A Noncentrosymmetric Metal-Free Borophosphate: Achieving a Large Birefringence and Excellent Stability by Covalent-Linkage.

Organic-inorganic hybrid linear and nonlinear optical (NLO) materials have received increasingly wide spread attention in recent years. Herein, the first hybrid noncentrosymmetric (NCS) borophosphate, (C5H6N)2B2O(HPO4)2 (4PBP), is rationally designed and synthesized by a covalent-linkage strategy. 4-pyridyl-boronic acid (4 PB) is considered as a bifunctional unit, which may effectively improve the optical properties and stability of the resultant material. On the one hand, 4 PB units are covalently linked with PO3(OH) groups via strong B-O-P connections, which significantly enhances the thermal stability of 4PBP (decomposition at 321, vs lower 200 °C of most of hybrid materials). On the other hand, the planar π-conjugated C5H6N units and their uniform layered arrangements represent large structural anisotropy and hyperpolarizability, achieving the largest birefringence (0.156 @ 546 nm) in the reported borophosphates and a second-harmonic generation response (0.7 × KDP). 4PBP also exhibits a wide transparency range (0.27-1.50 µm). This work not only provides a promising birefringent material, but also offers a practical covalent-attachment strategy for the rational design of new high-performance optical materials.

First report of Colletotrichum karstii causing anthracnose on soybean in China.

Soybean (Glycine max) is a significant grain and oil crop. Among the various challenges faced by soybean cultivation, anthracnose stands out as one of the most prevalent diseases. In June 2023, anthracnose symptoms on leaves characterized by irregular disease spots featuring gray-white centers and brown edges, along with many small black dots on their surface, were observed in a 20-hectare soybean (variety "Liu Yuehuang") field located in Luodian County (25°40'20″ N, 106°53'50″ E, 575 m), Guizhou Province, China. Around 30% of the 300 soybean plants examined were symptomatic, and a total of ten leaves were collected. Fragments (5×5 mm) from the edge of disease spots were sheared and surface-sterilized with 3% sodium hypochlorite and 75% ethanol for 60 s and 30 s, respectively. They were then flushed twice with sterile water, dried using sterile filter papers, finally placed on potato dextrose agar (PDA) and incubated at 28°C for two days. In total, 11 isolates with identical morphological characteristics were obtained. The colonies grown with white aerial mycelia on their surface; conidia were cylindrical, both ends are rounded, aseptate, hyaline, 11.0-14.0 (12.5) × 4.5-6.0 (5.0) µm (n = 30); appressoria were nearly ovoid, brown to black, 8.5-10.5 (9.5) × 5.5-7.5 (6.0) µm (n = 30). The morphological characteristics closely resembled the description of C. karstii (Damm et al., 2012). To further identify the isolates, chitin synthase (CHS-1), actin (ACT), beta-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the internal transcribed spacer (ITS) loci were amplified by using CHS-79F/CHS-345R, ACT-512F/ACT-783R (Carbone and Kohn, 1999), Bt2F/Bt2R (Woudenberg et al., 2009), GDF/GDR (Guerber et al., 2003) and ITS1/ITS4 (White et al., 1990) PCR primers, respectively. The BLAST results showed that the sequences of two representative strains, LD 2023048-1 and LD 2023048-2, were highly similar to those of strain C. karstii CGMCC3.14194 (ITS: OR342620 (99%) and OR342621 (99%) with HM585409, ACT: OR412337 (97%) and OR423341 (100%) with HM581995, CHS-1: OR423344 (99%,) and OR423345 (100%) with HM582023, GAPDH: OR423348 (98%) and OR423349 (98%) with HM585391, and TUB: OR423352 (99%) and OR423353 (99%) with HM585428). The phylogenetic tree combined five sequences showed that the two strains clustered into a branch of C. karstii CGMCC3.14194 with high support values. Thirty-day-old soybean plants (n = 10) (variety Liu Yuehuang) were separately sprayed with 1 × 105 spore suspensions/mL of the two strains by spray method, and plants sprayed with sterile distilled water were used as the negative control (n = 5). All the plants were then covered with plastic bags and cultured in the greenhouse (28℃, 80% humidity, 12 h light dark cycle). After ten days of inoculation, plants inoculated with C. karstii began to produce typical anthracnose symptoms, while the control remained asymptomatic. The confirmation of the reisolated pathogen as C. karstii was established through a comprehensive analysis of morphology and five sequencing loci. Pathogenicity tests were repeated three times. Anthracnose on soybean is caused by Colletotrichum spp. reported in China including C. truncatum (Hu et al., 2015), C. brevisporum (Shi et al., 2021) and C. fructicola (Xu et al., 2023). As far as we know, this study is the initial report of C. karstii inducing anthracnose on soybean to date, which establishes a fundamental reference for preventing and controlling this disease.

First report of Powdery Mildew Caused by Podosphaera xanthii on Siraitia grosvenorii in Guizhou, China.

Swingle (Siraitia grosvenorii), a member of the Cucurbitaceae family, stands out as a distinctive plant with both economic and medicinal significance. In October 2023, severe powdery mildew were observed on S. grosvenorii in Guiyang City (26.50°N; 106.66°E), Guizhou Province, China. About 80 % of the plants in the greenhouse showed powdery mildew symptoms. Three infected plant samples were selected for morphological and molecular analysis (GZAAS 23-0801, GZAAS 23-0802 and GZAAS 23-0803). The voucher specimens are deposited in the Key Laboratory of Agricultural Biotechnology of Guizhou Province. The symptoms initially manifested as irregular to nearly circular, small yellow spots, with distinct depressions as well as surfaces covered in white mycelium. Over time, these spots gradually expanded and merged patches. In the final stages, the entire leaves turned into yellow and withered. Microscopic observations showed that fungal hyphae were septate, branched, and flexuous to straight and 5 to 9 µm wide, and appressoria were indistinct to slightly nipple-shaped. Conidia were hyaline and ellipsoid to oval with fibrosin bodies and measured 31 to 43 × 18 to 24 µm (n = 50) with a length/width ratio of 1.3 to 2.3. Conidiophores were unbranched, straight, 120 to 268 × 14 to 22 µm (n = 30), producing two to five immature conidia in chains. Foot cells of conidiophores were cylindrical, 39 to 84 × 8 to 14 µm (n = 30), followed by one to three short cells. Short cells were cylindrical, 12 to 32 × 8 to 15 µm (n = 50). The morphological characteristics were identical with the previous description of Podosphaera xanthii (Braun and Cook, 2012). Total DNA was extracted from conidia and mycelia by the Chelex method (Walsh et al., 1991). The ribosomal DNA internal transcribed spacer (ITS) and nuclear ribosomal large subunit (LSU) were amplified by using the primers ITS1/ITS4 (White et al., 1990) and LSU1/LSU2 (Scholin et al., 1994), respectively. The ITS (OR825802, OR825803 and OR825804, respectively) and LSU (OR825805, OR825806 and OR825807, respectively) sequences of three isolates, were deposited in GenBank. The BLAST results revealed that both the ITS and LSU region sequence were 100% identical to those of P. xanthii (ITS: MF043939, MG754404 and KJ698669; LSU: OQ061319, AB936277and OP218411). Phylogenetic analyses of ITS and LSU sequences showed that our three isolates were clustered with P. xanthii (KX842351, LC270782 and LC270779) with high statistical support (ML/MP/BI: 100%/97%/1.00). Combined with their morphological characteristics, these three isolates were identified as P. xanthii. Pathogenicity tests were performed by gently brushing conidia onto the leaves of five healthy S. grosvenorii plants. Five non-inoculated plants were used as the control. All plants were maintained in a greenhouse at 25 ± 2°C. One week after inoculation, similar symptoms were observed in the inoculated plants, whereas no symptoms occurred on the control plants. By microscopic observation, the fungus on the inoculated plants was morphologically identical to those on originally diseased plants. Powdery mildew caused by P. xanthii has been reported on Vernonia cinerea (Wu et al., 2023), Vigna unguiculata (Zhang et al., 2023), Cucumis melo (Meesam et al., 2023). To our knowledge, this is the first report of powdery mildew caused by P. xanthii on S. grosvenorii in Guizhou, China. The occurrence of powdery mildew on S. grosvenorii may pose a potential threat to its large-scale cultivation. The pathogen could become a threat to other Cucurbitaceae members in the future.

Ultrasound-based deep learning in the establishment of a breast lesion risk stratification system: a multicenter study.

OBJECTIVES: To establish a breast lesion risk stratification system using ultrasound images to predict breast malignancy and assess Breast Imaging Reporting and Data System (BI-RADS) categories simultaneously. METHODS: This multicenter study prospectively collected a dataset of ultrasound images for 5012 patients at thirty-two hospitals from December 2018 to December 2020. A deep learning (DL) model was developed to conduct binary categorization (benign and malignant) and BI-RADS categories (2, 3, 4a, 4b, 4c, and 5) simultaneously. The training set of 4212 patients and the internal test set of 416 patients were from thirty hospitals. The remaining two hospitals with 384 patients were used as an external test set. Three experienced radiologists performed a reader study on 324 patients randomly selected from the test sets. We compared the performance of the DL model with that of three radiologists and the consensus of the three radiologists. RESULTS: In the external test set, the DL model achieved areas under the receiver operating characteristic curve (AUCs) of 0.980 and 0.945 for the binary categorization and six-way categorizations, respectively. In the reader study set, the DL BI-RADS categories achieved a similar AUC (0.901 vs. 0.933, p = 0.0632), sensitivity (90.98% vs. 95.90%, p = 0.1094), and accuracy (83.33% vs. 79.01%, p = 0.0541), but higher specificity (78.71% vs. 68.81%, p = 0.0012) than those of the consensus of the three radiologists. CONCLUSIONS: The DL model performed well in distinguishing benign from malignant breast lesions and yielded outcomes similar to experienced radiologists. This indicates the potential applicability of the DL model in clinical diagnosis. KEY POINTS: • The DL model can achieve binary categorization for benign and malignant breast lesions and six-way BI-RADS categorizations for categories 2, 3, 4a, 4b, 4c, and 5, simultaneously. • The DL model showed acceptable agreement with radiologists for the classification of breast lesions. • The DL model performed well in distinguishing benign from malignant breast lesions and had promise in helping reduce unnecessary biopsies of BI-RADS 4a lesions.

Sb4O3(TeO3)2(HSO4)(OH): An Antimony Tellurite Sulfate Exhibiting Large Optical Anisotropy Activated by Lone Pair Stereoactivity.

A new birefringent crystal of Sb4O3(TeO3)2(HSO4)(OH) was achieved by incorporating two stereochemically active lone pair (SCALP) cations of Sb(III) and Te(IV) into sulfates simultaneously. The Sb3+ and Te4+ ions display highly distorted coordination environments due to the SCALP effect. Sb4O3(TeO3)2(HSO4)(OH) displays a 3D structure composed of [Sb4O3(TeO3)2(OH)]∞+ layers bridged by [SO3(OH)]- tetrahedra. It possesses a large birefringence and a wide optical transparent range, making it a new UV birefringent crystal. First-principles calculation analysis suggests that the synergistic effect of the cooperation of SCALP effect of Sb3+ and Te4+ cations make a dominant contribution to the birefringence. The work highlights that units with SCALP cations have advantages in generating large optical anisotropy and are preferable structural units for designing novel birefringent materials.

Arbitrary superposition of plasmonic orbital angular momentum states with nanostructures.

A kind of plasmonic nanostructure is proposed that can generate the arbitrary superposition of orbital angular momentum (OAM) states in surface plasmons (SPs), which is achieved by combining the segmented spirals with nanoslit pairs. The structures can independently modulate both the phase and amplitude of SP waves, and thus enable the superposition of two OAM states with arbitrary topological charges (TCs) as well as free control of their relative amplitudes. Superposed states distributed over the entire Bloch sphere and hybrid superposed states with different TCs were constructed and experimentally demonstrated. This work will offer more opportunities for multifunctional plasmonic devices.

Production of monoclonal antibody against grouper (Epinephelus coioides) CD4-1 and the distribution of CD4-1+ cells.

CD4-a transmembrane glycoprotein molecule expressed on the surface of helper T (Th) cells-plays a central role in adaptive immune protection. In the current study, we developed a monoclonal antibody (mAb) against the grouper CD4-1. Western blotting and immunohistochemistry results revealed that the CD4-1 mAb could recognize the recombinant and natural protein of grouper CD4-1 as well as the CD4-1+ cells in the various tissues from grouper. Tissue distribution analyses revealed that the grouper CD4-1+ cells were expressed in all tissues tested in the healthy grouper, with greater localization in the thymus, head kidney, and spleen tissues. In addition, we tested the changes in the proportion of CD4-1+ cells in the thymus, head kidney, and the gills of grouper post the infection by C. irritans. Our data suggest that the CD4-1 mAb produced against grouper in the current study can be used as a tool to characterize CD4-1+ cells and to investigate the functions of the grouper CD4-1+ cells in the host response against pathogens infection.

Quantitative detection of parasitic ciliate Cryptocaryon irritans in seawater with an optimized sample processing method.

The protozoan Cryptocaryon irritans is one of the most important ectoparasites of marine fish, causing 'white spot disease' and mass mortality in aquaculture. To accurately predict disease outbreaks and develop prevention strategies, improved detection methods are required that are sensitive, convenient and rapid. In this study, a pair of specific primers based on the C. irritans 18S rRNA gene was developed and used in a real-time PCR (qPCR) assay. This assay was able to detect five theronts in 1 L of natural seawater. Furthermore, a linear model was established to analyse the log of Ct value and parasite abundance in seawater (y = -2.9623x + 24.2930), and the coefficient of determination (R2 ) value was 0.979. A lysis buffer was optimized for theront DNA extraction and used for storage sample. This method was superior to the commercial water DNA kit, and there was no significant degradation of DNA at room temperature for 24-96 hr. A dilution method was developed to manage qPCR inhibitors and used to investigate natural seawater samples in a net cage farm with diseased fish, and the findings were consistent with the actual situation. This study provides a valuable tool for assisting in the early monitoring and control of cryptocaryoniasis in aquaculture.

Sb6 O7 (SO4 )2 : A Promising Ultraviolet Nonlinear Optical Material with an Enhanced Second-Harmonic-Generation Response Activated by SbIII Lone-Pair Stereoactivity.

The stereochemical activity of lone pairs (SCALP) in a cation favors the formation of acentric materials and can enhance the second-harmonic-generation (SHG) response and/or the birefringence. By introducing functional SbIII into sulfates, an anhydrous sulfate of Sb6 O7 (SO4 )2 (1) is explored. Sb3+ cations are in seesaw configurations and in-phase aligned in a 3D asymmetric dense structure. Compound 1 exhibits an enhanced phase-matching SHG response, a moderate birefringence, a wide transparency window, and considerable environmental stabilities, which result in it being a promising UV nonlinear optical (NLO) material. Theoretical studies reveal that the stereoactive lone pairs on Sb3+ cations make the predominant contribution to the SHG effect. This work will attract more interest from scientists for research into SCALP-cation-based NLO materials.

Linear and Nonlinear Optical Properties of Centrosymmetric Sb4O5SO4 and Noncentrosymmetric Sb4O4(SO4)(OH)2 Induced by Lone Pair Stereoactivity.

Introducing stereochemically active lone-pair Sb3+ cations into sulfates, two three-dimensional (3D) antimony-sulfates, Sb4O5SO4 (1) and Sb4O(SO4)(OH)2 (2), were achieved under moderate hydrothermal conditions. Both structures are constructed by tetranuclear-{Sb4}-clusters-based layers and SO4 tetrahedra. However, owing to the different packing patterns of the layers, they display different characteristics: 1 exhibits a centrosymmetric structure while 2 possesses a noncentrosymmetric structure. UV-vis spectra show that they possess wide band gaps. Sb4O(SO4)(OH)2 is nonlinear optical (NLO) active with a second-harmonic generation (SHG) response of ∼1.2 times of KH2PO4, together with the phase-matchable capacity, endowing it a promising UV NLO material. The first-principle calculations were performed to elucidate the structure-property relationships. The results indicate that the lone pair stereoactivity of Sb3+ provides the large contribution to the macroscopic SHG effect.

IκB kinase α-1 and -2 regulate cytokine expression in the orange-spotted grouper (Epinephelus coioides).

IκB kinase (IKK) is the core regulator of the nuclear factor-κB (NF-κB) pathway, which is involved in cellular development and proliferation, as well as the inflammatory response. IKKα is an important subunit of the IKK complex. In this study, two IKKαs (EcIKKα-1 and -2) were characterized in E. coioides. Similar to IKKα of other species, EcIKKα-1 and -2 contained a kinase domain, a leucine zipper, a helix-loop-helix domain and a beta NF-κB essential modulator-binding domain. Sequence alignment indicated that EcIKKα-1 and -2 shared high degrees of sequence identity with IKKs from other species (about 63%-96%). EcIKKα-1 and -2 are widely expressed in all tissues, but have different expression profiles in normal groupers. Additionally, EcIKKα-1 and -2 responded rapidly to Cryptocaryon irritans infection at the local infection site (i.e., gill tissue), but there was no significant change in EcIKKα-2 expression. In GS cells, EcIKKα-1 was uniformly distributed in the cytoplasm, while EcIKKα-2 was observed uniformly both in the cytoplasm and nucleus. Both EcIKKα-1 and -2 were found to activate NF-κB, but the luciferase activity of EcIKKα-2 was twice that of EcIKKα-1. In addition, EcIKKα-1 and -2 can regulate the expression of immune-related cytokines (IL-1ß, IL-6, IL-8, IL-12 [p35 subunit], and TNF-α). These findings should prove helpful to further elucidate the innate immunity function of IKKα in fish.

Distribution of Mpeg1+ cells in healthy grouper (Epinephelus coioides) and after Cryptocaryon irritans infection.

Cryptocaryon irritans is an extremely harmful ciliated obligate parasite that is responsible for large economic losses in aquaculture. C. irritans infection can cause an insect-resistant immune response in fish, and many immune cells can be observed in the local infection site. However, it is unclear whether macrophages are involved in the host defense against C. irritans infection. The Mpeg1 protein can form pores and destroy the cell membrane of invading pathogens, and is also used as a macrophage-specific marker in mammals. Therefore, a polyclonal antibody against grouper recombinant Mpeg1a was produced to mark macrophages in this study, which could recognize both isoforms of Mpeg1 (Mpeg1a/b). Immunofluorescence revealed that EcMpeg1 positive cells were mostly distributed in the head kidney and spleen in healthy grouper. Immunofluorescence and immunohistochemistry showed that the number of EcMpeg1 positive cells increased in the gills after infection with C. irritans, implying that EcMpeg1 positive cells may be involved in the process of grouper resistance against C. irritans infection.

Orange-spotted grouper (Epinephelus coioides) NADPH oxidase: Cloning and expression analysis after Cryptocaryon irritans infection.

Phagocytic cells are activated to produce a large amount of reactive oxygen species (ROS) that kill pathogens quickly and efficiently through oxidation. NADPH oxidase is the main source of intracellular ROS. In the present study, five subunits of the phagocytic NADPH oxidase complex were identified in orange-spotted grouper (Epinephelus coioides). The open reading frame of grouper gp91phox, p22phox, p67phox, p47phox, and p40phox were 1,698 bp, 564 bp, 1,497 bp, 1,290 bp, and 1,050 bp, respectively, and encoded 565, 187, 498, 429, and 349 amino acids. Evolutionary analysis indicated that these proteins are evolutionarily homologous to the corresponding proteins of other fish and mammals, and contain conserved functional domains and sites that are important in mammals. In addition, real-time polymerase chain reaction analysis showed that the expression of these five genes was higher in immune-related tissues in normal grouper, and that these genes were up-regulated in gill and spleen after C. irritans infection, which suggests that these genes may be involved in the defense against C. irritans infection.

Application of Deep Learning in Quantitative Analysis of 2-Dimensional Ultrasound Imaging of Nonalcoholic Fatty Liver Disease.

OBJECTIVES: To verify the value of deep learning in diagnosing nonalcoholic fatty liver disease (NAFLD) by comparing 3 image-processing techniques. METHODS: A total of 240 participants were recruited and divided into 4 groups (normal, mild, moderate, and severe NAFLD groups), according to the definition and the ultrasound scoring system for NAFLD. Two-dimensional hepatic imaging was analyzed by the envelope signal, grayscale signal, and deep-learning index obtained by 3 image-processing techniques. The values of the 3 methods ranged from 0 to 65,535, 0 to 255, and 0 to 4, respectively. We compared the values among the 4 groups, draw receiver operating characteristic curves, and compared the area under the curve (AUC) values to identify the best image-processing technique. RESULTS: The envelope signal value, grayscale value, and deep-learning index had a significant difference between groups and increased with the severity of NAFLD (P < .05). The 3 methods showed good ability (AUC > 0.7) to identify NAFLD. Meanwhile, the deep-learning index showed the superior diagnostic ability in distinguishing moderate and severe NAFLD (AUC = 0.958). CONCLUSIONS: The envelope signal and grayscale values were vital parameters in the diagnosis of NAFLD. Furthermore, deep learning had the best sensitivity and specificity in assessing the severity of NAFLD.

Meta-analysis of single-stage versus two-staged management for concomitant gallstones and common bile duct stones.

OBJECTIVE: The purpose of this article was to compare the effectiveness and safety of single-stage (laparoscopic cholecystectomy [LC] plus laparoscopic common bile duct exploration [LCBDE]) with two-stage (LC plus endoscopic retrograde cholangiopancreatography (ERCP)/endoscopic sphincterotomy [EST]) in management for concomitant gallstones and common bile duct (CBD) stones. MATERIALS AND METHODS: Systematic review and meta-analysis of randomised controlled trials (RCTs) comparing outcomes following single-stage with two-stage management for concomitant gallstones and CBD stones published from 1990 to 2017 in PubMed, Embase and the Science Citation Index. The primary outcomes were stone clearance from the CBD, post-operative morbidity and mortality. The secondary outcomes were retained stone, conversion to other procedures, length of hospital stay and total operating time. Pooled risk ratio (RR) or weighted mean differences (WMD) with 95% confidence intervals (95% CIs) were calculated using either the fixed effects model or random effects model. RESULTS: Eleven RCTs studies were included in this analysis. These studies included a total of 1338 patients: 666 underwent LC + LCBDE and 672 underwent LC + ERCP/EST. The meta-analysis showed that no significant difference was noted between the two groups regarding CBD stone clearance (RR: 1.06; 95% CI: 0.99-1.14; P= 0.12), post-operative morbidity (RR: 1.03; 95% CI: 0.79-1.34; P= 0.81), mortality (RR: 0.30; 95% CI: 0.06-1.41; P= 0.13), retained stone (RR: 0.91; 95% CI: 0.57-1.47; P= 0.71), conversion to other procedures (RR: 0.80; 95% CI: 0.55-0.16; P= 0.23), length of hospital stay (WMD: 1.24, 95% CI: 3.57-1.09, P= 0.30), total operating time (WMD: 25.42, 95% CI: 22.38-73.22, P= 0.30). CONCLUSION: Single-stage is efficient and safe in the treatment of patients with concomitant gallstones and CBD stones while avoiding the second procedure. In selected patients, single-stage management for concomitant gallstones and CBD stones might be considered as the preferred approach. However, the findings have to be carefully interpreted due to the existence of heterogeneity, in addition, patient's condition, operator's experience also should be taken into account in making treatment decisions.

Effects of short-term fasting on the resistance of Nile tilapia (Oreochromis niloticus) to Streptococcus agalactiae infection.

Short-term feed deprivation or fasting is commonly experienced by aquaculture fish species and may be caused by seasonal variations, production strategies, or diseases. To assess the effects of fasting on the resistance of Nile tilapia to Streptococcus agalactiae infection, vaccinated and unvaccinated fish were fasted for zero, one, three, and seven days prior to infection. The cortisol levels of both vaccinated and unvaccinated fish first decreased and then increased significantly as fasting time increased. Liver glycogen, triglycerides, and total cholesterol decreased significantly after seven days of fasting, but glucose content did not vary significantly between fish fasted for three and seven days. Hexokinase (HK) and pyruvate kinase (PK) activity levels were lowest after seven days of fasting, while phosphoenolpyruvate carboxykinase (PEPCK) activity levels varied in opposition to those of HK and PK. Serum superoxide dismutase (SOD) and catalase (CAT) activity levels first increased and then decreased as fasting time increased; SOD activity was highest after three days of fasting. Interleukin-1beta (IL-1ß) and IL-6 mRNA expression levels first increased and then decreased significantly, peaking after three days of fasting. However, suppressor of cytokine signaling-1 (SOCS-1) mRNA expression levels were in opposition to those of IL-1ß and IL-6. Specific antibody levels did not vary significantly among unvaccinated fish fasted for different periods. Although specific antibody level first increased and then decreased in the vaccinated fish as fasting duration increased, there were no significant differences in the survival rates of fasted vaccinated fish after challenge with S. agalactiae. The final survival rates of vaccinated fish fasted for zero, one, three, and seven days were 86.67 ±â€¯5.44%, 80.00 ±â€¯3.14%, 88.89 ±â€¯6.28%, and 84.44 ±â€¯8.32%, respectively. Among the unvaccinated fish, the survival rate was highest (35.56 ±â€¯3.14%) in the fish fasted for three days and lowest (6.67 ±â€¯3.14%) in the fish fasted for seven days. Therefore, our results indicated that short-term fasting (three days) prior to an infection might increase the resistance of unvaccinated Nile tilapia to S. agalactiae.

Grouper (Epinephelus coioides) Mpeg1s: Molecular identification, expression analysis, and antimicrobial activity.

Macrophage expressed gene 1 (Mpeg1) is a molecule that can form pores and destroy the cell membrane of invading pathogens. In this study, we identified two Mpeg1 isoforms from the orange-spotted grouper (Epinephelus coioides) and named them EcMpeg1a and EcMpeg1b. Predicted proteins of the two EcMpeg1s contained a signal peptide, a conserved membrane attack complex/perforin (MACPF) domain, a transmembrane segment, and an intracellular region. Sequence alignment demonstrated that two EcMpeg1 proteins share a high sequence identity with that of other teleosts. Tissue distribution analysis showed that EcMpeg1s were expressed in all tissues tested in healthy grouper, with the highest expression in the head kidney and spleen. After infection with the ciliate parasite Cryptocaryon irritans, expression of the two EcMpeg1s was significantly upregulated in the spleen and gills. Furthermore, the recombinant EcMpeg1a showed antiparasitic and antibacterial activity against Gram-negative and -positive bacteria, whereas EcMpeg1b had an inhibitory effect only against Gram-positive bacteria. These results indicated that EcMpeg1s play an important role in the host response against invading pathogens.

Grouper (Epinephelus coioides) MyD88 adaptor-like (Mal): Molecular cloning, expression, and functionality.

Initiation of the innate immune response requires recognition of pathogen-associated molecular patterns by pathogen recognition receptors such as Toll-like receptors (TLRs). MyD88 adaptor-like (Mal) is an adaptor that responds to TLR activation and acts as a bridging adaptor for MyD88. In the present study, the open reading frame of Mal was identified in orange-spotted grouper (Epinephelus coioides), and named EcMal. It contained 831 bp encoding 276 aa, and was encoded by a 1299 bp DNA sequence with three exons and two introns. EcMal and the Mal sequence of other species shared different degrees of sequence identity, and clustered into the same group. EcMal was distributed in all tissues tested in healthy grouper, with the highest expression level in the head kidney. After infection with Cryptocaryon irritans, the expression level of EcMal was up-regulated in the gill and spleen. In addition, EcMal exhibited global cytosolic and nucleus localization, and could significantly activate NF-κB activity in grouper spleen cells.

GroEL, a novel vaccine candidate of piscine Streptococcus agalactiae identified by immunoproteome.

Streptococcus agalactiae is the major etiological agent of streptococcosis, which is responsible for huge economic losses in fishery, particularly in tilapia (Oreochromis niloticus) aquaculture. A research priority to control streptococcosis is to develop vaccines, so we sought to figure out the immunogenic proteins of S. agalactiae and screen the vaccine candidates for streptococcosis in the present study. Immunoproteomics, a technique involving two-dimensional gel electrophoresis (2-DE) followed by immunoblotting and mass spectrometry (MS), was employed to investigate the immunogenic proteins of S. agalactiae THN0901. Whole-cell soluble proteins were separated using 2-DE, and the immunogenic proteins were detected by western blotting using rabbit anti-S. agalactiae sera. A total of 17 immunoreactive spots on the soluble protein profile, corresponding to 15 different proteins, were identified by MALDI-TOF/TOF MS. Among the immunogenic proteins, GroEL attracted our attention as it was demonstrated to be immunogenic and protective against other streptococci. Nevertheless, to date, there have been no published reports on the immunogenicity and protective efficacy of GroEL against piscine S. agalactiae. Therefore, recombinant GroEL (rGroEL) was expressed in Escherichia coli BL21 (DE3) and purified by affinity chromatography. Immunization of tilapia with rGroEL resulted in an increase in antibody titers and conferred protection against S. agalactiae, with the relative percentage survival of 68.61 ±â€¯7.39%. The immunoproteome in the present study narrows the scope of vaccine candidates, and the evaluation of GroEL immunogenicity and protective efficacy shows that GroEL forms an ideal candidate molecule in subunit vaccine against S. agalactiae.

Molecular characteristics and functional study of tumor necrosis factor receptor-associated factor 2 from the orange-spotted grouper (Epinephelus coioides).

In mammals, tumor necrosis factor receptor-associated factor 2 (TRAF2) is a crucial intracellular adaptor protein, which performs a vital role in numerous signaling pathways that activate NF-κB, MAPKs, and IRFs. In the present study, three TRAF2 sequences were identified from the orange-spotted grouper (Epinephelus coioides), and named EcTRAF2-1, EcTRAF2-2, and EcTRAF2-3. These sequences contained conserved structure features that were similar to those of mammals. EcTRAF2-1 shared relatively low sequence identity with the other two EcTRAF2s. In healthy E. coioides, EcTRAF2s were widely expressed in all tissues tested, but with distinct expression profiles. After infection with Cryptocaryon irritans, EcTRAF2s was markedly upregulated in the gill and head kidney at most time points, implying that EcTRAF2s may be involved in host defense against C. irritans infection. In HEK293T cells, EcTRAF2s were scattered in the cytoplasm. EcTRAF2-1 and EcTRAF2-2 increased the activity of NF-κB, while EcTRAF2-3 reduced NF-κB activation mediated by EcTRAF2-1 implying that EcTRAF2-3 might be a negative regulator of EcTRAF2-1.