Thermal preparation of lysozyme-imprinted microspheres by using ionic liquid as a stabilizer.

Thermal preparation of lysozyme-imprinted microspheres was firstly investigated by using biocompatible ionic liquid (IL) as a thermal stabilizer. The imprinted microspheres made with IL could obtain the good recognition ability to template protein, whereas the imprinted polymer synthesized in the absence of it had a similar adsorption capacity to the non-imprinted one. Furthermore, the preparation conditions of imprinted polymers (MIPs) including the content of IL, temperature of polymerization, and types of functional monomers and crosslinkers were systematically analyzed via circular dichroism spectrum and activity assay. The results illustrated that using hydroxyethyl acrylate as the functional monomer, ethylene glycol dimethacrylate as the crosslinker, 5 % IL as the stabilizer, and 75 °C as the reaction temperature could retain the structure of template protein as much as possible. The obtained MIPs showed excellent recognition ability to the template protein with the separation factor and selectivity factor value of 4.30 and 2.21, respectively. Consequently, it is an effective way to accurately imprint and separate template protein by cooperatively using circular dichroism spectroscopy and activity assay during the preparation of protein MIPs. The method of utilizing IL to stabilizing protein at high temperature would offer a good opportunity for various technologies to improve the development of macromolecules imprinting.

Cytoplasmic Escape of Mitochondrial DNA Mediated by Mfn2 Downregulation Promotes Microglial Activation via cGas-Sting Axis in Spinal Cord Injury.

Neuroinflammation is associated with poor outcomes in patients with spinal cord injury (SCI). Recent studies have demonstrated that stimulator of interferon genes (Sting) plays a key role in inflammatory diseases. However, the role of Sting in SCI remains unclear. In the present study, it is found that increased Sting expression is mainly derived from activated microglia after SCI. Interestingly, knockout of Sting in microglia can improve the recovery of neurological function after SCI. Microglial Sting knockout restrains the polarization of microglia toward the M1 phenotype and alleviates neuronal death. Furthermore, it is found that the downregulation of mitofusin 2 (Mfn2) expression in microglial cells leads to an imbalance in mitochondrial fusion and division, inducing the release of mitochondrial DNA (mtDNA), which mediates the activation of the cGas-Sting signaling pathway and aggravates inflammatory response damage after SCI. A biomimetic microglial nanoparticle strategy to deliver MASM7 (named MSNs-MASM7@MI) is established. In vitro, MSNs-MASM7@MI showed no biological toxicity and effectively delivered MASM7. In vivo, MSNs-MASM7@MI improves nerve function after SCI. The study provides evidence that cGas-Sting signaling senses Mfn2-dependent mtDNA release and that its activation may play a key role in SCI. These findings provide new perspectives and potential therapeutic targets for SCI treatment.

[Cloning and functional analysis of Phyllostachys edulis MYB transcription factor PeMYB2].

MYB-type transcription factor is one of the largest families in plants, which plays important roles in accepting stress signals from environment and regulating the expression of stress-tolerant genes. In this paper, using homologous cloning and RACE technology, a MYB-type transcription factor, designated PeMYB2, was cloned from Phyllostachys edulis. The results of bioinformatics showed that PeMYB2 is a typical R2R3-MYB. It contained two tandem repeats in its N-terminus, and a membrane protein DUF3651 in its C-terminus. In addition, phylogenetic analysis indicated that PeMYB2 shared the highest homology with 85.98% to OsMYB18 protein from Oryza sativa spp. Japonica. In addition, a yeast one-hybrid assay showed that PeMYB2 could activate the expression of downstream genes. After PeMYB2 was transformed into Arabidopsis thaliana, seven PeMYB2 transgenic Arabidopsis lines were obtained. Phenotypic analysis of the transgenic and wild-type Arabidopsis showed that over-expression of PeMYB2 caused delayed flower or dwarfism in transgenic Arabidopsis. Under the abiotic stress conditions, such as salt and cold stresses, the over-expression of PeMYB2 in Arabidopsis had higher survival rate than the wild-type Arabidopsis. Expression analysis of saline stress response marker genes in the transgenic and wild-type plants under the salt stress condition showed that PeMYB2 regulated the expression of NXH1, SOS1, RD29A, and COR15A. As the result, PeMYB2 might play an important role in various responses to abiotic stresses in P. edulis.

[The apoptosis effect of antisense oligodeoxynucleotides targeting ATM on the laryngeal squamous cell carcinoma treated with radiation].

OBJECTIVE: To designed antisense oligodeoxynucleotides (AS-ODNs) to reduce the expression of ATM and to study its effect on the apoptosis of Hep-2 (human epidermoid laryngeal carcinoma) cells treated with radiation in vitro. METHODS: Experiment was divided into AS-Lipo, Sen-Lipo, Mis-Lipo, Lipo and Hep-2 group. The expression of ATM mRNA in Hep-2 cells was examined by real-time quantitative PCR. About 18 hours after transfection, they were irradiated simultaneously with different doses of X-ray radiation (0, 2, 4, 6, and 8 Gy) respectively. Clonogenic survival assay was carried out to detect the survival ability of Hep-2 cells after irradiation. After exposed to 4 Gy radiation, flow cytometry was carried out to analyze the cell apoptosis. RESULTS: The relative ATM mRNA expression in Hep-2 cells treated with ATM AS-ODNs was decreased to (11.03 +/- 2.51)% which was much lower than that of untreated cells (P < 0.05). After irradiation, the survival fraction (SF) of cells treated with ATM AS-ODNs was lower than that of other groups at the same dose of radiation. There was statistical significance between the group treated with ATM AS-ODNs and other groups (P < 0.05). The apoptotic rate for the group irradiated with ATM AS-ODNs was (30.7 +/- 1.31)%, which was significantly higher than that of others (P < 0.05). CONCLUSION: AS-ODNs of ATM reduce ATM mRNA expression and enhance Hep-2 cells apoptosis to radiation in vitro.

[RAPD analysis and construction of specific DNA probes between Pinellia ternata and Typhonium flagelliforme].

OBJECTIVE: To establish a molecular marking method to identify Pinellia ternata and Typhonium flagelliforme. METHODS: Twenty-two random oligonucleotide primers were used in RAPD analysis on the genomic DNA of two types of Pinellia ternata in Sichuan and two types of Typhonium flagelliforme in Guangxi. The special fragments were sequenced, marked as probes and then conducted Southern blot. RESULTS: A great deal of special bands was found between Pinellia ternata and Typhonium flagelliforme. A Pinellia ternata specific molecule was screened. CONCLUSION: RAPD analysis and specific DNA probes show potential value in the identification of Pinellia ternata and Typhonium flagelliforme.

[Inhibition of HBsAg and HBeAg expression by shRNA expressing vectors targeting HBV s and e gene].

AIM: To constructed the shRNA expressing vectors targeting HBsAg gene and HBeAg gene of HBV, Pgs1, Pgs2, Pgs3 and psiHBV4, psiHBV6 in order to prove their inhibitation on the HBV antigen expression in outlive HepG2. 2. 15 cells. METHODS: PTZ was used as negative control, both the shRNA expressing vectors targeting HBsAg and HBEAg gene of HBV was together transfected into HepG-2. 2. 15 cells with different combinations, and detected the expression liquid and the cultivated supernatant with MEIA after 24 h. RESULT: The group transfecting psiHbv4 and PgS2, psiHBV4 and PgS3 could significantly inhibit HBsAg and HBeAg expression compared with control group (P < 0.05) in the cell disruption liquid and the cultivated supernatant. But the group transfecting psiHBV5 and PgS1, psiHBV6 and PgS3 cannot significantly inhibit HBeAg expression (P > 0.05) in the cell disruption liquid. CONCLUSION: The shRNA expressing vectors targeting HBsAg and HBeAg gene of HBV psiHBV4 and PgS2, psiHBV4 and HBeS3 could significantly inhibit the antigen expression of HBV than only one.

Effect of S1P5 on proliferation and migration of human esophageal cancer cells.

AIM: To investigate the sphingosine 1-phosphate (S1P) receptor expression profile in human esophageal cancer cells and the effects of S1P5 on proliferation and migration of human esophageal cancer cells. METHODS: S1P receptor expression profile in human esophageal squamous cell carcinoma cell line Eca109 was detected by semi-quantitative reverse transcription polymerase chain reaction. Eca109 cells were stably transfected with S1P5-EGFP or control-EGFP constructs. The relation between the responses of cell proliferation and migration to S1P and S1P5 expression was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and migration assay, respectively. RESULTS: Both normal human esophageal mucosal epithelium and Eca109 cells expressed S1P1, S1P2, S1P3 and S1P5, respectively. Esophageal mucosal epithelium expressed S1P5 at a higher level than Eca109 cell line. S1P5 over-expressing Eca109 cells displayed spindle cell morphology with elongated and extended filopodia-like projections. The proliferation response of S1P5-transfected Eca109 cells was lower than that of control vector-transfected cells with or without S1P stimulation (P < 0.05 or 0.01). S1P significantly inhibited the migration of S1P5-transfected Eca109 cells (P < 0.001). However, without S1P in transwell lower chamber, the number of migrated S1P5-transfected Eca109 cells was greater than that of control vector-transfected Eca109 cells (P < 0.001). CONCLUSION: S1P binding to S1P5 inhibits the proliferation and migration of S1P5-transfected Eca109 cells. Esophageal cancer cells may down-regulate the expression of S1P5 to escape the inhibitory effect.

A comparison of different electrostatic potentials on prediction accuracy in CoMFA and CoMSIA studies.

Computational chemistry is playing an increasingly important role in drug design and discovery, structural biology, and quantitative structure-activity relationship (QSAR) studies. For QSAR work, selecting an appropriate and accurate method to assign the electrostatic potentials of each atom in a molecule is a critical first step. So far several commonly used methods are available to assign charges. However, no systematic comparison of the effects of electrostatic potentials on QSAR quality has been made. In this study, twelve semi-empirical and empirical charge-assigning methods, AM1, AM1-BCC, CFF, Del-Re, Formal, Gasteiger, Gasteiger-Hückel, Hückel, MMFF, PRODRG, Pullman, and VC2003 charges, have been compared for their performances in CoMFA and CoMSIA modeling using several standard datasets. Some charge assignment models, such as Del-Re, PRODRG, and Pullman, are limited to specific atom and bond types, and, therefore, were excluded from this study. Among the remaining nine methods, the Gasteiger-Hückel charge, though commonly used, performed poorly in prediction accuracy. The AM1-BCC method was better than most charge-assigning methods based on prediction accuracy, though it was not successful in yielding overall higher cross-validation correlation coefficient (q(2)) values than others. The CFF charge model worked the best in prediction accuracy when q(2) was used as the evaluation criterion. The results presented should help the selection of electrostatic potential models in CoMFA and CoMSIA studies.