The potential mechanism of concentrated mannan-oligosaccharide promoting goldfish's (Carassius auratus Linnaeus) resistance to Ichthyophthirius multifiliis invasion.

Because of the low host specificity, Ichthyophthirius multifiliis (Ich) can widely cause white spot disease in aquatic animals, which is extremely difficult to treat. Prior research has demonstrated a considerable impact of concentrated mannan-oligosaccharide (cMOS) on the prevention of white spot disease in goldfish, but the specific mechanism is still unknown. In this study, transcriptome sequencing, histological analysis, immunofluorescence analysis, phagocytosis activity assay and qRT-PCR assay were used to systematically reveal the potential mechanism of cMOS in supporting the resistance of goldfish (Carrasius auratus) to Ich invasion. According to the transcriptome analysis, the gill tissue of goldfish receiving the cMOS diet showed greater expression of mannose-receptor (MRC) related genes, higher phagocytosis activity, up-regulated expression of phagocytosis-related genes and inflammatory-related genes compared with the control, indicating that cMOS can have an effect on phagocytosis and non-specific immunity of goldfish. After the Ich challenge, transcriptome analysis revealed that cMOS fed goldfish displayed a higher level of phagocytic response, whereas non-cMOS fed goldfish displayed a greater inflammatory reaction. Besides, after Ich infection, cMOS-fed goldfish displayed greater phagocytosis activity, a stronger MRC positive signal, higher expression of genes associated with phagocytosis (ABCB2, C3, MRC), and lower expression of genes associated with inflammation (IL-1ß, IL-17, IL-8, TNF-α, NFKB). In conclusion, our experimental results suggest that cMOS may support phagocytosis by binding to MRC on the macrophage cell membrane and change the non-specific immunity of goldfish by stimulating cytokine expression. The results of this study provide new insights for the mechanism of cMOS on parasitic infection, and also suggest phagocytosis-related pathways may be potential targets for prevention of Ich infection.

Ammonia nitrogen stress damages the intestinal mucosal barrier of yellow catfish (Pelteobagrus fulvidraco) and induces intestinal inflammation.

Nitrogen from ammonia is one of the most common pollutants toxics to aquatic species in aquatic environment. The intestinal mucosa is one of the key mucosal defenses of aquatic species, and the accumulation of ammonia nitrogen in water environment will cause irreversible damage to intestinal function. In this study, histology, immunohistochemistry, ultrastructural pathology, enzyme activity analysis and qRT-PCR were performed to reveal the toxic effect of ammonia nitrogen stress on the intestine of Pelteobagrus fulvidraco. According to histological findings, ammonia nitrogen stress caused structural damage to the intestine and reduced the number of mucous cells. Enzyme activity analysis revealed that the activity of bactericidal substances (Lysozyme, alkaline phosphatase, and ACP) had decreased. The ultrastructure revealed sparse and shortened microvilli as well as badly degraded tight junctions. Immunohistochemistry for ZO-1 demonstrated an impaired intestinal mucosal barrier. Furthermore, qRT-PCR revealed that tight junction related genes (ZO-1, Occludin, Claudin-1) were downregulated, while the pore-forming protein Claudin-2 was upregulated. Furthermore, as ammonia nitrogen concentration grew, so did the positive signal of Zap-70 (T/NK cell) and the expression of inflammation-related genes (TNF, IL-1ß, IL-8, IL-10). In light of the above findings, we conclude that ammonia nitrogen stress damages intestinal mucosal barrier of Pelteobagrus fulvidraco and induces intestinal inflammation.

The Pathophysiological Changes and Clinical Effects of Tetramethylpyrazine in ICR Mice with Fluoride-Induced Hepatopathy.

The excessive intake of fluoride, one of the trace elements required to maintain health, leads to liver injury. Tetramethylpyrazine (TMP) is a kind of traditional Chinese medicine monomer with a good antioxidant and hepatoprotective function. The aim of this study was to investigate the effect of TMP on liver injury induced by acute fluorosis. A total of 60 1-month-old male ICR mice were selected. All mice were randomly divided into five groups: a control (K) group, a model (F) group, a low-dose (LT) group, a medium-dose (MT) group, and a high-dose (HT) group. The control and model groups were given distilled water, while 40 mg/kg (LT), 80 mg/kg (MT), or 160 mg/kg (HT) of TMP was fed by gavage for two weeks, with a maximum gavage volume for the mice of 0.2 mL/10 g/d. Except for the control group, all groups were given fluoride (35 mg/kg) by an intraperitoneal injection on the last day of the experiment. The results of this study showed that, compared with the model group, TMP alleviated the pathological changes in the liver induced by the fluoride and improved the ultrastructure of liver cells; TMP significantly decreased the levels of ALT, AST, and MDA (p < 0.05) and increased the levels of T-AOC, T-SOD, and GSH (p < 0.05). The results of mRNA detection showed that TMP significantly increased the mRNA expression levels of Nrf2, HO-1, CAT, GSH-Px, and SOD in the liver compared with the model group (p < 0.05). In conclusion, TMP can inhibit oxidative stress by activating the Nrf2 pathway and alleviate the liver injury induced by fluoride.

Immunosuppression and apoptosis activation mediated by p53-Bcl2/Bax signaling pathway -The potential mechanism of goldfish (Carassius auratus Linnaeus) gill disease caused by Myxobolus ampullicapsulatus.

Myxobolus, a major harmful type of myxospora, is one of the main parasitic pathogens of freshwater fish. Once myxoboliosis occurs, treatment can be extremely difficult. Therefore, clear understandings of the etiology of myxoboliosis and its pathological mechanism are keys for prevention and control. Here, histology, transmission electron microscopy, transcriptome study, tunel assay, and immunohistochemistry were carried out, revealing the morphology, pathological effects as well as host response mechanism of goldfish gill to Myxobolus ampullicapsulatus. Histological studies showed that the mature spores of Myxobolus ampullicapsulatus were composed of three parts, the spore shell, sporoplasm and bottle shaped polar capsule containing double S-shaped polar filaments. Transcriptome analysis revealed that Myxobolus ampullicapsulatus -infected (Myx) goldfish gills were characterized by apoptosis activation mediated by "p53 signaling pathway" with significantly up-regulated apoptosis-related differential genes dominated by p53-Bcl2/Bax signaling pathway. In addition, tunel assay revealed severe gill apoptosis in the Myx group. Transcriptome analysis also revealed that Myx group showed changes in immune response and significantly down-regulated immune-related differential genes. Beyond that, immunohistochemistry showed that there was no significant increase in the number of gill lymphocyte after parasite infection. These results suggest that the pathological mechanism of Myxobolus ampullicapsulatus infection on gills of goldfish may be related to apoptosis and immunosuppression. Subsequent qRT-PCR showed that apoptosis-related genes (Caspase3,Bad, Bax) and anti-inflammatory gene IL-10 were significantly increased, while immune-related pro-inflammatory genes (IL-1ß, IL-8) were markedly down-regulated, further verifying the transcriptome results. Based on the above results, we concluded that p53-Bcl2/Bax related networks that dominant the expression of apoptosis genes were activated while immunity was suppressed in the gills of Myxobolus ampullicapsulatus infected goldfish. Our study is not only of benefit to enrich the taxonomy of Myxobolus but also clarifies its pathogenic mechanism, thus providing targets for prevention and control of myxoboliosis.

cMOS enhanced the mucosal immune function of skin and gill of goldfish (Carassius auratus Linnaeus) to improve the resistance to Ichthyophthirius multifiliis infection.

METHODS: of supporting mucosal immune barrier integrity and prevention of some pathogenic infections in aquatic species, are key areas of active study, often focusing on feed additives. The objectives of this study were to explore the effects of feeding cMOS (concentrated mannan oligosaccharide) on the gill and skin mucosal barriers of goldfish (Carassius auratus Linnaeus) and evaluate health status during Ichthyophthirius multifiliis infection. After feeding the cMOS-containing diet for 60 days, Hematoxylin and eosin (H&E) staining showed greater length of gill lamella and thicker dermal dense layer, while Alcian Blue and Periodic acid-Schiff (AB-PAS) staining showed higher numbers of mucin cells in cMOS fed fish. Chemical analysis showed that fish fed cMOS had greater enzyme activity of lysozyme (LZM) and alkaline phosphatase (AKP) in gill and skin tissues, while qRT-PCR revealed higher expression of Muc-2 and IL-1ß, as well as lower expression of IL-10. After Ichthyophthirius multifiliis challenge, goldfish fed the cMOS diet had lower mortality and infection rates, as well as fewer visible white spots on the body surfaces. Histologically, the gill and skin of these fish presented less tissue damage and fewer parasites, and had a greater number of mucus cells. In addition, the expression of Muc-2 and IL-10 were notably higher while the expression of IL-1ß was significantly lower in cMOS fed goldfish than control fed fish. In this study, cMOS fed goldfish had stronger immune barrier function of skin and gill mucous, and better survival following Ichthyophthirius multifiliis infection.

Effect of Bacterial Infection on the Edibility of Aquatic Products: The Case of Crayfish (Procambarus clarkii) Infected With Citrobacter freundii.

Aquatic products are one of the world's essential protein sources whose quality and safety are threatened by bacterial diseases. This study investigated the possible effects of bacterial infection on the main edible part, the muscle, in the case of crayfish infected with Citrobacter freundii. The histopathological analysis confirmed that crayfish was sensitive to C. freundii and muscle was one of the target organs. The transcriptome results showed impaired intercellular junctions, downregulation of actin expression, and inhibition of metabolic pathways. Furthermore, transcriptomic results suggest that C. freundii mainly affect muscle structure and nutrition. Subsequent validation experiments confirmed structural damage and nutrient loss in C. freundii infected crayfish muscle. Besides, the spoilage tests showed that C. freundii did not accelerate muscle spoilage and the bacteria had a limited impact on food safety. Therefore, although C. freundii may not be a specific spoilage bacterium, it still affects the edible taste and nutritional value of crayfish muscle. The findings of this study might contribute to further research on C. freundii infection and provide a warning about the adverse effects of bacterial infection on aquatic products.

A High Starch Diet Alters the Composition of the Intestinal Microbiota of Largemouth Bass Micropterus salmoides, Which May Be Associated With the Development of Enteritis.

Starch is an inexpensive feed ingredient that has been widely used in fish feed. However, starch utilization by carnivorous fish is limited and excess starch is detrimental to the health of the organism. High starch diets often lead to liver damage, but the effects on the intestine are often overlooked. Therefore, in this study, two isonitrogenous and isolipidic semi-pure diets (NC: 0% α-starch, HC: 22% α-starch) were formulated and fed to largemouth bass (Micropterus salmoides) for 45 days. The effects of the high starch diet on the intestine of largemouth bass were comprehensively investigated by intestinal microbiota, histopathology, ultrastructural pathology, and enzymology analyses. Feeding the HC diet did not affect the growth of largemouth bass during the experimental period. However, the high starch diet led to a reduction in the diversity and abundance of intestinal microbiota in largemouth bass, with a significant increase in the abundance of harmful bacteria (Aeromonas) and a decrease in the abundance of beneficial bacteria (Clostridium, Lactobacillus, and Bifidobacterium). Feeding the HC diet caused the development of enteritis, with goblet cell hyperplasia, epithelial necrosis and detachment and inflammatory cell infiltration, and leading to enlarged apical openings and mitochondrial damage in goblet cells. Long-term feeding of the HC diet inhibited intestinal α-amylase activity. changes in the intestinal microbiota, such as an increase in Aeromonas and a decrease in Clostridium, Lactobacillus, and Bifidobacterium, may be closely related to the development of enteritis. Therefore, adding these beneficial bacteria as probiotics may be an effective way to prevent damage to the intestine of largemouth bass from a high carbohydrate diet. Our results suggest reducing the amount of starch added to the largemouth bass diets. This study provides a reference for protecting the largemouth bass gut during modern intensive culture.

Autolysis in Crustacean Tissues after Death: A Case Study Using the Procambarus clarkii Hepatopancreas.

Autolysis is an internal phenomenon following the death of an organism that leads to the degradation of tissues. In order to explore the initial stages of autolysis and attempt to establish reference standards for tissue changes after death, we studied the rapidly autolyzing tissue of the crayfish hepatopancreas. Samples from the hepatopancreas of crayfish were examined 0, 5, 10, 30, 60, and 120 minutes after death. Histological and ultrapathological examinations and evaluations and apoptotic cell counts were conducted to determine the initiation time and degree of autolysis. The results showed that autolysis in the hepatopancreas of crayfish began within 5 minutes. Initially, autolysis manifested in the swelling of hepatic tubular cells and the widening of mesenchyme. Cells undergoing autolysis showed severe organelle necrolysis. Based on these observations, tissue samples should be collected and preserved within five minutes to avoid interfering with histopathological diagnoses.

Protective effects of metallothionein and vitamin E in the trunk kidney and blood of cadmium poisoned Ctenopharyngodon idellus.

Cadmium (Cd), a substance with one of the most critical health hazard indices, can cause damage to both the blood and kidneys and accumulates in the body at last. The present work studied the toxicological effects of Cd and the therapeutic effects of metallothionein (MT) and vitamin E (VE) on the trunk kidney and blood of freshwater grass carp (Ctenopharyngodon idellus). Grass carp were divided into three groups: Cd + phosphate-buffered saline (PBS) group, Cd + VE group, and the Cd + MT group. Fish were injected with CdCl2 on the first day and then VE, MT, or PBS was administered 4 days post-injection. Fish not injected with Cd were used as a negative control. The blood and trunk kidney amassed Cd and suffered severe damage in the forms of organ toxicity cytotoxicity, and immunotoxicity. However, the MT reduced the Cd content in the trunk kidney and blood and partially stabilized the damaged organs. Treatment with VE, however, only demonstrated weaker protection against on Cd-induced toxicity. The results indicate that exogenous MT may play an essential role in restoring homeostasis of the Cd-poisoned urinary and circulatory system and that it may help eliminate Cd in aquatic animals.

Soy Isoflavones Ameliorate Fatty Acid Metabolism of Visceral Adipose Tissue by Increasing the AMPK Activity in Male Rats with Diet-Induced Obesity (DIO).

Soy isoflavones are natural active ingredients of soy plants that are beneficial to many metabolic diseases, especially obesity. Many studies have reported that obesity is closely related to visceral fatty acid metabolism, but the effect has not been well defined. In this study, we show that soy isoflavones improve visceral fatty acid metabolism in diet-induced obese male rats, which was indicated by reduced body weight and visceral fat cell area, as well as suppressed visceral fat synthesis and accelerated fat hydrolysis. We also found that common components of soy isoflavones, daidzein and genistein, were able to inhibit the lipid accumulation process in 3T3-L1 cells. Moreover, we showed that soy isoflavones can promote on AMP-activated protein kinase (AMPK) activity both in vivo and in vitro, which may be implicated in lipid metabolism regulation of soy isoflavones. Our study demonstrates the potential of soy isoflavones as a mechanism for regulating lipid homeostasis in visceral adipose tissue, proven to be beneficial for obesity treatment.

Caspase-11, a specific sensor for intracellular lipopolysaccharide recognition, mediates the non-canonical inflammatory pathway of pyroptosis.

Pyroptosis, a type of programmed cell death that along with inflammation, is mainly regulated by two main pathways, cysteinyl aspartate specific proteinase (caspase)-1-induced canonical inflammatory pathway and caspase-11-induced non-canonical inflammatory pathway. The non-canonical inflammatory pathway-induced pyroptosis is a unique immune response in response to gram-negative (G-) bacteria. It is induced by lipopolysaccharide (LPS) on the surface of G- bacteria. This activates caspase-11 which, in turn, activates a series of downstream proteins eventually forming protein pores on the cell membrane and inducing cell sacrificial processes. Caspase-11 belongs to the caspase family and is an homologous protein of caspase-1. It has the ability to specifically hydrolyze proteins, but it is still unclear how it regulates cell death caused by non-canonical inflammatory pathways. The present study describes a pathway that enables LPS to directly enter the cell and activate caspase-11, and the key role caspase-11 plays in the activation of pyroptosis and inflammation.

Impacts of Duck-Origin Parvovirus Infection on Cherry Valley Ducklings From the Perspective of Gut Microbiota.

Duck-origin goose parvovirus (D-GPV) is the causative agent of beak atrophy and dwarfism syndrome (BADS), characterized by growth retardation, skeletal dysplasia, and persistent diarrhea. However, the pathogenic mechanism of D-GPV remains undefined. Here, we first reported the gut microbiome diversity of D-GPV infected Cherry Valley ducks. In the investigation for the influence of D-GPV infection on gut microbiota through a period of infection, we found that D-GPV infection caused gut microbiota dysbiosis by reducing the prevalence of the dominant genera and decreasing microbial diversity. Furthermore, exfoliation of the intestinal epithelium, proliferation of lymphocytes, up-regulated mRNA expression of pro-inflammatory TNF-α, IL-1ß, IL-6, IL-17A, and IL-22 and down-regulated mRNA expression of anti-inflammatory IL-10 and IL-4 occurred when D-GPV targeted in cecal epithelium. In addition, the content of short chain fatty acids (SCFAs) in cecal contents was significantly reduced after D-GPV infection. Importantly, the disorder of pro-inflammatory and anti-inflammatory cytokines was associated with the decrease of SCFAs-producing bacteria and the enrichment of opportunistic pathogens. Collectively, the decrease of SCFAs and the enrichment of pathogen-containing gut communities promoted intestinal inflammatory injury. These results may provide a new insight that target the gut microbiota to understand the progression of BADS disease and to research the pathogenic mechanism of D-GPV.

Acinetobacter lwoffii, an emerging pathogen for fish in Schizothorax genus in China.

Acinetobacter lwoffii, a serious human pathogen, has been identified as a cause of nosocomial infections such as bacteremia, pneumonia and meningitis. There are only a few studies reporting A. lwoffii as a pathogen of fish. During 2016 and 2017, six bacterial strains, isolated from diseased fish of the Schizothorax genus, were identified as A. lwoffii by morphology, biochemical tests, 16S rDNA and gyrB gene sequencing analysis. One of these isolates was selected for experimental infection of Sclizothorax prenanti, Schizothorax davidi and Schizothorax wangchiachii, to confirm its pathogenicity. Experimentally infected fish showed similar symptoms to those observed in fish after natural outbreaks. Susceptibility of the isolates to 14 antibiotics was tested using a disc diffusion method; all isolates were resistant to cephalothin, aminoglycosides and ß-lactams, and sensitive only to some fluoroquinolones and tetracyclines. Histological examination revealed that A. lwoffii infection could cause pathological lesions in multiple organs and tissues, especially in liver, kidney, spleen and heart. These lesions included extensive haemorrhage, vacuolar degeneration, necrosis and inflammatory cell infiltration. To our knowledge, this is the first report on A. lwoffii as a virulent pathogen for fish of the Schizothorax genus.

Role of the Death Receptor and Endoplasmic Reticulum Stress Signaling Pathways in Polyphyllin I-Regulated Apoptosis of Human Hepatocellular Carcinoma HepG2 Cells.

Polyphyllin has been reported to exhibit anticancer effects against various types of cancer via the proapoptotic signaling pathway. The aim of the present study was to investigate the role of the endoplasmic reticulum stress and death receptor signaling pathways in PPI-induced apoptosis of human hepatocellular carcinoma HepG2 cells. Analysis demonstrated that PPI could significantly inhibit the proliferation and induce apoptosis of HepG2 cells in a dose- and time-dependent manner. Investigation into the molecular mechanism of PPI indicated that PPI notably mediated ER stress activation via IRE-1 overexpression and activation of the caspase-12 to protect HepG2 cells against apoptosis. In addition, PPI markedly induced the expression of death receptors signaling pathways-associated factors, including tumor necrosis factor (TNF) receptor 1/TNF-α and FAS/FASL. Additionally, suppression of the death receptor signaling pathways with a caspase-8 inhibitor, Z-IETD-FMK, revealed an increase in the death rate and apoptotic rate of HepG2 cells. Collectively, the findings of the present study suggested that the ER stress and death receptor signaling pathways were associated with PPI-induced HepG2 cell apoptosis; however, endoplasmic reticulum stress may serve a protective role in this process. The combination of PPI and Z-IETD-FMK may activate necroptosis, which enhances apoptosis. Therefore, the results of the present study may improve understanding regarding the roles of signaling pathways in PPI regulated apoptosis and contribute to the development of novel therapies for the treatment of HCC.

Molecular characterization of a p38 mitogen-activated protein kinase gene from Scylla paramamosain and its expression profiles during pathogenic challenge.

A novel p38 MAPK gene from S. paramamosain was cloned and characterized by rapid amplification of cDNA ends (RACE) technology. S. paramamosain p38 (Sp-p38) MAPK gene consists of an open reading frame of 1095bp encoding a 365-amino-acid protein, which showed close phylogenetic relationship to Litopenaeus vannamei p38 MAPK. The tissue distribution patterns showed that Sp-p38 MAPK was widely expressed in all examined tissues, with the highest expression in hemocytes and intestines. The expression levels of Sp-p38 MAPK in hemocytes was up-regulated post-stimulation, which reached the peak at 6h and 12h after bacteria (S. aureus and V. harveyi) and WSSV infection, respectively. In conclusion, our data contributed to define the biological characteristics of Sp-p38 MAPK and further demonstrated the critical role of Sp-p38 MAPK in vivo during the viral and bacterial infection.

Effects of α-enolase (ENO1) over-expression on malignant biological behaviors of AGS cells.

OBJECTIVE: To investigate the effects of α-Enolase (ENO1) over-expression on the proliferative and migratory abilities of AGS cells. METHODS: The target gene was cloned and mounted to the eukaryotic expression vector pcDNA3.1(+), then was transfected into gastric cancer cell lines AGS. mRNA and protein level of ENO1 in AGS cells were verified by real-time quantitative RT-PCR and Western Blot, respectively. The effects of over-expression of ENO1 on proliferative and migratory abilities of AGS cells were detected by the experiments of CCK-8, colony formation and wound healing assays. RESULTS: The eukaryotic expression vector pcDNA3.1(+)/eno1 was successfully constructed, and verified by sequencing. It was shown from the cell proliferation curves that the proliferative ability of AGS-ENO1 transfected group was higher than that of the control group after 72 hours (t = 3.44, P = 0.04), meanwhile, the number of the cell-colonies of the AGS-ENO1 group were significantly greater than that of the control group (t = 5.26, P = 0.01). For the ability of migration, it was significantly enhanced in the over-expression ENO1 cells than in the negative cells (t = 7.35, P < 0.001). CONCLUSION: The over-expression of ENO1 protein can enhance the abilities of proliferation and migration in gastric cancer cells of AGS, which indicates that ENO1 may be an important potential tumor-marker associated with the development of gastric cancer.

High-throughput cloning of human liver complete open reading frames using homologous recombination in Escherichia coli.

In this article, we describe a high-throughput cloning method, seamless enzyme-free cloning (SEFC), which allows one-step assembly of DNA fragments in vivo via homologous recombination in Escherichia coli. In the method, the desired open reading frame (ORF) is amplified by use of ORF-specific primers with flanking sequences identical to the two ends of a linearized vector. The polymerase chain reaction (PCR) product and the linearized vector are then cotransformed into E. coli cells, where the ORF is incorporated into the vector in vivo. SEFC is a simple, reliable, and inexpensive method of cloning in which PCR fragments are fused into expression vectors without unwanted amino acids or extra in vitro manipulations apart from the single PCR amplification step. Using this method, we successfully cloned human liver complete ORFs into the yeast AD and DB vectors and generated a clone resource of 4964 AD-ORFs and 4676 DB-ORFs in 3months. This approach will be useful for daily DNA cloning and for creating proteome-scale clone resources.