As internal protein elements , inteins are self-excised from their host protein and catalyze ligation of exteins with a peptide bond .They are widely found in all the three domains of the biological kingdom , and in viral proteins .Intein-mediated protein splicing is a posttranslational autocatalytic process that does not require auxiliary enzymes or cofactors . Protein splicing involves four intramolecular reactions and a small number of key catalytic residues in the intein and exteins . The development of expressed protein ligation ( EPL ) and protein trans-splicing ( PTS ) is assisted by inteins and protein splicing.This review introduces the biosynthesis of backbone-cyclized peptides libraries , and describes the applications of cyclic polypeptides and their prospective applications in the future .

Estrogen is involved in the process of postmenopausal osteoporosis, and the main functional cells are osteoclasts. However, the mechanism of estrogen on the osteoclasts is not yet fully understood. Here we demonstrate the effects of estrogen on osteoclasts in three aspects: morphology and structure, apoptosis of osteoclasts and differentiation of preosteoclasts, which give an overall explanation for effects of estrogen on osteoclasts.

Objective To investigate the clinical efficacy of radiofrequency surgical therapy combined with ozone for patients with discogenic low back pain. Methods The clinical data were collected in 120 patients treated with radiofrequen?cy and ozone for lumbar disc herniation at Tianjin Hospital from October 2013 to October 2015. Pain visual analogue scale (VAS) was used to patients at preoperative and postoperative 1 week, 1, 3, 6 and 12 months. Health survey (SF-36) score was used at preoperative and postoperative six months. The efficacy was evaluated by MacNab curative effect evaluation. Re?sults The preoperative VAS score was (7.02±0.64) points. The postoperative VAS scores were (3.13±0.32) points, (2.11± 0.67) points,(2.62±0.89) points,(2.37±0.34) points and (2.31±0.50) points at one week, one month, three months, six months and twelve months, respectively. The VAS scores were significantly decreased after surgery (P<0.05). Preoperative SF-36 score was (48.32 ± 7.46) points, which reached to the (82.03 ± 5.89) points six months after surgery (P < 0.05). After six months, the fineness rate reached to 89.17%evaluated by MacNab curative effect evaluation. Conclusion Radiofrequency combined with ozone treatment is an effective and reliable method for discogenic low back pain.

Objective To explore the angiogenesis&neovascularization effects of naringin treatment in ovariectomized rats’fracture healing. Methods Upper 1/3 transverse tibial fracture model 4 weeks later after ovariectomized were estimated and randomly divided into the naringin group and control group. Microfil perfusion technique was used to analysis the angiogene?sis situation at two weeks after bone fracture. HE staining was used to evaluate the level of angiogenesis&neovascularization of tis?sue from histological point of view. The relative expression of VEGF in the callus was identified by real?time polymerase chain re?action. Immunohistochemical technique was used to observe the vessel endothelial growth factor?2 in the callus of the two groups. Maximum fracture load was tested by three?point bend test. Results The vascular volume and vascular density were more in nar?ingin group than control group. The HE staining of the 2 week group slices shows that the VA, VN2 of the unit of high magnifica?tion vision of the naringin group was significantly larger compared to the control group. Real?time PCR revealed that the compara?tive expression of VEGF is more in naringin group than in control group; the positive number of VEGFR?2 is more in naringin group than in control group. Naringin can promote the maximum load of the callus. Conclusion Naringin can promote ovariecto?mized rats’angiogenesis&neovascularization in the early process of fracture healing. It may be act on the signaling pathway of VEGF/VEGFR?2.

It′s reported that RNA-binding proteins ( RBP) play key roles in post-transcriptional regulation of eukaryotic genes.The aberrations of RBP are associated with a large number of human disorders , particularly autoimmune and neuro-logic diseases .The interaction between RNA and proteins has been widely explored since the development of the method known as RNA immunoprecipitation with differential display or microarray analysis (RIP-ChIP) around the year of 2000. Since then, diverse derivatives of the RIP-ChIP, such as ultraviolet crosslinking and immunoprecipitation ( CLIP), high-throughput sequencing of CLIP cDNA library (HITS-CLIP), photoactivatable -ribonucleoside-enhanced crosslinking and immunoprecipitation ( PAR-CLIP) , and individual nucleotide resolution CLIP ( iCLIP ) have been developed .All these methods have some advantages over the original RIP-ChIP and greatly facilitate the study of RBP-RNA interactions .Addi-tionally , aided by the next-generation sequencing , transcriptome-wide identification of RBP target sites has become possible and the RNA-binding site resolution of RBP has also improved to some degree .We introduced the basic principles and processes of the interactions between proteins and RNA , focusing on the advantages , disadvantages and prospect of the present genome-wide version of CLIP .

Background Atrial electrical remodeling(AER)plays an important role in the pathogenesis and maintenance of atrialfibrillation.However,little is known about modulation of vagal activilty to AER.This study aimed to investigate the relationshipbetween vagal moduation and AER. Methods Twenty four adult mongrel dogs under general anesthesia were randomized into 3groups.Sympathetic activity was blocked by administration of metoprolol in 3 groups.The changes in vagal modulation to atria afterAER were observed in 10 dogs without vagal interruption in group A.The effects of vagal intervention on AER were investigated in 8dogs with administration of atropine in group B.The impact of aggressively vagal activity on AER was studied in 6 dogs with bilateralcervical vag sympathetic trunLks stimulation during AER in group C.Bilateral cervicall vagosympathetic trunks were decentralized.Multipolar catheters wereplaced into high right atria(RA),coronary sinus(CS)and rightventricle(RV).AER was induced by 600 bpmpacing through RA catheter for 30 minutes.Attial effective refractory period(ERP)and vulnerability window (VW)of atrial fibrillationwere measured with and without vagal stimulation before and after AER.Results In group A,ERP decreased significantly at baselineand during vagal stimulation after AER compared with that beforeAER(all P＜0.05).In group B,ERP remaind unchanged at baselineand vagal stimulation after AER compared with tbat before AER (all P＞0.05).In group C,ERP shortened significantly at baseline andvagal stimulation after AER compared with that before AER(all P＜0.05).ERP shortening after AER in Groups A and C increasedsignificantly than that in group B (all P＜0.05).Atrial fibrillation could not be induced at baseline(VW close to 0) before and after AERin three groups.VW became widen significantly during vagal stimulation after AER compared with that before AER in Groups A and C(all P＜0.05),while VW remained unchanged in group B (VW close to 0).Conclusions Short-term AER results in the decrease inERP.AER is accompanied by the increases in atrial vagal modulation.The increased vagal activity and vagal stimulation promote AER,thereby increase the susceptibility to atrial fibrillation.The interrupted vagal activity attenuates AER.thereby suppresses the atriaIfibrillation mediated by vagal stimutlation.

Objective To design and construct a new non-fusion soluble expression vector pTIG-mSUMO(small ubiq-uitin-related modifier) using the widely used solubility promoting protein SUMO and based on the translational coupling phenomenon in order to enable the non-fusion soluble expression of the broad-spectrum antiviral protein RA in Escherichia coli by pTIG-mSUMO.Methods The smt3 gene coding for SUMO protein was cloned from yeast genome DNA by PCR. After directed-site silent mutation to eliminate the EcoRⅠsite, the mutant mSUMO was inserted into pET-22b to obtain the translational coupling expression vector pTIG-mSUMO.The RA was subject to PCR amplification and cloned into the pTIG-mSUMO to obtain the expression plasmid pTIG-mSUMO/RA which was supposed to direct the soluble expression of RA by the translational coupling with mSUMO.Results A translational coupling expression vector pTIG-mSUMO which could di-rect/drive the SUMO and heterogonous protein non-fusion expression simultaneously was designed and constructed.The Western blotting result indicated that pTIG-mSUMO could direct the high-level expression of RA, around 40%of which was soluble.Conclusion A translational coupling expression vector pTIG-mSUMO is obtained.After coupling with SUMO, RA is highly expressed in E.coli and both the expression level and solubility are greatly improved.pTIG-mSUMO might contrib-ute to soluble expression of other proteins.

We reconstructed the erythromycin macrocyclic lactone (6-deoxyerythronolide B, 6dEB) synthesis pathway in Escherichia coli. We first cloned all the genes needed to synthesize the 6dEB into multi-gene co-expressed vectors. Then using the recognition sequences of isoschizomers Xba I/Spe I of vectors, we assembled the related genes into a series multiple-genes recombinant plasmids pBJ144, pBJ130. The recombinant plasmids pBJ144, pBJ130 were cotransformed into BAP1 to get the recombinant BAP1(pBJ144/pBJ130). SDS-PAGE analysis showed that individual genes were expressed correctly. After inducing at low temperature, adding propionate as substrate, we validated the crude product by mass spectrometry and the 6dEB yield was about 10 mg/L. These results indicated that the synthetic pathway of 6dEB was successfully assembled and reconstructed in Escherichia coli, which will greatly facilitate the reconstruction of whole erythromycin synthesis pathway and finally help to establish a stable research platform for developing of new derivatives of erythromycin and combinatorial biosynthesis of polyketide-type antibiotics.
Anti-Bacterial Agents ; biosynthesis ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Erythromycin ; analogs & derivatives ; biosynthesis ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics

Osteochondral defects caused by various factors are still difficult clinical problems. With the development of tissue engineering, the strategies and methods for repairing osteochondral defects in the past decade have made great progress, and some osteochondral tissue stratified stent products have gradually entered the clinical trial stage.. The related articles on tissue engineering for the treatment of osteochondral defects were retrieved by searching databases with key words osteochondral defects, cartilage repair and hierarchical scaffolds. This paper discussed the research status of hierarchical scaffolds in osteochondral tissue engineering during recent five years. In this work, the classification of hierarchical scaffold including monophasic scaffolds, bilayered scaffolds, multilayered scaffolds and gradient scaffolds, are summarized by comparing different experiment researches. Furthermore, the advantages and disadvantages of different types of hierarchical scaffolds were introduced through analyzing relevant studies. Monophasic scaffolds can support the adhesion and proliferation of osteoblasts and chondrocytes, but lack the inherent stratified structure features required for osteochondral regeneration.. Bilayered scaffolds consist of a chondral layer and subchondral layer which base on the biocompatibility of monophasic scaffolds. Biphasic scaffolds are significantly better than monophasic scaffolds in simulating natural cartilage, but the interface between chondral and subchondral layer is poor binding. Compared with bilayered scaffold, trilayered scaffolds are added with an intermediate layer which simulates the calcification of normal cartilage between the two layers, so as to obtain better connection of the bone and cartilage layer. Unlike hierarchical scaffolds, gradient scaffolds provide a gradient connection between the layers, which is more similar to the native osteochondral tissue. In the past five years, the development of osteochondral layered scaffolds mainly depended on the novel structure and fabrication methods of scaffolds. However, correlational clinical studies are quite few. Further high quality and large clinical studies are still required.

OBJECTIVE: To observe the conditions of sub-chronic crotonaldehyde exposure-induced pulmonary inflammation,oxidative stress and apoptosis in male rats,and to explore the related mechanisms. METHODS: The specific pathogen free Wistar male rats were randomly divided into control group,low-,medium-and high-dose crotonaldehyde groups,with 10 rats in each group. Rats were treated with crotonaldehyde at the concentrations of 0. 0,2. 5,4. 5 and 8. 5 mg/kg body weight by intra-gastric administration,once per day for 120 consecutive days. After the end of treatment,rats were sacrificed,the lungs were weighed and histopathological examination was performed. The levels of malondialdehyde( MDA),superoxide dismutase( SOD) and glutathione peroxidase( GSH-Px) in the serum of rats were determined by colorimetry. The relative expression of reactive oxygen species in lung tissues was detected by fluorescence probe. The apoptosis rate was detected by Tunel staining. The relative expression of B-cell lymphoma( Bcl)-2,Bcl-2 associated X protein( Bax) and cysteine aspartic acid protease-3( Caspase-3) proteins in lung tissue was detected by western blotting.RESULTS: The body weight of the rats in the high-dose group began to decrease after 30 days of exposure( P < 0. 05),and the body weight in the low-and medium-dose groups began to decrease at 90 days exposure( P < 0. 05),when compared with that of the control group at the same time. The body weight of the high-dose group was lower than that of the low-and medium-dose groups began to decrease at 90 days exposure( P < 0. 05). After exposure,the lung tissue of the three doses groups showed different degrees of inflammatory change in a dose-effect relationship. The level of serum MDA in rats increased with the treatment of crotonaldehyde( P < 0. 01). The activities of serum SOD and GSH-Px decreased with the treatment of crotonaldehyde( P < 0. 01). The relative expression of ROS and apoptosis rate in rat lung tissue increased with the treatment of crotonaldehyde( P < 0. 01). The relative expression of Bcl-2 protein and the ratio of Bcl-2/Bax in the lung tissue of rats decreased with the treatment of crotonaldehyde( P < 0. 01). The relative protein expression of Bax and Caspase-3 increased with the treatment of crotonaldehyde( P < 0. 01). CONCLUSION: Crotonaldehyde sub-chronic exposure can cause apoptosis in lung tissue by altering the oxidative balance,leading to inflammatory pathological changes in the lung.