A combination of genome-wide association study screening and SNaPshot for detecting sex-related SNPs and genes in Cynoglossus semilaevis.

Chinese tongue sole (Cynoglossus semilaevis) males and females exhibit great differences in growth rate and appearance. The species is heterogametic (ZW/ZZ) and has sex-reversed "pseudomales" that are genetically female and physiologically male. In this study, we identified eight sex-specific single nucleotide polymorphism (SNP) markers for the sex identification of C. semilaevis by using a combination of genome-wide association study (GWAS) screening and SnaPshot validation. Candidate SNPs were screened using genotyping by sequencing to perform GWAS of the differential SNPs between the sexes of C. semilaevis. The SNP loci were amplified using a multiplex PCR system and detected via SNaPshot, which enables multiplexing of up to 30-40 SNPs in a single assay and ensures high accuracy of the results. The molecular markers detected in our study were used to successfully identify normal males and pseudomales from 45 caught and 40 cultured C. semilaevis specimens. Linkage disequilibrium analysis showed that the eight SNP loci were related to each other, with a strong linkage. Moreover, we investigated the expression of prdm6 mRNA containing a missense SNP and confirmed that the gene is differentially expressed in the gonads of the different sexes of C. semilaevis; the expression of prdm6 mRNA was significantly higher in the males than in the females and pseudomales. This means prdm6 may be related to sex differentiation in C. semilaevis.

BML-111 suppresses TGF-ß1-induced lung fibroblast activation in vitro and decreases experimental pulmonary fibrosis in vivo.

Pulmonary fibrosis is an aggressive end­stage disease. Transforming growth factor­ß1 (TGF­ß1) mediates lung fibroblast activation and is essential for the progress of pulmonary fibrosis. BML­111, a lipoxinA4 (LXA4) receptor (ALX) agonist, has been reported to possess anti­ï¬brotic properties. The present study aimed to elucidate whether BML­111 inhibits TGF­ß1­induced mouse embryo lung fibroblast (NIH3T3 cell line) activation in vitro and bleomycin (BLM)­induced pulmonary fibrosis in vivo. In vitro experiments demonstrated that BML­111 treatment inhibits TGF­ß1­induced NIH3T3 cell viability and the expression of smooth muscle α actin (α­SMA), fibronectin and total collagen. Furthermore, this suppressive effect was associated with mothers against decapentaplegic homolog (Smad)2/3, extracellular signal­regulated kinase (ERK) and Akt phosphorylation interference. In vivo experiments revealed that BML­111 treatment markedly improved survival rate and ameliorated the destruction of lung tissue structure. It also reduced interleukin­1ß (IL­1ß), tumor necrosis factor­α (TNF­α) and TGF­ß1 expression in the BLM intratracheal mouse model. In addition, the expression ofα­SMA and extracellular matrix (ECM) deposition (total collagen, hydroxyproline and fibronectin) were also suppressed following BML­111 treatment. However, BOC­2, an antagonist of ALX, partially weakened the effects of BML­111. In conclusion, these results indicated that BML­111 inhibits TGF­ß1­induced fibroblasts activation and alleviates BLM­induced pulmonary fibrosis. Therefore, BML­111 may be used as a potential therapeutic agent for pulmonary fibrosis treatment.

Enzymatic synthesis of amoxicillin via a one-pot enzymatic hydrolysis and condensation cascade process in the presence of organic co-solvents.

A cascade reaction combining the enzymatic hydrolysis of Penicillin G potassium salt (PGK) with the kinetically controlled enzymatic coupling of in situ formed 6-aminopenicillanic acid (6-APA) with p-hydroxyphenylglycine methyl ester (D-HPGM) to give amoxicillin as the final product by using a single enzyme has been demonstrated successfully. Ethylene glycol (EG) was employed as a component of reaction buffer to improve the synthesis yield. Reaction parameters, including different cosolvents, EG content, the loading of immobilized penicillin G acylase (IPA), and reaction temperature and time were studied to evaluate their effects on the reaction. The best result of 55.2% yield was obtained from the reaction which was carried out in the mixed media containing 40% sodium dihydrogen phosphate buffer (apparent pH 6.0) and 60% EG (v/v), with the initial concentration 150 mM and 450 mM of PGK and D-HPGM, respectively, catalyzed by 50 IU/mL IPA at 25 degrees C for 10 h. The IPA could be recycled for nine batches without obviously losing of catalytic activity. The important strategy will have potential application in the beta-lactam antibiotics industry due to the advantages of saving the effort of isolating 6-APA, reducing usual enzymatic steps and the industrial cost of amoxicillin synthesis.

Regioselective synthesis of cyclodextrin mono-substituted conjugates of non-steroidal anti-inflammatory drugs at C-2 secondary hydroxyl by protease in non-aqueous media.

Three beta-cyclodextrin (beta-CD) conjugates of non-steroidal anti-inflammatory drugs were synthesized by enzymatic methods. Transesterification of beta-CD with vinyl ester of indomethacin, ketoprofen and etodolac was performed by the catalysis of alkaline protease from Bacillus subtilis in anhydrous DMF for 3 days. The drug molecules were selectively conjugated onto one of the secondary hydroxyl groups of beta-CD through ester-linkage to improve their poor water solubility and absorption characteristics. The products were characterized by ESI-MS, (1)H NMR and (13)C NMR. The structures of products with monoacylation occurring at the C-2 secondary hydroxyl groups of beta-CD were confirmed.