Molecular characterization and functional study of a galectin-9 from a teleost fish, Boleophthalmus pectinirostris.

Galectin-9, a tandem-repeat galectin, plays an important role in the regulation of innate immune response against various microbial infections. Here, galectin-9 from mudskipper (Boleophthalmus pectinirostris) was identified and named as BpGal-9. Putative BpGal-9 contains two conserved carbohydrate recognition domains (CRDs), one CRD within N-terminal (N-CRD) and the other one within C-terminal (C-CRD). Multi-alignment analysis indicated that BpGal-9 shared the highest amino acid sequence identity of 64.3 % with that of Southern platyfish (Xiphophorus maculatus). Phylogenetic analysis showed that BpGal-9 grouped tightly with other teleosts galectin-9 and was most closely related to that of Southern platyfish. BpGal-9 transcripts were more abundant in the intestine, and its expression upregulated significantly in the intestine, kidney, spleen, gills, and skin after Edwardsiella tarda infection. Meanwhile, BpGal-9 expression significantly increased in hemocytes and serum of mudskipper infected by E. tarda. The recombinant BpGal-9 (rBpGal-9) and rBpGal-9C-CRD could agglutinate all tested bacteria, whereas rBpGal-9N-CRD could only agglutinate three kinds of bacteria. When targeting the same bacteria, rBpGal-9 showed stronger agglutinating activities than rBpGal-9C-CRD or rBpGal-9N-CRD. In addition, the induction effect of three recombinant proteins on the mRNA expression of anti-inflammatory cytokines (BpIL-10 and BpTGF-ß) was better than that on the pro-inflammatory cytokines (BpIL-1ß and BpTNF-α). Our result suggested that the N-CRD and C-CRD of galectin-9 contribute differently to its multiple functions in innate immunity in teleosts.

Molecular characterization and antimicrobial activity of NK-lysin in black scraper (Thamnaconus modestus).

NK-lysin (NKL) is a positively charged antimicrobial peptide with broad-spectrum bactericidal activities. In this study, the cDNA sequence of NKL (TmNKL) from black scraper (Thamnaconus modestus) was cloned, which encodes a predicted polypeptide of 150 amino acids that contains a surfactant protein B domain with three disulfide bonds. Phylogenetically, TmNKL was most closely related to its teleost counterpart from tiger puffer (Takifugu rubripes). Expression analysis demonstrated that TmNKL transcripts were constitutively expressed in all tested tissues, with the highest expression levels in the gills. Its expression was significantly upregulated in the gills, head kidney, and spleen after infection with Vibrio parahaemolyticus. A linear peptide (TmNKLP40L) and a disulfide-type peptide (TmNKLP40O) were further synthesized and results showed that disulfide bonds are not essential for bactericidal activities of TmNKL, and that both forms of TmNKL exhibited potent bactericidal activities against 4 gram- negative bacteria, including V. parahaemolyticus, V. alginolyticus, Edwardsiella tarda, and V. harveyi. Observed antimicrobial activities are likely due to the effects of TmNKLP40L and TmNKLP40O treatment on disrupting the integrity of both inner and outer membrane of V. parahaemolyticus, resulting in hydrolysis of bacterial genomic DNA. Damaged cell membranes and leakage of intracellular contents were further confirmed using scanning and transmission microscopy. Moreover, administration of 1.0 µg/g TmNKLP40L or TmNKLP40O significantly decreased bacterial load in tissues and thus, pronouncedly enhanced the survival of V. parahaemolyticus-infected fish. Overall, our results demonstrated that TmNKL is a potent innate effector and provides protective effects against bacterial infection.

[Comparison of clinical and immunological characteristics between primary Sjögren's syndrome patients with positive and negative anti-SSB antibody].

OBJECTIVE: To analyze the differences of clinical manifestations and laboratory features between primary Sjögren's syndrome (pSS) patients with positive and negative anti-Sjögren's syndrome type B (SSB) antibody. METHODS: The clinical data of pSS patients hospitalized in Department of Rheumato-logy and Immunology, Peking University Third Hospital were retrospectively analyzed to investigate the differences of clinical and laboratory features between anti-SSB positive and negative groups. The t test, Mann-Whitney U test, Chi-square test and Fisher's exact probability were used for analysis. RESULTS: A total of 142 pSS patients were enrolled in this study, including 137 females and 5 males with a mean age of (54.8±13.3) years. The anti-SSB positive group included 44 patients accounting for 31.0% of the pSS patients. The anti-SSB positive pSS patients were younger at disease onset and at visit [age at visit: (50.9±14.5) years vs. (56.5±12.4) years; age at onset: (42.2±14.8) years vs. (49.5±15.3) years, P < 0.05]. The patients with anti-SSB positive more frequently presented with rash (29.5% vs. 14.3%, P < 0.05), enlargement of parotid glands (27.3% vs. 8.2%, P < 0.05), renal tubular acidosis (15.9% vs. 4.2%, P < 0.05), immune thrombocytopenia (9.1% vs. 1.0%, P < 0.05), rheumatoid factor (RF) positive (85.0% vs. 49.4%, P < 0.05), higher RF and antinuclear antibody (ANA) titers (median: 89.8 IU/mL vs. 20.5 IU/mL; median: 320 vs. 160, P < 0.05), anti-Sjögren's syndrome type A (SSA) antibody positive (97.7% vs. 64.3%, P < 0.05), elevation of γ globulin (71.4% vs. 38.5%, P < 0.05), higher levels of IgG (median: 21.0 g/L vs. 15.6 g/L, P < 0.05), higher proportions of CD3-CD19+ cells [(21.0±11.9)% vs. (13.7±9.6)%, P < 0.05] and lower proportions of CD3+ cells [(67.2±14.4)% vs. (76.6%±13.1)%, P < 0.05] than those negative. However, the anti-SSB positive group was less likely to show anti-mitochondrial antibodies (AMA)-M2 positivity (10.5% vs. 35.6%, P < 0.05). Glucocorticoids (90.9% vs. 73.5%, P < 0.05) and immunosuppressants (54.5% vs. 36.7%, P < 0.05) were more frequently used in anti-SSB positive pSS patients than those negative. CONCLUSION: The anti-SSB positive pSS patients were younger at disease onset while more frequently presenting with various symptoms, higher levels of other antibodies and activation of B cells than those negative. Glucocorticoids and immunosuppressants were more frequently used, indicating that anti-SSB positive group presented with a more severe clinal phenotype.

[Correlation of anti-phosphatidylserine/prothrombin antibodies with unexplained recurrent miscarriages].

OBJECTIVE: To investigate whether anti-phosphatidylserine/prothrombin antibodies and its IgG or IgM subtypes were correlated with unexplained recurrent miscarriages. METHODS: In our a single-center retrospective study, 283 patients with at least one unexplained miscarriage who visited the Third Hospital of Peking University between January 2021 and August 2023, aged between 18-40 years, and tested for anti-phosphatidylserine/prothrombin antibodies IgG or IgM subtypes, were included. The patients with either positive IgG or IgM anti-phosphatidylserine/prothrombin antibody were regarded as positive for anti-phosphatidylserine/prothrombin antibody. SPSS 26.0 software was used for statistical analysis. Chi-square test and Logistic regression analysis were used to study the correlation of anti-phosphatidylserine/prothrombin antibodies and its IgG or IgM subtypes with unexplained recurrent miscarriages. And the diagnostic sensitivity, specificity, the positive predictive value, the negative predictive value of anti-phosphatidylserine/prothrombin antibodies and its IgG or IgM subtypes in unexplained miscarriages was calculated with four-fold table. RESULTS: Chi-square analysis showed that anti-phosphatidylserine/prothrombin antibodies and its IgM subtypes were correlated with recurrent miscarriages (both P < 0.05), while the IgG subtype was not correlated with recurrent miscarriages (P>0.05). After adjusting with anticardiolipin antibodies, anti-ß2 glycoprotein antibodies, lupus anticoagulants, antinuclear antibodies, and age by Logistic regression analysis, anti-phosphatidylserine/prothrombin antibodies were correlated with unexplained recurrent miscarriages (OR=2.084, 95%CI 1.045-4.155, P < 0.05), and anti-phosphatidylserine/prothrombin antibody IgM subtypes were correlated with unexplained recurrent miscarriages (OR=2.368, 95%CI 1.187-4.722, P < 0.05).The sensitivity of anti-phosphatidylserine/prothrombin antibody in recurrent miscarriage was 65.43%, the specificity was 48.51%, the positive predictive value was 33.76%, and the negative predictive value was 77.78%. In the patients with recurrent miscarriages with negative classical antiphospholipid antibodies, the sensitivity of anti-phosphatidylserine/prothrombin antibody was 59.09%, the specificity was 63.23%, the positive predictive value was 40.63%, and the negative predictive value was 78.40%. The sensitivity of the anti-phosphatidylserine/prothrombin antibody IgM subtype for the diagnosis of recurrent miscarriage was 65.43%, the specificity was 50.99%, the positive predictive value was 34.87%, and the negative predictive value was 78.63%. CONCLUSION: Anti-phosphatidylserine/prothrombin antibody and IgM subtype antibody are correlated with unexplained recurrent miscarriages in patients with at least one unexplained miscarriage. Whether positive anti-phosphatidylserine/prothrombin antibody or IgM subtype could predict future unexplained recurrent miscarriages warrants a prospective study.

Molecular and functional characterization of a ladderlectin-like molecule from ayu (Plecoglossus altivelis).

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

Safety assessment of MPTA: An oral acute and 90-day sub-chronic toxicity study in Sprague-Dawley rats.

MPTA is a novel extract product derived from Macleaya cordata (Willd.) R. Br., which has good anti-inflammatory and antioxidant activity. The aim of this study was to investigate the acute oral toxicity and 90-day sub-chronic oral toxicity of MPTA. In the acute toxicity study, 50 SD rats of both sexes were randomly divided into 5 groups and dosed in a gradient from 197.53 mg/kg to 1000.00 mg/kg bw. Toxic effects were observed up to 14 days and LD50 was calculated. In a subchronic toxicity test, male and female SD rats were orally dosed repeatedly with 96.40, 19.28, 3.86 mg/kg bw of MPTA for 90 days. In addition, a control group was set up in the subchronic study. The acute toxicity test showed that the oral LD50 of MPTA was 481.99 mg/kg with a 95% confidence interval of 404.24-574.70 mg/kg. MPTA did not appear to induce toxic effects in the longer term in terms of food and water consumption, weight gain, haematological and clinical biochemical parameters and pathological examination. The first data on the potential toxicity of MPTA was provided to highlight the safety of short-term to longer-term oral administration of MPTA, and the experimental results yield and establish a NOEAL of 96.40 mg/kg/d for MPTA.

Molecular characterization and chemotaxis assay of a CC motif chemokine ligand 25 from Japanese sea bass (Lateolabrax japonicus).

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

Immune and gut bacterial successions of large yellow croaker (Larimichthys crocea) during Pseudomonas plecoglossicida infection.

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

A novel CC chemokine ligand 2 like gene from ayu Plecoglossus altivelis is involved in the innate immune response against to Vibrio anguillarum.

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

Molecular characterization of the NK-lysin in a teleost fish, Boleophthalmus pectinirostris: Antimicrobial activity and immunomodulatory activity on monocytes/macrophages.

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

CXCL16 upregulates RANKL expression in rheumatoid arthritis synovial fibroblasts through the JAK2/STAT3 and p38/MAPK signaling pathway.

OBJECTIVE: To explore the influence of chemokine, CXCL16, on the expression of the receptor activator nuclear factor κB ligand (RANKL) in rheumatoid arthritis (RA) fibroblast-like synoviocytes (RA-FLS). METHODS: The expression of CXCL16/CXCR6 and RANKL in RA or osteoarthritis (OA) patient synovia was examined by Western blot and immunohistochemistry. The serum concentration of CXCL16 and RANKL was measured by enzyme-linked immunosorbent assay (ELISA). RA-FLS were treated with recombinant CXCL16, and RANKL mRNA and protein were measured using PCR, Western blot and ELISA. RESULTS: The synovial expression of CXCL16, CXCR6, and RANKL was higher in RA patients than in patients with OA. The serum CXCL16 and RANKL levels were higher in RA patients compared with OA patients and healthy controls. CXCL16 correlated with erythrocyte sedimentation rate, C reactive protein, disease activity, serum rheumatoid factor, and RANKL. RA-FLS treated with CXCL16 showed markedly increased expression of RANKL. When STAT3 or p38 activation was blocked by an inhibitor, CXCL16 failed to upregulate RANKL expression. In contrast, inhibiting the Akt or Erk pathway did not achieve the same effect. CONCLUSIONS: CXCL16 upregulates RANKL expression in RA-FLS and these effects are mainly mediated by the JAK2/STAT3 and p38/MAPK signaling pathways.

Molecular characterization of an interleukin-4/13B homolog in grass carp (Ctenopharyngodon idella) and its role in fish against Aeromonas hydrophila infection.

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

Cathelicidin modulates the function of monocytes/macrophages via the P2X7 receptor in a teleost, Plecoglossus altivelis.

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

The MBD4 gene plays an important role in porcine adipocyte differentiation.

BACKGROUND: MBD4 (methyl-CpG binding domain protein 4) is an important G: T glycosylase that can identify T-G mismatches. It plays a role in active demethylation through base excision repair. Overexpression of MBD4 gene can cause the demethylation of numerous genes, and the remethylation of MBD4-associated genes can occur when the MBD4 gene is knocked out. To date, the functions and regulatory mechanisms of the MBD4 gene in the differentiation of porcine preadipocytes have not been clearly established. METHODS: Subcutaneous fat cells from 1- to 7-day-old Junmu-1 piglets were cultured in vitro, induced to differentiate, and then identified. A real-time fluorescence-based quantitative polymerase chain reaction (PCR) analysis was conducted to detect MBD4 messenger RNA (mRNA) expression. Cells were treated with MBD4-siRNA (small interfering RNA) and induced to differentiate. Changes in the lipid droplets were observed by oil red O staining. Changes in the mRNA and protein expression levels of MBD4 and the adipose differentiation-associated genes C/EBPα (CCAAT-enhancer-binding protein alpha), PPARγ (peroxisome proliferator-activated receptor gamma), and aP2 (adipocyte protein 2) were detected. In addition, the bisulfite sequencing method was used to detect changes in methylation in the promoters of certain genes associated with adipose differentiation. RESULTS: Levels of MBD4 mRNA and protein expression varied with time over the course of the porcine adipocyte differentiation, with the highest levels of this expression observed on day two of the differentiation process. After silencing MBD4 and inducing differentiation, the production of lipid droplets decreased, the mRNA expression levels of C/EBPα, PPARγ, and aP2 were significantly reduced, and DNA methylation modification levels were significantly elevated in the examined promoter regions. CONCLUSION: The silencing of the MBD4 gene can influence the DNA methylation levels of preadipocyte differentiation-related genes and subsequently inhibit the differentiation of porcine preadipocytes.

LECT2 improves the outcomes in ayu with Vibrio anguillarum infection via monocytes/macrophages.

Leukocyte cell-derived chemotaxin 2 (LECT2) is reported to be a cytokine involved in the immune response against pathogenic microorganisms in fish. However, its accurate function in whole fish remains unclear. In this study, we provide the first report on the effect of LECT2 on fish defenses against pathogens in vivo. The administration of recombinant LECT2 improved the survival rate of Vibrio anguillarum infected ayu. The bacterial burden of V. anguillarum infected ayu was decreased in LECT2-treated ayu blood, liver, spleen, and kidney compared with saline control. In bacteria-infected ayu, LECT2 treatment altered the mRNA expression of cytokines, including TNFα, IL-1ß, and IL-10, which are all important for the inflammatory response in fish. LECT2 treatment also reduced histological damage in bacteria-infected ayu, and increased peritoneal monocytes/macrophages in both healthy and infected ayu at 12 h post infection. When ayu monocytes/macrophages were depleted by clodronate-liposomes treatment, LECT2 treatment did not increase the survival rate of bacteria-infected fish compared with healthy control fish. Thus our results suggest that LECT2 can modulate host defense in ayu and mediate antibacterial protection against V. anguillarum through monocytes/macrophages.

Molecular characterization and functional analysis of two distinct liver-expressed antimicrobial peptide 2 (LEAP-2) genes in large yellow croaker (Larimichthys crocea).

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

Passive protective effect of chicken egg yolk immunoglobulins against experimental Vibrio anguillarum infection in ayu (Plecoglossus altivelis).

Oral administration of chicken egg yolk immunoglobulins (IgY) has attracted much attention as a means for controlling infectious diseases caused by microorganisms. This study evaluated the protective effect of IgY against Vibrio anguillarum infection in ayu, Plecoglossus altivelis. IgY was isolated from egg yolks laid by hens initially immunized with formalin-inactivated V. anguillarum. Lower mortality of ayu was observed in groups treated with anti-V. anguillarum IgY (aVIgY), compared with those treated with saline or with nonspecific IgY (nspIgY). All fish in saline-treated groups died within seven days after bacterial inoculation. The bacterial load in blood, liver, and spleen was significantly lower in fish treated with aVIgY than in fish treated with nspIgY. aVIgY treatment significantly reduced tumor necrosis factor-α (PaTNF-α), interleukin-1ß (PaIL-1ß), transforming growth factor-ß (PaTGF-ß), and leukocyte cell-derived chemotaxin-2 (PaLECT2) transcript levels in the head kidney, spleen, and liver of ayu challenged by V. anguillarum, compared with nspIgY treatment. The phagocytic activity of macrophages for V. anguillarum in the presence of specific IgY was significantly higher than that seen for nonspecific IgY. These results suggest that passive immunization by oral intubation with pathogen-specific IgY may provide a valuable treatment for V. anguillarum infection in ayu.

Inhibition of the interaction between microglial adenosine 2A receptor and NLRP3 inflammasome attenuates neuroinflammation posttraumatic brain injury.

AIMS: Adenosine 2A receptor (A2A R) is widely expressed in the brain and plays important roles in neuroinflammation, and the nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3) inflammasome is a crucial component of the innate immune system while the regulation of A2A R on it in the central nervous system (CNS) has not been clarified. METHODS: The effects of microglial A2A R on NLRP3 inflammasome assembly and activation were investigated in wild-type, A2A R- or NLRP3-knockout primary microglia with pharmacological treatment. Microglial A2A R or NLRP3 conditional knockout mice were used to interrogate the effects of this regulation on neuroinflammation posttraumatic brain injury (TBI). RESULTS: We found that A2A R directly interacted with NLRP3 and facilitated NLRP3 inflammasome assembly and activation in primary microglia while having no effects on mRNA levels of inflammasome components. Inhibition of the interaction via A2A R agonist or knockout attenuated inflammasome assembly and activation in vitro. In the TBI model, microglial A2A R and NLRP3 were co-expressed at high levels in microglia next to the peri-injured cortex, and abrogating of this interaction by microglial NLRP3 or A2A R conditional knockout attenuated the neurological deficits and neuropathology post-TBI via reducing the NLRP3 inflammasome activation. CONCLUSION: Our results demonstrated that inhibition of the interaction between A2A R and NLRP3 in microglia could mitigate the NLRP3 inflammasome assembly and activation and ameliorate the neuroinflammation post-TBI. It provides new insights into the effects of A2A R on neuroinflammation regulation post-TBI and offers a potential target for the treatment of NLRP3 inflammasome-related CNS diseases.

Subregions of the fusiform gyrus are differentially involved in the attentional mechanism supporting visual mental imagery in depression.

BACKGROUND: Impaired visual mental imagery is an important symptom of depression and has gradually become an intervention target for cognitive behavioral therapy. METHODS: Our study involved a total of 25 healthy controls (HC) and 23 individuals with moderate depressive symptoms (MD). This study explored the attentional mechanism supporting visual mental imagery impairments in depression using the Vividness of Visual Imagery Questionnaire (VVIQ), attentional network test (ANT), and resting-state functional magnetic resonance imaging (rs-fMRI). The intrinsic activity of attention-related regions relative to those supporting visual mental imagery was identified in depression patients. In addition, a meta-analysis was used to describe the cognitive function related to this intrinsic activity. RESULTS: The global correlation (GCOR) of the right anterior fusiform gyrus (FG) was decreased in depression patients. Attention-related areas were concentrated in the right posterior FG; the anterior and posterior functional connectivity (FC) of the FG was decreased in depression patients. Graph theoretic analysis showed that the degree of the right anterior FG was decreased, the degree of the anterior insula was increased, and the negative connection between these two regions was strengthened in depression patients. In addition, the degree of the right anterior FG, the FC between the subregions of the right FG, and the FC between the right anterior FG and insula were correlated with VVIQ scores; however, this correlation was not significant in depression patients. The meta-analysis suggested that the changes in the anterior FG in depressed patients may stem from difficulties of semantic memory retrieval. CONCLUSION: The changed intrinsic activity of subregions of the FG relative to the semantic memory retrieval may be associated with visual mental imagery impairments in depression.

Identification of a Hippocampus-to-Zona Incerta Projection involved in Motor Learning.

Motor learning (ML), which plays a fundamental role in growth and physical rehabilitation, involves different stages of learning and memory processes through different brain regions. However, the neural mechanisms that underlie ML are not sufficiently understood. Here, a previously unreported neuronal projection from the dorsal hippocampus (dHPC) to the zona incerta (ZI) involved in the regulation of ML behaviors is identified. Using recombinant adeno-associated virus, the projections to the ZI are surprisingly identified as originating from the dorsal dentate gyrus (DG) and CA1 subregions of the dHPC. Furthermore, projection-specific chemogenetic and optogenetic manipulation reveals that the projections from the dorsal CA1 to the ZI play key roles in the acquisition and consolidation of ML behaviors, whereas the projections from the dorsal DG to the ZI mediate the retrieval/retention of ML behaviors. The results reveal new projections from the dorsal DG and dorsal CA1 to the ZI involved in the regulation of ML and provide insight into the stages over which this regulation occurs.