In silico acquirement of the accurate residue details of protein on chromatographic media is a bottleneck in protein chromatography separation and purification. Here we developed a novel approach by coupling with H/D exchange and nuclear magnetic resonance to observe hen egg white lysozyme (HEWL) unfolding behavior adsorbed on cation exchange media (SP Sepharose FF). Analysis of 1D 1H-NMR shows that protein unfolding accelerated H/D exchange rate, leading to more loss of signal of amide hydrogen owing to exposure of residues and the more unfolding of protein. Analysis of two-dimensional hydrogen-hydrogen total correlation spectroscopy shows that lysozyme lost more signals and experienced great unfolding during its adsorption on media surface. However, for several distinct fragments, the protection degrees varied, the adsorbed lysozyme lost more signal intensity and was less protected at disorder structures (coil, bend, and turn), but was comparatively more protected against exchange at secondary structure domains (α-helix, β-sheet). Finally, the binding site was determined by electrostatic calculations using computer simulation methods in conjunction with hydrogen deuterium labeled protein and NMR. This study would help deeply understand the microscopic mechanism of protein chromatography and guide the purposely design of chromatographic process and media. Moreover, it also provide an effective tool to study the protein and biomaterials interaction in other applications.
Adsorption ; Amides ; Cations ; Computer Simulation ; Deuterium ; Hydrogen ; Magnetic Resonance Spectroscopy ; Muramidase ; chemistry ; Protein Structure, Secondary ; Protein Unfolding ; Proteins ; chemistry

BACKGROUND: Whether one-stage bone healing with red bone marrow or mesenchymal stem cell transplantation is needed? Whether closed reduction with intramedullary interlocking nailing combined with autologous red bone marrow transplantation can promote osteogenesis in the one-stage treatment of femoral comminuted fracture still remains unclear. OBJECTIVE: To explore the effects of closed reduction with intramedullary interlocking nailing and one-stage treatment with autologous red bone marrow transplantation on fracture healing. METHODS: Twenty healthy New Zealand rabbits were selected and anesthetized, and then the models of femoral comminuted fracture were established at the hinder limbs after throwing a 5 kg of hammer from 30 cm height. All rabbits underwent intramedullary fixation using Kirschner wire (diameter: 2 mm); the right limbs served as control groups, and the left limbs as transplantation group, subjected to the injection of autologous red bone marrow (1 mL) into the fracture region. Then the rabbits were respectively killed at 14, and 28 days after modeling, the thickness of thickest callus was measured on X-ray films, and the number of chondrocytes and relative area of bone trabecula in the fracture region were observed through hematoxylin-eosin staining under light microscope.RESULTS AND CONCLUSION: (1) The thickness of callus formation in the control group was significantly less than that in the transplantation group at 14 days after modeling (P < 0.01). (2) The number of chondrocytes in the transplantation group was significantly more than that in the control group at 14 days after modeling (P < 0.01),and the fracture healing in the transplantation group was faster that in the control group. (3) Compared with the transplantation group, the relative area bone trabecula in the fracture region in the control group was significantly reduced at 28 days after modeling (P < 0.01). (4) These finding indicate that red bone marrow transplantation promotes fracture healing in the intramedullary fixation for femoral comminuted fracture in rabbits.

A method was developed for analyzing the stable carbon isotope ratio of five volatile components ( Ethanol, Glycerol, Acetic acid, Ethyl lactate, 2-methyl-butanol ) in wine using gas chromatography-combustion-isotope ratio mass spectrometer ( GC-C-IRMS ) . The sample injection volume was less than 0. 5 μL, and the analytical time of each run was less than 14 min. The precision of this method was 0. 08‰-0. 25‰ for analyzing standards, while 0. 09‰-0. 36‰ for wine samples. Compared to element analysis-isotope ratio mass spectrometry ( EA-IRMS) results, the deviations were lower than 0. 5‰. Fifty-four wine samples from France, Australia, America and China were considered. The δ13 C of five volatile components were measured using GC-C-IRMS. Discriminant analysis ( DA) was employed for analyzing the geographical origin traceability of selected wine. The result indicated that δ13 C of volatile components could be used to distinguish the origin of wines. The method was shown to be effective in improving detection of the origin traceability of wine.

BACKGROUND:Along with the widespread application of biodegradable materials in the field of medicine and the in-depth research of biomechanics,the drawbacks of traditional medical metal materials are increasingly appearing.In recent years,researchers at home and abroad focus on biodegradable materials that are represented by high molecular polymer to seek new breakthroughs in the field of spinal instability.OBJECTIVE:To investigate biomechanical changes of polylactic acid-polyglycolic acid (PLGA) lumbar interbody fusion cage in the body and discusses its feasibility for treating segmental instability of the spine.METHODS:Forty-two healthy pigs (9 months old) were randomly divided into two groups (n=21),and L4/5 intervertebral disc nucleus pulposus was removed in all animals.In experimental group,PLGA lumbar interbody fusion cage filled with broken bone was implanted;and in control group,autologous bone was implanted.X-ray was performed to observe the fusion of operation segments at 4,12 and 72 weeks postoperatively.Feasibility of fibrous fusion was measured by biomechanical test.Histologically,bone graft fusion at the surgical site and material degradation were detected.RESULTS AND CONCLUSION:(1) Imaging examination:Bone graft fusion in two groups was not visible at 4 weeks after operation.Evidence of increasing fusion was found in the experimental group at 12 weeks after operation;a visible part of the bone bridge was found in the control group,in which there was one case of fusion.Degradation of the fusion cage with one case of fusion in experimental group was found after 72 weeks after operation,and two cases of fusion in the control group.(2) Biomechanical test:There was no difference in the spinal range of motion between the two groups in different states at 4 weeks after operation (P ＞ 0.05).The spinal range values of motion at most of the states at 72 weeks after operation were significantly lower than those at 4 weeks after operation.(3) Cell histology observation:With the passage of time,the materials in the experimental group degraded gradually;new bone grew slowly and then fast,with bone fusion step by step.Fusion results were similar in the two groups.Our experimental findings indicate that the PLGA lumbar fusion cage has good biocompatibility.In addition to the individual state (left flexion),the mechanical properties of the fusion cage are similar to that of autogenous bone,and the fusion cage enables the segmental reconstruction of the pig spine to the maximum extent.

BACKGROUND: Hepatic oval cells (HOCs) possess the potential of self-renewal, replication, and clone, proliferation and differentiation into mature hepatocytes under a certain condition. HOCs can be used as biomaterial for constructing biological artificial liver in vitro, employed for in vivo transplantation, as well as for tissue engineering as seed cells. HOCs can be widely used for improving clinical treatment of liver diseases. OBJECTIVE: To establish adult Wistar rat models of HOC proliferation, to perform/n vitro isolation and culture of HOCs, and to study the possibility of induction and differentiation of HOCs into hepatucytes. DESIGN: Observational study. SETTING: Institute of Gastroenterology, Nanfang Hospital, Southern Medical University. MATERIALS: Experiments were performed at the Laboratory of Institute of Gastroenterology of Nanfang Hospital from December 2003 to February 2006. Thirty-six healthy male Wistar rats aged 3-4 months (150-200 g) were provided by Experimental Animal Center of Southern Medical University. METHODS: Male Wistar rats were orally fed with ethionine received two-thirds partial hepatectomy (2/3 PH). HOCs were harvested and purified by two-steps perfusion and Percoll density gradient centrifugation, and then cultured in vitro and induced with hepatocyte growth factor (HGF), oncostatin M (OSM) and fibroblast growth factor-4 (FGF4). MAIN OUTCOME MEASURES: Identification and differentiation of HOCs. RESULTS: The concentration of HOCs was about 1.34×108 L-1 in each rat model after in vitro isolation. These cells were round, oval or polygon, about 1/6 1/3 the size of normal hepatocytes. The nucleus-cytoplasm ratio was relatively large. After 2 weeks, clone-like proliferation of HOCs could be observed. Laser scanning confocal microscopy indicated positive expression of stem cells markers Thy-1 and C-kit in cytoplasm and membrane of HOCs. Immunocytochemistry demonstrated positive stem cells marker alpha fetoprotein (AFP) in cytoplasm of HOCs. HOCs can stably passage and its shape gradually changed after inducing with HGF, OSM and FGF4. HOC volume became larger and HOCs lost their ability of sticking to the wall of culture flask. Apparent positive stain of cytoplasm albumin (Alb) was detected 14 days after induction, and the positive ratio increased along with the extension of inducing duration. Results of cytochemistry indicated a brown or black deposit after glucose-6-phosphotase (G-6-P) staining and red particles after periodic acid-Schiff (PAS) staining. CONCLUSION: Adult Wistar rat models of HOC proliferation are replicated by ethionine feeding combined with 2/3 PH. HOCs can be obtained through collagenase perfusion and Percoll density gradient centrifugation. Rat HOCs can be passaged and cultured in vitro. Under a certain condition, HOCs can be induced and differentiated into hepatocytes.

BACKGROUND:Stromal cellderived factor 1 in chemotactic migration of endogenous neural stem cells plays a very important role, but the specific migration mechanism is unclear
OBJECTIVE:To observe the effects of exogenous stromal cellderived factor 1 on chemotactic migration and proliferation of neural stem cells in the rat hippocampus
METHODS:Exogenous stromal cellderived factor 1 (5μL, 500μ/L) was injected into the hippocampus of Sprague-Dawley rats to establish animal models. Brain tissues were taken after days 3, 7, 14 and 21 of perfusion to prepare paraffin sections. Thereafter, nestin expression in the injection region and hippocampus was detected using immunohistochemical method. Experimental control and blank control groups were set.
RESULTS AND CONCLUSION:Paraffin section immunohistochemical results displayed the number of nestin-positive cells in the injection and the hippocampus was gradual y increased. At 3 and 7 days, nestin expression was a little and increased at 14 days, forming a migration tendency to the injection region. At 21 days, there were more nestin-positive cells in the injection area and hippocampus. However, there were no changes as above in the experimental control and blank control groups. The results showed that exogenous stromal cellderived factor 1 may induce the proliferation of neural stem cells in the hippocampus and may be involved in chemotactic migration of endogenous neural stem cells.

OBJECTIVETo investigate the mechanism by which exendin-4 promotes paracrine secretion of cytokines by adipose-derived stem cells (ADSCs).

METHODSIn vitro cultured SD rat ADSCs (fourth passage) with or without exendin-4 treatment underwent flow cytometry to characterize the surface markers. MTT assay was performed to assess the proliferation of the cells exposed to different concentrations (0-20 nm/L) of exendin-4, and the paracrine secretion of cytokines (bFGF, VEGF, HGF, and IGF-1) by the ADSCs was evaluated by qPCR. The changes in the expressions of p-Akt in the cells were analyzed by Western blotting and qPCR in response to exendin-4 (10 nm/L) with or without exposure to PI3K/Akt inhibitor LY-294002 (50 nm/L); bFGF, VEGF, HGF, and IGF-1 production in the cells were detected using ELISA kits.

RESULTSTreatment with exendin-4 for 12 h did not affect the surface marker profile of the ADSCs but promoted the cell proliferation (P<0.05). Exendin-4 significantly increased the mRNA expressions of VEGF, bFGF, HGF, and IGF-1 in a concentration-dependent manner, and 10 nm/L was the optimum concentration (P<0.05). Exendin-4 treatment resulted in significantly increased p-Akt expressions in the ADSCs, and PI3K/Akt inhibitor not only reversed such effects of exendin-4 on p-Akt but also diminished the exendin-4- mediated up-regulation of the paracrine cytokines.

CONCLUSIONExendin-4 can concentration-dependently promote the proliferative and paracrine capacities of ADSCs partially through the PI3K/Akt signaling pathway without affecting the surface marker profile of the cells.

Adipocytes ; cytology ; Animals ; Cell Proliferation ; Cells, Cultured ; Chromones ; Fibroblast Growth Factor 2 ; metabolism ; Hepatocyte Growth Factor ; metabolism ; Insulin-Like Growth Factor I ; metabolism ; Morpholines ; Peptides ; pharmacology ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Stem Cells ; cytology ; Up-Regulation ; Vascular Endothelial Growth Factor A ; metabolism ; Venoms ; pharmacology

Objective To analyze the NBS-LRR disease resistance gene family of Codonopsis pilosula(Franch.)Nannf.and explore the disease resistance mechanism,so as to solve the problem of root rot disease of C.pilosula and promote the breeding and industrial development of C.pilosula.Methods Based on the transcriptome data of C.pilosula in response to root rot pathogen,gene structure,phylogeny,gene interaction and expression pattern of C.pilosula NBS-LRR family genes were analyzed by using bioinformatics methods.Results 88 NBS-LRR family genes were successfully identified.Including N,NL,CN,CNL,TN,TNL and PN,there are 50,14,1,14,4,3 and 2 genes respectively.Their physical and chemical properties,gene structure,phylogeny,gene interaction and expression pattern were analyzed.The results showed that CNL subfamily genes of C.pilosula were amplified during evolution;CNL and TNL gene structures ar-relatively conservative;Expression pattern analysis showed that there were differences in temporal expression patterns of C.pilosula NBS-LRR family genes under F.oxysporum infection.The highly expressed genes DN64786c1g6,DN64786c1g5,DN48234c0g2,DN54844c1g2,DN59747c0g3,DN56071c1g8,DN64591c1g1,DN48464c1g1,DN59886c0g1 play an important role in regulating the disease resistance of C.pilosula.The DN54844c1g2 may interact with GLR family,and then participate in immune regulation by regulating Ca2+ influx;DN64786c1g5 may interact with cytc-1 and cytc-2,and then participate in the response of C.pilosula to root rot by participating in redox reaction;DN59747c0g3 may interact with MPK3 and play an important role in the response of C.pilosula to root rot by participating in map signal cascade,phosphorylating WRKY transcription factor and participating in hypersensitivity reaction(HR).Conclusion The identification and expression analysis of NBS-LRR family genes of C.pilosula is of great significance to explore the mechanism of root rot resistance and gene function of C.pilosula..

OBJECTIVETo evaluate the laparoscopy combined with transperineal extralevator abdominoperineal excision (TP-ELAPE) for locally advanced low rectal caner.

METHODSClinical data of 12 patients with locally advanced low rectal cancer undergoing laparoscopy combined with TP-ELAPE in our department from May 2013 to March 2015 were retrospectively analyzed. There were 8 male and 4 female patients with median aged of 63 (46 to 72) years. The median distance from tumor lower margin to anal verge was 3.5(2.0 to 4.0) cm. A self-made transanal suit for minimally invasive operation was used to make a sealed lacuna outside the sphincter, thus laparoscope can be applied to perform transperineal operation.

RESULTSAll the patients underwent operations successfully without conversion to open abdominal operation. The median operating time was 206 (180 to 280) minutes with perineal operating time 95(80 to 120) minutes. The median intraoperative blood loss was 120(50 to 200) ml. The median postoperative hospital stay was 12(9 to 18 ) days. Postoperative pathology revealed that all circumferential margins (CRM) were negative. The area of sample horizontal section was (2 824±463) mm(2), and of outer muscularis propria was(2 190±476) mm(2). Postoperative complications included chronic sacrococcygeal region pain in 2 cases, urinary retention in 3 cases, perineal wound infection in 1 case. No perineal seroma, perineal hernia, wound dehiscence and sinus tract formation were observed. Among 8 patients with preoperative normal sexual function, sexual dysfunction occurred in 2 patients. There was no local recurrence and metastasis during a median follow-up of 21(12 to 34) months.

CONCLUSIONLaparoscopy combined with TP-ELAPE has the potential to simplify the operation procedure for low rectal cancer, can ensure the radical treatment and safety of operation, and may be carried out in experienced centers.

Abdomen ; Aged ; Anal Canal ; Blood Loss, Surgical ; Digestive System Surgical Procedures ; methods ; Female ; Humans ; Laparoscopy ; Length of Stay ; Male ; Middle Aged ; Neoplasm Recurrence, Local ; Operative Time ; Perineum ; Postoperative Complications ; Postoperative Period ; Rectal Neoplasms ; surgery ; Rectum ; Retrospective Studies

Objective To study the effect and mechanism of Momordica anti-HIV protein of 30 ku (MAP30) on the migration of bladder cancer.Methods The IC50 of human bladder cancer 5637 and T24 cells was calculated by methyl thiazolyl tetrazolium (MTT) method.The migration ability of these two cells was evaluated by scratch migration test and Transwell cell migration test.The expression of migrating proteins such as matrix metalloproteinases (MMPs) and adhesion molecule N-cadherin were compared by Western blot.Results Scratch migration test:there were significant differences in migration rates of 5637 cells at 8 h and 22 h (P ＜ 0.05).There were significant differences in migration rates of T24 cells at 22 h (P ＜ 0.05),but no significant differences in migration rates at 8 h (P ＞ 0.05).The expression of Vimentin,Fibronectin,MMP-2,MMP-9 and N-Cadherin in 5637 cells and T24 cells of human bladder cancer decreased significantly after adding MAP30.The E-Cadherin expression in human bladder cancer 5637 cells were decreased,but no target band was detected in human bladder cancer T24 cells.Conclusions The ribosome-inactivating protein MAP30 can effectively inhibit the migration of human bladder cancer 5637 and T24 cells by inhibiting the EMT pathway and inhibiting the expression of MMPs.