Immunoprotective efficacy evaluation of OmpTS subunit vaccine against Aeromonas hydrophila infection in Megalobrama amblycephala.

Bacterial septicemia in freshwater fish is mainly caused by Aeromonas hydrophila infection, which affects the development of aquaculture industry. In the context of sustainable aquaculture, subunit vaccines are of great values because they play positive roles in reducing the overuse of antibiotics and protecting aquatic animals against bacterial infection. In this study, the recombinant outer membrane protein OmpTS of A. hydrophila were used as subunit vaccine to immunize Megalobrama amblycephala, and its immunoprotective effect and host immune responses were evaluated. The survival rates of the vaccinated groups after bacterial infection were significantly higher than that of the control group, especially of the OmpTS high-dose vaccinated group. The better protective effects of vaccinated groups might be attributed to the increased levels of serum IgM-specific antibody titer, the reduced relative abundance of A. hydrophila in various tissues, the increased number of immune-positive cells with different epitopes, the up-regulated expression levels of immune-related genes, and the enhanced activities of antibacterial enzymes. In conclusion, OmpTS subunit vaccine could strongly induce host immune responses in M. amblycephala, thereby enhancing both cellular and humoral immunity, which exhibited excellent and effective immunoprotective efficacy.

FP-GCN: Frequency Pyramid Graph Convolutional Network for Enhancing Pathological Gait Classification.

Gait, a manifestation of one's walking pattern, intricately reflects the harmonious interplay of various bodily systems, offering valuable insights into an individual's health status. However, the current study has shortcomings in the extraction of temporal and spatial dependencies in joint motion, resulting in inefficiencies in pathological gait classification. In this paper, we propose a Frequency Pyramid Graph Convolutional Network (FP-GCN), advocating to complement temporal analysis and further enhance spatial feature extraction. specifically, a spectral decomposition component is adopted to extract gait data with different time frames, which can enhance the detection of rhythmic patterns and velocity variations in human gait and allow a detailed analysis of the temporal features. Furthermore, a novel pyramidal feature extraction approach is developed to analyze the inter-sensor dependencies, which can integrate features from different pathways, enhancing both temporal and spatial feature extraction. Our experimentation on diverse datasets demonstrates the effectiveness of our approach. Notably, FP-GCN achieves an impressive accuracy of 98.78% on public datasets and 96.54% on proprietary data, surpassing existing methodologies and underscoring its potential for advancing pathological gait classification. In summary, our innovative FP-GCN contributes to advancing feature extraction and pathological gait recognition, which may offer potential advancements in healthcare provisions, especially in regions with limited access to medical resources and in home-care environments. This work lays the foundation for further exploration and underscores the importance of remote health monitoring, diagnosis, and personalized interventions.

Comparative Expression Profiling Reveals the Regulatory Effects of Dietary Mannan Oligosaccharides on the Intestinal Immune Response of Juvenile Megalobrama amblycephala against Aeromonas hydrophila Infection.

Mannan oligosaccharides (MOS) are functional oligosaccharides with beneficial effects on the non-specific immunity of Megalobrama amblycephala, but systematic studies on the immunomodulatory mechanisms of MOS are still lacking. To investigate the protective mechanisms of three different levels of dietary MOS supplementation on the intestinal immunity of juvenile M. amblycephala, comparative digital gene expression (DGE) profiling was performed. In this study, 622 differentially expressed genes (DEGs) were identified, while the similar expression tendency of 34 genes by qRT-PCR validated the accuracy of the DGE analyses. Gene Ontology (GO) enrichment revealed that the DEGs were mainly enriched in two functional categories of biological process and molecular function. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the DEGs were mainly related to complement and coagulation cascades, coagulation cascades, platelet activation, natural killer cell mediated cytotoxicity, Fc gamma R-mediated phagocytosis and antigen processing and presentation. In addition, the pro-inflammatory, apoptosis and tight junction-related genes were more significantly up-regulated upon infection in the dietary MOS groups to enhance host immune functions and maintain the stability of the intestinal barrier. These results will be helpful to clarify the regulatory mechanism of MOS on the intestinal immunity of M. amblycephala and lay the theoretical foundation for the prevention and protection of fish bacterial diseases.

Identification, expression patterns, evolutionary characteristics and recombinant protein activities analysis of CD209 gene from Megalobrama amblycephala.

CD209 is a type II transmembrane protein in the C-type lectin family, which is involved in the regulation of innate and adaptive immune system. Although it has been widely studied in mammals, but little has been reported about fish CD209 genes. In the present study, Megalobrama amblycephala CD209 (MaCD209) gene was cloned and characterized, its expression patterns, evolutionary characteristics, agglutinative and bacteriostatic activities were analyzed. These results showed that the open reading frame (ORF) of MaCD209 gene was 795 bp, encoding 264 aa, and the calculated molecular weight of the encoded protein was 29.7 kDa. MaCD209 was predicted to contain 2 N-glycosylation sites, 1 functional domain (C-LECT-DC-SIGN-like) and 1 transmembrane domain. Multiple sequence alignment showed that the amino acid sequence of MaCD209 was highly homologous with that of partial fishes, especially the highly conserved C-LECT-DC-SIGN-like domain and functional sites of CD209. Phylogenetic analysis showed that the CD209 genes from M. amblycephala and other cypriniformes fishes were clustered into one group, which was reliable and could be used for evolutionary analysis. Then, adaptive evolutionary analysis of teleost CD209 was conducted, and several positively selected sites were identified using site and branch-site models. Quantitative real-time PCR analysis showed that MaCD209 gene was highly expressed in the liver and heart. Moreover, the expression of MaCD209 was significantly induced upon Aeromonas hydrophila infection, with the peak levels at 4 h or 12 h post infection. The immunohistochemical analysis also revealed increased distribution of MaCD209 protein post bacterial infection. In addition, recombinant MaCD209 (rMaCD209) protein was prepared using a pET32a expression system, which showed excellent bacterial binding and agglutinative activities in a Ca2+-independent manner. However, rMaCD209 could only inhibit the proliferation of Escherichia coli rather than A. hydrophila. In conclusion, this study identified the MaCD209 gene, detected its expression and evolutionary characteristics, and evaluated the biological activities of rMaCD209 protein, which would provide a theoretical basis for understanding the evolution and functions of fish CD209 genes.

Molecular Characterization, Expression, Evolutionary Selection, and Biological Activity Analysis of CD68 Gene from Megalobrama amblycephala.

CD68 is a highly glycosylated transmembrane glycoprotein that belongs to the lysosome-associated membrane glycoprotein family and is involved in various immune processes. In this study, Megalobrama amblycephala CD68 (MaCD68) was cloned and characterized, and its expression patterns and evolutionary characteristics were analyzed. The coding region of MaCD68 was 987 bp, encoding 328 amino acids, and the predicted protein molecular weight was 34.9 kDa. MaCD68 contained two transmembrane helical structures and 18 predicted N-glycosylation sites. Multiple sequence alignments showed that the MaCD68 protein had high homology with other fish, and their functional sites were also highly conserved. Phylogenetic analysis revealed that MaCD68 and other cypriniformes fish clustered into one branch. Adaptive evolution analysis identified several positively selected sites of teleost CD68 using site and branch-site models, indicating that it was under positive selection pressure during evolution. Quantitative real-time reverse transcription polymerase chain reaction analysis showed that MaCD68 was highly expressed in the head kidney, spleen, and heart. After Aeromonas hydrophila infection, MaCD68 was significantly upregulated in all tested tissues, peaking at 12 h post-infection (hpi) in the kidney and head kidney and at 120 hpi in the liver and spleen, suggesting that MaCD68 participated in the innate immune response of the host against bacterial infection. Immunohistochemical and immunofluorescence analyses also showed that positive signals derived from the MaCD68 protein were further enhanced after bacterial and lipopolysaccharide treatment, which suggested that MaCD68 is involved in the immune response and could be used as a macrophage marker. Biological activity analysis indicated that recombinant MaCD68 (rMaCD68) protein had no agglutination or bactericidal effects on A. hydrophila but did have these effects on Escherichia coli. In conclusion, these results suggest that MaCD68 plays a vital role in the immune response against pathogens, which is helpful in understanding the immune responses and mechanisms of M. amblycephala.

Intelectin mediated phagocytosis and killing activity of macrophages in blunt snout bream (Megalobrama amblycephala).

Intelectin, a lectin discovered recently, has been identified in various vertebrate species, such as fish, amphibians, and mammals. In one of our previous studies, the efficient bacteria binding and agglutinating activity of the recombinant Megalobrama amblycephala intelectin protein (rMamINTL) and the enhanced immunopositive localization have been observed in the hepatic macrophage-like cells (kupffer cells) post Aeromonas hydrophila infection. Thus, the present study primarily focuses on the regulatory effects of rMamINTL on M. amblycephala macrophages. This study revealed a prominent LPS-binding activity of rMamINTL and a significantly increased phagocytosis of rMamINTL-treated A. hydrophila by M. amblycephala macrophages. However, the rMamINTL-treated M. amblycephala macrophages exhibited no evident regulatory effect on phagocytosis, whereas the enhanced killing activity of the rMamINTL-treated macrophages was observed, which may be attributed to the induced respiratory burst activity and the expression of inflammatory cytokines. In addition, the anti-proliferation effect of rMamINTL on two tumor cells was observed. However, its mechanism remains to be further studied. In short, these results show that MamINTL is a multifunctional immune protein with effective immunomodulatory activity.

Novel insights into the immune regulatory effects of ferritins from blunt snout bream, Megalobrama amblycephala.

Ferritins play vital roles in maintenance of iron homeostasis as iron storage proteins. Recently, the immune function of ferritins have attracted increasing attention, especially their roles in defense against pathogenic infections. However, the immune regulatory mechanism of fish ferritins are not well known. In the present study, comparative digital gene expression (DGE) profiling was performed to explore the regulatory effects of the Megalobrama amblycephala ferritins (MamFers) using MamFers overexpressed and control L8824 cells (Ctenopharyngodon idella hepatic cell line). Clean reads were aligned to the C. idella genome and differential expression analysis was conducted with representative differentially expressed genes pointed out. On that basis, further studies were performed to verify two pivotal regulated pathways in L8824 and EPC (Epithelioma Papulosum Cyprini cell line) cells, respectively. The results showed that NLRC5 (NOD-like Receptor Family CARD Domain Containing 5) mediated the regulation of MamFers on expression of MHC I (Major Histocompatibility Complex Class I) and its chaperone ß2M (Beta-2-Microglobulin) in L8824 cells. Then, ß2M further mediated the regulation of MamFers on hepcidin expression, indicating that MamFers regulated the expression of hepcidin via NLRC5/MHC I/ß2M axis. In addition, MamFers regulated the adhesion of Aeromonas hydrophila to EPC cells by regulating the expression of two extracellular matrix proteins Intgß1 (integrin ß1) and FN (fibronectin). In a word, the present study provided novel insights into the immune regulatory functions of fish ferritins.

Distribution of mannose receptor in blunt snout bream (Megalobrama amblycephala) during the embryonic development and its immune response to the challenge of Aeromonas hydrophila.

The mannose receptor (MR) is a type I transmembrane protein. Its ectodomain has eight C-type lectin-like domains, which are able to recognize and mediate the phagocytosis of a wide range of pathogens. Comprehensive studies have revealed that mammalian MR is widely distributed in the mononuclear phagocyte system (MPS, previously known as the reticuloendothelial system) and play a key role both in the physiological clearance and cell activation. Hitherto, neither the MR distribution, nor the function of clearance and cell activation has been investigated in fish. In the previous study, we have reported the full-length cDNA of blunt snout bream MR, analyzed its structure and relative mRNA expression during embryogenesis and in the liver, head kidney, spleen and intestine of fish after stimulation with killed Aeromonas hydrophila. In the present study, we developed a rabbit polyclonal antibody against MR and undertook a systematic survey of the expression of MR at the protein level by immunohistochemistry. To get more information about MR function, the mRNA expression of MR, pro-inflammatory factor TNF-α and anti-inflammatory factor ARG2 genes was measured by qRT-PCR in the liver, head kidney, and spleen after A. hydrophila challenge. We first observed MR expression in the yolk sac at the fertilized egg stage and possibly MR was expressed by early macrophages. We also showed the MR distribution in head kidney, body kidney, spleen, liver, intestine, muscle, brain, heart, and gills. Following A. hydrophila challenge the MR immunoreactive cells became more widespread in head kidney and spleen, which are the major reticuloendothelial systems of fish. The quantitative studies at mRNA levels showed that there exists a high correlation between MR expression and immune cytokine expressions after bacteria challenge.

Comparative analysis of two ferritin subunits from blunt snout bream (Megalobrama amblycephala): Characterization, expression, iron depriving and bacteriostatic activity.

Iron is an essential microelement for almost all living organisms, while an excess of iron is toxic, thus maintenance of iron homeostasis is vital. As iron storage protein, ferritin plays an important role in iron metabolism. In the present study, we cloned and characterized the ferritin H subunit from Megalobrama amblycephala, termed as MamFerH. An iron-responsive element (IRE) was predicted in the 5' untranslated region (UTR) of MamFerH, while its bulge structural was different from that of the reported ferritin M subunit (MamFerM). The MamFerH and MamFerM genes exhibited similar expression patterns during early development with specifically high expression post hatching, whereas their tissue expression patterns were different. Specifically, MamFerM was highly expressed in the spleen, liver and kidney, while MamFerH was predominantly expressed in the blood and brain, indicating their different functions. In addition, the expression of the two genes was induced upon Aeromonas hydrophila infection at both transcriptional and translational levels, and MamFerH was more efficient. Immunohistochemistry and immunofluorescence analysis confirmed their significant changes at protein level and distribution in the liver post infection, indicating their participation in host immune response. Furthermore, bacteriostatic experiment revealed that recombinant MamFerH displayed more significant inhibitory effect on the growth of A. hydrophila.

Characterization and expression analysis of an intelectin gene from Megalobrama amblycephala with excellent bacterial binding and agglutination activity.

Intelectin is a recently discovered lectin that plays vital roles in the innate immune response, iron metabolism and early embryogenesis. The structure, expression pattern and function of intelectin in mammals and amphibians have been well studied, while not well known in fish. In this study, we cloned a intelectin (MamINTL) gene from blunt snout bream (Megalobrama amblycephala), examined its expression patterns and explored its roles in innate immune response. The MamINTL cDNA encoded 312 amino acids, with a pro-protein of 34 kDa. Sequence analysis revealed the presence of a fibrinogen-related domain and eight conserved cysteine residues in the MamINTL. The MamINTL mRNA was detectable at various developmental stages, while it increased significantly post hatching. In healthy adult M. amblycephala, MamINTL was detected in various tissues with the highest expression in the liver. Upon challenge with Aeromonas hydrophila, significantly up-regulated expression of the MamINTL mRNA was observed in the liver, spleen, kidney, intestine and gill. In addition, increased level of MamINTL protein detected by Western Blotting was also observed in the liver, kidney and spleen, indicating the participation of MamINTL in the immune response. Immunohistochemistry analysis of the M. amblycephala liver sections showed significant changes in expression and location post infection. In addition, the recombinant MamINTL showed excellent binding and agglutination activity against GFP-expressed E. coli in a Ca2+-dependent manner. Generally, the present study provides clues for a better understanding of the characterization, expression patterns and functions of fish intelectins.

Mannose receptor mediated phagocytosis of bacteria in macrophages of blunt snout bream (Megalobrama amblycephala) in a Ca(2+)-dependent manner.

Mannose receptor (MR) is an important pattern-recognition receptor in macrophages and plays a critical role in immune responses. It is has been reported that mammalian macrophages are able to engulf a wide range of microorganisms mediated by Ca(2+)-dependent MR binding to terminal mannose residues which are frequently found on the pathogen surfaces. However, little is known about the MR-mediated phagocytosis in macrophages of fish. In this report, the distributions of MR in the macrophage and head kidney tissue from blunt snout bream were examined using MaMR specific antibody generated in our lab. Mannan and MaMR specific antibody inhibition experiments results collectively showed that MR was involved in the GFP-expressed E. coli engulfed in the macrophages, resulting in respiratory burst, nitric oxide production as well as inflammatory cytokines secretion, and the MaMR-mediated phagocytosis was Ca(2+)-dependent. These results will shed a new light on the immune functions of teleost MRs.

The Screening of the Protective Antigens of Aeromonas hydrophila Using the Reverse Vaccinology Approach: Potential Candidates for Subunit Vaccine Development.

The threat of bacterial septicemia caused by Aeromonas hydrophila infection to aquaculture growth can be prevented through vaccination, but differences among A. hydrophila strains may affect the effectiveness of non-conserved subunit vaccines or non-inactivated A. hydrophila vaccines, making the identification and development of conserved antigens crucial. In this study, a bioinformatics analysis of 4268 protein sequences encoded by the A. hydrophila J-1 strain whole genome was performed based on reverse vaccinology. The specific analysis included signal peptide prediction, transmembrane helical structure prediction, subcellular localization prediction, and antigenicity and adhesion evaluation, as well as interspecific and intraspecific homology comparison, thereby screening the 39 conserved proteins as candidate antigens for A. hydrophila vaccine. The 9 isolated A. hydrophila strains from diseased fish were categorized into 6 different molecular subtypes via enterobacterial repetitive intergenic consensus (ERIC)-PCR technology, and the coding regions of 39 identified candidate proteins were amplified via PCR and sequenced to verify their conservation in different subtypes of A. hydrophila and other Aeromonas species. In this way, conserved proteins were screened out according to the comparison results. Briefly, 16 proteins were highly conserved in different A. hydrophila subtypes, of which 2 proteins were highly conserved in Aeromonas species, which could be selected as candidate antigens for vaccines development, including type IV pilus secretin PilQ (AJE35401.1) and TolC family outer membrane protein (AJE35877.1). The present study screened the conserved antigens of A. hydrophila by using reverse vaccinology, which provided basic foundations for developing broad-spectrum protective vaccines of A. hydrophila.

Intelectin enhances the phagocytosis of macrophages via CDC42-WASF2-ARPC2 signaling axis in Megalobrama amblycephala.

Intelectin has been identified in various vertebrates and plays an important role in the host immune system. In our previous studies, recombinant Megalobrama amblycephala intelectin (rMaINTL) protein with excellent bacterial binding and agglutination activities enhances the phagocytic and killing activities of macrophages in M. amblycephala; however, the underlying regulatory mechanisms remain unclear. The present study showed that treatment with Aeromonas hydrophila and LPS induced the expression of rMaINTL in macrophages, and its level and distribution in macrophages or kidney tissue markedly increased after incubation or injection with rMaINTL. The cellular structure of macrophages was significantly affected after incubation with rMaINTL, resulting in an increased surface area and pseudopodia extension, which might contribute to enhancing the phagocytic ability of macrophages. Then, digital gene expression profiling analysis of the kidneys from rMaINTL-treated juvenile M. amblycephala identified some phagocytosis-related signaling factors that were enriched in pathways involved in the regulation of the actin cytoskeleton. In addition, qRT-PCR and western blotting verified that rMaINTL upregulated the expression of CDC42, WASF2, and ARPC2 in vitro and in vivo; however, the expression of these proteins was inhibited by a CDC42 inhibitor in macrophages. Moreover, CDC42 mediated the promotion of rMaINTL on actin polymerization by increasing the F-actin/G-actin ratio, which led to the extension of pseudopodia and remodeling of the macrophage cytoskeleton. Furthermore, the enhancement of macrophage phagocytosis by rMaINTL was blocked by the CDC42 inhibitor. These results suggested that rMaINTL induced the expression of CDC42 as well as the downstream signaling molecules WASF2 and ARPC2, thereby facilitating actin polymerization to promote cytoskeletal remodeling and phagocytosis. Overall, MaINTL enhanced the phagocytosis activity of macrophages in M. amblycephala via activation of the CDC42-WASF2-ARPC2 signaling axis.

Characterization and Expression Analysis of Genes from Megalobrama amblycephala Encoding Hemoglobins with Extracellular Microbicidal Activity.

Hemoglobin (Hb) usually comprises two α and two ß subunits, forming a tetramer responsible for oxygen transportation and storage. Few studies have elucidated fish hemoglobin immune functions. Megalobrama amblycephala is a freshwater-cultured fish prevalent in China. We identified two M. amblycephala hemoglobin subunits and analyzed their expression patterns and antibacterial activities. The respective full-length cDNA sequences of the M. amblycephala Hb α (MaHbα) and ß (MaHbß) subunits were 588 and 603 bp, encoding 143 and 148 amino acids. MaHbα and MaHbß were highly homologous to hemoglobins from other fish, displaying typical globin-like domains, most heme-binding sites, and tetramer interface regions highly conserved in teleosts. In phylogenetic analyses, the hemoglobin genes from M. amblycephala and other cypriniformes clustered into one branch, and those from other fishes and mammals clustered into other branches, revealing fish hemoglobin conservation. These M. amblycephala Hb subunits exhibit different expression patterns in various tissues and during development. MaHbα is mainly expressed in the blood and brain, while MaHbß gene expression is highest in the muscle. MaHbα expression was detectable and abundant post-fertilization, with levels fluctuating during the developmental stages. MaHbß expression began at 3 dph and gradually increased. Expression of both M. amblycephala Hb subunits was down-regulated in most examined tissues and time points post-Aeromonas hydrophila infection, which might be due to red blood cell (RBC) and hematopoietic organ damage. Synthetic MaHbα and MaHbß peptides showed excellent antimicrobial activities, which could inhibit survival and growth in five aquatic pathogens. Two M. amblycephala hemoglobin subunits were identified, and their expression patterns and antibacterial activities were analyzed, thereby providing a basis for the understanding of evolution and functions of fish hemoglobins.

Molecular characterization and expression patterns of CXCL8 gene from blunt snout bream (Megalobrama amblycephala) and its chemotactic effects on macrophages and neutrophils.

CXCL8 is a typical CXC-type chemokine, which mediates the migration of immune cells from blood vessels to the site of inflammation or injury to clear pathogenic microorganisms and repair damaged tissues. In this study, Megalobrama amblycephala CXCL8 (MaCXCL8) gene was identified and characterized. Sequence analysis showed that the deduced MaCXCL8 protein possessed the typical structure of CXCL8 from other species, with the characteristic CXC cysteine residues in the N-terminal and accompanied by a DLR motif (Asp-Leu-Arg motif). Phylogenetic analysis revealed that MaCXCL8 was homologous to that of Ctenopharyngodon idella and other cyprinid fishes. MaCXCL8 gene was expressed in all detected healthy tissues, with the highest expression levels in the spleen, and its expression was significantly up-regulated upon the challenge of Aeromonas hydrophila and Lipopolysaccharide (LPS) both in juvenile M. amblycephala tissues and primary macrophages. The immunohistochemical assay showed that MaCXCL8 was mainly distributed in the nucleus and cytoplasm, and its expression levels increased observably with the prolongation of bacterial infection. In addition, recombinant MaCXCL8 protein exhibited significant chemotactic effects on neutrophils and macrophages. In conclusion, MaCXCL8 is involved in the immune response of M. amblycephala, and these findings will be helpful to understand the biological roles of MaCXCL8 and provide a theoretical basis for the prevention and control of fish bacterial diseases.

Immunogenicity and protective efficacy of OmpA subunit vaccine against Aeromonas hydrophila infection in Megalobrama amblycephala: An effective alternative to the inactivated vaccine.

Aeromonas hydrophila is a kind of zoonotic pathogen, which can cause bacterial septicemia in fish and bring huge economic losses to global aquaculture. Outer membrane proteins (Omps) are conserved antigens of Aeromonas hydrophila, which can be developed as subunit vaccines. To evaluate the protective efficacy of inactivated vaccine and recombinant outer membrane protein A (OmpA) subunit vaccine against A. hydrophila in juvenile Megalobrama amblycephala, the present study investigated the immunogenicity and protective effects of both vaccines, as well as the non-specific and specific immune response of M. amblycephala. Compared with the non-vaccinated group, both inactivated and OmpA subunit vaccines improved the survival rate of M. amblycephala upon infection. The protective effects of OmpA vaccine groups were better than that of the inactivated vaccine groups, which should be attributed to the reduced bacterial load and enhanced host immunity in the vaccinated fish. ELISA assay showed that the titer of serum immunoglobulin M (IgM) specific to A. hydrophila up-regulated significantly in the OmpA subunit vaccine groups at 14 d post infection (dpi), which should contribute to better immune protective effects. In addition, vaccination enhanced host bactericidal abilities might also attribute to the regulation of the activities of hepatic and serum antimicrobial enzymes. Moreover, the expression of immune-related genes (SAA, iNOS, IL-1 ß, IL-6, IL-10, TNF α, C3, MHC I, MHC II, CD4, CD8, TCR α, IgM, IgD and IgZ) increased in all groups post infection, which was more significant in the vaccinated groups. Furthermore, the number of immunopositive cells exhibiting different epitopes (CD8, IgM, IgD and IgZ) that were detected by immunohistochemical assay had increased in the vaccinated groups post infection. These results show that vaccination effectively stimulated host immune response (especially OmpA vaccine groups). In conclusion, these results indicated that both the inactivated vaccine and OmpA subunit vaccine could protect juvenile M. amblycephala against A. hydrophila infection, of which OmpA subunit vaccine provided more effective immune protection and can be used as an ideal candidate for the A. hydrophila vaccine.

Dietary Mannan Oligosaccharides Enhance the Non-Specific Immunity, Intestinal Health, and Resistance Capacity of Juvenile Blunt Snout Bream (Megalobrama amblycephala) Against Aeromonas hydrophila.

Mannan oligosaccharides (MOS) have been studied and applied as a feed additive, whereas their regulation on the growth performance and immunity of aquatic animals lacks consensus. Furthermore, their immunoprotective effects on the freshwater fish Megalobrama amblycephala have not been sufficiently studied. Thus, we investigated the effects of dietary MOS of 0, 200, and 400 mg/kg on the growth performance, non-specific immunity, intestinal health, and resistance to Aeromonas hydrophila infection in juvenile M. amblycephala. The results showed that the weight gain rate of juvenile M. amblycephala was not significantly different after 8 weeks of feeding, whereas the feed conversion ratio decreased in the MOS group of 400 mg/kg. Moreover, dietary MOS increased the survival rate of juvenile M. amblycephala upon infection, which may be attributed to enhanced host immunity. For instance, dietary MOS increase host bactericidal and antioxidative abilities by regulating the activities of hepatic antimicrobial and antioxidant enzymes. In addition, MOS supplementation increased the number of intestinal goblet cells, and the intestine was protected from necrosis of the intestinal folds and disruption of the microvilli and junctional complexes, thus maintaining the stability of the intestinal epithelial barrier. The expression levels of M. amblycephala immune and tight junction-related genes increased after feeding dietary MOS for 8 weeks. However, the upregulated expression of immune and tight junction-related genes in the MOS supplemental groups was not as notable as that in the control group postinfection. Therefore, MOS supplementation might suppress the damage caused by excessive intestinal inflammation. Furthermore, dietary MOS affected the richness and composition of the gut microbiota, which improved the gut health of juvenile M. amblycephala by increasing the relative abundance of beneficial gut microbiota. Briefly, dietary MOS exhibited significant immune protective effects to juvenile M. amblycephala, which is a functional feed additive and immunostimulant.

COVID-19 Vaccination Acceptance in China after It Becomes Available: A Cross-Sectional Study.

The outbreak of coronavirus disease 2019 (COVID-19) has led to numerous tragic deaths all over the world. Great efforts have been made by worldwide nations for COVID-19 targeted vaccine development since the disease outbreak. In January 2021, the Chinese government started to provide free vaccination among nationwide communities, which was optional for citizens. As no evidence has been provided so far regarding COVID-19 vaccination acceptance since the initiation of nationwide vaccination, this study aims to investigate COVID-19 vaccination acceptance among Chinese citizens as well as its associated factors as an attempt to bridge such gap embedded in the current literature. An anonymous cross-sectional study was conducted online in March and April 2021 among adults, with the survey questionnaire designed based on the framework of the health belief model (HBM). Information on socio-demographics, risk perception, past pandemic-related experience, awareness of vaccination as well as acceptance of COVID-19 vaccination were collected. Chi-squared test and multi-level regression were performed to distinguish the acceptance between different groups as well as to identify the significant predictors. A total of 3940 participants completed the survey, with 90.6% of the participants reporting strong willingness to get vaccinated. A list of factors were found to be significantly associated with individuals' acceptance of vaccination, including the region of residence, ethnicity, annual income, whether or not they had experienced a major pandemic event in the past, risk perception of the COVID-19 as well as the awareness of receiving vaccination. Safety concerns about the vaccine (27.7%), concerns about receiving vaccination immediately after newly developed vaccines were released into the market (22.4%) as well as concerns about the potential side effects induced by vaccination (22.1%) were identified as the primary reasons of residents' resistance against vaccination. Overall, residents demonstrated strong willingness to receive vaccination against COVID-19 in China. However, the improvement of vaccination-related knowledge among Chinese residents should be highlighted as a critical strategy to facilitate the penetration of nationwide vaccination in order to ultimately achieve the establishment of herd immunity in China.

Novel insights into the immune regulatory effects of Megalobrama amblycephala intelectin on the phagocytosis and killing activity of macrophages.

Previous studies have found that the expression level of Megalobrama amblycephala intelectin (MaINTL) increased significantly post Aeromonas hydrophila infection, and recombinant MaINTL (rMaINTL) protein could activate macrophages and enhance the phagocytosis and killing activity of macrophages. In order to reveal the immune regulatory mechanisms of MaINTL, primary M. amblycephala macrophages were treated with endotoxin-removed rMaINTL and GST-tag proteins, then total RNA were extracted and used for comparative Digital Gene Expression Profiling (DGE). 1247 differentially expressed genes were identified by comparing rMaINTL and GST-tag treated macrophage groups, including 482 up-regulated unigenes and 765 down-regulated unigenes. In addition, eleven randomly selected differentially expressed genes were verified by qRT-PCR, and most of them shared the similar expression patterns as that of DGE results. GO enrichment revealed that the differentially expressed genes were mainly concentrated in the membrane part and cytoskeleton of cellular component, the binding and signal transducer activity of molecular function, the cellular process, regulation of biological process, signaling and localization of biological process, most of which might related with the phagocytosis and killing activity of macrophages. KEGG analysis revealed the activation and involvement of differentially expressed genes in immune related pathways, such as Tumor necrosis factor (TNF) signaling pathway, Interleukin 17 (IL-17) signaling pathway, Toll-like receptor signaling pathway, and NOD like receptor signaling pathway, etc. In these pathways, TNF-ɑ, Activator protein-1 (AP-1), Myeloid differentiation primary response protein MyD88 (MyD88), NF-kappa-B inhibitor alpha (ikBɑ) and other key signaling factors were significantly up-regulated. These results will be helpful to clarify the immune regulatory mechanisms of fish intelectin on macrophages, thus providing a theoretical basis for the prevention and control of fish bacterial diseases.

Topical application of phenytoin or nifedipine-loaded PLGA microspheres promotes periodontal regeneration in vivo.

OBJECTIVES: Gingival recession and alveolar bone loss are common manifestations of periodontitis. Periodontal regeneration is the ideal strategy for rehabilitating periodontal tissue defects and preventing tooth loss. The present study examined whether localized, topical application of gingival overgrowth-inducing drugs, phenytoin, nifedipine or cyclosporine, induces periodontal regeneration. METHODS: Polylactic-co-glycolic acid (PLGA) was used as the carrier for preparation of phenytoin, nifedipine or cyclosporine-loaded PLGA microspheres, using an oil-in-water emulsification technique. The drug-loaded microspheres were delivered to periodontal defects created on alveolar ridges mesial to the first maxillary molars of Sprague-Dawley rats. After eight weeks, the operation area in each rat, including the maxillary molars and periodontal tissues, was harvested and evaluated by micro-computed tomography, histochemical and immunohistochemical analyses. RESULTS: Physical parameters representative of periodontal regeneration, including the length of new alveolar bone (p < 0.01) and the area of new alveolar bone (p < 0.01) were significantly improved in the phenytoin group. Compared to other groups, the phenytoin group demonstrated increased expression of COL-1, VEGF-A, osteoblast and osteoclast markers (BMP-2, TGF-ß1, OCN and TRAP staining), as well as decreased expression of MMP-8. CONCLUSIONS: Results of the present study provided new evidence that localized, controlled release of phenytoin confers therapeutic benefits toward gingival recession and alveolar bone loss. Phenytoin appears to be a promising drug that promotes periodontal regeneration.