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Serine hydroxymethyltransferase 2 (SHMT2), a catalytic enzyme playing an important role in aerobic cellular respiration and mitochondrial metabolism, might be pivotal in self-renewal and differentiation of human pluripotent stem cells. Herein, we used the CRISPR/Cas9 editing system to construct a homozygous SHMT2 knockout (SHMT2-KO) human embryonic stem cell (hESC) line, exhibiting a normal karyotype, colony morphology, and high expression levels of pluripotent proteins. Furthermore, SHMT2 knockout did not impact the self-renewal ability or differentiation potential into three germ layers of hESCs. Accordingly, this cell line provides a valuable model for further assessing SHMT2 functions in human embryonic development.

Chicken feet, aplenty by-products in the chicken industry, are rich in collagen and contain abundant amino acids so that it can be used as an important source for the collagen market. Pepsin-soluble collagen (PSC) was extracted from chicken leg skin and explored the effects of single- and tri-frequency ultrasound on the self-assembly and vitro digestion characteristics. By the diverging and tri-frequency ultrasound reactor, PSC was treated with 20 kHz/270w (C20H5m), 40 kHz/270w (C40H5m), 60 kHz/270w (C60H5m), 20/40/60 kHz/90w × 3 (CtH5m) for 5 min. Results showed that ultrasound could accelerate the process of collagen self-assembly, and 60 kHz/270w was the fastest. Microfiber diameters of C60H5m were 65–89 nm, which was significantly lower than the control without ultrasound (80–161 nm). The digestion results indicated polypeptides with relative molecular weights founded in the range 200–5000 Da were exceeded 85%. The final digested product had the highest content of oligopeptide, consistent rheological properties, and elastic behavior. The cavitation and mechanical of ultrasound have effects on the self-assembly process and collagen gel structure and digestion characteristics, which is of great significance for the development of the chicken industry and collagen market.

S-adenosylhomocysteine/Methylthioadenosine nucleosidase (SAHN E.C.3.2.2.9) does not exist in mammalian cells but is essential for methyl recycling in numerous bacterial and protozoan species. Inhibition of this enzyme could limit synthesis of autoinducers of bacterial quorum sensing (QS), and hence, causes reduction in biofilm formation and may attenuate virulence. In this study, sahn deletion mutant of E. coli MG1655, sahn-complemented strain, and SANH-overexpressing strain were established and used to identify the secretion of autoinducer-2 (AI-2) and biofilm formation. The results indicated that deletion of the sahn gene abolished the production of the QS signal AI-2 and biofilm formation in mutant strain MG1655-Δsahn. And the complementation strain MG1655-Δsahn (pET-28a-sahn) showed restored production of AI-2 and biofilm formation, which indicates that the sahn gene plays an important role in bacterial quorum sensing. The recombinant SAHN protein was overexpressed and purified. The enzymatic activity of SAHN was successfully determined by a coupling-enzyme analysis based on xanthine oxidase, with the Vmax and Km of SAHN enzymatic reaction confirmed. Given that sahn is essential for the quorum sensing of both Gram-negative and Gram-positive bacteria, SAHN could be a potential target for wide-spectrum antibiotics.

Human S-adenosyl-homocysteine hydrolase (SAHH, E.C.3.3.1.1) has been considered to be an attractive target for the design of medicines to treat human disease, because of its important role in regulating biological methylation reactions to catalyse the reversible hydrolysis of S-adenosylhomocysteine (SAH) to adenosine (Ado) and l-homocysteine (Hcy). In this study, SAHH protein was successfully cloned and purified with optimized, Pichia pastoris (P. pastoris) expression system. The biological activity results revealed that, among the tested compounds screened by ChemMapper and SciFinder Scholar, 4-(3-hydroxyprop-1-en-1-yl)-2-methoxyphenol (coniferyl alcohol, CAS: 458-35-5, ZINC: 12359045) exhibited the highest inhibition against rSAHH (IC50= 34 nM). Molecular docking studies showed that coniferyl alcohol was well docked into the active cavity of SAHH. And several H-bonds formed between them, which stabilized coniferyl alcohol in the active site of rSAHH with a proper conformation.

A new variant of pseudorabies virus (PRV) with high pathogenicity has been prevalent in many swineherds vaccinated with Bartha-K61 in China since 2011. Several gene-deleted vaccine candidates have been developed based on new emerging PRV variants. PRV-AH, a new emerging PRV strain from Anhui Province, was isolated in our laboratory in 2013. In the present study, rPRV-AH-gI−/gE− and rPRV-AH-gI−/gE−/gC+ were generated based on PRV-AH by homologous recombination. The growth kinetics of rPRV-AH-gI−/gE− and rPRV-AH-gI−/gE−/gC+ were similar to their parental strains. Compared with the commercial inactivated vaccine of Ea strain, the immune efficacy of the inactivated vaccine based on recombinant viruses was evaluated in mice and weaned pigs. The result showed that the level of neutralizing antibody in mice immunized with rPRV-AH-gI−/gE−/gC+ was higher compared with those immunized with rPRV-AH-gI−/gE- at a dose of 106 TCID50 at 8 weeks post initial immunization (p

A series of novel 3-substituted N-methylcarbazole-imidazolium salt derivatives has been prepared and evaluated in vitro against a panel of tumor cell lines (Hep G-2, Hela and PC12). The results suggest that the presence of substituted 2-methyl-imidazole or imidazole ring and substitution of the imidazolyl-3-position with a naphthylacyl or 4-bromophenacyl group were important for improving cytotoxic activity. Compounds 17, 18, 27, and 28 with 4-bromophenacyl and naphthylacyl groups displayed good activities with IC50 values of 0.09-7.20 μm against three tumor cell lines investigated and more active than DDP. Compound 35 exhibited cytotoxic activity selectively against Hela cell.

Background Several studies have suggested that caffeic acid phenethyl ester (CAPE) can induce the expression of hypoxia inducible factor-1α (HIF-1α) protein. We determined whether CAPE has a novel function in improving the homing and engraftment of haematopoietic stem/progenitor cells (HSPCs) by regulating HIF-1α gene expression in the bone marrow (BM) niche. Methods For survival experiments, lethally irradiated C57BL/6 mice were injected with a low number of BM mononuclear cells (MNCs) and CAPE according to the indicated schedule. Homing efficiency analysis was conducted using flow cytometry and colony-forming unit (CFU) assays. The influence of intraperitoneal injection of CAPE on short-term and long-term engraftment of HSPCs was evaluated using competitive and non-competitive mouse transplantation models. To investigate the mechanism by which CAPE enhanced HSPC homing, we performed these experiments including Q-PCR, western blot, immunohistochemistry and CFU assays after in-vivo HIF-1α activity blockade. Results CAPE injection significantly increased the survival rate of recipient mice after lethal irradiation and transplantation of a low number of BM MNCs. Using HSPC homing assays, we found that CAPE notably increased donor HSPC homing to recipient BM. The subsequent short-term and long-term engraftment of transplanted HSPCs was also improved by the optimal schedule of CAPE administration. Mechanistically, we found that CAPE upregulated the expression of HIF-1α, vascular endothelial growth factor-A (VEGF-A) and stromal cell-derived factor 1α (SDF-1α). The HIF-1α inhibitor PX-478 blocked CAPE-enhanced HSPC homing, which supported the idea that HIF-1α is a key target of CAPE. Conclusions Our results showed that CAPE administration facilitated HSPC homing and engraftment, and this effect was primarily dependent on HIF-1α activation and upregulation of SDF-1α and VEGF-A expression in the BM niche. Electronic supplementary material The online version of this article (doi:10.1186/s13287-017-0708-x) contains supplementary material, which is available to authorized users.

For the development of an efficient intestinal delivery system for Porcine interferon-α (PoIFN-α), the understanding of transport mechanisms of which in the intestinal cell is essential. In this study, we investigated the absorption mechanisms of PoIFN-α in intestine cells. Caco-2 cells and fluorescein isothiocyanate-labeled (FITC)-PoIFN-α were used to explore the whole transport process, including endocytosis, intracellular trafficking, exocytosis, and transcytosis. Via various techniques, the transport pathways of PoIFN-α in Caco-2 cells and the mechanisms were clarified. Firstly, the endocytosis of PoIFN-α by Caco-2 cells was time, concentration and temperature dependence. And the lipid raft/caveolae endocytosis was the most likely endocytic pathway for PoIFN-α. Secondly, both Golgi apparatus and lysosome were involved in the intracellular trafficking of PoIFN-α. Thirdly, the treatment of indomethacin resulted in a significant decrease of exocytosis of PoIFN-α, indicating the participation of cyclooxygenase. Finally, to evaluate the efficiency of PoIFN-α transport, the transepithelial electrical resistance (TEER) value was measured to investigate the tight junctional integrity of the cell monolayers. The fluorescence microscope results revealed that the transport of PoIFN-α across the Caco-2 cell monolayers was restricted. In conclusion, this study depicts a probable picture of PoIFN-α transport in Caco-2 cells characterized by non-specificity, partial energy-dependency and low transcytosis.

Cocaine- and amphetamine-regulated transcript (CART) peptide plays a pivotal role in neuroprotection against stroke-related brain injury. However, the regulatory mechanism on CART transcription, especially the repression mechanism, is not fully understood. Here, we show that the transcriptional repressor neuron-restrictive silencer elements (NRSF, also known as REST) represses CART expression through direct binding to two NRSF-binding elements (NRSEs) in the CART promoter and intron 1 (named pNRSE and iNRSE, respectively). EMSA show that NRSF binds to pNRSE and iNRSE directly in vitro. ChIP assays show that NRSF recruits differential co-repressor complexes including CoREST and HDAC1 to these NRSEs. The presence of both NRSEs is required for efficient repression of CART transcription as indicated by reporter gene assays. NRSF overexpression antagonizes forskolin-mediated up-regulation of CART mRNA and protein. Ischemia insult triggered by oxygen-glucose deprivation (OGD) enhances NRSF mRNA levels and then NRSF antagonizes the CREB signaling on CART activation, leading to augmented cell death. Depletion of NRSF in combination with forskolin treatment increases neuronal survival after ischemic insult. These findings reveal a novel dual NRSE mechanism by which NRSF represses CART expression and suggest that NRSF may serve as a therapeutic target for stroke treatment.

Forsythia suspensa extracts, isolated from the air-dried fruit of Forsythia suspensa, is a common traditional Chinese medicine recorded in Chinese Pharmacopeia. To evaluate the in vitro antibacterial activity of Forsythia suspensa extract, the minimum inhibitory concentration (MIC) was determined using a microtitre assay. For in vivo investigation, 50 Kunming mice were used, groups 1 to 4 (10 per group) were inoculated intraperitoneally with 1 ml of S. suis (LD50 CFU/ml) and treated orally 3 times per day for 7 days with: group 1,group 2, group 3 and group 4. Another group (group 5) of 10 mice were control group. Serial dilutions of the lung homogenates were counted after an 18 hour incubation at 37°C.Ethanol extracts of Forsythia suspensa showed significant bacteriostatic activity against all tested strains of S. suis with MICs all greater than 1.562 mg/ml, especially the amoxicillin-resistant strains. When tested in vivo, a 1:4 mixture of amoxicillin and Forsythia suspense extract had the greatest protective effect on mice against S. suis, followed by Forsythia suspensa extract alone.