FOXO1 represses MCL1 transcription to regulate the function of vascular smooth muscle cells in intracranial aneurysm.

Intracranial aneurysm (IA) is a pathological dilation of the cerebral arteries. Vascular smooth muscle cell (VSMC) dysfunction assumes a role in IA development. In this context, this study probed the role of FOXO1 in human brain VSMC (HBVSMC) function via MCL1. FOXO1 and MCL1 expression in arterial wall tissues from IA patients and inflammatory cytokines (IL-1ß, TNF-α, and IL-6) levels in the serum of IA patients were, respectively, detected with qRT-PCR and ELISA. Pearson's correlation analysis was utilized to analyze the correlation between FOXO1 and MCL1. After FOXO1 and/or MCL1 were overexpressed in HBVSMCs, caspase-3 and Cyt-c protein expression were examined by western blot, cell proliferation by CCK-8 and EdU assays, and cell apoptosis by flow cytometry. IL-1ß, TNF-α, and IL-6 levels were assessed in the supernatant of HBVSMCs with ELISA. Dual-luciferase gene reporter and ChIP assays were conducted to evaluate the binding of FOXO1 to MCL1. FOXO1 expression was high and MCL expression was low in arterial wall tissues from IA patients, and IL-1ß, TNF-α, and IL-6 levels were high in the serum of IA patients. There was an inverse correlation between FOXO1 and MCL1 mRNA levels. Moreover, FOXO1 bound to the MCL1 promoter to decrease MCL1 transcription. In addition, FOXO1 overexpression augmented cell apoptosis, caspase-3 and Cyt-c protein expression, and IL-1ß, TNF-α, and IL-6 secretion, while reducing cell proliferation in HBVSMCs, which was abrogated by further MCL1 overexpression. FOXO1 impeded MCL1 transcription to curb HBVSMC proliferation and facilitate their apoptosis and inflammation.

Production and sequence validation of a complete full length ORF collection for the pathogenic bacterium Vibrio cholerae.

Cholera, an infectious disease with global impact, is caused by pathogenic strains of the bacterium Vibrio cholerae. High-throughput functional proteomics technologies now offer the opportunity to investigate all aspects of the proteome, which has led to an increased demand for comprehensive protein expression clone resources. Genome-scale reagents for cholera would encourage comprehensive analyses of immune responses and systems-wide functional studies that could lead to improved vaccine and therapeutic strategies. Here, we report the production of the FLEXGene clone set for V. cholerae O1 biovar eltor str. N16961: a complete-genome collection of ORF clones. This collection includes 3,761 sequence-verified clones from 3,887 targeted ORFs (97%). The ORFs were captured in a recombinational cloning vector to facilitate high-throughput transfer of ORF inserts into suitable expression vectors. To demonstrate its application, approximately 15% of the collection was transferred into the relevant expression vector and used to produce a protein microarray by transcribing, translating, and capturing the proteins in situ on the array surface with 92% success. In a second application, a method to screen for protein triggers of Toll-like receptors (TLRs) was developed. We tested in vitro-synthesized proteins for their ability to stimulate TLR5 in A549 cells. This approach appropriately identified FlaC, and previously uncharacterized TLR5 agonist activities. These data suggest that the genome-scale, fully sequenced ORF collection reported here will be useful for high-throughput functional proteomic assays, immune response studies, structure biology, and other applications.

The role of matrix metalloproteinase 14 polymorphisms in susceptibility to intervertebral disc degeneration in the Chinese Han population.

INTRODUCTION: Matrix metalloproteinase 14 (MMP14) plays an important role in the pathophysiology of intervertebral disc degeneration (IVDD). The present study aimed to determine whether two single nucleotide polymorphisms (-378 T/C and -364 G/T) of MMP14 were associated with the risk and severity of IVDD in the Chinese Han population. MATERIAL AND METHODS: A total of 908 patients with IVDD and 906 healthy controls were enrolled in this study. The grade of disc degeneration was determined according to Schneiderman's classification for magnetic resonance imaging. The polymorphisms of MMP14 were genotyped using polymerase chain reaction and direct sequencing. RESULTS: The genotype distribution of -364G/T did not show a significant difference between IVDD patients and healthy controls. The frequencies of the -378T/C and CC genotypes were significantly lower among IVDD patients compared with healthy controls (p < 0.001); unconditional logistic regression analysis revealed that the CT and CC genotypes were significantly associated with a decreased risk of IVDD compared with the TT genotype (p < 0.001). Patients with IVDD showed significantly higher frequencies of the T allele at -378T/C than healthy controls (p < 0.001). In addition, the -375 CC genotype, as well as the C allele, was associated with lower degenerative grades of IVDD compared with the TT genotype and the T allele, respectively (both p < 0.001). CONCLUSIONS: The -378T/C polymorphism of MMP14 may be associated with the risk and severity of IVDD in the Chinese Han population. It shows potential to become a biomarker to predict risk and severity of IVDD.

Comparison of rivaroxaban and parnaparin for preventing venous thromboembolism after lumbar spine surgery.

BACKGROUND: The aim of this study was to evaluate the effectiveness and safety of rivaroxaban for preventing venous thromboembolism (VTE) after lumbar spine surgery. METHODS: In this randomized, controlled study, 665 patients who underwent lumbar surgery were randomly assigned to receive either rivaroxaban or parnaparin. Rivaroxaban and parnaparin were used for preventing postoperative venous thrombosis. The occurrence of postoperative efficacy endpoint events (venous thrombosis) and safety endpoint events (hemorrhage) was compared for each group. RESULTS: Efficacy endpoint results: in the rivaroxaban group, there were 6 thrombotic events (1.7%), 2 cases with severe VTE (0.6%), and 3 cases with symptomatic VTE (0.9%). In the parnaparin group, there were 10 thrombotic events (3.1%), 4 cases with severe VTE (1.2%), and 6 cases with symptomatic VTE (1.9%). Safety endpoint results: in the rivaroxaban group, there were 21 cases with bleeding events (6.2%), 2 cases with severe bleeding (0.6%), and 19 cases with non-severe bleeding (5.6%). In the parnaparin group, there were 21 bleeding events (6.2%), 1 case with severe bleeding (0.3%), and 16 cases with non-severe bleeding (4.9%). The incidences of thromboembolic events, including severe and symptomatic VTE, were not significantly different between the two groups (P > 0.05). Bleeding event rates, including severe and non-severe bleeding, were also not significantly different. CONCLUSIONS: Rivaroxaban proved to be equally effective as parnaparin for anticoagulation therapy, with both drugs exhibiting a similar prevention effect against postoperative VTE after lumbar spine surgery, without increasing the risk of postoperative bleeding.

Management of subaxial cervical facet dislocation through anterior approach monitored by spinal cord evoked potential.

STUDY DESIGN: Retrospective case series. OBJECTIVE: To discuss the clinical efficacy of anterior cervical surgery of decompression, reduction, stabilization, and fusion in treating subaxial cervical facet dislocation without spinal cord injury or with mild spinal cord injury monitored by spinal cord evoked potential. SUMMARY OF BACKGROUND DATA: The optimal treatment of lower cervical facet dislocation has been controversial. Because of the risk of iatrogenic damage of neurological function, it is challenging for surgeons to manage the lower cervical facet dislocation without or with mild spinal cord injury. To avoid the risks, more secure strategy need to be designed. METHODS: A retrospective study was performed on 17 cases of subaxial cervical facet dislocation without spinal cord injury or with mild spinal cord injury treated by anterior cervical surgery under spinal cord evoked potential monitor from January 2008 to June 2012. There were 12 males, 5 females, with a mean age of 40.1 years (from 21 to 73 yr). Dislocation sites: 1 in C3-C4, 2 in C4-C5, 6 in C5-C6, 8 in C6-C7; 10 cases with unilateral cervical facet dislocation, 7 cases with bilateral dislocation. Thirteen patients were preoperatively classified as grade D and 4 as E according to Frankel standard. All patients were followed up for average of 16 months. RESULTS: All operations were completed successfully. Postoperative radiographs showed that the sequence and curvature of the cervical spine were well recovered. And, evidence of intervertebral fusion was observed at 3 months in all cases. No redislocation or symptoms of spinal cord injury occurred. Thirteen cases with mild spinal cord injury recovered at 1 month after operation. CONCLUSION: Anterior cervical surgery of decompression, reduction, stabilization, and fusion monitored by spinal cord evoked potential is an effective and safe method for treatment of subaxial cervical facet dislocation without or with mild spinal cord injury. LEVEL OF EVIDENCE: 4.

Bone cement enhanced pedicle screw fixation combined with vertebroplasty for elderly patients with malignant spinal tumors.

BACKGROUND: Older patients with malignant spinal tumors are difficult to treat because they have many co-morbidities including osteoporosis. The purpose of this research is to discuss the technique and clinical outcome of bone cement enhanced pedicle screw fixation combined with vertebroplasty (the Sandwich Procedure) for elderly patients with severe osteoporosis and malignant spinal tumors. METHODS: This study includes 28 consecutive elderly patients with malignant thoracic or lumbar spinal tumors. There were nine patients with myelomas, and 19 patients with metastatic bone tumors. The Sandwich Procedure began with curettage of the tumor and a vertebroplasty with bone cement (polymethyl methacrylate, PMMA), followed by PMMA enhanced pedicle screw fixation. Patients were evaluated with the visual analogue scale (VAS), oswestry disability index (ODI), American Spinal Cord Injury Association (ASIA) neurological function classification, and the radiographic degree of kyphosis (Cobb angle). Data were analyzed using paired t-test to compare the pre- and post-operative values. The complications, local recurrences, and the survival status were also recorded. RESULTS: There was no operative mortality, and the mean operative time was 210 minutes (range 150 - 250 minutes). The average blood loss was 1550 ml (range 650 - 3300 ml). The average amount of cement for vertebroplasty was 3.6 ml (range 3 -5 ml). The VAS, ODI, and ASIA scores were significantly improved after surgery (P < 0.05). However, we found no differences between the pre and post-operative Cobb angles. The shortest survival time was 3 months, and we found no evidence of local recurrence in this group of patients. CONCLUSION: The Sandwich Procedure is a safe operation and provides symptomatic relief in these difficult patients, permitting further treatment with chemotherapy or radiotherapy.

Risk factor analysis of proximal junctional kyphosis after posterior fusion in patients with idiopathic scoliosis.

STUDY DESIGN: A retrospective analysis of 150 adolescents who underwent spinal fusion for idiopathic scoliosis. OBJECTIVE: To analyse the incidence of the postoperative proximal junctional kyphosis after posterior fusion to the upper thoracic vertebra in adolescents with idiopathic scoliosis and to explore its risk factors. SUMMARY OF BACKGROUND DATA: The reported incidence of the proximal junctional kyphosis after the posterior fusion in patients with idiopathic scoliosis varies depending on surgical methods and strategies adopted by the institution. METHODS: The changes in the Cobb angle of the proximal junctional kyphosis on the lateral spine X-ray were measured and the presence of PJK was recorded. The risk factors were screened using statistical analysis. RESULTS: PJK occurred in 35 out of 123 patients with an overall incidence of 28%. Among them, 28 patients (80%) experienced PJK within 1.5 years after surgery. The PJK-inducing factors included greater than 10 degrees intraoperative decrease in thoracic kyphosis, thoracoplasty, the use of a pedicle screw at the top vertebra, autogenous bone graft and fusion to the lower lumbar vertebra (below L2). CONCLUSIONS: There is a high incidence of postoperative proximal junctional kyphosis after posterior fusion to the upper thoracic vertebra within 1.5 years after surgery in adolescents with idiopathic scoliosis. In order to reduce its incidence, the risk factors for PJK should be carefully evaluated before surgery.

A biomedically enriched collection of 7000 human ORF clones.

We report the production and availability of over 7000 fully sequence verified plasmid ORF clones representing over 3400 unique human genes. These ORF clones were derived using the human MGC collection as template and were produced in two formats: with and without stop codons. Thus, this collection supports the production of either native protein or proteins with fusion tags added to either or both ends. The template clones used to generate this collection were enriched in three ways. First, gene redundancy was removed. Second, clones were selected to represent the best available GenBank reference sequence. Finally, a literature-based software tool was used to evaluate the list of target genes to ensure that it broadly reflected biomedical research interests. The target gene list was compared with 4000 human diseases and over 8500 biological and chemical MeSH classes in approximately 15 Million publications recorded in PubMed at the time of analysis. The outcome of this analysis revealed that relative to the genome and the MGC collection, this collection is enriched for the presence of genes with published associations with a wide range of diseases and biomedical terms without displaying a particular bias towards any single disease or concept. Thus, this collection is likely to be a powerful resource for researchers who wish to study protein function in a set of genes with documented biomedical significance.

Building a human kinase gene repository: bioinformatics, molecular cloning, and functional validation.

Kinases catalyze the phosphorylation of proteins, lipids, sugars, nucleosides, and other important cellular metabolites and play key regulatory roles in all aspects of eukaryotic cell physiology. Here, we describe the mining of public databases to collect the sequence information of all identified human kinase genes and the cloning of the corresponding ORFs. We identified 663 genes, 511 encoding protein kinases, and 152 encoding nonprotein kinases. We describe the successful cloning and sequence verification of 270 of these genes. Subcloning of this gene set in mammalian expression vectors and their use in high-throughput cell-based screens allowed the validation of the clones at the level of expression and the identification of previously uncharacterized modulators of the survivin promoter. Moreover, expressions of the kinase genes in bacteria, followed by autophosphorylation assays, identified 21 protein kinases that showed autocatalytic activity. The work described here will facilitate the functional assaying of this important gene family in phenotypic screens and their use in biochemical and structural studies.

Proteome-scale purification of human proteins from bacteria.

The completion of the human genome project and the development of high-throughput approaches herald a dramatic acceleration in the pace of biological research. One of the most compelling next steps will be learning the functional roles of all proteins. Achievement of this goal depends in part on the rapid expression and isolation of proteins at large scale. We exploited recombinational cloning to facilitate the development of methods for the high-throughput purification of human proteins. cDNAs were introduced into a master vector from which they could be rapidly transferred into a variety of protein expression vectors for further analysis. A test set of 32 sequence-verified human cDNAs of various sizes and activities was moved into four different expression vectors encoding different affinity-purification tags. By means of an automatable 2-hr protein purification procedure, all 128 proteins were purified and subsequently characterized for yield, purity, and steps at which losses occurred. Under denaturing conditions when the His6 tag was used, 84% of samples were purified. Under nondenaturing conditions, both the glutathione S-transferase and maltose-binding protein tags were successful in 81% of samples. The developed methods were applied to a larger set of 336 randomly selected cDNAs. Sixty percent of these proteins were successfully purified under denaturing conditions and 82% of these under nondenaturing conditions. A relational database, FLEXProt, was built to compare properties of proteins that were successfully purified and proteins that were not. We observed that some domains in the Pfam database were found almost exclusively in proteins that were successfully purified and thus may have predictive character.