Specific Inhibition of Tumor Cells by Oncogenic <i>EGFR</i> Specific Silencing by RNA interference

<div><p>Anticancer agents that have minimal effects on normal cells and tissues are ideal cancer drugs. Here, we show specific inhibition of human cancer cells carrying oncogenic mutations in the epidermal growth factor receptor (EGFR) gene by means of oncogenic allele-specific RNA interference (RNAi), both <i>in vivo</i> and <i>in vitro</i>. The allele-specific RNAi (ASP-RNAi) treatment did not affect normal cells or tissues that had no target oncogenic allele, whereas the suppression of a normal <i>EGFR</i> allele by a conventional <i>in vivo</i> RNAi caused adverse effects, i.e., normal EGFR is vital. Taken together, our current findings suggest that specific inhibition of oncogenic <i>EGFR</i> alleles without affecting the normal <i>EGFR</i> allele may provide a safe treatment approach for cancer patients and that ASP-RNAi treatment may be capable of becoming a safe and effective, anticancer treatment method. </p> </div

Effects of ASP-RNAi on tumor growth.

<p>(<b>A</b>) Schematic drawing of the experimental plan. s.c.: subcutaneous. (<b>B</b>) <i>In vivo</i> luminescent imaging. Subcutaneous tumor models were established with PC-3/luc cells and siRNAs at indicated doses were administered according to the experimental plan (A). Tumors were monitored by an IVIS imaging system. (<b>C</b>) Tumor growth. Luminescent intensities of the tumors treated with siRNAs were quantified and analyzed (mean ± SDs, n = 5 mice/group). (<b>D</b>) Wet weight of tumors treated with siRNAs. Three weeks after treatment (Day 28), the treated tumors were isolated and their wet weight was measured [mean ± SDs; n = 5 mice/group; *<i>P</i> < 0.05 by Dunnett’s test (vs. siControl)]. (<b>E</b>) Western blot analysis. Subcutaneous tumors were treated with 2.0 mg/kg b.w. of si747/49_3D8 (red box) or siControl (open box). Three days after treatment (Day 10), the treated tumors were isolated and examined by Western blotting using indicated antibodies. (<b>F</b>) Plasma biochemical parameters in mice treated with siRNAs. Plasma specimens were prepared from treated mice at Day 28, and subjected to plasma biochemical analyses to examine alkaline phosphatase (ALP), total protein (TP), GPT, GOT, and total (T-), direct (D-) and indirect (I-) bilirubin (BIL). Significant difference in each parameter was examined by Dunnett’s test as in D. n.s., no significant difference. (<b>G</b>) Body weight. Body weight of the treated mice was measured at Day 28. Statistical analysis was carried out as in F.</p

Adverse effects of knockdown of normal <i>Egfr</i> in normal ICR mice.

<p>(<b>A</b>) Plasma analyses. siEgfr siRNA targeting the wild-type mouse <i>Egfr</i> gene, another siRNA (as indicated), or delivery vehicle was administered 3 times, on Days 1, 3, and 5, to 10-week-old ICR mice via the lateral tail vein. The day after administration, measurement of body weight was carried out (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073214#pone.0073214.s019" target="_blank">Table S5</a>). Two days after the last administration, the mice were subjected to hematological analyses (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073214#pone.0073214.s020" target="_blank">Table S6</a>) followed by separation of blood plasma. The plasma specimens were examined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073214#pone-0073214-g002" target="_blank">Figure 2</a>. Examined biochemical parameters are indicated (n=4 mice/group; mean ± SDs; * <i>P</i><0.05 by Tukey-Kramer test; n.s., no statistical significance). (<b>B</b>) TUNEL assay. Cryosections of intestinal tissue were prepared from ICR mice that had been treated with the indicated siRNAs; cryosections were examined using a TUNEL assay. A marked increase in intestinal apoptosis was detected in the siEgfr-treated ICR mice.</p

Effects of systemic siRNA administration on tumor growth in lung cancer models.

<p>(<b>A</b>) <i>In vivo</i> luminescent imaging. PC-3/luc cells were intravenously administered to nude mice and examined using an IVIS imaging system 5 days after cell injection. PC-3/luc-positive mice were randomly divided into two groups, and subjected to systemic administration of indicated siRNAs twice (Day 5 and 7) via the lateral tail vein. The treated mice were examined using the IVIS imaging system; photographic images of luminescent signals at Day 5 and 10 (after cell injection) are shown (left panels). Box plots represent the luminescent intensities of the treated mice at Day 5 and 10 (right panels) [n = 5 mice/group; *<i>P</i> < 0.05 by Student’s <i>t</i>-test (two-tailed); n.s., no significant difference]. (<b>B</b>) Lung tissues isolated from siRNA-treated mice. Lung tissues were isolated from PC-3-bearing mice treated with the indicated siRNAs, and the wet weights were measured [mean ± SDs; n=5 mice/group; *<i>P</i> < 0.05 by Student’s <i>t</i>-test (two-tailed)]. (<b>C</b>) Histological analysis of lung tissues. Cryosections of lung tissue were prepared from PC3-bearing mice treated with the indicated siRNAs; cryosections were stained with conventional hematoxylin and eosin solution. (<b>D</b>) <i>In vivo</i> imaging of active caspase. Six hours after administration of the indicated siRNAs on Day 5, VivoGlo Caspase 3/7 substrate (Promega) was administrated intraperitoneally to the treated mice; <i>in vivo</i> imaging and subsequent imaging analysis were carried out. Box plots represent luminescent intensities [n = 6 mice/group; *<i>P</i> < 0.05 by Student’s <i>t</i>-test (two-tailed)].</p

Effects of gefitinib on tumor growth.

<p>(<b>A</b>) Schematic drawing of the experimental plan. p.o.: per os (oral administration). (<b>B</b>) <i>In vivo</i> luminescent imaging. Subcutaneous tumor models were established with PC-3/luc cells as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073214#pone-0073214-g002" target="_blank">Figure 2</a>, and gefitinib was administered at the indicated doses. Photographic images of luminescent signals at Day 7 and 28 after the inoculation of PC-3/luc cells are shown. (<b>C</b>) Tumor growth. Luminescent intensities of the tumors treated with gefitinib were quantified and analyzed (n = 5 tumor models/group). Error bars represent SDs. (<b>D</b>) Wet weight of the tumors treated with gefitinib. Three weeks after treatment (Day 28), the treated tumors were isolated and wet weight was measured. A significant difference against the vehicle control group (open bar) is indicated by an asterisk [*<i>P</i> < 0.05 by Student’s <i>t</i>-test (two-tailed)]. Error bars represent SDs. (<b>E</b>) Examination of plasma biochemical parameters in the treated mice. Plasma specimens were prepared from the mice at Day 28, and subjected to plasma biochemical analyses as in Figures 2 and 4 [n = 5 mice/group; mean ± SDs; <i>P</i> < 0.05 by Dunnett’s test (vs. siControl); n.s., no significant difference]. (<b>F</b>) Body weight. Body weight of the treated mice was measured at Day28 [n = 5 mice/group; mean ± SDs; *<i>P</i> < 0.05 by Dunnett’s test (vs. siControl)]. (<b>G</b>) TUNEL assay. Cryosections of intestinal tissues were prepared from model mice treated with gefitinib (0, 50, 100 mg/kg b.w.) at Day 28 as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073214#pone-0073214-g004" target="_blank">Figure 4</a>, and the cryosections were examined by a TUNEL assay.</p

Additional file 1 of Effects of others’ gaze and facial expression on an observer’s microsaccades and their association with ADHD tendencies

Additional file 1: Supplementary Figure 1. Eye movements and microsaccade detections during fixation. Supplementary Figure 2. Average microsaccadic rates and the function of detection threshold λ. Supplementary Figure 3. Relationship between the peak velocity and the amplitude of detected microsaccades and their histograms

What Time Periods of the Day Are Concerning for Parents of Children with Attention Deficit Hyperactivity Disorder?

<div><p>Background/Aim</p><p>The questionnaire-children with difficulties (QCD) is a parent-assessed questionnaire designed to evaluate a child’s difficulties in functioning during specific time periods of the day. In this study, the QCD was applied to determine the time periods of the day that are concerning for the parents of children with attention deficit hyperactivity disorder (ADHD). The results were compared with those for a community sample.</p> <p>Methods</p><p>Elementary and junior high school students with ADHD (243 boys, 55 girls) and a community sample of children (518 boys, 618 girls) were enrolled in this study. Their behaviors were assessed by the QCD, the ADHD-rating scale (ADHD-RS), and the Oppositional Defiant Behavior Inventory (ODBI). The effects of gender (boy/girl) and diagnosis (ADHD/community sample) on the total QCD score were analyzed across each school grade (elementary/junior high school). Correlation coefficients between QCD and ADHD-RS/ODBI scores were analyzed.</p> <p>Results</p><p>The QCD score for the ADHD group was significantly lower than that for the community sample (P < 0.001). There were significantly strong correlations between “evening” and ADHD-RS and ODBI scores for all children with ADHD (r > 0.41, P < 0.001) and between “night” and inattention and oppositional symptoms for the girls with ADHD (r > 0.40, P < 0.001).</p> <p>Conclusions</p><p>Parents reported that children with ADHD faced greater difficulties in completing basic daily activities compared with the community controls, particularly in the evening. Furthermore, these difficulties were related to the severity of ADHD symptoms. The parents’ perceptions depended on the gender, ADHD and oppositional symptoms, and the time period of the day. This study determined that children with ADHD face greater difficulties in daily functioning compared with community sample children, that these difficulties are time-dependent, and that these difficulties were particularly experienced in the evening.</p> </div

Investigation of Rare Single-Nucleotide <i>PCDH15</i> Variants in Schizophrenia and Autism Spectrum Disorders

<div><p>Both schizophrenia (SCZ) and autism spectrum disorders (ASD) are neuropsychiatric disorders with overlapping genetic etiology. <i>Protocadherin 15</i> (<i>PCDH15</i>), which encodes a member of the cadherin super family that contributes to neural development and function, has been cited as a risk gene for neuropsychiatric disorders. Recently, rare variants of large effect have been paid attention to understand the etiopathology of these complex disorders. Thus, we evaluated the impacts of rare, single-nucleotide variants (SNVs) in <i>PCDH15</i> on SCZ or ASD. First, we conducted coding exon-targeted resequencing of <i>PCDH15</i> with next-generation sequencing technology in 562 Japanese patients (370 SCZ and 192 ASD) and detected 16 heterozygous SNVs. We then performed association analyses on 2,096 cases (1,714 SCZ and 382 ASD) and 1,917 controls with six novel variants of these 16 SNVs. Of these six variants, four (p.R219K, p.T281A, p.D642N, c.3010-1G>C) were ultra-rare variants (minor allele frequency < 0.0005) that may increase disease susceptibility. Finally, no statistically significant association between any of these rare, heterozygous <i>PCDH15</i> point variants and SCZ or ASD was found. Our results suggest that a larger sample size of resequencing subjects is necessary to detect associations between rare <i>PCDH15</i> variants and neuropsychiatric disorders.</p></div

Data_Sheet_1_Validation and Factor Analysis of the Japanese Version of the Highs Scale in Perinatal Women.docx

<p>Background: The Highs scale has been developed to evaluate hypomanic symptoms in the first postpartum week. However, it has not been elucidated whether this scale is also applicable to pregnant women. To address this issue, we confirmed the factor structure, reliability, and validity of the Japanese version of the Highs scale for pregnant and postpartum women.</p><p>Methods: 418 women provided effective responses to both the Highs scale and the Edinburgh Postnatal Depression Scale (EPDS) during early pregnancy (before week 25), late pregnancy (around week 36), at 5 days and at 1 month after delivery. Subjects were randomly divided into two groups, and exploratory and confirmatory factor analyses were performed for each group. Cronbach's alpha was calculated and the correlation of the Highs scale with EPDS was analyzed. The correlation between the subscales was analyzed at four time points, and the correlation of subscales between the four time points was confirmed.</p><p>Results: This scale was found to have the two-factor structure with elation and agitation subscales. The two subscales had reasonable internal consistency at all time points (Cronbach's alpha range: Factor 1, 0.696–0.758; Factor 2, 0.553–0.694). The overall scale had reasonable internal consistency at all time points (Cronbach's alpha range: 0.672–0.738). Based on the correlation analysis of the two subscales and EPDS, discriminative and convergent validity were indicated at all time points, confirming the construct validity of the Highs scale. Subscale scores showed a significant correlation with EPDS at all time points (r = 0.388, 0.384, 0.498, and 0.442, p < 0.01).</p><p>Conclusions: The Japanese version of the Highs scale is reliable and valid, and can be applied for evaluating the hypomanic symptoms during pregnancy and postpartum period.</p

Rare <i>PCDH15</i> SNVs identified in this study.

<p>Rare <i>PCDH15</i> SNVs identified in this study.</p