membrane. Therefore, our results suggest that the extracellular domain of MjLTL1 might be released from the cell surface as a soluble protein by MjADAM, and function as an opsonin involved in the antibacterial immune responses in shrimp.

Antibacterial activity of serine protease inhibitor 1 from kuruma shrimp Marsupenaeus japonicus.

Serine protease inhibitors (Serpins) are a large family of protease inhibitors involved in many critical biological processes such as blood coagulation, fibrinolysis, programmed cell death, development, and innate immunity. We identified MjSerp1, a serpin in the kuruma shrimp Marsupenaeus japonicus. The MjSerp1 cDNA has a 1239 bp open reading frame (ORF) that encodes a 412-amino acid protein with a 23 aa signal peptide and a classic serpin domain. MjSerp1 has a calculated molecular mass of 46.3 kDa and a predicted isoelectric point of 5.51. MjSerp1 is mainly expressed in the hepatopancreas and the intestines, and is moderately expressed in hemocytes. Expression pattern analysis indicated that MjSerp1 is upregulated in the hepatopancreas after Vibrio anguillarum challenge. rMjSerp1 inhibits three Gram-positive bacteria and two Gram-negative bacteria, but does not inhibit phenoloxidase activity. The microorganism binding assay showed that rMjSerp1 closely binds to both Gram-positive and Gram-negative bacteria. MjSerp1 also exhibits inhibitory activity against microbial serine proteases, such as subtilisin A and proteinase K, indicating that MjSerp1 acts as a microbial serine protease inhibitor. rMjSerp1 injection into shrimp enhances V. anguillarum clearance, but MjSerp1 knockdown through RNA interference impairs Vibrio clearance in vivo. These results indicate that MjSerp1 functions as a direct effector in the bacterial clearance of M. japonicus. All together, our findings provide novel evidences for the serine protease inhibitor in shrimp immunity.