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The pathogenic effects of type 2 diabetes on bone tissue are gaining attention, but the cellular and molecular mechanisms underlying osteoimmunology are still unclear in diabetes-related bone diseases. We delineated the single-cell transcriptome of bone marrow cells from both wide type and type 2 diabetes mice, which provided the first detailed global profile of bone marrow cells and revealed a distinct bone immune microenvironment at the genetic level under type 2 diabetic condition. It was observed that osteoclast activity was inhibited due to a dysregulated cytokine network, which ultimately led to decreased osteoclast formation and differentiation. In type 2 diabetes mice, a specific Cd36+ cluster (cluster 18, monocytes/macrophages 2) was identified as the precursor of osteoclasts with diminished differentiation potential. AP-1 was demonstrated to be the key transcription factor in the underlying mechanism.

Introduction: Emerging evidence suggests that long-term nucleos(t)ide analogue (NA) therapy can be ceased in a selective group of chronic hepatitis B (CHB). This is being gradually implemented in clinical practice. Case Presentation: A 68-year-old man known with a chronic hepatitis B e antigen-positive hepatitis B infection without signs of advanced liver fibrosis or cirrhosis was admitted with acute liver failure. Two months prior to his admission, he ceased his NA therapy. During the admission, NA therapy was restarted, but the liver function worsened. The patient was put on the high-urgency liver transplantation waiting list, and the next day, he was successfully transplanted. However, the patient died 17 days later due to hemorrhagic shock that resulted from intra-abdominal bleeding and acute pancreatitis. Conclusion: Current guidelines suggest that NA therapy can be discontinued in a selective group of CHB patients. However, these guidelines suggest different stopping and follow-up criteria. This case illustrates that NA withdrawal is not without risks and that these differences in recommendations may lead to inadequate management and eventually a fatal outcome.

Lymphocystis disease is frequently prevalent and transmissible in various teleost species worldwide due to lymphocystis disease virus (LCDV) infection, causing unsightly growths of benign lymphocystis nodules in fish and resulting in huge economic losses to aquaculture industry. However, the molecular mechanism of lymphocystis formation is unclear. In this study, LCDV was firstly detected in naturally infected flounder (Paralichthys olivaceus) by PCR, histopathological, and immunological techniques. To further understand lymphocystis formation, transcriptome sequencing of skin nodule tissue was performed by using healthy flounder skin as a control. In total, RNA-seq produced 99.36%-99.71% clean reads of raw reads, of which 91.11%-92.89% reads were successfully matched to the flounder genome. The transcriptome data showed good reproducibility between samples, with 3781 up-regulated and 2280 down-regulated differentially expressed genes. GSEA analysis revealed activation of Wnt signaling pathway, Hedgehog signaling pathway, Cell cycle, and Basal cell carcinoma associated with nodule formation. These pathways were analyzed to interact with multiple viral infection and tumor formation pathways. Heat map and protein interaction analysis revealed that these pathways regulated the expression of cell cycle-related genes such as ccnd1 and ccnd2 through key genes including ctnnb1, lef1, tcf3, gli2, and gli3 to promote cell proliferation. Additionally, cGMP-PKG signaling pathway, Calcium signaling pathway, ECM-receptor interaction, and Cytokine-cytokine receptor interaction associated with nodule formation were significantly down-regulated. Among these pathways, tnfsf12, tnfrsf1a, and tnfrsf19, associated with pro-apoptosis, and vdac2, which promotes viral replication by inhibiting apoptosis, were significantly up-regulated. Visual analysis revealed significant down-regulation of cytc, which expresses the pro-apoptotic protein cytochrome C, as well as phb and phb2, which have anti-tumor activity, however, casp3 was significantly up-regulated. Moreover, bcl9, bcl11a, and bcl-xl, which promote cell proliferation and inhibit apoptosis, were significantly upregulated, as were fgfr1, fgfr2, and fgfr3, which are related to tumor formation. Furthermore, RNA-seq data were validated by qRT-PCR, and LCDV copy numbers and expression patterns of focused genes in various tissues were also investigated. These results clarified the pathways and differentially expressed genes associated with lymphocystis nodule development caused by LCDV infection in flounder for the first time, providing a new breakthrough in molecular mechanisms of lymphocystis formation in fish.

The Major histocompatibility complex (Mhc) is an important molecule for antigen presenting and binds to T cell receptors, activating T lymphocytes and triggering specific immune responses. To investigate the role of MhcII in adaptive immunity, in this study, mhcIIα and mhcIIβ of flounder (Paralichthys olivaceus) were cloned, polyclonal antibodies (Abs) against their extracellular regions were produced, respectively, and their distribution on cells and tissues and expression patterns, which varied by antigen stimulation or pathogen infection, were investigated. The results showed that the open reading frame (ORF) of mhcIIα is 708 bp, including 235 amino acids (aa); and the ORF of mhcIIβ is 741 bp, encoding 246aa. The mhcIIα and mhcIIβ were significantly expressed in gills, spleen, and peripheral blood leukocytes (PBLs). Their antibodies could specifically recognize eukaryotic expressed MhcIIα and MhcIIβ. MhcIIα+ and MhcIIβ+ cells were 30.2 ± 2.9% of the percentage in peripheral blood leukocytes. MhcII molecules were co-localized with CD83 and IgM on leukocytes, respectively, but not on CD4+ or CD8+ T lymphocyte subpopulations. The expression of both mhcIIα and mhcIIβ were significantly upregulated in flounder after bacteria and virus challenges. The percentages of MhcII+ cells, MhcII+/CD83+, and MhcII+/IgM+ double-positive cells increased significantly after PHA and ConA stimulation, respectively; they varied significantly in PBLs after polyI:C stimulation, and no variations were found after LPS treatment. In the meantime, variations in MhcII+ cells were consistent with that of CD4+ T lymphocytes. These results suggest that MhcII, mainly expressed in B cells and dendritic cells, play an essential role in antigen presentation, and respond significantly to exogenous antigens and T cell-dependent antigens. These results may provide an important reference for the study of cellular immunity in teleosts.

Vibrio anguillarum (V. anguillarum) is a bacterium that seriously harms flounder and other aquaculture species. Vaccination is an effective means of preventing vibriosis and is mainly administered by intraperitoneal injection. Effective antigen processing at the initial stage of immunization is essential to elicit adaptive immune responses and improve vaccine efficacy. To understand the early immune response of flounder caused by inactivated V. anguillarum, we detected the transcriptome profiles of the cells in the peritoneal cavity (PoPerCs) after inactivated V. anguillarum immunization. More than 10 billion high-quality reads were obtained, of which about 89.33% were successfully mapped to the reference genome of flounder. A total of 1985, 3072, 4001, and 5476 differentially expressed genes were captured at 6, 12, 24, and 48 h post immunization, respectively. The hub module correlated with the immunization time was identified by WGCNA. GO and KEGG analysis showed that hub module genes were abundantly expressed in various immune-related aspects, including the response to stimuli, the immune system process, signal transducer activity, autophagy, the NOD-like receptor signaling pathway, the toll-like receptor signaling pathway, the T cell receptor signaling pathway, and Th17 cell differentiation. Additionally, genes related to Th cell differentiation are presented as heatmaps. These genes constitute a complex immune regulatory network, mainly involved in pathogen recognition, antigen processing and presentation, and Th cell differentiation. The results of this study provide the first transcriptome profile of PoPerCs associated with inactivated V. anguillarum immunity and lay a solid foundation for further studies on effective V. anguillarum vaccines.

Myeloperoxidase (MPO) is a cationic leukocyte haloperoxidase and together with other proteins, they possess activities against various microorganisms and are involved in extracellular trap (ET) formation. The present work describes the gene and deduced protein sequences, and functions of MPO in flounder (PoMPO). The PoMPO possesses a 2313 bp open reading frame (ORF) that encodes a protein of 770 amino acids. The highest PoMPO mRNA expression levels were found in the head kidney, followed by peritoneal cells, gill, spleen, skin, muscle, and liver. PoMPO was expressed in MHCII+ and GCSFR+ cells which indicated that PoMPO mainly is expressed in flounder macrophages and granulocytes. Bacterial lipopolysaccharide-stimulated peritoneal leukocytes showed an increased protein level of PoMPO while it seemed that LPS also promoted the migration of MPO+ cells from the head kidney into the peripheral blood and peritoneal cavity. After phorbol 12-myristate 13-acetate (PMA) or bacterial stimulation, flounder leukocytes produced typical ET structures containing DNA with decoration by MPO. The ETs containing DNA and PoMPO effectively inhibited the proliferation of ET-trapped bacteria. Blocking PoMPO with antibodies decreased the enzymatic activity, which attenuated the antibacterial activity of ETs. This study pinpoints the involvement of ETs in flounder innate responses to pathogens.

GATA3 is a transcription factor that plays an important role in T cell lineage differentiation and T-helper 2 (Th2) type immune responses. In this study, we developed two rat antibodies against Atlantic salmon GATA-3 (anti-rSsGATA-3a and anti-rSsGATA-3b, respectively). The western blotting and immunofluorescence results showed that anti-rSsGATA-3b antibodies recognized endogenous SsGATA-3 proteins, while the anti-rSsGATA-3a antibodies did not bind SsGATA-3. Immunohistochemical analysis revealed that SsGATA-3 positive cells were detected in all tissues tested, with relatively high number of immune reactive cells in the gills and spleen. Furthermore, the immunohistochemical study revealed that SsGATA-3 was expressed in pillar cells, epithelial cells, chondrocytes, perichondrium cells, and some undifferentiated basal cells. In addition, we determined 577 bp of the upstream promoter sequence of SsIL-4/13a and found four motifs that matched SsGATA-3 binding sites. The promoter regions of SsIL-4/13a were assessed by transfecting four deletion reporter constructs and SsGATA-3 overexpression plasmids. The result showed that SsGATA-3 enhanced the activity of SsIL-4/13a promoters within the region ranging from -317 to -302 bp upstream of the transcriptional start site. Antibodies against Th2 markers such as GATA-3 are valuable in addressing the diversity of T cell responses in fish.

The protective immune response produced by fish after vaccination is crucial for vaccine effectiveness. Our previous studies have shown inactivated vaccine against Edwardsiella tarda can induce immune response in flounder (Paralichthys olivaceus). To elucidate the protective immune response at the genetic level, in this study, flounder was immunized with inactivated E. tarda for 5 weeks, and then they were challenged with E. tarda. The spleen was dissected at 7th day post immunization, 1st and 7th day post challenge, respectively. Transcriptome analysis showed that average of 46 million clean reads were obtained per library, while percentage of clean reads being mapped to reference genome was more than 89% in all cases, which suggested good quality of samples. As for differentially expressed genes (DEGs) identification in inactivated E. tarda groups, at 7th day post immunization, 1422 DEGs were identified and significantly enriched in innate immune-related pathways, such as Phagosome, Cell adhesion molecules and NF-kappa B signaling pathway; At 1st post challenge day, 1210 DEGs were identified and enriched to Antigen processing and presentation and Cell adhesion molecules, indicating that the pathogen was rapidly recognized and delivered; At 7th post challenge day, 1929 DEGs were identified, belonged to Toll-like receptor signaling pathway, Antigen processing and presentation, Th1 and Th2 cell differentiation and Th17 cell differentiation. Compared to 7th post immunization day, 73 immune-associated DEGs were identified at 1st post challenge day. Protein-protein interaction networks analysis revealed 11 hub genes (TLR7, TLR3, CXCR4, IFIH1, TLR8 etc), associated with recognition of pathogens and activation of innate immunity; while for 7th post challenge day, 141 immune-associated DEGs were identified. 30 hub genes (IL6, STAT1, HSP90A.1, TLR7, IL12β etc) were associated with stimulation of lymphocyte differentiation and activation of cellular immunity. Ten immune-related genes were randomly selected for RT-qPCR validation at each time point. In conclusion, data revealed protection of flounder against E. tarda infection by inactivated vaccine is mediated via immediate recognition of pathogen and subsequently activation of cellular immunity. Results give new aspect for vaccine protection cascades, is good references for vaccine evaluation.

Hemocytes play central roles in shrimp immune system, whereas whose subclasses have not yet been completely defined. At present, the morphological classification of hemocytes is inadequate to classify the complete hemocyte repertoire and elucidate the functions and differentiation and maturation processes. Based on single-cell RNA sequencing (scRNA-seq) of hemocytes in healthy Litopenaeus vannamei, combined with RNA-FISH and flow cytometric sorting, we identified three hemocyte clusters including TGase+ cells, CTL+ cells and Crustin+ cells, and further determined their functional properties, potential differentiation trajectory and correspondence with morphological subpopulations. The TGase+ cells were mainly responsible for the coagulation, exhibiting distinguishable characteristics of hyalinocyte, and appeared to be developmentally arrested at an early stage of hemocyte differentiation. The CTL+ cells and Crustin+ cells arrested at terminal stages of differentiation mainly participated in recognizing foreign pathogens and initiating immune defense responses, owning distinctive features of granule-containing hemocytes. Furthermore, we have revealed the functional sub-clusters of three hemocyte clusters and their potential differentiation pathways according to the expression of genes involved in cell cycle, cell differentiation and immune response, and the successive differentiation and maturation of hyalinocytes to granule-containing hemocytes have also mapped. The results revealed the diversity of shrimp hemocytes and provide new theoretical rationale for hemocyte classification, which also facilitate systematic research on crustacean immunity.

Hirame novirhabdovirus (HIRRV) is one of most serious viral pathogens causing significant economic losses to the flounder (Paralichthys olivaceus)-farming industry. Previous studies have shown that the outbreak of HIRRV is highly temperature-dependent, and revealed the viral replication was significantly affected by the antiviral response of flounders under different temperatures. In the present study, the proteome and phosphoproteome was used to analyze the different antiviral responses in the HIRRV-infected flounder under 10 °C and 20 °C. Post viral infection, 472 differentially expressed proteins (DEPs) were identified in the spleen of flounder under 10 °C, which related to NOD-like receptor signaling pathway, RIG-I-like receptor signaling pathway, RNA transport and so on. Under 20 °C, 652 DEPs were identified and involved in focal adhesion, regulation of actin cytoskeleton, phagosome, NOD-like receptor signaling pathway and RIG-I-like receptor signaling pathway. Phosphoproteome analysis showed that 675 differentially expressed phosphoproteins (DEPPs) were identified in the viral infected spleen under 10 °C and significantly enriched in Spliceosome, signaling pathway, necroptosis and RNA transport. Under 20 °C, 1304 DEPPs were identified and significantly enriched to Proteasome, VEGF signaling pathway, apoptosis, Spliceosome, mTOR signaling pathway, mRNA surveillance pathway, and RNA transport. To be noted, the proteins and phosphoproteins involved in interferon production and signaling showed significant upregulations in the viral infected flounder under 20 °C compared with that under 10 °C. Furthermore, the temporal expression profiles of eight selected antiviral-related mRNA including IRF3, IRF7, IKKβ, TBK1, IFIT1, IFI44, MX1 and ISG15 were detected by qRT-PCR, which showed a significantly stronger response at early infection under 20 °C. These results provided fundamental resources for subsequent in-depth research on the HIRRV infection mechanism and the antiviral immunity of flounder, and also gives evidences for the high mortality of HIRRV-infected flounder under low temperature.

Streptococcus iniae is a severe Gram-positive pathogen that can infect a wide range of freshwater and marine fish species. In continuation of our earlier studies on the development of S. iniae vaccine candidates, pyruvate dehydrogenase E1 subunit alpha (PDHA1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were highly efficacious in protecting flounder (Paralichthys olivaceus) against S. iniae. In the present study, to investigate the potential of multi-epitope vaccination strategy to prevent flounder against S. iniae infection, the liner B-cell epitopes of PDHA1 and GAPDH proteins were predicted using a bioinformatics approach and were identified by immunoassay, and recombinant B-cell multi-epitopes of PDHA1 and GAPDH (rMEPIP and rMEPIG) containing immunodominant epitope-concentrated domains were expressed in Escherichia coli BL21 (DE3) and were used as a subunit vaccine to immunize healthy flounder, while recombinant PDHA1 (rPDHA1), GAPDH (rGAPDH) and formalin-inactivated S. iniae (FKC) served as controls. Then, the immunoprotection efficacy of rMEPIP and rMEPIG was evaluated by determining the percentages of CD4-1+, CD4-2+, CD8β+ T lymphocytes and surface-IgM-positive (sIgM+) lymphocytes in peripheral blood leucocytes (PBLs), spleen leucocytes (SPLs) and head kidney leucocytes (HKLs), as well as total IgM, specific IgM, and relative percentage survival (RPS) post immunization, respectively. It was found that fish immunized with rPDHA1, rGAPDH, rMEPIP, rMEPIG and FKC showed significant increases in sIgM+, CD4-1+, CD4-2+, and CD8β+ lymphocytes and production of total IgM and specific IgM against S. iniae or recombinant proteins rPDHA1 and rGAPDH, which indicated the activation of humoral and cellular immune responses after vaccination. Moreover, RPS rate of the multi-epitope vaccine rMEPIP and rMEPIG groups reached 74.07% and 77.78%, higher than that of rPDHA1 and rGAPDH (62.96% and 66.67%) and KFC (48.15%). These results demonstrated that B-cell multi-epitope protein vaccination, rMEPIP and rMEPIG, could give a better protective effect against S. iniae infection, which provided a promising strategy to design the efficient vaccine in teleost fish.

The CD80/86 molecule is one of the important co-stimulatory ligands and involves antigen-specific immune responses by ligating with CD28 and then delivering the required second signal to T-cell activation. In this study, a CD80/86 homolog was identified, and its expression characteristics were studied in flounder (Paralichthys olivaceus). The open reading frame (ORF) of CD80/86 is 906 bp, encoding 301 aa, and the extracellular amino acid sequence encoded two IgV- and IgC-like structural domains; fCD80/86 is highly expressed in head kidney, peripheral blood leukocytes (PBLs), and spleen, and has relatively high expression in muscle. Antibodies specific for CD80/86 were produced, and CD80/86 was colocalized with MHCII+, CD40+, and CD83+ leukocytes but not with IgM+, CD3+, or CD4+ lymphocytes. The cloned CD80/86 in flounder shares conserved structural features with its mammalian counterparts and is mainly distributed on antigen-presenting cells. Based on these data, CD80/86 as an adjuvant to enhance the immune response of DNA vaccine was investigated. A bicistronic DNA vaccine expressing both CD80/86 and the outer membrane protein (OmpK) of Vibrio anguillarum (p-OmpK-CD80/86) was successfully constructed. After immunization, p-OmpK-CD80/86 could induce the upregulation of the proportion of IgM+ and CD4+ cells in flounder, compared to the p-OmpK- or p-CD80/86-immunized group; CD28 genes were significantly induced in the p-CD80/86 and p-OmpK-CD80/86 groups. Compared to the p-OmpK group, the higher expression of CD83, MHCI, CD4, CD8, and IL-2 was detected at the injection site. The relative percent survival (RPS) produced by p-OmpK-CD80/86 is 66.11% following the V. anguillarum challenge, while the RPS of p-OmpK or p-CD80/86 is 46.30% and 5.56%, respectively. The results revealed that CD80/86 is mainly found in antigen-presenting cells, and could help elicit humoral immune responses in teleost through the CD80/86-CD28 signaling pathway involving CD4+ lymphocytes.

Secretory immunoglobulin (SIg) is crucial for mucosal surface defenses, but the transepithelial secretion of SIg mediated by polymeric immunoglobulin receptor (pIgR) is not clarified in fish. We previously found that flounder (Paralichthys olivaceus) pIgR (fpIgR) and secretory IgM (SIgM) increased in gut mucus post-vaccination. Here, the fpIgR-positive signal was mainly observed in the intestinal epithelium, whereas the IgM-positive signal was mainly distributed in the lamina propria, before immunization. IgM signals increased in the lamina propria and then in the epithelium after immunization with inactivated Vibrio anguillarum, and co-localization between IgM and fpIgR in the epithelium was determined, while the presence of EdU+IgM+ cells in the lamina propria identified the proliferative B cells, revealing that the secretion and transepithelial transport of SIgM locally occurred in the gut of flounder. Subsequently, we established an in-vitro model of transfected MDCK cells that stably expressed the fpIgR. After a recombinant eukaryotic expression plasmid (pCIneoEGFP-fpIgR) was constructed and transfected into MDCK cells, stable expression of the fpIgR in transfected MDCK-fpIgR cells was confirmed, and the tightness and integrity of the polarized cell monolayers grown on Transwells were evaluated. Afterward, the serum IgM of flounder was purified as a binding ligand and placed in the lower compartment of Transwells. An ~800-kDa protein band in the upper compartment was shown to be IgM- and fpIgR-positive, and IgM-positive fluorescence was seen in MDCK-fpIgR cells but not in MDCK-mock cells. Hence, the fpIgR helped polymeric IgM to pass across MDCK-fpIgR cells via transcytosis in a basolateral-to-apical fashion. These new findings provide a better understanding of the pathways shaping mucosal IgM responses and the local mucosal immune mechanisms in teleosts.

Adjuvants are used to increase the strength, quality, and duration of the immune response of vaccines. Neutrophils are the first immune cells that arrive at the injection site and can release DNA fibers together with granular proteins, so-called neutrophil extracellular traps (NETs), to entrap microbes in a sticky matrix of extracellular chromatin and microbicidal agents. Similar extracellular structures were also released by macrophages, mast cells, and eosinophils and are now generalized as “ETs.” Here we demonstrated that Alum adjuvant stimulation led to peritoneal cells swarming and ET release in vitro. Moreover, compared to antigen stimulation alone, ET release was significantly increased after stimulation with antigen-mixed adjuvants and in a time- and dose-dependent manner. In vivo, we were able to monitor and quantify the continuous changes of the ET release in the same fish by using the small animal in vivo imaging instrument at different times during the early stages after intraperitoneal immunization. The results showed that the fluorescence signal of ETs in the peritoneum increased from 0 to 12 h after injection and then gradually decreased. The fluorescence signals came from extracellular DNA fibers, which are sensitive to DNase I and confirmed by microscopy of peritoneal fluid ex vivo. In summary, this study introduced a new method for detecting ETs in the peritoneum of fish in vivo and indicated that ET formation is involved in the immune response at the early stage after intraperitoneal immunization to vaccines.

Hirame novirhabdovirus (HIRRV) is an ongoing threat to the aquaculture industry. The water temperature for the onset of HIRRV is below 15°C, the peak is about 10°C, but no mortality is observed over 20°C. Previous studies found the positive signal of matrix protein of HIRRV (HIRRV-M) was detected in the peripheral blood leukocytes of viral-infected flounder. Flow cytometry and indirect immunofluorescence assay showed that HIRRV-M was detected in mIgM+ B lymphocytes in viral-infected flounder maintained at 10°C and 20°C, and 22% mIgM+ B lymphocytes are infected at 10°C while 13% are infected at 20°C, indicating that HIRRV could invade into mIgM+ B lymphocytes. Absolute quantitative RT-PCR showed that the viral copies in mIgM+ B lymphocytes were significantly increased at 24 h post infection (hpi) both at 10°C and 20°C, but the viral copies in 10°C infection group were significantly higher than that in 20°C infection group at 72 hpi and 96 hpi. Furthermore, the B lymphocytes were sorted from HIRRV-infected flounder maintained at 10°C and 20°C for RNA-seq. The results showed that the differentially expression genes in mIgM+ B lymphocyte of healthy flounder at 10°C and 20°C were mainly enriched in metabolic pathways. Lipid metabolism and Amino acid metabolism were enhanced at 10°C, while Glucose metabolism was enhanced at 20°C. In contrast, HIRRV infection at 10°C induced the up-regulation of the Complement and coagulation cascades, FcγR-mediated phagocytosis, Platelets activation, Leukocyte transendothelial migration and Natural killer cell mediated cytotoxicity pathways at 72 hpi. HIRRV infection at 20°C induced the up-regulation of the Antigen processing and presentation pathway at 72 hpi. Subsequently, the temporal expression patterns of 16 genes involved in Antigen processing and presentation pathway were investigated by qRT-PCR, and results showed that the pathway was significantly activated by HIRRV infection at 20°C but inhibited at 10°C. In conclusion, HIRRV could invade into mIgM+ B lymphocytes and elicit differential immune response under 10°C and 20°C, which provide a deep insight into the antiviral response in mIgM+ B lymphocytes.

Fc receptor (FcR) is an important opsonin receptor on the surface of immune cells, playing an important role in antibody-dependent cell-mediated immunity. Our previous work found that the FcR of flounder showed a marked expression response in phagocytizing IgM+ B cell, which suggested that FcR might participate in regulating Ig-opsonized phagocytosis. In this paper, in order to elucidate the potential role of FcR in mediating phagocytosis of IgM+ B cell, flounder anti-E. tarda serum was prepared and complement-inactivated for the use of E. tarda opsonization, and the sera of healthy flounder were used as control. Flow cytometric analysis showed that the phagocytosis rates of antiserum-opsonized E. tarda in peripheral blood mIgM+ B lymphocytes were significantly higher than the control group, and higher phagocytosis rates of mIgM+ B lymphocyte could be detected with an increasing incubation time ranging from 1 to 5 h. The phagocytosis rates of antiserum-opsonized E. tarda by mIgM+ B lymphocyte for an incubation time of 1, 3 or 5 h were 51.1, 63.0, and 77.5% respectively, which were significantly higher than the phagocytosis rates in the control groups with 40.2, 50.9, and 63.8%, respectively. While the Fc fragment of IgM on the surface of opsonized E. tarda was blocked by rabbit anti-flounder IgM polyclonal antibodies, phagocytosis rates of mIgM+ B lymphocyte decreased significantly compared with the unblocked group. Moreover, the proportion of mIgM+ B lymphocytes with higher intracellular reactive oxygen species (ROS) levels rose to 32.1% from the control level of 23.0% after phagocytosis of antiserum-opsonized E. tarda. FcγRII and Syk were found to be significantly upregulated, while FcγRIII was significantly downregulated in the mIgM+ B lymphocytes post phagocytosis. Furthermore, when FcγRII of mIgM+ B lymphocytes was blocked by the prepared antibodies, their phagocytosis rate of antiserum-opsonized E. tarda was 39.0%, which was significantly lower than the unblocked group of 54.0%. These results demonstrate that FcR plays a critical role in mediating phagocytosis and bactericidal activity of mIgM+ B lymphocytes, which would facilitate an improved understanding of the regulatory roles of FcR in phagocytosis of teleost B lymphocytes.

Dendritic cells (DCs) are the professional antigen presenting cells, which play important roles in regulating the immune response and tolerance. In mammals, CD83 is considered as a maturation marker for DCs, which is a type I membrane glycoprotein. In this research, the gene sequence of the CD83 in Paralichthys olivaceus was obtained on NCBI, the sequence characteristics were studied and CD83 was detected in the head kidney, spleen, gut, gill, liver and muscle, with lower expression in the liver and muscle and higher expression in the head kidney and spleen. And then the CD83 recombinant protein was expressed and the specific antibodies (Abs) were prepared, transiently expressed CD83 eukaryotic proteins could be identified by western blot and indirect immunofluorescence. Then single cell suspensions of head kidney were obtained and DCs were collected by density gradient centrifugation after 7 days of cultivation. Giemsa staining revealed that the nuclei were kidney and dumbbell shaped, and the cell surfaces had dendritic or pseudopod-like protrusions. In addition, the DCs could phagocytose fluorescent microspheres, and the phagocytosis rate was 79.27±1.01%. After incubation with allogeneic lymphocytes, it was found that the lymphocytes clustered and proliferated significantly with the ratio ranging from 39.0% to 59.8% in a dose-dependent manner, which proved that the DCs could elicit mixed lymphocyte responses. After LPS immunization in vivo, the relative expression of CD83 in the head kidney and spleen showed a trend of increasing and then decreasing, peaking at 6 h. Meanwhile, the expression of CD83, MHC II, CD80/86 and CD40 were significantly up-regulated in DCs after stimulated with LPS for 24 h in vitro. And then IIF revealed that CD83+ cells could be detected in the LPS-stimulated group using CD83 Abs, while no positive signal was detected in the control group, which suggested that CD83 protein may be considered as a specific marker for the maturation of DCs in teleost.

The nerve necrosis virus (NNV), a pathogen of viral nervous necrosis disease in several important mariculture economic fish species, causes economic loss. Its nonstructural protein B2 encoded by the sub-genomic RNA3 affects the amplification of the virus. In this study, the B2 protein was recombinantly expressed, the polyclonal antibodies were produced and the dynamics of the B2 protein and genomes were measured in vivo and in vitro after NNV infection. Then, the effects of the overexpressed B2 protein on virus proliferation were investigated. The results showed that the polyclonal antibodies can recognize the B2 protein in both SSN-1 cells and the brain/eye of the grouper. The RNA3 expression significantly increased at 12 h and kept rising till the end of the experiment; it was 106.9 copies/μL at 120 h. The B2 protein could be first detected at 3 h post-infection, which was earlier than the capsid protein was first detected (12 h post-infection). The B2 protein can be detected in the brain, eye and heart on day 3 and the copy number of genomes reached a maximum at 6 d post-infection. There was a low expression of NNV genomes in the liver, spleen and kidney, and no virus was detected in the gill, stomach and intestine. In the meantime, the B2 protein was successfully expressed in GF-1 cells and significantly enhanced virus proliferation, which produced an earlier cytopathic effect and higher cell death rates after 3 d post-infection than the control. In conclusion, the B2 protein acts as an early expressed protein during virus replication and proliferation and is involved in the early infection of NNV. The results may provide insight into the early stage of virus infection and prevention of the disease.

CD28 is well known as a critical T-cell costimulatory receptor involved in T cell activation by binding to its ligands. In this study, CD28 was cloned, and its expression profiles were characterized in flounder (Paralichthys olivaceus); variations of CD28+ cells after being stimulated with different types of antigens and the function of the CD28 costimulatory pathway on T-cell activation were investigated in vitro. fCD28 consists of four exons and three introns, and the full-length cDNA of fCD28 was 675-bp encoded 224 amino acids. The conserved motif (121TFPPPF126) binding to the CD80/86 ligand exists in the Ig-superfamily homology domain. The high expression of fCD28 is in gills, PBLs, head kidney, and spleen. CD28+ cells were co-localized with CD4+ T lymphocytes but not on IgM+ B lymphocyte cells. Moreover, the expression of CD28 was significantly varied in flounder after being stimulated by keyhole limpet hemocyanin (KLH) at both the transcriptional and cellular levels, while no significant differences were observed between lipopolysaccharide (LPS) stimulation and the control group. Notably, treatment of PBLs cultured in vitro with CD28 molecule-specific antibody (anti-CD28 Abs) and PHA produced more cell colonies and stimulated the proliferation of cultured leukocytes compared to PHA stimulation alone and the control group, and a higher level of IL-2 was detected in the culture medium. Meanwhile, anti-CD28 Abs increased the percent of CD28+ cells (10.41 ± 1.35%), CD4+ T lymphocytes (18.32 ± 2.15%), and CD28+/CD4+ double-positive cells (6.24 ± 1.52%). This effect also resulted in significant variations in the genes of cell membrane-bound molecules, cytokines, and related signaling pathways in cultured leukocytes, with significant changes in the genes of interleukin-2 (IL-2) and nuclear factor of activated T cells (NFAT) in the early stages of culture, and the expression of other molecules increased over time. These results proved the localization of the CD28 molecule on T lymphocytes in flounder, and anti-CD28 may act as the B7 ligand involved in T cell activation after antigen stimulation. These data provide a basis for a more in-depth study of the mechanism of the CD28 costimulatory pathway in T cell activation.

The peritoneal cavity plays an important role in the immune response, and intraperitoneal administration is an ideal vaccination route in fish. However, immune responses in the peritoneal cavity of teleost fish are still not completely characterized. This study characterized the morphology of peritoneal cavity cells (PerC cells) and their composition in flounder (Paralichthys olivaceus). Flow cytometric analysis of the resident PerC cells revealed two populations varying in granularity and size. One population, approximately 15.43% ± 1.8%, was smaller with a lower granularity, designated as lymphocytes. The other population of the cells, about 78.17% ± 3.52%, was larger with higher granularity and was designated as myeloid cells. The results of cytochemical staining and transmission electron microscopy indicated that peritoneal cavity in flounder normally contains a resident population of leukocytes dominated by granulocytes, macrophages, dendritic cells, and lymphocytes. The percentages of IgM+, CD4+, G-CSFR+, MHCII+, and CD83+ leukocytes among PerC cells determined by flow cytometry were 3.13% ± 0.4%, 2.83% ± 0.53%, 21.12% ± 1.44%, 27.11% ± 3.30%, and 19.64% ± 0.31%, respectively. Further, the changes in IgM+, CD4+, G-CSFR+, MHCII+, and CD83+ leukocytes in flounder after Vibrio anguillarum infection and immunization were compared. The composition changed rapidly after the infection or vaccination treatment and included two stages, a non-specific stage dominated by phagocytes and a specific immune stage dominated by lymphocytes. Due to the virulence effectors of bacteria, the infected group exhibited a more intense and complicated PerC cells immune response than that of the immunization group. Following our previous study, this is the first report on the morphology and composition of PerC cells and the early activation of PerC cells in flounder response to V. anguillarum infection and vaccination.

T-bet, a T-box family member, is a transcription factor essential for the differentiation of naive CD4+ T cells into Th1 cells that are involved in both innate and adaptive immune responses. In this study, the transcription factor T-bet of flounder (Paralichthys olivaceus) was cloned and characterized, and its expression profile after infection was analyzed. T-bet+ cells were identified in flounder, and the expression and localization of T-bet in T lymphocyte subsets and B lymphocytes were investigated. Finally, the proliferation of T-bet+ cells, T lymphocyte subsets, and B lymphocytes were studied after stimulation with IFN-γ, IL-2, and IL-6, respectively, and the variations of some transcription factors and cytokines in CD4+ T lymphocyte subsets were detected. The results showed that T-bet in flounder consists of 619 aa with a conserved T-box DNA binding domain. T-bet was abundantly expressed in the spleen, head kidney, and heart, and it was significantly upregulated after infection with Vibrio anguillarum, Edwardsiella tarda, and Hirame rhabdovirus, especially in the group of Edwardsiella tarda. A polyclonal antibody against recombinant protein of T-bet was prepared, which specifically recognized the natural T-bet molecule in flounder. T-bet+ cells were found to be distributed in the lymphocytes of peripheral blood, spleen, and head kidney, with the highest proportion in spleen, and the positive signals of T-bet occurred in the cell nucleus. T-bet was also detected in the sorted CD4-1+, CD4-2+, CD8+ T lymphocytes, and IgM+ B lymphocytes. In addition, T-bet+ cells, coordinated with CD4-1+ and CD4-2+ T lymphocytes, were proliferated after stimulation with IFN-γ, IL-2, and IL-6. Especially in sorted CD4-1+ and CD4-2+ T lymphocytes, IFN-γ and IL-2 were able to upregulate the expression of T-bet, forming a positive feedback loop in Th1-type cytokine secretion. These results suggest that T-bet may act as a master transcription factor regulating flounder CD4+ T lymphocytes involved in a Th1-type immune response.

Nervous necrosis virus (NNV) can cause enormous economic losses in mariculture. Vaccines are promising ways to control the disease. In this study: the interferon regulatory factor 3 (IRF3) gene of pearl gentian grouper was cloned and functionally analyzed; then a bicistronic DNA vaccine encoding both capsid protein (CP) and IRF3 was constructed; then the cellular, humoral, and local immune responses in the grouper after immunization were investigated; and then the protective effects after the NNV challenge were investigated. The results showed that the vaccine successfully expressed CP and IRF3. After immunization, the lymphocytes were recruited at the injection site in the muscles. The percentage of sIgM+ lymphocytes in the head, kidney, and spleen significantly increased and peaked at 28.8 ± 3.1% and 42.6 ± 4.2% at the 3rd to 4th weeks. Six immune-related genes were significantly up-regulated. In the meantime, the total antibodies, anti-NNV specific antibodies, and neutralizing antibody titers in serum increased. After the challenge with 105, 106 or 107 TCID50/fish, the relative percent survival rate was 81.25%, 73.91%, and 66.67%, respectively. In 106 TCID50/fish groups, the percentages of sIgM+ lymphocytes, antibodies, and the viral load were investigated. In conclusion, the bicistronic vaccine significantly induced humoral and cellular responses in pearl gentian grouper and provided effective protection against NVV infection.

This paper proposes an H∞ observer based on descriptor systems to estimate the state of charge (SOC). The battery’s open-current voltage is chosen as a generalized state variable, thereby avoiding the artificial derivative calculation of the algebraic equation for the SOC. Furthermore, the observer’s dynamic performance is saved. To decrease the impacts of the uncertain noise and parameter perturbations, nonlinear H∞ theory is implemented to design the observer. The sufficient conditions for the H∞ observer to guarantee the disturbance suppression performance index are given and proved by the Lyapunov stability theory. This paper systematically gives the design steps of battery SOC H∞ observers. The simulation results highlight the accuracy, transient performance, and robustness of the presented method.

Interleukin-2 (IL-2) is an important immunomodulatory cytokine that primarily promotes the activation, proliferation, and differentiation of CD4+ T helper subsets and CD4+ T regulatory cells. In our previous studies, IL-2 and IL-2 receptor beta (IL-2Rβ) genes of flounder (Paralichthys olivaceus) were cloned, and IL-2Rβ molecules expressed on both B and T lymphocytes were identified. In the present study, the interaction of flounder IL-2 (fIL-2) with the IL-2 receptor beta (fIL-2Rβ) was investigated. The proportion of CD4+ T lymphocytes and IL-2Rβ+ cells were detected both in vivo and in vitro. Firstly, the binding of recombinant flounder IL-2 protein (rfIL-2) and rfIL-2Rβ was verified by pull-down assay and enzyme-linked immunosorbent assay. Indirect immunofluorescence assay showed that rfIL-2 enhanced the proliferation of CD4+ and IL-2Rβ+ cells in the gill and spleen. Furthermore, CD4-1+, CD4-2+ T lymphocytes and IL-2Rβ+ cells were significantly upregulated in cultured peripheral blood lymphocytes (PBLs) with addition of rfIL-2, as shown by Flow cytometry. The related genes were examined by Q-PCR in cultured PBLs with added rfIL-2. The results showed that the IL-2–IL-2R interaction induced upregulated expression of T lymphocyte surface makers, Th1-related cytokines or transcription factors, and critical genes of the IL-2 signaling pathway. In addition, these IL-2–elicited biological functions and immune responses were downregulated by blocked with anti–rfIL-2Rβ and anti–rfIL-2 Abs, showing that IL-2Rβ plays an indispensable role in IL-2 elicited biological function. Our results demonstrated that the interaction between IL-2 and IL-2Rβ showed its potential to enhance the proliferation of CD4+ T lymphocytes in flounder. As found in mammals, a Th1-mediated mechanism regulated by this interaction exists in teleost.

The mammalian target of rapamycin (mTOR) is an evolutionarily highly conserved atypical serine/threonine protein kinase, which regulates cell growth, proliferation, apoptosis, autophagy, and metabolism. As a regulatory protein, Raptor is awfully important for the stability and function of mTOR complex 1 (mTORC1). However, the studies about how Raptor/mTORC1 participates in and regulates immune response in lower vertebrates are still limited. In this study, we investigated the regulation of immune response by the Raptor/mTORC1 signaling pathway in rockfish Sebastes schlegelii. Sebastes schlegelii Raptor (Ss-Raptor) is a highly conserved protein during the evolution, in both primary and tertiary structure. Ss-Raptor mRNA was widely distributed in various tissues of rockfish and has a relative higher expression in spleen and blood. After infected by Micrococcus luteus or Listonella anguillarum, mRNA expression of Ss-Raptor rapidly increased within 48 h. Once Raptor/mTORC1 signaling was blocked by rapamycin, expression of the pro-inflammatory cytokines IL-1β and IL-8 was severely impaired, suggesting potential regulatory role of Raptor/mTORC1 signaling in the innate immune response of rockfish. In addition, Raptor/mTORC1 pathway participated in lymphocyte activation of rockfish through promoting 4EBP1 and S6 phosphorylation. Inhibition of Raptor/mTORC1 signaling crippled the lymphocyte expansion during primary adaptive immune response, manifesting by the decrease of lymphoid organ weight and lymphocyte numbers. More importantly, inhibition of Raptor/mTORC1 signaling impaired the lymphocyte mediated cytotoxic response, and made the fish more vulnerable to the bacterial infection. Together, our results suggested that Raptor and its tightly regulated mTORC1 signaling acts as modulatory center to regulate both innate and lymphocyte-mediated adaptive immune response during bacterial infection. This research has shed new light on regulatory mechanism of teleost immune response, and provide helpful evidences to understand the evolution of immune system.

Oxidative stress is an important cause of dopaminergic (DA) neuron apoptosis in Parkinson’s disease (PD). Mesenchymal stromal cells (MSCs) possess antioxidative features. In this study, we investigated whether MSCs could reduce oxidative stress and protect DA neurons from apoptosis by intravenous (I.V.) injection in the early stage of a 6-hydroxydopamine (6-OHDA)-induced PD model. MSCs were injected into the tail vein of mice, and behavioral tests, immunofluorescence staining, western blot, and oxidative stress levels were assessed at different time points. After 6-OHDA exposure, DA neuron apoptosis was detected, together with severe oxidative stress in brain and periphery. Compared with the non-transplanted sham controls, motor function in the 6-OHDA-lesioned group after I.V. injection of MSCs was significantly improved, and the levels of DA neuron apoptosis and oxidative stress decreased. The results demonstrate that MSCs can rescue DA neurons from ongoing apoptosis by reducing oxidative stress, and provide insights on developing new therapeutic strategies to offset the degenerative process of PD.

Most defensins are cationic antimicrobial peptides with broad-spectrum killing activity against bacteria, fungi and enveloped viruses. However, it should be recognized that there are some non-cationic β-defensins in organisms, which need to be further studied. In this study, a new spliced isoform of anionic β-defensin from flounder (Paralichthys olivaceus, fBD) was identified, and its antibiosis, chemotaxis and modulation of phagocytosis were examined. In addition, the contributions of fBD to the antimicrobial activity of extracellular traps (ETs) were also analyzed. The recombinant fBD (rfBD) could effectively inhibit the growth of Gram-positive bacteria (S. aureus, Micrococcus luteus) and Gram-negative bacteria (E. coli, V. alginolyticus, V. anguillarum). An indirect immunofluorescence assay showed that the fBD was co-localized in the extracellular traps released by the leukocytes. When the ETs were blocked with antibodies against rfBD, the proliferation of S. aureus and E. coli incubated with ETs tended to increase compared with that in the control group. In addition, the results obtained by flow cytometry showed that the rfBD could significantly chemoattract leukocytes and increase phagocytic activity in vitro. In conclusion, this study provides new insights into the biological function of anionic defensins, which can serve as one of the important effectors in extracellular traps and as a bridge between innate and adaptive immunity in teleosts.

The general understanding has been that only adaptive immunity is capable of immunological memory, but this concept has been challenged in recent years by studies showing that innate immune systems can mount resistance to reinfection—as the innate immune system can adapt its function following an insult. Innate immune training offers an attractive approach in intensive fish larval rearing, especially since the adaptive immune system is not fully developed. Trained innate immunity will potentially favor robust fish in terms of resistance to viral and bacterial diseases. So-called immunostimulants such as ß-glucans have for decades been used both in laboratories and in intensive fish aquaculture. Treatment of fish by ß-glucans (and by other substances with pathogen-associated molecular patterns) often induces activation of non-specific/innate immune mechanisms and induces higher disease resistance. The reported effects of e.g., ß-glucans fit nicely into the concept “trained innate immunity,” but the research on fish does not yet include analysis of epigenetic changes that may be a prerequisite for long-lasting trained innate immunity. In this “perspective,” we will discuss how in practical terms and based on prior knowledge one can introduce innate immune training in brood stock fish, and their offspring, and whether innate immune training by ß-glucans is a viable approach in larval aquaculture.

Abstract The recent explosive outbreak of Zika virus (ZIKV) infection has been reported in South and Central America and the Caribbean. Neonatal microcephaly associated with ZIKV infection has already caused a public health emergency of international concern. No specific vaccines or drugs are currently available to treat ZIKV infection. The ZIKV helicase, which plays a pivotal role in viral RNA replication, is an attractive target for therapy. We determined the crystal structures of ZIKV helicase-ATP-Mn2+ and ZIKV helicase-RNA. This is the first structure of any flavivirus helicase bound to ATP. Comparisons with related flavivirus helicases have shown that although the critical P-loop in the active site has variable conformations among different species, it adopts an identical mode to recognize ATP/Mn2+. The structure of ZIKV helicase-RNA has revealed that upon RNA binding, rotations of the motor domains can cause significant conformational changes. Strikingly, although ZIKV and dengue virus (DENV) apo-helicases share conserved residues for RNA binding, their different manners of motor domain rotations result in distinct individual modes for RNA recognition. It suggests that flavivirus helicases could have evolved a conserved engine to convert chemical energy from nucleoside triphosphate to mechanical energy for RNA unwinding, but different motor domain rotations result in variable RNA recognition modes to adapt to individual viral replication.

Clostridium perfringens α toxin (CPA) is an important virulence factor that causes livestock hemorrhagic enteritis and food poisoning by contaminated meat products. In this study, the nano-silica microspheres combined with smartphone image processing technology was developed to realize real-time CPA detection. First, the N-terminal and C-terminal domain of the CPA toxin (CPAC3 and CPAN) and their anti-sera were prepared. The silica microspheres coupled with the antibody of CPAC3 was prepared to capture the toxin that existed in the detection sample and the fluorescent-labeled antibody of CPAN was incubated. Moreover, the fluorescent pictures of gray value were performed in a cell phone app, corresponding to toxin concentration. The new assay takes 90 min to perform and can detect CPA as little as 32.8 ng/mL. Our results showed a sensitive, stable, and convenient CPA detection system, which provides a novel detection method of native CPA in foods.

The overexpression of GATA-3, T-bet and TGF-ß may theoretically induce IL-4/A, IFN-γ and IL-17A expression, respectively. Whether this also applies to fish is not yet known. The plasmid vectors encoding reporter gene (RFP)-tagged T-bet, GATA-3 and TGF-ß were used as overexpression tools, transfected into cells or injected intramuscularly to monitor the expression of IFN-γ, IL-4/13A and IL-17A. In addition, the fish were either experimentally challenged with Vibrio anguillarum (VA group) or Piscirickettsia salmonis (PS group). The reporter gene (RFP) inserted upstream of the GATA-3, T-bet and TGF-ß genes, was observed in muscle cell nuclei and in inflammatory cells after intramuscular (i.m.) injection. PS group: following the injection of GATA-3 and T-bet-encoding plasmids, the expression of GATA-3 and T-bet was high at the injection site. The spleen expression of IFN-γ, following the injection of a T-bet-encoding plasmid, was significantly higher on day 2. VA group: The T-bet and GATA-3-overexpressing fish expressed high T-bet and GATA-3 mRNA levels in the muscles and on day 4 post-challenge. The expression of TGF-ß in the muscles of fish injected with TGF-ß-encoding plasmids was significantly higher on days 7 (8 days pre-challenge) and 19 (4 days after challenge). The protective effects of the overexpression of T-bet, GATA-3 and TGF-ß on both bacterial infections were negligible.

Edwardsiella tarda is a Gram-negative bacterium that can infect a broad range of hosts including humans and fish. Accumulating evidences have indicated that E. tarda is able to survive and replicate in host phagocytes. However, the pathways involved in the intracellular infection of E. tarda are unclear. In this study, we examined the entry and endocytic trafficking of E. tarda in the mouse macrophage cell line RAW264.7. We found that E. tarda entered RAW264.7 and multiplied intracellularly in a robust manner. Cellular invasion of E. tarda was significantly impaired by inhibition of clathrin- and caveolin-mediated endocytic pathways and by inhibition of endosome acidification, but not by inhibition of macropinocytosis. Consistently, RAW264.7-infecting E. tarda was co-localized with clathrin, caveolin, and hallmarks of early and late endosomes, and intracellular E. tarda was found to exist in acid organelles. In addition, E. tarda in RAW264.7 was associated with actin and microtubule, and blocking of the functions of these cytoskeletons by inhibitors significantly decreased E. tarda infection. Furthermore, formaldehyde-killed E. tarda exhibited routes of cellular uptake and intracellular trafficking similar to that of live E. tarda. Together these results provide the first evidence that entry of live E. tarda into macrophages is probably a passive, virulence-independent process of phagocytosis effected by clathrin- and caveolin-mediated endocytosis and cytoskeletons, and that the intracellular traffic of E. tarda involves endosomes and endolysosomes.

It is well appreciated that ethnicity influences the natural history and immune responses during a chronic hepatitis B infection. In this study, we explore the effect of ethnicity and treatment cessation on Hepatitis B surface Antigen (HBsAg) seroclearance in patients with Nucleos(t)ide Analogue (NA)-induced Hepatitis B e Antigen (HBeAg) seroconversion. We performed a multi-ethnic, multicentric observational cohort study. The analyzed cohort consisted of 178 mono-infected, predominantly male (75.3%) chronic hepatitis B patients of mixed ethnicity (44.4% Asians, 48.9% Caucasians) with nucleos(t)ide analogue-induced HBeAg seroconversion. Treatment was withdrawn in 105 patients and continued in 73, leading to HBsAg loss in 14 patients off- and 16 patients on-treatment, respectively. Overall, HBsAg loss rates were not affected by treatment cessation (hazard ratio 1.45, p = 0.372), regardless of consolidation treatment duration. Caucasian ethnicity was associated with an increased chance of HBsAg loss (hazard ratio 6.70, p = 0.001), but hepatitis B virus genotype was not (p = 0.812). In conclusion, ethnicity is the most important determinant for HBsAg loss after NA-induced HBeAg seroconversion, with up to six-fold higher HBsAg loss rates in Caucasians compared to Asians, irrespective of treatment cessation and consolidation treatment duration.

Pseudomonas fluorescens is a common bacterial pathogen to a wide range of aquaculture animals including various species of fish. In this study, we employed proteomic analysis and identified filamentous hemagglutinin (FHA) as an iron-responsive protein secreted by TSS, a pathogenic P. fluorescens isolate. In vitro study showed that compared to the wild type, the fha mutant TSSfha (i) exhibited a largely similar vegetative growth profile but significantly retarded in the ability of biofilm growth and producing extracellular matrix, (ii) displayed no apparent flagella and motility, (iii) was defective in the attachment to host cells and unable to form self-aggregation, (iv) displayed markedly reduced capacity of hemagglutination and surviving in host serum. In vivo infection analysis revealed that TSSfha was significantly attenuated in the ability of dissemination in fish tissues and inducing host mortality, and that antibody blocking of the natural FHA produced by the wild type TSS impaired the infectivity of the pathogen. Furthermore, when introduced into turbot as a subunit vaccine, recombinant FHA elicited a significant protection against lethal TSS challenge. Taken together, these results indicate for the first time that P. fluorescens FHA is a key virulence factor essential to multiple biological processes associated with pathogenicity.

Nitrobenzene (NB) as essential products and raw materials in industrial production is a highly toxic and prevalent water pollutant that threatens human health and eco-safety. Photocatalytic methods are widely applied in the field of environmental remediation due to their unique photoexcited redox capacity, which provides a great potential for photoreduction of NB. Here, ultrafast photoreduction and detoxification of NB were explored by establishing a biohybrid photocatalytic system with mild biomineralization. It was discovered that the whole-cell hybrid system developed by biomineralization of CdS nanoparticles on the cell surface of Shewanella oneidensis MR-1 (SW) exhibited high photoreduction activity on NB. Strikingly, SW-CdS exhibited a first-order photoreduction rate constant (k) of 1.1 h–1, which was 54 times higher than that with S. oneidensis MR-1 (metabolic reduction). Impressively, this photoreduction rate achieved by SW-CdS was the highest photoreduction rate of NB ever reached by various nanoparticles. In addition, the photoreduction products obtained showed toxicity even lower than that of the traditional reduction product (aniline) for NB detoxification. Further analysis revealed that the biomineralized CdS nanoparticles served as the main catalytical center and contributed to the ultrahigh photoreduction efficiency of SW-CdS. This work provided a simple, ultrafast, and low-toxic method for the photoreduction and detoxification of NB, which would be promising for application in environmental remediation. [ABSTRACT FROM AUTHOR]