Elevated expression of the RNA-binding motif protein 43 predicts poor prognosis in esophageal squamous cell carcinoma.

RNA-binding proteins (RBPs) play crucial roles in the post-transcriptional regulation of mRNA during numerous physiological and pathological processes, including tumor genesis and development. However, the role of RNA-binding motif protein 43 (RBM43) in esophageal squamous cell carcinoma (ESCC) has not been reported so far. The current study was the first to evaluate RBM43 protein expression by immunohistochemistry (IHC) in an independent cohort of 207 patients with ESCC, to explore its potential prognostic value and clinical relevance in ESCC. The results indicated that RBM43 protein levels were significantly elevated in ESCC tissues and increased RBM43 expression was associated with age and N categories. In addition, ESCC patients with high expression of RBM43 had shorter overall survival (OS) and disease-free survival (DFS) than those with low RBM43 expression. Furthermore, when survival analyses were conducted at different clinical stages, overexpression of RBM43 was significantly correlated with shortened survival in patients with ESCC at early stages (TNM stage I-II and N0 stage). Cox regression analysis further proved that high RBM43 expression was an independent predictor of poor prognosis in ESCC patients. In conclusion, increased expression of RBM43 is correlated with malignant attributes to ESCC and predicts unfavorable prognosis, suggesting an effective prognostic biomarker and potential therapeutic target for ESCC.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) participates in anti-lipopolysaccharide factors (ALFs) gene expression in mud crab.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a key cytoplasm signal adaptor that mediates signals activated by tumor necrosis factor receptor (TNFR) superfamily and the Interleukin-1 receptor/Toll-like receptor (IL-1/TLR) superfamily. The full-length 2492 bp TRAF6 (Sp-TRAF6) from Scylla paramamosain contains 1800 bp of open reading frame (ORF) encoding 598 amino acids, including an N-terminal RING-type zinc finger, two TRAF-type zinc fingers and a conserved C-terminal meprin and TRAF homology (MATH) domain. Multiple alignment analysis shows that the putative amino acid sequence of Sp-TRAf6 has highest identity of 88% with Pt-TRAF6 from Portunus trituberculatus, while the similarity of Sp-TRAF6 with other crustacean sequences was 54-55%. RT-PCR analysis indicated that Sp-TRAF6 transcripts were predominantly expressed in the hepatopancreas and stomach, whereas it was barely detected in the heart and hemocytes in our study. Moreover, Sp-TRAF6 transcripts were significantly up-regulated after Vibrio parahemolyticus and LPS challenges. RNA interference assay was carried out used by siRNA to investigate the genes expression patterns regulated by Sp-TRAF6. The qRT-PCR results showed that silencing Sp-TRAF6 gene could inhibit SpALF1, SpALF2, SpALF5 and SpALF6 expression in hemocytes, while inhibit SpALF1, SpALF3, SpALF4, SpALF5 and SpALF6 expression in hepatopancreas. Taken together, the acute-phase response to immune challenges and the inhibition of SpALFs gene expression indicate that Sp-TRAF6 plays an important role in host defense against pathogen invasions via regulation of ALF gene expression in S. paramamosain.

[Immunoreactivity and immunogenicity analysis of the recombinant cathepsin L-like protease of Fasciola hepatica in SD rats].

OBJECTIVE: To analyze the immunoreactivity of recombinant cathepsin L-like proteases (CatL) protein of Fasciola hepatica and its immunogenicity in SD rats. METHODS: The E. coli BL21(DE3) cells harbouring recombinant plasmid pET30a-FhCatL were inoculated in LB medium, and the protein expression was induced with IPTG. The recombinant protein FhCatL was analyzed by SDS-PAGE and the immunoreactivity was identified by Western blotting with sera from Fasciola hepatica-infected goat as the primary antibody. Twenty SD rats were randomly divided into immunized group and adjuvant control group. SD rats in immunized group were injected subcutaneously with 200 microg of purified FhCatL protein. All the rats received three immunizations at 3-week intervals. The adjuvant control group with 10 SD rats received only adjuvants emulsified with the same amount of PBS. Serum samples were collected at the day before the second and final immunization, 3, 6, and 9 weeks after the final immunization. The IgG antibody of rats' sera was examined by indirect ELISA and spleen lymphocyte proliferation (SLP) was tested by methyl thiazolyl tetrazolium (MTT). RESULTS: The molecular weight of purified FhCatL was about Mr 42,000. The recombinant FhCatL was recognized by pool sera of goats naturally infected with F. hepatica. The titer of specific antibody IgG in SD rats induced by the recombinant protein against CatL protein was significantly higher than that of the control, and the antibody titer reached the peak at three weeks after the final immunization (1 : 102,400). The stimulation index of splenocytes in immunized group was 2.176 +/- 0.047, which was significantly higher than that of the control (1.171 +/- 0.032) (P<0.05). CONCLUSION: The recombinant FhCatL protein bears stronger immnoreactivity and immunogenicity.

Enhancement of the immune response and protection against Vibrio parahaemolyticus by indigenous probiotic Bacillus strains in mud crab (Scylla paramamosain).

In a previous study, bacterial communities of the intestine in three populations of crabs (wild crabs, pond-raised healthy crabs and diseased crabs) were probed by culture-independent methods. In this study, we examined the intestinal communities of the crabs by bacterial cultivation with a variety of media. A total of 135 bacterial strains were isolated from three populations of mud crabs. The strains were screened for antagonistic activity against Vibrio parahaemolyticus using an agar spot assay. Antagonistic strains were then identified by 16S rRNA gene sequence analysis. Three strains (Bacillus subtilis DCU, Bacillus pumilus BP, Bacillus cereus HL7) with the strongest antagonistic activity were further evaluated for their probiotic characteristics. The results showed that two (BP and DCU) of them were able to survive low pH and high bile concentrations, showed good adherence characteristics and a broad spectrum of antibiotic resistance. The probiotic effects were then tested by feeding juvenile mud crabs (Scylla paramamosain) with foods supplemented with 10(5) CFU/g of BP or DCU for 30 days before being subjected to an immersion challenge with V. parahaemolyticus for 48 h. The treated crabs showed significantly higher expression levels of immune related genes (CAT, proPO and SOD) and activities of respiratory burst than that in controlled groups. Crabs treated with BP and DCU supplemented diets exhibited survival rates of 76.67% and 78.33%, respectively, whereas survival rate was 54.88% in crabs not treated with the probiotics. The data showed that indigenous mud-associated microbiota, such as DCU and BP, have potential application in controlling pathogenic Vibriosis in mud crab aquaculture.

[Immunogenicity analysis of recombinant GST protein of Fasciola hepatica in SD rats].

OBJECTIVE: To analyze the immunogenicity of recombinant glutathione S-transferase protein of Fasciola hepatica (FhGST) in SD rats. METHODS: The recombinant expression plasmid pET30a-FhGST was transformed into E. coli BL21 (DE3) cells and induced with IPTG for protein expression. The recombinant protein FhGST was analyzed by SDS-PAGE and identified by Western blotting. Twenty SD rats were randomly divided into two groups: immunized group and adjuvant control group. SD rats in immunized group were injected subcutaneously with 200 microg of purified FhGST protein. The adjuvant control group with 10 SD rats received only adjuvants emulsified with PBS. All the rats received three immunizations at 3-week intervals. Serum samples were collected at pre-immunization, the day after each immunization, 3 weeks and 6 weeks after the final immunization. The IgG antibody of rats' sera was examined by indirect ELISA and spleen lymphocyte proliferation (SLP) was tested by MTT. RESULTS: The molecular weight of purified FhGST was about M(r) 31 300. The recombinant FhGST was recognized by pool sera of goats naturally infected with F. hepatica. The recombinant protein induced specific antibody IgG against GST protein in SD rats significantly higher than that of the control, and the antibody titer reached the peak at 9 weeks after the first immunization (GMT 1:89 144). FhGST protein significantly enhanced the growth and proliferation of rat splenocytes. CONCLUSION: The recombinant FhGST protein induces specific immune response in SD rats.

[Cloning, expression and immunogenicity analysis of cathepsin L-like protease of Fasciola hepatica].

OBJECTIVE: To clone and express the cathepsin L-like protease gene of Fasciola hepatica (FhCL) and investigate the immunogenicity of the recombinant FhCL protein. METHODS: Specific primers were designed according to the reported FhCL gene in GenBank. Using total RNA from adult worms of F. hepatica, FhCL gene was amplified by RT-PCR. The PCR product was cloned into pMD18-T vector and then subcloned into pET30a(+) vector. The recombinant plasmid was transformed into E. coli BL21 (DE3) and followed by expression of the protein induced by IPTG. The expression situation of recombinant FhCL was analyzed by SDS-PAGE. Its immunoresponse to the sera of infected goat and the antisera of SD rats against FhCL was examined by Western blotting analysis. RESULTS: PCR and double enzyme digestion showed that the FhCL gene fragment was about 1,000 bp in length. The constructed recombinant plasmid pET30a (+)-FhCL was identified by sequencing. The recombinant protein (Mr 42,000) was expressed in the form of inclusion body. The protein was recognized respectively by the sera of infected goat and the sera from rat immunized with FhCL. CONCLUSION: The recombinant plasmid pET30a(+)-FhCL has been constructed, which shows high antigenicity.

Reproduction response of Chinese mitten-handed crab (Eriocheir sinensis) fed different sources of dietary lipid.

The effect of feeding three semi-purified diets containing different lipid sources (anchovy oil, soybean oil and pork lard) on fecundity, hatchability and egg fatty acid composition of Chinese mitten-handed crab (Eriocheir sinensis) broodstock was compared with a fresh clam diet in a 6-month feeding trial. Broodstock crabs fed the diet containing pork lard showed poor fecundity and low hatchability. Crabs fed the diet containing soybean oil showed improved fecundity; however, no significant improvement in hatchability was observed. Broodstock fed the diet containing anchovy oil showed the highest fecundity and egg hatchability. Eggs from broodstock fed anchovy oil as sole dietary lipid had a higher n-3 polyunsaturated fatty acid (PUFA) content (33.3%) compared with those of crabs fed diets with soybean oil (20.1%) and pork lard (16.3%) as lipid sources. The results indicate a close correlation between: (1) the 20:5n-3 content of the egg lipid and fecundity; (2) the 22:6n-3 content and hatchability; and (3) fecundity, hatchability and n-3/n-6 fatty acid ratio. The results also suggest that each of these n-3 HUFAs may play different and specific roles in crab reproduction and that either must be adequate in the broodstock diet.