Overexpression of miRNA-433-5p protects acute spinal cord injury through activating MAPK1.

OBJECTIVE: The aim of this study was to clarify the role of microRNA-433-5p (miRNA-433-5p) in influencing pathological lesions following acute spinal cord injury (SCI) by targeting mitogen-activated protein kinase 1 (MAPK1). PATIENTS AND METHODS: SCI model was successfully established in mice by performing hitting injury procedures. Serum levels of miRNA-433-5p and MAPK1 in SCI patients and mice were determined. Grip strengths of both forelimbs in SCI mice and controls were determined. Dual-Luciferase reporter gene assay was applied to verify the binding relation between miRNA-433-5p and MAPK1. After overexpression of miRNA-433-5p and MAPK1 in vivo, the grip strength changes in SCI mice were assessed. Furthermore, the protein level of inflammatory factor iNOS in 293T cells influenced by miRNA-433-5p and MAPK1 was detected by Western blot. RESULTS: MiRNA-433-5p was significantly downregulated in the serum of SCI patients and mice, whereas MAPK1 was up-regulated. Grip strengths of SCI mice were significantly lower than those of controls at different postoperative time points. However, this could be markedly reversed by the in vivo overexpression of miRNA-433-5p. Western blot indicated that the protein level of iNOS was remarkably downregulated in 293T cells overexpressing miRNA-433-5p. MAPK1 was confirmed as the target of miRNA-433-5p, whose expression level was negatively regulated by miRNA-433-5p. Importantly, MAPK1 partially reversed the protective role of miRNA-433-5p in grip strength of SCI mice and inflammatory response at post-SCI. CONCLUSIONS: Overexpression of miRNA-433-5p protects SCI-induced motor dysfunction and inflammatory response by targeting MAPK1.

Anomalous phase behavior of first-order fluid-liquid phase transition in phosphorus.

Although the existence of liquid-liquid phase transition has become more and more convincing, whether it will terminate at a critical point and what is the order parameter are still open. To explore these questions, we revisit the fluid-liquid phase transition (FLPT) in phosphorus (P) and study its phase behavior by performing extensive first-principles molecular dynamics simulations. The FLPT observed in experiments is well reproduced, and a fluid-liquid critical point (FLCP) at T = 3000 ∼ 3500 K, P = 1.5-2.0 Kbar is found. With decreasing temperature from the FLCP along the transition line, the density difference (Δρ) between two coexisting phases first increases from zero and then anomalously decreases; however, the entropy difference (ΔS) continuously increases from zero. These features suggest that an order parameter containing contributions from both the density and the entropy is needed to describe the FLPT in P, and at least at low temperatures, the entropy, instead of the density, governs the FLPT.

In vivo study of microarc oxidation coated biodegradable magnesium plate to heal bone fracture defect of 3mm width.

Microarc oxidation (MAO) coated magnesium (Mg) with improved corrosion resistance appeal increasing interests as a revolutionary biodegradable metal for fractured bone fixing implants application. However, the in vivo corrosion degradation of the implants and bone healing response are not well understood, which is highly required in clinic. In the present work, 10µm and 20µm thick biocompatible MAO coatings mainly composed of MgO, Mg2SiO4, CaSiO3 and Mg3(PO4)2 phases were fabricated on AZ31 magnesium alloy. The electrochemical tests indicated an improved corrosion resistance of magnesium by the MAO coatings. The 10µm and 20µm coated and uncoated magnesium plates were separately implanted into the radius bone fracture site of adult New Zealand white rabbits using a 3mm width bone fracture defect model to investigate the magnesium implants degradation and uninhibited bone healing. Taking advantage of the good biocompatibility of the MAO coatings, no adverse effects were detected through the blood test and histological examination. The implantation groups of coated and uncoated magnesium plates were both observed the promoting effect of bone fracture healing compared with the simple fracture group without implant. The releasing Mg2+ by the degradation of implants into the fracture site improved the bone fracture healing, which is attributed to the magnesium promoting CGRP-mediated osteogenic differentiation. Mg degradation and bone fracture healing promoting must be tailored by microarc oxidation coating with different thickness for potential clinic application.

[Long-term outcomes of off-pump coronary artery bypass grafting].

Objective: To investigate the long-term outcomes of off-pump coronary artery bypass grafting (OPCABG). Methods: Clinical data of 1 129 consecutive patients ( 937 males and 192 females) with coronary artery disease receiving OPCABG at Department of Cardiovascular Surgery, Chinese PLA General Hospital between January 2000 and December 2015 was retrospectively analyzed.The age of patients ranged from 29 to 83 years, with a mean age of (62.0±9.6) years. The follow-up data of the patients, including the graft patency and repeated revascularization rate, were analyzed. Results: Of the 1 129 patients analyzed, 1 059 cases (93.8%) were available for follow-up for 29-192 months[with a mean time of (95.6±34.1) months]. The 5-year, 10-year, 15-year and 16-year graft patency rate of arterial graft was 96.1%, 95.4%, 93.7% and 93.2%, respectively. The 5-year, 10-year, 15-year and 16-year graft patency rate of venous graft was 92.8%, 81.4%, 70.9% and 68.3%, respectively. During the follow-up, 69 (6.11%) patients underwent repeated revascularization procedures. Conclusion: OPCABG is safe and effective with a good long-term graft patency rate.

Relationship between structural order and water-like anomalies in metastable liquid silicon: Ab initio molecular dynamics.

The relationship between structural order and water-like anomalies in tetrahedral liquids is still open. Here, first-principle molecular dynamics are performed to study it in metastable liquid Si. It is found that in T-P phase diagram, there indeed exists a structural anomaly region, which encloses density anomaly but not diffusivity anomaly. This is consistent with that of SW Si and BKS SiO2 but different from that of SPC/E water. Two-body excess entropy anomaly can neither capture the diffusivity, structural, and density anomalies, as it can in a two-scale potential fluid. In structural anomaly region, tetrahedrality order qtetra (measuring the extent to which an atom and its four nearest neighbours adopt tetrahedral arrangement) and translational order ttrans (measuring the tendency of two atoms to adopt preferential separation) are not perfectly correlated, which is different from that in SW Si and renders it impossible to use the isotaxis line to quantify the degree of structural order needed for water-like anomalies to occur. Along the isotherm of critical temperature Tc, ttrans/qtetra is approximately linear with pressure. With decreasing pressure along the isotherm below Tc, ttrans/qtetra departs downward from the line, while it is the opposite case above Tc.

Expression of Magnaporthe oryzae genes encoding cysteine-rich proteins secreted during nitrogen starvation and interaction with its host, Oryza sativa.

Previous studies have shown that the blast fungus, Magnaporthe oryzae, may experience nitrogen starvation during infection of its plant host (rice,Oryza sativa). Here, we studied the expression of seven genes encoding cysteine-rich proteins with N-terminal signal peptides during nitrogen limitation and throughout the infection process. Some genes were upregulated to a greater extent in weak pathogenic strains than in strong pathogenic strains when they were cultured in complete media, and the expression of some genes was higher in both weak and strong pathogenic strains cultured in 1/10-N and nitrogen starvation media. Furthermore, the expression of these genes was upregulated to different extents in the early stages of M. oryzae infection. These data demonstrate that the genes of interest are highly expressed in weak and strong pathogenic strains cultured under nitrogen limitation and at the early stage of the infection process. This indicates that cysteine-rich secreted proteins in the blast fungus might be involved in establishing disease in the host and that they are sensitive to nitrogen levels. Thus, their role in sensing nitrogen availability within the host is implied, which provides a basis for further functional identification of these genes and their products during plant infection.

Construction and expression of prokaryotic expression vectors fused with genes of Magnaporthe oryzae effector proteins and mCherry.

The aim of the current study was to investigate the prokaryotic expression of the Magnaporthe oryzae effector genes BAS1 and BAS4 fused to the fluorescent protein mCherry. Based on previous polymorphic analysis of BAS1 and BAS4 in rice blast strains using PCR, blast strains containing the PCR products of BAS1 and BAS4 were selected for liquid culture for total RNA extraction. For PCR analysis, cDNA was selected as a template to amplify the coding region of BAS1 and BAS4, the plasmid pXY201 was selected as template to amplify the mCherry sequence, and the three sequences were cloned into pMD®19-T vectors. Positive recombinant plasmids were digested using two restriction enzymes and the cleaved fragments of BAS1 and mCherry and BAS4 and mCherry were ligated to pGEX-4T-1 vectors and expression was induced using IPTG. The PCR results showed that the sequence sizes of BAS1, BAS4, and mCherry were 348, 309, and 711 bp, respectively, and these were cloned into pMD®19-T vectors. After digestion and gel purification, the fragments of BAS1 and mCherry, BAS4 and mCherry were ligated into pGEX-4T-1 vectors and expressed in Escherichia coli BL21 competent cells. The expressed proteins were approximately 60 kDa, corresponding to their theoretical size. Prokaryotic expression products of BAS1 and BAS4 fused to mCherry were presented in this study, providing a base for constructing prokaryotic expression vectors of pathogen effector genes fused to mCherry, which will contribute to further study of the subcellular localization, function, and protein interactions of these effectors.

Construction of overexpression vectors of Magnaporthe oryzae genes BAS1 and BAS4 fusion to mCherry and screening of overexpression strains.

The aim of this study was to construct overexpression vectors and selecting strains of the Magnaporthe oryzae effectors BAS1 and BAS4. Primer pairs of BAS1, BAS4, and mCherry were designed based on their known nucleotide sequences. The coding sequences of BAS1 and BAS4 were amplified, and the pXY201 plasmid was selected as a template to amplify the mCherry sequence. Fragments of BAS1 and mCherry, and BAS4 and mCherry were ligated into the pCAMBIA1302 vector. The recombinant pCAMBIA-BAS1-mCherry and pCAMBIA-BAS4-mCherry plasmids were transformed into E. coli DH5α competent cells. Transformants were screened by PCR, and plasmids from the positive transformants were extracted by enzymatic digestion to obtain pCAMBIA-BAS1-mCherry and pCAMBIA-BAS4-mCherry. The pCAMBIA-BAS1-mCherry and pCAMBIA-BAS4-mCherry plasmids were transformed into protoplasts of rice blast strains and the transformed strains were screened by PCR using primer pairs against the hygromycin gene. The result showed that the PCR products corresponded with the theoretical sizes. RT-PCR was used to analyze the expression of BAS1 and BAS4 in five transformed strains of BAS1 and BAS4, and the result showed that the higher expression level of the two genes was occurred in five transformant strains comparing to wild-type strain A3467-40 (the strain containing BAS1 and BAS4), but there was no difference among the five overexpression strains. The sporulation and spore germination of transformed strains was higher than in wild type strain, and there was no difference in the germination time. Construction of overexpression vectors and strains of M. oryzae effectors BAS1 and BAS4 provide reference material for other new effectors.

Celecoxib inhibits ß-catenin-dependent survival of the human osteosarcoma MG-63 cell line.

Cyclo-oxygenase (COX)-2 inhibitors may exert antitumour effects through COX-2-independent mechanisms. This study investigated the effects of the COX-2 inhibitor celecoxib on the viability of the human osteosarcoma MG-63 cell line and its ß-catenin signalling pathway. Cell viability and apoptosis were examined in celecoxib-treated cells or after ß-catenin knockdown in vitro. Analyses were performed to detect glycogen synthase kinase (GSK)-3ß, phosphorylated GSK-3ß, ß-catenin, c-Myc and cyclin D1 proteins, and mRNA levels of ß-catenin, c-Myc and CCND1 (encoding cyclin D1). ß-Catenin was shown to be required for MG63 cell survival and celecoxib exerted an inhibitory effect on the viability of cultured MG-63 cells in a time- and dose-dependent manner. ß-Catenin protein decreased in the cytosol and nucleus following celecoxib treatment (from 6 h after initiation of treatment onwards; lowest protein levels were reached at > 72 h). Significant reductions in ß-catenin, c-Myc and CCND1 mRNA were observed. Celecoxib inhibited MG-63 cell viability, possibly by activating GSK-3ß and inhibiting ß-catenin-dependent gene transcription, suggesting a role for celecoxib in osteosarcoma treatment.

Cross-cultural adaptation, reliability and validity of the Chinese version of the Fear Avoidance Beliefs Questionnaire.

The Fear Avoidance Beliefs Questionnaire (FABQ) was translated and cross-culturally adapted for China. Its psychometric properties were then evaluated in Chinese-speaking patients with low-back pain and the scales were tested for internal consistency, reproducibility, ceiling-and-floor effects, construct validity and responsiveness. A total of 15 patients were selected for pre-testing and a further 230 patients completed the FABQ (and other scales) at baseline and 14 days later. A test-retest reliability analysis was carried out on 61 of the 230 patients. The FABQ was found to be easily understood. Explorative factor analysis by principal components analysis, yielded a two-factor model for the FABQ, relating to work and physical activity, and this was confirmed by confirmatory factor analysis using structural equation modelling. The FABQ yielded high values for internal consistency and reproducibility; no ceiling-and-floor effects were detected. Generally, the FABQ scales and baseline variables were weakly correlated. Cohen's effect size was 0.22 and responsiveness was low. It was concluded that the translation and adaptation of the FABQ into Chinese was successful; the scales had acceptable factor structure, internal consistency, test-retest reliability and construct validity.

Transplanted bone morphogenetic protein/poly(lactic-co-glycolic acid) delayed-release microcysts combined with rat micromorselized bone and collagen for bone tissue engineering.

This study was designed to optimize the preparation of delayed-release microcysts containing bone morphogenetic protein 2 (BMP-2) combined with poly(lactic-co-glycolic acid) (PLGA) and to investigate their osteogenic properties when combined with rat autologous micromorselized bone and collagen. Rat autologous micromorselized bone, collagen and BMP-2/PLGA delayed-release microcysts were implanted in various combinations into the rat gluteus maximus muscle sack model. The following post-operative measurements were made: general observations of the implant site, histological observations, osteogenesis measurements and alkaline phosphatase activity. Autologous micromorselized bone combined with collagen and BMP-2/PLGA delayed-release microcysts demonstrated significantly superior osteogenic properties than any of the other combinations of these three components. These findings suggest that micromorselized bone combined with collagen and BMP-2/PLGA delayed-release microcysts could reduce the quantity of BMP-2 and autologous bone required for these procedures, making their use feasible in human bone restoration.

Functional reconstruction of maxilla with pedicled buccal fat pad flap, prefabricated titanium mesh and autologous bone grafts.

The aim of this study was to evaluate the use of pedicled buccal fat pad flap (PBFPF), prefabricated titanium mesh and autologous bone graft in maxillary reconstruction. Seventeen patients with a unilateral class I-III maxillary defect were involved. Preoperatively, a solid model was manufactured based on virtual maxillectomy and reconstruction of the abnormal maxilla. Intraoperatively, PBFPF was applied to repair the soft-tissue defect, serving as nasal lining and the receiving bed for bone grafts. Titanium mesh was prefabricated on the solid model and then, together with bone grafts from iliac crest, fixed to residual bones to reconstruct the hard-tissue defect. Postoperative aesthetic appearance and function were followed up. No exposure of titanium mesh, leakage or oronasal regurgitation occurred. Of the patients with a class I or II defect 91% (10/11) and of those with a class III defect 50% (3/6) gained a good appearance. Fifteen patients were articulate. Eleven patients received dental rehabilitation and had a normal diet. PBFPF with prefabricated titanium mesh and autologous bone grafts is a reliable option for reconstruction of unilateral maxillary defects of class I and II, but this method alone should be used cautiously in defects of class III and beyond.

Microbial transformation of 3-hydroxy-5,6-cyclopropanocholestanes--an alternative route to 6-methylsteroids.

Incubation of 3beta-hydroxy-5,6alpha-cyclopropano-5alpha-cholestane (4), 3beta-hydroxy-5,6beta-cyclopropano-5beta-cholestane (5), and 3beta-hydroxy-5,6alpha-cyclopropano-5alpha-cholest-7-e ne (6) with Mycobacterium sp. (NRRL B-3805) gave a mixture of side chain cleaved 17-keto steroids as the major products in 52, 57, and 69% yields, respectively. Among these 17-keto steroids, the cyclopropyl ring eliminated product, androst-4-ene-3,17-dione (9), was isolated in 6, 4, and 8% yields, respectively. A cyclopropyl ring migration product, 6alpha,7alpha-cyclopropanoandrost-4-ene-3,17-dione (16), was isolated from the incubation mixture of 6 in 4% yield, also 10% yield of 16 was obtained when 5, 6alpha-cyclopropano-5alpha-androst-7-ene-3,17-dione (12) was incubated. The cyclopropyl ring opening and subsequent reduction followed by oxidation of the two major biotransformation products, 5, 6beta-cyclopropano-5beta-androsta-3,17-dione (10) and 5, 6alpha-cyclopropano-5alpha-androsta-3,17-dione (7), gave 6beta- and 6alpha-methylandrost-4-ene-3,17-dione in 60, and 45% yields, respectively.

Microbial transformation of dihydrosarsasapogenin with Mycobacterium sp.

Microbial transformation of sarsasapogenin (1) with Mycobacterium sp. (NRRL B-3805) gave 25(S)-neospirost-4-en-3-one (2) as the sole product in 62% yield. Incubation of dihydrosarsasapogenin (3) led to the isolation of seven products in 0.5 (4), 6.6 (5), 5 (6), 16 (7), 1 (8), 1 (9), and 4.5% (10) yields, respectively, while 15% of 3 was recovered. Among these products, 8 and 9 were C22 steroids, and 10 was a C19 steroid. Isolation of these C19 and C22 steroids indicated that this microorganism is capable of cleaving the ether linkage between C-16 and C-22 in 3. In addition, 12 alpha-hydroxylation was also observed in all these three metabolites.

Consecutive reaction kinetics involving distributed fraction of methanogens in fluidized-bed bioreactors.

A kinetic model involving the distributed fractions of acidogens and methanogens is proposed. To determine the fluxes and biochemical reaction rates of the substrate sucrose and its intermediates, volatile fatty acids (VFAs) in bulk liquid and within the biofilm, a kinetic model was developed by combining the solid-phase model with the liquid-phase model. The predicted substrate removal efficiencies of the conventional and tapered fluidized-bed bioreactors (CFB, TFBs) are in good agreement with the experimental results. The biofilm thickness in TFBs are thicker than that in CFB, resulting in performance enhancement with TFBs. The simulated results obtained from the kinetic model show that methanogenesis is the rate-limiting step of degradation of the simple organic compound (sucrose), and the chemical oxygen demand (COD) concentration in the effluent is mainly contributed by the intermediates VFAs. The distributed fractions of acidogens and methanogens determined experimentally are 0.4 and 0.6, respectively.

[Therapeutic effect of carboplatin and etoposide combinative therapy on 123 lung cancer cases].

The clinical effect of Carboplatin and Etoposide (CE regimen) on 58 cases of small cell lung cancer (SCLC) and 65 cases of non small cell lung cancer (NSCLC) was observed, and its influence on immunity was investigated simultaneously. The result showed that the overall response rate of SCLC group was 72.4% (42/58). Among them, the response rate of primary treatment was 81.0% (31/38), and that of secondary treatment was 30.0% In NSCLC group, the response rate of primary treatment was 30.0% (6/20). In NSCLS group, the rate of primary treatment was 22.9% (8/35), and that of secondary treatment was 13.3% (4/30). From the result it was found that the response rate of primary treatment was higher than that of secondary treatment, and that the main side-effect of CE regimen was mild leukopenia, and the other side-effects included gastrointestinal symptoms, alopecia and mild hepatic function changes. The influence of CE regimen on immunity was temporary, and will soon recover. Repeating the regimen is reasonable. So we think that CE regimen is one of the best regimens for SCLC patients.