Deferoxamine/magnesium modified ß-tricalcium phosphate promotes the bone regeneration in osteoporotic rats.

Recently, Deferoxamine (DFO) and magnesium (Mg) have been identified as critical factors for angiogenesis and bone formation. However, in current research studies, there is a lack of focus on whether DFO plus Mg can affect the regeneration of ß-tricalcium phosphate (ß-TCP) in osteoporosis and through what biological mechanisms. Therefore, the present work was aimed to preparation and evaluate the effect of Deferoxamine/magnesium modified ß-tricalcium phosphate promotes (DFO/Mg-TCP) in ovariectomized rats model and preliminary exploration of possible mechanisms. The MC3T3-E1 cells were co-cultured with the exudate of DFO/Mg-TCP and induced to osteogenesis, and the cell viability, osteogenic activity were observed by Cell Counting Kit-8(CCK-8), Alkaline Phosphatase (ALP) staining, Alizarin Red Staining (RES) and Western Blot. In vitro experiments, CCK-8, ALP and ARS staining results show that the mineralization and osteogenic activity of MC3T3-E1increased significantly after intervention by DFO/Mg-TCP, as well as a higher levels of protein expressions including VEGF, OC, Runx-2 and HIF-1α. In vivo experiment, Micro-CT and Histological analysis evaluation show that DFO/Mg-TCP treatment presented the stronger effect on bone regeneration, bone mineralization and biomaterial degradation, when compared with OVX+Mg-TCP group and OVX+TCP group, as well as a higher VEGF, OC, Runx-2 and HIF-1α gene expression. The present study indicates that treatment with DFO/Mg-TCP was associated with increased regeneration by enhancing the function of osteoblasts in an OVX rat.

A new method of monitoring catheter-directed thrombolysis for deep venous thrombosis-application of D-dimer and fibrinogen testing.

BACKGROUND: Catheter-directed thrombolysis (CDT) is one of the main treatment methods for acute deep venous thrombosis (DVT), which has the characteristics of long treatment time and large dosage of thrombolytic drugs. In the absence of good monitoring methods, problems such as low thrombolytic efficiency and high risk of bleeding are easy to occur. OBJECTIVE: To evaluate the value of D-dimer (D-D) and fibrinogen (FIB) testing as a thrombolysis-monitoring method during CDT for acute DVT. METHODS: Twenty patients with acute DVT were divided into group A and group B. During CDT, the D-D and FIB testing every 8 h were used in group A, and the venography and FIB testing every 24 h in group B. The thrombolysis rate, thrombolysis time, urokinase dosage, and X-ray radiation dose were compared. RESULTS: The thrombolysis rate in group A was significantly higher than that in group B (p < 0.05), but the number of venography and radiation dose were significantly lower than those in group B (p < 0.05). CONCLUSION: D-D and FIB testing can improve the thrombolysis rate, reduce the risk of bleeding, and decrease the number of angiograms and X-ray radiation dose during CDT.

Role of serum cystatin C in the prediction of contrast-induced nephropathy after intra-arterial interventions.

BACKGROUND: The diagnosis of contrast-induced nephropathy (CIN) is usually based on changes in serum creatinine (sCr). However, sCr has poor sensitivity as a biomarker of kidney injury. The aim of this study was to investigate the usefulness of serum cystatin C (sCysC) to predict CIN after intra-arterial interventions. METHODS: A total of 360 consecutive patients underwent intra-arterial procedures using digital subtraction angiography. SCr, sCysC, and estimated glomerular filtration rate were measured at 1 to 2 days before and at 48, 72 h, and 7 days after the procedure. RESULTS: Thirty-one patients (8.61%) developed CIN. Receiver operating characteristic (ROC) curve analysis showed that pre-operative sCysC levels had good discriminatory power (area under the curve [AUC] = 0.634; 95% confidence interval [CI] = 0.526-0.743) for evaluating the risk of CIN after an endovascular procedure, with a sensitivity of 53.33% and specificity of 73.70%. ROC analysis showed that sCysC at 48 h after contrast medium administration was predictive of CIN after an endovascular procedure (AUC = 0.735; 95% CI = 0.647-0.822) with satisfactory sensitivity of 74.20% and specificity of 63.90%. Diabetes mellitus was an independent risk factor for CIN (odds ratio = 2.778; 95% CI = 1.045-7.382; P = 0.040). CONCLUSIONS: SCysC is an appropriate biomarker to predict the occurrence of CIN. Baseline sCysC before an intervention is useful to obtain a preliminary estimate of the risk of CIN. A 48-h cut-off value of sCysC of 0.99 mg/L after an endovascular procedure may help to rule out patients at lower risk of CIN.

LINC00205 modulates the expression of EPHX1 through the inhibition of miR-184 in hepatocellular carcinoma as a ceRNA.

Several studies have shown that low expression of epoxide hydrolase 1 (EPHX1) is closely associated with varying human cancers, including hepatocellular carcinoma (HCC). This study aims to explore the potential mechanism of EPHX1 silencing and revealed a novel regulatory pathway in the pathogenesis of HCC. In this study, micro ribonucleic acid (miR)-184 was predicted and validated to be a regulator of EPHX1 through experiments, and its expression was negatively correlated with the messenger RNA (mRNA) levels of EPHX1 in primary tumors. Elevation of EPHX1 suppressed cell proliferation and migration as well as cell cycle progression, and induced apoptosis, while downregulation of miR-184 exhibited the opposite effect on cellular processes. Moreover, LINC00205 interacted with miR-184 and was markedly downregulated in tumors. The effects of the miR-184 inhibitor on cell proliferation, apoptosis, and migration were reversed in part by the transfection with LINC00205 small interfering RNAs. In addition, LINC00205 acted as a molecular sponge to positively regulate the mRNA and protein levels of EPHX1 via regulating miR-184. The tumorigenicity of HCC cells was enhanced by LINC00205 shRNA but diminished by overexpression of EPHX1 in vivo. Clinically, the EPHX1 expression in patients with HCC was markedly downregulated. Taken together, the results of this study suggest that as a competing endogenous RNA, LINC00205 may regulate EPHX1 by inhibiting miR-184 in the progression of HCC and that targeting the LINC00205/miR-184/EPHX1 axis may provide a treatment protocol for patients.

miR-505 acts as a tumor suppressor in gastric cancer progression through targeting HMGB1.

Gastric cancer (GC) is a frequent type of malignant tumor worldwide. GC metastasis results in the majority of clinical treatment failures. MicroRNAs (miRNA) are identified to exhibit crucial roles in GC. Our current study aimed to explore the biological roles of miR-505 in GC progression. It was observed that miR-505 was robustly decreased in GC cells compared with human normal gastric epithelial GES-1 cells. Overexpression of miR-505 was able to repress GC progression in AGS and BGC-823 cells. In addition, high-mobility group box 1 (HMGB1) has been identified as a crucial oncogene in several cancer types. By carrying out bioinformatics analysis, HMGB1 was predicted as a direct target of miR-505. Meanwhile, HMGB1 was found to be significantly increased in GC cells and it was confirmed in our study that miR-505 can directly target HMGB1 in vitro. miR-505 mimics can inhibit HMGB1 messenger RNA and protein expression dramatically. Subsequently, knockdown of HMGB1 can inhibit GC cell proliferation, colony formation, and induce cell apoptosis. Furthermore, HMGB1 silence suppressed GC cell migration and invasion greatly in vitro. Finally, it was validated that miR-505 can inhibit GC progression by targeting HMGB1 in vivo. Taken these together, it was indicated that miR-505/HMGB1 axis was involved in the development of GC. miR-505 can serve as a potential prognostic indicator in GC therapy.

[Role of BET Bromodomain in Hematopoietic Differentiation from hESCs].

OBJECTIVE: To explore the role of bromodomain and extra terminal (BET) bromodomain in hematopoietic differentiation from human enbryonic stem cells (hESC). METHODS: The effect of BET hematopoietic inhibitor I-BET151 on hematopoietic differentiation from hESC was detected by using a monolayer hematopoietic defferentiation model, immunofluorescence, flow cytometry and real-time PCR; moreover the role of I-BET151 in process of hematopoietic differentiation was explored by adding I-BET151 in different differentiation stages. RESULTS: The analysis results of immunofluorescence, flow cytometry and real-time PCR showed that I-BET 151 significantly inhibited the generation of CD43 positive hematopoietic stem and progenitor cells (HSPCs). It was found that the addition of I-BET 151 in different stages, including APLNR+ lateral plate mesoderm production, CD34+CD31+ hemogenic endothelium (HEP) generation and endothelial-to-hematopoietic transition, significantly suppressed the generation of CD43 positive hematopoietic progenitor cells. CONCLUSION: I-BET 151 inhibites hematopoietic differentiation from hESCs at several stages, suggesting that the BET bromodomain plays important roles in multiple stages of hematopoietic differentiation from hESCs.

Identification of four differentially methylated genes as prognostic signatures for stage I lung adenocarcinoma.

BACKGROUND: Lung adenocarcinoma (LUAD) is the main subtype of non-small cell lung cancer with a low survival prognosis. We aimed to generate a prognostic model for the postoperative recurrence of LUAD. METHODS: The methylated DNA data of LUAD patients were downloaded from the Cancer Genome Atlas (TCGA). The differentially methylated genes were identified and protein-protein interacting network was constructed, with which prognostic signature of this cancer was generated. Survival and functional pathways analysis w used to evaluate the clustering ability of the prognostic signature. RESULTS: We identified 151 differentially methylated genes related to relapse-free survival of patients with LUAD. Nine hub genes were identified in PPI network, with which 4 gene pair signature was selected as prognostic signature. The potential functions of 6 genes (JDP2, SERPINA5, PLG, SEMG2, RFX5, and POLR3B) in the 4-gene pair signature were enriched in intracellular protein synthesis and transportation. CONCLUSION: The four gene pair signature can predict the prognosis of patients with stage I LUAD. Our study provides a reference for patients with postoperative adjuvant therapy.

Efficient generation of targeted and controlled mutational events in porcine cells using nuclease-directed homologous recombination.

BACKGROUND: Nuclease-based genome editing has rapidly sped the creation of new models of human disease. These techniques also hold great promise for the future of clinical xenotransplantation and cell-based therapies for cancer or immunodeficient pathology. However, to fully realize the potential of nuclease editing tools, the efficiency and precision of their application must be optimized. The object of this study was to use nonintegrating selection and nuclease-directed homologous recombination to efficiently control the genetic modification of the porcine genome. METHODS: Clustered randomly integrating spaced palindromic repeats and associated Cas9 protein (CRISPR/Cas9)-directed mutagenesis with a single-guide RNA target was designed to target the alpha-1,3-galactosyltransferase locus (GGTA1) of the porcine genome. A vector expressing a single-guide RNA, Cas9 protein, and green fluorescent protein was used to increase plasmid-delivered mutational efficiency when coupled with fluorescence sorting. Single and double-strand DNA oligonucleotides with a restriction site replacing the start codon were created with variable homology lengths surrounding the mutational event site. Finally, a transgene construct was flanked with 50 base pairs of homology directed immediately 5' to a nuclease cut site. These products were introduced to cells with a constant concentration of CRISPR/cas9 vector. Phenotype-specific mutational efficiency was measured by flow cytometer. Controlled homologous insertion was measured by Sanger sequence, restriction enzyme digest and flow cytometry. RESULTS: Expression of a fluorescence protein on the Cas9 vector functioned as a nonintegrating selection marker. Selection by this marker increased phenotype-silencing mutation rates from 3.5% to 82% (P = 0.0002). Insertion or deletion mutation increased from 11% to 96% (P = 0.0007). Co-transfection with homologous DNA oligonucleotides increased the aggregate phenotype-silencing mutation rates up to 22% and increased biallelic events. Single-strand DNA was twice as efficient as double-strand DNA. Furthermore, nuclease-mediated insertion by homology-directed repair successfully drove locus-specific transgene expression in the porcine genome. CONCLUSIONS: A nonintegrating selection strategy based on fluorescence expression can increase the mutational efficiency of the CRISPR/Cas9 system. The precision of this system can be increased by the addition of a very short homologous template sequence and can serve as a method for locus-specific transgene delivery. Together these strategies may be used to efficiently control mutational events. This system may be used to better use the potential of nuclease-mediated genomic editing.

Association of STAT4 rs7574865 polymorphism with susceptibility to inflammatory bowel disease: A systematic review and meta-analysis.

OBJECTIVE: Association of Signal transducers and activators of transcription-4 (STAT4) gene polymorphism with susceptibility to inflammatory bowel disease have been investigated in a number of epidemiological studies, but the results are inclusive. The aim of this meta-analysis was to more precisely estimate the relationship. METHODS: The databases of Pubmed and CBM updated to October, 2014 were retrieved. Random- or fixed-effect model was used to estimate odd radio (OR) and corresponding 95% confidence interval (95%CI) on the basis of heterogeneity. RESULTS: Seven articles containing 2196 Crohn's disease (CD) cases, 1588 ulcerative colitis (UC) cases and 4126 controls were identified. We detected a significant association between STAT4 rs7574865 polymorphism and IBD susceptibility in overall population (GG vs. GT+TT, OR=0.855, 95% CI=0.760-0.962, P=0.009), but not in Caucasian and Asian population, respectively. No association was detected between rs7574865 polymorphism and CD susceptibility in overall, Asian and Caucasian population, respectively. Interestingly, a significant association was detected between rs7574865 with UC susceptibility in overall population (G vs. T, OR=0.881, 95% CI=0.798-0.972, P=0.012; GG vs. GT+TT, OR=0.788, 95% CI=0.679-0.914, P=0.002; GG vs. TT, OR=0.683, 95% CI=0.498-0.937, P=0.018) and Caucasians (GG vs. GT+TT, OR=0.833, 95% CI=0.701-0.990, P=0.038; GG+GT vs. TT, OR=0.667, 95% CI=0.456-0.975, P=0.037; GG vs. TT, OR=0.636, 95% CI=0.433-0.934, P=0.021), respectively, and a possible association was found in Asian population (GG vs. GT+TT, OR=0.709, 95% CI=0.503-0.998, P=0.049). CONCLUSIONS: STAT4 rs7574865 gene is IBD risk factor, and this gene polymorphism is associated with UC susceptibility, especially in Caucasians. To confirm these findings, further studies with more sample size are required for a definitive conclusion.

Development of a cytokine-producing immortalized murine Kupffer cell line.

Kupffer cells (KC) play a critical role in both liver physiology and the pathogenesis of various liver diseases. Isolated primary KC have a limited lifespan in culture, and due to the relatively low number obtained, limit their study in vitro. Here, a cytokine-producing immortalized KC (ImKC) line was established from transgenic mice that express the thermolabile mutant tsA58 of the Simian virus 40 large T antigen under the control of the H-2k(b) promoter. Primary KC were obtained using a three step procedure: liver perfusion, centrifugal elutriation, and sorting for F4/80⁺ cells. ImKC were identified within the small-intermediate population of KC that maintained stable expression of F4/80, and the surface antigens CD11b, CD14 and TLR4. ImKC grow at IFNγ-independent manner at 37°C and exhibited a doubling time of ∼24 h when cultured in RPMI 1640 with 5% FBS. Our observations indicate that both activation of telomerase and expression of P53 are markedly increased, suggesting that enhanced telomerase activity and P53 expression may contribute to the immortalization of this cell population. ImKC cells maintained a high capacity to phagocytose FITC-latex beads, and bind/phagocytose erythrocytes. In addition, similar to primary KC, ImKC responded to stimulation with lipopolysaccharide (LPS: 0.1-1µg/ml) by upregulating mRNA levels of TNFα (23-fold), IL-6 (28-fold), and IL-1ß (1459-fold), as measured by qRT-PCR. Protein levels of TNFα and IL-6 were also increased, 10-fold and 12-fold, respectively. Reactive oxygen species (ROS) and nitric oxide (NO) production were significantly enhanced in ImKC following an LPS challenge. Furthermore, LPS elicited a marked increase in mitogen activated protein kinase (MAPK) phospho-(ERK1/2, JNK) and NF-κB p50 with decreased IκBα in ImKC, as assessed by Western blot. Collectively, these results demonstrate that the ImKC line retains critical characteristics of primary KC, and thus provides a useful tool to assess the role of KC in liver injury and chronic diseases.

Contribution of environment and genetics to pancreatic cancer susceptibility.

Several risk factors have been identified as potential contributors to pancreatic cancer development, including environmental and lifestyle factors, such as smoking, drinking and diet, and medical conditions such as diabetes and pancreatitis, all of which generate oxidative stress and DNA damage. Oxidative stress status can be modified by environmental factors and also by an individual's unique genetic makeup. Here we examined the contribution of environment and genetics to an individual's level of oxidative stress, DNA damage and susceptibility to pancreatic cancer in a pilot study using three groups of subjects: a newly diagnosed pancreatic cancer group, a healthy genetically-unrelated control group living with the case subject, and a healthy genetically-related control group which does not reside with the subject. Oxidative stress and DNA damage was evaluated by measuring total antioxidant capacity, direct and oxidative DNA damage by Comet assay, and malondialdehyde levels. Direct DNA damage was significantly elevated in pancreatic cancer patients (age and sex adjusted mean ± standard error: 1.00 ± 0.05) versus both healthy unrelated and related controls (0.70 ± 0.06, p<0.001 and 0.82 ± 0.07, p = 0.046, respectively). Analysis of 22 selected SNPs in oxidative stress and DNA damage genes revealed that CYP2A6 L160H was associated with pancreatic cancer. In addition, DNA damage was found to be associated with TNFA -308G>A and ERCC4 R415Q polymorphisms. These results suggest that measurement of DNA damage, as well as select SNPs, may provide an important screening tool to identify individuals at risk for development of pancreatic cancer.

Immortalized porcine liver sinusoidal endothelial cells: an in vitro model of xenotransplantation-induced thrombocytopenia.

BACKGROUND: Xenotransplantation has the potential to solve the critical shortage of human organs available for allotransplantation. The major barrier to porcine liver xenotransplantation is sequestration of human platelets causing thrombocytopenia. Porcine liver sinusoidal endothelial cells (LSEC) bind and phagocytose human platelets at least in part through binding of the asialoglycoprotein receptor 1 (ASGR1). Our purpose was to generate an immortalized porcine LSEC (iLSEC) line that mimics primary LSEC in ASGR1 expression and phagocytosis of human platelets. Porcine iLSEC would enable continued study of xenotransplantation-induced thrombocytopenia in vitro with fewer animals sacrificed. METHODS: Primary domestic porcine LSEC were transduced with lentiviral vector expressing the large and small T antigen of SV40 (SV40 TAg). The phenotype and genotype of the immortalized LSEC were compared with primary LSEC. RESULTS: A total of eight clones expressing SV40 TAg were isolated, and one clone was subcultured and analyzed for growth, phenotype, and function during passages 15-40. Expression of the SV40 TAg was confirmed by confocal microscopy and western blot. MTS cell proliferation assay demonstrated that the clone rapidly grew in culture medium with 2-10% fetal bovine serum. iLSEC expressed the endothelial cell marker, CD31, as determined by confocal microscopy and flow cytometry. Activation of iLSEC by treatment with lipopolysaccharide (LPS) resulted in upregulation of the inflammatory cytokine interleukin 6 (IL 6) by qPCR and ELISA. iLSEC phagocytosed human serum albumin and latex beads as measured by flow cytometry. Human platelets were phagocytosed by immortalized porcine LSEC. CONCLUSIONS: Immortalized porcine LSEC retain a phagocytic phenotype, making them a good model for the study of xenotransplantation-induced thrombocytopenia and may provide further insight into the phagocytic role of LSEC.

Structure analysis and comparative characterization of the cytochrome c' and flavocytochrome c from thermophilic purple photosynthetic bacterium Thermochromatium tepidum.

The thermodynamic and spectroscopic properties of two soluble electron transport proteins, cytochrome (Cyt) c' and flavocytochrome c, isolated from thermophilic purple sulfur bacterium Thermochromatium (Tch.) tepidum were examined and compared with those of the corresponding proteins from a closely related mesophilic bacterium Allochromatium (Alc.) vinosum. These proteins share sequence identities of 82% for the cytochromes c' and 86% for the flavocytochromes c. Crystal structures of the two proteins have been determined at high resolutions. Differential scanning calorimetry and denaturing experiments show that both proteins from Tch. tepidum are thermally and structurally much more stable than their mesophilic counterparts. The denaturation temperature of Tch. tepidum Cyt c' was 22 °C higher than that of Alc. vinosum Cyt c', and the midpoints of denaturation using guanidine hydrochloride were 2.0 and 1.2 M for the Tch. tepidum and Alc. vinosum flavocytochromes c, respectively. The enhanced stabilities can be interpreted on the basis of the structural and sequence information obtained in this study: increased number of hydrogen bonds formed between main chain nitrogen and oxygen atoms, more compact structures and reduced number of glycine residues. Many residues with large side chains in Alc. vinosum Cyt c' are substituted by alanines in Tch. tepidum Cyt c'. Both proteins from Tch. tepidum exhibit high structural similarities to their counterparts from Alc. vinosum, and the different residues between the corresponding proteins are mainly located on the surface and exposed to the solvent. Water molecules are found in the heme vicinity of Tch. tepidum Cyt c' and form hydrogen bonds with the heme ligand and C-terminal charged residues. Similar bound waters are also found in the vicinity of one heme group in the diheme subunit of Tch. tepidum flavocytochrome c. Electron density map of the Tch. tepidum flavocytochrome c clearly revealed the presence of disulfur atoms positioned between two cysteine residues at the active site near the FAD prosthetic group. The result strongly suggests that flavocytochrome c is involved in the sulfide oxidation in vivo. Detailed discussion is given on the relationships between the crystal structures and the spectroscopic properties observed for these proteins.

Metal cations modulate the bacteriochlorophyll-protein interaction in the light-harvesting 1 core complex from Thermochromatium tepidum.

The light-harvesting 1 reaction center (LH1-RC) complex from Thermochromatium (Tch.) tepidum exhibits unusual Q(y) absorption by LH1 bacteriochlorophyll-a (BChl-a) molecules at 915nm, and the transition energy is finely modulated by the binding of metal cations to the LH1 polypeptides. Here, we demonstrate the metal-dependent interactions between BChl-a and the polypeptides within the intact LH1-RC complexes by near-infrared Raman spectroscopy. The wild-type LH1-RC (B915) exhibited Raman bands for the C3-acetyl and C13-keto CO stretching modes at 1637 and 1675cm(-1), respectively. The corresponding bands appeared at 1643 and 1673cm(-1) when Ca(2+) was biosynthetically replaced with Sr(2+) (B888) or at 1647 and 1669cm(-1) in the mesophilic counterpart, Allochromatium vinosum. These results indicate the significant difference in the BChl-polypeptide interactions between B915 and B888 and between B915 and the mesophilic counterpart. The removal of the original metal cations from B915 and B888 resulted in marked band shifts of the C3-acetyl/C13-carbonyl νCO modes to ~1645/~1670cm(-1), supporting a model in which the metal cations are involved in the fine-tuning of the hydrogen bonding between the BChl-a and LH1-polypeptides. Interestingly, the interaction modes were almost identical between the Ca(2+)-depleted B915 and Sr(2+)-depleted B888 and between B915 and Ca(2+)-substituted B888, despite the significant differences in their LH1 Q(y) peak positions and the denaturing temperatures, as revealed by differential scanning calorimetry. These results suggest that not only the BChl-polypeptide interactions but some structural origin may be involved in the unusual Q(y) red-shift and the enhanced thermal stability of the LH1-RC complexes from Tch. tepidum.

Gene sequencing and characterization of the light-harvesting complex 2 from thermophilic purple sulfur bacterium Thermochromatium tepidum.

In this study, gene sequences coding for the light-harvesting (LH) 2 polypeptides from a thermophilic purple sulfur bacterium Thermochromatium tepidum are reported and characterization of the LH2 complex is described. Three sets of pucBA genes have been identified, and the gene products have been analyzed by electrophoresis and reversed-phase chromatography. The result shows that all of the genes are expressed but the distribution of the expression is not uniform. The gene products undergo post-translational modification, where two of the ß-polypeptides appear to be N-terminally methylated. Absorption spectrum of the purified LH2 complex exhibits Q (y) transitions at 800 and 854 nm in dodecyl ß-maltopyranoside solution, and the circular dichroism spectrum shows a "molischianum"-like characteristic. No spectral change was observed for the LH2 when the bacterium was cultured under different conditions of light intensity. In lauryl dimethylamine N-oxide (LDAO) solution, significant changes in the absorption spectrum were observed. The B850 peak decreased and blue-shifted with increasing the LDAO concentration, whereas the B800 intensity increased without change in the peak position. The spectral changes can be partially or almost completely reversed by addition of metal ions, and the divalent cations seem to be more effective. The results indicate that ionic interactions may exist between LH2, detergent molecules and metal ions. Possible mechanisms involved in the detergent- and cation-induced spectral changes are discussed.

Examination of the putative Ca2+-binding site in the light-harvesting complex 1 of thermophilic purple sulfur bacterium Thermochromatium tepidum.

The core light-harvesting complex (LH1) of purple sulfur photosynthetic bacterium Thermochromatium tepidum exhibits an unusual absorption maximum at 915 nm for the Q (y) transition, and is highly stable when copurified with reaction center (RC) in a LH1-RC complex form. In previous studies, we demonstrated that the calcium ions are involved in both the large red shift and the enhanced thermal stability, and possible Ca(2+)-binding sites were proposed. In this study, we further examine the putative binding sites in the LH1 polypeptides using purified chromatophores. Incubation of the chromatophores in the presence of EDTA revealed no substantial change in the absorption maximum of LH1 Q (y) transition, whereas further addition of detergents to the chromatophores-EDTA solution resulted in a blue-shift for the LH1 Q (y) peak with the final position at 892 nm. The change of the LH1 Q (y) peak to shorter wavelengths was relatively slow compared to that of the purified LH1-RC complex. The blue-shifted LH1 Q (y) transition in chromatophores can be restored to its original position by addition of Ca(2+) ions. The results suggest that the Ca(2+)-binding site is exposed on the inner surface of chromatophores, corresponding to the C-terminal region of LH1. An Asp-rich fragment in the LH1 α-polypeptide is considered to form a crucial part of the binding network. The slow response of LH1 Q (y) transition upon exposure to EDTA is discussed in terms of the membrane environment in the chromatophores.

[The effect of the thoracic cage on the vertebral axial rotation of adolescent idiopathic scoliosis: a biomechanical study].

OBJECTIVE: To investigate the effect of the rib cage on the vertebral axial rotation of adolescent idiopathic scoliosis under axial load condition. METHODS: Three dimensional finite element model of adolescent idiopathic scoliosis included and excluded thoracic cage was built based on the data of computer tomography. The model was imported into the preprocessor of the ANSYS 8.0 software for assigning boundary and loading conditions. Then the axial loading condition was simulated after entering the solution modular. The magnitude and direction of each vertebral axial rotation of the scoliotic spine were read and analyzed in the postprocessor of the ANSYS software. RESULTS: The rib cage had a significant influence on the axial rotation of the vertebra above the structural curve and had no influence on the axial rotation of the lumbar and sacral vertebra. The effect of the thoracic cage on the axial rotation of the apical vertebra was limited. Under different loading conditions, the apical vertebra of both models rotated in the same direction. The magnitude of the vertebral rotation of both models has no statistical significance. CONCLUSIONS: Adolescent idiopathic scoliosis can lead to the anatomical changes of the vertebra and the thoracic cage. The corresponding changes of biomechanical features of the scoliotic spine and rib cage would occur. The deformed thoracic cage could not maintain the rotation stability as the normal one.

Role of APE1 in differentiated neuroblastoma SH-SY5Y cells in response to oxidative stress: use of APE1 small molecule inhibitors to delineate APE1 functions.

Oxidative DNA damage has been implicated in a number of central nervous system pathologies. The base excision repair (BER) pathway is one of the most important cellular protection mechanisms that respond to oxidative DNA damage. Human apurinic (apyrimidinic) endonuclease/redox effector factor (APE1/Ref-1 or APE1) is an essential enzyme in the BER pathway and is expressed in both mitotic and post-mitotic cells in humans. In neurons, a reduction of APE1 expression increases chemotherapy-induced cytotoxicity, while overexpression of APE1 protects cells against the cytotoxicity. However, given the multiple functions of APE1, knockdown of total APE1 is not completely informative of whether it is the redox or DNA repair activity, or interactions with other proteins. Therefore, the use of selective small molecules that can block each function independent of the other is of great benefit in ascertaining APE1 function in post-mitotic cells. In this study, we chose differentiated SH-SY5Y cells as our post-mitotic cell line model to investigate whether a drug-induced decrease in APE1 DNA repair or redox activity contributes to the growth and survival of post-mitotic cells under oxidative DNA damaging conditions. Here, we demonstrate that overexpression of WT-APE1 or C65-APE1 (repair competent) results in significant increase in cell viability after exposure to H(2)O(2). However, the 177/226-APE1 (repair deficient) did not show a protective effect. This phenomenon was further confirmed by the use of methoxyamine (MX), which blocks the repair activity of APE1 that results in enhanced cell killing and apoptosis in differentiated SH-SY5Y cells and in neuronal cultures after oxidative DNA damaging treatments. Blocking APE1 redox function by a small molecule inhibitor, BQP did not decrease viability of SH-SY5Y cells or neuronal cultures following oxidative DNA damaging treatments. Our results demonstrate that the DNA repair function of APE1 contributes to the survival of nondividing post-mitotic cells following oxidative DNA damage.

[Combined pedicle subtraction osteotomy and polysegmental closing wedge osteotomy for correction of the severe thoracolumbar kyphotic deformity in ankylosing spondylitis].

OBJECTIVE: To study retrospectively the efficacy and complications of combined pedicle subtraction osteotomy (PSO) and polysegmental closing wedge osteotomy for correction of the severe rigid thoracolumbar kyphotic deformity in ankylosing spondylitis (AS). METHODS: A total of 8 consecutive male patients with AS and severe thoracolumbar kyphotic deformity (mean age 32 years, range 28 - 46) were involved in this study from August 2004 to June 2007. The average preoperative Cobb angle of thoracic spine (T(1)-T(12)) was 96 degrees (range, 80 degrees - 112 degrees ), the mean preoperative angle of lumbar lordosis (L(1)-S(1)) was 10 degrees (5 degrees - 15 degrees ). The mean chin-brow angle was 47 degrees (range, 40 degrees - 58 degrees ). The average gaze angle was 43 degrees (range, 32 degrees - 50 degrees ). After preoperative assessment, single-level PSO was performed in L(3) vertebrae and two-level polysegmental closing wedge osteotomy was performed in thoracolumbar vertebrae (T(12)-L(1), L(1-2)). Radiographic and clinical results and complications were assessed. RESULTS: The surgical time was (298.1 +/- 20.7) minutes and blood loss during the procedure was (1588.8 +/- 171.6) ml. The follow-up period was (11.5 +/- 7.7) months. The postoperative angle and the amount of correction of the thoracic and lumbar spine were 76.1 degrees +/- 9.6 degrees , 20.3 degrees +/- 1.1 degrees and 48.4 degrees +/- 4.7 degrees , 38.4 degrees +/- 4.7 degrees respectively. The postoperative chin-brow and gaze angle was 16.5 degrees +/- 4.6 degrees and 73.0 degrees +/- 5.2 degrees , respectively. The amount of correction for sagittal balance was (12.3 +/- 1.6) cm. No nerve, vascular injury, stress fracture and coronal decompensation occurred in the patients. CONCLUSIONS: Combined PSO and polysegmental closing wedge osteotomy by posterior approach only is safe and effective for correction of the severe rigid thoracolumbar kyphotic deformity in AS. The visual field is significantly improved after surgery.

Specific Ca2+-binding motif in the LH1 complex from photosynthetic bacterium Thermochromatium tepidum as revealed by optical spectroscopy and structural modeling.

Native and Ca(2+)-depleted light-harvesting-reaction center core complexes (LH1-RC) from the photosynthetic bacterium Thermochromatium (Tch.) tepidum exhibit maximal LH1-Q(y) absorption at 915 and 889 nm, respectively. To understand the structural origins of the spectral variation, we performed spectroscopic and structure modeling investigations. For the 889 nm form of LH1-RC, bacteriochlorophyll a (BChl a) in the native form was found by means of near-infrared Fourier-transform Raman spectroscopy, a higher degree of macrocycle distortion and a stronger hydrogen bond with the beta-Trp(-8) residue. SWISS-MODEL structure modeling suggests the presence of a specific coordination motif of Ca(2+) at the C-terminus of the alpha-subunit of LH1, while MODELLER reveals the tilt of alpha- and beta-polypeptides with reference to the structural template, as well as a change in the concentric orientation of BChl a molecules, both of which may be connected to the long-wavelength LH1-Q(y) absorption of the 915 nm form. The carotenoid spirilloxanthin shows a twisted all-trans configuration in both forms of LH1 as evidenced by the resonance Raman spectroscopic results. With regard to the thermal stability, the 915 nm form was shown by the use of temperature-dependent fluorescence spectroscopy to be approximately 20 K more stable than the 889 nm form, which may be ascribed to the specific Ca(2+)-binding motif of LH1.