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Abstract To investigate the effects of radiation exposure due to the Fukushima nuclear power plant accident, following the disaster Fukushima Prefecture launched thyroid ultrasound examinations of residents who were generally younger than 18 years at the time of the earthquake. As the rate of pediatric thyroid cancer was higher than expected, we conducted biological dose assessment based on the frequency of translocated chromosome (Tr) aberrations using peripheral blood lymphocytes. Tr formation frequency was compared among the thyroid cancer (n = 38, median age 18 years, age range 12–26 years), thyroid-related disease (n = 30, median age 21 years, age range 15–28 years), and healthy controls (n = 31, median age 22 years, age range 20–23 years) groups. Tr aberration frequency was initially significantly higher in the thyroid cancer than in the other two groups; however, differences among the groups disappeared after adjusting for history of CT scan, as 92%, 67%, and 28% of those in the thyroid cancer, thyroid-related disease, and control groups, respectively, had undergone CT previously. Therefore, the significant difference in the initial number of Tr formations is presumably due to radiation exposure from CT. Accordingly, the effects of medical exposure on the chromosomes of children and adolescents should be noted.

There have been no systematic epidemiological evaluations of the relationship between thyroid autoimmunity and the clinical background of young patients with thyroid nodules. We aimed to clarify the clinical features associated with thyroglobulin or thyroperoxidase antibodies (thyroid autoantibodies [Tabs]) in children and young adults with nodules. We performed a cross-sectional study using data from 3,018 participants of 3-29 years of age with nodules, including thyroid cancer, from the Fukushima Health Management Survey. After stratification of the data for body mass index (BMI) and the bilateral width and thickness of the area (BWTAR) as indicators of thyroid volume for age, sex, body surface area (BSA), and sex-adjusted standard deviation score (SDS), trend analyses were performed. A logistic regression analysis was performed using tab-positivity as an objective variable. The overall prevalence of tab-positivity is 13.9%. It was high in females (17%), participants with diffuse goiter (DG) (19.2%), and those with papillary thyroid carcinoma (PTC) (12.8%). The age- and sex-adjusted odds ratios (95% confidence intervals) for BMI-SDS, BWTAR-SDS, presence of DG, diagnosis of PTC, and TSH concentrations were 0.962 (0.863-1.073), 1.263 (1.171-1.361), 7.357 (4.816-11.239), 2.787 (1.965-4.014), and 1.403 (1.257-1.564), respectively. Tab positivity was independently associated with a large thyroid, the presence of DG, the presence of PTC, and a high TSH concentration in patients with nodules. Based on the systematic epidemiologic evidence shown in young patients, Tab positivity might complement ultrasonography for the assessment of the thyroid function and identification of malignancy in younger patients with asymptomatic thyroid nodules.

The main cause of diffuse thyroid goiter is autoimmune chronic thyroiditis, otherwise known as Hashimoto's thyroiditis. Thyroid hormones play pivotal roles in growth and development during childhood. However, the prevalence of diffuse goiter and the relationships between diffuse goiter, thyroid volume, cysts and nodules, and anthropometric measurements in children are not well known. Among 789,459 participants who participated in thyroid ultrasound examinations, 320,206 participants (male: 161,728; female: 158,478) aged 1-23 years were analyzed. Logistic regression analyses were conducted to calculate the odds ratios of the standard deviation score of body mass index (BMI-SDS), the SDS of bilateral width multiplied thickness area (BWTAR-SDS) as a provisional determination of thyroid volume, and the presence of nodules or cysts for positive diffuse goiter compared with negative diffuse goiter after correction for sex and age. The prevalence of diffuse goiter increased in a female-dominant manner with aging. Compared with the absence of diffuse goiter, the age- and sex-adjusted odds ratios (95% confidence intervals) for BMI-SDS (1 SD), BWTAR-SDS (1 SD), cysts, and nodules were 1.24 (1.21-1.27), 3.21 (3.13-3.29), 0.53 (0.50-0.58), and 1.38 (1.17-1.64), respectively. The odds ratios of nodules for positive diffuse goiter were 4.18 (1.08-16.08), 1.76 (1.01-3.07), 1.80 (1.32-2.45), and 1.34 (1.08-1.67) in the age groups 1-7, 8-11, 12-15, and 16-23 years, respectively. The age-dependent increase in the prevalence of diffuse goiter was independently associated with increased BMI and positive prevalence of nodules in young individuals.

In response to concerns about health due to radiation exposure, the Fukushima Prefecture launched the Thyroid Ultrasound Examination program for residents aged 0-18 years at the time of the earthquake. Herein, we considered the confounding factors involved in the regional differences in the development of thyroid cancer. In this study, the 242 065 individuals who participated in both first- and second-round surveys were classified into four groups by address according to their air radiation dose. The number of participants diagnosed as malignant or suspicious for malignancy by cytological examination were 17, 38, 10 and 4 with detection rates of 53.8, 27.8, 21.7 and 14.5 per 100 000 participants in Regions 1, 2, 3 and 4, respectively. Sex (P = 0.0400), age at the time of the primary examination (P < 0.0001) and interval between the first- and second-round surveys (P < 0.0001) were significantly different among the four regions, and these were suspected to be confounding factors affecting regional differences in malignant nodule detection rates. In addition, significant regional differences were observed in the participation rate in the confirmatory examination (P = 0.0037) and the fine needle aspiration cytology implementation rate (P = 0.0037), which could be potential biases. No significant regional differences in the detection of malignant nodules were found in the multivariate logistic regression analysis after adjusting for the survey interval alone or for sex, age and survey interval. The confounding factors and biases identified in this study that may have important impacts on thyroid cancer detection rate should be fully considered in future studies., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Japanese Radiation Research Society and Japanese Society for Radiation Oncology.)

In thyroid cancer, it has been suggested that PD-L1 overexpression is associated with some clinicopathological factors and prognosis. The aim of this study is to characterize the expression of PD-L1, the presence of the BRAF V600E mutation, as well as cellular and humoral immunity in thyroid cancer, and to investigate the factors that predict the effectiveness of anti-PD-L1 antibody therapy. Blood samples were collected from 33 patients who were newly diagnosed with thyroid cancer after surgery or biopsy. PD-L1 expression, BRAF V600E mutation, and CD8+ expression were examined by immunohistological staining using clinical thyroid cancer specimens. With a PD-L1 staining cut-off value of 1%, 13 (39.4%) patients were classified as PD-L1 positive. Stimulation Index (SI) is an indicator of T cell activation. PD-L1 expression was significantly correlated with low SI level ( p = 0.046). Moreover, BRAF V600E mutation was detected in 24 of the 33 (72.7%) patients, and was significantly associated with PD-L1 expression ( p = 0.047). In addition, enhanced CD8+ expression was significantly associated with PD-L1 expression ( p = 0.003). Multivariate analyses confirmed that high CRP levels ( p = 0.039) were independently and significantly associated with poor progression-free survival. These findings suggest that elevated PD-L1 status can be a prognostic indicator for survival in patients with thyroid cancer when comprehensively assessed using the expression of CD8+, the presence of BRAF V600E mutation and the patient's immune status.

We previously described the thyroid volume, which was calculated by measuring the thyroid width, thickness, and longitudinal length using ultrasonography, in children and adolescents. We have proposed a simplified method for quantitatively assessing the thyroid size, to overcome the inaccuracy and challenges in measuring the longitudinal length of the thyroid. Based on measurements of 317,847 (girls: 156,913, boys: 160,934) children and adolescents, we calculated sex-specific means and standard deviations of thyroid width and thickness, and of the cross-sectional area computed by multiplying them, for every age and 0.1 m 2 of body surface area, after ensuring normal distribution with Box-Cox transformation. Multivariate regression analysis revealed that female sex, age, and body surface area were independently associated with areas of each thyroid lobe. Our novel method may be useful in quantitatively assessing the thyroid size, and appropriately diagnosing pathological conditions, such as hypoplasia, atrophy, and enlargement of the thyroid gland, in children and adolescents., Competing Interests: The authors declare no conflict of interest, in relation to this study. HO is a member of the “Department of Mesenchymal Stem Cell Research”, endowed by Alfresa Corporation., (2023©The Japanese Society for Pediatric Endocrinology.)

The Great East Japan Earthquake on March 11, 2011, and the subsequent tsunami caused an accident at the Fukushima Daiichi Nuclear Power Plant, in which extensive damage to the nuclear power reactors resulted in massive radioactive contamination. Fukushima Prefecture implemented the Thyroid Ultrasound Examination (TUE) program as part of the Fukushima Health Management Survey project in response to residents' anxieties about health risks due to radiation exposure for residents aged 0-18 years at the time of the nuclear accident. This program consisted of the primary examination and the confirmatory examination. In the primary examination, thyroid nodules and cysts were examined using portable ultrasound apparatuses. The confirmatory examination was performed to have clinical or cytological diagnosis. As of June 30, 2021, 116, 71, 31, 36, and 9 examinees in the first, second, third, and fourth round of surveys, and the survey at age 25 years, respectively, were determined to have nodules cytologically diagnosed as malignant or suspicious for malignancy. The confirmatory examination of the fourth-round survey and the primary and confirmatory examination of fifth-round survey are currently in progress. Together with the low thyroid absorbed radiation dose estimated in the United Nations Scientific Committee on the Effects of Atomic Radiation 2020 report, our results suggested that the increased incidence of childhood thyroid cancer in Fukushima Prefecture was not caused by radiation exposure, but rather by the highly sensitive detection method. As detailed in this review, there were ongoing challenges in our program, such as actions against the risk of overdiagnosis and psychological support for participants and their families.

Background: After the first-round (Preliminary Baseline Survey) ultrasound-based examination for thyroid cancer in response to the accident at the Fukushima Daiichi Nuclear Power Plant in 2011, two rounds of surveys (Full-scale Survey) have been carried out in Fukushima Prefecture. Using the data from these surveys, the geographical distribution of thyroid cancer incidence over 6 or 7 years after the disaster was examined., Methods: Children and adolescents who underwent the ultrasound-based examinations in the second- and/or third-round (Full-scale) survey in addition to the first-round survey were included. With a discrete survival model, we computed age, sex, and body mass index standardized incidence ratios (SIRs) for municipalities. Then, we employed spatial statistics to assess geographic clustering tendency in SIRs and Poisson regression to assess the association of SIRs with the municipal average absorbed dose to the thyroid gland at the 59-municipality level., Results: Throughout the second- and third-round surveys, 99 thyroid cancer cases were diagnosed in the study population of 252,502 individuals. Both flexibly shaped spatial scan statistics and maximized excess events test did not detect statistically significant spatial clustering (P = 0.17 and 0.54, respectively). Poisson regression showed no significant dose-response relationship: the estimated relative risks of lowest, middle-low, middle-high, and highest areas were 1.16 (95% confidence interval [CI], 0.52-2.59), 0.55 (95% CI, 0.31-0.97), 1.05 (95% CI, 0.79-1.40), and 1.24 (95% CI, 0.89-1.74)., Conclusion: There was no statistical support for geographic clustering or regional association with radiation dose measures of the thyroid cancer incidence in the cohort followed up to the third-round survey (fiscal years 2016-2017) in Fukushima Prefecture.

Background and Purpose The Thyroid Ultrasound Examination (TUE) program is conducted as part of the Fukushima Health Management Survey. Following the established criteria, examinees are called in for a secondary confirmation examination, which may induce high anxiety related to a thyroid cancer for both the examinees and their families. Therefore, Fukushima Medical University created the Thyroid Support Team to reduce anxiety. The purpose of this study is to analyze the psychosocial support for examinees and their families through two types of records, and to clarify the current issues and determine future directions of support.Materials and methods We analyzed 223 records of support for the first visit of examinees who attended the secondary confirmatory examination, conducted at Fukushima Medical University from September 2018 to March 2019.Results During the first visit, frequent topics and questions brought up by the examinees and their families were about the "Thyroid Ultrasound Examination (TUE) program" and "Examination findings". The Thyroid Support Team members assisted them by "Responding to questions", "Confirming the doctor's explanation" and "Providing information". The percentage of people with high anxiety decreased in both examinees and their family members after the examination. The level of anxiety was lower among those who had already taken the secondary confirmatory examination. Family members' anxiety was significantly higher than that of the examinees, and anxiety levels were highly correlated between examinees and their families.Conclusion The psychosocial support for examinees and their families was important in reducing their anxiety.　Currently there are changes in social conditions and various opinions concerning the TUE.　Thus, careful explanation and the need for decision-making supports for the examinees and their families increased. Also, we should take into account the aging of the examinees and expanding the available psychosocial support.

The Fukushima Daiichi Nuclear Power Plant accident occurred on March 11 2011, following the Great East Japan Earthquake and tsunami. Radioactive materials, including I-131, were released into the environment after the accident. Shortly after, the prefectural government initiated the Fukushima Health Management Survey for monitoring the long-term health conditions of the residents of Fukushima Prefecture. In the survey, thyroid ultrasonography was scheduled for all people aged 18 years or younger who were living in Fukushima Prefecture at the time of disaster. The total number of examinees was approximately 370,000 in the Preliminary Baseline Survey (PBLS), and 380,000 in the first Full-scale Survey (FSS). First, thyroid ultrasonography was performed as the Primary Examination. When a thyroid nodule that meets the fine needle aspiration cytology (FNAC) guideline is detected, thyroid FNAC is performed. By the end of June 2017, the cytological specimens of 187 examinees had been interpreted as Malignant or Suspicious for Malignancy (SFM). In this article, the cytological results of whole categories are presented using the criteria of The Bethesda System for Reporting Thyroid Cytopathology. The total numbers of examinees with SFM or Malignant in PBLS and at the first FSS were 106 (62.0%) and 71 (38.0%), respectively. The data of the cytological results of SFM and Malignant were already reported. However, this is the first report of cytological data from categories other than SFM and Malignant. The results of the current study will contribute to future research into the thyroid conditions of children and adolescents.

The identification of thyroid cancers among children after the Chernobyl nuclear power plant accident propelled concerns regarding long-term radiation effects on thyroid cancer in children affected by the Fukushima Daiichi nuclear power plant accident in Fukushima, Japan. Herein we consider the potential association between absorbed dose in the thyroid and the risk of developing thyroid cancer as detected by ultrasonography on 300 473 children and adolescents aged 0-18 years in Fukushima. The absorbed dose mentioned in the present study indicates the sum of that from external exposure and that from internally deposited radionuclides. We grouped participants according to estimated absorbed doses in each of 59 municipalities in Fukushima Prefecture, based on The United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR) 2013 report. The 59 municipalities were assigned to quartiles by dose. We limited our analyses to participants aged ≥6 years because only one case of thyroid cancer was observed in participants aged ≤5 years; 164 299 participants were included in the final analysis. Compared with the lowest dose quartile, the age- and sex-adjusted rate ratios (95% confidence intervals) for the low-middle, high-middle and highest quartiles were 2.00 (0.84-4.80), 1.34 (0.50-3.59) and 1.42 (0.55-3.67) for the 6-14-year-old groups and 1.99 (0.70-5.70), 0.54 (0.13-2.31) and 0.51 (0.12-2.15) for the >15-year-old group, respectively. No dose-dependent pattern emerged from the geographical distribution of absorbed doses by municipality, as estimated by UNSCEAR, and the detection of thyroid cancer among participants within 4-6 years after the accident. Ongoing surveillance might further clarify the effects of low-dose radiation exposure on thyroid cancer in Fukushima., (© The Author(s) 2020. Published by Oxford University Press on behalf of The Japanese Radiation Research Society and Japanese Society for Radiation Oncology.)

Background: The tumor-node-metastasis (TNM) classification system to categorized anaplastic thyroid cancer (ATC) was revised., Methods: The revised system was evaluated using a large database of ATC patients., Results: A total of 757 patients were analyzed. The proportion and median overall survival values (OS: months) for each T category were T1 ( n = 8, 1.1%, 12.5), T2 ( n = 43, 5.7%, 10.9), T3a ( n = 117, 15.5%, 5.7), T3b ( n = 438, 57.9%, 3.9), and T4 ( n = 151, 19.9%, 5.0). The OS of the N0 and N1 patients were 5.9 and 4.3, respectively (log-rank p < 0.01). Sixty-three (58.3%) patients migrated from stage IV A to IV B by revision based on the existence of nodal involvement and 422 patients (55.7%) were stratified into stage IV B, without a worsening of their OS (6.1), leaving 45 patients (5.9%) in stage IV A with fair OS (15.8). The hazard ratios for the survival of the patients of stage IV B compared to stage IV A increased from 1.1 to 2.1 by the revision. No change was made for stage IV C ( n = 290, 38.8%, 2.8)., Conclusion: The revised TNM system clearly indicated the prognoses of ATC patients by extracting rare patients with fair prognoses as having stage IV A disease and categorized many heterogeneous patients in stage IV B., Competing Interests: The authors declare no conflict of interest.

The Great East Japan Earthquake, which occurred on March 11, 2011, and its subsequent Fukushima Daiichi Nuclear Power Plant accident, prompted implementation of the Thyroid Ultrasound Examination (TUE) program as a part of the Fukushima Health Management Survey. The purpose of this program is to support residents of Fukushima Prefecture, and to analyze the health effects of the released radionuclides. Regardless of relatively high participation rates and a well-planned diagnostic flow, it is conceivable that not all thyroid cancer cases can be detected by the TUE program. The aims of the present study were to identify and characterize these "outside" cases, targeting patients at Fukushima Medical University (FMU) Hospital. As of June 30, 2017, we have successfully identified 11 outside cases. These corresponded to 5.7% of the 194 subjects who were identified as having thyroid cancer or suspected thyroid cancer in the TUE program. Although the outside subjects of other institutes were not investigated, the present study may have identified the majority of outside subjects in Japan, considering that FMU Hospital treats a large number of thyroid cancer subjects. Furthermore, the characteristics of the 11 subjects were not different from those of the subjects identified in the TUE program. These findings confirm that the TUE program was able to identify subjects of thyroid cancer adequately and sufficiently.

Nucleos(t)ide analog (NA) therapy has proven effective in treating chronic hepatitis B. However, NAs frequently result in viral relapse after the cessation of therapy. This is because NAs cannot fully eliminate the viral episomal covalently closed circular DNA (cccDNA) in the nucleus. In this study, we identified small molecular compounds that control host factors related to viral replication using in silico screening with simulated annealing based on bioinformatics for protein-ligand flexible docking. Twelve chemical compound candidates for alpha-glucosidase (AG) inhibitors were identified from a library of chemical compounds and used to treat fresh human hepatocytes infected with HBV. They were then monitored for their anti-viral effects. HBV replication was inhibited by one candidate (1-[3-(4-tert-butylcyclohexyl)oxy-2-hydroxypropyl]-2,2,6,6-tetramethylpiperidin-4-ol) in a dose-dependent manner. This compound significantly reduced ccc DNA production, compared to Entecavir (p < 0.05), and had a lower anti-AG effect. Gene expression analysis and structural analysis of this compound showed that its inhibitive effect on HBV was via interaction with Sp1. The nuclear transcription factor Sp1 acts on multiple regions of HBV to suppress HBV replication. Identifying candidates that control nuclear transcription factors facilitate the development of novel therapies. Drugs with a mechanism different from NA are promising for the elimination of HBV.

Potential inhibitors of a target biomolecule, NAD-dependent deacetylase Sirtuin 1, were identified by a contest-based approach, in which participants were asked to propose a prioritized list of 400 compounds from a designated compound library containing 2.5 million compounds using in silico methods and scoring. Our aim was to identify target enzyme inhibitors and to benchmark computer-aided drug discovery methods under the same experimental conditions. Collecting compound lists derived from various methods is advantageous for aggregating compounds with structurally diversified properties compared with the use of a single method. The inhibitory action on Sirtuin 1 of approximately half of the proposed compounds was experimentally accessed. Ultimately, seven structurally diverse compounds were identified.

Thyroid ultrasound screening of young residents in Fukushima Prefecture, Japan, showed a high detection rate of papillary thyroid carcinoma (PTC). Detailed morphological analysis of these tumors was not presented to date. This study sets out to evaluate changes in histopathological and invasive characteristics of Fukushima PTC with time after the nuclear accident of March 2011 in all available cases and in different age subgroups. Histological specimens of 115 PTCs from patients aged 18 years or younger at the time of the Fukushima Dai-ichi Nuclear Power Plant accident, who underwent surgical resection at Fukushima Medical University during 2012-2016, were reviewed. Patients were divided into those treated during the first 4 years after the accident (n = 78, shorter-onset) or later (n = 37, longer-onset). The whole group and 3 age subgroups: children (aged less than 15 years), adolescents (aged from 15 to less than 19 years), and young adults (aged from 19 years) at surgery were analyzed. No statistically significant time-related changes in tumor structure or invasiveness were found in the whole group or in age-matched subgroups. Statistically significant age-related downtrend was observed for intrathyroid spread in the whole group of patients. The absence of temporal changes in tumor morphological characteristics and tumor invasiveness strongly suggests common etiology of the shorter- and longer-onset Fukushima PTCs, which are unlikely related to the effect of exposure to very low doses of radiation., (© 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

While thyroid is considered to be a dormant organ, when required, it can regenerate through increased cell proliferation. However, the mechanism for regeneration remains unknown. Nkx2-1(fl/fl);TPO-cre mouse thyroids exhibit a very disorganized appearance because their thyroids continuously degenerate and regenerate. In mouse thyroids, a cluster of cells are found near the tracheal cartilage and muscle, which are positive for expression of NKX2-1, the master transcription factor governing thyroid development and function. In the present study, we propose that this cluster of NKX2-1-positive cells may be the precursor cells that mature to become thyroid follicular cells, forming thyroid follicles. We also found that phosphorylation of AKT is induced by NKX2-1 in the proposed thyroid progenitor-like side-population cell-derived thyroid cell line (SPTL) cells, suggesting the possibility that NKX2-1 plays a role in differentiation through the modulation of AKT signaling. This study revealed that Nkx2-1(fl/fl);TPO-cre mice provide a suitable model to study in vivo regeneration and folliculogenesis of the thyroid.

We propose a new iterative screening contest method to identify target protein inhibitors. After conducting a compound screening contest in 2014, we report results acquired from a contest held in 2015 in this study. Our aims were to identify target enzyme inhibitors and to benchmark a variety of computer-aided drug discovery methods under identical experimental conditions. In both contests, we employed the tyrosine-protein kinase Yes as an example target protein. Participating groups virtually screened possible inhibitors from a library containing 2.4 million compounds. Compounds were ranked based on functional scores obtained using their respective methods, and the top 181 compounds from each group were selected. Our results from the 2015 contest show an improved hit rate when compared to results from the 2014 contest. In addition, we have successfully identified a statistically-warranted method for identifying target inhibitors. Quantitative analysis of the most successful method gave additional insights into important characteristics of the method used.

Background: Non-small cell lung cancer (NSCLC) remains a lethal disease despite many proposed treatments. Recent studies have indicated that epigenetic therapy, which targets epigenetic effects, might be a new therapeutic methodology for NSCLC. However, it is not clear which objects (e.g., genes) this treatment specifically targets. Secreted frizzled-related proteins (SFRPs) are promising candidates for epigenetic therapy in many cancers, but there have been no reports of SFRPs targeted by epigenetic therapy for NSCLC., Methods: This study performed a meta-analysis of reprogrammed NSCLC cell lines instead of the direct examination of epigenetic therapy treatment to identify epigenetic therapy targets. In addition, mRNA expression/promoter methylation profiles were processed by recently proposed principal component analysis based unsupervised feature extraction and categorical regression analysis based feature extraction., Results: The Wnt/β-catenin signalling pathway was extensively enriched among 32 genes identified by feature extraction. Among the genes identified, SFRP1 was specifically indicated to target β-catenin, and thus might be targeted by epigenetic therapy in NSCLC cell lines. A histone deacetylase inhibitor might reactivate SFRP1 based upon the re-analysis of a public domain data set. Numerical computation validated the binding of SFRP1 to WNT1 to suppress Wnt signalling pathway activation in NSCLC., Conclusions: The meta-analysis of reprogrammed NSCLC cell lines identified SFRP1 as a promising target of epigenetic therapy for NSCLC.

Hepatitis B virus (HBV) chronically infects millions of people worldwide and is a major cause of serious liver diseases, including liver cirrhosis and liver cancer. In our previous study, in silico screening was used to isolate new anti-viral compounds predicted to bind to the HBV capsid. Four of the isolated compounds have been reported to suppress the cellular multiplication of HBV experimentally. In the present study, molecular dynamics simulations of the HBV capsid were performed under rotational symmetry boundary conditions, to clarify how the structure and dynamics of the capsid are affected at the atomic level by the binding of one of the isolated compounds, C13. Two simulations of the free HBV capsid, two further simulations of the capsid-C13 complex, and one simulation of the capsid-AT-130 complex were performed. For statistical confidence, each set of simulations was repeated by five times, changing the simulation conditions. C13 continued to bind at the predicted binding site during the simulations, supporting the hypothesis that C13 is a capsid-binding compound. The structure and dynamics of the HBV capsid were greatly influenced by the binding and release of C13, and these effects were essentially identical to those seen for AT-130, indicating that C13 likely inhibits the function of the HBV capsid.

A search of broader range of chemical space is important for drug discovery. Different methods of computer-aided drug discovery (CADD) are known to propose compounds in different chemical spaces as hit molecules for the same target protein. This study aimed at using multiple CADD methods through open innovation to achieve a level of hit molecule diversity that is not achievable with any particular single method. We held a compound proposal contest, in which multiple research groups participated and predicted inhibitors of tyrosine-protein kinase Yes. This showed whether collective knowledge based on individual approaches helped to obtain hit compounds from a broad range of chemical space and whether the contest-based approach was effective.

Background: Feature extraction (FE) is difficult, particularly if there are more features than samples, as small sample numbers often result in biased outcomes or overfitting. Furthermore, multiple sample classes often complicate FE because evaluating performance, which is usual in supervised FE, is generally harder than the two-class problem. Developing sample classification independent unsupervised methods would solve many of these problems., Results: Two principal component analysis (PCA)-based FE, specifically, variational Bayes PCA (VBPCA) was extended to perform unsupervised FE, and together with conventional PCA (CPCA)-based unsupervised FE, were tested as sample classification independent unsupervised FE methods. VBPCA- and CPCA-based unsupervised FE both performed well when applied to simulated data, and a posttraumatic stress disorder (PTSD)-mediated heart disease data set that had multiple categorical class observations in mRNA/microRNA expression of stressed mouse heart. A critical set of PTSD miRNAs/mRNAs were identified that show aberrant expression between treatment and control samples, and significant, negative correlation with one another. Moreover, greater stability and biological feasibility than conventional supervised FE was also demonstrated. Based on the results obtained, in silico drug discovery was performed as translational validation of the methods., Conclusions: Our two proposed unsupervised FE methods (CPCA- and VBPCA-based) worked well on simulated data, and outperformed two conventional supervised FE methods on a real data set. Thus, these two methods have suggested equivalence for FE on categorical multiclass data sets, with potential translational utility for in silico drug discovery.

Background: Non-small cell lung cancer (NSCLC) remains lethal despite the development of numerous drug therapy technologies. About 85% to 90% of lung cancers are NSCLC and the 5-year survival rate is at best still below 50%. Thus, it is important to find drugable target genes for NSCLC to develop an effective therapy for NSCLC., Results: Integrated analysis of publically available gene expression and promoter methylation patterns of two highly aggressive NSCLC cell lines generated by in vivo selection was performed. We selected eleven critical genes that may mediate metastasis using recently proposed principal component analysis based unsupervised feature extraction. The eleven selected genes were significantly related to cancer diagnosis. The tertiary protein structure of the selected genes was inferred by Full Automatic Modeling System, a profile-based protein structure inference software, to determine protein functions and to specify genes that could be potential drug targets., Conclusions: We identified eleven potentially critical genes that may mediate NSCLC metastasis using bioinformatic analysis of publically available data sets. These genes are potential target genes for the therapy of NSCLC. Among the eleven genes, TINAGL1 and B3GALNT1 are possible candidates for drug compounds that inhibit their gene expression.

Background: Aberrant DNA methylation is often associated with cancers. Thus, screening genes with cancer-associated aberrant DNA methylation is a useful method to identify candidate cancer-causing genes. Aberrant DNA methylation is also genotype dependent. Thus, the selection of genes with genotype-specific aberrant DNA methylation in cancers is potentially important for tailor-made medicine. The selected genes are important candidate drug targets., Results: The recently proposed principal component analysis based selection of genes with aberrant DNA methylation was applied to genotype and DNA methylation patterns in squamous cell carcinoma measured using single nucleotide polymorphism (SNP) arrays. SNPs that are frequently found in cancers are usually highly methylated, and the genes that were selected using this method were reported previously to be related to cancers. Thus, genes with genotype-specific DNA methylation patterns will be good therapeutic candidates. The tertiary structures of the proteins encoded by the selected genes were successfully inferred using two profile-based protein structure servers, FAMS and Phyre2. Candidate drugs for three of these proteins, tyrosine kinase receptor (ALK), EGLN3 protein, and NUAK family SNF1-like kinase 1 (NUAK1), were identified by ChooseLD., Conclusions: We detected genes with genotype-specific DNA methylation in squamous cell carcinoma that are candidate drug targets. Using in silico drug discovery, we successfully identified several candidate drugs for the ALK, EGLN3 and NUAK1 genes that displayed genotype-specific DNA methylation.

Suppression of autophagy is always accompanied by marked accumulation of p62, a selective autophagy substrate. Because p62 interacts with the Nrf2-binding site on Keap1, which is a Cullin 3-based ubiquitin ligase adapter protein, autophagy deficiency causes competitive inhibition of the Nrf2-Keap1 interaction, resulting in stabilization of Nrf2 followed by transcriptional activation of Nrf2 target genes. Herein, we show that liver-specific autophagy-deficient mice harbor adenomas linked to both the formation of p62- and Keap1-positive cellular aggregates and induction of Nrf2 targets. Importantly, similar aggregates were identified in more than 25% of human hepatocellular carcinomas (HCC), and induction of Nrf2 target genes was recognized in most of these tumors. Gene targeting of p62 in an HCC cell line markedly abrogates the anchorage-independent growth, whereas forced expression of p62, but not a Keap1 interaction-defective mutant, resulted in recovery of the growth defect. These results indicate the involvement of persistent activation of Nrf2 through the accumulation of p62 in hepatoma development.

We have devised a power function (PF) that can predict the accuracy of a three-dimensional (3D) structure model of a protein using only amino acid sequence alignments. This Power Function (PF) consists of three parts; (1) the length of a model, (2) a homology identity percent value and (3) the agreement rate between PSI-PRED secondary structure prediction and the secondary structure judgment of a reference protein. The PF value is mathematically computed from the execution process of homology search tools, such as FASTA or various BLAST programs, to obtain the amino acid sequence alignments. There is a high correlation between the global distance test-total score (GDT&#95;TS) value of the protein model based upon the PF score and the GDT&#95;TS(MAX) value used as an index of protein modeling accuracy in the international contest Critical Assessment of Techniques for Protein Structure Prediction (CASP). Accordingly, the PF method is valuable for constructing a highly accurate model without wasteful calculations of homology modeling that is normally performed by an iterative method to move the main chain and side chains in the modeling process. Moreover, a model with higher accuracy can be obtained by combining the models ordered by the PF score with models sorted by the size of the CIRCLE score. The CIRCLE software is a 3D-1D program, in which energetic stabilization is estimated based upon the experimental environment of each amino acid residue in the protein solution or protein crystals.

Almost all proteins express their biological functions through the structural conformation of their specific amino acid sequences. Therefore, acquiring the three-dimensional structures of proteins is very important to elucidate the role of a particular protein. We had built protein structure model databases, which is called RIKEN FAMSBASE (http://famshelp.gsc.riken.jp/famsbase/). The RIKEN FAMSBASE is a genome-wide protein structure model database that contains a large number of protein models from many organisms. The HUMAN FAMSBASE that is one part of the RIKEN FAMSBASE contains many protein models for human genes, which are significant in the pharmaceutical and medicinal fields. We have now implemented an update of the human protein modeling database consisting of 242918 constructed models against the number of 20743 human protein sequences with an improved modeling method called Full Automatic protein Modeling System Developed (FAMSD). The results of our benchmark test of the FAMSD method indicated that it has an excellent capability to pack amino acid side-chains with correct torsion angles in addition to the main-chain, while avoiding the formation of atom-atom collisions that are not found in experimental structures. This new protein structure model database for human genes, which is named HUMAN FAMSD-BASE, is open to the public as a component part of the RIKEN FAMSBASE at http://mammalia.gsc.riken.jp/human&#95;famsd/. A significant improvement of the HUMAN FAMSD-BASE in comparison with the preceding HUMAN FAMSBASE was verified in the benchmark test of this paper. The HUMAN FAMSD-BASE will have an important impact on the progress of biological science.

The prediction of a protein three-dimensional (3D) structure is one of the most important challenges in computational structural biology. We have developed an automatic protein 3D structure prediction method called FAMSD. FAMSD is based on a comparative modeling method which consists of the following four steps: (1) generating and selecting sequence alignments between target and template proteins; (2) constructing 3D structure models based on each selected alignment; (3) selecting the best 3D structure model and (4) refining the selected model. In the FAMSD method, sequence alignment programs such as a series of BLAST programs, SP3 and SPARKS2 programs, the homology modeling program FAMS (Full Automatic Modeling System), the model quality estimation program CIRCLE and the molecular dynamics program APRICOT were used in combination to construct high quality protein models. To assess the FAMSD method we have participated in the 8th Critical Assessment of Techniques for Protein Structure Prediction (CASP8) experiment. The results of our original assessment indicate that the FAMSD method offers excellent capability in packing side-chains with the correct torsion angles while avoiding the formation of atom-atom collisions. Since side-chain packing plays a significant role in defining the biological function of proteins, this method is a valuable resource in biological, pharmaceutical and medicinal research efforts.

We report a novel method, ChooseLD (CHOOse biological information Semi-Empirically on the Ligand Docking), which uses simulated annealing (SA) based on bioinformatics for protein-ligand flexible docking. The fingerprint alignment score (FPAScore) value is used to determine the docking conformation of the ligand. This method includes the matching of chemical descriptors such as fingerprints (FPs) and the root mean square deviation (rmsd) calculation of the coordinates of atoms of the chemical descriptors. Here, the FPAScore optimization for the translation and rotation of a rigid body is performed using the Metropolis Monte Carlo method. Our ChooseLD method will find wide application in the field of biochemistry and medicine to improve the search for new drugs targeting various proteins implicated in diseases.

Background: Vaccine therapy targeting tumor antigens recognized by cytotoxic T cells (CTL) has been tried extensively. However, in a cancer-bearing state, the Th1/Th2 balance shifts to Th2 dominance, and this has been the obstacle to vaccine therapy to induce the CTL. DC1/DC2 subsets have also been reported to control the differentiation of Th subsets. The key to tumor immunotherapy is how to activate the DC1-Th1 lineage., Patients and Methods: Six normal adults and 14 patients with gastric or colorectal cancers, who gave informed consent, were studied. The Th1/Th2 and DC1/DC2 ratios were determined by FACS. IL-12 and IL-10 production from PBMC were measured by ELISA., Results: The Th1/Th2 and DC1/DC2 ratios were all significantly lower in the patients with gastric or colorectal cancers compared to normal adults. After protein-bound polysaccharide K (PSK) therapy in cancer patients, the Th1/Th2 balance shifted to Th1 dominance and the DC1/DC2 balance to DC1 dominance. IL-10 production was significantly decreased by PSK therapy., Conclusion: In the cancer-bearing state, the Th1/Th2 and DC/1/DC2 balance becomes Th2- and DC2-dominant. PSK therapy results in a shift of the Th1/Th2 and DC1/DC2 balance towards Th1 and DC1 dominance. We plan to examine whether combining dendritic cells (DC) vaccination therapy with oral PSK enhances the induction of T cell and DC differentiation in cancer patients.

To accelerate the development of drugs against severe acute respiratory syndrome (SARS), we constructed a homology model of the SARS coronavirus main protease using our modeling software, FAMS Ligand&Complex, and released it before the X-ray structure was solved. The X-ray structure showed our model as accurately predicted and useful for structure based drug design.

We report here 7 new mutations in the ADAMTS13 gene responsible for Upshaw-Schulman syndrome (USS), a catastrophic phenotype of congenital thrombotic thrombocytopenic purpura, by analyzing 5 Japanese families. There were 3 mutations that occurred at exon-intron boundaries: 414+1G>A at intron 4, 686+1G>A at intron 6, and 1244+2T>G at intron 10 (numbered from the A of the initiation Met codon), and we confirmed that 2 of these mutations produced aberrantly spliced messenger RNAs (mRNAs). The remaining 4 mutations were missense mutations: R193W, I673F, C908Y, and R1123C. In expression experiments using HeLa cells, all mutants showed no or a marginal secretion of ADAMTS13. Taken together with the findings in our recent report we determined the responsible mutations in a total of 7 Japanese patients with USS with a uniform clinical picture of severe neonatal hyperbilirubinemia, and in their family members, based on ADAMTS13 gene analysis. Of these patients, 2 were homozygotes and 5 were compound heterozygotes. The parents of one homozygote were related (cousins), while those of the other were not. Molecular models of the metalloprotease, fifth domain of thrombospondin 1 (Tsp1-5), and Tsp1-8 domains of ADAMTS13 suggest that the missense mutations could cause structural defects in the mutants.

Transforming growth factor-beta (TGF-beta) has growth-stimulating effects on mesenchymal cells and several tumor cell lines. The signaling pathway for this effect is, however, not well understood. We examined how TGF-beta stimulates proliferation of MG63 human osteosarcoma cells. Two distinct type I receptors for TGF-beta, ALK-1 and ALK-5, were expressed and functional in MG63 cells. Of these two receptors, ALK-5 appears to be responsible for the growth stimulation because expression of constitutively active ALK-5, but not ALK-1, stimulated proliferation of MG63 cells. SB-431542 (0.3 microM), a novel inhibitor of ALK4/5/7 kinase, suppressed TGF-beta-induced growth stimulation. DNA microarray analysis as well as quantitative real-time PCR analysis of RNAs from TGF-beta-treated cells demonstrated that several growth factors, including platelet-derived growth factor AA, were induced in response to TGF-beta in MG63 cells. Gleevec (1 microM) as well as AG1296 (5 microM) inhibited TGF-beta-induced growth stimulation of MG63 cells, suggesting that platelet-derived growth factor AA was mainly responsible for the growth-stimulatory effect of TGF-beta. We also examined the mechanisms of perturbation of growth-suppressing signaling in MG63 cells. We found that expression of c-Myc, which is down-regulated by TGF-beta in many other cells, was up-regulated in MG63 cells, suggesting that up-regulation of c-Myc expression may be the mechanism canceling growth-suppressing signaling of TGF-beta in MG63 cells.

Enlarged FAMSBASE is a relational database of comparative protein structure models for the whole genome of 41 species, presented in the GTOP database. The models are calculated by Full Automatic Modeling System (FAMS). Enlarged FAMSBASE provides a wide range of query keys, such as name of ORF (open reading frame), ORF keywords, Protein Data Bank (PDB) ID, PDB heterogen atoms and sequence similarity. Heterogen atoms in PDB include cofactors, ligands and other factors that interact with proteins, and are a good starting point for analyzing interactions between proteins and other molecules. The data may also work as a template for drug design. The present number of ORFs with protein 3D models in FAMSBASE is 183 805, and the database includes an average of three models for each ORF. FAMSBASE is available at http://famsbase.bio.nagoya-u.ac.jp/famsbase/.

A recently identified ribonucleotide reductase (RR), p53R2, is directly regulated by p53 for supplying nucleotides to repair damaged DNA. We examined the role of this p53R2-dependent pathway for DNA synthesis in a p53-regulated cell cycle checkpoint, comparing it to R2-dependent DNA synthesis. The elevation of DNA synthesis activity through RR in response to gamma-irradiation was closely correlated with the level of expression of p53R2 but not of R2. The p53R2 product accumulated in nuclei, whereas R2 levels in cytoplasm decreased. We found a point mutation of p53R2 in cancer cell line HCT116, which resulted in loss of RR activity. In those cells, DNA damage-inducible apoptotic cell death was enhanced through transcriptional activation of p53AIP1. The results suggest that p53R2-dependent DNA synthesis plays a pivotal role in cell survival by repairing damaged DNA in the nucleus and that dysfunction of this pathway might result in activation of p53-dependent apoptosis to eliminate dangerous cells.

A novel method is described, which uses changes in NMR chemical shifts to characterise the structural change in a protein with pressure. Melittin in methanol is a small alpha-helical protein, and its chemical shifts change linearly and reversibly with pressure between 1 and 2000 bar. An improved relationship between structure and HN shift has been calculated, and used to drive a molecular dynamics-based calculation of the change in structure. With pressure, the helix is compressed, with the H-O distance of the NH-O=C hydrogen bonds decreased by 0.021 +/- 0.039 A, leading to an overall compression along the entire helix of about 0.4 A, corresponding to a static compressibility of 6 x 10(-6) bar(-1). The backbone dihedral angles phi and psi are altered by no more than +/- 3 degrees for most residues with a negative correlation coefficient of -0.85 between phi(i) and psi(i - 1), indicating that the local conformation alters to maintain hydrogen bonds in good geometries. The method is shown to be capable of calculating structural change with high precision, and the results agree with structural changes determined using other methodologies.

Several complementary NMR approaches were used to study the interaction of mastoparan, a 14-residue peptide toxin from wasp venom, with lipid membranes. First, the 3D structure of mastoparan was determined using 1H-NMR spectroscopy in perdeuterated (SDS-d25) micelles. NOESY experiments and distance geometry calculations yielded a straight amphiphilic alpha-helix with high-order parameters, and the chemical shifts of the amide protons showed a characteristic periodicity of 3-4 residues. Secondly, solid-state 2H-NMR spectoscopy was used to describe the binding of mastoparan to lipid bilayers, composed of headgroup-deuterated dimyristoylglycerophosphocholine (DMPC-d4) and dimyristoylphosphatidylglycerol (DMPG). By correlating the deuterium quadrupole splittings of the alpha-segments and beta-segments, it was possible to differentiate the electrostatically induced structural response of the choline headgroup from dynamic effects induced by the peptide. A partial phase separation was observed, leading to a DMPG-rich phase and a DMPG-depleted phase, each containing some mastoparan. Finally, the insertion and orientation of a specifically 15N-labeled mastoparan (at position Ala10) in the bilayer environment was investigated by solid-state 15N-NMR spectroscopy, using macroscopically oriented samples. Two distinct orientational states were observed for the mastoparan helix, namely an in-plane and a trans-membrane alignment. The two populations of 90% in-plane and 10% trans-membrane helices are characterized by a mosaic spread of +/- 30 degrees and +/- 10 degrees, respectively. The biological activity of mastoparan is discussed in terms of a pore-forming model, as the peptide is known to be able to induce nonlamellar phases and facilitate a flip-flop between the monolayers.

The structure of [Arg8]vasopressin methylenedithioether ([AVP]CH2) has been determined in dimethylsulfoxide-d6. Two-dimensional DQF-COSY and NOESY spectra were measured and used to derive angle and distance constraints for restrained molecular dynamics (MD) calculations. In the MD trajectory, two types of beta-turn structure were found in the region from Tyr2 to Asn5, suggesting an equilibrium between type-I and type-II' beta-turn structures. When Halpha chemical shifts were used as an additional constraint, the type-I turn was favoured. To validate this result, an independent energy minimization procedure was used, using differences between calculated and observed chemical shifts. The two approaches gave essentially identical results. It is therefore concluded that the type-I turn predominates in solution. Analysis of calculated chemical shift contributions suggests that the beta-turn structure found in AVP is well preserved in [AVP]CH2, although the pressin ring size is expanded.

We have constructed an extensive database of 13C C alpha and C beta chemical shifts in proteins of solution, for proteins of which a high-resolution crystal structure exists, and for which the crystal structure has been shown to be essentially identical to the solution structure. There is no systematic effect of temperature, reference compound, or pH on reported shifts, but there appear to be differences in reported shifts arising from referencing differences of up to 4.2 ppm. The major factor affecting chemical shifts is the backbone geometry, which causes differences of ca. 4 ppm between typical alpha-helix and beta-sheet geometries for C alpha, and of ca. 2 ppm for C beta. The side-chain dihedral angle chi 1 has an effect of up to 0.5 ppm on the C alpha shift, particularly for amino acids with branched side-chains at C beta. Hydrogen bonding to main-chain atoms has an effect of up to 0.9 ppm, which depends on the main-chain conformation. The sequence of the protein and ring-current shifts from aromatic rings have an insignificant effect (except for residues following proline). There are significant differences between different amino acid types in the backbone geometry dependence; the amino acids can be grouped together into five different groups with different phi, psi shielding surfaces. The overall fit of individual residues to a single non-residue-specific surface, incorporating the effects of hydrogen bonding and chi 1 angle, is 0.96 ppm for both C alpha and C beta. The results from this study are broadly similar to those from ab initio studies, but there are some differences which could merit further attention.

The structure of the bee venom peptide melittin has been determined in water at pH 6.0, 50 mM sodium phosphate, at various temperatures. At all temperatures studied, the peptide is tetrameric, and consists of two helical regions (residues 2-11 and 13-23) with an unstructured C-terminal region. The connection between the helices (residues 11-13) is well defined. At 30 degrees C, the structure of the monomeric unit has been characterised using NOEs, and a family of structures is presented (root-mean-square deviation to the mean structure 1.4 A over the structured residues), with low NOE violations and good stereochemistry. The angle between the helices is 46+/-13 degrees, and the structure is very similar to the previously determined crystal structure of the aqueous tetramer. The peptide forms a tetramer that is made up from a dimer of dimers. The structure of the dimeric unit has been determined, using a novel joint refinement of intermonomer NOEs and chemical shifts. The relative position of the monomeric units in the dimer is different from that in the crystal, with less direct contact between monomers. As the temperature is raised to 70 degrees C, the peptide remains tetrameric, but the monomer units start to separate, as shown by a reduction in intermonomer NOE intensities and chemical shifts. The structural changes have been characterised: over the temperature range studied, the monomers separate by approximately 2.0 A. This movement may have implications for the mechanism by which melittin inserts into membranes.