Expression and prognostic relevance of centromere protein A in primary osteosarcoma.

Centromere protein A (CENP-A) is one of the fundamental components of the human active kinetochore and plays important roles in cell-cycle regulation, cell survival, and genetic stability. The aim of the present study was to explore the expression and prognostic significance of CENP-A in osteosarcoma. The results of real-time quantitative PCR and Western blotting analysis revealed an enhanced expression of CENP-A in osteosarcomas relative to adjacent non-tumorous bone tissues at both mRNA and protein levels. Immunohistochemically, 72 of the 123 osteosarcoma specimens (58.5%) had high expression of CENP-A. CENP-A overexpression was significantly correlated with tumor size (P=0.002), poor response to neoadjuvant chemotherapy (P=0.016), local recurrence/lung metastasis (P=0.001), high Ki-67 index (P=0.004), and P53 positivity (P=0.005). Median overall and recurrence-free survival time was significantly shorter in patients with high-CENP-A osteosarcomas than in those with low-CENP-A osteosarcomas. Multivariate analysis identified CENP-A as an independent poor prognostic factor for osteosarcoma. In conclusion, our results demonstrate that elevated CENP-A expression is significantly associated with osteosarcoma progression and has an independent prognostic value in predicting overall and recurrence-free survival for patients with osteosarcoma.

A universal pedicle screw and V-rod system for lumbar isthmic spondylolysis: a retrospective analysis of 21 cases.

OBJECTIVE: To investigate the surgical outcome of a universal pedicle screw-V rod system and isthmic bone grafting for isthmic spondylolysis. METHODS: Twenty-four patients with isthmic spondylolysis at L5 and grade 0-I spondylolisthesis (Meyerding classification) received isthmic bone graft and stabilization using the universal pedicle screw-V rod system. Back pain was evaluated using the visual analog scale (VAS) and time to bone healing, improvement in spondylolisthesis and intervertebral space height at L5/S1 and L4/L5 were assessed. RESULTS: Twenty-one patients were followed up for 24 months and included in the analysis. Back pain was markedly improved at 3 months postoperatively with a statistical difference in VAS scores compared with preoperative VAS scores (P<0.001). The VAS scores were 0 to 3 at 6 months postoperatively in all patients and no back pain was reported in all patients except 2 patients who complained of back pain after prolonged sitting. X-ray examination showed a bone graft healing time of 3 to 12 months. Grade I spondylolisthesis improved to grade 0 in 4 patients and no noticeable change was observed in the remaining 17 cases. The intervertebral space height at L5/S1 was statistically increased (P<0.05) while no statistically significant change was seen at L4/L5. There was no statistically significant difference in the ROM of the intervertebral disks of L5/S1 and L4/5 before and after surgery. CONCLUSIONS: The universal pedicle screw-V rod system and isthmic bone grafting directly repairs isthmic spondylolysis and reduces back pain, prevents anterior displacement of the diseased segment and maintains intervertebral space height, thus offering a promising alternative to current approaches for isthmic spondylolysis.

[Pulmonary toxicity in high-fat diet SD rats induced by intravenous injection of multi-walled carbon nanotubes].

OBJECTIVE: To observe the pulmonary toxicity of multi-walled carbon nanotubes (MWCNTs) in high-fat diet SD rats. METHODS: One hundred forty male SD rats were randomly divided into 6 groups. The normal control group, high-fat diet model group, vehicle group, and group treated with low dose of MWCNTs consisted of 30 rats, respectively, which were divided in 3 subgroups (10 rats each subgroup), respectively. The groups treated with medium and high loses of MWCNTs consisted of 10 rats, respectively. All the animals were exposed to high-fat-diet except for the control group which was given with normal diet. Before intravenous exposure, the high-fat diet model group, vehicle group, and three MWCNTs treated groups were gavaged with 700 thousand U/kg Vit D3 for three days, then given with high-fat-diet. The vehicle group was exposed to normal saline containing 1% Tween 80 and the low exposure group was exposed to MWCNTs at the dose of 50 µg/kg by tail vein injection twice a week for 8, 12 or 16 weeks. Other tow exposure groups were exposed to MWCNTs at the doses of 100, and 200 µg/kg by tail vein injection twice a week, respectively for 16 weeks. The lungs were from the executed rats, the lung indexes were calculated, the pathological changes of lungs were examined under light microscope after HE staining. qRT-PCR assay was utilized to detect the expression levels of pro-inflammation cytokines IL-1ß (IL-1ß) and TNF-α mRNA in the lungs. RESULTS: As compared with the vehicle group, the lung indexes in groups exposed to 100 and 200 µg/kg MWCNTs increased significantly (P < 0.05). It was found under light microscope that the MWCNTs were accumulated in lungs of three exposure groups in 16 weeks after exposure, including pneumorrhagia, alveolar walls thicken, fibrosis, and granulomas. As compared with the vehicle group, the levels of IL-1ß mRNA in group exposed to 50 µg/kg MWCNTs for 12 weeks and the groups exposed to 50, 100 and 200 µg/kg MWCNTs for 16 weeks decreased significantly (P < 0.05). As compared with the vehicle group, the levels of TNF-α mRNA in the groups exposed to 50 µg/kg MWCNTs for 8 and 16 weeks increased significantly (P < 0.05), the level of TNF-α mRNA in the groups exposed to 50 µg/kg MWCNTs for 12 weeks decreased significantly (P < 0.05). As compared with the vehicle group, the level of TNF-α mRNA in the groups exposed to 200 µg/kg MWCNTs for 16 weeks reduced significantly (P < 0.05). CONCLUSION: The MWCNTs accumulation and chronic inflammatory changes were found in the lungs of rats exposed to MWCNTs by tail vein injection.

[Effects of multiwall carbon nano-onions on platelet aggregation and hemostatic function].

OBJECTIVE: To observe the effects of multiwall carbon nano-onions (MWCNOs) on platelet aggregation and hemostatic function. METHODS: The platelet aggregation was determined with Born's method at different concentration of MWCNOs (0, 0.2, 2.0, 20.0 microg/ml) in vitro. Twenty male SD rats were randomly divided into 4 groups which were exposed to 0, 2, 4 and 8 mg/kg MWCNOs, respectively. Then platelet count, platelet aggregation, activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), bleeding time (BT) and platelet count (PC) were measured at 12 h after receiving tail intravenous injection of MWCNOs. The effects of MWCNOs (4 mg/kg) on platelet aggregation and platelet count at different time points were observed. RESULTS: In vitro, MWCNOs exhibited the potent inhibitory effects on rat platelet aggregation caused by ADP in a concentration-dependent manner. The platelet aggregation in the highest dosage of 20.0 microg/ml group was 50.0% +/- 6.9% which was significantly lower than that (73.2% +/- 4.3%) in control group (P<0.01). In vivo, the highest inhibitory was up to 20.4%, but there was no significant difference, as compared with control group. MWCNOs did not affect the APTT, PT, TT, BT and PC. CONCLUSION: Under this experimental condition, MWCNOs might inhibit platelet aggregation but not affect hemostatic function.

[Construction of quantitative real-time PCR detection system of transgenic tomato line Zeneca B,Da,F].

OBJECTIVE: To construct the plasmid reference molecules for detection of transgenic tomato line Zeneca B,Da,F using quantitative real-time PCR(qPCR). METHODS: Three plasmid reference molecules pEasy-T3-APX, pEasy-T3-16A and pEasy-T3-16S were cloned based on reverse genetics, which contain the target fragments of tomato endogenous reference gene apx (ERG-apx), gene-specific sequence of pg(GS-pg) and construct-specific sequence of vectors pJR16S/pJR16A (CS-16S/CS-16A) of Zeneca B,Da,F, respectively. Primers and Taqman probes were designed by Beacon Designer 7.5.The specificity, sensitivity, reproducibility and the limit of detection(LOD) of the qualitative and quantitative PCR system based on the plasmid reference molecules were evaluated. PicoGreen was used to measure the DNA concentration of the plasmid reference molecules. Two sets of samples containing 1% or 0.1% (w/w) pEasy-T3-16A or pEasy-T3-16S mixed with pEasy-T3- APX as background DNA were prepared for evaluating the efficacy of the qPCR system. RESULTS: The target fragments for qPCR detection were anchored, ERG-apx 108 bp, GS-pg 108 bp , CS-16S 109 bp and CS-16A 102 bp. The three plasmid reference molecules were confirmed at the expected sizes by restriction enzyme digestion. The qPCR results showed that the RSD of reproducibility were 0.2% to1.5%, LOD was 25 copies, R2 values for these standard curves were 0.994 ~0.998 and amplification efficiencies were 93.3%~102.4%.The bias between the test and true values of two sets of mixed samples ranged from -9.3% to 14.7% after adjusting by conversion factors(Cf). CONCLUSION: The plasmid reference molecules and qPCR system for qualitative and quantitative detection of transgenic tomato line Zeneca B,Da,F have been established successfully.

[Oxidative damage of single-walled carbon nanotubes in striaturn and hippocampi of mice].

OBJECTIVE: To study the oxidative damage of SWCNTs in striaturn and hippocampi of mice. METHODS: Forty male ICR mice were divided into experiment group (12.5 mg/kg SWCNTs) and control group (saline containing 0.1% Tween80) randomly. Each group was subdivided into 1, 7, 14 and 28 days group, 5 mice in each subgroup, then treated with tail intravenous injection for 5 continuous days. The striatum and hippocampus were isolated on the ice bath and homogenized in saline. SOD, GSH-Px, and MDA in the supernatants were measured with xanthine oxidize, GSH consumption in enzymatic reaction and TBA methods. RESULTS: After exposure to 12.5 mg/kg SWCNTs for 5 d, SOD activity in striaturn and hippocampi decreased on 1st day and reached the minimum on 7th day, then increased gradually. The SOD activity in the SWCNTs treatment groups on 7th day were significantly decreased when compared to control (P < 0.05). Comparison with control group, GSH-Px activity in striaturn obviously decreased on 7th day then increased on 14th day, the difference between 7th day and 14th day was significantly (P < 0.05). GHS-Px activity in the hippocampi in SWCNTs group on 7th day and 14th day was significantly lower than that in control group (P < 0.05), then increased to the level of control group on 28th day. MDA contents of striaturn and hippocampi in SWCNTs group reduced on 1st day, then gradually increased on 7th day and 14th day, then reduced, MDA contents on7th day and 14th day n SWCNTs group were significantly higher than that in control group (P < 0.05). CONCLUSIONS: The results of present study indicated that SWCNTs could decrease antioxidase activity and increase the Lipid peroxide in striaturn and hippocampi of mice.

[Posterior short-segment pedicle screw fixation combined with vertebroplasty for the treatment of thoracolumbar burst fractures].

OBJECTIVE: To evaluate the clinical efficacy of posterior short-segment pedicle screw fixation combined reduction of central end-plate by end-plate expand clamp and vertebroplasty with calcium phosphate cement for the treatment of thoracolumbar burst fractures. METHODS: Thirty patients with thoracolumbar burst fractures were treated with posterior pedicle screw fixation combined reduction of central end-plate by end-plate expand clamp and vertebroplasty with calcium phosphate cement. There were 22 males and 8 females with an average of 47 years (range from 25 to 71 years). Segment of fracture had 1 case in T11, 5 in T12, 14 in L1, 8 in L2, 2 in L3, 1 in L4, 1 in L5. According to Denis typing, 4 cases were type A, 25 type B, 1 type C, 1 type D, 1 type E. The relative anterior height of vertebral body and the vertebral angle were measured before and after operation and during the follow-up period (8 months after operation) through X-ray, the failure of internal fixation and recurrent kyphosis were evaluated during the follow-up period. Vertebral intracorporeal gap was measured by CT images after operation and the central end-plate fracture and reduction were observed by the reconstructed CT images (sagittal and coronary) before and after operation. RESULTS: All patients were followed up from 8 to 15 months with an average of 12 months. Relative anterior height of vertebral body and vertebral angle were well restored after operation, and there was no significant change between after operation and 8 months after operation. Preoperative,postoperative and 8 months after operation, relative anterior height of vertebral body respectively was 40.1%, 98.2%, 97.8%, vertebral angle respectively was 18.30, 2.70, 3.20. No failure of internal fixation and recurrent kyphosis were found during the follow-up period. The vertebral intracorporeal gap was about 3.1% and reduction of central endplate fracture was satisfactory. CONCLUSION: Posterior short-segment pedicle screw fixation combined with vertebroplasty with calcium phosphate cement can provide excellent reduction of post-traumatic segmental kyphosis and restore vertebral body height in the fracture level, prevent the failure of internal fixation which is an ideal method for the treatment of thoracolumbar burst fractures.

[Clinical research of delayed hyperextension injury concomitance spinal cord injury of cervical spine].

OBJECTIVE: To discuss Clinic feature and turnover of delayed hyperextension injury concomitance spinal cord injury of cervical spine. METHODS: The clinic data of 30 patients delayed hyperextension injury of cervical spine were reviewed and analyzed. Course of disease was from 3 months to 8 years. Thirty patients were divided into three groups according to course of disease. The first group, 3 - 6 months, 17 cases; the second group, 6 - 12 months, 8 cases; the third group, 12 months-8 years, 5 cases. Neurological function improvement rates were evaluated according to the JOA scores at preoperative, 3 months and 1 year post operation, and complications were observed in three groups. Twenty-six cases were treated with anterior decompression, bone graft and plate fixation. Four cases were treated with posterior decompression, bone graft and plate fixation. RESULTS: The patient number of the three group exist with statistical significance. Thirty patients were followed up for 18 - 39 months, 23 months on average. Neurological function recovery rates were 23.8% in the first group, 53.9% in the second group and 54.3% in the third group at 1 year post operation. JOA scores of the first group and the second group with statistical significance at 3 months and 1 year post operation. JOA scores of the first group and the third group with statistical significance at 3 months and 1 year post operation. JOA scores of the second group and the third group without statistical significance at 3 months and 1 year post operation. CONCLUSIONS: Incidence of hyperextension injury associated with spinal cord injury of cervical spine would degrade along with course of disease prolong. If delayed spinal cord injury occurred earlier, the patient's condition was severer and badly improvement rate.

[Optimization of tomato genetic transformation, kanamycin-resistant screening and seed selection].

OBJECTIVE: To optimize the process of tomato genetic transformation, screening and seed selection using multiepitope antigenic gene (MAG) and truncated major surface antigen 1 (tSAG1) of Toxoplasma gondii as the target insert genes. METHODS: Tomato high-frequency regeneration system was optimized with different choices of media and explants. The genetic transformation procedure was optimized using different tomato cultivars, explants, culture temperatures, media and acetosyringone (AS) supplements. Three concentrations of kanamycin were utilized for resistant selection of the transgenic candidate roots. The selected lines were trained, transplanted to soil and grown in a greenhouse till maturity. Sterile seeding using kanamycin-incorporated medium was conducted for screening transgenic tomato generations. RESULTS AND CONCLUSION: Cotyledons were better than hypocotyls as the regeneration explants. The regeneration rate of cotyledons reached 98% (59/60) using the optimized regeneration medium ZB3. The culture medium and temperature were the key factors for tomato transgenic shoot induction. The number of transgenic buds increased significantly at the appropriate temperature condition (23-/+1 degrees celsius;), and AS of 100 micromol/L in the medium before inoculation also significantly raised transformation rate. The budding rate of Zhongshu No.5 cotyledons was 35% (28/81) using the medium ZB2 under (23-/+1) degrees celsius;. Kanamycin at 80 mg/L was optimal for transgenic plantlet rooting selection with the rooting rate of 48% (31/65). 117 transgenic lines were obtained. Non-transgenic tomato plant growth, especially the root and elongation, was inhibited obviously with kanamycin at 100 mg/L or above, and the roots became purple and lacked lateral roots. The transgenic tomato seeds could be selected effectively with kanamycin at 150 mg/L.