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Simple Summary: The outbreak of Hirame novirhabdovirus is significantly temperature dependent. The aim of this study was to identify differential responses of flounder to HIRRV infection at different temperatures by proteome and phosphoproteome. Post HIRRV infection under 10 °C and 20 °C, the enriched immune-related DEPs were both involved in RLR and NLR signaling pathways, apoptosis, phagosome and lysosome, and the DEPPs were also both enriched in spliceosome, mTOR signaling pathway and RNA transport. Compared with the group under 10 °C, the proteins and phosphoproteins involved in interferon production and signaling showed stronger response to infection under 20 °C. qRT-PCR assay showed that eight antiviral-related mRNA including IRF3, IRF7, IKKβ, TBK1, IFIT1, IFI44, MX1 and ISG15 displayed significantly stronger and quicker response at early infection under 20 °C. This study provided a comprehensive understanding of signaling alterations and differential antiviral responses of flounder to HIRRV infection under different temperatures. 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. [ABSTRACT FROM AUTHOR]

Which factors are associated with the risk of clinical pregnancy loss in women with polycystic ovary syndrome (PCOS) undergoing IVF? Case–control study nested in a multicentre randomized trial comparing live birth rates between fresh and frozen embryo transfer in women with PCOS. Women with the outcome of clinical pregnancy loss were selected as the case group, those with live birth as the control group. Parameters before IVF treatment and variables during ovarian stimulation and embryo transfer were compared. Women with clinical pregnancy loss had higher maternal body mass index (BMI, P = 0.010), anti-Müllerian hormone (AMH, P = 0.032), 2-h glucose concentration after 75 g oral glucose tolerance test (OGTT, P = 0.025), and a higher proportion of fresh embryo transfers (P = 0.001). There were significant interactions between the types of transfer and antral follicle count (AFC, P = 0.013), 2-h glucose concentration after OGTT (P = 0.024) on clinical pregnancy loss in PCOS, indicating that these factors may have different effects on pregnancy loss after fresh versus frozen embryo transfer. When the multivariable logistic regression analysis was stratified by the fresh or frozen embryo transfer, AFC (adjusted odds ratio [aOR] 1.03, 95% confidence interval [CI] 1.01–1.05) was a risk factor for clinical pregnancy loss after fresh embryo transfer, while 2-hour glucose concentration after OGTT (aOR 1.13, 95% CI 1.01–1.25) was associated with clinical pregnancy loss in frozen embryo transfer (FET) cycles. In women with PCOS, fresh embryo transfer, higher BMI, AFC and 2-h glucose concentration after OGTT were risk factors for clinical pregnancy loss. FET may be a better choice to decrease the risk of clinical pregnancy loss, especially for those with higher AFC. During FET, 2-h glucose after OGTT appears to be associated with clinical pregnancy loss and warrants close monitoring. [ABSTRACT FROM AUTHOR]

Spleen tyrosine kinase (Syk) is a non-receptor protein tyrosine kinase, and it mediates downstream signaling of FcR-mediated immune responses. Our previous work revealed that the expression of Syk was significantly up-regulated in flounder mIgM+ B lymphocytes after phagocytosis of antiserum-opsonized Edwardsiella tarda , which suggested Syk might be involved in Ig-opsonized phagocytosis. In this paper, phospho-Syk (pSyk) inhibitor was used to investigate the potential role of phosphorylated Syk in FcR-mediated phagocytosis of IgM+ B cells. Indirect immunofluorescence assay (IFA) and Western blotting showed that the level of phosphorylated Syk in the mIgM+ B lymphocytes treated with pSyk inhibitor was significantly lower compared to the control group after stimulation with flounder antiserum. Flow cytometry analysis showed that after 3 h incubation with antiserum-opsonized E. tarda , the phagocytosis rates of mIgM+ B lymphocytes from peripheral blood, spleen and head kidney pre-treated with pSyk inhibitor were 48.1%, 40.1% and 43.6% respectively, which were significantly lower than that of the control groups with 58.7%, 53.2% and 57.4%, respectively. And likewise, after pSyk inhibitor treatment, the proportions of mIgM+ B lymphocytes with higher intracellular reactive oxygen species (ROS) levels in peripheral blood, spleen and head kidney decreased to 15.2%, 12.0% and 12.1% from the control level of 26.5%, 25.9% and 26.3%, respectively. Moreover, the expression of three genes affected by pSyk, including phospholipase Cγ1 (PLCγ1), phospholipase Cγ2 (PLCγ2) and phosphatidylinositol 3 kinase (PI3K) were found to be significantly down-regulated in pSyk inhibitor-treated mIgM+ B lymphocytes post phagocytosis. These results suggest that pSyk plays a key role in FcR-mediated phagocytosis and bactericidal activity of mIgM+ B lymphocytes, which promotes further understanding of the regulatory role of pSyk in teleost B cells phagocytosis. • The first study to find that the activated FcR could induce phosphorylation of Syk in flounder mIgM + B lymphocytes. • The pSyk played a key role in FcR-mediated phagocytosis and bactericidal activity of flounder mIgM+ B lymphocytes. • The pSyk was essential for ADCP-related downstream signaling in flounder mIgM+ B lymphocytes. [ABSTRACT FROM AUTHOR]

Objective: To assess whether the between-group difference in singleton birth weight following frozen vs. fresh embryo transfer varied with infant sex.Design: A post hoc exploratory secondary analysis of data from three multicenter randomized trials compared the live birth rates between freeze-only vs. fresh embryo transfer.Setting: Academic fertility centers.Patient(s): A total of 1,886 women who achieved singleton live birth after a frozen or fresh embryo transfer during these trials were included.Intervention(s): Women underwent either a frozen or fresh embryo transfer.Main Outcome Measure(s): Mean birth weight, large for gestational age (LGA), and small for gestational age (SGA).Result(s): There was an interaction between the types of embryo transfer and infant sex on the birth weight and on the incidences of LGA and SGA. Among male infants, compared with singletons following fresh embryo transfer, singletons following frozen embryo transfer had higher mean birth weights (3,520.6 ± 526.1 vs. 3,345.1 ± 524.9 g), a higher risk of being LGA (25.2% vs. 15.7%), and a lower risk of being SGA (3.3% vs. 6.1%). However, among the female infants, no statistically significant difference was found in the mean birth weight (3,336.5 ± 514.8 vs. 3,299.5 ± 485.0 g) or the risks of being LGA (18.8% vs. 15.7%) or SGA (5.2% vs. 6.0%) between frozen and fresh embryo transfer.Conclusion(s): Male singletons born after frozen embryo transfer were more likely to have a higher birth weight than those born after fresh embryo transfer. [ABSTRACT FROM AUTHOR]

Protein phosphorylation and dephosphorylation are the most common and important regulatory mechanisms in signal transduction, which play a vital role in immune defense response. Our previous study has found the level of tyrosine phosphorylation was significantly changed in the hemocytes of Fenneropenaeus chinensis upon white spot syndrome virus (WSSV) infection. In order to explore the relationship between protein phosphorylation and WSSV infection, the quantitative phosphoproteomics was employed to identify differential phosphorylated proteins in hemocytes of F. chinensis before and after WSSV infection, and elucidate the role of key differential phosphorylated proteins in WSSV infection process. The results showed that a total of 147 differential phosphorylated proteins were identified in the hemocytes, including 64 phosphorylated proteins and 83 dephosphorylated proteins, which were mostly enriched in pyruvate metabolism, TCA cycle, glycolysis, and ribosomal biosynthesis. Functional analysis of differential phosphorylated proteins showed that they were involved in cell apoptosis, cell phagocytosis, cell metabolism and antiviral infection. A total of 236 differential phosphorylation sites were found, including 91 modified sites in the phosphorylation proteins and 145 modified sites in the dephosphorylation proteins. Motif analysis showed that these phosphorylation sites could activate mitogen-activated protein kinase, P70 S6 kinase and other kinases in hemocytes. Moveover, the phosphorylation levels of eukaryotic protein initiation factor 4E binding proteins and histone H3 were further determined by ELISA and Western blotting, which both exhibited a significant increase post WSSV infection and reach their peak levels at 6 and 12 h, respectively. Moreover, we found that lactate, a metabolite closely related to pyruvate metabolism, TCA cycle and glycolysis, was significantly increased in the hemocytes after WSSV infection. This study revealed the protein phosphorylation response in hemocytes of F. chinensis to WSSV infection, which help to clarify the response characteristics and virus resistance mechanism of hemocytes in F. chinensis , and also facilitate further understanding of the interaction between WSSV and shrimp hemocytes. • The phosphorylation response of hemocytes was determined at the early WSSV infection stage. • 147 differential phosphorylated proteins were identified in the WSSV-infected hemocytes. • Differential phosphoproteins were significantly enriched in energy metabolism. • The lactate concentration in hemocytes was significantly increased after WSSV infection. [ABSTRACT FROM AUTHOR]

This study presents a new multiplex lateral flow immunoassay (LFIA) for the detection of aflatoxin B 1 (AFB 1), deoxynivalenol (DON), and zearalenone (ZEN). The multi-LFIA utilized phycocyanin (PC), a protein with excellent photophysical properties, as a fluorescent indicator for quantifying the three mycotoxins on a lateral flow strip platform. PC and monoclonal antibodies (mAbs) against AFB 1 , DON, and ZEN were immobilized onto carboxylated latex nanospheres (LNS) through a one-step coupling process. The resulting conjugates with strong red fluorescence were then used for multiplex sensing of AFB 1 , DON, and ZEN in wheat, corn, and animal feed. The visual results of the assay were obtained using LED UV lights coupled with a smartphone in less than 25 min. Under the optimal conditions, the new multi-LFIA achieved minimum detection limits of 1.04, 1.6, and 2.08 ng/mL for AFB 1 , 2.2, 6.45, and 2.9 ng/mL for DON, and 1.74, 1.67, and 2.11 ng/mL for ZEN in wheat, corn, and feed, respectively. The proposed method demonstrated high selectivity for AFB 1 , DON, and ZEN and showed strong correlation with LC-MS/MS results in spiked samples. In summary, the proposed method is feasible, rapid, and could serve as a highly sensitive platform for detecting AFB 1 , DON, and ZEN and other analytes in a single format. • A new method for multi-target mycotoxin detection was developed using PC as a source of fluorescence signal. • PC and anti-AFB1, DON, or ZEN mAbs were immobilized on the surface of latex nanospheres. • Conjugates with strong signal were developed and used to detect AFB1, DON, and ZEN in wheat, corn, and animal feed. • The natural fluorescent protein PC demonstrated promising potential for multiplex mycotoxin detection. [ABSTRACT FROM AUTHOR]

Plant salt tolerance relies on a coordinated functioning of different tissues and organs. Salinity tissue tolerance is one of the key traits that confer plant adaptation to saline environment. This trait implies maintenance low cytosolic Na+/K+ ratio in metabolically active cellular compartments. In this study, we used Nitraria sibirica Pall. a perennial woody halophyte species, to understand the mechanistic basis of its salinity tissue tolerance. The results showed that the growth of seedlings was stimulated by 100–200 mM NaCl treatment. The ions distribution analysis showed that the leaves act as an Na+ sink, while the plant roots possess superior K+ retention. The excessive Na+ absorbed from the soil was mainly transported to the shoot and was eventuallysequestrated into mesophyll vacuoles in the leaves. As a result, N. sibirica could keep the optimal balance of K+/Na+ at a tissue- and cell-specific level under saline condition. To enable this, N. sibirica increased both vacuolar H+-ATPase and H+-PPase enzymes activities and up-regulated the expressions of NsVHA , NsVP1 and NsNHX1 genes. Vacuolar Na+ sequestration in the leaf mesophyll, mediated by NsVHA , NsVP1 and NsNHX1 , reduced the Na+ concentration in cytosol and inhibited further K+ loss. Meanwhile, N. sibirica enhanced the Two Pore K+ expression at the transcriptional level to promote K+ efflux from vacuole into cytoplasm, assisting in maintaining cytosolic K+ homeostasis. It is concluded that the tissue tolerance traits such as vacuolar Na+ sequestration and intracellular K+ homeostasis are critical to confer adaptation of N. sibirica to soil salinity. [ABSTRACT FROM AUTHOR]

Nitraria sibirica Pall., a typical halophyte that can survive under extreme drought conditions and in saline-alkali environments, exhibits strong salt tolerance and environmental adaptability. Understanding the mechanism of molecular and physiological metabolic response to salt stress of plant will better promote the cultivation and use of halophytes. To explore the mechanism of molecular and physiological metabolic of N. sibirica response to salt stress, two-month-old seedlings were treated with 0, 100, and 400 mM NaCl. The results showed that the differentially expressed genes between 100 and 400 mmol L−1 NaCl and unsalted treatment showed significant enrichment in GO terms such as binding, cell wall, extemal encapsulating structure, extracellular region and nucleotide binding. KEGG enrichment analysis found that NaCl treatment had a significant effect on the metabolic pathways in N. sibirica leaves, which mainly including plant-pathogen interaction, amino acid metabolism of the beta alanine, arginine, proline and glycine metabolism, carbon metabolism of glycolysis, gluconeogenesis, galactose, starch and sucrose metabolism, plant hormone signal transduction and spliceosome. Metabolomics analysis found that the differential metabolites between the unsalted treatment and the NaCl treatment are mainly amino acids (proline, aspartic acid, methionine, etc.), organic acids (oxaloacetic acid, fumaric acid, nicotinic acid, etc.) and polyhydric alcohols (inositol, ribitol, etc.), etc. KEGG annotation and enrichment analysis showed that 100 mmol L−1 NaCl treatment had a greater effect on the sulfur metabolism, cysteine and methionine metabolism in N. sibirica leaves, while various amino acid metabolism, TCA cycle, photosynthetic carbon fixation and sulfur metabolism and other metabolic pathways have been significantly affected by 400 mmol L−1 NaCl treatment. Correlation analysis of differential genes in transcriptome and differential metabolites in metabolome have found that the genes of AMY2, BAM1, GPAT3, ASP1, CML38 and RPL4 and the metabolites of L-cysteine, proline, 4-aminobutyric acid and oxaloacetate played an important role in N. sibirica salt tolerance control. This is a further improvement of the salt tolerance mechanism of N. sibirica, and it will provide a theoretical basis and technical support for treatment of saline-alkali soil and the cultivation of halophytes. [ABSTRACT FROM AUTHOR]

Hirame novirhabdovirus (HIRRV), as a highly pathogenic fish virus, is frequently prevalent in a variety of aquaculture fish in recent years, which seriously threatens the healthy development of aquaculture industry. Epidemiological studies show that the outbreak of HIRRV is obviously temperature dependent. Virus proliferation experiments in vitro and in vivo at different temperatures indicate the antiviral response of flounder is a main reason affect the replication of HIRRV. The RNA-Seq was used to analyze the different antiviral response in flounder which infected with HIRRV at different temperatures, the experiment set two temperatures of 10 °C and 20 °C. The flounder infected with HIRRV by artificial injection, and the spleens were collected at 24 h after infection. Meanwhile, the fish injected with EPC supernatant at different temperatures were set as control groups. It can obtain four pairwise comparison groups if determine a single variable, and the differentially expressed genes were further selected. The results showed that after infection with HIRRV at 10 °C and 20 °C, the differentially expressed genes in spleen were significantly enriched in inflammatory and immune-related pathways like Arachidonic acid metabolism, Cytokine-cytokine receptor interaction, Toll-like receptor (TLR) signaling pathway, RIG-I-like receptor (RLRs) signaling pathway, NOD-like receptor (NLR) signaling pathway and Cytosolic DNA-sensing pathway etc. In addition, the expression of phagocytes, lysosomes, endocytosis related genes were significantly upregulated at high temperature whether HIRRV positive or not. But compared to the infected flounder at 10 °C, some genes of RLRs signaling pathway were significantly upregulated at 20 °C, it can be speculated that RLRs pathway may be related to the anti-HIRRV response of flounder. Therefore, key genes of RLRs signaling pathway including mda5 , lgp2 , mita , mavs , irf3 , irf7 , ifn I-3 and ifn-γ were selected, and the temporal expression patterns of these genes in infected flounder at different temperatures were further detected by qRT-PCR. The results showed that HIRRV infection can significantly stimulate and activate the RLRs pathway of flounder, and the response level of this pathway was significantly higher at 20 °C than 10 °C. In general, this study provides important data for the further study about the pathogenesis of HIRRV infection in flounder. • The mortality of HIRRV-infected flounder at 10 °C was significantly higher than it at 20 °C. • The differential antiviral responses of flounder to HIRRV at 10 and 20 °C were determined by RNA-seq. • The flounder showed significantly different immune responses against HIRRV infection under different temperatures. • HIRRV infection can significantly activate the RLRs pathway of flounder especially at high temperatures. [ABSTRACT FROM AUTHOR]

Glycoprotein is an important immunogenic protein of Hirame novirhabdovirus (HIRRV). In this study, the full-length and N-/C-terminal portions of glycoprotein were recombinantly expressed (rG, rGn and rGc protein), and the induced immune responses were investigated in flounder (Paralichthys olivaceus) model. The results showed that compared to PBS control, rG, rGn and rGc proteins and inactivated HIRRV suspension (iVS) could all stimulate significant increases of flounder CD4-1+, CD4-2+ T lymphocytes and surface IgM positive (sIgM+) B lymphocytes in peripheral blood, spleen and head kidney (p < 0.05). However, no significant differences of the percentages of CD4-1+ or CD4-2+ T lymphocytes were observed among three protein vaccination groups (p > 0.05). iVS could induce the highest mean levels of CD4+ T lymphocytes in peripheral blood and spleen. For sIgM+ B lymphocytes, the average peak percentages in rG and rGc groups were higher than rGn group. Moreover, significant increases of specific serum IgM against HIRRV or rG protein were observed in iVS, rG, rGn and rGc groups, but rG group exhibited the highest mean level. Furthermore, rG protein induced the highest titer of neutralizing antibodies against HIRRV, followed by iVS. Meanwhile, the challenge test showed that the relative percent survival (RPS) of rG, rGn, rGc and iVS groups were 75.0%, 35.7%, 53.6% and 60.7%, respectively. These results revealed that the full-length G protein would be a more effective subunit vaccine candidate against HIRRV infection. • The full-length and N-/C-terminal portions of HIRRV-G induced different immune responses in flounder. • The flounder vaccinated with rG had the highest level of specific serum antibodies against HIRRV and rG. • The flounder vaccinated with rG exhibited the highest neutralizing antibodies titer against HIRRV. • The rG protein conferred the highest RPS against HIRRV and followed by inactivated HIRRV. [ABSTRACT FROM AUTHOR]

Previous studies have demonstrated that white spot syndrome virus (WSSV) could induce hemocytes apoptosis in shrimps, however the inter-relationship between apoptotic process and the WSSV infection status is still currently underexplored. In the present work, the apoptosis and the viral proliferation in hemocytes of Litopenaeus vannamei were simultaneously investigated post WSSV infection by two-color immunofluorescence flow cytometry and real-time quantitative PCR. The apoptotic hemocytes of WSSV-infected shrimp was significantly increased at 12 h post infection (hpi), whereas underwent a slight decline at 24 hpi subsequently. Since 24 hpi, the apoptotic rate of hemocytes in the WSSV-infected shrimp exhibited a rapid and significant increase, and reached the peak level at 48 hpi with the ratio of 18.1 ± 2.0%. Meanwhile, the percentage of WSSV-infected hemocytes and WSSV copies in hemocytes significantly increased at 24 hpi and maintained at a high level afterwards. With the rapid increase of hemocytes apoptosis, hemocyte density in hemolymph decreased dramatically to less than 20% of the mean value of control. Co-localization assay showed that the apoptotic WSSV-infected hemocytes occupied the dominant proportion of total apoptotic hemocytes, which reached the peak at 48 hpi with 12.6 ± 1.5%. The expression profiles of seven pro-apoptotic genes and two apoptosis-inhibiting genes showed significant differential responses at different stages of WSSV infection, reflecting the interplay between the virus and the host immune response. Our results demonstrated that the apoptotic response of shrimp hemocytes could be significantly influenced by the WSSV infection process, which might provide an insight into deeper relationships between viral infection and apoptosis. • The apoptotic hemocytes increased markedly with the rapid proliferation of WSSV. • WSSV-infected hemocytes occupied the dominant proportion of total apoptotic hemocytes. • Apoptosis-related genes showed differential responses at different stages of WSSV infection. • Apoptotic process has a close relationship with WSSV infection status. [ABSTRACT FROM AUTHOR]

Mycotoxins and pesticides are prevalent in cereal food. It is difficult to detect these two kinds of hazard factors simultaneously in rapid assay. In order to find a solution to the problem, carbamates and aflatoxins were selected in this study to establish a rapid, on-site, and quantitative paper sensor. Two novel monoclonal antibodies (mAbs) against carbaryl and carbofuran (1D2 and G11) were developed. The IC50 values (half maximal inhibitory concentration) were 0.8 ng/mL and 217.6 ng/mL for carbaryl and carbofuran, respectively. Based on the sensitive and specific mAbs, a multi-TRFICA (time-resolved fluorescence) paper sensor was developed, which simultaneously detected six types of hazardous chemicals, including AFB 1 , AFB 2 , AFG 1 , AFG 2 , carbaryl, and carbofuran. A universal sample pretreatment method for mycotoxins and pesticides was explored to apply on established competitive indirect enzyme-linked immunosorbent assay (icELISA) and multi-TRFICA-paper sensor. The established paper sensor can be easily observed with naked eyes, qualitatively under a UV lamp, and quantitated using a home-made device. It exhibited a calculated limit of quantity for AFTs, carbaryl, and carbofuran of 0.03, 0.02, and 60.2 ng/mL in corn samples, respectively. The spiking-recoveries and real sample studies proved that multi-TRFICA-paper sensor is an accurate, sensitive, and high throughput detection method for simple and low-cost analysis in corn samples. Image 1 • A paper sensor to detect pesticides and mycotoxins: simultaneity from sample pretreatment to detection. • Sensitive and specific monoclonal antibodies against carbaryl and carbofuran were developed, respectively. • A cost and resource-efficient microscope-based subcloning method was developed in the hybridomas screening. • The on-site paper sensor demonstrated simultaneous quantification of six hazard chemicals rapidly and accurately. [ABSTRACT FROM AUTHOR]

Interleukin-2 (IL-2) is mainly produced by CD4+ T helper lymphocytes, which is an important immunomodulatory cytokine that primarily promotes activation, proliferation and differentiation of T cells. In the present study, flounder (Paralichthys olivaceus) interleukin 2 homologue (poIL-2) was identified for the first time, and its expression patterns were characterized in healthy, virus- or bacteria-infected flounder. The full-length cDNA sequences of poIL-2 was 989 bp with an open reading frame of 423 bp coding a polypeptide of 140 amino acids (aa). The deduced aa sequences shared low similarities (<53%) with other known fish IL-2s. Multiple alignment of aa sequences revealed that poIL-2 own the classical IL-2 family signature of "C-X(3)-EL-X(2)-(T/V)-(V/M/L)-(K/T/R)-X-EC" and "DS-X-(F/L)Y(A/T/S)P". In healthy flounder, IL-2 mRNA was highly expressed in PBLs, spleen and hindgut, and moderately expressed in gill, trunk kidney and stomach. PHA, LPS and Con-A could effectively induce poIL-2 expression in primary cultured peripheral blood leukocytes in vitro. poIL-2 transcripts were significantly up-regulated in spleen, kidney, gill and hindgut post infections with Edwardsiella tarda and Hirame novirhabdovirus (HIRRV). The eukaryotic expression vector encoding poIL-2 (pcIL-2) was constructed and intramuscularly injected, which could be successfully expressed in flounders and induced significantly higher expressions of six immune related genes including poIL-2, β -defensin, CD4-1, CD8α, IFN-γ and TNF-α compared with the injection with control plasmid. Moreover, pretreatment with pcIL-2 could markedly increase the survival rate of flounder challenged with HIRRV. Our results demonstrated that poIL-2 plays an important role in the induction of immune responses and immune defense against bacterial and virus infection, which indicated its potential use as an immunopotentiator to prevent diseases in flounder. • An IL-2 homologue was cloned and characterized in flounder. • The poIL-2 could be significantly elicited by mitogens in vivo and microbial infections in vitro. • Injection of pcIL-2 could upregulate the expressions of six immune-related genes. • pcIL-2 could enhance the resistance of flounder against HIRRV infection. [ABSTRACT FROM AUTHOR]

B cells have been found to have phagocytic activity in recent years, but the studies exploring the regulation mechanisms are still lacking to date. In the present study, the recombinant interleukin-10 (rIL-10) was obtained to study the function of IL-10 on phagocytosis of flounder (Paralichthys olivaceus) mIgM+ B lymphocytes. Flow cytometric analysis showed that IL-10 significantly enhanced the phagocytosis of Edwardsiella tarda but not Lactococcus lactis by mIgM+ B lymphocytes. Moreover, significantly higher intracellular ROS levels were detected in mIgM+ B lymphocytes following rIL-10 stimulation. The qRT-PCR analysis showed that rIL-10 could upregulate the expressions of IL- 10Rb and Stat3 in mIgM+ B lymphocytes, suggesting that IL-10 might modulate the phagocytosis of mIgM+ B lymphocytes by activating IL-10R and Stat3. In addition, we also found that the enhancing effect of IL-10 on phagocytosis and intracellular ROS levels of mIgM+ B lymphocytes were suppressed by the administration of niclosamide. These results collectively demonstrated that IL-10 enhanced mIgM+ B lymphocyte-mediated phagocytosis of E. tarda and intracellular bactericidal ability, and IL-10R and Stat3 might play a curial role in the regulation of IL-10-stimulated phagocytosis, which would deepen our understanding of regulation mechanism of B cell phagocytosis. • IL-10 enhanced phagocytosis of E. tarda but not L. lactis by mIgM+ B lymphocytes. • IL-10 enhanced the intracellular ROS activity of mIgM+ B lymphocytes. • High purity of flounder mIgM+ B lymphocytes were successfully sorted. • IL-10 modulated the phagocytosis of mIgM+ B lymphocytes via IL-10R and Stat3. [ABSTRACT FROM AUTHOR]

Biotoxins, particularly mycotoxins, marine toxins, and bacterial food toxins, have emerged as major threats in food, feed, seafood, and medicine fields. They have potential carcinogenic, teratogenic, and mutagenic effects on humans, occasionally causing high mortality and morbidity. One of the evident issues is related to the ever-growing use of fast food and demand for prepared food without proper acknowledgment of associated toxins. Hence, developing better methods for monitoring biotoxins is of great significance. Recently, biosensors have been increasingly used due to their simple and quick detection procedure, less sample volume, low cost, and so on. This study aimed to extrapolate the application of biosensors in biotoxin detection in recent years, with particular emphasis on their optical, electrochemical, piezoelectric, and photothermal applications. Further, different detection platforms were integrated as multimodal combination biosensors for biotoxins to meet the requirement of highly accurate technologies. This study examined the major challenges faced by biosensors and provided novel insights for improving the technology in food safety. • The evident concerns were related to the demand for prepared food without proper acknowledgment of biotoxins. • Recent progress in the analysis of biotoxins as threats was reviewed. • Different kinds of composite nanomaterials were investigated to develop biosensors. • Multimodal technologies, for example, smart biosensors, are needed to tackle this challenge. [ABSTRACT FROM AUTHOR]

In order to investigate the dynamic distribution of antigen in different tissues post vaccination, an absolute real-time quantitative PCR was employed to detect the amount of antigen in flounder (Paralichthys olivaceus) post intraperitoneal (i.p.) injection with three concentrations (107, 108, 109 CFU ml−1) of formalin-inactivated Edwardsiella tarda bacterin. The results showed that the amount of uptaken antigen quickly increased and then decreased in different tissues. The peak occurred first in the spleen and head kidney at 6–9 h after injection, and in the liver and blood at 9–15 h, then in the gill, intestine and skin at 15–24 h, finally in the muscle at 24–36 h. The amount of antigen was highest in the spleen and head kidney, followed by the blood, liver and gill, and lowest in the intestine, skin and muscle. Among the three concentration groups, the amount of antigen increased with the increasing concentration of the vaccine in the blood, liver, gill, intestine, skin and muscle, except for the spleen and head kidney, in which more antigens were found in the 108 CFU ml−1 group than that in 109 CFU ml−1 group. Moreover, IIFA and western blotting was performed to examine the tissue distribution of antigen at 9 h after vaccination with 108 CFU ml−1 formalin-inactivated E. tarda. The bacteria were mainly observed in the spleen and head kidney, then the liver, gill and blood, and least in the intestine, skin and muscle, which was roughly in accordance with the results of absolute qPCR. Furthermore, the expressions of CD4-1, MHC IIα, CD8α and MHC Iα in different tissues were detected by RT-qPCR, and the expression levels of these genes were highest in the spleen and head kidney, then in the blood, gill, liver, and lowest in the intestine, skin and muscle. All these results provided useful information for dynamic transportation of antigen uptake post vaccination, and also deepened the understanding of immune response to the injection vaccination. • The dynamic changes of uptaken antigen among tissues have been explored. • The spleen and head kidney are the major tissues to take up antigen. • The highest amount of antigen was taken up in the 108 CFU ml−1 vaccination group. • Antigen presentation related genes were directly affected by the amount of antigen. [ABSTRACT FROM AUTHOR]

Abstract Vibrio ichthyoenteri is a marine pathogen that primarily causes bacterial enteritis of flatfish. In order to screen out the effective vaccine candidate proteins, five outer membrane proteins (OMPs) of V. ichthyoenteri , including OmpV, OmpU, OmpT, Omp1 and Omp2, were selected and recombinantly expressed in Escherichia coli. The result of western blotting showed that all of these recombinant proteins could be recognized by flounder anti- V. ichthyoenteri antibodies. The immune protective effect of the five rOMPs were also investigated, and the relative percent survival (RPS) of rOmpV, rOmpU, rOmpT, rOmp1 and rOmp2 was 69.2%, 46.2%, 76.9%, 65.4% and 30.8%, respectively. The RPS of rOmpT was significantly higher than inactivated V. ichthyoenteri (57.7%) and other rOMPs, so the immune responses of flounder induced by rOmpT were further investigated. The results showed that both the production of specific serum antibodies and the proliferation of sIg + lymphocytes could be significantly induced by rOmpT. Meanwhile, the MHCIIα , INF-γ , CD4-1 , and IL-1β genes were significantly up-regulated after immunization with rOmpT. These results demonstrated that rOmpT could induce strong innate and humoral immune response in flounder and provided effective immune protection against V. ichthyoenteri challenge, which indicated that OmpT is a promising vaccine candidate against V. ichthyoenteri infection. Highlights • OmpT of V. ichthyoenteri is an immunogenic protein. • The RPS evoked by rOmpT was the highest compared with the other OMPs and the inactivated V. ichthyoenteri. • ROmpT could activate the innate and humoral immune responses of flounder. [ABSTRACT FROM AUTHOR]

Abstract Previous studies have demonstrated that protein tyrosine phosphorylation plays an important role in WSSV infection. In the present work, in order to further elucidate the potential role of protein tyrosine phosphorylation in white spot syndrome virus (WSSV) infection. The expression variation of tyrosine phosphorylated proteins in hemocytes of shrimp (Litopenaeus vannamei) after WSSV infection were examined by flow cytometric immunofluorescence assay (FCIFA) and enzyme linked immunosorbent assay (ELISA), and results showed that the level of protein tyrosine phosphorylation in hemocytes fluctuated significantly after WSSV infection and exhibited two peaks at 6 and 24 h post infection (hpi). Meanwhile, tyrosine phosphorylated proteins in hemocytes after WSSV infection were also detected by cell immunofluorescence, and results showed that the fluorescence intensity in hemocytes was altered with the course of WSSV infection and showed stronger fluorescent signals at 6 and 24 hpi compared to other time points. Furthermore, two dimensional gel electrophoresis (2-DE) and 2-DE western blotting were applied to identify the differentially expressed tyrosine phosphorylated proteins in hemocytes before and after WSSV infection. The result of 2-DE western blotting showed that there were nine tyrosine phosphorylated proteins in the hemocytes of healthy shrimp, whereas twenty-one tyrosine phosphorylated proteins were detected in the hemocytes of shrimp at 6hpi. Then, the differential tyrosine phosphorylated proteins were analyzed by Mass Spectrometry (MS), and eight of them were identified to be sodium/potassium-transporting ATPase subunit alpha, ubiquitin/ribosomal L40 fusion protein, actin-D, phosphopyruvate hydratase, beta-actin, ATP synthase subunit beta, receptor for activated protein kinase c1 and protein disulfide-isomerase. Moreover, the expression levels of sodium/potassium-transporting ATPase subunit alpha, ubiquitin/ribosomal L40 fusion protein, phosphopyruvate hydratase, ATP synthase subunit beta , receptor for activated protein kinase c1 and protein disulfide-isomerase were examined to be up-regulated post WSSV infection by quantitative real-time RT-PCR. Taken together, these results demonstrated that protein tyrosine phosphorylation was involved in the process of WSSV infection, which might play an important role in the immune response to WSSV infection in shrimp. Highlights • The level of protein tyrosine phosphorylation in hemocyte exhibited two peaks post WSSV infection. • Tyrosine phosphorylated proteins were detected in hemocytes by cell immunofluorescence. • 12 differentially tyrosine phosphorylated proteins were identified post WSSV infection. [ABSTRACT FROM AUTHOR]

Highlights • The mIgT and sIgT genes of turbot are cloned and characterized. • The sIgT and mIgT were differentially expressed in different tissues of healthy turbot. • The mIgT were mainly induced in systematic organs post injection and immersion vaccinations. • The sIgT were mainly induced in mucosa-associated tissues post vaccinations. Abstract Immunoglobulins (Igs) are humoral mediators playing the prevailing role in the innate and adaptive immunities of jawed vertebrates and provide obligatory duties to protect the organism from a wide variety of pathogens. In the present study, the membrane-bound and secretory immunoglobulin T (mIgT and sIgT) genes of turbot (Scophthalmus maximus) were cloned for the first time with the intention of better understanding the IgT functions. The mIgT cDNA is 2, 049 bp in length including a leader region, a variable region, four constant regions and a transmembrane region (TM), while the 1, 932 bp sIgT cDNA lacks the transmembrane region. In healthy turbot, the total IgT was highly expressed in gill, spleen and liver followed by peripheral blood leucocytes (PBL), skin and hindgut, and then in stomach, head kidney, trunk kidney, midgut and foregut. The expression levels of sIgT in PBL, gill, skin and spleen were much higher than mIgT. Furthermore, the expression profiles of turbot mIgT and sIgT were investigated post vaccination with formalin-inactivated Vibrio anguillarum via intraperitoneal injection and immersion, and the results showed that the expressions of mIgT and sIgT were both significantly induced by two administration routes, whereas intraperitoneal injection mostly induced mIgT expression in systematic organs including head kidney, spleen and trunk kidney, and the immersion vaccination elicited a much stronger response of sIgT in mucosa-associated tissues including gills, liver, hindgut and skin. Taken together, these results demonstrated that mIgT and sIgT were differentially expressed in different tissues and both responded positively to the vaccinations in turbot, and indicated that IgT-secreting plasma cells are abundant in mucosa-associated tissues and played important roles in mucosal immunity of turbot. [ABSTRACT FROM AUTHOR]

Cytokines have the potential as adjuvants for the application of vaccines in mammals. However, the adjuvant potential of teleost cytokines was limited. In the present work, the adjuvant effects of four recombinant cytokines including IL-1β, IL-8, TNF-α and G-CSF on E. tarda subunit vaccine rOmpV were comparatively investigated in flounder ( Paralichthys olivaceus ). Compared with control, the levels of specific serum antibodies and IgM + B lymphocytes were significantly enhanced by rIL-1β, rIL-8 and rG-CSF, whereas rIL-1β and rIL-8 induced significantly higher levels than rG-CSF. All four cytokines enhanced the expression of genes involved in humoral and/or cellular immunities, whereas rIL-1β and rIL-8 induced highest levels of genes involved in humoral immunities and cellular immunities, respectively. Compared to the relative percent survivals (RPS) of control group (40%) and rOmpV plus rG-CSF group (54%), rOmpV plus rIL-1β or rIL-8 produced higher RPS of 75% and 68%, respectively. Our results indicated that rIL-1β and rIL-8 are promising adjuvants for subunit vaccines against E. tarda . [ABSTRACT FROM AUTHOR]

Simple Summary: The Major histocompatibility complex (Mhc) plays an essential role in antigen presentation as part of the adaptive immune system. To investigate the role of MhcII in adaptive immunity, this study cloned the mhcIIα and mhcIIβ of flounder (Paralichthys olivaceus) and examined their distribution and expression patterns, which varied by antigen stimulation or pathogens infection. The analyses showed that they were significantly expressed in gills, spleen, and peripheral blood leukocytes (PBLs), and MhcII molecules were co-localized with CD83 and IgM on leukocytes, respectively. The expression of both mhcIIα and mhcIIβ were significantly upregulated in flounder after infection. The percentages of MhcII+ cells, MhcII+/CD83+, and MhcII+/IgM+ cells increased significantly after PHA and ConA stimulation, respectively; they varied significantly in PBLs after polyI:C stimulation and no obvious variations were found after LPS treatment. These results suggested that MhcII, mainly expressed in B cells and dendritic cells, responded significantly to exogenous antigens and T cell-dependent antigens. In conclusion, MhcII was associated with cellular immunity in teleosts. 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. [ABSTRACT FROM AUTHOR]

Abstract B cells in some fish were recently found to have potent phagocytic activities. Sea bass ( Lateolabrax japonicus ) as an important economical marine fish species, it could be used as an appropriate model to study the functions of B cells in phagocytosis. In the paper, three positive hybridomas designated as 1E11, 2H4 and 3F3 secreting monoclonal antibodies (MAbs) against sea bass immunoglobulin M (IgM) were produced and used as research tools. Indirect enzyme-linked immunosorbent assay showed that all the three MAbs had a high binding capacity with sea bass serum IgM. Western blotting analysis showed that all the three MAbs were specific for the heavy chain of sea bass IgM. Indirect immunofluorescence assay (IFA) analysis suggested that both MAbs 1E11 and 2H4 could recognize membrane-bound IgM (mIgM) molecule of sea bass. Specificity analysis showed that three MAbs had no cross-reactions with other six teleosts IgMs. Flow cytometric analysis exhibited that the percentages of sea bass mIgM + lymphocytes in peripheral blood, spleen and pronephros were 25.6%, 21.1%, and 17.5%, respectively. Moreover, we found that the mIgM + lymphocytes of sea bass could phagocytose fluorescence microspheres and Lactococcus lactis , but lower phagocytosis rates of L. lactis was observed. These results demonstrated that the MAbs produced in this paper could be used as tools to study secretory IgM and mIgM + lymphocytes of sea bass, and mIgM + lymphocytes might also play an important role in innate immunity of sea bass. Highlights • MAbs against IgM of sea bass are produced and characterized. • The produced MAbs are special for the sIgM and mIgM of sea bass. • The produced MAbs don't cross-react with other six teleosts' IgMs. • The mIgM + lymphocytes of sea bass possess phagocytic capacity. • The phagocytosis rates of L. lactis are lower than those of fluorescence microsphere. [ABSTRACT FROM AUTHOR]

Intramuscular (i.m.) injection is one of the common delivery methods of vaccination in aquaculture, which could induce an ideal immune protection to fish. In the present study, the olive flounders ( Paralichthys olivaceus ) were injected intramuscularly with 200 μl of three concentrations of formalin-inactivated Edwardsiella tarda bacterin (10 7 , 10 8 , 10 9 CFU ml −1 ) to investigate the transportation and dynamic distribution of antigen uptake in tissues by absolute real-time quantitative PCR (qPCR). The amount of uptaken antigen increased firstly, and then decreased. The peak occurred first in the blood at 6–9 h after i.m. injection, and in the spleen and head kidney at 9–15 h, then in the liver, gill and muscle at 15–24 h, finally in the skin and intestine at 36 h. The amount of uptaken antigen was highest in the head kidney, followed by in the spleen, blood, gill, and liver, and lowest in the muscle, skin and intestine. Among the three dose groups, the amount of uptaken antigen in all tested tissues became higher with the increasing dose of injected bacterin. Moreover, the tissue distribution of antigen uptake was investigated by indirect immunofluorescence assay (IIFA) at 15 h after i.m. injection with 200 μl of 10 8 CFU ml −1 E. tarda bacterin. The distribution of antigen was mainly observed in the head kidney, then in the spleen, blood, liver, gill and muscle, and least in the skin and intestine, which correlated with the results of absolute qPCR detection. Furthermore, the expression levels of MHC Iα, MHC IIα, CD4-1 and CD8α were detected by RT-qPCR. The expression of these four genes peaked highest in the head kidney, followed by in the spleen, liver, blood and gill, and lowest in the muscle, skin and intestine, and the levels increased in parallel with the increasing dose of injected vaccine. All these results provided an important insight into the dynamic transportation of antigen uptake, and also deepened the understanding of immune response to the i.m. injection. [ABSTRACT FROM AUTHOR]

Edwardsiella tarda has become one of the most severe fish pathogens throughout the world. Thus, studies on the design and production of highly protective vaccines against this pathogen, as well as the mechanisms of vaccine-induced disease resistance are urgently needed. In this study, the NADP-dependent isocitrate dehydrogenase (IDH) of E. tarda was recombinantly expressed and flounder ( Paralichthys olivaceus ) anti-rIDH serum was prepared. Also, the outer membrane proteins (OMPs) of E. tarda were extracted and analyzed by western blotting. The results showed that flounder anti-rIDH serum could specifically recognize a 44 kDa protein of E. tarda OMPs, which was identified to be the native IDH of E. tarda by mass spectrometric. Consistently, immunogold electron microscopy showed that IDH could be detected on the membrane of E. tarda . Then, the vaccine potential of rIDH was tested in a flounder model, and the results showed that rIDH produced a relative percent survival (RPS) of 73.3%, which was significantly higher than that produced by formalin killed E. tarda cells. Immunological analysis showed that rIDH could induce the proliferation of rIDH-specific sIg+ lymphocytes, which resulted in the production of anti- E. tarda antibodies. Accordingly, serum bactericidal activity assay showed that the serum of rIDH vaccinated fish exhibited the highest bactericidal activity compared with other groups. qRT-PCR analysis showed that rIDH could enhance the expressions of IFN-γ, NKEF, IL-6, MHCIα, CD4-1 and CD8α. Moreover, the bacterial burden was also detected in vaccinated fish after challenge, which showed that the number of E. tarda cells in spleen of rIDH group was significantly lower than other groups. All these results suggested that rIDH is a promising vaccine candidate against E. tarda infection. [ABSTRACT FROM AUTHOR]

Developing a rapid, accurate and quantitative method for detecting white spot syndrome virus (WSSV) is extremely urgent and critical for reducing the risk of white spot disease outbreaks. In the present work, an optimized double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for quantitative detection of WSSV. The method employed rabbit polyclonal antibodies against WSSV as the capture antibody and previously produced anti-WSSV monoclonal antibodies as the detector antibody. A standard curve of the log concentration of WSSV versus OD value was established, which was linear in the concentration range of 120–7680 ng/mL, and the linear regression equation was y = 0.166x − 0.151. Viral proteins in different tissues of crayfish ( Procambarus clarkia ) post artificial infection with WSSV were quantitatively measured using the DAS-ELISA. WSSV proliferated quickly within 60 h post infection and gradually slowed down afterwards. According to the linear regression relationship, the viral proteins in hemolymph, gut and gonad were firstly able to be quantified at 24 h post infection with the concentrations of 186, 158 and 128 ng/mL, respectively. These three tissues also contained higher viral proteins than the gill, heart, hepatopancreas and muscle during the entire infection period. The viral protein concentration in gut reached the highest level of 6220 ng/mL at 72 h post infection. Real time quantitative PCR was also used to detect the dynamic change of viral copies in crayfish hemolymph post WSSV infection, with similar results for both assays. The developed DAS-ELISA could detect WSSV propagation from initial to moribund stage in infected crayfish and demonstrated potential application for diagnosis of WSSV. [ABSTRACT FROM AUTHOR]

Vibrio ichthyoenteri was an important causative agent of bacterial enteritis in flounder ( Paralichthys olivaceus ). Outer membrane protein A (OmpA) of Gram-negative pathogen was a major cell surface antigen. In the present study, OmpA of V. ichthyoenteri was recombinantly expressed in Escherichia coli , and the immunogenicity of OmpA was identified by western blotting using flounder anti-rOmpA and anti- V. ichthyoenteri antibodies. The vaccine potential of rOmpA was tested in a flounder model, and a high relative percentage of survival rate was obtained with 73.1% after challenge with V. ichthyoenteri . Meanwhile, the immune response of flounder induced by rOmpA was also investigated, and the results showed that the sIg + lymphocytes in blood, spleen, and pronephros significantly proliferated, and the peak levels occurred at week 4 after immunization. Moreover, rOmpA could induce higher levels of specific serum antibodies than the control group after immunization, and the peak level occurred at week 5 after immunization. Meanwhile, qRT-PCR analysis showed that the expressions of CD4-1, CD8α, IL-1β, IFN-γ, MHCIα and MHCIIα genes were significantly up-regulated after immunization with rOmpA. Taking together, these results demonstrated that rOmpA could evoke highly protective effects against V. ichthyoenteri challenge and induce strong immune response of flounder, which indicated that OmpA was a promising vaccine candidate. [ABSTRACT FROM AUTHOR]

Fumonisins (FBs) are widely found in rice, maize, peanut, wheat, and other agricultural products. These have been detected using a chromatography technique, whereas the rapid assay by a highly sensitive monoclonal antibody is minimally reported. Herein, a highly sensitive monoclonal antibody (7A11) was successfully developed by hybridoma technique. The 50% inhibition concentration (IC 50 ) of 7A11 monoclonal antibody was 0.32 ng/mL in an optimized buffer. The cross-reactivity between fumonisin B 1 and fumonisin B 2 , B 3 was 4.3% and 12.8%, respectively. Based on the newly developed 7A11 antibody, a high sensitivity indirect competitive enzyme-linked immunosorbent assay (icELISA) and gold nanoparticles-based gray imaging quantification immunoassay (GNPs-GI) were established. The analytical range of icELISA was 0.08–1.38 ng/mL and that for GNPs-GI was 0.24–15 ng/mL. Both the methods showed adequate recoveries (80.0–105.8% for icELISA and 78.5–115.2% for GNPs-GI) in spiked samples. Compared to high-performance liquid chromatography (HPLC), icELISA and GNPs-GI indicated reliability that could be used for further detection of fumonisin B 1 in agricultural products. [ABSTRACT FROM AUTHOR]

Hirame rhabdovirus (HIRRV) is a rhabdovirus that causes severe disease in fish. The mortality due to HIRRV infection occurs at temperatures below 15 °C, but no mortality is observed over 20 °C. In this study, Japanese flounder ( Paralichthys olivaceus ) was artificially infected with the HIRRV CNPo2015 strain at 10 °C or 20 °C. Absolute quantitative real-time PCR was employed to examine the viral replication in spleens after HIRRV infection. Expression profiles of four interferon-related genes (type I IFN, Mx, ISG15, MDA5) and two proinflammatory genes (TNF-α and IL-1β) were also investigated by quantitative real-time PCR. Results showed that viral copies in spleens increased gradually over time and peaked at 72 h post infection (hpi) in the 10 °C group, while viral copies in the 20 °C group increased within 24 hpi, but afterwards decreased to very low levels. Moreover, the expressions of IFNs in the 10 °C group reached the highest levels at 72 hpi, whereas their peak levels appeared much earlier in the 20 °C group, at 12 hpi. The expression levels of TNF-α and IL-1β in the 10 °C group peaked at 12 hpi and then quickly declined. However, the two genes were highly expressed during 6–24 hpi in the 20 °C group. Based on these findings, we concluded that HIRRV infection induced an efficient antiviral immune response at 20 °C, which might inhibit the viral transcription at early stages and finally prevent HIRRV infection. [ABSTRACT FROM AUTHOR]

In this work, the two positive hybridomas designated as 1B2 and 2G9 secreting monoclonal antibodies (MAbs) against half-smooth tongue sole IgM were produced and then cloned by limited dilution. Western blotting showed that MAbs 1B2 and 2G9 could specifically react with the heavy and light chains of IgM, respectively. The indirect immunofluorescence assay (IFA) showed that green fluorescent signals were detected on the membrane of lymphocytes, which indicated that membrane-bound IgM (mIgM) could be recognized by both MAbs 1B2 and 2G9. Specificity analysis showed that no cross-reactions were detected between IgM of half-smooth tongue sole and other five teleosts IgMs using MAbs 1B2 and 2G9. Flow cytometric analysis (FCA) demonstrated that the percentages of mIgM+ lymphocytes in peripheral blood, spleen and pronephros of health half-smooth tongue sole were 21.1%, 17.6% and 16.1%, respectively. Moreover, the phagocytosis rates of fluorescence microspheres by mIgM+ lymphocytes in isolated leukocytes from peripheral blood, spleen and pronephros of health half-smooth tongue sole were determined to be 4.6 ± 0.5%, 3.6 ± 0.4% and 4.2 ± 0.3% by FCA, respectively, and the proportions of mIgM+ phagocytic lymphocytes in total mIgM+ lymphocytes in the peripheral blood, spleen and pronephros were calculated to be 21.6 ± 1.1%, 20.6 ± 1.8% and 26.0 ± 0.8%, respectively. These results demonstrated that the MAbs produced in this study could specifically recognize the serum IgM and mIgM on B lymphocytes of half-smooth tongue sole. Meanwhile, partial mIgM+ lymphocytes have the phagocytic capacity, which indicates mIgM+ lymphocyte play important role in innate immune response. [ABSTRACT FROM AUTHOR]

Immunoglobulin D (IgD) is considered to be an enigmatic Ig molecule because of the lack understanding of its immunological functions. In the present study, a partial δ region of the flounder IgD was recombinantly expressed, purified and used as an immunogen to produce monoclonal antibodies (MAbs) against the H chain of flounder IgD. After fusion, a total of 97 hybridomas were generated and observed under an inverted microscope One of the hybridomas, designated 5G7, gave strong positive results in an indirect enzyme-linked immunosorbent assay (ELISA) and was cloned and subcloned by limiting dilution. Western blot analysis showed that MAb 5G7 could specifically recognize a 118 kDa protein from peripheral blood lymphocytes (PBLs), which was identified to be the H chain of flounder IgD by mass spectrometric analysis. Indirect immunofluorescence assay tests (IIFAT) showed that specific fluorescence signals were observed on the membranes of the PBLs, which suggests that MAb 5G7 could recognize the membrane-bound IgD molecule. Moreover, only the subset of IgD+/IgM + B cells were observed in the PBLs of healthy flounder when tested by flow cytometry analysis. Consistent with the results of flow cytometry, a double immunofluorescence assay test (DIFAT) showed that the positive lymphocytes were stained with both green and red fluorescence signals, which represent the IgM+/IgD + lymphocytes subset. These results demonstrate that the produced MAb 5G7 could specifically recognize the flounder IgD, which provides a useful tool to study the functions of flounder IgD. [ABSTRACT FROM AUTHOR]

The CD3 complex is an important cell surface marker of T lymphocytes and essential for T lymphocytes activation in higher vertebrates. In the present work, the CD3ε of Japanese flounder ( Paralichthys olivaceus ) was recombinantly expressed in E. coli BL21 (DE3) and used as an immunogen to produce mouse anti-rCD3ε polyclonal antibodies, which could specifically recognize a 20 kDa protein in the membrane proteins of peripheral blood lymphocytes (PBL) of Japanese flounder by co-immunoprecipitation assay. Mass spectrometric analysis showed the 20 kDa protein was the native CD3ε of Japanese flounder. Both the flow cytometric analysis and double immunofluorescence assay (DIFA) showed that the CD3 + T lymphocytes could be identified specifically by the mouse anti-rCD3ε polyclonal antibodies, which didn't cross-react with the sIgM + lymphocytes. Immunohistochemistry showed that CD3 + T lymphocytes could be detected in gill, skin, stomach, intestine, spleen, liver, head-kidney and mid-kidney. Flow cytometric analysis showed the percentages of CD3 + T lymphocytes in the PBL, spleen lymphocytes (SL) and head-kidney lymphocytes (HKL) of Japanese flounder increased rapidly after immunization with formalin-inactivated Edwardsiella tarda , and reached their peak levels at 5th day with 12.6%, 9.7% and 8.7%, respectively, and then decreased gradually. These results suggested that CD3 + T lymphocytes play important roles in mucosal and cell-mediated immunity, and the results would deepen our understanding on the roles of teleost T lymphocytes in the immune response. [ABSTRACT FROM AUTHOR]

Outer membrane protein C is a porin that resides in the outer membrane, and it has been identified as an antigenic protein in many bacteria, such as Escherichia coli , Salmonella typhi , Aeromonas hydrophila and Edwardsiella tarda . In the present work, we aimed at evaluating the immune protective potential of E. tarda OmpC as a DNA vaccine. For this purpose, a recombinant DNA plasmid encoding OmpC (pCG-OmpC) gene was constructed and its vaccine potential was analyzed in a flounder ( Paralichthys olivaceus ) model. The results showed that pCG-OmpC produced a relative percent survival (RPS) of 55% following E. tarda challenge at 6 w post vaccination. Moreover, OmpC transcripts and OmpC-GFP fusion protein could be detected in flounder after immunization with pCG-OmpC, and OmpC-GFP fusion protein could also be detected in HINAE cells after transfection with pCG-OmpC. Meanwhile, the immune responses induced by pCG-OmpC were investigated, and the results showed that pCG-OmpC could significantly induce the production of specific serum antibodies and the proliferation of sIg + lymphocytes in blood, spleen and pronephros. qRT-PCR analysis showed that the expressions of TLR5M, MyD88, NF-κB, MHCIα, MHCIIα, CD4-1, CD8α, IL-1β and TNF-α genes were significantly induced after vaccinated with pCG-OmpC, while there was no significant difference in expressions of TLR2 and IL-6 between pCG-OmpC and PBS injected fish. Taking together, these results indicated that pCG-OmpC could induce strong innate, humorol and cellular immune responses of flounder, and finally evoked highly protective effects, suggesting that pCG-OmpC was a promising DNA vaccine candidate. [ABSTRACT FROM AUTHOR]

To obtain effective vaccine candidates for Edwardsiella tarda , six outer membrane proteins (OMPs) of E. tarda were selected and recombinantly expressed. Western blotting analysis showed that flounder anti- E. tarda serum could recognize rOmpV, rOmpK, rOmpX and rOmpI, but not rOmpH and rOmpR. Immunization of flounder with different rOMPs resulted in high levels of protection following the E. tarda challenge with relative percent survivals (RPS) of 92.1% (rOmpI), 84.2% (rOmpX), 68.4% (rOmpK), 60.5% (rOmpV), 57.9% (rOmpH) and 39.5% (rOmpR). Meanwhile, the immune response of flounder induced by rOMPs was investigated, and the results showed that: (1) The levels of specific antibodies against E. tarda induced by rOmpI and rOmpX were significantly higher at weeks 3 and 4 post-vaccination than those of the other rOMPs; (2) all six rOMPs could induce the proliferation of sIg+ lymphocytes, and the peaks of sIg+ lymphocytes in the blood and spleen of rOmpI-, rOmpX- and rOmpH-vaccinated fish were significantly higher than those of other rOMPs; (3) all six rOMPs could induce up-regulation of the MHC IIα, CD4-1, CD4-2, CD8α, CD8β, IL-1β, TNF-α and IFN-γ genes. Among them, the mRNA levels of the CD4-1 and CD4-2 genes induced by rOmpI and rOmpX were generally higher than those induced by the other rOMPs, while there was no significant difference in the expression of TLR2, TLR5M and MHC Iα between the rOMPs-vaccinated group and control group. These results demonstrate that rOmpI and rOmpX could produce a RPS of over 80% against the challenge of E. tarda and induce stronger immune responses in flounder, which indicates that OmpI and OmpX are promising vaccine candidates against E. tarda infection. [ABSTRACT FROM AUTHOR]

Our previous work has demonstrated that the immune response of Japanese flounder was associated with the concentration of formalin-inactivated Edwardsiella tarda and immersion time. In order to further investigate the influence of immersion vaccine dose and bath time on the antigen uptake, formalin-killed Edwardsiella tarda bacterin was prepared and adjusted to four concentrations (10 9 , 10 8 , 10 7 , 10 6 cfu ml −1 ) for 30, 60 and 90 min immersion in Japanese flounder model, respectively. Absolute quantitative real-time PCR was employed to examine the bacterin uptake in gill, skin, spleen and kidney at 3 and 6 h post vaccination. The results showed that the antigen uptaken in gills and skin were significant higher than spleen and kidney, and the antigen amounts in gill and skin both declined from 3 to 6 h, whereas the antigen amounts in spleen and kidney gradually increased. Significant higher antigen amounts were detected in 10 9 -30, 10 9 -60, 10 8 -60, 10 8 -90 and 10 8 -90 groups than other groups (P < 0.05), especially the 10 8 -60min group displayed the highest antigen uptaken. Meanwhile, the expression profiles of antigen recognization and presentation genes (MHCⅡα, TcRα, CD4-1), immunoglobulins (IgM, IgT), inflammatory cytokines (IL-1β, IL-6), heat shock protein 70 (HSP70) and c-type lysozyme were analyzed using real-time PCR. On the whole, the transcription levels of the eight genes exhibited to be higher in 10 7 -90, 10 8 and 10 9 cfu ml −1 groups than other groups (P < 0.05), especially the 10 8 -60 group displayed the highest up-regulation. These results demonstrated that immersion with formalin-inactivated E. tarda, especially under 10 8 -60 min condition could efficiently enhance the antigen uptake and the expression of immune-related genes, which provided evidences for an enhanced vaccination effects under an optimized combination of vaccine dose and immersion time. [ABSTRACT FROM AUTHOR]

• NH 4 F was employed to preparate TiO 2. • NH 4 F-assisted preparation improve the specific surface area of TiO 2. • Separation of photoinduced carriers is significantly boosted. • NH 4 F-TiO 2 exhibits higher photocatalytic activity than TiO 2. In this study, TiO 2 photocatalyst was prepared by a hydrothermal method with the assistance of NH 4 F. The prepared samples were characterized by surface photovoltage spectrum (SPS), UV–vis diffusion reflectance spectrometry (DRS), X-ray diffraction (XRD), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS). The results show that NH 4 F-assisted preparation can enlarge the light absorption range of TiO 2 , improve the specific surface area, and effectively enhance the separation efficiency of photogenerated carriers of TiO 2. The catalytic activity of NH 4 F-TiO 2 for degradation of rhodamine B (RhB) under a xenon lamp was studied. The results show that the catalytic activity of NH 4 F-TiO 2 is 2.6 times higher than that of the blank TiO 2. [ABSTRACT FROM AUTHOR]

In the present study, molecular chaperone GroEL of Edwardsiella tarda was found both react with flounder ( Paralichthys olivaceus ) anti-recombinant GroEL (rGroEL) and anti- E. tarda antibodies, and it was both detected in the outer membrane and the secretome. To evaluate the vaccine potential of GroEL, pCI-neo-GFP-GroEL (pCG-GroEL) was prepared and used to vaccinate healthy flounder, and immune protective effects were investigated. The results showed that pCG-GroEL produced an RPS of 60% following E. tarda challenge at week 5 after immunization. Moreover, GroEL transcripts and GFP-tagged GroEL could be detected in vaccinated flounder after immunization with pCG-GroEL, and the GFP-tagged GroEL could be also detected in HINAE cells after transfection with pCG-GroEL. Meanwhile, the immune response of flounder induced by pCG-GroEL was investigated, and the results showed that: (1) the levels of specific serum antibodies against E. tarda induced by pCG-GroEL were significantly higher than other groups at 3–5 week post vaccination; (2) pCG-GroEL could induce the proliferation of sIg+ lymphocytes, and the levels of sIg+ lymphocytes in blood, spleen and pronephros of pCG-GroEL vaccinated fish were significantly increased compared with the PBS vaccinated fish at 3 w, 2 w and 2 w after immunization, respectively; (3) MHCIα, MHCIIα, CD4-1, CD8α, IL-1β and TNF-α were significantly induced by immunization with pCG-GroEL, and the mRNA levels of CD4-1 and CD8α rapidly increased in pCG-GroEL vaccinated fish after challenge with E. tarda . Taking together, pCG-GroEL could elicit highly protective effects against E. tarda and induce strong immune response of flounder, suggesting that GroEL was a promising vaccine candidate. [ABSTRACT FROM AUTHOR]

Antigen uptake is a critical process for activation of the immune system, and therefore the ability to enhance antigen uptake is a primary consideration in the development of an immersion vaccination of fish. In the present work, flounders ( Paralichthys olivaceus ) were immersed in three hyperosmotic solutions with 40, 50 and 60‰ salinities, then transferred into seawater of normal salinity (i.e. 30‰) containing formalin-inactivated Edwardsiella tarda for 30 min. The antigen uptake in vaccinated flounder was determined using an absolute quantitative PCR (qPCR). The results showed significantly higher antigen uptake in the tissues of flounders immersed in solutions with 50‰ and 60‰ salinity compared to the control group directly immersed in vaccine (DI) ( P < 0.05), and the highest amount of antigen was detected in flounders immersed in the 50‰ salinity solution, whereas there was no significant difference in antigen uptake between the 40‰ salinity group and the DI group ( P > 0.05). A rapid and significant increase in antigen uptake was detected in the mucosal-associated tissues including the gill, skin and intestine ( P < 0.05) compared with the spleen, kidney and liver. Antigen uptake in the gill and skin both peaked at 30 min post immersion, which was significantly higher than the levels of uptake measured in the other tissues ( P < 0.05), and then quickly declined. In contrast, antigen uptake in the spleen, kidney and liver gradually increased 3 h post immersion (hpi). The expression profiles of four antigen presentation-related immune genes (MHC Iα, MHC IIα, CD4-1 and CD8α) were investigated after immersion. These four genes showed a significantly stronger response in the immersed flounders exposed to 50‰ salinity compared with the DI group ( P < 0.05). In the mucosal-associated tissues, the expression of MHC Iα and CD8α genes peaked at 24 hpi, while the expression of MHC IIα and CD4-1 genes showed up-regulation in the gill and skin and reached the peak in these tissues at 48 hpi. The expressions of the four genes were also up-regulated in spleen, kidney and liver, but reached peak expression in these tissues at 48–72 hpi. The results demonstrated that hyperosmotic immersion, notably 50‰ salinity significantly enhanced antigen uptake and the expression of selected genes associated with antigen presentation, providing evidence for an enhanced immune activation of the fish’s immune response by the hyperosmotic immersion treatment prior to vaccination. [ABSTRACT FROM AUTHOR]

[Display omitted] • La doped Bi 2 WO 6 with rich oxygen vacancies was prepared. • The separation rate of photo-generated carriers is largely enhanced. • The level of O 2 – is significantly boosted. • La-Bi 2 WO 6 exhibits higher activity than the reference Bi 2 WO 6. To ameliorate the photocatalytic performance of Bi 2 WO 6 , La-doped Bi 2 WO 6 photocatalysts were prepared by a hydrothermal method using glacial acetic acid as solvent. The samples were carefully characterized by serval analytic methods. X ray photoelectron spectroscopy (XPS) and low-temperature electron spin-resonance (ESR) spectroscopy demonstrate that La-doped Bi 2 WO 6 photocatalysts have rich oxygen vacancies (OVs). Elemental mapping and XPS confirm the existence of La in Bi 2 WO 6. Doping La into Bi 2 WO 6 significantly boosts the level of OVs, separation of photoinduced carriers and the generation of superoxide radical (O 2 –). Photocatalytic performance of the samples was evaluated by removal of Rhodamine B (RhB) under simulated sunlight irradiation. The results indicate that the activity of La-Bi 2 WO 6 is higher than the reference Bi 2 WO 6. When the content of La is 0.6%, the degradation rate constant of RhB on the 0.6% La-Bi 2 WO 6 is 1.83 times of that of the blank Bi 2 WO 6. Doping La into Bi 2 WO 6 is a reliable approach to promote the photocatalytic activity of Bi 2 WO 6. [ABSTRACT FROM AUTHOR]

For purpose of ameliorating the photocatalytic capability of TiO 2 , TiO 2 photocatalysts were facilely fabricated by a hydrothermal strategy through the assistance of polyaniline (PANI). TiO 2 samples prepared (PANI-TiO 2) were characterized in detail by miscellaneous characterization approaches. The consequences affirm that PANI-assisted preparation can construct tunable oxygen vacancy (OVs), notably heightening the photocatalytic capability of TiO 2. Photocatalytic degradation of rhodamine B (RhB) and ciprofloxacin (CIP) were carried out under Xenon lamp irradiation. Compared with the blank TiO 2 , PANI-TiO 2 photocatalysts show superior photocatalytic capability to the blank TiO 2. When the weight proportion of PANI/TiO 2 is 4%, the degradation efficiency of RhB and CIP reaches 92.6% and 89.9% respectively, and the first-order kinetic constant of RhB and CIP over the sample is 3.7 and 1.7 times of that on the reference TiO 2. This demonstration offers a potent reference for fabrication of effective photocatalysts for destruction of organic contaminants. • PANI was employed to assist preparation of TiO 2. • PANI-assisted preparation can construct tunable OVs. • Separation of photoinduced carriers was significantly boosted. • PANI-TiO 2 exhibits higher photocatalytic activity than TiO 2. [ABSTRACT FROM AUTHOR]

In previous work, small ubiquitin-like modifier (SUMO) in hemocytes of Chinese shrimp Fenneropenaeus chinensis was found to be up-regulated post-white spot syndrome virus (WSSV) infection using proteomic approach. However, the role of SUMO in viral infection is still unclear. In the present work, full length cDNAs of SUMO (FcSUMO) and SUMO-conjugating enzyme E2 UBC9 (FcUBC9) were cloned from F. chinensis using rapid amplification of cDNA ends approach. The open reading frame (ORF) of FcSUMO encoded a 93 amino acids peptide with the predicted molecular weight (M.W) of 10.55 kDa, and the UBC9 ORF encoded a 160 amino acids peptide with the predicted M.W of 18.35 kDa. By quantitative real-time RT-PCR, higher mRNA transcription levels of FcSUMO and FcUBC9 were detected in hemocytes and ovary of F. chinensis, and the two genes were significantly up-regulated post WSSV infection. Subsequently, the recombinant proteins of FcSUMO and FcUBC9 were expressed in Escherichia coli BL21 (DE3), and employed as immunogens for the production of polyclonal antibody (PAb). Indirect immunofluorescence assay revealed that the FcSUMO and UBC9 proteins were mainly located in the hemocytes nuclei. By western blotting, a 13.5 kDa protein and a 18.7 kDa protein in hemocytes were recognized by the PAb against SUMO or UBC9 respectively. Furthermore, gene silencing of FcSUMO and FcUBC9 were performed using RNA interference, and the results showed that the number of WSSV copies and the viral gene expressions were inhibited by knockdown of either SUMO or UBC9, and the mortalities of shrimp were also reduced. These results indicated that FcSUMO and FcUBC9 played important roles in WSSV infection. [ABSTRACT FROM AUTHOR]

In the optimization of immersion strategy, vaccine concentration and immersion time are two key factors needed to be considered, which largely determined the immune efficacy. In this work, the healthy flounder were vaccinated in formalin-killed Edwardsiella tarda at four concentrations (10 6 , 10 7 , 10 8 , 10 9 CFU ml −1 ) for three immersion times (30, 60, 90 min), respectively. At the 6th week post vaccination, the flounders were challenged with live E. tarda , and the relative percent survival (RPS) of flounder in 10 9 –30, 10 8 –60, 10 8 –90 and 10 7 –90 min groups were 70%, 78%, 74% and 65%, respectively, which were much higher than the other vaccination groups. Meanwhile, the sIg + cells in the leucocytes of peripheral blood (PBL), spleen (SL), head kidney (HKL) were monitored by flow cytometry, and the specific sera and mucosal antibodies were measured by indirect ELISA for 6 weeks. The results showed that the proportions of sIg + cells in PBL, SL and HKL of vaccinated fish were significantly higher than the untreated fish since the 2nd week ( P < 0.05), and the fish in 10 9 –30, 10 8 –60, 10 8 –90 and 10 7 –90 min groups exhibited stronger responses than other groups, especially the 10 8 –60 min group displayed the strongest response, which reached the peaks (54.63% in PBL, 37.21% in SL, 36.51% in HKL) at the 5th week. ELISA assay showed that the kinetics of specific antibodies in sera were similar to the variation of sIg + cells, and the higher antibody levels were also detected in the four groups with higher RPS and stronger sIg+ response, whereas the mucosal antibody showed a faster response, which were significantly higher than the control since the 1st week ( P > 0.05). These results demonstrated that the higher RPS was closely associated with stronger immune response, and immersion with formalin-inactivated E. tarda under 10 8 CFU ml −1 for 60 min induced the highest immune response of flounder against E. tarda bacterin, which might be applied for the control of edwardsiellosis in flounder. [ABSTRACT FROM AUTHOR]

• An electrochemical immunosensor based on anti-idiotypic nanobody was devised. • The study offers a strategy for nontoxic detection using an alternative antigen. • An anti-idiotypic nanobody with satisfactory sensitivity was generated. • The nanosensor exhibited good detection performance in aflatoxin M1 analysis. This study aimed to devise a nontoxic electrochemical immunosensor to quantitatively determine aflatoxin M 1 by chronoamperometry with novel anti-idiotypic nanobody-functionalized screen-printed carbon electrodes (SPCEs). Anti-idiotype nanobodies (AIdnb) were developed to replace the high toxic chemically synthesized antigen. AIdnb was immobilized on the surface of SPCE via covalent coupling as capture reagent. The functionalized SPCEs were followed by characterization using electrochemical impedance spectroscopy, fourier-transform infrared spectroscopy, transmission electron microscopy mapping, and atomic force microscopy. After optimizing experimental parameters, the assembled immunosensor exhibited a good linearity range of 0.25–5.0 ng/mL, with the limit of detection of 0.09 ng/mL. The immunosensor showed a satisfactory selectivity to AFM 1 , without interference from analogs, including zearalenone, ochratoxin, and fumonisin B 1. For practical application, the developed immunosensor was validated using real spiked samples with the recovery range 82.0%–108.0% and relative standard deviation (RSD) 10.1%–13.0%, indicating that it could be used in milk samples. [ABSTRACT FROM AUTHOR]

[Display omitted] • P123-Ag 2 MoO 4 has stronger visible light-responsive capacity than Ag 2 MoO 4. • The level of O 2 – is significantly increase. • The separation efficiency of photoinduced charge has been enhanced. • P123-Ag 2 MoO 4 displays higher activity than the blank Ag 2 MoO 4o. To improve photocatalytic activity of Ag 2 MoO 4 , in this study, Ag 2 MoO 4 photocatalyst was successfully prepared by a hydrothermal method assisted with EO 20 PO 70 EO 20 (P123). The prepared Ag 2 MoO 4 photocatalyst (P123-Ag 2 MoO 4) was characterized by some typical analytical methods. The results show that P123-assisted preparation can boost the specific surface area of Ag 2 MoO 4 , promote the introduction of oxygen vacancies (OVs) in Ag 2 MoO 4 , and accelerate the separation of photoinduced carriers and facilitate the generation of O 2 –, therefore improving the photocatalytic activity of Ag 2 MoO 4. Under UV light irradiation, catalytic performance of the photocatalysts was evaluated by degradation of methyl orange (MO). When the mass ratio of P123/Ag 2 MoO 4 is 4%, the photocatalytic activity of Ag 2 MoO 4 is 10.1 times higher than that of the blank Ag 2 MoO 4. Considering the results, photocatalytic enhancement mechanism was elaborated. This study provides a facile and effective preparation method to boost the photocatalytic activity of Ag 2 MoO 4 photocatalyst. [ABSTRACT FROM AUTHOR]

The 27.8kDa membrane protein from flounder (Paralichthys olivaceus) gill (FG) cells was previously identified as a putative cellular receptor involved in lymphocystis disease virus (LCDV) infection. In this paper, the expression of receptor-27.8kDa (27.8R) and LCDV loads in FG cells and hirame natural embryo (HINAE) cells were investigated upon LCDV infection and anti-27.8R monoclonal antibody (MAb) treatment. The results showed the 27.8R was expressed and co-localized with LCDV in both FG and HINAE cell surface. After LCDV infection, the expression of 27.8R exhibited a dose-dependent up-regulation with the increasing of LCDV titers, and demonstrated a tendency to increase firstly and then decrease during a time course up to 9 days; LCDV copies showed a similar variation trend to the 27.8R expression, however, it reached the highest level later than did the 27.8R expression. Additionally, the 27.8R expression and LCDV copies in FG cells were higher than those in HINAE cells. In the presence of increasing concentration of the anti-27.8R MAbs, the up-regulation of 27.8R expression and the copy numbers of LCDV significantly declined post LCDV infection, and the cytopathic effect induced by LCDV in the two cell lines was accordingly reduced, indicating anti-27.8R MAbs pre-incubation could inhibit the up-regulation of 27.8R expression and LCDV infection. These results suggested that LCDV infection could induce up-regulation of 27.8R expression, which in turn increased susceptibility and availability of FG and HINAE cells for LCDV entry, providing important new insights into the LCDV replication cycle and the interaction between this virus and the host cells. [ABSTRACT FROM AUTHOR]

Aflatoxin B1 poses grave threats to food and feed safety due to its strong carcinogenesis and toxicity, thus requiring ultrasensitive rapid on-site determination. Herein, a portable immunosensor based on chromatographic time-resolved fluoroimmunoassay was developed for sensitive and on-site determination of aflatoxin B1 in food and feed samples. Chromatographic time-resolved fluoroimmunoassay offered a magnified positive signal and low signal-to-noise ratio in time-resolved mode due to the absence of noise interference caused by excitation light sources. Compared with the immunosensing performance in previous studies, this platform demonstrated a wider dynamic range of 0.2-60 μg/kg, lower limit of detection from 0.06 to 0.12 µg/kg, and considerable recovery from 80.5% to 116.7% for different food and feed sample matrices. It was found to be little cross-reactivity with other aflatoxins (B2, G1, G2, and M1). In the case of determination of aflatoxin B1 in peanuts, corn, soy sauce, vegetable oil, and mouse feed, excellent agreement was found when compared with aflatoxin B1 determination via the conversational high-performance liquid chromatography method. The chromatographic time-resolved fluoroimmunoassay affords a powerful alternative for rapid on-site determination of aflatoxin B1 and holds a promise for food safety in consideration of practical food safety and environmental monitoring. [ABSTRACT FROM AUTHOR]

[Display omitted] In this study, monoclinic phase bismuth vanadate (BiOV 4) photocatalyst with unique hollow microsphere morphology was successfully prepared by a hydrothermal method in the existence of sodium dodecyl benzene sulfonate (SDBS). The prepared photocatalysts were characterized by X-ray diffraction (XRD), scanning electron (SEM) and X-ray photoelectron spectrometer (XPS) and UV–vis diffuse reflectance spectroscopy (UV–vis DRS). Experimental results show that SDBS definitely changes the microstructure of BiVO 4 , which is allocated to the template role of SDBS in the preparation process. Moreover, the hydrothermal treatment time is also of crucial importance in affecting the structure and morphology of the photocatalysts, and the optimal hydrothermal treatment time for the formation of hollow microsphere is 24 h. Furthermore, the feasible growth mechanism for hollow microsphere was elaborated. Enriched oxygen vacancies (OVs) are introduced into BiOV 4 prepared with SDBS, largely elevating the separation efficiency of photo-generated charges. Under visible light irradiation, the photocatalytic activities of BiOV 4 for destruction of rhodamine (RhB) were evaluated. The photocatalytic degradation rate constant of RhB on the 3SBVO is 2.23 times of that on the blank BiOV 4 as the mass ratio of SDBS/BiOV 4 is 3 %. Photocatalytic degradation mechanism of BiVO 4 toward detoxification of organic pollutants was presented. [ABSTRACT FROM AUTHOR]

Mycotoxins pose a grave global threat to human life and health by contaminating food and feed and cause enormous losses in healthcare and trading. Trace mycotoxin concentrations and diverse matrices in food make identification and measurement challenges, necessitating highly specific and sensitive detection methods. Electrochemical (EC) sensors are characterized by simple operation, outstanding sensitivity, low cost, and facile miniaturization and have become a promising strategy for addressing specificity and sensitivity in detection. Recent studies on EC sensors for mycotoxin detection for food safety are reviewed here. First, we summarize the fabrication of EC sensors and techniques with enhanced specificity and sensitivity. Then, we review state‐of‐the‐art EC sensors for detecting major mycotoxins. Challenges and opportunities for this technology are further discussed. Finally, in‐depth information is provided on using EC sensors to detect mycotoxins for food safety, as well as the development of EC sensors for academic study and practical application. [ABSTRACT FROM AUTHOR]

Interleukin 2 (IL-2) is an immunoregulatory cytokine that plays significant role in the activation and proliferation of immune cells. In teleost, the functions of IL-2 signaling on the proliferation and differentiation of T lymphocytes were well documented. However, there is still unclear about the effects of IL-2 signaling on B cell immunity in fish. Hence, in this study, full-length transcriptome sequencing was performed to investigate the activation of IL-2 on flounder (Paralichthys olivaceus) lymphocytes in vitro , the effects of IL-2 on the immunity of B cells after its receptors (IL-2Rβ or IL-2Rγ) blocked were further investigated. The results shown that the differentially expressed genes in lymphocytes after IL-2 stimulation were annotated to the pathways related to the immune response of B cells. The percentages of mIgM+ B cells were increased, and the capacities of antibody secretion and phagocytosis of B cells were enhanced after IL-2 stimulation. However, the function of IL-2 on B lymphocytes immunity was significantly inhibited after IL-2 receptors were blocked, especially after IL-2Rβ was blocked. Collectively, we can conclude that IL-2 is able to promote the proliferation of B lymphocytes, antibody secretion, and enhance their phagocytosis in flounder, and these effects are mediated through IL-2/IL-2R signaling. • Full-length transcriptome sequencing was performed to analyze the activation pathways of IL-2 on flounder lymphocytes. • The effects of IL-2 on the immunity of B cells after IL-2Rβ or IL-2Rγ blocked were further investigated. • IL-2 promotes B cells proliferation, antibody secretion, and enhances their phagocytosis in flounder. • The activation of B cells by IL-2 is mediated through IL-2-IL-2R signaling. [ABSTRACT FROM AUTHOR]

To elucidate molecular responses of shrimp hemocytes to white spot syndrome virus (WSSV) infection, two-dimensional gel electrophoresis was applied to investigate differentially expressed proteins in hemocytes of Chinese shrimp (Fenneropenaeus chinensis) at 24 h post infection (hpi). Approximately 580 protein spots were detected in hemocytes of healthy and WSSV-infected shrimps. Quantitative intensity analysis revealed 26 protein spots were significantly up-regulated, and 19 spots were significantly down-regulated. By mass spectrometry, small ubiquitin-like modifier (SUMO) 1, cytosolic MnSOD, triosephosphate isomerase, tubulin alpha-1 chain, microtubule-actin cross-linking factor 1, nuclear receptor E75 protein, vacuolar ATP synthase subunit B L form, inositol 1,4,5-trisphosphate receptor, arginine kinase, etc., amounting to 33 differentially modulated proteins were identified successfully. According to Gene Ontology annotation, the identified proteins were classified into nine categories, consisting of immune related proteins, stimulus response proteins, proteins involved in glucose metabolic process, cytoskeleton proteins, DNA or protein binding proteins, proteins involved in steroid hormone mediated signal pathway, ATP synthases, proteins involved in transmembrane transport and ungrouped proteins. Meanwhile, the expression profiles of three up-regulated proteins (SUMO, heat shock protein 70, and arginine kinase) and one down-regulated protein (prophenoloxidase) were further analyzed by real-time RT-PCR at the transcription level after WSSV infection. The results showed that SUMO and heat shock protein 70 were significantly up-regulated at each sampling time point, while arginine kinase was significantly up-regulated at 12 and 24 hpi. In contrast, prophenoloxidase was significantly down-regulated at each sampling time point. The results of this work provided preliminary data on proteins in shrimp hemocytes involved in WSSV infection. [ABSTRACT FROM AUTHOR]

A monoclonal antibody (MAb) 6H7 raised specifically against granulocytes of scallop (Chlamys farreri) was employed to observe granulocyte occurrence successively in blastulae, gastrulae, trochophore larvae, D-shape larvae, umbo-veliger larvae and creeping larvae of C. farreri by immunohistochemistry assay contrasted with H&E stain using semi-thin sections. Moreover, the reactivity of the MAb with granulocytes of C. farreri, Bay scallop Argopecten irradians, Japanese scallop Patinopecten yessoensis, Blue mussel Mytilus edulis, Pacific oyster Crassostrea gigas and Manila clam Ruditapes philippinarum, was detected by immunofluorescence assay (IFA) with differential interference contrast and fluorescent microscopy and flow cytometric immunofluorescence assay (FCIFA). The results showed that positive signals were first observed at D-shape larval stage, about 28 h post fertilization, after that, umbo-veliger larvae exhibited the positive cells with a diameter of 3–5 μm distributed in velum, digestive gland and esophagus. Then in creeping larvae, the number of positive cells increased with average diameter of 5–7 μm, and widely distributed in foot, digestive gland, gills and adductor muscles. No positive signal was found in blastulae, gastrulae and trochophore larvae. The results of IFA and FCIFA showed MAb 6H7 reacted to granulocytes of C. farreri, A. irradians, P. yessoensis and C. gigas, and the positive percentage reactivity were 53 ± 2.5%, 15 ± 2.5%, 12 ± 2.1% and 19 ± 2.1%, respectively, however, no cross-reaction was detected in hemocytes of R. philippinarum and M. edulis. [ABSTRACT FROM AUTHOR]