Effects of dietary chitosan oligosaccharide on the growth, intestinal microbiota and immunity of juvenile red claw crayfish (Cherax quadricarinatus).

This study aimed to evaluate the potential benefits of chitosan oligosaccharide (COS) on red claw crayfish (Cherax quadricarinatus) and explore its underlying mechanisms. The crayfish were randomly divided into six groups, and the diets were supplemented with COS at levels of 0 (C0), 0.2 (C1), 0.4 (C2), 0.6 (C3), 0.8 (C4), and 1 (C5) g kg-1. Treatment with COS significantly improved the growth performance of the crayfish with a higher weight gain rate (WGR) and specific growth rate (SGR) in the C2 group compared to the C0 group. Additionally, the content of crude protein in the crayfish muscles in the C1 group was significantly higher than that of the C0 group. Regarding non-specific immunity, the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and alkaline phosphatase (AKP), and the levels of expression of the genes related to immunity (SOD; anti-lipopolysaccharide factor [ALF]; thioredoxin1 [Trx1]; C-type lysozyme, [C-LZM]; and GSH-Px) in the hepatopancreas and hemolymph increased significantly (P < 0.05) after supplementation with 0.4 g kg-1 of COS, while the content of malondialdehyde (MDA) decreased (P < 0.05). The survival rate of C. quadricarinatus increased (P < 0.05) in the C2, C3, C4, and C5 groups after the challenge with Aeromonas hydrophila. This study found that COS has the potential to modulate the composition of the intestinal microbiota and significantly reduce the abundance of species of the phylum Proteobacteria and the genera Aeromonas and Vibrio in the gut of C. quadricarinatus, while the abundance of bacteria in the phylum Firmicutes and the genus Candidatus_Hepatoplasma improved significantly. This study suggests that the inclusion of COS in the diet of C. quadricarinatus can enhance growth, boost immunity, and increase resistance to infection with A. hydrophila, especially when supplemented at 0.4-0.8 g kg-1.

Effects of dietary astaxanthin on growth performance, muscle composition, non-specific immunity, gene expression, and ammonia resistance of juvenile ivory shell (Babylonia areolate).

Astaxanthin is one of the important immunopotentators in aquaculture. However, little is known about the physiological changes and stress resistance effects of astaxanthin in marine gastropods. In this study, the effects of different astaxanthin concentrations (0, 25, 50, 75, and 100 mg/kg) on the growth, muscle composition, immune function, and resistance to ammonia stress in Babylonia areolata were investigated after three months of rearing. With the increase in astaxanthin content, the weight gain rate (WGR), specific growth rate (SGR), and survival rate (SR) of B. areolata showed an increasing trend. The 75-100 mg/kg group was significantly higher than the control group (0 mg/kg). There was no significant difference in the flesh shell ratio (FSR), viscerosomatic index (VSI), and soft tissue index (STI) of the experimental groups. Astaxanthin (75 mg/kg) significantly increased muscle crude protein content and increased hepatopancreas alkaline phosphatase (AKP), superoxide dismutase (SOD), and catalase (CAT) activity. Astaxanthin (75-100 mg/kg) significantly increased the total antioxidant capacity (T-AOC) and acid phosphatase (ACP) of the hepatopancreas and decreased the malondialdehyde (MDA) content of B. areolata. Astaxanthin significantly induced the expression levels of functional genes, such as SOD, Cu/ZnSOD, ferritin, ACP, and CYC in hepatopancreas and increased the survival rate of B. areolata under ammonia stress. The addition of 75-100 mg/kg astaxanthin to the feed improved the growth performance, muscle composition, immune function, and resistance to ammonia stress of B. areolata.

Effects of antimicrobial peptides from dietary Hermetia illucens larvae on the growth, immunity, gene expression, intestinal microbiota and resistance to Aeromonas hydrophila of juvenile red claw crayfish (Cherax quadricarinatus).

Antimicrobial peptides (AMPs), which are widely present in animals and plants, have a broad distribution, strong broad-spectrum antibacterial activity, low likelihood of developing drug resistance, high thermal stability and antiviral properties. The present study investigated the effects of adding AMPs from Hermetia illucens larvae on the growth performance, muscle composition, antioxidant capacity, immune response, gene expression, antibacterial ability and intestinal microbiota of Cherax quadricarinatus (red claw crayfish). Five experimental diets were prepared by adding 50 (M1), 100 (M2), 150 (M3) and 200 (M4) mg/kg of crude AMP extract from H. illucens larvae to the basal diet feed, which was also used as the control (M0). After an eight-week feeding experiment, it was discovered that the addition of 100-150 mg/kg of H. illucens larvae AMPs to the feed significantly improved the weight gain rate and specific growth rate of C. quadricarinatus. Furthermore, the addition of H. illucens larvae AMPs to the feed had no significant effect on the moisture content, crude protein, crude fat and ash content of the C. quadricarinatus muscle. The addition of 100-150 mg/kg of H. illucens larvae AMPs in the feed also increased the antioxidant capacity, nonspecific immune enzyme activity and related gene expression levels in C. quadricarinatus, thereby enhancing their antioxidant capacity and immune function. The H. illucens larvae AMPs improved the structure and composition of the intestinal microbiota of C. quadricarinatus, increasing the microbial community diversity of the crayfish gut. Finally, the addition of 100-150 mg/kg of H. illucens larvae AMPs in the feed enhanced the resistance of C. quadricarinatus against Aeromonas hydrophila, improving the survival rate of the crayfish. Based on the aforementioned findings, it is recommended that H. illucens larvae AMPs be incorporated into the C. quadricarinatus feed at a concentration of 100-150 mg/kg.

New insights into the regulation mechanism of Pacific white shrimp (Litopenaeus vannamei) hepatopancreas under 4-nonylphenol exposure using transcriptome analysis.

4-Nonylphenol (4-NP) is one of the common endocrine-disrupting chemicals (EDCs) in estuaries and coastal zones, which can exert detrimental effects on the physiological function of aquatic organisms. However, the molecular response triggered by 4-NP remains largely unknown in Pacific white shrimp (Litopenaeus vannamei). In this study, transcriptomic analysis was performed to investigate the underlying mechanisms of 4-NP toxicity in the hepatopancreas of L. vannamei. Nine RNA-Seq libraries were generated from L. vannamei at 0 h, 24 h, and 48 h following exposure to 4-NP. Compared with 0 h vs 24 h, 962 up- and 463 down-regulated differentially expressed genes (DEGs) were identified, indicating that many genes in L. vannamei were induced to resist adverse circumstances by 4-NP exposure. In contrast, 902 up- and 1027 down-regulated DEGs were revealed in the comparison of 0 h vs 48 h, demonstrating that prolonged exposure to the stress from 4-NP resulted in more inhibited genes. To validate the accuracy of the transcriptome data, eight DEGs were selected for quantitative real-time polymerase chain reaction (qRT-PCR), which were consistent with the RNA-Seq results. Through KEGG pathway enrichment analysis, three specific pathways related to hormonal effects and endocrine function of L. vannamei were enriched significantly, including tyrosine metabolism, insect hormone biosynthesis, and melanogenesis. After 4-NP stress, genes involved in tyrosine metabolism (Tyr) and melanogenesis pathway (AC, CBP, Wnt, Frizzled, Tcf, and Ras) were induced to promote melanin pigment to help shrimp resist adverse environments. In the insect hormone biosynthesis, ALDH, CYP15A1, CYP15A1/C1, and JHE genes were activated to synthesize juvenile hormone (JH), while Spook, Phm, Sad, and CYP18A1 were induced to generate molting hormone. There is an enhanced interaction between the molting hormone and JH, with JH playing a dominant role and maintaining its "classic status quo action". Our study demonstrated that 4-NP exposure led to impairments of biological functions in L. vannamei hepatopancreas. The genes and pathways identified provide novel insights into the molecular mechanisms underlying 4-NP toxicity effects in prawns and enrich the information on the toxicity mechanism of crustaceans in response to EDCs exposure.

New insights into the regulation mechanism of red claw crayfish (Cherax quadricarinatus) hepatopancreas under air exposure using transcriptome analysis.

Red claw crayfish (Cherax quadricarinatus) is an important freshwater shrimp species worldwide with enormous economic value. Waterless transportation is an inherent feature of red claw crayfish transportation. However, the high mortality of red claw crayfish is a severe problem in the aquaculture of crayfish after waterless transportation. In this study, we investigated the responses of the hepatopancreas from the red claw crayfish undergoing air exposure stress and normal conditions on transcriptome levels. We used Illumina-based RNA sequencing (RNA-Seq) to perform a transcriptome analysis from the hepatopancreas of red claw crayfish challenged by air exposure. An average of 57,148,800 clean reads per library was obtained, and 33,567 unigenes could be predicted and classified according to their homology with matches in the National Center for Biotechnology Information (NCBI) non-redundant protein sequences (Nr), Gene Ontology (GO), a manually annotated and reviewed protein sequence database (Swiss-Prot), protein families (Pfam), Clusters of Orthologous Groups (COG) of proteins, and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. 690 and 3407 differentially expressed genes (DEGs) were identified between the two stress stages of the red claw crayfish. More DEGs were identified in 12 h, indicating that gene expressions were largely changed at 12 h. Some immune-related pathways and genes were identified according to KEGG and GO enrichment analysis. A total of 12 DEGs involved in immune response and trehalose mechanism were verified by quantitative real-time-polymerase chain reaction (qRT-PCR). The results indicated that the red claw crayfish might counteract the stress of air exposure at the transcriptomic level by increasing expression levels of antioxidant-, immune-, and trehalose metabolism-related genes. These transcriptome results from the hepatopancreas provide significant insights into the influence mechanism of air exposure to the trehalose mechanism and immune response in the red claw crayfish.

Effects of Elephantopus scaber extract on growth, proximate composition, immunity, intestinal microbiota and resistance of the GIFT strain of Nile tilapia Oreochromis niloticus to Streptococcus agalactiae.

This study aimed to investigate the effects of Elephantopus scaber extract on the GIFT (genetic improvement of farmed tilapia) strain of Nile tilapia Oreochromis niloticus. A total of 800 tilapia with an initial body weight of 1.34 ± 0.09 g each were randomly divided into five groups. The tilapia in the control group (E0 group) were fed on a basal diet only. Meanwhile, tilapia in the four experimental groups were fed on a basal diet supplemented with 1 g/kg (E1 group), 3 g/kg (E2 group), 5 g/kg (E3 group), and 7 g/kg (E4 group) of E. scaber extract for 10 weeks. Results showed that the survival rate was higher in the experimental groups than in the control group. Compared with the control group, some growth parameters (FW, WGR, SGR, VSI, and HSI) were significantly improved in the E1 group and E2 group. The crude lipid content in the dorsal muscle and liver was lower in the E1 group than in the control group. After E. scaber extract supplementation, activities of immunity-related enzymes (ACP, AKP, T-AOC, SOD, CAT, GSH-Px and LZM) in plasma, liver, spleen and head kidney, and expressions of immunity-related genes (IL-1ß, IFN-γ, TNF-α, and CCL-3) in liver, spleen and head kidney showed various degrees of improvement, while MDA content and Hsp70 expression level were decreased. The survival rate of tilapia increased in all the supplementation groups after Streptococcus agalactiae treatment. E. scaber extract addition changed the species composition, abundance, and diversity of intestinal microbiota in tilapia. These results demonstrate that E. scaber extract supplementation in diet can improve the growth, immunity, and disease resistance of GIFT against S. agalactiae. E. scaber extract supplementation can also change intestinal microbiota and reduce crude lipid content in dorsal muscle and liver. The above indicators show that the optimal dose of E. scaber extract for GIFT is 1 g/kg.

Synergism between the phosphatidylinositol 3-kinase p110ß isoform inhibitor AZD6482 and the mixed lineage kinase 3 inhibitor URMC-099 on the blockade of glioblastoma cell motility and focal adhesion formation.

BACKGROUND: Glioblastoma multiforme, the most aggressive and malignant primary brain tumor, is characterized by rapid growth and extensive infiltration to neighboring normal brain parenchyma. Our previous studies delineated a crosstalk between PI3K/Akt and JNK signaling pathways, and a moderate anti-glioblastoma synergism caused by the combined inhibition of PI3K p110ß (PI3Kß) isoform and JNK. However, this combination strategy is not potent enough. MLK3, an upstream regulator of ERK and JNK, may replace JNK to exert stronger synergism with PI3Kß. METHODS: To develop a new combination strategy with stronger synergism, the expression pattern and roles of MLK3 in glioblastoma patient's specimens and cell lines were firstly investigated. Then glioblastoma cells and xenografts in nude mice were treated with the PI3Kß inhibitor AZD6482 and the MLK3 inhibitor URMC-099 alone or in combination to evaluate their combination effects on tumor cell growth and motility. The combination effects on cytoskeletal structures such as lamellipodia and focal adhesions were also evaluated. RESULTS: MLK3 protein was overexpressed in both newly diagnosed and relapsing glioblastoma patients' specimens. Silencing of MLK3 using siRNA duplexes significantly suppressed migration and invasion, but promoted attachment of glioblastoma cells. Combined inhibition of PI3Kß and MLK3 exhibited synergistic inhibitory effects on glioblastoma cell proliferation, migration and invasion, as well as the formation of lamellipodia and focal adhesions. Furthermore, combination of AZD6482 and URMC-099 effectively decreased glioblastoma xenograft growth in nude mice. Glioblastoma cells treated with this drug combination showed reduced phosphorylation of Akt and ERK, and decreased protein expression of ROCK2 and Zyxin. CONCLUSION: Taken together, combination of AZD6482 and URMC-099 showed strong synergistic anti-tumor effects on glioblastoma in vitro and in vivo. Our findings suggest that combined inhibition of PI3Kß and MLK3 may serve as an attractive therapeutic approach for glioblastoma multiforme.

Effects of dietary trehalose on growth, trehalose content, non-specific immunity, gene expression and desiccation resistance of juvenile red claw crayfish (Cherax quadricarinatus).

This study was performed to investigate the effects of dietary trehalose on growth, muscle composition, non-specific immune responses, gene expression and desiccation resistance of juvenile red claw crayfish (Cherax quadricarinatus). A total of 540 (body weight of 0.41 ± 0.05) crayfish were randomly divided into six groups for a feeding experiment. Six diets with trehalose levels at 0 (Diet 1), 1 (Diet 2), 2 (Diet 3), 5 (Diet 4), 10 (Diet 5) and 15 (Diet 6) g kg-1 were prepared to feed juvenile red claw crayfish for 8 weeks. The results showed that the weight gain rate (WGR) and specific growth rate (SGR) of crayfish in Diet 4, Diet 5 and Diet 6 groups were significantly improved compared with the control group (Diet 1). Muscle crude protein contents of crayfish fed Diet 4, Diet 5 and Diet 6 were significantly higher than those of the control group. The activities of superoxide dismutase (SOD) and alkaline phosphatase (AKP) in hepatopancreas and hemolymph of crayfish for Diet 4, Diet 5, and Diet 6 groups were significantly increased while malondialdehyde (MDA) content was significantly reduced when compared with the control. The total antioxidant capacity (T-AOC), catalase (CAT) and glutathione peroxidase (GPx) activities in the hepatopancreas and hemolymph of crayfish fed Diet 5 and Diet 6 were significantly higher than those in the control group. However, acid phosphatase (ACP) activity was not significantly different among all experimental groups. The hepatopancreas and intestine trehalose contents of crayfish showed an upward trend with the increase of dietary trehalose levels. Compared with the control group, supplementation of 5-15 g kg-1 trehalose in the feed up-regulated the expression levels of GPx, C-type lysozyme (C-LZM), antilipolysacchride factor (ALF), facilitated trehalose transporter homolog isoform X2 (Tret1-2) and facilitated trehalose transporter isoform X4 (Tret1-4) mRNA. In addition, supplementation of 5-15 g kg-1 trehalose in the feed could improve the survival rate of red claw crayfish under desiccation stress. These results suggested that supplementation of 5-15 g kg-1 trehalose in feed could significantly improve the growth performance, muscle protein, non-specific immunity and desiccation resistance of juvenile red claw crayfish.

Characteristics of H7N9 avian influenza pneumonia: a retrospective analysis of 17 cases.

BACKGROUND: H7N9 avian influenza is an infection of public health concern, in part because of its high mortality rate and pandemic potential. AIMS: To describe the clinical features of H7N9 avian influenza and the response to treatment. METHODS: Clinical, radiological and histopathological data, and treatment-related of H7N9-infected patients hospitalised during 2014-2017 were extracted and analysed. RESULTS: A total of 17 H7N9 patients (three females; mean age, 58.4 ± 13.7 years) was identified; of these six died. All patients presented with fever and productive cough; four patients had haemoptysis and 13 had chest distress and/or shortness of breath. Early subnormal white blood cell count and elevation of serum liver enzymes were common. Multilobar patchy shadows, rapid progression to ground-glass opacities, air bronchograms and consolidation were the most common imaging findings. Histopathological examination of lung tissue of three patients who died showed severe alveolar epithelial cell damage, with inflammatory exudation into the alveolar space and hyaline membrane formation; widened alveolar septae, prominent inflammatory cell infiltration; and hyperplasia of pneumocytes. Viral inclusions were found in the lung tissue of two patients. All patients received antiviral drugs (oseltamivir ± peramivir). Four patients carried the rs12252-C/C interferon-induced transmembrane protein-3 (IFITM3) genotype, while the others had the C/T genotype. CONCLUSIONS: H7N9 virus infection causes human influenza-like symptoms, but may rapidly progress to severe pneumonia and even death. Clinicians should be alert to the possibility of H7N9 infection in high-risk patients. The presence of the IFITM3 rs12252-C genotype may predict severe illness.

[Transcriptome and quality evaluation of Sichuan Dao-di herbs Zanthoxylum bungeanum].

"Huajiao" is dried ripe fruit peel of Zanthoxylum bungeanum or Z. schinifolium, is konwn as geoherbs, especially the "Dahongpao" cultivated in Hanyuan, Maoxian and Jiulong of Sichuan province. However, the genetic basis of Dao-di "Huajiao" is virtually unknown. The transcriptome of the fruit and leaf from Sichuan(Hanyuan, Jiulong, Lixian, Maoxian), Gansu(Wudu) province and Shaanxi(Fengxian) province was sequenced. Trinity de novo assembling resulted in a total of 177 616 unigenes. Through the KEGG, NR, SwissProt, Trembl, KOG/COG, GO, Pfam database comparision 106 644 annotated Unigene finally, 4 574 deferentially expressed genes were found in fruit between Sichuan and other provinces, including 3 740 up-regulated genes and 834 down-regulated genes. Among the up-regulated genes, 27 up-regulated genes were raleted to terpenoids, and 8 up-regulated genes were related to isoquinoline alkaloid bio-synthesis. Furthermore, it was also showed remarkable differences in groups which enrichment ratio of the diffe-rent expressed gene compared. The different expressed genes were annotated by the KEGG database into plant-pathogen interaction, plant hormone signal transduction and phenylpropanoid biosynthesis in fruit and leaf, but isoflavonoid bio-synthesis and betaine bio-synthesis were significantly different in fruit and leaf. The study laid a certain reference basis for comparison of quality and different expressed gene of Z. bungeanum from different groups.

Thioredoxin H (TrxH) contributes to adversity adaptation and pathogenicity of Edwardsiella piscicida.

Thioredoxins (Trxs) play an important role in defending against oxidative stress and keeping disulfide bonding correct to maintain protein function. Edwardsiella piscicida, a severe fish pathogen, has been shown to encode several thioredoxins including TrxA, TrxC, and TrxH, but their biological roles remain unknown. In this study, we characterized TrxH of E. piscicida (named TrxHEp) and examined its expression and function. TrxHEp is composed of 125 residues and possesses typical thioredoxin H motifs. Expression of trxHEp was upregulated under conditions of oxidative stress, iron starvation, low pH, and during infection of host cells. trxHEp expression was also regulated by ferric uptake regulator (Fur), an important global regulatory of E. piscicida. Compared to the wild type TX01, a markerless trxHEp in-frame mutant strain TX01∆trxH exhibited markedly compromised tolerance of the pathogen to hydrogen peroxide, acid stress, and iron deficiency. Deletion of trxHEp significantly retarded bacterial biofilm growth and decreased resistance against serum killing. Pathogenicity analysis shows that the inactivation of trxHEp significantly impaired the ability of E. piscicida to invade host cells, reproduce in macrophages, and infect host tissues. Introduction of a trans-expressed trxH gene restored the lost virulence of TX01∆trxH. There is likely to be a complex relationship of functional complementation or expression regulation between TrxH and another two thioredoxins, TrxA and TrxC, of E. piscicida. This is the first functional report of TrxH in fish pathogens, and the findings suggest that TrxHEp is essential for coping with adverse circumstances and contributes to host infection of E. piscicida.

Major histocompatibility complex class I (MHC Iα) of Japanese flounder (Paralichthys olivaceus) plays a critical role in defense against intracellular pathogen infection.

The major histocompatibility complex (MHC) is a highly polymorphic region of the vertebrate genome that plays a critical role in initiating immune responses towards invading pathogens. It is well known that MHC I molecules play a central role in the immune response to viruses. However, rare literatures were reported the role of MHC I in the resistance to intracellular bacteria. Sequences of MHC Iα were identified in multiple teleost species, including Japanese flounder (Paralichthys olivaceus), however, the immunological function of MHC Iα remain largely unknown. In this study, we examined the expression profile and biological activity of an MHC Iα homologue, PoMHC Iα, from P. olivaceus. Structural analysis showed that PoMHC Iα possesses conserved structural characteristics of MHC Iα proteins, including MHC_I domain, IGc1 domain, transmembrane region. Expression of PoMHC Iα was upregulated in a time-dependent manner by extracellular and intracellular bacterial pathogens and viral pathogen infection. Different expression patterns were exhibited in response to the infection of different types of microbial pathogens in different immune tissues. Recombinant PoMHC Iα increased the capability of host cells to defense against intracellular pathogen Edwardsiella tarda infection and enhanced the expression of immune related genes. The knockdown of PoMHC Iα attenuated the ability of cells to eliminate E. tarda, which was sustained by the in vivo results that overexpression of PoMHC Iα promoted the host defense against invading E. tarda. Antigen uptake assay indicated PoMHC Iα participated in cells antigen presentation. Collectively, this study is the first report that MHC Iα plays an important role in immune defense against intracellular bacterial pathogen in teleost. Taken together, these findings add new insights into the biological function of teleost MHC Iα and emphasize the importance of MHC I gene products for the control of E. tarda infection.

cDNA cloning and expression analysis of glutaredoxin (Grx) 2 in the Pacific white shrimp Litopenaeus vannamei.

Glutaredoxin (Grx) is a class molecule oxidoreductase, which can regulate the redox state of proteins and plays a key role in antioxidant defense. However, the informations of Grx cDNA sequences and their functions are lack in decapod crustacea. In the present study, the cDNA of LvGrx 2 was cloned from the Pacific white shrimp, Litopenaeus vannamei. The open reading frame (ORF) of LvGrx 2 was 360 bp, which encoded a polypeptide of 119 amino acids. The molecular mass of the predicted protein is 12.87 kDa with an estimated pI of 8.22. Sequence alignment showed that the amino acid sequence of LvGrx 2 shares 59%, 59% and 58% identity with that of the coelacanth Latimeria chalumnae, the plateau frog Nanorana parkeri and the half-smooth tongue sole Cynoglossus semilaevis, respectively. Quantitative real-time PCR analysis revealed that LvGrx 2 were detected in a wide range of tissues, with highest expression in gill, hepatopancrea and intestine, and weakest expression in muscle. The expression responses of LvGrx 2 were analyzed in hepatopancrea and gill after ammonia-N stress or lipopolysaccharide (LPS) injection. During ammonia-N exposure, the LvGrx 2 transcriptions in hepatopancrea and gill significantly up-regulated, and the peak value appeared after 12 h and 24 h exposure respectively. After LPS injection, expression levels of LvGrx 2 in hepatopancrea obviously increased in the early and late stages, while LvGrx 2 transcription in gill sharply up-regulated in the middle period. These results suggest that LvGrx 2 may play a vital role in shrimp defense system against environmental stress and pathogen infection. RNA interference experiment was designed to further probe roles of LvGrx 2 during ammonia-N exposure. Ammonia-N induced obvious improvement in expression levels of LvGrx 2, LvGrx 3, GPx, GST and Trx, accompanied by increases of protein carbonyl and malondialdehyde (MDA) contents. However, transcription of GPx and GST were much weaker in LvGrx 2 interfered-shrimp, and oxidative damage in both lipid and protein were more serious. These results further suggest that LvGrx 2 in shrimp participates in oxidative defence and regulation of antioxidant system.

Histochemical distribution of four types of enzymes and mucous cells in the intestine of koi carp (Cyprinus carpio var. koi).

The main purpose of this study was to investigate the distribution of acid phosphatase (ACP), alkaline phosphatase (ALP), non-specific esterase (NSE), peroxidase (POD), and mucous cells in the intestine of the koi carp Cyprinus carpio var. koi. ACP activity was located in the striated border, enterocytes, and lamina propria of the anterior and middle intestines. The ACP activity in the anterior intestine was higher than that in the middle and posterior intestines. ALP existed in the striated border of enterocytes and lamina propria, serosa, muscular layer, and the junction between muscular layer and submucosa layer of the intestine. The ALP activity in the anterior intestine was higher than that in the middle and posterior intestines. NSE activity was localized in the cytoplasm of enterocytes in the whole intestine, and the middle intestine showed the lower NSE activity than the anterior and posterior intestines. POD activity was localized in the blood cells of the lamina propria and cytoplasm of enterocytes in all intestinal segments. The POD activity among the anterior, middle, and posterior intestines was non-significantly different. Alcian blue periodic acid-Schiff histochemical results revealed three types of mucous cells in the intestine. The total number of mucous cells and percentage of type I cells among the anterior, middle, and posterior intestines were non-significantly different. The percentage of the type II cells was the highest in the posterior intestine, while the lowest in the anterior intestine. The percentage of the type III cells was the highest in the anterior intestine, while the lowest in the posterior intestine.

Prediction of hepatic necroinflammatory activity in patients with chronic hepatitis B by a simple noninvasive model.

BACKGROUND: A model was constructed using clinical and serum variables to discriminate between chronic hepatitis B (CHB) patients with and without significant necroinflammatory activity (score 4-18 vs. score 0-3). METHODS: Consecutive CHB patients who underwent liver biopsy were divided into two sequential groups: a training group (n = 401) and a validation group (n = 401). Multivariate analysis identified alanine aminotransferase, γ-glutamyltransferase, prothrombin time and albumin as independent predictors of necroinflammatory activity. RESULTS: The area under the receiver operating characteristic curve was 0.826 for the training group and 0.847 for the validation group. Using a cut-off score of H ≤ 0.375, significant necroinflammatory activity (score 4-18) was excluded with high accuracy [78.2% negative predictive value (NPV), 72% positive predictive value (PPV), and 90.8% sensitivity] in 238 (59.4%) of 401 patients in the training group and with the same certainty (88.1% NPV, 61.2% PPV, and 95.1% sensitivity) among 204 (50.9%) of 401 patients in the validation group. Similarly, applying a cut-off score of H > 0.720, significant necroinflammatory activity was correctly identified with high accuracy (90.8% PPV, 57.7% NPV, and 92.0% specificity) in 150 (37.4%) of 401 patients in the training group and with the same certainty (91.8% PPV, 64.6% NPV, and 95.4% specificity) in 188 (46.9%) of 401 patients in the validation group. CONCLUSIONS: A predictive model based on easily accessible variables identified CHB patients with and without significant necroinflammatory activity with a high degree of accuracy. This model may decrease the need for liver biopsy for necroinflammatory activity grading in 72.1% of CHB patients.

Transcriptome profiling of immune-responsive genes in the intestine of Cynoglossus semilaevis Günther challenged with Shewanella algae.

To better understand gene expression in the intestine after Shewanella algae infection and provide insights into its immune roles in the tongue sole, Cynoglossus semilaevis, sequencing-based high-throughput RNA analysis (RNA-Seq) for the intestines between the control group and 12 h post-injection group was performed. After assembly, there was an average of 23,957,159 raw sequencing reads, and 23,943,491 clean reads were obtained after filtering out low-quality reads. Then, 383 differentially expressed genes (DEGs) in the intestines in response to S. algae infection were identified. Subsequently, gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the DEGs were conducted to further explore their functions. Among all of the pathways involved, sixteen pathways were related to the immune system, among which the complement and coagulation cascades pathway was the most prominent for immunity-related DEGs, followed by the leukocyte transendothelial migration pathway. Furthermore, the expression levels of twelve selected DEGs in the immune-related pathways were identified by quantitative real-time polymerase chain reaction, substantiating the reliability and reproducibility of the RNA-Seq results. In summary, this study represents an important genomic resource for understanding the potential immune role of the tongue sole intestine from the perspective of gene expression.

Flow cytometic analysis of Penaeus monodon haemocyte responses to poly I:C.

This study was aimed at investigating the cellular responses of Penaeus monodon haemocytes to poly I:C stimulation using flow cytometric assay. Total haemocyte count (THC), percentages of different haemocyte subpopulations [hyaline cells (HC), semigranular cells (SGC) and granular cells (GC)], non-specific esterase activity (EA), total reactive oxygen species/reactive nitrogen species (ROS/RNS) production, nitric oxide (NO) production, apoptotic haemocyte ratio and plasmic phenoloxidase (PO) activity were determined in poly I:C-injected shrimp. Results showed that poly I:C at a low dose (5 µg shrimp-1) caused obvious increases in THC, GC proportion, ROS/RNS production and NO production, but had no significant effect on EA, apoptosis and PO activity. In the early stage of poly I:C injection at a high dose (20 µg shrimp-1), THC and GC proportion improvements could also be observed, suggesting that GC might be induced to release from hemocytopoietic or other tissues to participate in immune response, and this subpopulation might be the main cell type involved in the cellular defence against virus. In the later period, proportions of both GC and SGC reduced paralleled by THC reduction, indicating that depletion of GC and SGC was mainly contributed to the reduced count of circulating haemocyte. Obvious increases in ROS/RNS production and NO production were induced in haemocyte of shrimp under a high dose of poly I:C stimulation, but only slight rise of EA and suppression of PO activity could be observed in poly I:C-stimulated shrimp, suggesting that ROS/RNS-dependent system was vital in the immune defence of shrimp against virus. On the other hand, increase of apoptotic haemocyte ratio and THC reduction were presented after the drastic increases of ROS/RNS and NO productions, implying that the stimulated ROS/RNS might be excess and harmful, and was the major factor for the haemocyte apoptosis and depletion. THC recovered after 48 h injection, while haemocyte apoptosis also returned to the control level, suggesting that apoptosis might be contributed to eliminate damaged, weak or infected haemocytes to renew the circulating haemocytes, and it could be considered as an important defending strategy against virus.

Oxidative burst activity in haemocytes of the freshwater prawn Macrobrachium rosenbergii.

Oxidative burst, release of reactive oxygen species/reactive nitrogen species (ROS/RNS) contributed to microorganisms killing, is a vital immune response of crustacean haemocyte. Three morphologic haemocyte types (hyaline cells, HC; semigranular cells, SGC; granular cells, GC) have been defined in crustaceans, and found to play different roles in immune defense. However, oxidative burst activities of different haemocyte subpopulations in crustaceans are currently not documented. In the present study, we investigated the oxidative burst activities of the three haemocyte types in the freshwater prawn Macrobrachium rosenbergii using the common ROS fluorescent probe dichlorofluorescin-diacetate (DCFH-DA). Nitric oxide (NO) donor sodium nitroprusside (SNP) improved the DCF fluorescence in haemocytes, while NO scavenger C-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) and NO-synthase inhibitor NG-monomethyl-l-arginine (L-NMMA) reduced the fluorescence, suggesting that DCF fluorescence intensity could also be modified by intracellular NO level and activity of NO-synthase pathway. ROS/RNS was also produced in the untreated haemocytes. GC contained most non-induced ROS/RNS production, while oxidative activity of HC was rather weak. No significant impact of PMA could be observed on ROS/RNS level in all the three cell types. Both zymosan A (ZA) and lipopolysaccharide (LPS) significantly triggered the production of ROS/RNS in SGC and GC, whereas they had no effect on those of HC, suggesting that SGC and GC were the primary cell types involved in pathogens killing by ROS/RNS pathway. Cytochalasin B (Cyt B) inhibited the ZA-induced ROS/RNS production, but could not change the ROS/RNS level stimulated by LPS. For unstimulated haemocytes, ROS/RNS productions decreased 29.6%, 44.1% and 48.6% in SGC, and decreased 44.5%, 28.4% and 57.3% in GC, in the presence of L-NMMA, Fccp and DPI respectively, whereas apocynin could not modulate DCF fluorescence in both SGC and GC, suggesting that mitochondrial oxidative pathway was relatively more dominant in SGC, and NO-synthase (NOS) pathway appeared more active in GC. For LPS-stimulated haemocytes, oxidative activities decreased 22.9%, 42.9%, 29.6% and 60.0% in SGC, and reduced 40.6%, 25.2%, 26.7% and 70.6% in GC with the presence of L-NMMA, apocynin, Fccp and DPI respectively, suggesting that NADPH-oxidase (NOX) pathway in both SGC and GC was activated by LPS, and it became the predominant oxidative pathway in stimulated SGC, while NOS pathway was the relative main source for ROS/RNS production in stimulated GC.

MicroRNA-203 promotes liver regeneration after partial hepatectomy in cirrhotic rats.

BACKGROUND: Liver resection or partial hepatectomy (PH) is still the most commonly used therapeutic option for hepatocellular carcinoma (HCC) at present. However, the impaired regenerative ability induced by the accompanied liver cirrhosis is an important risk factor of posthepatectomy liver failure, and posthepatectomy liver failure is a feared complication that accounts for up to 75% of mortality after extensive liver resection. MicroRNA(miR)-203 is a tumor suppressor of HCC and may act as a positive intermediary in A20-enhanced interleukin (IL-6)/signal transducer and activator of transcription 3 (STAT3) pro-proliferative signals, which may promote liver regeneration after PH. However, its direct pro-proliferative effect on cirrhotic liver after hepatectomy is unknown. MATERIALS AND METHODS: Liver cirrhosis was induced by intraperitoneal injection of 50% CCl4-olive oil solution in adult male Wistar rat. Rats with liver cirrhosis received portal vein injection of physiological saline, miR-203 lentivirus, or control empty lentivirus, and then 70% PH was performed under ether anesthesia 7 d later. Liver samples were harvested at 0, 24, 36, and 72 h after 70% PH. Hepatic expressions of cyclin D1 and Ki67 were checked to evaluate the liver regenerative ability. Hepatic expressions of IL-6, suppressor of cytokine signaling 3 (SOCS3), and phospho-STAT3 were also tested to clarify the mechanisms of miR-203 in liver regeneration. RESULTS: The regeneration of miR-203 overexpression cirrhotic liver after 70% PH was enhanced and peaked at 24 and 36 h after 70% PH. The cyclin D1-positive liver cells/high-power field (HPF) in miR-203 overexpression liver markedly increased at 24 and 36 h after 70% PH compared with 0-h samples. When comparing with the control groups, cyclin D1-positive liver cells/HPF in miR-203 overexpression liver were also significantly increased at 24 and 36 h after 70% PH. A similar result of the Ki67-positive liver cells/HPF was achieved at 36 h after 70% PH. The hepatic expression of IL-6 showed a rising tendency after 70% PH, and the levels of IL-6 are significantly higher in miR-203 overexpression livers. Hepatic expression of SOCS3 was negatively expressed with hepatic miR-203 expression level, and the reduced expression of SOCS3 facilitated the phosphorylation of STAT3. CONCLUSIONS: By targeting SOCS3 and then enhancing proliferating IL-6/STAT3 signaling pathway, hepatic overexpression of miR-203 can facilitate the initiation of liver regeneration and enhance the potency of liver regeneration after 70% PH in cirrhotic rat. Together with the tumor suppressive effect on HCC, miR-203 would be an ideal candidate for promoting liver regeneration in HCC patients undergoing liver resection without the risk of tumorigenesis or cancer recurrence.

Various cellular responses of different shrimp haemocyte subpopulations to lipopolysaccharide stimulation.

Different haemocyte types have been reported to play diverse roles in immune defense of shrimp. To investigate the roles of the three haemocyte types [hyaline cells (HC), semigranular cells (SGC) and granular cells (GC)] of shrimp in immune responses against lipopolysaccharide (LPS), percentage, non-specific esterase activity (EA), reactive oxygen species (ROS) production and nitric oxide (NO) production of the three haemocyte subpopulations were analyzed in LPS-injected Penaeus monodon using flow cytometry. Results showed that percentage of HC increased after 3 h injection, and returned to the original level after 48 h. Proportion of SGC and GC reduced after 6-36 h and 3-12 h respectively, and recovered to the initial level after 48 and 24 h respectively. Loss of SGC and GC might be related to degranulation to release proPO system, and degranulation of GC seemed more sensitive to LPS stimulation. EA of both HC and SGC improved after 3-6 h injection, while EA of GC was induced after 3-24 h. No significant effect of LPS injection could be found in ROS production and NO production of HC. Enhanced ROS levels was observed in SGC and GC after 3-24 h and 3-36 h respectively, and NO production of SGC and GC improved after 3-48 h injection. These results demonstrated that SGC and GC possessed strong capabilities for LPS-induced EA, ROS production and NO production, while HC only displayed EA response to LPS, suggesting that GC and SGC play the main role in immune defense of shrimp against Gram-negative bacteria.