A novel high-throughput (HTP) cloning strategy for site-directed designed chimeragenesis and mutation using the Gateway cloning system

There is an increasing demand for easy, high-throughput (HTP) methods for protein engineering to support advances in the development of structural biology, bioinformatics and drug design. Here, we describe an N- and C-terminal cloning method utilizing Gateway cloning technology that we have adopted for chimeric and mutant genes production as well as domain shuffling. This method involves only three steps: PCR, in vitro recombination and transformation. All three processes consist of simple handling, mixing and incubation steps. We have characterized this novel HTP method on 96 targets with >90% success. Here, we also discuss an N- and C-terminal cloning method for domain shuffling and a combination of mutation and chimeragenesis with two types of plasmid vectors

LPA, C1P, and S1P in IPF

Idiopathic pulmonary fibrosis (IPF) is the most common idiopathic interstitial pneumonias. Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are signaling lipids that evoke growth factor-like responses to many cells. Recent studies revealed the involvement of LPA and S1P in the pathology of IPF. In this study, we determined LPA, S1P and ceramide 1-phosphate (C1P) in peripheral blood plasma of IPF patients, and examined correlation to the vital capacity of lung (VC), an indicator of development of fibrosis. Blood plasma samples were taken from eleven patients with IPF and seven healthy volunteers. The lipids of the sample were extracted and subjected to liquid chromatography-tandem mass spectrometry for analysis. Results showed that there is a significant negative correlation between VC and plasma LPA levels, indicating that IPF patients with advanced fibrosis had higher concentration of LPA in their plasma. Average of S1P levels were significantly higher in IPF patients than those in healthy subjects. Although it is not statistically significant, a similar correlation trend that observed in LPA levels also found between VC and S1P levels. These results indicated that plasma LPA and S1P may be associated with deterioration of pulmonary function of IPF patients

The history of Traube\u27s stethoscope in Japan, America and Europe through literatures and photographs, and research into the spiritual bonds between midwives and mothers

[Purpose] In this study, we examined the history of Traube (tubular stethoscope), which has been used by maternity nurses since the 1800\u27s for the diagnosis and practice of midwifery. In addition, we determined the contemporary significance of the bond between maternity nurses and pregnant women through Traube, based on the literature on midwifery in which maternity nurses provided support to pregnant women using this medical instrument. [Method] The analysis was based on historical data related to midwifery. The primary and secondary parameters were identified by consulting the Japan Medical Abstracts Society website and library book storerooms and by searching and extracting relevant books from the libraries of medical departments at historical universities and the National Diet Library in Japan. [Result] The data collected from the 1800\u27s to 2000\u27s were divided into three areas : age, region, and contents of description. We prepared chronological tables on the history and photographs of Traube in Japan, America, and Europe, using data extracted over a period of time. The data describing the use of Traube to listen to fetal heart tones in pregnant women was divided into two areas, i.e., description and age, to prepare a historical chronological table. [Discussion] During the postmodern age, birth delivery at home or in residential regions was the origin of manual midwifery. Maternity nurses encouraged pregnant women using their hands, eyes, ears, and mind, based on the five senses. This practice has been preserved over the years through the diagnosis and practice of midwifery by listening to the fetal heart tones using Traube. Maternity nurses deepened their love for pregnant women and their unborn child using the warmth of their hands and Traube and in combination with their mind

Processes for Instilling Confidence in Mothers to Continue BreastFeeding

This study clarifies the process through which mothers gain confidence during continued breastfeeding. Using an interview guide, semi-structured interviews were conducted with eight mothers. The interviews focused on the process through which mothers gain confidence during continued breastfeeding. The data were analyzed using constant comparative analysis, drawing on Saiki\u27s grounded theory approach. The average age of the particpants was 31.3 (+-4.34). The mothers displayed desire and enthusiasm from the time of their pregnancy as they . Then, as the mothers , they moved from having the perception that {communication by way of breastfeeding is something special} to achieving a sense of oneness by feeling that they were [always together with their infant during breastfeeding]. The mothers succeeded in through consultations with midwives and other medical professionals. Moreover, as the mothers came to {understand the simplicity and virtue of breasts by investing effort in breastfeeding}, they {recognized the possibility of feeding the infant only breast milk}, affording them a degree of . On the other hand, {early anxiety about breastfeeding} led to increased and fatigue among the mothers, as well as a sense of . In addition, the notion that {doing nothing would lead to the use of powdered milk} led mothers to experience . Both before and after giving birth, the mothers maintained a strong desire and enthusiasm as they , as well as a strong sense of oneness with their infant because they were [always together with their infant during breastfeeding]. Repeated breastfeeding helped the mothers acquire the ability to care for themselves, affording them a degree of emotional comfort, which, in turn, helped them develop confidence as they recognized that breastfeeding is possible precisely because it involves engagement with their own child

Qualitative analysis of emotional process mothers go through before becoming confident with breast-feeding

Objective: The Healthy Parents and Children 21 Project in Japan has set an objective to achieve a 60% breast-feeding rate among mothers who have one-month-old children. Using qualitative analysis, this study clarifies the emotional process that mothers experience before they feel confident with breast-feeding. Methods: Semi-structured interviews were conducted with four mothers who were breast-feeding children ranging in age from 2 to 24 months, on their emotional transitions before they felt confident with breast-feeding, and analysis was performed with the use of Saiki\u27s grounded theory approach. With the agreement of the appropriate ethics committee, we explained our objective and protocol, and obtained final approval for the research in writing. Results: The average age of the mothers was 31.8 (+-4.3) years old, and the average interview time was 35.0 (+-17.3) minutes. On analyzing the emotional process the mothers we identified 【single-minded continuation of breast-feeding】 as the core category, and five sub-categories such as >, >, and > We created a single integrated diagram. Discussion: The mothers had had a strong desire to breast-feed even during pregnancy and >. Once they began breast-feeding the mothers gained a sense of security and confidence as they felt "relieved while breast milk was being released from their body". These feelings encouraged them to "single-mindedly continue to breast-feed", and "keeping up with it led them to appreciate the simplicity and beauty of breast-feeding" Conclusion: The Surveyed mothers developed affection for their children through mutual interactions between them by means of breast-feeding. Realizing that their breast milk is adequate to feed their children gave them the confidence to know that their bodies alone can support the growth of their children. The results of this study indicate the need for environments and support that help mothers remain motivated to breast-feed their children

ショウガイジ オ モツ ハハオヤ ノ セイシンテキ ケンコウド 2 ニュウヨウジキ ト ガクレイキ ノ ヒカク

本研究の目的は、障害児を持つ母親の精神的健康度の現状を明らかにし、必要な支援内容に関する指針を得ることである。 対象者はE県の通園事業・通園施設を利用する乳幼児の母親247名とE県立養護学校に通学する児童を持つ母親98名である。母親の属性(年齢、就労状況、世帯構造等)と、「育児ストレス・コーピング」、「育児バーンアウト」、「孤独感」について調査し分析した。さらに、同研究(I)で明らかにした他の指標(「母親の健康関連QOL」「育児ソーシャルサポート」「父親の育児サポート認知」「育児負担感」)との関連性について分析した。また、乳幼児と児童の母親の違いについて分析し、障害児を持つ母親の精神的健康度を高めるための示唆を得た。 障害児を持つ母親の健康関連QOLを高めるためには、乳幼児を持つ母親の場合、育児ソーシャルサポートが逃避的コーピングを回避し、さらに父親の育児サポートが調整的コーピング行動を惹起し、育児負担感や、育児バーンアウト、孤独感を抑制することが示された。一方、児童の母親の場合は健康関連QOLと育児ソーシャルサポートや、父親の育児サポートとは直接的な関連性がないことが明らかになった。しかし、育児負担感や育児バーンアウト、孤独感とは強い相関が認められた。また、育児負担感を軽減し育児バーンアウトを抑制するためには、父親の育児サポートが大きな要因となっていることが明らかになった。また、孤独感は児童の母親の場合は全ての尺度と関連があったが、乳幼児の場合は、父親のサポート認知とは関連が認められないという違いが見られた。The purpose of this study is to investigate condition of mental health on mothers who have disabled preschool/school age children and to provide useful information for developing the assistance guideline for them. 247 mothers who utilize nursery schools for the disabled and 98 mothers who use schools for the disabled attended this study. Questionnaire was consisted of attributes of mothers (age, employment condition, family structure), coping type to parenting stress, parenting burnout, and loneliness. After analyzing these data, following findings were found. For mothers who have preschool disabled children, parenting-related social supports prevented their negative coping (i.e. escape). And parenting support from fathers reduce their parenting stress, parenting burnout and loneliness. For mothers who have school age disabled children, parenting-related social support and parenting support from fathers do not have positive influences on their condition of mental health

ショウガイジ オ モツ ハハオヤ ノ セイシンテキ ケンコウド 1 ニュウヨウジキ ト ガクレイキ ノ ヒカク

本研究の目的は、障害児を持つ母親の精神的健康度の現状を明らかにし、必要な支援内容に関する指針を得ることである。 対象者はE県の通園事業、通園施設を利用する乳幼児の母親247名とE県立養護学校に通学する児童を持つ母親98名である。母親の属性(年齢、就労状況、世帯構造等)と子どもの属性(年齢、性、障害の種類と程度)および「母親の健康関連QOL」、「育児ソーシャルサポート」、母親による「父親の育児サポート認知」、「育児負担感」を調査し分析した。さらに乳幼児と児童の母親の違いについて考察した。 その結果、障害児を持つ母親のQOLを高めるためには、母親の育児負担感を軽減する必要があることが示された。また、児童の母親にとっては父親の育児サポートが育児負担感を軽減することが示唆された。乳幼児の母親のQOLは、育児ソーシャルサポート、父親のサポートによって高められることが示された。The purpose of this study is to examine the level of mental health of mothers with disabled children in order to develop guidelines for the assistance necessary to help them. The subjects were 247 mothers with disabled infants utilizing the institutes of nursery schools for disabled infants in E prefecture and 98 mothers of disabled children attending an E prefectural school for the disabled. This study examined and analyzed the attributes of mothers (age, employment status and household structure), the attributes of children (age, sex, and degree and kind of disability),the quality of life (QOL) of mothers, the social support for raising children, the level of support from fathers, and the parenting stress of mother. This study also examined the difference between mothers of children and those of infants. The results of this study indicate that to increase the QOL of mothers, parenting stress of mother need to be reduced. Moreover, this study suggests that the QOL of mothers be improved by the support of fathers and the social support for raising disabled children

Breastfeeding experiences of Japanese women living in Perth, Australia

The aim of this study was to document the breastfeeding practices of Japanese-Australian mothers living in Perth. A cross-sectional survey of mothers who had delivered babies in Japan or Australia or both was carried out on a sample of 163 mothers recruited through Japanese social and cultural groups in Perth and by a 'snowball' technique. Factors involved in the decision to breastfeed were analysed using multivariate regression analysis. The main outcome measures were the initiation and duration of breastfeeding and cultural beliefs about breastfeeding. Breastfeeding initiation rates of the Japanese- Australian mothers in Japan and in Australia were higher than for other Australians and are consistent with breastfeeding rates in Japan. In Australia, 65% of Japanese-Australian mothers were still breastfeeding at six months. The most common reason for the decision to cease breastfeeding was 'insufficient breastmilk'. The significant factors in breastfeeding duration were 'the time the infant was introduced to infant formula', 'the time when the feeding decision was made', 'doctors support breastfeeding' and 'the mother received enough help from hospital staff'; these were positively associated with the duration of breastfeeding. Japanese mothers take a lot of notice of advice given by health professionals about infant feeding practices

Functional analyses of the CENP-O complex in mice

   During cell division, it is important to ensure that the genomic information is faithfully transmitted to daughter cells. To achieve correct segregation ofchromosomes, specialized apparatus are built and complexly regulated duringeukaryotic cell division. The kinetochore is one of these apparatus. The kinetochoreforms a large structure on centromere DNA and plays important roles in mitoticprogression by microtubule attachment. The kinetochore of higher eukaryotes has atrilaminar structure that consists of the inner plate, outer plate and midzone. Theinner plate functions as the foundation of kinetochore formation. While a largenumber of proteins that localize to the inner kinetochore have been identifiedrecently, precise functions of each kinetochore protein are poorly understood andlittle is known about their functional role especially in organismal context.The CENP-O complex that consists of CENP-50/U, O, P, Q, and R constitutivelylocalizes into the kinetochore and is required for recovery from spindle damage inchicken DT40 cells. While DT40 cells with KO of most proteins localized in innerkinetochore die, DT40 cells lacking CENP-O complex proteins are still viable. Therefore, we focus on functional role of the CENP-O complex proteins inorganismal context.   Previous study showed that mouse ES cells and mice lacking CENP-50/U diewithin several days after depletion of the protein and during early development,respectively, while chicken DT40 cells with KO of CENP-50/U are viable. Thisobservation suggests that the CENP-O complex plays an essential role in the earlydevelopment of mice. Notably, knockout phenotype of CENP-R in DT40 cells isdifferent from that of other CENP-O complex proteins in some aspects andkinetochore localization of CENP-R occurs downstream of other complex proteins.CENP-R may play a different role from that of other CENP-O complex proteins. It isimportant to examine function of CENP-O complex including CENP-R inorganismal context.   In this study, ES cell lines and mice lacking CENP-R were created to elucidate the function of CENP-O complex proteins in mice, and their phenotypes were compared with that of CENP-50/U.  Although mice with knock out of CENP-50/U died during early embryogenesis, CENP-R null mice were viable and normally produced offspring, suggesting that CENP-R is not essential in mice and has different role from that of other CENP-Ocomplex proteins.   To elucidate that the lethality of CENP-50/U deficient mice is caused at a cellular level, ES cells lacking CENP-50/U or R were generated. Although ES cells with KOof CENP-50 /U were not viable and died within several days, ES cells lackingCENP-R were normally propagated. In addition, mitotic index and the number ofanaphase with lagging chromosomes were increased in ES cells lacking CENP-50/U.These results suggest that the lethality of mice lacking CENP-50/U is caused at acellular level, and each cell in mice lacking CENP-50/U dies due to some mitoticdefects. I also confirmed that the kinetochore structure and the dependencies ofkinetochore localization of CENP-O complex proteins are conserved betweenchicken DT40 cells and mice ES cells.   Chicken DT40 cells are derived from chicken B cells, and DT40 cells lackingCENP-50/U are viable. Therefore, I expected that the viabilities of mice cells lackingCENP-50/U are different among cell types. To examine this, I generated inducibleknockout mice, in which CENP-50/U is removed by ERT2-conjugated Crerecombinase. I isolated mouse embryonic fibroblast cells and B-cells from adultinducible knockout mice of CENP-50/U. As a result, both mouse B cells and mouseembryonic fibroblast cells were viable and normally propagated even afterCENP-50/U was removed from kinetochore by OHT addition although ES cellslacking CENP-50/U are lethal. I concluded that the importance of CENP-50/U inmitosis is different among cell types in mice.   In living organism, there are various cell divisions such as meiosis, segmentation of fertilized egg or asymmetric cell division in which machinery of chromosome segregation, growth rate, mitotic regulation are different in detail. In addition, the frequency of chromosome missegregation is different among cell types.It is possible that the kinetochore has a function in strict regulation of theseparticular cell divisions, however, little is known about kinetochore functions andvariety in organismal context. In this study, I showed that the importance ofCENP-50/U differs among cell types. From this result, I suggested that CENP-50/Umay be required for these strict regulations of mitosis in mice