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Futai K.,Medical and Biological Laboratories Corporation | Terasaka N.,ETH Zurich | Katoh T.,University of Tokyo | Suga H.,University of Tokyo
Methods | Year: 2016

Detection of rare small RNA species whose sizes are overlapping with tRNAs often suffers from insufficient sensitivity due to the overwhelming abundance of tRNAs. We here report a method, named tRid (tRNA r. id), for removing abundant tRNAs from small RNA fractions regardless of tRNA sequence species. By means of tRid, we are able to selectively enrich small RNAs which have been previously difficult to access due to mass existence of tRNAs in such fractions. A flexible tRNA-acylation ribozyme, known as flexizyme, is a key tool where the total tRNAs are aminoacylated with N-biotinylated phenylalanine regardless of tRNA sequences, and therefore the biotin-tagged tRNAs could be readily removed from the small RNA fractions by the use of streptavidin-immobilized magnetic beads. Next generation sequencing of the isolated small RNA fraction revealed that small RNAs with less than 200. nt were effectively enriched, allowing us to identify previously unknown small RNAs in HeLa and E. coli. © 2016. Source


Hollevoet K.,Ghent University | Nackaerts K.,University Hospital Gasthuisberg | Thimpont J.,Occupational Diseases Fund | Germonpre P.,University of Antwerp | And 7 more authors.
American Journal of Respiratory and Critical Care Medicine | Year: 2010

Rationale: Soluble mesothelin (SM) is currently the reference serum biomarker of malignant pleural mesothelioma (MPM). Megakaryocyte potentiating factor (MPF), which originates from the same precursor protein, is potentially more sensitive, yet lacks validation. Objectives: To analyze the diagnostic performance of MPF as anMPM biomarker and compare this performance with SM. Methods: A total of 507 participants were enrolled in six cohorts: healthy control subjects (n = 101), healthy asbestos-exposed individuals (n589), andpatients with benign asbestos-related disease (n5123), benign respiratory disease (n=46), lung cancer (n=63), and MPM (n = 85). Sera were analyzed for SM and MPF levels using the Mesomark and Human MPF ELISA kit, respectively. Measurements and Main Results: SM and MPF levels differed signifi-cantly between patients with MPM and participants from each other cohort (P < 0.001). Receiver operating characteristics curve analysis did not reveal a significant difference between both markers in area under curve (AUC) for distinguishing MPM from all cohorts jointly (SM = 0.871, MPF = 0.849; P = 0.28). At 95% specificity, SM and MPF had a sensitivity of 64% (cutoff = 2.00 nmol/L) and 68% (cutoff = 12.38 ng/ml), respectively. Combining both markers did not improve the diagnostic performance. Conclusions: Inthis prospective multicenter study,MPFis validated as a highly performant MPM biomarker. The similar AUC values of SM and MPF, together with the limited difference in sensitivity, show that both serum biomarkers have an equivalent diagnostic performance. Source


Sasaki T.,Keio University | Shiohama A.,Keio University | Kubo A.,Keio University | Kawasaki H.,Keio University | And 7 more authors.
Journal of Allergy and Clinical Immunology | Year: 2013

Background Flaky tail (ma/ma Flgft/ft) mice have a frameshift mutation in the filaggrin (Flgft) gene and are widely used as a model of human atopic dermatitis associated with FLG mutations. These mice possess another recessive hair mutation, matted (ma), and develop spontaneous dermatitis under specific pathogen-free conditions, whereas genetically engineered Flg-/- mice do not. Objective We identified and characterized the gene responsible for the matted hair and dermatitis phenotype in flaky tail mice. Methods We narrowed down the responsible region by backcrossing ma/ma mice with wild-type mice and identified the mutation using next-generation DNA sequencing. We attempted to rescue the matted phenotype by introducing the wild-type matted transgene. We characterized the responsible gene product by using whole-mount immunostaining of epidermal sheets. Results We demonstrated that ma, but not Flgft, was responsible for the dermatitis phenotype and corresponded to a Tmem79 gene nonsense mutation (c.840C>G, p.Y280*), which encoded a 5-transmembrane protein. Exogenous Tmem79 expression rescued the matted hair and dermatitis phenotype of Tmem79 ma/ma mice. Tmem79 was mainly expressed in the trans-Golgi network in stratum granulosum cells in the epidermis in both mice and humans. The Tmem79ma/ma mutation impaired the lamellar granule secretory system, which resulted in altered stratum corneum formation and a subsequent spontaneous dermatitis phenotype. Conclusions The Tmem79ma/ma mutation is responsible for the spontaneous dermatitis phenotype in matted mice, probably as a result of impaired lamellar granule secretory system and altered stratum corneum barrier function. © 2013 American Academy of Allergy, Asthma & Immunology. Source


Iba Y.,Health Science University | Fujii Y.,RIKEN | Ohshima N.,Health Science University | Sumida T.,RIKEN | And 8 more authors.
Journal of Virology | Year: 2014

Neutralizing antibodies that target the hemagglutinin of influenza virus either inhibit binding of hemagglutinin to cellular receptors or prevent the low-pH-induced conformational change in hemagglutinin required for membrane fusion. In general, the former type of antibody binds to the globular head formed by HA1 and has narrow strain specificity, while the latter type binds to the stem mainly formed by HA2 and has broad strain specificity. In the present study, we analyzed the epitope and function of a broadly neutralizing human antibody against H3N2 viruses, F005-126. The crystal structure of F005-126 Fab in complex with hemagglutinin revealed that the antibody binds to the globular head, spans a cleft formed by two hemagglutinin monomers in a hemagglutinin trimer, and cross-links them. It recognizes two peptide portions (sites L and R) and a glycan linked to asparagine at residue 285 using three complementarity-determining regions and framework 3 in the heavy chain. Binding of the antibody to sites L (residues 171 to 173, 239, and 240) and R (residues 91, 92, 270 to 273, 284, and 285) is mediated mainly by van der Waals contacts with the main chains of the peptides in these sites and secondarily by hydrogen bonds with a few side chains of conserved sequences in HA1. Furthermore, the glycan recognized by F005-126 is conserved among H3N2 viruses. F005-126 has the ability to prevent low-pH-induced conformational changes in hemagglutinin. The newly identified conserved epitope, including the glycan, should be immunogenic in humans and may induce production of broadly neutralizing antibodies against H3 viruses. © 2014, American Society for Microbiology. Source


Hollevoet K.,Ghent University | Nackaerts K.,University Hospital Gasthuisberg | Thas O.,Ghent University | Thimpont J.,Occupational Diseases Fund | And 8 more authors.
Chest | Year: 2012

Background: Soluble mesothelin (SM) and megakaryocyte potentiating factor (MPF) are serum biomarkers of mesothelioma. This study examined the effect of clinical covariates on biomarkers levels and their diagnostic and prognostic value. Methods: Five hundred ninety-four participants were enrolled in a multicenter study, including 106 patients with mesothelioma and 488 control subjects. Multiple linear regression analyses were used to identify which covariates were independently associated with SM and MPF levels. The effect of these covariates on the diagnostic accuracy was evaluated with receiver operating characteristics curve analysis. In patients with mesothelioma, survival analysis was performed with Cox regression. Results: SM and MPF levels were independently associated with age, glomerular filtration rate (GFR), and BMI in control subjects and with GFR and tumor stage in patients with mesothelioma. The diagnostic accuracy of SM and MPF was significantly affected by the distribution of these covariates in the study population. The patients with mesothelioma were best discriminated from the control subjects with either the youngest age, the highest GFR, or the largest BMI. Furthermore, the control subjects were significantly better differentiated from stage II to IV than from stage I mesothelioma. MPF, not SM, was an independent negative prognostic factor, but only if adjusted for the biomarker-associated covariates. Conclusions: SM and MPF levels were affected by the same clinical covariates, which also had a significant impact on their diagnostic and prognostic value. To improve the interpretation of biomarker results, age, GFR, and BMI should be routinely recorded. Approaches to account for these covariates require further validation, as does the prognostic value of SM and MPF. ©2012 American College of Chest Physicians. Source

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