[Preparation of humanized anti-tissue factor antibody and its mechanism of killing colon cancer cells and inhibiting cell migration].

Objective To investigate the killing effect of humanized antibodies targeting tissue factors on colon cancer cells as well as migration-inhibiting effect. Methods Humanized anti-tissue factor antibody was purified by Protein A and gel filtration chromatography from cultured CHO-5G4.1 cells that highly express the antibody. The purity was detected by capillary SDS-PAGE. Anticoagulant activity was assessed using the prothrombin time test. Killing effect of the antibody on SW620 and SW480 colorectal cancer cells was tested using antibody-dependent cell-mediated cytotoxicity (ADCC). The effect of antibodies on cell migration was investigated using TranswellTM assay. Gelatin zymography and Western blotting were used to detect the changes of matrix metalloproteinase 2 (MMP2), MMP9, focal adhesion kinase (FAK) and phosphorylated FAK (p-FAK) after treatment with humanized anti-tissue factor antibody. Results The purity of the humanized anti-tissue factor antibody was estimated 96.9% by the two-step method. The purified antibody showed an obvious anticoagulant activity. The antibody treatment had a significant killing effect on colorectal cancer cells through ADCC and a significant inhibiting effect (99%) on cell migration. The antibody significantly inhibited expression of MMP2 and p-FAK. Conclusion The humanized anti-tissue factor antibody can effectively kill tumor cells through ADCC and inhibit cancer cell migration, which is possibly mediated by MMP2 expression through suppression of FAK signaling.

Design, Synthesis, and Preliminary Bioactivity Evaluation of 2,7-Substituted Carbazole Derivatives as Potent Autotaxin Inhibitors and Antitumor Agents†.

BACKGROUND: Autotaxin-LPA signaling has been implicated in cancer progression, and targeted for the discovery of cancer therapeutic agents. OBJECTIVE: Potential ATX inhibitors were synthesized to develop novel leading compounds and effective anticancer agents. METHODS: The present work designs and synthesizes a series of 2,7-subsitituted carbazole derivatives with different terminal groups R [R = -Cl (I), -COOH (II), -B(OH)2 (III), or -PO(OH)2 (I-IV)]. The inhibition of these compounds on the enzymatic activity of ATX was measured using FS-3 and Bis-pNpp as substrates, and the cytotoxicity of these compounds was evaluated using SW620, SW480, PANC-1, and SKOV-3 human carcinoma cells. Furthermore, the binding of leading compound with ATX was analyzed by molecular docking. RESULTS: Compound III was shown to be a promising antitumor candidate by demonstrating both good inhibition of ATX enzymatic activity and high cytotoxicity against human cancer cell lines. Molecular docking study shows that compound III is located in a pocket, which mainly comprises amino acids 209 to 316 in domain 2 of ATX, and binds with these residues of ATX through van der Waals, conventional hydrogen bonds, and hydrophobic interactions. CONCLUSION: Compound III with the terminal group R = -B(OH)2 has the most potent inhibitory effect with the greatest cytotoxicity to cancer cells. Moreover, the docking model provides a structural basis for the future optimization of promising antitumor compounds.

A whole-genome sequence study identifies genetic risk factors for neuromyelitis optica.

Neuromyelitis optica (NMO) is a rare autoimmune disease that affects the optic nerve and spinal cord. Most NMO patients ( > 70%) are seropositive for circulating autoantibodies against aquaporin 4 (NMO-IgG+). Here, we meta-analyze whole-genome sequences from 86 NMO cases and 460 controls with genome-wide SNP array from 129 NMO cases and 784 controls to test for association with SNPs and copy number variation (total N = 215 NMO cases, 1244 controls). We identify two independent signals in the major histocompatibility complex (MHC) region associated with NMO-IgG+, one of which may be explained by structural variation in the complement component 4 genes. Mendelian Randomization analysis reveals a significant causal effect of known systemic lupus erythematosus (SLE), but not multiple sclerosis (MS), risk variants in NMO-IgG+. Our results suggest that genetic variants in the MHC region contribute to the etiology of NMO-IgG+ and that NMO-IgG+ is genetically more similar to SLE than MS.

Improving genetic diagnosis in Mendelian disease with transcriptome sequencing.

Exome and whole-genome sequencing are becoming increasingly routine approaches in Mendelian disease diagnosis. Despite their success, the current diagnostic rate for genomic analyses across a variety of rare diseases is approximately 25 to 50%. We explore the utility of transcriptome sequencing [RNA sequencing (RNA-seq)] as a complementary diagnostic tool in a cohort of 50 patients with genetically undiagnosed rare muscle disorders. We describe an integrated approach to analyze patient muscle RNA-seq, leveraging an analysis framework focused on the detection of transcript-level changes that are unique to the patient compared to more than 180 control skeletal muscle samples. We demonstrate the power of RNA-seq to validate candidate splice-disrupting mutations and to identify splice-altering variants in both exonic and deep intronic regions, yielding an overall diagnosis rate of 35%. We also report the discovery of a highly recurrent de novo intronic mutation in COL6A1 that results in a dominantly acting splice-gain event, disrupting the critical glycine repeat motif of the triple helical domain. We identify this pathogenic variant in a total of 27 genetically unsolved patients in an external collagen VI-like dystrophy cohort, thus explaining approximately 25% of patients clinically suggestive of having collagen VI dystrophy in whom prior genetic analysis is negative. Overall, this study represents a large systematic application of transcriptome sequencing to rare disease diagnosis and highlights its utility for the detection and interpretation of variants missed by current standard diagnostic approaches.

Analysis of protein-coding genetic variation in 60,706 humans.

Large-scale reference data sets of human genetic variation are critical for the medical and functional interpretation of DNA sequence changes. Here we describe the aggregation and analysis of high-quality exome (protein-coding region) DNA sequence data for 60,706 individuals of diverse ancestries generated as part of the Exome Aggregation Consortium (ExAC). This catalogue of human genetic diversity contains an average of one variant every eight bases of the exome, and provides direct evidence for the presence of widespread mutational recurrence. We have used this catalogue to calculate objective metrics of pathogenicity for sequence variants, and to identify genes subject to strong selection against various classes of mutation; identifying 3,230 genes with near-complete depletion of predicted protein-truncating variants, with 72% of these genes having no currently established human disease phenotype. Finally, we demonstrate that these data can be used for the efficient filtering of candidate disease-causing variants, and for the discovery of human 'knockout' variants in protein-coding genes.

[Preparation and characterization of a monoclonal antibody against human tissue factor with anticoagulation activity].

OBJECTIVE: To prepare and characterize a monoclonal antibody (mAb) against human tissue factor (hTF) with anticoagulation activity. METHODS: BALB/c mice were immunized with truncated recombinant protein (rhTF243). Hybridoma cell lines were generated from cell fusion, and screened using indirect ELISA and prothrombin time (PT). After ascites was developed in BALB/c mice, antibody titers were determined using indirect ELISA. Western blotting was performed to study the antibody specificity. Anticoagulant activity of the antibody was detected by PT assay. RESULTS: A mAb to hTF with excellent anticoagulation activity was identified. Its immunoglobulin subclass belonged to IgG1. Titer of ascites fluid was 1:200 000. Western blotting and PT analysis confirmed the specificity and anticoagulant activity of the antibody. The mAb reacted specifically to both recombinant hTF243 and natural TF on SW620 colon cancer cell surface. CONCLUSION: A hTF mAb with anticoagulation activity and high specificity has been successfully prepared.

Human genomics. Effect of predicted protein-truncating genetic variants on the human transcriptome.

Accurate prediction of the functional effect of genetic variation is critical for clinical genome interpretation. We systematically characterized the transcriptome effects of protein-truncating variants, a class of variants expected to have profound effects on gene function, using data from the Genotype-Tissue Expression (GTEx) and Geuvadis projects. We quantitated tissue-specific and positional effects on nonsense-mediated transcript decay and present an improved predictive model for this decay. We directly measured the effect of variants both proximal and distal to splice junctions. Furthermore, we found that robustness to heterozygous gene inactivation is not due to dosage compensation. Our results illustrate the value of transcriptome data in the functional interpretation of genetic variants.

[Application of electric coagulation treatment via bronchoscopy in the management of congenital vallecular cyst in children].

OBJECTIVE: To discuss the effect of electric coagulation through bronchoscopy in diagnosis and treatment of congenital vallecular cyst in children. METHOD: Ten cases of congenital vallecular cyst in the study with age ranged from 21 days to 4 years and 10 months were treated with electric coagulation through bronchoscopy. The therapeutic effect was evaluated by endoscopic and clinical manifestation. And all the patients were followed-up for 6-12 months. RESULT: All the patients obtained 3-5 times electric coagulation. After the operation, the cyst decreased in size, epiglottis softening was subsided, uplift uncompression, dyspnea and laryngeal stridor were improved obviously. After follow-up periods of 6-12 months, no capsule wall were left, and the activity of the epiglottis resumed.No severe complication was found in any patient. CONCLUSION: Electric coagulation through bronchoscopy is a simple, effective and safe method to treat congenital vallecular cyst in children.

Research on the structure and performance of bacterial magnetic nanoparticles.

Magnetite nanocrystals have been widely used in many fields. Recently, a new magnetite nanocrystals, called magnetosome, has been found in magnetotactic bacteria. In this article, we researched on the properties of magnetosomes in detail, such as crystalline, morphology, crystal-size distributions, vitro cytotoxicity, and magnetic properties and quantified primary amino groups on the magnetosomes membrane surface by fluorescamine assay for the first time. From the results, it was clear that magnetosomes have more potential in the biomedical applications than synthetic magnetite.

Reconstructing phylogenetic networks using maximum parsimony.

Phylogenies - the evolutionary histories of groups of organisms - are one of the most widely used tools throughout the life sciences, as well as objects of research within systematics, evolutionary biology, epidemiology, etc. Almost every tool devised to date to reconstruct phylogenies produces trees; yet it is widely understood and accepted that trees oversimplify the evolutionary histories of many groups of organims, most prominently bacteria (because of horizontal gene transfer) and plants (because of hybrid speciation). Various methods and criteria have been introduced for phylogenetic tree reconstruction. Parsimony is one of the most widely used and studied criteria, and various accurate and efficient heuristics for reconstructing trees based on parsimony have been devised. Jotun Hein suggested a straightforward extension of the parsimony criterion to phylogenetic networks. In this paper we formalize this concept, and provide the first experimental study of the quality of parsimony as a criterion for constructing and evaluating phylogenetic networks. Our results show that, when extended to phylogenetic networks, the parsimony criterion produces promising results. In a great majority of the cases in our experiments, the parsimony criterion accurately predicts the numbers and placements of non-tree events.