Corrigendum: The effect and underlying mechanism of yeast ß-glucan on antiviral resistance of zebrafish against spring viremia of carp virus infection.

[This corrects the article DOI: 10.3389/fimmu.2022.1031962.].

Deciphering the gut microbiome of grass carp through multi-omics approach.

BACKGROUND: Aquaculture plays an important role in global protein supplies and food security. The ban on antibiotics as feed additive proposes urgent need to develop alternatives. Gut microbiota plays important roles in the metabolism and immunity of fish and has the potential to give rise to novel solutions for challenges confronted by fish culture. However, our understanding of fish gut microbiome is still lacking. RESULTS: We identified 575,856 non-redundant genes by metagenomic sequencing of the intestinal content samples of grass carp. Taxonomic and functional annotation of the gene catalogue revealed specificity of the gut microbiome of grass carp compared with mammals. Co-occurrence analysis indicated exclusive relations between the genera belonging to Proteobacteria and Fusobacteria/Firmicutes/Bacteroidetes, suggesting two independent ecological groups of the microbiota. The association pattern of Proteobacteria with the gene expression modules of fish gut and the liver was consistently opposite to that of Fusobacteria, Firmicutes, and Bacteroidetes, implying differential functionality of Proteobacteria and Fusobacteria/Firmicutes/Bacteroidetes. Therefore, the two ecological groups were considered as two functional groups, i.e., Functional Group 1: Proteobacteria and Functional Group 2: Fusobacteria/Firmicutes/Bacteroidetes. Further analysis revealed that the two functional groups differ in genetic capacity for carbohydrate utilization, virulence factors, and antibiotic resistance. Finally, we proposed that the ratio of "Functional Group 2/Functional Group 1" can be used as a biomarker that efficiently reflects the structural and functional characteristics of the microbiota of grass carp. CONCLUSIONS: The gene catalogue is an important resource for investigating the gut microbiome of grass carp. Multi-omics analysis provides insights into functional implications of the main phyla that comprise the fish microbiota and shed lights on targets for microbiota regulation. Video Abstract.

Association between CT-based adipose variables, preoperative blood biochemical indicators and pathological T stage of clear cell renal cell carcinoma.

Background: Clear cell renal cell carcinoma (ccRCC) is corelated with tumor-associated material (TAM), coagulation system and adipocyte tissue, but the relationships between them have been inconsistent. Our study aimed to explore the cut-off intervals of variables that are non-linearly related to ccRCC pathological T stage for providing clues to understand these discrepancies, and to effectively preoperative risk stratification. Methods: This retrospective analysis included 218 ccRCC patients with a clear pathological T stage between January 1st, 2014, and November 30th, 2021. The patients were categorized into two cohorts based on their pathological T stage: low T stage (T1 and T2) and high T stage (T3 and T4). Abdominal and perirenal fat variables were measured based on preoperative CT images. Blood biochemical indexes from the last time before surgery were also collected. The generalized sum model was used to identify cut-off intervals for nonlinear variables. Results: In specific intervals, fibrinogen levels (FIB) (2.63-4.06 g/L) and platelet (PLT) counts (>200.34 × 109/L) were significantly positively correlated with T stage, while PLT counts (<200.34 × 109/L) were significantly negatively correlated with T stage. Additionally, tumor-associated material exhibited varying degrees of positive correlation with T stage at different cut-off intervals (cut-off value: 90.556 U/mL). Conclusion: Preoperative PLT, FIB and TAM are nonlinearly related to pathological T stage. This study is the first to provide specific cut-off intervals for preoperative variables that are nonlinearly related to ccRCC T stage. These intervals can aid in the risk stratification of ccRCC patients before surgery, allowing for developing a more personalized treatment planning.

Clinico-radiologic subtypes and therapeutic observation of acute Marchiafava-Bignami disease.

We aimed to investigate the clinico-radiologic features of acute Marchiafava-Bignami disease (MBD) and its evolutionary process after effective treatment through subgroup comparison. The clinical and MRI data of 23 patients with acute MBD were retrospectively analyzed and divided into type A (12 cases, with entire callosal involvement) and type B (11 cases, with focal callosal involvement). The clinical assessments and MRI findings (before and after treatment) were compared between the two subtypes. Compared with type B, type A had lower MoCA (Montreal Cognitive Assessment) scores at admission (16.50 ± 1.73 vs 18.27 ± 1.68, P = 0.021) and were more common with extracallosal involvement (66.67% vs 18.18%, P = 0.036) and longer illness duration (18.3 ± 2.1 days vs 15.6 ± 2.4 days, P = 0.012). During the treatment, the residual lesion in the splenium was more common in type A (58.33% vs 9.09%, P = 0.027). After treatment, the MoCa scores of both subtypes gradually increased (P < 0.001), and the callosal and extracallasal lesions disappeared completely. Clinico-radiologic typing of acute MBD is related to the severity of early symptoms, but not to the prognosis. Complete clinico-radiologic recovery is possible for both subtypes with combined treatment. The clinico-radiologic reversibility is helpful for accurate diagnosis and therapeutic evaluation.

Synthesis of lipoic acid ferulate and evaluation of its ability to preserve fish oil from oxidation during accelerated storage.

Lipoic acid ferulate (LAF) was synthesized and its anti-free radical ability in vitro was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonicacid) (ABTS) assays. Protective effects of LAF in stabilizing fish oil were tested, compared to antioxidants such as lipoic acid, ferulic acid and tert-butylhydroxyquinone (TBHQ) by measuring peroxide values, thiobarbituric acid reactants, p-anisidine values, nuclear magnetic resonance (NMR) spectra and gas chromatography-mass spectrometry (GC-MS) spectra of fish oil during accelerated storage (12 days, 80 °C). The inhibitory effect of these antioxidants on fish oil oxidation followed the order TBHQ ≧ LAF > ferulic acid > lipoic acid. In addition, the omega-3 polyunsaturated fatty acids were the first to be oxidized. The formation of oxidation products followed a first-order kinetic model, and the addition of LAF effectively reduced the reaction rate constants. Therefore, LAF can effectively slow down the formation of oxidative products and prolong the shelf life of fish oil.

Dietary cultured supernatant mixture of Cetobacterium somerae and Lactococcus lactis improved liver and gut health, and gut microbiota homeostasis of zebrafish fed with high-fat diet.

Postbiotics have the ability to improve host metabolic disorders and immunity. In order to explore whether the postbiotics SWFC (cultured supernatant mixture of Cetobacterium somerae and Lactococcus lactis) repaired the adverse effects caused by feeding of high-fat diet (HFD), zebrafish were selected as the experimental animal and fed for 6 weeks, with dietary HFD as the control group, and HFD containing 0.3 g/kg and 0.4 g/kg SWFC as the treatment groups. The results indicated that addition of SWFC in the diet at a level of 0.3 and 0.4 g/kg didn't affect the growth performance of zebrafish (P > 0.05). Supplementation of dietary SWFC0.3 relieved lipid metabolism disorders through significant increasing in the expression of pparα and cpt1, and decreasing the expression of cebpα, pparγ, acc1 and dgat-2 genes (P < 0.05). Moreover, the content of triacylglycerol was markedly lower in the liver of zebrafish grouped under SWFC0.3 (P < 0.05). Dietary SWFC0.3 also improved the antioxidant capacity via increasing the expression level of ho-1, sod and gstr genes, and significant inducing malondialdehyde content in the liver of zebrafish (P < 0.05). Besides, dietary SWFC0.3 also notably improved the expression level of lysozyme, c3a, defbl1 and defbl2 (P < 0.05). The expression level of pro-inflammatory factors (nf-κb, tnf-α, and il-1ß) were significantly decreased and the expression level of anti-inflammatory factor (il-10) was markedly increased in the postbiotics 0.3 g/kg group (P < 0.05). Feeding with SWFC0.3 supplemented diet for 6 weeks improved the homeostasis of gut microbiota and increased the survival rate of zebrafish after challenged with Aeromonus veronii Hm091 (P < 0.01). It was worth noting that the positive effect of dietary SWFC at a level of 0.3 g/kg was considerably better than that of 0.4 g/kg. This may imply that the effectiveness and use of postbiotics is limited by dosage.

Central and peripheral pulmonary sclerosing pneumocytomas: multi-phase CT study and comparison with Ki-67.

BACKGROUND: This study aimed to evaluate the multi-phase CT findings of central and peripheral pulmonary sclerosing pneumocytomas (PSPs) and compared them with Ki-67 to reveal their neoplastic nature. PATIENTS AND METHODS: Multi-phase CT and clinical data of 33 PSPs (15 central PSPs and 18 peripheral PSPs) were retrospectively analyzed and compared their multi-phase CT features and Ki-67 levels. RESULTS: For quantitative indicators, central PSPs were larger than peripheral PSPs (10.39 ± 3.25 cm3 vs. 4.65 ± 2.61 cm3, P = 0.013), and tumor size was negatively correlated with acceleration index (r = -0.845, P < 0.001). The peak enhancement of central PSPs appeared in the delayed phase, with a longer time to peak enhancement (TTP, 100.81 ± 19.01 s), lower acceleration index (0.63 ± 0.17), progressive enhancement, and higher Ki-67 level. The peak enhancement of peripheral PSPs appeared in the venous phase, with the shorter TTP (62.67 ± 20.96 s, P < 0.001), higher acceleration index (0.99 ± 0.25, P < 0.001), enhancement washout, and lower Ki-67 level. For qualitative indicators, the overlying vessel sign (86.67% vs. 44.44%, P = 0.027), prominent pulmonary artery sign (73.33% vs. 27.78%, P = 0.015), and obstructive inflammation/atelectasis (26.67% vs. 0%, P = 0.033) were more common in central PSPs, while peripheral PSPs were more common with halo sign (38.89% vs. 6.67%, P = 0.046). CONCLUSIONS: The location of PSP is a possible contributing factor to its diverse imaging-pathological findings. The tumor size, multi-phase enhancement, qualitative signs, and Ki-67 were different between central and peripheral PSPs. Combined tumor size, multi-phase findings, and Ki-67 level are helpful to reveal the nature of the borderline tumor.

The effect of stabilized culture of Lactobacillus rhamnosus GCC-3 on gut and liver health, and anti-viral immunity of zebrafish.

Probiotics are promising antibiotics alternatives to improve growth and disease resistance of cultured fish. Our study aimed to investigate the effect of dietary stabilized culture of Lactobacillus rhamnosus GCC-3 on growth performance, gut and liver health and anti-viral ability of zebrafish (Danio rerio). Zebrafish (0.161 ± 0.001 g) were fed control and the experimental diet containing 1% GCC-3 culture (1 × 107 CFU/g diet) for four weeks. Growth performance and gut and liver health parameters were monitored after four weeks feeding. The gut microbiota was analyzed by 16S rRNA gene sequencing. In another experiment, zebrafish (0.212 ± 0.001 g) were fed with basal or GCC-3 diets and challenged by spring viremia of carp virus (SVCV) at the end of feeding. The antiviral immune response was evaluated at 2nd and 4th days post SVCV infection and survival rate was calculated 14 days after challenge. The results showed that adding 1% GCC-3 significantly improved growth performance of zebrafish (P < 0.05). The intestinal expression of hypoxia-inducible factor Hif-1α, tight junction protein ZO-1α and ZO-1ß was significantly up-regulated in 1% GCC-3 group compared with control (P < 0.05). Besides, 1% GCC-3 decreased the content of MDA and increased total antioxidant capacity in the intestine, and the relative expression of SOD, GST and Gpxa was improved. The abundance of Proteobacteria was reduced while Firmicutes was enriched in the intestinal microbiota of 1% GCC-3 group compared with control (P < 0.05). Zebrafish fed 1% GCC-3 showed higher survival rate after SVCV challenge. Accordingly, the expression of antiviral genes in the spleen was increased at 2nd and 4th days post infection. In conclusion, our results indicate that dietary 1% GCC-3 supplementation can improve gut and liver health as well as antiviral immunity of zebrafish.

The direct and gut microbiota-mediated effects of dietary bile acids on the improvement of gut barriers in largemouth bass (Micropterus salmoides).

Fish gut barrier damage under intensive culture model is a significant concern for aquaculture industry. This study aimed to investigate the effects of bile acids (BAs) on gut barriers in Micropterus salmoides. A germ-free (GF) zebrafish model was employed to elucidate the effects of the direct stimulation of BAs and the indirect regulations mediated by the gut microbiota on gut barrier functions. Four diets were formulated with BAs supplemented at 0, 150, 300 and 450 mg/kg, and these 4 diets were defined as control, BA150, BA300 and BA450, respectively. After 5 weeks of feeding experiment, the survival rate of fish fed with BA300 diet was increased (P < 0.05). Histological analysis revealed an improvement of gut structural integrity in the BA150 and BA300 groups. Compared with the control group, the expression of genes related to chemical barrier (mucin, lysozyme and complement 1) and physical barrier (occludin and claudin-4) was increased in the BA150 and BA300 groups (P < 0.05), and the expression of genes related to immunological barrier (interleukin [IL]-6, tumor growth factor ß, IL-10, macrophage galactose-type lectin and immunoglobulin M [IgM]) was significantly increased in the BA300 group (P < 0.05), but the expression of genes related to chemical barrier (hepcidin) and immunological barrier (IL-1ß, tumor necrosis factor-α, IL-6 and arginase) was significantly decreased in the BA450 group (P < 0.05). Gut microbiota composition analysis revealed that the abundance of Firmicutes was augmented prominently in the BA150 and BA300 groups (P < 0.05), while that of Actinobacteriota and Proteobacteria showed a downward trend in the BA150 and BA300 groups (P > 0.05). The results of the gut microbiota transferring experiment demonstrated an upregulation of gut barrier-related genes, including immunoglobulin Z/T (IgZ/T), IL-6, IL-1ß and IL-10, by the gut microbiota transferred from the BA300 group compared with the control (P < 0.05). Feeding the BA300 diet directly to GF zebrafish resulted in enhanced expression of IgM, IgZ/T, lysozyme, occludin-2, IL-6 and IL-10 (P < 0.05). In conclusion, BAs can improve the gut barriers of fish through both direct and indirect effects mediated by the gut microbiota.

The positive effects of postbiotic (SWF concentration®) supplemented diet on skin mucus, liver, gut health, the structure and function of gut microbiota of common carp (Cyprinus carpio) fed with high-fat diet.

Postbiotics are an emerging research interest in recent years, which shows that metabolites, lysate extracts, cell wall components and even culture supernatants of probiotics can also exhibit significant prebiotic effects. In this study postbiotic stress worry free concentration® (SWFC) were prepared from the composition of culture supernatant of Cetobacterium somerae and Lactococcus lactis. The positive effects of SWFC supplemented diets on the growth performance, skin mucus, liver and gut health, and intestinal microbiota profile of Cyprinus carpio fed with high fat diets were investigated. 180 C. carpio with an average body weight of (3.01 ± 0.01) g were selected and randomly divided into three groups. They were fed with one of the three experimental diets supplemented with SWFC of 0 (control), 0.2 and 0.3 g/kg for 98 days, afterwards indexes were detected. The results revealed that, addition of SWFC had no significant effect on growth performance of C. carpio, while it can improve the health of the fish remarkably. In addition, SWFC improved mucosal C3, T-AOC, SOD activities, and decreased lipid peroxidation product MDA level, which were notably better than those in the control group (P < 0.05). In terms of the liver health systems, C. carpio fed on the diet supplemented with 0.2 g/kg of SWFC, showed significant improvement of the liver injured by HFD and reduce the contents of serum ALT and AST, and liver TAG (P < 0.05; P < 0.01). The expression of inflammation-related and lipid synthesis genes revealed that SWFC0.2 group could noteworthy enhance antioxidant capacity, reduced the expression of pro-inflammatory factors (TNF-α, IL-1ß) and lipid synthesis genes (ACC, FAS, PPAR-ß, PPAR-γ), and up-regulated the expression of anti-inflammatory factors (TGF-ß). Additionally, intestinal morphology arose inflammatory cell infiltration, while intestinal integrity was better in SWFC groups compared with the control. Furthermore, the contents of serum LPS and LBP were remarkably lower in the SWFC0.2 group compared with the control (P < 0.01). The mRNA expression of genes related to gut health indicated that SWFC supplementation noteworthy up-regulated the expression of antioxidant (Nrf2, CAT, GPX), immune (Hepcidin, IL-10) and tight junction protein-related (ZO-1, Occludin). Simultaneously, the results of GF-zebrafish showed that the relative expression of anti-inflammatory genes (IL-1ß, TGF-ß) and antioxidant related genes (Nrf2, HO-1) were significantly up-regulated in SWFC groups. Data on intestinal microbiota profile verified that, at the phylum level, the abundance of Fusobacteria was remarkably elevated in the SWFC groups (P < 0.05), whereas the abundance of Firmicutes was declined noteworthy in SWFC0.2 and SWFC0.3 compared to the control group (P < 0.05; P < 0.01) respectively. At the genus level, the abundance of Cetobacterium in the SWFC groups were notably higher than those in the control group (P < 0.05), while the Vibrio content in the SWFC groups was significantly decreased (P < 0.05). PCoA result indicated that the intestinal microflora of SWFC0.2 group was abundant and diverse. Our results elucidate that dietary supplementation of SWFC protects C. carpio from HFD induced inflammatory response and oxidative stress, ameliorate skin mucus, liver and gut health, and improve the gut microbiota balance. Therefore, SWFC could be considered as an improving-fish-health additive, when supplemented to aquatic animal feed. With regards to how SWFC regulates the immunity and inflammatory responses and which signal transductions are involved remains unclear and more scientific evidences are needed to address these issues.

Nuclease treatment enhanced the ameliorative effect of yeast culture on epidermal mucus, hepatic lipid metabolism, inflammation response and gut microbiota in high-fat diet-fed zebrafish.

As a functional feed additive, yeast cultures are rich in nucleotides, and adding extra nuclease can significantly increase the content of nucleotides in yeast culture. In this experiment, the effects on growth, epidermal mucus, liver and intestinal health of zebrafish were evaluated by supplementing the yeast culture or nuclease-treated yeast culture with a high-fat diet (HFD). One-month-old zebrafish were fed four diets: normal diet (NORM), HFD, yeast culture diet (YC), and nuclease-treated yeast culture diet (YC (N)) for three weeks. Results showed that the complement 4 activity of the epidermal mucus in YC (N) group was significantly higher than those in HFD and YC groups (P < 0.05). The YC and YC (N) significantly reduced the content of hepatic triglyceride caused by HFD (P < 0.05). Moreover, compared with the YC group, the YC (N) significantly increased the expression of lipolysis genes, such as PPARα, PGC1α, ACOX3 (P < 0.05). Compared with the YC group, the YC (N) group significantly increased the expression of liver pro-inflammatory factors TNFα, IL-6, IL-1ß and anti-inflammatory factors TGFß, IL-10 (P < 0.05). The diet YC and YC (N) significantly improved the height of the intestinal villus (P < 0.05). Compared with the HFD group, the YC (N) group significantly increased the expression of intestinal pro-inflammatory factors TNFα, IL-6 and anti-inflammatory factors TGFß, IL-10 (P < 0.05). The YC (N) group significantly decreased the abundance of intestinal Proteobacteria and Acinetobacter, and increased the abundance of intestinal Actinobacteria, Mycobacterium and Rhodobacter (P < 0.05). In conclusion, compared with the supplement of yeast culture, nuclease treated yeast culture can further alleviate the adverse effects of HFD on liver and intestinal health, and be used as feed additives for the nutritional and immune regulation of fish.

The effect and underlying mechanism of yeast ß-glucan on antiviral resistance of zebrafish against spring viremia of carp virus infection.

ß-glucan has been used as immunostimulant for fish. However, the effect of yeast ß-glucan on viral infections has been less studied in fish. In this study, we investigated the effects of ß-glucan on the resistance of zebrafish against spring viraemia of carp virus (SVCV) and elucidated the underlying mechanisms. Zebrafish were fed with a control diet or diet supplemented with 0.01% and 0.025% ß-glucan for 2 weeks, and were challenged by SVCV. Zebrafish embryonic fibroblast (ZF4) cells were treated with 5 µg/mL ß-glucan and were infected by SVCV. We further investigated the effect of ß-glucan on autophagy level post SVCV infection. The intestinal microbiota was evaluated by 16S rRNA gene pyrosequencing. Results showed that dietary supplementation of 0.025% ß-glucan significantly increased survival rate of zebrafish compared with control group after SVCV challenge (P < 0.05). Dietary ß-glucan significantly increased the expression of genes related to type I IFN antiviral immune pathway in the spleen of zebrafish after viral infection, including type I IFN genes (ifnφ1, ifnφ2, ifnφ3), IFN-stimulated genes (mxb, mxc), as well as other genes involved in the IFN signaling pathway, including tlr7, rig1, mavs, irf3 and irf7. Morpholino knockdown of type I IFN receptors dampened the antiviral effect of ß-glucan in zebrafish larvae, indicating that ß-glucan-mediated antiviral function was at least partially dependent on IFN immune response. Furthermore, ß-glucan can inhibit the replication of SVCV in ZF4 cells. However, ß-glucan did not stimulate type I IFN antiviral response in ZF4 cells, and the antiviral effect of ß-glucan in ZF4 was independent of Myd88. Interestingly, ß-glucan induced autophagy in ZF4 cells after SVCV infection. Inhibition of autophagy blocked the antiviral effect of ß-glucan in ZF4 cells. Lastly, dietary ß-glucan changed the composition of intestinal microbiota in zebrafish, with reduced abundance of Proteobacteria and an enrichment of Fusobacteria and Firmicutes. To sum up, our results indicate that the ß-glucan enhanced resistance of zebrafish against SVCV and the mechanism involved stimulation of type I IFN antiviral immune response of fish after viral infection.

Study about the combination strategy of Bacillus subtilis wt55 with AiiO-AIO6 to improve the resistance of zebrafish to Aeromonas veronii infection.

Disease problems will seriously restrict the sustainable development of aquaculture, and the environmental-friendly prevention strategies are urgently needed. Probiotics and quorum-quenching enzyme are innovative strategies to control bacterial diseases. Firstly, the bacteriostatic activity of Bacillus subtilis wt55 strain and quenching enzyme AiiO-AIO6 on the growth of Aeromonas veronii were tested in vitro, and the results showed wt55 inhibit the growth of A. veronii, but AiiO-AIO6 did not. Then, the synergistic effects of simple combination of B. subtilis wt55 and AiiO-AIO6 were evaluated next. The results showed this combination could improve the survival rate and significantly reduce the number of invasive A. veronii in gut after challenge compared to the other groups, corresponding to the lower intestinal alkaline phosphatase activity. One of its effect mechanisms is the combination could inhibit the growth of A. veronii in vitro; the other is direct immersion of germ-free zebrafish proved AiiO-AIO6 did not directly regulate the innate immune response of the host, but wt55 did it, and the simple combination group could significantly reduce the expression of nuclear factor kappa-B (NF-κB) and proinflammatory cytokine interleukin-1ß (IL-1ß), increase the expression of lysozyme gene; and the third is intestinal microbiota also plays a regulatory role: the gut microbiota from combination group could significantly inhibit the expression of IL-1ß and NF-κB, and increased the expression of transforming growth factor-ß (TGF-ß) and lysozyme. Given the effectiveness of this simple combination, a B. subtilis quorum-quenching recombinant expression strain in which AiiO-AIO6 was surface displayed on the spores and secreted by vegetative cells was built. The results showed that the survival rate after challenge was lower than that of the group treated with AiiO-AIO6 or wt55 alone, and the expression of proinflammatory cytokine IL-1ß and NF-κB were significantly higher. Our study demonstrated the effectiveness of B. subtilis and AiiO-AIO6 simple combination and established an efficient B. subtilis expression system.

Comparison of foreign language anxiety based on four language skills in Chinese college students.

BACKGROUND: Numerous studies have established that foreign language anxiety (FLA) has a significant impact on learners' language acquisition and performance. FLA is a unique form of anxiety that occurs in response to a certain circumstance. Even though a growing amount of research has extended to the examination of skill-based anxiety in specific, such as listening, speaking, reading, and writing, little used corresponding skilled-based FLA scales to assess learners' skill-based FLA at one group of learners. To fill a void in this line of research, the study aimed to explore learners' primary FLA by comparing their four language skill-specific FLAs with four different skill-based FLA scales. Additionally, we wished to investigate the variables that influence learners' FLA. METHODS: All participants in the study were first-year medical students. Individual instructors delivered and collected a total of 1023 questionnaires using an open questionnaire platform during normal English lessons in the mid-semester. SPSS 24.0 (Statistical Package for Social Science 24 version) was used to analyze all statistics. Internal validity tests were undertaken on each scale to ascertain the component structure of certain modified scales. The study employed the independent sample t-test and a statistical description to investigate students' major FLA and its variables. RESULTS: With a mean value of 106.863, the predominant FLA arouses from English listening anxiety. English reading anxiety was the lowest, with a mean score of 62.726. Male and female students both demonstrated the greatest degree of FLA in English listening and the least anxiety in English reading. However, their mean difference was not statistically significant (t = 1.220). By comparing the mean scores for four skill-based FLAs across language proficiency groups, it became clear that the scores for the medium were much higher than the average, with average scores of ESA: 91.988, ELA: 106.864, EWA: 74.157, ERA: 62.726, respectively, and the higher-level group scored lower than the average. Students' prior English learning achievements are negatively connected with their FLA, with r values of -.207 (ELA), -.143 (EWA), and - .204 (ERA). The self-evaluation of students' English listening, writing, and reading abilities was considerably adversely connected with FLA, but the self-evaluation of their English-speaking abilities was favorably correlated with FLA. CONCLUSION: A comparison of students' FLA revealed that the primary skill-based FLA is related to English listening anxiety. Regardless of gender or language proficiency level, FLA was mainly driven by foreign language listening anxiety in all individuals. Prior language achievement and students' self-evaluation are associated with their FLA.

Dietary supplementation of exopolysaccharides from Lactobacillus rhamnosus GCC-3 improved the resistance of zebrafish against spring viremia of carp virus infection.

Spring viremia of carp virus (SVCV) can cause high mortality of fish. The aim of this study was to investigate the effects of Lactobacillus rhamnosus GCC-3 exopolysaccharides (GCC-3 EPS) on zebrafish (Danio rerio) infected with SVCV and elucidate the underlying mechanisms. Zebrafish were fed with a control diet or diet supplemented with 0.5% and 1% of GCC-3 EPS for 2 weeks. The results showed that supplementation of GCC-3 EPS significantly improved the survival rate of zebrafish compared with the control group. In addition, dietary 0.5% and 1% GCC-3 EPS significantly up-regulated the expression of genes related to type I interferon (IFN) antiviral immunity. Consistent with in vivo results, GCC-3 EPS significantly inhibited SVCV replication in zebrafish embryonic fibroblast (ZF4) cells while significantly increased the expression of type I IFN signaling pathway related genes. Furthermore, knocking down TANK-binding kinase 1 significantly blocked the antiviral effect of GCC-3 EPS. Dietary GCC-3 EPS improved gut microbiota, and the culture supernatant of GCC-3 EPS-associated microbiota significantly inhibited SVCV replication in ZF4 cells compared with the control-microbiota counterpart. In conclusion, our results indicate that dietary GCC-3 EPS can improve the resistance of zebrafish against SVCV infection, and the mechanism may involve enhanced type I interferon signaling.

Nuclease-Treated Stabilized Fermentation Product of Cetobacterium somerae Improves Growth, Non-specific Immunity, and Liver Health of Zebrafish (Danio rerio).

High-fat diets (HFD) are harmful to fish health. Probiotics are commonly utilized to improve fish nutrition metabolism, immune response, and health. Nucleic acids of the probiotic bacterium can be hydrolyzed by nuclease to generate nucleotides. The present study aimed to evaluate the effects of stabilized fermentation product of nuclease-treated Cetobacterium somerae XMX-1 [XMX-1 (N)] on growth, non-specific immunity, and liver health of zebrafish (Danio rerio). Compared to the HFD group, 100 g/kg XMX-1 (N) significantly increased weight gain and decreased feed conversion ratio (FCR). However, 5 or 10 g/kg XMX-1 (N) had no influence on zebrafish growth. In addition, supplementation of 100 g/kg XMX-1 (N) significantly increased lysozyme activity and total antioxidant capacity in skin mucus, and the expression of inflammation related genes interleukin 1 beta (IL-1ß), interleukin 10 (IL-10), and interleukin 6 (IL-6) in the gut as well as fatty acid oxidation related genes uncoupling protein 2 (UCP2) and proliferator-activated receptor γ coactivator 1α (PGC1α) in the liver, while decreased the content of hepatic triacylglycerol (TAG) in zebrafish. The gene sequencing, 16S rRNA, showed that 100 g/kg XMX-1 (N) enhanced the relative abundance of Firmicutes while lowered Proteobacteria and Actinobacteria. 10 g/kg XMX-1 (N) significantly increased lysozyme activity and complement component 4 (C4) in skin mucus, and intestinal expression of inflammation-related genes. In the 5 g/kg XMX-1 (N) group, however, only an increase in C4 level in skin mucus was observed. Together, these results reveal that dietary supplementation with nuclease-treated C. somerae XMX-1 (N) has a dose-dependent beneficial effect on fish health.

Dietary Succinate Impacts the Nutritional Metabolism, Protein Succinylation and Gut Microbiota of Zebrafish.

Succinate is widely used in the food and feed industry as an acidulant, flavoring additive, and antimicrobial agent. This study investigated the effects of dietary succinate on growth, energy budget, nutritional metabolism, protein succinylation, and gut microbiota composition of zebrafish. Zebrafish were fed a control-check (0% succinate) or four succinate-supplemented diets (0.05, 0.10, 0.15, and 0.2%) for 4 weeks. The results showed that dietary succinate at the 0.15% additive amount (S0.15) can optimally promote weight gain and feed intake. Whole body protein, fat, and energy deposition increased in the S0.15 group. Fasting plasma glucose level decreased in fish fed the S0.15 diet, along with improved glucose tolerance. Lipid synthesis in the intestine, liver, and muscle increased with S0.15 feeding. Diet with 0.15% succinate inhibited intestinal gluconeogenesis but promoted hepatic gluconeogenesis. Glycogen synthesis increased in the liver and muscle of S0.15-fed fish. Glycolysis was increased in the muscle of S0.15-fed fish. In addition, 0.15% succinate-supplemented diet inhibited protein degradation in the intestine, liver, and muscle. Interestingly, different protein succinylation patterns in the intestine and liver were observed in fish fed the S0.15 diet. Intestinal proteins with increased succinylation levels were enriched in the tricarboxylic acid cycle while proteins with decreased succinylation levels were enriched in pathways related to fatty acid and amino acid degradation. Hepatic proteins with increased succinylation levels were enriched in oxidative phosphorylation while proteins with decreased succinylation levels were enriched in the processes of protein processing and transport in the endoplasmic reticulum. Finally, fish fed the S0.15 diet had a higher abundance of Proteobacteria but a lower abundance of Fusobacteria and Cetobacterium. In conclusion, dietary succinate could promote growth and feed intake, promote lipid anabolism, improve glucose homeostasis, and spare protein. The effects of succinate on nutritional metabolism are associated with alterations in the levels of metabolic intermediates, transcriptional regulation, and protein succinylation levels. However, hepatic fat accumulation and gut microbiota dysbiosis induced by dietary succinate suggest potential risks of succinate application as a feed additive for fish. This study would be beneficial in understanding the application of succinate as an aquatic feed additive.

Partially replacing dietary fish meal by Saccharomyces cerevisiae culture improve growth performance, immunity, disease resistance, composition and function of intestinal microbiota in channel catfish (Ictalurus punctatus).

The aim of the present study was to investigate the partial replacement of fish meal by Saccharomyces cerevisiae culture on growth performance, immunity, composition and function of intestinal microbiota and disease resistance in channel catfish (Ictalurus punctatus). Two equal nitrogen and energy diets were prepared including a basal diet (containing 10% fish meal, Control) and an experimental diet (replacing 20% of the fish meal of the basal diet with yeast culture, RFM). Channel catfish were fed with the diets for 12 weeks. The results showed that weight gain and condition factor were significantly increased, and FCR was significantly decreased in RFM group (P < 0.05). The gene expression of intestinal HIF1α was significantly increased in RFM group (P < 0.05), while the expressions of NF-κB in the intestine and liver were significantly decreased (P < 0.05). The relative abundance of Firmicutes tended to increase, and the Turicibacter had an upward trend (0.05 < P < 0.2). In addition, the survival rate of channel catfish was significantly increased in RFM group after challenged with Aeromonas veronii Hm091 and Aeromonas hydrophila NJ-1 (P < 0.05). Compared with intestinal microbiota of channel catfish of control group, intestinal microbiota of channel catfish of RFM group significantly increased the expression of HIF1α, and decreased the expression of IL-1ß and TNF-α (P < 0.05) in germ-free zebrafish. Intestinal microbiota induced by RFM diet also significantly increased disease resistance to Aeromonas veronii Hm091 and Aeromonas hydrophila NJ-1. In conclusion, replacement of fish meal by the yeast culture improved the growth, immunity and disease resistance of channel catfish, and intestinal microbiota of channel catfish induced by the yeast culture played a critical role in these effects.

Excess DHA Induces Liver Injury via Lipid Peroxidation and Gut Microbiota-Derived Lipopolysaccharide in Zebrafish.

Being highly unsaturated, n-3 long-chain polyunsaturated fatty acids (LC-PUFAs) are prone to lipid peroxidation. In this study, zebrafish were fed with low-fat diet (LFD), high-fat diet (HFD), or 2% DHA-supplemented HFD (HFDHA2.0). To study the possible negative effects of the high level of dietary DHA, growth rates, blood chemistry, liver histology, hepatic oxidative stress, apoptosis, and inflammatory processes were assessed. The cell studies were used to quantify the effects of DHA and antioxidant on cellular lipid peroxidation and viability. The possible interaction between gut microbiota and zebrafish host was evaluated in vitro. HFDHA2.0 had no effect on hepatic lipid level but induced liver injury, oxidative stress, and hepatocellular apoptosis, including intrinsic and death receptor-induced apoptosis. Besides, the inclusion of 2% DHA in HFD increased the abundance of Proteobacteria in gut microbiota and serum endotoxin level. In the zebrafish liver cell model, DHA activated intrinsic apoptosis while the antioxidant 4-hydroxy-Tempo (tempo) inhibited the pro-apoptotic negative effects of DHA. The apoptosis induced by lipopolysaccharide (LPS) was unaffected by the addition of tempo. In conclusion, the excess DHA supplementation generates hepatocellular apoptosis-related injury to the liver. The processes might propagate along at least two routes, involving lipid peroxidation and gut microbiota-generated LPS.

Effect of dietary supplementation of Cetobacterium somerae XMX-1 fermentation product on gut and liver health and resistance against bacterial infection of the genetically improved farmed tilapia (GIFT, Oreochromis niloticus).

The purpose of this study was to evaluate the effects of Cetobacterium somerae XMX-1 fermentation product on gut and liver health and resistance against bacterial infection in genetically improved farmed tilapia (GIFT, Oreochromis niloticus). Fingerling GIFTs (n = 120; initial weight 1.33 ± 0.00 g) were randomly assigned to twelve 90-L tanks (four tanks per diet, 10 fish per tank) with three groups: control group (basal high fat diet), 1% XMX-1 group and 2% XMX-1 group (basal diet supplemented with 10 and 20 g XMX-1/kg feed respectively). After 49 days feeding trial, the growth performance and gut and liver health parameters of tilapia were evaluated. Also the gut microbiota and virome were detected by sequencing. 2% XMX-1 fermentation product had no effect on growth performance. For gut health, the expression of hypoxia-inducible factor-lα (Hif-1α) tend to increase in 1% XMX-1 group (P = 0.053). The expression of intestinal interleukin-6 (IL-6) and tumor growth factor ß (TGF-ß) was significantly down-regulated in 1% and 2% XMX-1 groups (P < 0.05), and the intestinal expression of interleukin-1ß (IL-1ß) had a trend to decrease (P = 0.08) in 1% XMX-1 group versus control. 1% and 2% XMX-1 groups also increased the intestinal expression of tight junction genes Claudin (P = 0.06 and 0.07, respectively). For liver health, XMX-1 fermentation product significantly decreased liver TAG (P < 0.05). Furthermore, the hepatic expression of lipid synthesis gene fatty acid synthase (FAS) was significantly decreased and the expression of lipid catabolism related-gene uncoupling protein 2 (UCP2) was significantly increased in 1% XMX-1 and 2% XMX-1 groups (P < 0.01). And the hepatic expression of IL-1ß and IL-6 significantly decreased in 1% XMX-1 and 2% XMX-1 groups (P < 0.05). XMX-1 fermentation product increased the abundance of Fusobacteria in the gut microbiota and 2% XMX-1 group led to alteration in the virome composition at family level. Lastly, the time of tilapia death post Aeromoans challenge was delayed in 1% XMX-1 and 2% XMX-1 groups compared with control. To sum up, our results show that the dietary supplementation of XMX-1 fermentation product can improve the gut and liver health as well as the resistance against pathogenic bacteria of tilapia.