DBDMH-Promoted Methylthiolation in DMSO: A Metal-Free Protocol to Methyl Sulfur Compounds with Multifunctional Groups.

Organic thioethers play an important role in the discovery of drugs and natural products. However, the green synthesis of organic sulfide compounds remains a challenging task. The convenient and efficient synthesis of 5-alkoxy-3-halo-4-methylthio-2(5H)-furanones from DMSO is performed via the mediation of 1,3-dibromo-5,5-dimethylhydantoin (DBDMH), affording a facile route for the sulfur-functionalization of 3,4-dihalo-2(5H)-furanones under transition metal-free conditions. This new approach has demonstrated the functionalization of non-aromatic Csp2-X-type halides with unique structures containing C-X, C-O, C=O and C=C bonds. Compared with traditional synthesis methods using transition metal catalysts with ligands, this reaction has many advantages, such as the lower temperature, the shorter reaction time, the wide substrate range and good functional group tolerance. Notably, DMSO plays multiple roles, and is simultaneously used as an odorless methylthiolating reagent and safe solvent.

[Myrislignan Induces Apoptosis in Gastric Cancer Cell Line Through PI3K/AKT Signaling Pathway].

Objective: To investigate the effect of myrislignan (MYR) on the apoptosis of gastric cancer cell line and its relationship with phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. Methods: The gastric cells (SGC-7901) were treated with MYR at different concentrations, i.e., 0, 25, 50, 100, and 200 µmol/L, for 48 h and 72 h and the effect of MYR on the proliferation of SGC-7901 cells was measured by CCK-8 assay. Then, SGC-7901 cells were treated with different concentrations of MYR at 50, 100, and 200 µmol/L for 48 h. Meanwhile, a normal control group and a dimethyl sulfoxide (DMSO) solvent control group (0.1% DMSO) were established. Flow cytometry was used to determine the apoptosis rate of SGC-7901 cells. The protein expression levels of PI3K, AKT, Bcl-2-associated X protein (BAX), cysteine-dependent aspartate-specifc protease-3 (Caspase-3), and Caspase-9 were determined by Western blot. Then, PI3K activator (20 µmol/mL) was used to treat SGC-7901 cells for 48 h in 4 groups, the control group, 0.1% DMSO group, MYR group, and MYR+PI3K activator group, and the effect on MYR's induction of apoptosis and regulation of the protein expression levels of PI3K, AKT, BAX, Caspase-3, and Caspase-9 in SGC-7901 cells. Results: Compared with the control group, MYR at 50, 100 and 200 µmol/L inhibited the proliferation of gastric cancer cells, increased the apoptosis rate, down-regulated the protein expression levels of PI3K and AKT, and up-regulated the protein expression levels of BAX, Caspase-3, and Caspase-9 in a dose-dependent manner ( P<0.05). However, PI3K activator attenuated MYR-induced apoptosis in gastric cancer cells and MYR's regulation of PI3K, AKT, BAX, Caspase-3, and Caspase-9 protein expression ( P<0.05). Conclusion: MYR induces the expression of BAX, Caspase-3, and Caspase-9 proteins by inhibiting the PI3K/AKT signaling pathway, thereby promoting the apoptosis of gastric cancer cells.

[Therapeutic Effect of Artesunate on Influenza A Viral Pneumonia].

Objective: To investigate the therapeutic effect of artesunate (ART) on influenza A viral pneumonia. Methods: A total of 36 mice were evenly and randomly assigned to six groups, a normal control group (C group), a solvent control group (M group, 10% DMSO), a positive drug group (P group, oseltamivir, 1.25 mg/kg/day), ART high-dose group (ART-G group, 120 mg/kg/day), ART medium-dose group (ART-Z group, 60 mg/kg/day), and ART low-dose group (ART-D group, 30 mg/kg/day). Except for group C, which did not receive any influenza A virus intervention or intraperitoneal injection, mice in the five other groups were infected with influenza A virus through intranasal drip. Then, after 12 hours, mice in the five other groups received intraperitoneal injection of the assigned drugs and dosage once a day. The signs, body weight, and survival of the mice were observed over the course of treatment. After 7 days of treatment, the lung tissue of the mice was collected and weighed, and the lung index was calculated accordingly. HE staining was performed to observe the pathological changes in the lung tissue. The mRNA and protein expression levels of Toll-like receptor 4 (TLR4), nuclear factor kappa-B (NF-κB [p65]), tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and IL-1ß were examined with RT-qPCR and Western blot, respectively. Results: Compared with those in C group, mice in the M group had worse physical signs and lower body mass and survival, increased lung index, severe pathological changes in lung tissue, and increased levels of TLR4, NF-κB (p65), TNF-α, IL-6 and IL-1ß mRNA and protein expression in their lung tissue ( P<0.05). Compared with those in M group, the mice in the ART groups had better physical signs, higher body mass and survival rate, decreased lung index, improvement of pathological changes in the lung tissue, and decreased levels of level of TLR4, NF-κB (p65), TNF-α, IL-6 and IL-1ß mRNA and protein expression in the lung tissue ( P<0.05). Furthermore, the most prominent changes in these indexes were observed in the ART-G group. Conclusion: ART has therapeutic effects on influenza A viral pneumonia, and the mechanisms are related to the inhibition of TLR4/p65 signaling pathway activation and anti-inflammation.

Furanonyl amino acid derivatives as hemostatic drugs: design, synthesis and hemostasis performance.

Using 3,4-dihalo-2(5H)-furanones and easily available hemostatic drugs, such as tranexamic acid (TA), 4-aminomethylbenzoic acid (ABA), aminocaproic acid (AA) as starting materials, serial multi-functional molecules 2(5H)-furanonyl amino acids are designed by the combination of different pharmacophores, and successfully synthesized by a transition metal-free Michael addition-elimination reaction. The reaction is carried out under mild conditions with ethanol-dichloromethane as solvent and only stirring at room temperature for 24 h, and the yield can be up to 91%. All products are well characterized by infrared spectroscopy (IR), nuclear magnetic resonance (NMR), high-resolution mass spectra (HRMS). Ten typical target compounds among them are selected out for the experiments of hemostasis performance by the evaluation of in vitro clot formation model and liver hemorrhage model. The test results show that, their hemostasis effect is better than the original drugs. Especially the target compound G, a TA derivative from 5-borneoloxy-3,4-dibromo-2(5H)-furanone, has the best hemostasis effect among all the tested compounds. These obtained target molecules are expected to be used as multi-functional hemostatic drugs.

N-alkylation briefly constructs tunable multifunctional sensor materials: Multianalyte detection and reversible adsorption.

A series of N-alkyl-substituted polybenzimidazoles (SPBIs), synthesized by simple condensation and N-alkylation, act as functional materials with tunable microstructures and sensing performance. For their controllable morphologies, the formation of nano-/microspheres is observed at the n(RBr)/n(PBI) feed ratio of 5:1. Products with different degrees of alkylation can recognize metal ions and nitroaromatic compounds (NACs). For example, SPBI-c, obtained at the feed ratio of 1:1, can selectively detect Cu2+, Fe3+, and NACs. By contrast, SPBI-a, obtained at the feed ratio of 0.1:1, can exclusively detect Cu2+ with high sensitivity. Their sensing mechanisms have been studied by FT-IR spectroscopy, SEM, XPS, and DFT calculations. Interestingly, the SPBIs can adsorb Cu2+ in solution and show good recyclability. These results demonstrate that polymeric materials with both sensing and adsorption applications can be realized by regulating the alkylation extent of the main chain, thus providing a new approach for the facile synthesis of multifunctional materials.

Simple inorganic base promoted C-N and C-C formation: synthesis of benzo[4,5]imidazo[1,2-a]pyridines as functional AIEgens used for detecting picric acid.

Metal-free catalyzed intermolecular tandem Michael addition/cyclization has been developed for the synthesis of benzo[4,5]imidazo[1,2-a]pyridines from α-bromocinnamaldehyde and 2-substituted benzimidazoles. The reaction promoted by a simple inorganic base displays moderate to good yields and good functional group tolerance. The optical properties of some typical products have been investigated. We found that, due to the presence of the benzene ring at the C1-position of benzo[4,5]imidazo[1,2-a]pyridines which restricts intramolecular motion, as a new type of aggregation-induced emission (AIE) luminogen (AIEgen), they show very good solid-state fluorescence with quantum yields up to 88.80%. Importantly, the AIE performance of compound 3b can be useful to detect the nitroaromatic explosive picric acid (PA) with a detection limit and quenching constant of 42.5 nM and 7.27 × 104 M-M, respectively.

[Monoclonal Antibody Against N Terminal 439-451 Epitopes of Anti-Mullerian Hormone and Its Properties].

OBJECTIVE: To prepare the specific monoclonal antibody against the N-terminal specific epitope peptide of anti-mullerian hormone (AMH) and to identify its specificity. METHODS: Using bioinformatics analysis software to predict the specific peptide fragment of AMH. Then synthesized four antigenic epitope peptide segments of mature N-terminal region of AMH as the screening target antigen. Synthesized AMH wholegene.Using the prokaryotic expression system to abtain recombinant AMH protein. Immunized BALB/c mice with the recombinant AMH, and prepared mouse spleen cells for fusing with SP/20 cells. Preparation of AMH monoclonal antibody by hybridoma technology. The monoclonal antibodies against AMH were screened by using four N-terminal epitope peptides (1: 439-451 RGRDPRGPGRAQ, 2: 273-285 PPRPSAELEESPP, 3: 42-54 DLDWPPGSPQEPL, 4: 494-506 WPQSDRNPRYGNH) as antigens, and indirect ELISA and Western blot were used to identify the antigen binding characteristics of the selected monoclonal antibodies. RESULTS: Two hybridoma cell lines with stable anti-AMH-1 and anti-AMH-2 antibody activities were screened. The two antibodies were named anti-AMH-1 and anti-AMH-2 respectively. The antibody titers were 1∶12 000 and 1∶1 600 after purification. Western blot confirmed that the two McAbs recognized different antigens. Anti-AMH-1 could not only recognize the N-terminal 439-451 epitope peptide of AMH, but also recognize the amino acid sequence of recombinant AMH, as well as the ovarian tissue. Anti-AMH-2 could recognize recombinant AMH and ovarian tissue. CONCLUSION: Two monoclonal antibodies against N-terminal specific epitopes of human AMH were successfully constructed.

Streptomyces reniochalinae sp. nov. and Streptomyces diacarni sp. nov., from marine sponges.

Two marine actinomycete strains, LHW50302T and LHW51701T, were isolated from marine sponges collected in Sansha, Hainan Province, China. The morphological, chemotaxonomic and phylogenetic characteristics were consistent with their classification in the genus Streptomyces. The strains formed hooked and looped chains of arthrospores with smooth surfaces. The cell-wall hydrolysates of the strains contained ll-diaminopimelic acid as the diagnostic diamino acid. MK-9(H8) was the predominant menaquinone. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. Major fatty acids of the strains were iso-C16 : 0, anteiso-C15 : 0 and anteiso-C17 : 0. The 16S rRNA gene sequences indicated that the strains clustered together with Streptomyces albus CGMCC 4.1640T and Streptomyces qinglanensis CGMCC 4.6825T. Multilocus sequence analysis (MLSA) confirmed their relationship. Genome relatedness in forms of average nucleotide identity, digital DNA-DNA hybridization value and MLSA evolutionary distance between each of the strains and its closest relatives showed that they belonged to distinct species. On the basis of these results, strains LHW50302T and LHW51701T belong to two novel species in the genus Streptomyces, for which the names Streptomyces reniochalinae sp. nov. (type strain LHW50302T=CCTCC AA 2018013T=DSM 106194T) and Streptomyces diacarni sp. nov. (type strain LHW51701T=CCTCC AA 2018017T=DSM 106126T) are proposed, respectively.

Dysivillosins A-D, Unusual Anti-allergic Meroterpenoids from the Marine Sponge Dysidea villosa.

Four unusual meroterpenoids, dysivillosins A-D (1-4), were isolated from an organic extract of the marine sponge Dysidea villosa collected from the South China Sea. Their planar structures were determined by 1D and 2D NMR and HRESIMS techniques, while the relative and absolute configurations were elucidated by NOESY experiments and comparison between the calculated and experimental ECD spectra. To the best of our knowledge, dysivillosins A-D are the first examples of terpene-polyketide-pyridine hybrid metabolites from the nature. Anti-allergic activity evaluation showed that compounds 1-4 potently inhibited the release of ß-hexosaminidase, a marker of degranulation, in a dose-dependent manner with IC50 values of 8.2-19.9 µM. Additionally, the four meroterpenoids could downregulate the production of lipid mediator leukotrienes B4 (LTB4) and pro-inflammatory cytokine interleukin-4 (IL-4) in the antigen-stimulated RBL-2H3 mast cells. Further biological investigations revealed that dysivillosin A (1) could suppress the phosphorylation of Syk and PLCγ1 in IgE/FcɛRI/Syk signaling pathway, which resulted in the inhibition of degranulation and the downregulation of LTB4 and IL-4 production in mast cells.

Unusual Anti-allergic Diterpenoids from the Marine Sponge Hippospongia lachne.

Hipposponlachnins A (1) and B (2), possessing an unprecedented tetracyclo [9.3.0.02,8.03,7] tetradecane ring system, and the probable biogenetic precursor [3, (1R*,2E,4R*,7E,10S*,11S*,12R*)-10, 18-diacetoxydolabella-2,7-dien-6-one] of 1‒2 were isolated from the South China Sea marine sponge Hippospongia lachne. The structures of the novel compounds were determined using integrated spectroscopic methods in combination with single-crystal X-ray diffraction analysis. Compounds 1‒2 showed potent inhibitory activity on the release of ß-hexosaminidase, a biomarker for degranulation, as well as the production of pro-inflammatory cytokine IL-4 and lipid mediator LTB4 in DNP-IgE-stimulated RBL-2H3 cells.

[The New Bacteria Expressing Recombinant Multi-epitope Vaccine against Helicobacter pylori and Its Microbiological Characteristics].

OJECTIVE: To construct the engineering bacteria with recombinant plasmid expressing the multi-epitope vaccine which composed of Helicobacter pylori urea membrane channel protein (UreI), Helicobacter pylori urease B subunit (UreB) and cholera toxin B subunit (CTB), and then to study it's microbiological characteristics. METHODS: The sequence contains some dominant epitopes of Helicobacter pylori UreI and UreB was designed, and ctB was added at the N-terminal, all the sequence were linked by flexible linkers. Codon optimization was done according to Escherichia coli (E. coli) BL21 (DE3) bias, the optimized sequence was designated BIB. BIB sequence was synthesized and cloned into plasmid pET28a(+). The recombinant plasmid was confirmed by restriction enzyme digestion and DNA sequencing. The recombinant protein BIB was expressed in E. coli BL21 (DE3) and analyzed by Western blot. RESULTS: The plasmid of pET28a(+)/BIB was constructed successfully, confirmed by restriction enzyme digestion and DNA sequencing. The recombinant protein BIB with relative molecular mass about 33 x 10(3) could be produced by E. coli BL21 (DE3) and was detected by Western blot. The relative molecular mass and N-terminal amino acid sequence of BIB were 100% identity with the design. CONCLUTION: The engineering bacteria with recombinant plasmid expressing the multi-epitope vaccine against Helicobacter pylori was constructed successfully. The recombinant protein BIB can be identified by anti-Sydney strain 1 of Helicobacter pylori (H. pylori SS1) polyclonal antibody and anti-CTB monoclonal antibody, which demonstrated that BIB has the expected antigenicity.

Antitumor effect and mechanism of action of a tumor-targeting recombinant human tumor necrosis factor-α fusion protein mediated by urokinase.

The aim of this study was to investigate the effect of the tumor­targeting recombinant human tumor necrosis factor (rhTNF)­α fusion protein mediated by urokinase on Sl80 tumor­bearing mice, as well as to explore its mechanisms of action. Furthermore, the study aimed to observe the effect of the protein on liver and kidney function. rhTNF­α fusion protein prokaryotic expression vectors were constructed using genetic engineering techniques, and were introduced into Escherichia coli. Expression of the fusion protein was induced, and it was then separated and purified in order to determine its cytotoxic activity on L929 cells. Kunming mice were randomly divided into four groups after being inoculated with S180 tumor cells. The groups were then injected with saline (control group, group S), or saline with 0.1 µg/ml fusion protein (low dose group, group L), 0.2 µg/ml fusion protein (middle dose group, group M) or 0.3 µg/ml (high dose group, group H). The mice were sacrificed after 12 days and liver [mg/kg; (liver weight/body weight) x 1,000] and kidney [mg/kg; (kidney weight/body weight) x 1,000] indices, tumor weight, the percentage reduction in mean tumor size, and the levels of alanine transaminase (ALT), albumin (ALB), creatinine (Cr) and blood urea nitrogen (BUN) in each group of mice were determined. In addition, the levels of urokinase­type plasminogen activator (uPA), the expression of bcl­2, bax and vascular endothelial growth factor (VEGF), and the percentage of apoptotic cells were measured with an enzyme­linked immunosorbent assay, streptavidin­biotin complex of immunohistochemistry and terminal deoxynucleotidyl transferase­mediated dUTP nick end labeling, respectively. The fusion protein significantly inhibited the growth of S180 tumor cells in vivo in a dose­dependent manner. With an increase in the dose of fusion protein, ALT, uPA, bcl­2 and VEGF levels decreased, and ALB levels increased. However, liver and kidney indices and bax expression were not significantly altered. Cr and BUN levels did not change significantly in the low and middle dose groups, but did increase in the high dose group. Compared with the control group, the percentage of apoptotic cells in the high­dose group was significantly higher. In conclusion, the fusion protein significantly inhibited S180 tumor growth in a mouse model, possibly by reducing the levels of uPA, bcl­2 and VEGF. There was a mildly toxic effect on the kidneys with the high dose, but a protective effect in the liver.

[Identification of Chinese herbal medicines based on terahertz spectroscopy analysis].

In order to study the feasibility of the identification of Chinese herbal medicines based on terahertz spectroscopy, the optical characteristic of astragalus, angelica, eucommia and three kinds of astragalus samples with different impurities in the frequency range 0.2-2.2 THz were researched by terahertz time-domain spectroscopy (THz-TDS), and their time-domain spectra, the frequency spectra and the absorption spectra were obtained at room temperature. The results indicated that the time-domain spectra, frequency-domain spectra and absorption spectra of astragalus, angelica, and eucommia have large differences in such a frequency range, the frequency-domain spectra and absorption spectra of three kinds of astragalus samples with different impurities are similar but there exists distinct difference. These researches proved that it is feasible to use terahertz time-domain spectroscopy to identify Chinese herbal medicine and provided a new method for Chinese herbal medicine quality control.

[The research on the construction and characteristics of recombinant engineering bacteria with multi-epitope of Helicobacter pylori].

OBJECTIVE: To construct the multi epitope prokaryotic expression plasmid and appropriate engineering bacteria expressing the multi-epitope fusion protein of urea membrane channel protein (UreI), urease B subunit (UreB) and adhesin (HpaA) of Helicobacter pylori, then study its microbiological characteristics. METHODS: The target sequence contains multi epitope gene sequence of Helicobacter pylori were designed and synthesized, subsequently; it was subcloned into the expression vector pET28a (+), confirmed by restriction enzyme digestion and DNA sequencing. The fusion protein rIBA was expressed in E. coli Rosseta (DE3) and analyzed by Western blot. RESULTS: The plasmid of pET28a(+)/IBA was constructed successfully, confirmed by endonuclease digestion and sequence analyze. The expressed rIBA protein with relative molecular mass about 40 x 10(3) and can be detected by Western blot. CONCLUSION: The prokaryotic engineering bacteria expression multi-epitope of the Helicobacter pylori was constructed successfully. The recombinant protein rIBA expressed by the engineering bacteria can be identified by Sydney strain 1 of Helicobacter pylori (H. pylori SS1) specific antibody IgY, which demonstrated that the rIBA has high correlation with H. pylori SS1.

Expression of CD40 and growth-inhibitory activity of CD40 agonist in ovarian carcinoma cells.

The CD40 receptor is a member of the tumour necrosis factor receptor family and is widely expressed on various cell types. The antitumour activity of CD40 agonist antibody has been observed in B-cell-derived malignancies, but its activity on ovarian cancer remains unclear. However, in this paper, we first confirmed that the anti-CD40 agonist antibody could inhibit the growth of ovarian cancer cells and induce apoptosis. This study investigated the expression of CD40 by ovarian carcinoma tissues and cell lines, at the same time, we evaluated the effect of a recombinant soluble human CD40L (rshCD40L) and an anti-CD40 agonist antibody on cell growth and apoptosis. Flow cytometry and immunohistochemistry assay demonstrated that CD40 was expressed on ovarian carcinoma cell lines and primary ovarian carcinoma cells derived from ascites, as well as on ovarian carcinoma tissues. The growth inhibition of rshCD40L and the anti-CD40 agonist antibody on ovarian carcinoma cells was examined by MTT assay, and the proportion of apoptotic tumour cells was analysed by flow cytometry and Hoechst staining. Our study showed that CD40 was expressed on all ovarian carcinoma cell lines and was examined in 86.2% (162/188) of ovarian cancer tissue samples, but not in normal ovarian tissues (n = 20). Treatment with rshCD40L or anti-CD40 agonist antibody significantly inhibited ovarian carcinoma cell growth and induced apoptosis. Theses results suggest that CD40 is expressed on ovarian carcinoma cells, moreover, that rshCD40L and anti-CD40 agonist antibody have therapeutic potential to inhibit human ovarian cancer growth.

The effect of miR-7 on behavior and global protein expression in glioma cell lines.

Malignant glioma is a common cancer of the nervous system. Despite recent research efforts in cancer therapy, the prognosis of patients with malignant glioma has remained dismal. MicroRNAs are noncoding RNAs that inhibit the expression of their targets in a sequence-specific manner, and a few have been shown to act as oncogenes or tumor suppressors. Here, we aimed at exploring the precise biological role of microRNA-7 (miR-7) and the global protein changes in glioma cell lines transiently transfected with miR-7. Transfection of miR-7 into glioma cell lines causes inhibition of cell migration and invasion and suppression of tumorigenesis. Moreover, ectopic expression of miR-7 inhibits lung metastases of glioma in vivo. Among 65 protein spots with differential expression separated by 2-DE, 37 proteins were successfully identified by MS/MS analysis. Of those, the 25 downregulated proteins, which include 14-3-3ζ, eukaryotic translation initiation factor 5A (EIF5A), and annexin A4, may be downstream targets of miR-7, a finding that could elucidate some aspects of the behavior of glioma cells at the protein level. In conclusion, the absence of miR-7 function could cause downstream molecules to switch on or off, resulting in glioma development, invasion, and metastases. MiR-7-based gene treatment may be a novel anti-invasion therapeutic strategy for malignant glioma.

[A isolation and identification of an antioxidant from Monochoria vaginalis].

With solvent extraction, a total of four fractions were separated from 75% alcohol extract of Monochoria vaginalis, and their antioxidative activities were measured by iodine method. The results showed that among the fractions, n-butanol fraction exhibited the highest antioxidative activity, which was not only significantly higher than CK (water), but also equivalent to the natural antioxidant tea polyphenols and synthetic antioxidant butylated hydroxyl toluene (BHT). A compound was isolated from the n-butanol fraction by using column chromatography, and identified as stigmasterol 3-O-beta-D-glucopyranoside. Its antioxidative activity estimated by the determination of the percentage of scavenged free radicals indicated that this compound exhibited a higher activity than BHT in scavenging free radicals.