Interleukin-12 receptor ß2 from grass carp: Molecular characterization and its involvement in Aeromonas hydrophila-induced intestinal inflammation.

Interleukin-12 receptor ß2 (IL-12Rß2) is a signaling subunit of heterodimeric receptors for IL-12 and IL-35. It plays important regulatory functions in the development of Th1 cells and in the expression of inflammatory cytokines in mammals and other higher vertebrates. However, little is known about IL-12Rß2 in teleost fish. In this work, we have cloned and characterized IL-12Rß2 from grass carp (Ctenopharyngodon idella). The full-length cDNA of grass carp IL-12Rß2 is 2875 bp, which encodes a mature protein with 741 amino acids. This mature protein contains three fibronectin type III domains, a transmembrane helix, and CXW and WSXWS-like motifs that are characteristic of the type I cytokine receptor family. Phylogenetic analysis revealed that cyprinid fish IL-12Rß2 formed a single branch, clearly separated from those of other vertebrates. We expressed and purified a recombinant grass carp IL-12Rß2 protein containing major antigenic regions, which was used to raise a polyclonal antibody. The specificity of the antibody was assessed by Western blotting analysis of whole cell lysates from Escherichia coli cells expressing the recombinant IL-12Rß2, grass carp intestinal intraepithelial lymphocytes, and cultured C. idella kidney cells. To explore the potential regulatory role of IL-12Rß2 in inflammation, we generated an intestinal inflammation model by anal intubation of fish with Aeromonas hydrophila. Immunohistochemical staining of the inflamed intestines revealed that IL-12Rß2 expression is consistent with inflammatory cell recruitment during intestinal inflammation. Real-time quantitative PCR revealed that IL-12Rß2 is widely expressed in normal tissues and is up-regulated in most tissues after infecting with A. hydrophila. We found that IL-12Rß2, IL-12p35, and interferon-γ were expressed in similar patterns in the intestines during inflammation. Taken together, our results suggest that IL-12Rß2 is involved in the regulation of intestinal inflammation.

[Comparison of chemical constituents between Cyathula offinalis and adulterant and their admixture by HPLC characteristic chromatogram fingerprint combined with chemometrics].

Cyathula capitate is the main adulterant of C.offinalis. According to the literature reported, there are obvious differences in properties, taste and pharmacological activity between C. capitate and C.offinalis. Therefore, C. capitate can only be used as a local conventional medicine and can't be a substitute for C. offinalis. Since the appearance of C.capitata is very similar to the C.offinalis and the content of cyasterone also can reach the limit of the current pharmacopoeia standard, the C.capitata is mostly sold in the form of impersonation oradmixture, which seriously affected the safety of the clinical medication and the development of the genuine crude drugs. In view of this, HPLC characteristic fingerprint was used to reveal the difference of multi-ingredients of C. offinalis, C. capitata and their admixture. According to the HPLC chromatogram of C.offinalis, C. capitata. and their admixture, 65 different components were obtained to set up a peak area data matrix of 26×65, which was applied to perform the characteristic peak difference analysis, similarity analysis, hierarchical clustering analysis HCA and principal component analysis (PCA). Characteristic peak difference analysis showed that the characteristic peaks of C. capitata and their admixture are more and higher respond than those of C. offinalis. The 9 characteristic peaks were used to distinguish C. capitata, 2 of which were used to distinguish C. offinalis mixed with 5% C. capitata. UV spectra of 9 characteristic peaks are mostly similar to the end absorption spectra of saponins, indicating that C. capitata may contain a large amount of saponins. By the reference fingerprint of C.offinalis established, the similarity analysis showed that the similarity degree of C. offinalis are higher than 0.942, while the similarity degree of C. capitata, C.offinalis mixed with 5% C. capitata are less than 0.383 and 0.399. C.offinalis, C. capitata, C.offinalis mixed with 5% C. capitata could be obviously divided into 3 classes by HCA and PCA. These results showed that there are obvious difference in the chemical composition of C. offinalis, C. capitata and their admixture, which could provide evidence for their identification.

Nutrition effects on the biofilm immobilization and 3,5-DNBA degradation of Comamonas testosteroni A3 during bioaugmentation treatment.

The survival of inoculated microbes is critical for successful bioaugmentation in wastewater treatment. The influence of readily available nutrients (RANs) on the colonization of two functional bacteria, Pseudomonas putida M9, a strong biofilm-forming strain, and Comamonas testosteroni A3, a 3,5-dinitrobenzoic acid (3,5-DNBA)-degrading strain, in biofilms was studied with 3,5-dinitrobenoic acid synthetic wastewater (DCMM) complemented with various ratios of Luria-Bertani broth (LB). With the increase in LB rate, the biofilm biomass was increased, the percentage of gfp-labeled M9 measured in the mixed culture enhanced, and also M9 became dominant. In laboratory-scale sequencing batch biofilm reactors, with the increase in 3,5-DNBA concentration and extension of the running time, the 3,5-DNBA removal in DCMM wastewater complemented with RANs tended to be more efficient and its removal rates increased gradually over the experimental period. Our study demonstrated that supplementing RANs could be a useful strategy for enhancing colonization of degrading bacteria in wastewater treatment systems.

Bioaugmentation of a sequencing batch biofilm reactor with Comamonas testosteroni and Bacillus cereus and their impact on reactor bacterial communities.

The immobilization of microorganisms is essential for efficient bioaugmentation systems. The performance of Bacillus cereus G5 as biofilm-forming bacteria and Comamonas testosteroni A3 a 3,5 dinitrobenzoic acid (DNB)-degrading strain] in laboratory-scale sequencing batch biofilm reactors (SBBRs) treating DNB synthetic wastewater has been examined. The microbial diversity in the reactors was also explored. The reactor R3 inoculated with B. cereus G5 and C. testosteroni A3 together not only improved the removal of contaminants, but also exhibited obvious resistance to shock loading with DNB during later operations. Pyrosequencing was used to evaluate bacterial communities in three reactors. Comamonas was predominant in the reactor R3, indicating the effect of G5 in promoting immobilization of A3 cells in biofilms. Those microbial resources, e.g.G5, which can stimulate the self-immobilization of the degrading bacteria offer a novel strategy for immobilization of degraders in bioaugmentation systems and show broader application prospects.

The roles of AMPK-mediated autophagy and mitochondrial autophagy in a mouse model of imiquimod-induced psoriasis.

BACKGROUND: Psoriasis is a systemic inflammatory disease characterized by epidermal hyperplasia and skin inflammatory infiltrates. Inactivation of AMPK has been shown to decrease autophagy, thereby inhibiting elimination of inflammatory factors and harmful substances, and aggravating psoriasis. However, the molecular mechanism through which AMPK affects psoriasis remains to be further explored. In this study, we investigated whether AMPK regulates autophagy through the ULK1/Atg7 signaling pathway and regulates mitochondrial autophagy through the PINK1/Parkin signaling pathway, thereby affecting a mouse model of psoriasis. METHODS: Imiquimod was used to induce psoriasis-like lesions on the backs of mice. The severity of skin lesions in psoriatic mice was evaluated with the skin inflammation severity score, and epidermal thickness was measured on the basis of H&E staining. RT-PCR, western blotting and immunofluorescence staining were used to detect indicators of autophagy and mitochondrial autophagy. RESULTS: AMPK activity was inhibited in the psoriasis mouse model, the autophagy-associated proteins ULK1/Atg7 were inhibited, and the mitochondrial autophagy proteins PINK1/Parkin were also decreased. Results indicated that autophagy and mitochondrial autophagy were inhibited in the mouse model. When AMPK signaling was upregulated, ULK1/Atg7 and PINK1/Parkin were upregulated, autophagy and mitochondrial autophagy increased, and skin lesions in the mouse model were alleviated. ULK1/Atg7 and PINK1/Parkin were down-regulated when AMPK signaling was downregulated, and psoriasis-like skin lesions were aggravated in mice. These results indicated that AMPK regulates autophagy through the ULK1/Atg7 signaling pathway and regulates mitochondrial autophagy through the PINK1/Parkin signaling pathway, thus affecting the prognosis of psoriasis in the mouse model. CONCLUSION: AMPK affects the prognosis of psoriasis in a mouse model by regulating autophagy and mitochondrial autophagy.

Identification of four genes associated with cutaneous metastatic melanoma.

BACKGROUND: Cutaneous melanoma is an aggressive cancer with increasing incidence and mortality rates worldwide. Metastasis is one of the primary elements that influence the prognosis of patients with cutaneous melanoma. This study aims to clarify the potential mechanism underlying the low survival rate of metastatic melanoma and to search for novel target genes to improve the survival rate of patients with metastatic tumors. METHODS: Gene expression dataset and clinical data were downloaded from The Cancer Genome Atlas portal. Differentially expressed genes (DEGs) were identified, and their functions were studied through gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses. Survival and multivariate Cox regression analyses were used to screen out candidate genes that could affect the prognosis of patients with metastatic melanoma. RESULTS: After a series of comprehensive statistical analysis, 464 DEGs were identified between primary tumor tissues and metastatic tissues. Survival and multivariate Cox regression analyses revealed four vital genes, namely, POU2AF1, ITGAL, CXCR2P1, and MZB1, that affect the prognosis of patients with metastatic melanoma. CONCLUSION: This study provides a new direction for studying the pathogenesis of metastatic melanoma. The genes related to cutaneous metastatic melanoma that affect the overall survival time of patients were identified.

Bioaugmentation of strain Methylobacterium sp. C1 towards p-nitrophenol removal with broad spectrum coaggregating bacteria in sequencing batch biofilm reactors.

This work was conducted in order to evaluate an instance of bioaugmentation, namely, the addition of a novel p-nitrophenol (PNP)-degrading bacterium Methylobacterium sp. C1 coaggregated with two other broad-spectrum coaggregating strains (Bacillus megaterium T1 and Bacillus cereus G5) within sequence batch biofilm reactors (SBBRs). Results showed that biofilms consisting of C1 and coaggregating bacteria were resistant to shock loads and were more efficient at PNP removal. High-throughput sequencing data revealed that biofilms formed in the presence of the coaggregating bacteria demonstrated greater microbial diversity. These results suggest that broad-spectrum coaggregating bacteria may be capable of mediating the immobilization of exogenous degrading bacteria into biofilms, rendering them more resistant to toxic compounds and environmental stresses. This represents the first attempt to assess the bioaugmentation of PNP-contaminated wastewater treatment through the utilization of broad-spectrum coaggregating bacteria.

Isolation and characterization of broad spectrum coaggregating bacteria from different water systems for potential use in bioaugmentation.

The bridging bacteria with broad-spectrum coaggregation ability play an important role during multispecies-biofilm development. In this study, through a visual and semi-quantitative assay, twenty-two bacterial strains with aggregation ability were obtained from 8 different water environments, and these strains were assigned to 7 genera according to their 16S rDNA and they were Aeromonas, Bacillus, Comamonas, Exiguobacterium, Pseudomonas, Shewanella and Comamonas. Furthermore, all possible 231 pairwise combinations among these 22 strains were explored for coaggregation ability by spectrophotometric assay. Among all these strains, it was found that Bacillus cereus G5 and Bacillus megaterium T1 coaggregated with themajority of assayed other strains, 90.5% (19 of 21 strains) and 76.2% respectively (17 of 21 strains) at a higher coaggregation rates (A.I. greater than 50%), indicating they have a broad-spectrum coaggregation property. The images of coaggregates also confirmed the coexistence of G5 and T1 with their partner strains. Biofilm biomass development of G5 cocultured with each of its partner strains were further evaluateded. The results showed that 15 of 21 strains, when paired with G5, developed greater biofilm biomass than the monocultures. Furthermore, the images from both fluorescence microscopy and scanning electron microscopy (SEM) demonstrated that G5 and A3-GFP (a 3,5-dinitrobenzoic acid-degrading strain, staining with gfp),could develop a typical spatial structure of dual-species biofilm when cocultured. These results suggested that bridging-bacteria with a broad spectrum coaggregating ability, such as G5,could mediate the integration of exogenous degrading bacteria into biofilms and contribute to the bioaugmentation treatment.

A functional polymorphism of TGFBR2 is associated with risk of breast cancer with ER(+), PR(+), ER(+)PR(+) and HER2(-) expression in women.

Little is known about the correlation between TGFBR2 G-875A and breast cancer risk. Moreover, the associations of the expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2) in breast cancer tissues with the TGFB1 C-509T, T+29C and TGFBR2 G-875A polymorphisms remain to be determined. In this study, we genotyped for TGFB1 C-509T, T+29C and TGFBR2 G-875A in fresh surgically resected tissues (n=82) and archived paraffin-embedded specimens (n=88) from 170 patients with breast cancer, as well as peripheral blood samples from 178 cancer-free female individuals. Evaluation of ER, PR and HER2 expression was performed using immunohistochemical staining. Logistic regression analysis was carried out to determine the risk of breast cancer by calculating the odds ratios (ORs) and their 95% confidence intervals (CIs). As a result, no difference was observed in the TGFB1 C-509T, T+29C genotype and allele frequencies between patients and controls. However, the frequency of the TGFBR2 -875A allele was marginally higher in cancer-free female individuals than that of women with breast cancer (24.2 vs. 17.9%, P=0.05). Notably, when stratification was performed by ER, PR and HER2 expression, the TGFBR2 -875A allele was found to correlate significantly to a decreased risk of breast cancer with ER(+) (OR=0.57, 95% CI 0.35-0.92), PR(+) (OR=0.54, 95% CI 0.34-0.88), ER(+)PR(+) (OR=0.55, 95% CI 0.33-0.92) and HER2(-) (OR=0.55, 95% CI 0.34-0.88) under a dominant genetic model. In conclusion, this is the first study to suggest that the TGFBR2 -875A allele modifies predisposition to breast cancer with an expression of ER(+), PR(+), ER(+)PR(+) and HER2(-).

Characterization and application of two novel monoclonal antibodies against human CXCR4: cell proliferation and migration regulation for glioma cell line in vitro by CXCR4/SDF-1alpha signal.

CXCR4/SDF-1alpha signal is essential for cell development, hematopoiesis, organogenesis, and vascularization as well as leukocyte trafficking. Many published reports have highlighted CXCR4 as a target in HIV infection and in cancer metastasis. In this study, we generated two specific monoclonal antibodies (MAbs 6H7 and 7D4) against human CXCR4 and found that they could recognize different antigen epitopes identified by 12G5, a commercially available anti-CXCR4 MAb. With the two MAbs, we detected the expression pattern of CXCR4 on T lymphocytes and human tumor cell lines by FCM (flow cytometry), as well as glioma tissues by immunohistochemical staining. The results showed widespread CXCR4 expression patterns in tumor cell lines and tissues and an inducible expression in CD4(+) T lymphocytes. Furthermore, we demonstrated that both of the MAbs have different functions on glioma cell line proliferation and migration in vitro. MAb 6H7 could synergistically enhance glioma cell line U251 cell proliferation induced by SDF-1alpha, while MAb 7D4 showed a blocking effect. Despite the difference in proliferation, both antibodies could attenuate chemotaxis of U251 cell induced by SDF-1alpha. Taken together, the two novel antibodies may be of great value to explore the mechanisms of SDF-1alpha CXCR4 signal in tumor cells metastasis.

[Construction of the transfected cell line expressing the human CXCR4 gene and study of its biological function].

AIM: To construct the tranfected cell line expressing the human CXCR4 gene and to study the biological function. METHODS: The total RNA was isolated from peripheral blood mononuclear cell (PBMC) with TRIzol, and the CXCR4 gene was amplified by RT-PCR, then digested with restriction endonuclease Pst I and EcoR I, and inserted into retrovirus vector pEGZ-Term. The recombinant vector together with its two helper virus vectors was co-transfected into the package cells 293T with LipofectAMINE 2000. Then the supernatant of the 293T cell culture was used to infect L929 cells, the cell clones stably expressing the CXCR4 molecule were screened in the presence of Zeocin (500 mg/L) after 72 h cultivation. RESULTS: It was found that the full-length of CXCR4 gene was successfully cloned, and the recombinant retrovirus vector carrying the CXCR4 gene was constructed. The CXCR4 cDNA transfected L929 cell could stably express the human CXCR4 on the cell membrane, and the migration ability of transfected cells was well evidenced in the transwell system induced by SDF-1alpha after the transfection with CXCR4. CONCLUSION: The CXCR4 transfected L929 cell line was successfully established, and it can make the basis for the further research.