The effect of phosphorylation on the conformational dynamics and allostery of the association of death-associated protein kinase with calmodulin.

Protein phosphorylation plays an important role in the signal transduction and is capable of regulation of cell activity. The death-associated protein kinase 1 (DAPK1), as a Ser/Thr kinase, interacts with calmodulin (CaM) to regulate apoptotic and autophagic signaling. Autophosphorylation of DAPK1 at Ser308 located at the autoregulatory domain (ARD) blocks CaM binding and inhibits kinase catalytic activity. However, the mechanism underlying the influence of Ser308 phosphorylation (pS308) on the DAPK1 activity remains unclear. Here, we performed multiple, microsecond length molecular dynamics (MD) simulations, the molecular mechanics generalized Born/surface area (MM-GBSA) binding free energy calculations, principal component analysis, and dynamic cross-correlation analysis to unravel the conformational dynamics and allostery of the DAPK1 - CaM interaction triggered by the pS308 at the ARD. MD simulations showed that pS308 affected the conformational stability of the DAPK1 - CaM complex. Further energetic and structural exploration revealed that pS308 weakened the association of the phosphorylated DAPK1 to CaM, which lowered the susceptibility of DAPK1 to be activated by CaM. This result can provide mechanistic insights into the molecular underpinning through which the DAPK1 kinase activity is modulated by the auto-phosphorylation.Communicated by Ramaswamy H. Sarma.

Clinical outcomes of short rod technique in posterior lumbar interbody fusion surgery: a minimum of 2-year follow-up.

PURPOSE: We present for the first time a novel entry point of pedicle screws (Short Rod Technique, SRT), which can avoid superior facet violation and has been verified as a safe screw placement method. The purpose of this study is to determine the clinical outcomes of SRT in posterior lumbar interbody fusion (PLIF) surgery. METHODS: We retrospectively analyzed the clinical outcomes of 89 patients who received SRT and 109 patients who received PLIF surgery with regular entry points of pedicle screws with a minimum of 2 years of follow-ups. Patients were divided into three groups according to the number of fusion segments, and the clinical outcomes of the three groups were compared. RESULTS: The length of the wound and the length of rods were significantly shorter in the each SRT group. Less intraoperative blood loss was observed in the SRT group in patients with a single segment and two segments fusions, but not in three segments fusions. Fewer degenerations of the upper adjacent segment were observed in the SRT group in patients with a single segment and three segments fusions. In addition, less postoperative wound pain related to PLIF surgery was observed in the SRT group in patients with two and three segments fusions. CONCLUSION: SRT has been validated as an effective technique with good clinical outcomes, especially for reducing the occurrence of upper ASD in PLIF surgery with a single segment and three segments. The present study provides spinal surgeons with a novel method for performing PLIF surgery.

Sagittal imaging study of the lumbar spine with the short rod technique.

PURPOSE: The short rod technique (SRT) is a novel method for lumbar pedicle screw placement to reduce surgical trauma and avoid damage to the facet joint and articular surface. The core concept is to change the entry point and angle of the screw on the vertebrae at both ends in the sagittal plane to shorten the length of the longitudinal rods. The purpose of this study is to determine the sagittal screw angle (SSA) and its safe Maximum (MAX) value on each lumbar vertebra for the SRT and to observe the shortening effect on the longitudinal rods. METHODS: A total of 152 healthy adults were investigated by measuring the lumbar spine lateral view images. The SSA and MAX-SSA were measured with SRT as reference to the conventional placement technique method. The distance between the entry points of the proximal and distal vertebrae was measured to compare the changes in the length of the longitudinal rods using the two screw placement techniques. RESULTS: + SSA increased from L1 to L4, and -SSA increased from L2 to L5, in which the -SSA of L2, L3, and L4 were significantly greater than those of + SSA (P < 0.05). + MAX-SSA at L1-L4 was 23.26 ± 3.54°, 23.68 ± 3.37°, 24.12 ± 3.29°, and 24.26 ± 3.42°, respectively. -MAX-SSA at L2-L5 was 36.25 ± 3.26°, 38.26 ± 3.73°, 38.62 ± 3.63° and 37.33 ± 3.31°, respectively. Theoretical reductions by calculation for the 2-segment lumbar pedicles were: L1-2: 9 mm, L2-3: 9.29 mm, L3-4: 6.23 mm, and L4-5: 7.08 mm; And the 3-segment lumbar pedicles were: L1-3: 16.97 mm, L2-4: 16.73 mm, L3-5, and 18.24 mm, respectively. CONCLUSIONS: The application of the SRT to lumbar pedicles is a safe screw placement method that can significantly shorten the length of the used longitudinal rods.

RHOA inhibits chondrogenic differentiation of mesenchymal stem cells in adolescent idiopathic scoliosis.

PURPOSE: The etiology of adolescent idiopathic scoliosis (AIS) remains unclear. The chondrogenic differentiation of mesenchymal stem cells (MSCs) is important in AIS, and the Ras homolog gene family member A (RHOA) is associated with chondrogenesis. The purpose of this study was to explore the effect of RHOA on the chondrogenic differentiation of MSCs in AIS. METHODS: We isolated MSCs from patients with AIS (AIS MSCs) and individuals without AIS (control MSCs). The inhibitor Y27632 was used to inhibit the function of RHOA/ROCK signaling, and plasmid-based overexpression and siRNA-mediated knockdown were used to manipulate RHOA expression. CCK-8 was used to detect cell viability. The phosphorylation levels of LIMK1, MLC2 and cofilin were detected by Western blotting. The mRNA expression of aggrecan, SOX9, and COL2A1 were confirmed using RT-PCR. Immunofluorescence was used to analyze F-actin and collagen II. Alcian blue staining was performed to assess the secretion of glycosaminoglycans (GAGs). RESULTS: We found that RHOA was significantly upregulated in AIS MSCs, and the phosphorylation levels of LIMK1, MLC2, and cofilin were increased. The mRNA expressions of aggrecan, SOX9, and COL2A1 were notably reduced in AIS MSCs. However, these effects were abolished by Y27632 treatment and RHOA knockdown in AIS MSCs. In addition, RHOA knockdown in AIS MSCs increased the content of collagen II and GAGs. RHOA overexpression in the control MSCs markedly activated the RHOA/ROCK signaling and decreased the expression of aggrecan, SOX9, and COL2A1, F-actin, and GAGs. CONCLUSION: RHOA regulates the chondrogenic differentiation ability of MSCs in AIS via the RHOA/ROCK signaling pathway and this regulation may involve SOX9.

Comparison of robot versus fluoroscopy-assisted pedicle screw instrumentation in adolescent idiopathic scoliosis surgery: A retrospective study.

The aim of this study was to explore whether a robot-assisted (RA) technique has advantages over the conventional fluoroscopy-assisted (FA) technique in clinical and radiological outcomes and whether it could decrease the incidence of mis-implantations of pedicle screws in adolescent idiopathic scoliosis (AIS) correction surgery. A total of 101 patients with AIS were recruited (RA group: 45 patients underwent RA screw insertion; FA group: 56 patients underwent FA screw insertion). When comparing the radiological data between the two groups, the major and secondary curves were both corrected proficiently with no difference in Cobb angle comparison at the last follow-up, suggesting that both the RA technique and the FA technique could lead to efficient radiographic correction and similar clinical outcomes (all, p > 0.05). In the RA group, operation time, blood loss, and transfusion volume were significantly greater than those in the FA group, while the accuracy of screw implantations in patients with AIS with a thoracic scoliotic curve in the RA group was higher than that in the FA group. In conclusion, both the RA and FA techniques could approach proficient radiographic correction and similar clinical outcomes in AIS surgery. Compared with the conventional fluoroscopy technique, the RA technique might improve the accuracy of screw implantations in patients with AIS with a thoracic scoliotic curve, while the increased operation time, blood loss, and transfusion volume might be the disadvantages due to the preliminary stage of the learning curve.

Assessment of sagittal spinopelvic alignment in asymptomatic Chinese juveniles and adolescents: a large cohort study and comparative meta-analysis.

STUDY DESIGN: Retrospective study and comparative meta-analysis. OBJECTIVE: To document the sagittal spinopelvic alignment in a large cohort study in asymptomatic Chinese juveniles and adolescents, and to explore whether these parameters were different from various regions using meta-analysis. METHODS: Medical records of 656 asymptomatic Chinese juveniles and adolescents were reviewed, whose mean age was 13.14 ± 3.41 years old, including 254 male and 402 female volunteers. Demographic and lateral radiological parameters were evaluated. Furthermore, a systematic online search was performed to identify eligible studies. Weight mean difference (WMD) with 95% confidence interval (CI) were used to evaluate whether these sagittal parameters were different from various regions. RESULTS: The mean value of sagittal spinopelvic alignment in this study was calculated and analyzed respectively. Significant differences of PI (34.20 ± 4.00 vs. 43.18 ± 7.12, P < 0.001) and PT (3.99 ± 6.04 vs. 8.42 ± 7.08, P < 0.001) were found between juveniles and adolescents. A total of 17 studies were recruited for meta-analysis. For juvenile populations, TK, PI and SS of Caucasians were significantly larger than those of our study (all P < 0.001). As for adolescent populations, PI (P = 0.017), TK (P = 0.017) and SS (P < 0.001) of Caucasians was found to be greater when compared with that of our study. All in all, TK, PI and SS in Chinese pre-adult populations were significantly smaller than those populations in Caucasian regions (all P < 0.001). CONCLUSION: Our study was the first large-scale study that reported the mean values of sagittal parameters in asymptomatic Chinese juveniles and adolescents. There were significant differences in TK, PI and SS between our study and other previous reported populations, which reminded us for using specific mean values in different populations when restoring a relatively normal sagittal spinopelvic balance in spinal deformity.

Clinical Application of a New Assessment Tool for Myelopathy Hand Using Virtual Reality.

STUDY DESIGN: A cross-sectional study. OBJECTIVE: To assess the effectiveness of a new assessment tool, myelopathy-hand functional evaluation system (MFES), in evaluating the hand dysfunction of patients with cervical myelopathy in the 10-second grip-and-release test (10 second G-R test). SUMMARY OF BACKGROUND DATA: Clumsy fingers movement is a common symptom of myelopathy patients. Evaluating the impaired hand function can provide a strong basis in assessing the severity of myelopathy. Currently, no objective and effective evaluation method is widely accepted in clinical practice. METHODS: MFES mainly consists of a pair of wise-gloves and a computer with software. One hundred and ninety-eight consecutive participants were asked to wear the wise-gloves and then perform 10 seconds G-R test. The movements of each finger were recorded by MFES and converted into waveforms. Relevant waveform parameters were measured and analyzed. The Japanese Orthopedics Association (JOA) scores of each patient were marked and the maximum spinal cord compression (MSCC) was measured on midsagittal T2-weighted magnetic resonance imaging (MRI). RESULTS: Myelopathy patients had a lower number of G-R cycles and a longer time per cycle than healthy subjects. There were significant differences in adduction and abduction time in patients with JOA scores greater than 6, but not in healthy subjects and patients with JOA scores less than 6. The waveforms of ulnar three fingers in myelopathy patients were lower and wider than those in healthy individuals. The average ratio value of wave height to wave width (a/b) could quantitatively reflect such differences of waveforms. According to receiver operating characteristic (ROC) curve analysis, the optimal threshold value of the normal average ratio was more than 1.92. The average a/b value was correlated with the JOA scores of the motor function in the upper extremities (r = 0.842). CONCLUSION: MFES appears to be an objective and quantitative assessment tool for patients with cervical myelopathy. LEVEL OF EVIDENCE: 3.

Upregulation of cGMP-dependent Protein Kinase (PRKG1) in the Development of Adolescent Idiopathic Scoliosis.

OBJECTIVE: To explore the molecular regulatory mechanisms underlying fibroblast differentiation and dysfunction in the development of adolescent idiopathic scoliosis (AIS) in an effort to identify candidate therapeutic targets for AIS. METHODS: The GSE110359 dataset, obtained from the bone marrow stromal cells of 12 AIS patients and five healthy controls, was retrieved from the GEO database. The data were preprocessed and differentially expressed genes (DEGs) were identified. KEGG pathway and Gene Ontology (GO)-Biological Process (BP) enrichment analyses were performed to identify the function of the DEGs. A protein-protein interaction (PPI) and a microRNA-transcription factor (TF)-target co-regulatory network were constructed to identify hub genes in the development of AIS. In addition, hub DEGs were evaluated by quantitative PCR (qPCR) and immunohistochemical staining. RESULTS: A total of 188 DEGs including 100 up-regulated and 88 down-regulated genes were obtained. The up-regulated DEGs were related to "p53 signaling pathway", "FoxO signaling pathway", and "cGMP-PKG signaling pathway" terms, while the down-regulated DEGs were significantly enriched in seven terms including "protein processing in endoplasmic reticulum". The key up-regulated genes, PRKG1, CCNG2, and KAT2B, and the key down-regulated genes, MAP2K1 and DUSP6, were identified by the PPI and miRNA-TF-Target regulatory network analyses. mRNA expression patterns for PRKG1, DUSP6, and KAT2B were successfully verified by qPCR. In addition, PRKG1 protein levels were found to be elevated during the immunohistochemical analysis. CONCLUSION: Increased expression of PRKG1 in AIS patients might be an attractive therapeutic target for AIS. However, further gain or loss-of-function studies should be conducted.

MicroRNA-300 Regulates the Ubiquitination of PTEN through the CRL4BDCAF13 E3 Ligase in Osteosarcoma Cells.

Cullins, critical members of the cullin-RING ubiquitin ligases (CRLs), are often aberrantly expressed in different cancers. However, the underlying mechanisms regarding aberrant expression of these cullins and the specific substrates of CRLs in different cancers are mostly unknown. Here, we demonstrate that overexpressed CUL4B in human osteosarcoma cells forms an E3 complex with DNA damage binding protein 1 (DDB1) and DDB1- and CUL4-associated factor 13 (DCAF13). In vitro and in vivo analyses indicated that the CRL4BDCAF13 E3 ligase specifically recognized the tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) for degradation, and disruption of this E3 ligase resulted in PTEN accumulation. Further analyses indicated that miR-300 directly targeted the 3' UTR of CUL4B, and DNA hypermethylation of a CpG island in the miR-300 promoter region contributed to the downregulation of miR-300. Interestingly, ectopic expression of miR-300 or treatment with 5-AZA-2'-deoxycytidine, a DNA methylation inhibitor, decreased the stability of CRL4BDCAF13 E3 ligase and reduced PTEN ubiquitination. By applying in vitro screening to identify small molecules that specifically inhibit CUL4B-DDB1 interaction, we found that TSC01131 could greatly inhibit osteosarcoma cell growth and could disrupt the stability of the CRL4BDCAF13 E3 ligase. Collectively, our findings shed new light on the molecular mechanism of CUL4B function and might also provide a new avenue for osteosarcoma therapy.

Preparation and Assessment of an Individualized Navigation Template for Lower Cervical Anterior Transpedicular Screw Insertion Using a Three-Dimensional Printing Technique.

STUDY DESIGN: Prospective trial. OBJECTIVE: To establish an individualized navigation template for safe and accurate insertion of lower cervical anterior transpedicular screw (ATPS) based on a three-dimensional (3D) printing technique. SUMMARY OF BACKGROUND DATA: Conventional screw insertion manually under fluoroscopy easily leading to deviation of ATPS screw channel, cervical instrumentation procedures demand the need for a precise technique for screw placement. METHODS: Twenty adult cervical spine specimens (10 men and 10 women, with a mean age of 50.29 ±â€Š6.98) were selected for computed tomography pre- and postoperatively. A 3D lower cervical spine model was reconstructed using Mimics software to measure the screw-related parameters and generate a reverse template with optimal screw channel as well as a prototype using 3D printing. Assisted by the navigation template, bilateral ATPS were inserted into the cadavers. RESULTS: The mean outer width and height of pedicle were 5.31 ±â€Š1.23 and 6.78 ±â€Š1.10 mm, respectively. The average length, sagittal, and axial angles of the optimal screw channel obtained through the optimal entry point were 36.34 ±â€Š4.39 mm, 40.67°â€Š±â€Š5.10°, and 93.7°â€Š±â€Š7.96°, respectively. The adjustable safe ranges of sagittal and axial angles were 3.89°â€Š±â€Š1.13° and 5.64°â€Š±â€Š0.97°, respectively. The axial and sagittal accuracies of the 200 screws were 99.5% and 97%, respectively. The average deviations of the actual entry point and the preset opening in the X, Y, and Z axes were 0.39 ±â€Š0.43, 0.21 ±â€Š0.41, and 0.29 ±â€Š0.14 mm, respectively (P > 0.05). CONCLUSION: An individualized ATPS navigation template was developed using Mimics software and 3D printing prototyping, based on computed tomography, for highly accurate screw insertion. LEVEL OF EVIDENCE: 4.

Autophagic degradation of FOXO3a represses the expression of PUMA to block cell apoptosis in cisplatin-resistant osteosarcoma cells.

Autophagy and apoptosis are the two major modes of cell death, and autophagy usually inhibits apoptosis. The current understanding has shown that there is a complex crosstalk between the components of these two pathways. Here, we describe a transcriptional mechanism that links autophagy to apoptosis. We show that the cisplatin-resistant MG63-R12 and U2OS-R5 osteosarcoma sublines, in comparison to their parental MG63 and U2OS cells, respectively, exhibit increased autophagy but decreased apoptosis levels after treatment with cisplatin. We then used a microarray assay to examine the gene expression changes in these two cisplatin-resistant sublines and found that the expression of the transcription factor FOXO3a was dramatically decreased. Pharmacological treatment with either 3-methyladenine to inhibit autophagy or with rapamycin to activate autophagy in these two cisplatin-resistant sublines resulted in the accumulation or degradation of FOXO3a, respectively. Ectopic expression of FOXO3a in MG63-R12 and U2OS-R5 cells significantly enhanced cell sensitivity to cisplatin through a mechanism in which FOXO3a directly binds to the PUMA promoter and activates its expression, as well as its downstream event, the intrinsic apoptosis pathway. Importantly, this overexpression resulted in tumor growth inhibition in vivo. In conclusion, our results provide new insights into the molecular link between autophagy and apoptosis that involves a FOXO3a-mediated transcriptional mechanism. Importantly, our results may facilitate the development of therapeutic strategies for osteosarcoma patients who have become resistant to cisplatin therapy.

CRL4BDCAF11 E3 ligase targets p21 for degradation to control cell cycle progression in human osteosarcoma cells.

Cell cycle progression in mammals is strictly controlled by a number of cyclin-dependent kinases (CDKs) and CDK inhibitors (CKIs), the expression of which is often dysregulated in cancer cells. Our previous work revealed that Cullin 4B (CUL4B), a critical component of the Cullin4B-RING E3 ligase complex (CRL4B), is overexpressed in human osteosarcoma cells through an unknown mechanism. Here, we demonstrated that CUL4B forms an E3 ligase with RBX1 (RING-box 1), DDB1 (DNA damage binding protein 1), and DCAF11 (DDB1 and CUL4 associated factor 11) in human osteosarcoma cells. In vitro and in vivo ubiquitination analyses indicated that CRL4BDCAF11 E3 ligase was able to specifically ubiquitinate a CDK inhibitor-p21Cip1 at K16, K154, K161 and K163 but not at K75 and K141. Knocking down any component of the CRL4BDCAF11 complex, including CUL4B, DDB1 or DCAF11, using short hairpin RNAs (shRNAs) attenuated the ubiquitination level of p21Cip1, inhibited osteosarcoma cell proliferation, led to cell cycle arrest at S phase, and decreased colony formation rate. Taken together, our data suggest that the CRL4BDCAF11 complex represents a unique E3 ligase that promotes the ubiquitination of p21Cip1 and regulates cell cycle progression in human osteosarcoma cells.

Evaluation of DTI Parameter Ratios and Diffusion Tensor Tractography Grading in the Diagnosis and Prognosis Prediction of Cervical Spondylotic Myelopathy.

STUDY DESIGN: Prospective cohort. OBJECTIVE: To explore the correlations between diffusion tensor imaging (DTI) ratios and diffusion tensor tractography (DTT) grading with clinical symptoms and outcomes of cervical spondylotic myelopathy (CSM). SUMMARY OF BACKGROUND DATA: In addition to magnetic resonance imaging (MRI) and computed tomography, DTI may be useful in understanding pathophysiology of spinal cord in earlier stages of the CSM but it may be confounded by age and cervical level, and previous studies had small sample sizes. METHODS: Controls (n = 36) and patients with CSM (n = 93) underwent magnetic resonance imaging (MRI) and DTI at the Changhai Hospital of Shanghai between September 2011 and March 2013. Apparent diffusion coefficient (ADC), fractional anisotropy (FA) of white matter (WM), and central grey matter (GM) were assessed. Patients were divided into three MRI grades: no abnormal signals; increased T2WI; and increased T2WI and low T1WI. DTT images were divided into three grades: no abnormal signals; abnormal local signal cord and disordered fiber tracts; and distortion of the spinal cord and interrupted fiber tracts. RESULTS: FA and ADC both correlated with age in all three bilateral WM funiculi and GM, whereas FA and ADC ratios only showed correlation with age in the ventral funiculus (VF) and central GM. Differences were observed in ADC ratios and FA ratios from different Japanese Orthopedic Association (JOA) score subgroups and JOA recovery subgroups. For the three WM funiculi and GM, correlations between DTI ratios, JOA scores, and JOA recovery rates were consistently higher than those between DTI values, JOA scores, and JOA recovery rates (all P < 0.05). MRI grading was correlated with the JOA scores (r = -0.674, P < 0.001) but not JOA recovery rates (r = -0.197, P = 0.058), whereas DTT grading was correlated with both JOA scores (r = -0.813, P < 0.001) and JOA recovery rate (r = -0.429, P < 0.001). CONCLUSION: DTI parameter ratios seemed to be less influenced by age than raw DTI results and could be more valuable than absolute DTI parameters for the evaluation of CSM. DTT grading is more valuable than MRI grading for diagnosis and prognostic prediction in CSM patients. LEVEL OF EVIDENCE: 3.

A Novel High Mechanical Property PLGA Composite Matrix Loaded with Nanodiamond-Phospholipid Compound for Bone Tissue Engineering.

A potential bone tissue engineering material was produced from a biodegradable polymer, poly(lactic-co-glycolic acid) (PLGA), loaded with nanodiamond phospholipid compound (NDPC) via physical mixing. On the basis of hydrophobic effects and physical absorption, we modified the original hydrophilic surface of the nanodiamond (NDs) with phospholipids to be amphipathic, forming a typical core-shell structure. The ND-phospholipid weight ratio was optimized to generate sample NDPC50 (i.e., ND-phospholipid weight ratio of 100:50), and NDPC50 was able to be dispersed in a PLGA matrix at up to 20 wt %. Compared to a pure PLGA matrix, the introduction of 10 wt % of NDPC (i.e., sample NDPC50-PF10) resulted in a significant improvement in the material's mechanical and surface properties, including a decrease in the water contact angle from 80 to 55°, an approximately 100% increase in the Young's modulus, and an approximate 550% increase in hardness, thus closely resembling that of human cortical bone. As a novel matrix supporting human osteoblast (hFOB1.19) growth, NDPC50-PFs with different amounts of NDPC50 demonstrated no negative effects on cell proliferation and osteogenic differentiation. Furthermore, we focused on the behaviors of NDPC-PFs implanted into mice for 8 weeks and found that NDPC-PFs induced acceptable immune response and can reduce the rapid biodegradation of PLGA matrix. Our results represent the first in vivo research on ND (or NDPC) as nanofillers in a polymer matrix for bone tissue engineering. The high mechanical properties, good in vitro and in vivo biocompatibility, and increased mineralization capability suggest that biodegradable PLGA composite matrices loaded with NDPC may potentially be useful for a variety of biomedical applications, especially bone tissue engineering.

Uric Acid Promotes Osteogenic Differentiation and Inhibits Adipogenic Differentiation of Human Bone Mesenchymal Stem Cells.

To investigate the effect of uric acid on the osteogenic and adipogenic differentiation of human bone mesenchymal stem cells (hBMSCs). The hBMSCs were isolated from bone marrow of six healthy donors. Cell morphology was observed by microscopy and cell surface markers (CD44 and CD34) of hBMSCs were analyzed by immunofluorescence. Cell morphology and immunofluorescence analysis showed that hBMSCs were successfully isolated from bone marrow. The number of hBMSCs in uric acid groups was higher than that in the control group on day 3, 4, and 5. Alizarin red staining showed that number of calcium nodules in uric acid groups was more than that of the control group. Oil red-O staining showed that the number of red fat vacuoles decreased with the increased concentration of uric acid. In summary, uric acid could promote the proliferation and osteogenic differentiation of hBMSCs while inhibit adipogenic differentiation of hBMSCs.

Age-related changes of the diffusion tensor imaging parameters of the normal cervical spinal cord.

BACKGROUND: The diffusion tensor imaging (DTI) parameters of the cervical spinal cord (CSC) changes with age. However, previous studies only examined specific CSC areas. OBJECTIVES: To analyze the DTI parameters in all intervertebral space levels of the whole normal CSC and to study the impact of age on these parameters in a Chinese population. METHODS: Thirty-six healthy subjects aged 20-77 years were recruited. DTI parameters were calculated for gray matter (GM) and white matter (WM) funiculi in all the CSC intervertebral spaces (C1/2-C6/7). Age-related changes of DTI parameters were analyzed for the GM and WM funiculi. RESULTS: Fractional anisotropy (FA) and mean diffusivity (MD) were lower in GM than in WM. MD and FA values were lower in the WM in the lower CSC compared with the upper CSC (all P<0.05), but no difference was observed in GM. In ventral funiculi, MD increased with age, while FA decreased (all P<0.001). In lateral and dorsal funiculi, MD and FA decreased with age (all P<0.001). In GM, MD and FA decreased with age (all P<0.001). Significant age-related changes were observed in FA and MD from GM and WM funiculi. FA was correlated with age in all funiculi (ventral: r=-0.733; lateral: r=-0.468; dorsal: r=-0.607; GM: r=-0.724; all P<0.01). CONCLUSION: Important changes in MD and FA were observed with advancing age at all levels of CSC in Chinese patients. DTI parameters may be useful to assess CSC pathology, but the influence of age and segments need to be taken into account in diagnosis.

CUL4B promotes proliferation and inhibits apoptosis of human osteosarcoma cells.

Cullin 4B (CUL4B) is a component of the Cullin4B-Ring E3 ligase complex (CRL4B) that functions in proteolysis and is implicated in tumorigenesis. Here, we report that CUL4B is associated with tumorigenesis by promoting proliferation and inhibiting apoptosis of human osteosarcoma cells. We performed RNA interference (RNAi) with a lentiviral vector system to silence the CUL4B gene using osteosarcoma SAOS-2 cells. The negative control included the normal target cells infected with the negative control virus whereas the knockdown cells included the normal target cells transfected with the RNAi target virus. We assessed the inhibition resulting from the decreased expression of the CUL4B gene on the proliferation rate of SAOS-2 cells, and also evaluated the cell cycle distribution, apoptosis and clonability. Compared with the negative control, the CUL4B gene expression was significantly inhibited in the SAOS-2 cells at the mRNA and protein levels in the knockdown group (P<0.01). Furthermore, in the knockdown group, the cell proliferation rate and clonability were also significantly inhibited (P<0.01). The apoptosis rate increased significantly (P<0.05). A significant decrease in the number of cells in the G1 phase (P<0.01) and significant increases in the S (P<0.01) and G2 phases (P<0.05) were observed. The silencing of CUL4B gene expression can effectively inhibit osteosarcoma cell proliferation and induce apoptosis. These findings may provide a novel biomarker for the treatment of osteosarcoma.

[A modified goat model of acute spinal cord compression injury from a percutaneous balloon catheter: method feasibility and preliminary observation].

OBJECTIVE: To establish a goat model of acute spinal cord compression injury through a modified percutaneous technique with a Foley double-lumen urine catheter and explore the method feasibility and preliminary observation. METHODS: Twelve adult male Chongming goats were randomly divided into 3 groups:control (A, n = 4), 0.5 ml compression (B, n = 4) and 1 ml compression (C, n = 4). After local anesthesia, all animals received epidural balloon catheter (5Fr) insertion via a percutaneous trans-lumbosacral interlaminar space technique that mimicked the method used in vascular access for angiography. The balloon catheter was advanced under fluoroscopic guidance until its distal tip reached the middle of T6 level.One week later, for groups B and C, the balloon was inflated by half-strength contrast material, 0.5 ml and 1 ml, respectively. The balloon was left inflated for 30 min and then deflated. The images of computed tomography (CT) and magnetic resonance (MR) were taken before and after surgical procedures.Quantitative assessment of spine canal occupying rate was accomplished by an off-line software program based on CT results. Motor function was assessed by the modified Tarlov scale. Two animals of each group were sacrificed after a total observation period of 48 h and 72 h respectively.Spinal cords from the injured level were then obtained for pathologic examinations. RESULTS: All animals underwent successful catheterization occupying 6.8%±0.7% (Group A), 6.7%±0.7% (Group B) and 6.6%±0.6% (Group C) of spine canal respectively. After inflation, the occupying rate of groups B and C achieved 43.4%±2.5% and 88.1%±2.3% respectively.Ventral compression of spinal cord was noted on MR images.Hindlimb movement remained normal after catheter insertion in all groups. All animals in group B and C became paraplegic after inflation. And a positive correlation existed between injection volume and Tarlov score. Pathological findings confirmed neuron atrophy, increased gap around neurons, mild demyelination and vacuolar degeneration both in groups B and C at 48 h after injury. Pathological changes deteriorated at 72 h after injury. CONCLUSION: The results of behavioral evaluation, radiographic images and pathological examination reveal an evidence of acute spinal cord injury. Percutaneous epidural balloon catheter insertion differs from previous techniques by avoiding surgical exposure and associated artifacts, yet it offers injury mechanisms similar to those of human spinal cord injury. As a new means of modeling spinal cord injury in animals, this technique has many potential applications.