Relation of Developmental Disorder Tendency and Internalizing Problems in Higher Grade Elementary School Children

The purpose of this study was to examine whether developmental disorder tendency such as ADHD and ASD tendency are related to internalizing problems through teacher teaching attitudes and school life events, targeting higher grade elementary school children enrolled in regular classes. Higher grade elementary school children (N=206) completed a self-report questionnaire concerning their teacher teaching attitudes, their daily experiences with schoolwork and their relationships with friends and classroom teacher at school, their self-esteem, and internalizing problems. Their homeroom teachers (N=9) rated each children’s developmental disorder tendency. As a result of path analysis, it became clear that developmental disorder tendency to be related to internalizing problems through mediating teacher teaching attitudes, school life events, and self-esteem.井上弥先生・樋口聡先生退職記念特集号本研究は，2018年度に広島大学教育学部に提出した卒業論文を加筆・修正したものです

Template properties of mutagenic cytosine analogues in reverse transcription

We have studied the mutagenic properties of ribonucleotide analogues by reverse transcription to understand their potential as antiretroviral agents by mutagenesis of the viral genome. The templating properties of nucleotide analogues including 6-(β-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido[4,5-c](1,2)oxazin-7-one, N(4)-hydroxycytidine, N(4)-methoxycytidine, N(4)-methylcytidine and 4-semicarbazidocytidine, which have been reported to exhibit ambiguous base pairing properties, were examined. We have synthesized RNA templates using T3 RNA polymerase, and investigated the specificity of the incorporation of deoxyribonucleoside triphosphates opposite these cytidine analogues in RNA by HIV and AMV reverse transcriptases. Except for N(4)-methylcytidine, both enzymes incorporated both dAMP and dGMP opposite these analogues in RNA. This indicates that they would be highly mutagenic if present in viral RNA. To study the basis of the differences among the analogues in the incorporation ratios of dAMP to dGMP, we have carried out kinetic analysis of incorporation opposite the analogues at a defined position in RNA templates. In addition, we examined whether the triphosphates of these analogues were incorporated competitively into RNA by human RNA polymerase II. Our present data supports the view that these cytidine analogues are mutagenic when incorporated into RNA, and that they may therefore be considered as candidates for antiviral agents by causing mutations to the retroviral genome

Spin colossal magnetoresistance in an antiferromagnetic insulator

Colossal magnetoresistance (CMR) refers to a large change in electrical conductivity induced by a magnetic field in the vicinity of a metal–insulator transition and has inspired extensive studies for decades1,2. Here we demonstrate an analogous spin effect near the Néel temperature, TN = 296 K, of the antiferromagnetic insulator Cr2O3. Using a yttrium iron garnet YIG/Cr2O3/Pt trilayer, we injected a spin current from the YIG into the Cr2O3 layer and collected, via the inverse spin Hall effect, the spin signal transmitted into the heavy metal Pt. We observed a two orders of magnitude difference in the transmitted spin current within 14 K of the Néel temperature. This transition between spin conducting and non-conducting states was also modulated by a magnetic field in isothermal conditions. This effect, which we term spin colossal magnetoresistance (SCMR), has the potential to simplify the design of fundamental spintronics components, for instance, by enabling the realization of spin-current switches or spin-current-based memories

Tightly regulated and homogeneous transgene expression in human adipose-derived mesenchymal stem cells by lentivirus with tet-off system.

Genetic modification of human adipose tissue-derived multilineage progenitor cells (hADMPCs) is highly valuable for their exploitation in therapeutic applications. Here, we have developed a novel single tet-off lentiviral vector platform. This vector combines (1) a modified tetracycline (tet)-response element composite promoter, (2) a multi-cistronic strategy to express an improved version of the tet-controlled transactivator and the blasticidin resistance gene under the control of a ubiquitous promoter, and (3) acceptor sites for easy recombination cloning of the gene of interest. In the present study, we used the cytomegalovirus (CMV) or the elongation factor 1 α (EF-1α) promoter as the ubiquitous promoter, and EGFP was introduced as the gene of interest. hADMPCs transduced with a lentiviral vector carrying either the CMV promoter or the EF-1α promoter were effectively selected by blasticidin without affecting their stem cell properties, and EGFP expression was strictly regulated by doxycycline (Dox) treatment in these cells. However, the single tet-off lentiviral vector carrying the EF-1α promoter provided more homogenous expression of EGFP in hADMPCs. Intriguingly, differentiated cells from these Dox-responsive cell lines constitutively expressed EGFP only in the absence of Dox. This single tet-off lentiviral vector thus provides an important tool for applied research on hADMPCs

Blasticidin selection of hADMPCs transduced with single tet-off lentiviral vector platform.

<p>hADMPCs were transduced with pTRE-EGFP-CMV-tTA-2A-Bsd (CMV) or pTRE-EGFP-EF-tTA-2A-Bsd (EF) at m.o.i. of 250. The cells were treated with 4 µg/mL blasticidin and 1 µg/mL Dox for 2 weeks. Then, the cells were cultured in the absence (Dox (-)) or presence (Dox (+)) of 1 µg/mL Dox for 4 days, and analyzed under a microscope (A) and flow cytometer (B). The cells were treated with 100 nM TSA (TSA), 5 µM 5-aza-dC (aza-dC), or both for 48 h before analyzed by flow cytometer. (C) A representative fluorescence histogram of EGFP. (D) The median fluorescence intensities of the EGFP-expressing populations. Error bars represent the standard error of 3 independent analyses. **, P<0.01 (Student's t test). Scale bar, 200 µm.</p

Differentiation potential of Dox-responsive hADMPCs.

<p>Dox-responsive hADMPCs were differentiated into adipocytes (A, E), osteocytes (B, F), chondrocytes (C, G), and neuronal cells (D, H). (A–D) Phase contrast (ph) and fluorescent (GFP) images. Dox-responsive hADMPCs were differentiated in the absence of Dox (Dox(−)) or in the presence of 1 µg/mL Dox (Dox(+)) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066274#s2" target="_blank">material and methods</a> section. (E–I) Confirmation of differentiated cells by oil red O staining for adipocytes (E), alizarin red staining for osteocytes (F), immunohistochemical staining against collagen II for chondrocytes (G), and immunohistochemical staining against β3-tubulin for neuronal cells (H). The percentages of differentiated cells to each cell type were calculated by the computerized image analysis (I). Cells that were not induced to differentiate (non-induced) were used as a negative control. CMV; hADMPCs transduced with pTRE-EGFP-CMV-tTA-2A-Bsd, EF; hADMPCs transduced with pTRE-EGFP-EF-tTA-2A-Bsd. Scale bar, 50 µm.</p

Schematic drawings of the single lentiviral vectors for tet-off system used in this work.

<p>(A) Gateway-compatible destination vectors containing <i>att</i>R recombination sites flanking a <i>ccd</i>B gene and a chloramphenicol-resistance gene, which allows an easy and rapid shuttling of gene of interest flanked by attL sites into the destination vectors using the Gateway LR recombination reaction. They also have an improved version of tetracycline-controlled transactivator (tTA) linked to the blasticidin resistant (Bsd) gene by the Thosea asigna virus 2A (2A) peptide sequence, whose expression is regulated by the CMV or EF-1α promoter. In the present study, we constructed an entry vector encoding EGFP flanked by attL, resulting in a destination clone, pTRE-EGFP-CMV-tTA-2A-Bsd or pTRE-EGFP-EF-tTA-2A-Bsd (B). In the absence of doxycycline (Dox), tTA-2A binds to the TRE-Tight promoter and activates EGFP transcription. For more details, see the Results section. CMV pro, CMV promoter; LTR, long terminal repeats; ψ, packaging signal; RRE, rev response elements; cPPT, central polypurine tract; TRE, tet-responsive element; Cm^R, chloramphenicol resistance; tTA, tetracycline-controlled transactivator; Bsd, blasticidin resistance; WPRE, woodchuck hepatitis virus posttranscriptional control element; SIN, self-inactivating. (C) (D) hADMPCs were transduced with pTRE-EGFP-CMV-tTA-2A-Bsd or pTRE-EGFP-EF-tTA-2A-Bsd at m.o.i. of 250. Four days after transduction, the cells were divided into 2 populations; with 1 µg/mL of Dox (Dox (+)) and without Dox (Dox (-)). (C) Fluorescent and phase contrast images. Scale bar, 200 µm. (D) Log fluorescence histograms of EGFP by flow cytometry analysis. (E) The whole cell lysates from hADMPCs transduced with pTRE-EGFP-CMV-tTA-2A-Bsd or pTRE-EGFP-EF-tTA-2A-Bsd were subjected to western blotting to monitor the cleavage efficiency of tTA-2A-Bsd proteins. A primary antibody against TetR was used to detect either tTA-2A-Bsd (non-cleaved form) or tTA-2A (cleaved form). Asterisk indicates a nonspecific band.</p

Comparison of a Variable-Number Tandem-Repeat (VNTR) Method for Typing Mycobacterium avium with Mycobacterial Interspersed Repetitive-Unit-VNTR and IS1245 Restriction Fragment Length Polymorphism Typing▿ †

Mycobacterium avium complex (MAC) infections are increasing annually in various countries, including Japan, but the route of transmission and pathophysiology of the infection remain unclear. Currently, a variable-number tandem-repeat (VNTR) typing method using the Mycobacterium avium tandem repeat (MATR) loci (MATR-VNTR) is employed in Japan for epidemiological studies using clinical isolates of M. avium. In this study, the usefulness of this MATR-VNTR typing method was compared with that of the IS1245-restriction fragment length polymorphism (IS1245-RFLP) typing method and a mycobacterial interspersed repetitive-unit (MIRU)-VNTR typing method reported previously (V. C. Thibault, M. Grayon, M. L. Boschiroli, C. Hubbans, P. Overduin, K. Stevenson, M. C. Gutierrez, P. Supply, and F. Biet, J. Clin. Microbiol. 45:2404-2410, 2007). Seventy clinical isolates identified as M. avium from human immunodeficiency virus-negative patients with MAC infections were used. MATR-VNTR typing using 15 loci distinguished 56 patterns of different allele profiles, yielding a Hunter-Gaston discriminatory index (HGDI) of 0.990. However, IS1245-RFLP and MIRU-VNTR typing yielded HGDIs of 0.960 and 0.949, respectively, indicating that MATR-VNTR has an excellent discriminatory power compared with MIRU-VNTR and IS1245-RFLP typing. Moreover, concomitant use of the MATR-VNTR method and IS1245-RFLP typing increased the HGDI to 0.999. MATR-VNTR typing is inexpensive and easy to perform and could thus be useful in establishing a digital multifacility database that will greatly contribute to the clarification of the transmission route and pathophysiology of M. avium infections

Expression pattern of surface cell markers on Dox-responsive hADMPCs.

<p>Dox-responsive hADMPCs after selection by blasticidin were cultured in the absence (Dox(-)) or presence (Dox (+)) of 1 µg/mL Dox for 4 days. Expression of the different surface markers were analyzed by flow cytometry and compared to the expression by a parental hADMPCs. They were stained with PE-coupled antibodies against CD13, CD29, CD34, CD44, CD73, CD90, CD105, and CD166. Histogram of a PE-coupled mouse IgG1 κ isotype control is shown in gray. CMV; hADMPCs transduced with pTRE-EGFP-CMV-tTA-2A-Bsd, EF; hADMPCs transduced with pTRE-EGFP-EF-tTA-2A-Bsd.</p