Laccase Lac-W detoxifies aflatoxin B1 and degrades five other major mycotoxins in the absence of redox mediators.

A multicopper oxidase Lac-W from Weizmannia coagulans 36D1 was identified and characterized as a laccase (Lac-W) with a robust enzymatic activity, which was used in various mycotoxins degradation. We demonstrated that Lac-W could directly degrade six major mycotoxins in the absence of redox mediators in pH 9.0, 24h static incubation at room temperature, including aflatoxin B1 (AFB1, 88%), zearalenone (60%), deoxynivalenol (34%), T-2 toxin (19%), fumonisin B1 (18%), and ochratoxin A (12%). The optimal condition for Lac-W to degrade AFB1 was 30 °C, pH 9.0, enzyme-substrate ratio 3U/µg in 24h static condition. Furthermore, we characterized aflatoxin Q1 as a Lac-W-mediated degradation product of AFB1 using UHPLC-MS/MS. Interestingly, degradation products of AFB1 failed to generate cell death and apoptosis of intestinal porcine epithelial cells. Finally, our molecular docking simulation results revealed that the substrate-binding pocket of Lac-W was large enough to allow the entry of six mycotoxins with different structures, and their degradation rates were positively correlated to their interacting affinity with Lac-W. In summary, the unique properties of the Lac-W make it a great candidate for detoxifying multiple mycotoxins contaminated food and feed cost-effectively and eco-friendly. Our study provides new insights into development of versatile enzymes which could simultaneously degrade multiple mycotoxins.

miR-944 inhibits lung adenocarcinoma tumorigenesis by targeting STAT1 interaction.

Lung adenocarcinoma (LAC) is a leading cause of cancer-associated mortalities, particularly in developed countries. The aberrant expression of microRNAs (miRNAs) has been proven to regulate numerous diseases in the past two decades. miRNAs have been identified in almost all human cancer types. In the present study, the role of miR-944 in LAC proliferation was examined. It was identified that miR-944 was downregulated in LAC tissues and cells, and miR-944 overexpression inhibited A549 and H1299 cell proliferation, as determined by the Cell Counting Kit-8 and colony formation assay. Signal transducer and activator of transcription 1 (STAT1) was upregulated in LAC tissues and cells. Kaplan-Meier analysis demonstrated that the 5-year overall survival in patients with high STAT1 levels was significantly reduced, compared with patients with negative and low STAT1 expression. STAT1 was the direct target of miR-944. Additionally, a miR-944 mimic inhibited A549 cell growth in vitro. Collectively, these data demonstrate that miR-944 serves a pivotal role in LAC tumor growth by targeting STAT1. The data obtained indicated that miR-944 may be a novel biomarker and could result in potential therapies for LAC.

Larvicidal activity of lansiumamide B from the seeds of Clausena lansium against Aedes albopictus (Diptera: Culicidae).

The larvicidal activity of crude petroleum ether, toluene, n-butanol, ethyl acetate, acetone, and methanol extracts of the seeds of Clausena lansium was assayed for their toxicities against the early fourth instar larvae of Aedes albopictus. The larval mortality was observed after 24-h exposure. The LC(50) value of petroleum ether extract was 22.99 ppm, showing the best larvicidal activity among all six solvent extracts. A cinnamon amide compound lansiumamide B (N-methyl-N-cis-styrylcinnamamide) was isolated from the petroleum ether extract by column chromatographic method, which exhibited a strong larvicidal activity against the early fourth instar larvae of A. albopictus with LC(50) and LC(90) values of 0.45 and 2.19 ppm, respectively. The structure was elucidated by (1)H NMR, (13)C NMR spectral data. The larvicidal activity against mosquito of lansiumamide B from the seed of C. lansium was evaluated for the first time.

[Regulation of NF-kappaB signaling pathway by poxvirus].

Poxviruses, a type of ds-DNA viruses which mainly target at the epithelial cell, are the pathogens of human and animals. During the revolution of poxviruses, the viruses encode multiple proteins that regulate the immune system to monitor the viral reproductive cycle in host cells. The nuclear kappa B (NF-kappaB) pathway is essential to signal transcription in the innate immune system. Therefore, poxviruses have adopted different strategies to elude immune detection and destruction regulated by NF-kappaB. Further research in this field would help us develop preventive and therapeutic preparation for pox. Given the renewed interest in poxvirus, we review the current understanding of how the various classes of poxviralimmunomodulatory proteins target and manipulate the NF-kappaB pathway.

In vivo recovery effect of silibinin treatment on streptozotocin-induced diabetic mice is associated with the modulations of Sirt-1 expression and autophagy in pancreatic ß-cell.

Improper adjustments of autophagy and silent information regulator 1 (Sirt-1) expression were reported to be closely associated with metabolic disorders. In this study, we examined the roles of Sirt-1 and autophagy in streptozotocin-induced diabetes mellitus, assessed the relationship between autophagy and Sirt-1, and investigated the protective mechanism of silibinin. Diabetes was induced in 6-week-old mice by intravenous injection of streptozotocin (150 mg/kg/day, for 2 weeks). In the treatment groups, silibinin (50 mg/kg/day, intramuscular injection, for 8 weeks) or inhibitors (50 mg/kg/day, subcutaneous injection, for 8 weeks) were given. Diabetic control animals received vehicle for the same time. Compared with diabetic controls, silibinin or autophagy inhibitor, 3-methyladenine, treated mice showed decreased levels of glycosylated hemoglobin A1C (P < 0.01), serum triglyceride (P < 0.01), cholesterol (P < 0.01), blood glucose (P < 0.05), autophagy (P < 0.05), and apoptosis ratio (P < 0.05) of pancreatic ß-cells. Systemic administration of silibinin reversed streptozotocin-induced downregulation of Sirt-1 expression. Sirt-1 may play a role in regulating the physiological level of autophagy and is associated with loss of pancreatic ß-cells and metabolic biochemical disorders. Through promoting Sirt-1 expression and recovering autophagy physiologically, silibinin may reverse hyperglycemia and repair damaged pancreatic ß-cells.

[Identification of the binding site on glycophorin A for Plasmodium falciparum EBA-175].

OBJECTIVE: To identify the binding site on glycophorin A (GPA) for EBA-175 to provide clue for developing short peptide vaccine and therapeutic agents against Plasmodium falciparum. METHODS: With the recombinant protein of EBA-175 as the target molecule, the mimetic peptides of GPA were screened from a 12-mer random peptide library. Three rounds of biopanning were carried out, and enzyme-linked immunosorbent assay (ELISA), competitive ELISA, Dot-ELISA and Western blotting used to evaluate the binding between the phage-borne peptides and EBA-175. The insert DNA sequences of positive clones were determined and their amino acid sequences deduced. RESULTS: Thirty clones from the third round were randomly selected, of which 27 were found positive by sandwich ELISA. Competitive ELISA proved that most of the phage-borne peptides could competitively inhibit the binding of antibody (EBA-175 Ab) with EBA-175. Analysis of DNA and amino acid sequences indicated that 24 positive phage clones contained the conservative sequence of IRR, which was highly homologous with the 114-116 amino acids of GPA. CONCLUSION: These phage-displayed peptides can bind with EBA-175, and the amino acid sequence IRR might play an important role in the binding between EBA-175 and GPA.

[Preparation and characterization of monoclonal antibodies against erythrocyte-binding antigen 175 of Plasmodium falciparum].

OBJECTIVE: To prepare and characterize the monoclonal antibodies (mAbs) against erythrocyte-binding antigen 175 of Plasmodium falciparum (EBA-175). METHODS: BALB/ c mice were immunized with purified recombinant EBA-175 and mAbs against EBA-175 were prepared by means of hybridoma technique. Enzyme-linked immunosorbent assay (ELISA) and Western blotting were employed for characterization of the mAbs. RESULTS: Six McAbs against EBA-175 antigen were obtained, 5 of which were identified as IgG1 and one as IgG2a. The titer of these aAbs was 1:12,800 to 1:25,600 in the ascites and 1:256 to 1:512 in supernatant, and ELISA demonstrated specific binding of the 4 mAbs (1F3, 2H5, 4A1 and 4H9)with Plasmodium falciparum. Three of these mAbs recognized the protein of EBA-175 as shown by Western blotting. CONCLUSION: Six hybridoma cell lines secreting the mAbs against EBA175 with high specificity are successfully established.

[Screening and preliminary identification of mimetic peptides of Plasmodium falciparum-infected erythrocyte membrane surface protein 1].

OBJECTIVE: To screen and identify mimetic peptides of Plasmodium falciparun-infected erythrocyte membrane surface protein 1 in order to explore anti-adhesive agent against cerebral malaria. METHODS: Phage-borne peptide KLYLIAEGSVAA was used as panning targets to select target binders in a disulfide-constrained heptapeptides library. Three rounds of biopanning were carried out and then ELISA and competitive ELISA were used to evaluate the binding character between phage-borne peptides and ICAM-1. The insert DNA sequences of positive clones were determined and their amino acid sequences were deduced. RESULTS: After three-round panning, 22 clones were randomly chosen from the third panning and analyzed. Three clones showed positive interaction with ICAM-1, and two of them possessed the amino acid sequence C-ITAVPVR-C, the other one was C-DIMGGYN-C. These peptides specifically inhibited the binding of 15.2 antibody to ICAM-1 detected by competitive ELISA. CONCLUSION: Two kinds of mimetic peptides of PfEMP-1 have been obtained, which can bind with ICAM-1 specifically.

[Immunogenicity of a multiple epitope DNA vaccine against hepatitis B virus].

AIM: To study the immunogenicity of a multiple epitope DNA vaccine against hepatitis B virus(HBV). METHODS: A multiple epitope HBV antigen gene BPT was synthesized and cloned into eukaryotic expression vector pcDNA3.1 and then BALB/c mice were immunized with the DNA vaccine. The specific humoral and cellular immune responses were detected by indirect ELISA, cytotoxicity of CTL, and lymphocyte proliferation. The immunized mice were also observed for the possible toxicity and side effects after administration of the DNA vaccine. RESULTS: Immunization with pcDNA3.1/BPT elicited high-level antigen-specific IgG and antigen-specific CTL response, and stimulated lymphocyte proliferation. RT-PCR analysis of spleen lymphocytes showed that levels of IL-12 mRNA in immunized mice were notably higher than that in control mice. CONCLUSION: The multiple epitope HBV DNA vaccine can induce specific humoral and cellular immune responses, which lays a certain foundation for development of prophylactic and therapeutic HBV vaccine.

[Refolding and purification of Plasmodium falciparum glutamate dehydrogenase fusion protein].

OBJECTIVE: To establish the method for renaturation and purification of the fusion protein of Plasmodium falfiparum (FCC1/HN ) glutamate dehydrogenase (GDH) with glutathione S-transferase (GST). METHODS: The recombinant plasmid GDH/pGEX-4T-1, encoding the full-length GDH gene, was transformed into E.coli BL21 (DE3) to achieve IPTG-induced high expression of GDH/GST in the form of inclusion bodies identified by SDS-PAGE. After denaturation with 8 mol/L urea, the inclusion bodies were subjected to 3 different renaturation methods, namely Sephacryl S-200 chromatography, dialysis and dilution, for refolding of the fusion protein. The refolded GDH/GST was then purified by different chromatographic approaches. RESULTS: SDS-PAGE analysis showed that the expression GDH/GST fusion protein mounted up to approximately 25% of the total bacterial protein. The dilution was better than the other two methods for the refolding of the fusion protein, with the optimized renaturation condition necessitating the presence of 20 mmol/L Tris-HCl and 1 mmol/L EDTA at pH8.5 with GSSG/GSH ratio of 1 10, which resulted in a recovery rate exceeding 90%. Two-step ion exchange chromotography was optimal for purification of the fusion protein. CONCLUSION: The high-purity and biologically active GDH/GST can be acquired by dilution renaturation followed by two-step ion exchange chromatography.

[Preparation and identification of phage-displayed ICAM-1-mimic peptide].

AIM: To obtain bioactive ICAM-1 mimetic peptide. METHODS: Phages displaying P1 and P2 were prepared by phage amplication and PEG precipitation. The binding between phage-displayed peptides and anti-ICAM-1 mAb 15.2 was evaluated by sandwich ELISA and competitive ELISA. Bioactivities of P1 and P2 was detected by immunohistochemical staining. RESULTS: Phage-displayed peptides P1 and P2 could specifically bind to mAb 15.2, and the binding could be competitively inhibited by ICAM-1. Immunohistochemical staining showed that P1 and P2 could mimic the binding of ICAM-1 to its receptor LFA-1. CONCLUSION: Phage-displayed peptides P1 and P2 are bioactive just as native ICAM-1.

[Establishment of biopanning model of phage display peptide library in the blood vessels of excised human osteosarcoma vasculature and its significance].

OBJECTIVE: To establish an in vivo biopanning model of phage display peptide library in the blood vessels contained in surgically removed human osteosarcoma. METHOD: In 28 patients with osteosarcoma, digital subtraction angiography (DSA) of the involved limb was performed preoperatively to understand the approximate status of the arteries in the tumors. The tumors were then surgically removed and carefully trimmed, perfused via a simulated Langendorff perfusion apparatus with the indexes as the pH value, temperature and O2 partial pressure monitored in the blood vessels. A 12-meres phage display peptide library was biopanned to isolate peptides capable of homing specifically to the excised osteosarcoma. RESULTS: In vivo biopanning models were successfully established in all the excised tumors, which could be directly used in perfusion experiment and the indexes monitored in the blood vessels in the tumors were comparable to those of living tissues. Some high-affinity peptides specific to the blood vessels in osteosarcoma were obtained with the motif of RLTR. CONCLUSION: In vivo biopanning model simulating the Langendorff perfusion apparatus can be easily established which is instrumental for the application of phage display technology in human living tissues and may facilitate the study of targeted chemotherapy of osteosarcoma.

[Identification of the binding site on ICAM-1 for red blood cells infected by Plasmodium falciparum].

OBJECTIVE: To identify the binding site on ICAM-1 to PRBCs in order to explore anti-adhesive agent against cerebral malaria. METHODS: Monoclonal antibody 15.2 against ICAM-1 domain 1 was chosen as target molecule to screen mimetic peptides of ICAM-1 from a 12-mer random peptide library. Three rounds of biopanning were carried out and then ELISA, competitive ELISA, dot-ELISA and Western blotting were used to evaluate the binding character between phage-borne peptides and McAb 15.2. The insert DNA sequences of positive clones were determined and their amino acid sequences were deduced. RESULTS: Thirty clones from the third round were randomly selected, and 26 of them were found positive by sandwich ELISA. The competitive ELISA test proved that most phage-borne peptides could competitively inhibit the binding of antibody (15.2 McAb) with ICAM-1. Analysis of DNA and amino acid sequences indicated that over a half positive phage clones expressed 12-mer peptide KLYLIAEGSVAA. Comparison of peptide K(XX) L(XXX) GSV with the 64-73 aa of primary sequence of ICAM-1 showed a 50% homogeneity. CONCLUSION: These peptides displayed by phage may be analogs of ICAM-1, K..L...GSV probably plays a significant role on the binding reaction of ICAM-1 and PRBCs.