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

Auxin/AID versus conventional knockouts: Distinguishing the roles of CENP-T/W in mitotic kinetochore assembly and stability

Most studies using knockout technologies to examine protein function have relied either on shutting off transcription (conventional conditional knockouts with tetracycline-regulated gene expression or gene disruption) or destroying the mature mRNA (RNAi technology). In both cases, the target protein is lost at a rate determined by its intrinsic half-life. Thus, protein levels typically fall over at least 1–3 days, and cells continue to cycle while exposed to a decreasing concentration of the protein. Here we characterise the kinetochore proteome of mitotic chromosomes isolated from a cell line in which the essential kinetochore protein CENP-T is present as an auxin-inducible degron (AID) fusion protein that is fully functional and able to support the viability of the cells. Stripping of the protein from chromosomes in early mitosis via targeted proteasomal degradation reveals the dependency of other proteins on CENP-T for their maintenance in kinetochores. We compare these results with the kinetochore proteome of conventional CENP-T/W knockouts. As the cell cycle is mostly formed from G1, S and G2 phases a gradual loss of CENP-T/W levels is more likely to reflect dependencies associated with kinetochore assembly pre-mitosis and upon entry into mitosis. Interestingly, a putative super-complex involving Rod-Zw10-zwilch (RZZ complex), Spindly, Mad1/Mad2 and CENP-E requires the function of CENP-T/W during kinetochore assembly for its stable association with the outer kinetochore, but once assembled remains associated with chromosomes after stripping of CENP-T during mitosis. This study highlights the different roles core kinetochore components may play in the assembly of kinetochores (upon entry into mitosis) versus the maintenance of specific components (during mitosis)

Mitotic chromosomes are compacted laterally by KIF4 and condensin and axially by topoisomerase IIα

© 2012 Samejima et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication dateMitotic chromosome formation involves a relatively minor condensation of the chromatin volume coupled with a dramatic reorganization into the characteristic "X" shape. Here we report results of a detailed morphological analysis, which revealed that chromokinesin KIF4 cooperated in a parallel pathway with condensin complexes to promote the lateral compaction of chromatid arms. In this analysis, KIF4 and condensin were mutually dependent for their dynamic localization on the chromatid axes. Depletion of either caused sister chromatids to expand and compromised the "intrinsic structure" of the chromosomes (defined in an in vitro assay), with loss of condensin showing stronger effects. Simultaneous depletion of KIF4 and condensin caused complete loss of chromosome morphology. In these experiments, topoisomerase IIα contributed to shaping mitotic chromosomes by promoting the shortening of the chromatid axes and apparently acting in opposition to the actions of KIF4 and condensins. These three proteins are major determinants in shaping the characteristic mitotic chromosome morphology

The effective nursing educational plan for the elderly at hospital discharge. - through the questionnaires for the nurses and the elderly patients -

近年の急速な高齢化社会にともない複数の疾病や種々の障害をもちながら自宅で療養する高齢者が増加してきている。このような状況でいったん入院療養した高齢者が自宅へ帰り生活をしていくにはかなりの困難が予測される。高齢者が退院し自宅での生活にスムーズに適応できるか否かは看護婦の退院指導の良否に関わってくる。そこで指導側の看護婦の指導計画や指導方法、内容について調査すると共に受け手の患者の指導希望内容との比較を行い有効な退院指導のあり方を検討した。患者の心配事や援助や相談の希望内容と看護婦の重要とする指導内容はほぼ同様の内容であった。その内容は日常生活に関すること、病気や健康に関することが多かった。看護婦の退院指導の計画でカンファレンスをしている看護婦はしていない看護婦に比べ指導内容の経済的なこと、趣味や生きがいに関する事、生活環境、家族の協力に関することを重要とすると回答したものが有意に多かった。The elderly with chronic disease or handicaps who receive nursing home care is currently increasing. They may have some difficulties in their daily life after hospital discharge. The proper nursing educational plan may determine their post-hospital life without anxiety. The purpose of this study is to find out the effective nursing educational plan for them. Therefore, we investigated the contents of the nursing educational plan for their hospital discharge and what elderly patients need in their post-hospital life. As a result, both contents were similar. Those were about their daily life, disease, health. And there were significantly defferences between well-discussed plan and no discussed plan in the items of economy, hobbies, purpose of life, home environment, family cooperation. Results from this study suggest that more detailed discussion among nurses at hospital discharge is necessary to determine the individual nursing educational plan of elderly patients in their post-hospital life

Dust from Comet 209P/LINEAR during its 2014 Return: Parent Body of a New Meteor Shower, the May Camelopardalids

We report a new observation of the Jupiter-family comet 209P/LINEAR during its 2014 return. The comet is recognized as a dust source of a new meteor shower, the May Camelopardalids. 209P/LINEAR was apparently inactive at a heliocentric distance rh = 1.6 au and showed weak activity at rh < 1.4 au. We found an active region of <0.001% of the entire nuclear surface during the comet's dormant phase. An edge-on image suggests that particles up to 1 cm in size (with an uncertainty of factor 3-5) were ejected following a differential power-law size distribution with index q=-3.25+-0.10. We derived a mass loss rate of 2-10 kg/s during the active phase and a total mass of ~5x10^7 kg during the 2014 return. The ejection terminal velocity of millimeter- to centimeter-sized particles was 1-4 m/s, which is comparable to the escape velocity from the nucleus (1.4 m/s). These results imply that such large meteoric particles marginally escaped from the highly dormant comet nucleus via the gas drag force only within a few months of the perihelion passage.Comment: 18 pages, 4 figures, accepted on 2014 December 11 for publication in the Astrophysical Journal Letter