Development of an integrative database with 499 novel microsatellite markers for Macaca fascicularis

<p>Abstract</p> <p>Background</p> <p>Cynomolgus macaques (<it>Macaca fascicularis</it>) are a valuable resource for linkage studies of genetic disorders, but their microsatellite markers are not sufficient. In genetic studies, a prerequisite for mapping genes is development of a genome-wide set of microsatellite markers in target organisms. A whole genome sequence and its annotation also facilitate identification of markers for causative mutations. The aim of this study is to establish hundreds of microsatellite markers and to develop an integrative cynomolgus macaque genome database with a variety of datasets including marker and gene information that will be useful for further genetic analyses in this species.</p> <p>Results</p> <p>We investigated the level of polymorphisms in cynomolgus monkeys for 671 microsatellite markers that are covered by our established Bacterial Artificial Chromosome (BAC) clones. Four hundred and ninety-nine (74.4%) of the markers were found to be polymorphic using standard PCR analysis. The average number of alleles and average expected heterozygosity at these polymorphic loci in ten cynomolgus macaques were 8.20 and 0.75, respectively.</p> <p>Conclusion</p> <p>BAC clones and novel microsatellite markers were assigned to the rhesus genome sequence and linked with our cynomolgus macaque cDNA database (QFbase). Our novel microsatellite marker set and genomic database will be valuable integrative resources in analyzing genetic disorders in cynomolgus macaques.</p

Collection of Macaca fascicularis cDNAs derived from bone marrow, kidney, liver, pancreas, spleen, and thymus

<p>Abstract</p> <p>Background</p> <p>Consolidating transcriptome data of non-human primates is essential to annotate primate genome sequences, and will facilitate research using non-human primates in the genomic era. <it>Macaca fascicularis </it>is a macaque monkey that is commonly used for biomedical and ecological research.</p> <p>Findings</p> <p>We constructed cDNA libraries of <it>Macaca fascicularis</it>, derived from tissues obtained from bone marrow, liver, pancreas, spleen, and thymus of a young male, and kidney of a young female. In total, 5'-end sequences of 56,856 clones were determined. Including the previously established cDNA libraries from brain and testis, we have isolated 112,587 cDNAs of <it>Macaca fascicularis</it>, which correspond to 56% of the curated human reference genes.</p> <p>Conclusion</p> <p>These sequences were deposited in the public sequence database as well as in-house macaque genome database <url>http://genebank.nibio.go.jp/qfbase/</url>. These data will become valuable resources for identifying functional parts of the genome of macaque monkeys in future studies.</p

MOLECULAR DISCRIMINATION BETWEEN INDIVIDUAL METACERCARIAE OF PARAGONIMUS HETEROTREMUS AND P. WESTERMANI OCCURRING IN THAILAND

Abstract. To accurately discriminate between individual metacercariae of Paragonimus heterotremus and P. westermani occurring in Thailand, polymerase chain reaction (PCR)-based molecular methods were established and subjected to an evaluation. We first amplified and sequenced the second internal transcribed spacer (ITS2) region of the nuclear ribosomal DNA of the two species. Based on their nucleotide differences, P. heterotremus and P. westermani were unequivocally discriminated from each other. These nucleotide differences were further utilized to select the ApaL1 endonuclease site for PCR-restriction fragment length polymorphism (PCR-RFLP) analyses and to design species-specific primers for multiplex PCR reactions. Both PCR-RFLP and multiplex PCR methods allowed a more rapid and labor-effective species discrimination. Furthermore, the multiplex PCR method enabled the most efficient discrimination because species identification involved a single round of PCR in a single tube. In Thailand, P. heterotremus is the only species affecting humans. Thus, the methods established in the present study can be used as reliable tools to identify the lung fluke metacercariae that cause human disease. primers. All of these methods utilize nucleotide differences in the second internal transcribed spacer (ITS2) of the nuclear ribosomal DNA (rDNA) for dicrimination between the two species. In the present study, we focused on the lung flukes occurring in Thailand and applied the methods for species discrimination between individual metacercariae of P. heterotremus and P. westermani. MATERIALS AND METHODS Parasite material and DNA isolation The metacercariae of P. heterotremus and P. westermani DNA amplification, restriction digestion and sequencing The rDNA region spanning the ITS2 from individual metacercariae of the two species was amplified by PCR using the primers, 3S (forward, 5&apos;-GGTACCGGTGGATCACTCGGCTCGTG-3&apos;) and A28 (reverse, 5&apos;-GGGATCCTGGTTAGTTTCTTTT CCTCCGC-3&apos;). These primers were designed on the basis of the conserved rDNA sequences of the Schistosoma specie

Dengue virus type 2 unresponsive to the current PCR primer; : construction of a new PCR primer to detect all strains of Dengue virus type 2.

We found that one strain of dengue virus (Trinidad 1751; TR) did not respond to the PCR primer for Jamaica/83. We investigated such property with other 10 strains of dengue virus type 2 and found 2 more unresponsive strains. All 3 strains were isolated from the central America. To detect the envelope gene of those 3 strains by PCR, we synthesized primers based on TR strain as the reference sequence. Using these primers, we could detect the 3 strains by PCR at the usual annealing temperature. We recommed the new primer for diagnosis of DEN 2

Molecular Characterization of Cryptosporidium Isolates Obtained from Human and Bovine Infections in Japan

Cryptosporidiumポリスレオニン遺伝子のPCR/RFLP分析により、わが国で分離されたヒト由来Cryptosporidium22株は、ヒト型C.parvum(genotype1)、動物型C.parvum(genotype2)、トリ型C.meleagridis(genotype3)の3遺伝子型に明瞭に型別された。ヒト由来genotype3のC.meleagridis分離株は、Cryptosporidium18S　rRNA遺伝子のシークエンス分析により同定され、その存在が本邦で初めて確認された

New Lyssavirus Genotype from the Lesser Mouse-eared Bat (Myotis blythi), Kyrghyzstan

The Aravan virus was isolated from a Lesser Mouse-eared Bat (Myotis blythi) in the Osh region of Kyrghyzstan, central Asia, in 1991. We determined the complete sequence of the nucleoprotein (N) gene and compared it with those of 26 representative lyssaviruses obtained from databases. The Aravan virus was distinguished from seven distinct genotypes on the basis of nucleotide and amino acid identity. Phylogenetic analysis based on both nucleotide and amino acid sequences showed that the Aravan virus was more closely related to genotypes 4, 5, and—to a lesser extent—6, which circulates among insectivorus bats in Europe and Africa. The Aravan virus does not belong to any of the seven known genotypes of lyssaviruses, namely, rabies, Lagos bat, Mokola, and Duvenhage viruses and European bat lyssavirus 1, European bat lyssavirus 2, and Australian bat lyssavirus. Based on these data, we propose a new genotype for the Lyssavirus genus

地域在住自立高齢者を対象にした体力測定会への参加希望者における閉じこもりリスクと孤独感との関連

　Purpose：This study was conducted for the purpose of examining the risk of becoming homebound and the correlation of loneliness in the community-dwelling older adults volunteering to participate in a physical f itness measurement program．　Method：The subjects of this study consisted of 638 older adults dwelling in Kameoka City who volunteered to participate in a physical fitness measurement program in 2012 and desired also to participate in a similar program held roughly one year and a half years later．A survey was conducted in the form of a questionnaire survey that was mailed to the subjects．The contents of the survey consisted of questions relating to gender，age，household composition，frequency of meetings with separately dwelling family members，number of neighbors they can rely on，tasks within the home，presence of a hobby，participation in group activities，subjective view of health，subjective physical strength，risk of depression，risk of becoming homebound，assessment using the Japanese language version of the UCLA loneliness scale（ 3rd edition）（ to be simply referred to as ""loneliness""） and assessment using the Life Satisfaction Index K（ to be abbreviated as LSIK）．Analyses consisted of an intergroup comparison of loneliness scores for each parameter．This was followed by an analysis of covariance using those parameters for which there was a correlation with loneliness as covariates in order to examine the correlation between risk of becoming homebound and loneliness．　Results：539 of the subjects submitted valid responses to the survey （response rate：84.5%）．The analysis of the risk of becoming homebound indicated that 20 subjects were homebound（ 3.7%），90 subjects were at risk of becoming homebound （16.7%） and 429 subjects were not homebound （79.6%）． According to the results of an intergroup comparison of loneliness scores for each survey parameter，signif icant differences（ p ＜0.001） were observed for gender，number of neighbors the subjects can rely on，subjective view of health，subjective physical strength，risk of depression， tasks within the home，presence of a hobby，participation in group activities and LSIK score． The average loneliness scores of each risk of becoming homebound were 36.2 ± 8.9 in the nonhomebound group，41.9 ± 9.8 in the group at risk of becoming homebound，and 45.8 ± 8.7 in the homebound group，with scores becoming higher as the tendency to become homebound worsened． The correlation between the two was such that significant differences （ p =0.007） were observed when the above parameters were used as covariates，and loneliness was greater in the group at risk of becoming homebound than in the non-homebound group（ p =0.047）．　Discussion：This study demonstrated that persons at risk of becoming homebound are present even among relatively healthy older adults expressing a strong desire to continue to participate in physical f itness measurement programs like the subjects of this study，and that there is already a strong sense of loneliness among subjects of the group at risk of becoming homebound． Efforts to eliminate this sense of loneliness targeted at older adults at risk of becoming homebound who are already demonstrating a decrease in frequency of leaving their homes are expected to lead to effective countermeasures for preventing these persons from becoming homebound．　目　的：地域在住自立高齢者を対象とした体力測定会への参加希望者における閉じこもりリスクの程度と孤独感との関連を明らかにすることを目的とした。　方　法：亀岡市在住高齢者で2012 年の体力測定会に参加し，その約1 年半後の体力測定会に参加を希望した638 名を対象とした。調査は郵送法による質問紙調査を行った。質問項目は性別，年齢，世帯構成，別居家族と会う頻度，近所に頼れる人の人数，家庭内の仕事，趣味の有無，グループ活動への参加，主観的健康感，主観的体力，うつのリスク，閉じこもりリスク，日本語版UCLA 孤独感尺度（第3 版）（以下，孤独感），生活満足度尺度K（以下，LSIK）である。分析は各調査項目において孤独感得点の群間比較を行った。次に，孤独感と関連のあった項目を共変量とした共変量分散分析を行い，閉じこもりリスクと孤独感の関連を明らかにした。　結　果：有効回答者539 名（有効回答率84.5%）であった。閉じこもりリスクの分布は，閉じこもり群20 名（3.7%），閉じこもり予備群90 名（16.7%），非閉じこもり群429 名（79.6%）であった。調査項目ごとの孤独感得点の群間比較では性別，近所に頼れる人の人数，主観的健康感，主観的体力，うつのリスク，家庭内の仕事，趣味の有無，グループ活動への参加，LSIK に有意差が認められた（ p ＜ 0.001）。閉じこもりリスク別の孤独感得点の平均は，非閉じこもり群36.2 ± 8.9 点，閉じこもり予備群41.9 ± 9.8点，閉じこもり群45.8 ± 8.7 点と閉じこもり傾向に伴い高まった。両者の関連では先の項目を共変量としても有意差があり（ p =0.007），閉じこもり予備群の孤独感が非閉じこもり群に比べて有意に高かった（ p =0.047）。　考　察：体力測定会に継続参加可能な意欲の高い比較的健康な高齢者においても，閉じこもりリスク保有者が存在し閉じこもり予備群で孤独感が高いことが示された。閉じこもり予備群のうちから，孤独感を解消させるような働きかけを行うことが閉じこもり予防対策につながると期待される。原

A 2-year follow-up study on falls and related factors among community-dwelling elderly individuals

　Purpose：We conducted a 2-year follow-up study on falls and related causes experienced by localregion, independent elderly citizens．　Method：We analyzed a total of 533 subjects, all independent elderly citizens living in Kameoka City in Kyoto Prefecture who participated in both the 2011 Everyday Life Sphere Needs Assessment and the 2013 Physical Strength Measurements studies． Our study focused on the 25 items included as basic attributes in the 2011 Everyday Life Sphere Needs Assessment．After performing an analysis of the relationships between gender, age, and fall experience, we employed the existence of falls as a subordinate variable, age as a covariant, and each of the 25 basic-attribute items as independent variables in a multivariate logistic regression analysis to grasp the causes related to falls． Furthermore, we used 7 determinate items and a batch-input logistic regression analysis method to analyze the effect of an independent life style on falls．　Results：In the 2-year follow-up study, 35.5% of the subjects experienced at least one fall．The age of the subjects experiencing falls was slightly higher in the female group, compared with the males, but no significant difference was demonstrated．A significant difference was demonstrated in the ratio of falls in the highly-aged elderly group, compared with the lower-aged elderly group． In regard to the relationship between falls and each of the determinate items, in the male group, significant differences were demonstrated for motility, oral functions, memory lapses, and the tendency for depression, and especially the motility function showed a strong causal relationship, even with suppression of the other causes． In the female group, a significant relationship was demonstrated between subjects with a tendency for depression and subjects with a high instrumental activities of daily living index, and with suppression of the other causes in the analysis, a significant relationship was demonstrated between subjects with a low oral function index and a high instrumental activities of daily living index．　Conclusion：It was suggested that even the independent elderly citizens were predisposed to fall down by aging． In addition, sex deference of factor related to falls was observed, and thus sexspecific support policy for fall prevention that considered a characteristic and a background by sex is necessary．　目的：地域在住の自立高齢者を対象に2 年間の追跡調査を行い，転倒経験の有無とその関連要因を分析した。　方法：京都府亀岡市在住の自立高齢者で，2011 年の日常生活圏域ニーズ調査と2013 年の体力測定の両方に参加した533 人について分析した。調査内容は，基本属性の他，日常生活圏域ニーズ調査25項目を用いて，①運動機能，②低栄養，③口腔機能，④閉じこもり，⑤物忘れ，⑥うつ傾向，⑦手段的日常生活動作（IADL）の7 つの判定項目を設定した。分析は，性，年齢と転倒経験との関連を分析した後，転倒有無を従属変数，年齢を共変量，各判定項目それぞれを独立変数とするロジスティック回帰分析により転倒に関連する要因を把握した。また，7 つの判定項目を一括投入するロジスティック回帰分析により転倒への独立した影響を分析した。　結果：追跡2年間において，少なくとも1回以上転倒のあった者は35.5%であった。女性は男性より転倒した者がやや高率であったが有意差はなく，後期高齢者は前期高齢者より有意に高率であった。転倒と各判定項目との関連は，男性では，運動機能，口腔機能，物忘れ，うつ傾向において有意差がみられ，とくに運動機能は，他の要因の影響を調整しても強い関連を示した。女性では，うつ傾向とIADLの高い者に有意な関連がみられ，他の要因の影響を調整した分析では口腔機能の低い者，IADL の高い者に有意な関連がみられた。　考察：地域在住の自立した高齢者であっても，加齢により転倒しやすくなることが示唆された。また，転倒に関連する要因において，男性と女性とで異なった様相を呈し，性による特性や背景を考慮した転倒予防対策が必要であると推察された。原

Reactivation of CDX2 in Gastric Cancer as Mark for Gene Silencing Memory

To explore the epigenetic mechanism that reactivates CDX2 (a homeobox transcription factor that serves as a tumor-suppressor gene) in intestinal-type gastric cancer during cancer progression, we examined the methylation status of the CDX2 gene promoter and the expression pattern of methyl-CpG binding protein-2 (MeCP2). From archives of the pathology records of surgically excised advanced stomach cancer cases in the Department of Molecular Pathology, Ehime University in a past decate (n=265), 10 cases of intestinal-type tubular adenocarcinoma, well-differentiated type (wel) with minor poorly-differentiated adenocarcinoma (por) components were selected. The expression pattern of CDX2, MUC2 and MeCP2 in these 10 cases was analyzed by immunohistochemistry. The cancerous and non-cancerous areas were selectively obtained by microdissection, and the methylation status of the CDX2 promoter of each area was assessed by methylation-specific polymerase chain reaction (MSP). In all 10 cases, CDX2 expression was clearly observed in the nucleus of the non-cancerous background of the intestinal metaplasic area, where the unmethylation pattern of the CDX2 gene promoter prevailed with reduced MeCP2 expression. In this metaplastic area, CDX2 expression was co-localized with its target gene, MUC2. CDX