GC/MS based metabolomics: development of a data mining system for metabolite identification by using soft independent modeling of class analogy (SIMCA)

<p>Abstract</p> <p>Background</p> <p>The goal of metabolomics analyses is a comprehensive and systematic understanding of all metabolites in biological samples. Many useful platforms have been developed to achieve this goal. Gas chromatography coupled to mass spectrometry (GC/MS) is a well-established analytical method in metabolomics study, and 200 to 500 peaks are routinely observed with one biological sample. However, only ~100 metabolites can be identified, and the remaining peaks are left as "unknowns".</p> <p>Result</p> <p>We present an algorithm that acquires more extensive metabolite information. Pearson's product-moment correlation coefficient and the Soft Independent Modeling of Class Analogy (SIMCA) method were combined to automatically identify and annotate unknown peaks, which tend to be missed in routine studies that employ manual processing.</p> <p>Conclusions</p> <p>Our data mining system can offer a wealth of metabolite information quickly and easily, and it provides new insights, particularly into food quality evaluation and prediction.</p

Concentration Distribution of Particles in Solid-Liquid Two-Phase Flow Through Vertical Pipe

The concentration distributions of particles were measured in a vertical pipe for both an upward and a doward flow. When the stream Reynolds number was low, the profile for the upward flow was opposite to that for the downward flow. The profiles of the concentration distributions under various conditions were classified into several kinds of modes. They were summarized into a figure by means of the stream Reynolds number and the particle Reynolds number. Two possible forces were suggested in order to explain the general distribution of partices

Unique microbial ecosystems of Antarctica

第6回極域科学シンポジウム[OB] 極域生物圏11月17日（火）　統計数理研究所 セミナー室1（D305

Supplementary material for the article: Fukushi, K.; Fujita, Y.; Nonogaki, J.; Tsujimoto, J.-I.; Hattori, T.; Inui, H.; Beškoski, V. P.; Hotta, H.; Hayashi, M.; Nakano, T. Capillary Zone Electrophoresis Determination of Fluoride in Seawater Using Transient Isotachophoresis. Analytical and Bioanalytical Chemistry 2018, 410 (6), 1825–1831. https://doi.org/10.1007/s00216-017-0838-0

Supplementary material for: [https://doi.org/10.1007/s00216-017-0838-0]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2083

Measurement of femoral axial offset

Purpose to examine the accuracy and reproducibility of the femoral axial offset measured from the retrocondylar plane by computed tomography (CT). Bone specimens of the femur of 15 males and 15 females were analyzed. CT imaging was performed and data of the coordinates were collected (center of femoral head, center of an ellipse around greater trochanter, center of an ellipse around the base of femoral neck, posterior edge of great trochanter, and both posterior condyles). The angle between the line connecting center of the femoral head and center of an ellipse around greater trochanter and the line connecting both posterior condyles was set as anteversion 1. The angle between the line connecting the center of femoral head and center of an ellipse around base of the femoral neck and the line connecting both posterior condyles was set as anteversion 2. The femoral axial offset was measured from the retrocondylar plane. Measurements were performed three times on the same subject, and intrarater reliability (ICC) was determined. In addition, interrater reliability (ICC) was determined by comparing data from three raters. The mean value for anteversion 1 was 20.1° for males and 22.7° for females. The values for anteversion 2 were 16.0° and 19.9° for males and females, respectively. Offset was 34.0 and 33.4 mm in males and females, respectively. Intrarater ICC and interrater ICC exceeded 0.81 for both methods, suggesting that the method of measurement was reliable. Accuracy and reproducibility of the measurement of femoral axial offset from the retrocondylar plane were high

Bcl2 Deficiency Activates FoxO through Akt Inactivation and Accelerates Osteoblast Differentiation

Osteoblast apoptosis plays an important role in bone development and maintenance, and is in part responsible for osteoporosis in sex steroid deficiency, glucocorticoid excess, and aging. Although Bcl2 subfamily proteins, including Bcl2 and Bcl-XL, inhibit apoptosis, the physiological significance of Bcl2 in osteoblast differentiation has not been fully elucidated. To investigate this, we examined Bcl2-deficient (Bcl2(-/-)) mice. In Bcl2(-/-) mice, bromodeoxyuridine (BrdU)-positive osteoblasts were reduced in number, while terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive osteoblasts were increased. Unexpectedly, osteoblast differentiation was accelerated in Bcl2(-/-) mice as shown by the early appearance of osteocalcin-positive osteoblasts. Osteoblast differentiation was also accelerated in vitro when primary osteoblasts were seeded at a high concentration to minimize the reduction of the cell density by apoptosis during culture. FoxO transcription factors, whose activities are negatively regulated through the phosphorylation by Akt, play important roles in multiple cell events, including proliferation, death, differentiation, longevity, and stress response. Expressions of FasL, Gadd45a, and Bim, which are regulated by FoxOs, were upregulated; the expression and activity of FoxOs were enhanced; and the phosphorylation of Akt and that of FoxO1 and FoxO3a by Akt were reduced in Bcl2(-/-) calvariae. Further, the levels of p53 mRNA and protein were increased, and the expression of p53-target genes, Pten and Igfbp3 whose proteins inhibit Akt activation, was upregulated in Bcl2(-/-) calvariae. However, Pten but not Igfbp3 was upregulated in Bcl2(-/-) primary osteoblasts, and p53 induced Pten but not Igfbp3 in vitro. Silencing of either FoxO1 or FoxO3a inhibited and constitutively-active FoxO3a enhanced osteoblast differentiation. These findings suggest that Bcl2 deficiency induces and activates FoxOs through Akt inactivation, at least in part, by upregulating Pten expression through p53 in osteoblasts, and that the enhanced expression and activities of FoxOs may be one of the causes of accelerated osteoblast differentiation in Bcl2(-/-) mice

Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis

<p>Abstract</p> <p>Background</p> <p>Various protein expression systems, such as <it>Escherichia coli </it>(<it>E. coli</it>), <it>Saccharomyces cerevisiae </it>(<it>S. cerevisiae</it>), <it>Pichia pastoris </it>(<it>P. pastoris</it>), insect cells and mammalian cell lines, have been developed for the synthesis of G protein-coupled receptors (GPCRs) for structural studies. Recently, the crystal structures of four recombinant human GPCRs, namely β₂adrenergic receptor, adenosine A_2areceptor, CXCR4 and dopamine D3 receptor, were successfully determined using an insect cell expression system. GPCRs expressed in insect cells are believed to undergo mammalian-like posttranscriptional modifications and have similar functional properties than in mammals. Crystal structures of GPCRs have not yet been solved using yeast expression systems. In the present study, <it>P. pastoris </it>and insect cell expression systems for the human muscarinic acetylcholine receptor M2 subtype (CHRM2) were developed and the quantity and quality of CHRM2 synthesized by both expression systems were compared for the application in structural studies.</p> <p>Results</p> <p>The ideal conditions for the expression of CHRM2 in <it>P. pastoris </it>were 60 hr at 20°C in a buffer of pH 7.0. The specific activity of the expressed CHRM2 was 28.9 pmol/mg of membrane protein as determined by binding assays using [³H]-quinuclidinyl benzilate (QNB). Although the specific activity of the protein produced by <it>P. pastoris </it>was lower than that of Sf9 insect cells, CHRM2 yield in <it>P. pastoris </it>was 2-fold higher than in Sf9 insect cells because <it>P. pastoris </it>was cultured at high cell density. The dissociation constant (Kd) for QNB in <it>P. pastoris </it>was 101.14 ± 15.07 pM, which was similar to that in Sf9 insect cells (86.23 ± 8.57 pM). There were no differences in the binding affinity of CHRM2 for QNB between <it>P. pastoris </it>and Sf9 insect cells.</p> <p>Conclusion</p> <p>Compared to insect cells, <it>P. pastoris </it>is easier to handle, can be grown at lower cost, and can be expressed quicker at a large scale. Yeast, <it>P. pastoris</it>, and insect cells are all effective expression systems for GPCRs. The results of the present study strongly suggested that protein expression in <it>P. pastoris </it>can be applied to the structural and biochemical studies of GPCRs.</p

Proteasomal degradation of BRAHMA promotes Boron tolerance in Arabidopsis

High levels of boron (B) induce DNA double-strand breaks (DSBs) in eukaryotes, including plants. Here we show a molecular pathway of high B-induced DSBs by characterizing Arabidopsis thaliana hypersensitive to excess boron mutants. Molecular analysis of the mutants revealed that degradation of a SWItch/Sucrose Non-Fermentable subunit, BRAHMA (BRM), by a 26S proteasome (26SP) with specific subunits is a key process for ameliorating high-B-induced DSBs. We also found that high-B treatment induces histone hyperacetylation, which increases susceptibility to DSBs. BRM binds to acetylated histone residues and opens chromatin. Accordingly, we propose that the 26SP limits chromatin opening by BRM in conjunction with histone hyperacetylation to maintain chromatin stability and avoid DSB formation under high-B conditions. Interestingly, a positive correlation between the extent of histone acetylation and DSB formation is evident in human cultured cells, suggesting that the mechanism of DSB induction is also valid in animals