Pichia pastoris composition expressed aerolysin mutant of Aeromonas veronii as an oral vaccine evaluated in zebrafish (Danio rerio).

Vaccines are one of the most practical means to stop the spreading of Aeromonas veronii in aquaculture. In this study, virulence factor aerolysin mutant NTaer which has lost its hemolytic activity was used as a target antigen. Pichia pastoris constitutive secretory expression NTaer (GS115-NTaer) was used as a potential safe oral vaccine to evaluate its effectiveness on zebrafish immunity. The result shows that vaccination of GS115- NTaer for four weeks did not affect the growth performance of the host, while eliciting an effective immune protective response. Compared with the control group, the GS115-NTaer could significantly up-regulate the relative expression level of the intestinal tight junction protein 1α (TJP1α) gene, and significantly increased the contents of lysozyme (LYZ), complement C3 and C4 in the gut, indicating that the innate immune response of the fish was activated. The relative gene expression levels of macrophage-expressed gene 1 (MPEG1) and T cell receptor (TCR-α) in the gut, and MPEG1, CD4, CD8, TCR-α, GATA3, and T-bet in the spleen were all increased significantly, indicating that the cellular immune response of the fish was activated. Furthermore, the contents of serum IgM and intestinal mucosa IgZ antibodies were significantly increased, which showed that humoral immunity was also activated. Moreover, inoculation with GS115-NTaer significantly changed the structure of gut microbiota. In particular, the relative ratio of (Firmicutes + Fusobacteriota + Bacteroidota)/Proteobacteria was significantly higher than that of the control and GS115 groups. Lastly, the vaccinated fish were challenged with A. veronii, and the relative percent survival of GS115 and the GS115-NTear groups was 14.28% and 33.43%. This improvement of immunity was not only due to the specific immune response but also attributed to the improvement of innate immunity and the gut microbiota which was demonstrated by the germ-free zebrafish model. Collectively, this study provides information on the effectiveness of GS115-NTear as an oral vaccine for the green prevention and control of A. veronii infection in fish aquaculture.

Lactobacillus rhamnosus GG triggers intestinal epithelium injury in zebrafish revealing host dependent beneficial effects.

Lactobacillus rhamnosus GG (LGG), the well-characterized human-derived probiotic strain, possesses excellent properties in the maintenance of intestinal homeostasis, immunoregulation and defense against gastrointestinal pathogens in mammals. Here, we demonstrate that the SpaC pilin of LGG causes intestinal epithelium injury by inducing cell pyroptosis and gut microbial dysbiosis in zebrafish. Dietary SpaC activates Caspase-3-GSDMEa pathways in the intestinal epithelium, promotes intestinal pyroptosis and increases lipopolysaccharide (LPS)-producing gut microbes in zebrafish. The increased LPS subsequently activates Gaspy2-GSDMEb pyroptosis pathway. Further analysis reveals the Caspase-3-GSDMEa pyroptosis is initiated by the species-specific recognition of SpaC by TLR4ba, which accounts for the species-specificity of the SpaC-inducing intestinal pyroptosis in zebrafish. The observed pyroptosis-driven gut injury and microbial dysbiosis by LGG in zebrafish suggest that host-specific beneficial/harmful mechanisms are critical safety issues when applying probiotics derived from other host species and need more attention.

An Unexpected Alteration Colonic Mucus Appearance in the Constipation Model via an Intestinal Microenvironment.

Due to the lack of research between the inner layers in the structure of colonic mucous and the metabolism of fatty acid in the constipation model, we aim to determine the changes in the mucous phenotype of the colonic glycocalyx and the microbial community structure following treatment with Rhubarb extract in our research. The constipation and treatment models are generated using adult male C57BL/6N mice. We perform light microscopy and transmission electron microscopy (TEM) to detect a Muc2-rich inner mucus layer attached to mice colon under different conditions. In addition, 16S rDNA sequencing is performed to examine the intestinal flora. According to TEM images, we demonstrate that Rhubarb can promote mucin secretion and find direct evidence of dendritic structure-linked mucus structures with its assembly into a lamellar network in a pore size distribution in the isolated colon section. Moreover, the diversity of intestinal flora has noticeable changes in constipated mice. The present study characterizes a dendritic structure and persistent cross-links have significant changes accompanied by the alteration of intestinal flora in feces in models of constipation and pretreatment with Rhubarb extract.

Surface-Displayed Amuc_1100 From Akkermansia muciniphila on Lactococcus lactis ZHY1 Improves Hepatic Steatosis and Intestinal Health in High-Fat-Fed Zebrafish.

Fatty liver and intestinal barrier damage were widespread in most farmed fish, which severely restrict the development of aquaculture. Therefore, there was an urgent need to develop green feed additives to maintain host liver and intestinal health. In this study, a probiotic pili-like protein, Amuc_1100 (AM protein), was anchored to the surface of Lactococcus lactis ZHY1, and the effects of the recombinant bacteria AM-ZHY1 on liver fat accumulation and intestinal health were evaluated. Zebrafish were fed a basal diet, high-fat diet, and high-fat diet with AM-ZHY1 (108 cfu/g) or control bacteria ZHY1 for 4 weeks. Treatment with AM-ZHY1 significantly reduced hepatic steatosis in zebrafish. Quantitative PCR (qPCR) detection showed that the expression of the lipogenesis [peroxisome-proliferator-activated receptors (PPARγ), sterol regulatory element-binding proteins-1c (SREBP-1c), fatty acid synthase (FAS), and acetyl-CoA carboxylase 1 (ACC1)] and lipid transport genes (CD36 and FABP6) in the liver were significantly downregulated (p < 0.05), indicating that AM-ZHY1 could reduce liver fat accumulation by inhibiting lipid synthesis and absorption. Moreover, supplementing AM-ZHY1 to a high-fat diet could significantly reduce serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, indicating that liver injury caused by high-fat diets was improved. The expression of tumor necrosis factor (TNF)-a and interleukin (IL)-6 in the liver decreased significantly (p < 0.05), while IL-1ß and IL-10 did not change significantly in the AM-ZHY1 group. Compared to the high-fat diet-fed group, the AM-ZHY1 group, but not the ZHY1 group, significantly increased the expression of intestinal tight junction (TJ) proteins (TJP1a, claudina, claudin7, claudin7b, claudin11a, claudin12, and claudin15a; p < 0.05). Compared to the high-fat diet group, the Proteobacteria and Fusobacteria were significantly reduced and increased in the AM-ZHY1 group, respectively. In conclusion, the recombinant bacteria AM-ZHY1 has the capacity to maintain intestinal health by protecting intestinal integrity and improving intestinal flora structure and improving fatty liver disease by inhibiting lipid synthesis and absorption. This study will lay a foundation for the application of AM protein in improving abnormal fat deposition and restoring the intestinal barrier in fish.

Rhubarb extract relieves constipation by stimulating mucus production in the colon and altering the intestinal flora.

BACKGROUND: Constipation, mainly characterized by the difficulty in defecation, is a clinical symptom caused by a variety of factors. It can be manifested as normal or slow colonic transport abnormalities, which can occur alone or concurrently with defecation disorders. As there is not uniform definition and assessment standard, no clear plan could be used for the treatment of constipation. Although rhubarb, a traditional Chinese medicine, plays a therapeutic role in diseases involving constipation symptoms, the detailed mechanism of it in treating constipation remains unclear. METHODS: A model of constipation-induced by diphenoxylate was prepared. Immunofluorescent staining was used to detect the expression of mucin 2 (MUC2), calnexin and chymase in colon. Western blotting was used to detect changes of tryptase and calnexin in the colon. And real-time polymerase chain reaction (PCR) was utilized to detect the changes of immunoglobulin-binding protein (Bip), X-box binding protein 1 (Xbp1) and C/EBP homologous protein (CHOP) of colonic goblet cells in mRNA levels. ELISA and biochemical kits were utilized to detect the changes of MUC2, Trefoil factor 3 (TFF3), acetylcholine, histamine and C-C motif chemokine ligand 5 (CCL5) in the colon. And the changes of colonic mucosa and intestinal flora of constipation model mice caused by rhubarb extract (RE) were analyzed to identify the mechanism of RE on the treatment of constipation. RESULTS: RE promotes the secretion of colonic mucus by recruiting mast cells and enhancing the content of histamine and Ach in the mice colon. In the process, RE causes up-regulation of Bip and CHOP mRNA expression and down-regulation of Xbp1 and Xbp1s mRNA expression that induces ER stress of colonic epithelium associated with changes in the intestinal flora diversity and short-chain fatty acids content. CONCLUSION: RE could relieve constipation by promoting the secretion of colonic mucus via mast cells activation and improving the intestinal microenvironment.

Ability of prebiotic polysaccharides to activate a HIF1α-antimicrobial peptide axis determines liver injury risk in zebrafish.

Natural polysaccharides have received much attention for their ability to ameliorate hepatic steatosis induced by high-fat diet. However, the potential risks of their use have been less investigated. Here, we show that the exopolysaccharides (EPS) from Lactobacillus rhamnosus GG (LGG) and L. casei BL23 reduce hepatic steatosis in zebrafish fed a high-fat diet, while BL23 EPS, but not LGG EPS, induce liver inflammation and injury. This is due to the fact that BL23 EPS induces gut microbial dysbiosis, while LGG EPS promotes microbial homeostasis. We find that LGG EPS, but not BL23 EPS, can directly activate intestinal HIF1α, and increased HIF1α boosts local antimicrobial peptide expression to facilitate microbial homeostasis, explaining the distinct compositions of LGG EPS- and BL23 EPS-associated microbiota. Finally, we find that liver injury risk is not confined to Lactobacillus-derived EPS but extends to other types of commonly used natural polysaccharides, depending on their HIF1α activation efficiency.

Function and dysfunction of plasma cells in intestine.

As the main player in humoral immunity, antibodies play indispensable roles in the body's immune system. Plasma cells (PCs), as antibody factories, are important contributors to humoral immunity. PCs, recognized by their unique marker CD138, are always discovered in the medullary cords of spleen and lymph nodes and in bone marrow and mucosal lymphoid tissue. This article will review the origin and differentiation of PCs, characteristics of short- and long-lived PCs, and the secretion of antibodies, such as IgA, IgM, and IgG. PCs play a crucial role in the maintenance of intestinal homeostasis using immunomodulation though complex mechanisms. Clearly, PCs play functional roles in maintaining intestinal health, but more details are needed to fully understand all the other effects of intestinal PCs.

[Effect of Denitrification and Phosphorus Removal Microorganisms in Activated Sludge Bulking Caused by Filamentous Bacteria].

Activated sludge bulking or foaming caused by filamentous bacteria is a frequent problem in the operation and management of wastewater treatment plants. To clarify the effect of filamentous bacteria sludge bulking on the functional flora in the biological denitrification and phosphorus removal system, morphological identification and Illumina MiSeq sequencing were applied to investigate the distribution of key micro-flora from the non-bulking period, sludge bulking period, and biological foaming period in five municipal wastewater treatment plants. The results showed that the sludge bulking and biological foaming were caused by Microthrix parvicella when the maximum contents were 6% and 38%, respectively. The main bacteria for denitrification and phosphorus removal were Nitrosomonas, Nitrospira, Thauera, and Candidatus Accumulibacter phosphatis. Compared to the non-bulking period, the relative abundance of AOB and PAO was significantly decreased when the maximum contents were 54% and 47%, respectively, during the bulking period. In addition, the relative abundance of denitrifying bacteria was significantly increased when the maximum content was 73%. The fluctuation of micro-flora for denitrification and phosphorus removal was affected by the activated sludge bulking and was related to the treatment process and physiological characteristics of the bacteria.

Functional macrophages and gastrointestinal disorders.

Macrophages (MΦ) differentiate from blood monocytes and participate in innate and adaptive immunity. Because of their abilities to recognize pathogens and activate bactericidal activities, MΦ are always discovered at the site of immune defense. MΦ in the intestine are unique, such that in the healthy intestine, they possess complex mechanisms to protect the gut from inflammation. In these complex mechanisms, they produce anti-inflammatory cytokines, such as interleukin-10 and transforming growth factor-ß, and inhibit the inflammatory pathways mediated by Toll-like receptors. It has been demonstrated that resident MΦ play a crucial role in maintaining intestinal homeostasis, and they can be recognized by their unique markers. Nonetheless, in the inflamed intestine, the function of MΦ will change because of environmental variation, which may be one of the mechanisms of inflammatory bowel disease (IBD). We provide further explanation about these mechanisms in our review. In addition, we review recent discoveries that MΦ may be involved in the development of gastrointestinal tumors. We will highlight the possible therapeutic targets for the management of IBD and gastrointestinal tumors, and we also discuss why more details are needed to fully understand all other effects of intestinal MΦ.

[Application of Stir Bar Sorptive Extraction and Gas Chromatograph Mass Spectrometer to the Phthalic Acid Esters Analysis in Seawater].

For the determination of trace amounts of phthalic acid esters (PAEs) in complex seawater matrix, a stir bar sorptive extraction gas chromatography mass spectrometry (SBSE-GC-MS) method was established. Dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), butyl benzyl phthalate (BBP), dibutyl phthalate (2-ethylhexyl) phthalate (DEHP) and dioctyl phthalate (DOP) were selected as study objects. The effects of extraction time, amount of methanol, amount of sodium chloride, desorption time and desorption solvent were optimized. The method of SBSE-GC-MS was validated through recoveries and relative standard deviation. The optimal extraction time was 2 h. The optimal methanol content was 10%. The optimal sodium chloride content was 5% . The optimal desorption time was 50 min. The optimal desorption solvent was the mixture of methanol to acetonitrile (4:1, volume: volume). The linear relationship between the peak area and the concentration of PAEs was relevant. The correlation coefficients were greater than 0.997. The detection limits were between 0.25 and 174.42 ng x L(-1). The recoveries of different concentrations were between 56.97% and 124.22% . The relative standard deviations were between 0.41% and 14.39%. On the basis of the method, several estuaries water sample of Jiaozhou Bay were detected. DEP was detected in all samples, and the concentration of BBP, DEHP and DOP were much higher than the rest.