a new technique called “t-SNE” that visualizes high-dimensional data by giving each datapoint a location in a two or three-dimensional map. (Maaten and Hinton, 2008 PDF)
Most researchers are already familiar with another dimensionality reduction algorithm, Principle Components Analysis (PCA) also available in R2 and explained in more detail in the Principle Components Analysis tutorial. Both PCA and t-SNE reduce the dimension while maintaining the structure of high dimensional data, however, PCA can only capture linear structures. t-SNE on the other hand captures both linear and non-linear relations and preserves local distances in high dimensions while reducing the information to 2 dimensions (an XY plot). (16. t-SNE: high dimensionality reduction in R2 How to find groups in your dataset using t-SNE. r2-tutorials.readthedocs.io)
そもそもなぜ次元削減をする必要があるの？
Computers have no problem processing that many dimensions. However, we humans are limited to three dimensions. Computers still need us (thankfully), so we often need ways to effectively visualize high-dimensional data before handing it over to the computer. (An illustrated introduction to the t-SNE algorithm By Cyrille Rossant March 3, 2015)
（上記サイトはヴィジュアルに非常にわかりやすくt-SNEの説明をしています）
遺伝子解析にはなぜPCAよりもt-SNEが適しているの？
First, although PCA minimizes global reconstruction error, it may not preserve local proximities of points. In visualizing gene expression data, we are typically more interested in resolving nearby clusters than in preserving the correct distance relationships between genes with very different patterns of expression. But the optimization criterion of PCA results in the opposite priority: the relationship of distant points is depicted as accurately as possible, while small inter-point distances can be distorted. Second, there may be no single linear projection that gives a good view of the data: in such a case, all linear projection methods will fail. (An intuitive graphical visualization technique for the interrogation of transcriptome data. Bushati et al., 2011. Nucleic Acids ResearchVolume 39, Issue 17,Pages 7380–7389)
t-SNEの使い方の注意は？
Following are a few common fallacies to avoid while interpreting the results of t-SNE:
For the algorithm to execute properly, the perplexity should be smaller than the number of points. Also, the suggested perplexity is in the range of (5 to 50)
Sometimes, different runs with same hyper parameters may produce different results.
…
(Comprehensive Guide on t-SNE algorithm with implementation in R & Python
SAURABH.JAJU2, JANUARY 22, 2017)
t-SNEの短所は？
t-SNE has three potential weaknesses: (1) it is unclear how t-SNE performs on general dimensionality reduction tasks, (2) the relatively local nature of t-SNE makes it sensitive to the curse of the intrinsic dimensionality of the data, and (3) t-SNE is not guaranteed to converge to a global optimum of its cost function. (Maaten and Hinton, 2008 PDF)
ゲノムデータ（遺伝子発現プロファイルの解析）にt-SNEが使われるようになったのはいつ頃から？
自分が調べた限り、下記の論文よりも古い論文が見つかりませんでした。
Here, we test the recently developed nonlinear dimensionality reduction algorithm, t -statistic Stochastic Neighbor Embedding ( t -SNE) ( 8 ), on a variety of real-world transcriptome data sets. (An intuitive graphical visualization technique for the interrogation of transcriptome data. Nucleic Acids Res. 2011 Sep 1;39(17):7380-9.
We tested seven DRTs applied to four microarray cancer datasets and ran four clustering algorithms using the original and reduced datasets. … On the other hand, t-distributed Stochastic Embedding (t-SNE) and Laplacian Eigenmaps (LE) achieved good results for all datasets. (Comparative study on dimension reduction techniques for cluster analysis of microarray data. Date of Conference: 31 July-5 Aug. 2011 ieeexplore.ieee.org)
どんなデータに使えるの？
Question: why PCA for RNA-Seq but tSNE for scRNA-seq? (biostars.org)
Question: What to use: PCA or tSNE dimension reduction in DESeq2 analysis? (support.bioconductor.org)
t-SNEを実際に使うには？（生物学研究者向け）
Rを用いてt-SNE
A step-by-step workflow for low-level analysis of single-cell RNA-seq data with Bioconductor (bioconductor.org)
A step-by-step workflow for low-level analysis of single-cell RNA-seq data Aaron T.L. Lun, et al. F1000Research Software tool article
The Rtsne module in Array Studio will allow the user to cluster different cells with UMI counts, using the Rtsne package in R (arrayserver.com)
Seurat is an R package designed for QC, analysis, and exploration of single cell RNA-seq data. Seurat aims to enable users to identify and interpret sources of heterogeneity from single cell transcriptomic measurements, and to integrate diverse types of single cell data. (satijalab.org)
Identifying and Characterizing Subpopulations Using Single Cell RNA-seq Data (hms-dbmi.github.io)
シングルセルRNA-seqガイド
A practical guide to single-cell RNA-sequencing for biomedical research and clinical applications (Haque et al., Genome Med. 2017; 9: 75)
Visualization and analysis of single-cell RNA-seq data by kernel-based similarity learning. Bo Wang, Junjie Zhu, Emma Pierson, Daniele Ramazzotti & Serafim Batzoglou Nature Methods volume 14, pages 414–416 (2017) 新手法の提案 We present single-cell interpretation via multikernel learning (SIMLR), an analytic framework and software which learns a similarity measure from single-cell RNA-seq data in order to perform dimension reduction, clustering and visualization. 既存の手法との比較 On seven published data sets, we benchmark SIMLR against state-of-the-art methods.
CIDR: Ultrafast and accurate clustering through imputation for single-cell RNA-seq data. Lin P, Troup M, Ho JW. Genome Biol. 2017 Mar 28;18(1):59. 新手法の提案 Most existing dimensionality reduction and clustering packages for single-cell RNA-seq (scRNA-seq) data deal with dropouts by heavy modeling and computational machinery. Here, we introduce CIDR (Clustering through Imputation and Dimensionality Reduction), an ultrafast algorithm that uses a novel yet very simple implicit imputation approach to alleviate the impact of dropouts in scRNA-seq data in a principled manner. 従来の手法t-SNEなどとの比較 Using a range of simulated and real data, we show that CIDR improves the standard principal component analysis and outperforms the state-of-the-art methods, namely t-SNE, ZIFA, and RaceID, in terms of clustering accuracy. 代表的な結果の図
Visualization and cellular hierarchy inference of single-cell data using SPADE. Anchang B, Hart TD, Bendall SC, Qiu P, Bjornson Z, Linderman M, Nolan GP, Plevritis SK. Nat Protoc. 2016 Jul;11(7):1264-79. 新たなデータ可視化手法の提案 we describe the use of Spanning-tree Progression Analysis of Density-normalized Events (SPADE), a density-based algorithm for visualizing single-cell data and enabling cellular hierarchy inference among subpopulations of similar cells. 別のデータ可視化手法であるt-SNEとの比較 We compare SPADE with recently developed single-cell visualization approaches based on the t-distribution stochastic neighborhood embedding (t-SNE) algorithm.
SAIC: an iterative clustering approach for analysis of single cell RNA-seq data. Yang L, Liu J, Lu Q, Riggs AD, Wu X. BMC Genomics. 2017 Oct 3;18(Suppl 6):689. 解析の重要性 An important step in the single–cell transcriptome analysis is to identify distinct cell groups that have different gene expression patterns. 従来の手法の問題点 Many studies rely on principal component analysis (PCA) with arbitrary parameters to identify the genes that will be used to cluster the singlecells. 新手法の提案 We have developed a novel algorithm, called SAIC (Singlecell Analysis via Iterative Clustering), that identifies the optimal set of signature genes to separate singlecells into distinct groups. データ可視化のステップでのt-SNEの利用 We applied the SAIC algorithm to one simulated dataset and two published single cell datasets. After signature genes selection, the results were evaluated by Davies-Bouldins index and then visualized using both a t-SNE 2D–plot and an unsupervised hierarchical clustering heatmap.
Specific loci in the mouse genome that allow stable, reliable, and ubiquitous transgene expression in the absence of adverse effects like Rosa26, Hprt1, or Col1A1, have become essential tools in the performance of genetic studies. Safe harbor loci allow comparable analysis between different lines of mice in isogenic contexts. ((Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus Ordovas et al. JoVE)
Expression of a randomly integrated transgene is unpredictable and tends to be unstable over time due to epigenetic effects. Further, random integration often yields multiple integrants per cell, and this can result in the disruption or activation of host cell genes. Such unintended side effects produce a non-isogenic experimental setting, and this can confound data analysis and interpretation. (DeKelver et al., Genome Res. 2010. 20: 1133-1142)
ヒト細胞においてもトランス遺伝子を挿入できる、類似のセーフ・ハーバー部位が報告されました（DeKelver, et al., 2010）。これはアデノ随伴ウイルス（AAV）が通常、第19染色体上のPP1R12Cという特定のゲノム座位に組込まれるとの報告に端を発しています。AAVS1としても知られる本座位へAAVを組み込んだ場合でも、初代培養細胞や不死化細胞、人工多能性幹細胞（iPSC）および胚性幹細胞などをはじめとする種々の細胞に目に見える表現型の変化を生じることはありません。 (セーフ・ハーバー部位への目的遺伝子組込み用ゲノム編集ツール 実験検証 コスモ・バイオ GeneCopoeia 技術情報)
用例
Here we describe a strategy to genetically modify human iPS cells at ‘safe harbor’ sites in the genome, which fulfill five criteria based on their position relative to contiguous coding genes, microRNAs and ultraconserved regions. (Genomic safe harbors permit high β-globin transgene expression in thalassemia induced pluripotent stem cells. Nat Biotechnol. 2011 Jan;29(1):73-8.)
Nuclease, and using these nucleases, secretory tissues which they derived, cloned, and a method of expressing a transgene from the safe harbor locus in animals. (The methods and compositions for controlling transgene expression Google Patents JP2014526279A)
Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome. DeKelver et al., Genome Res. 2010. 20: 1133-1142)
Disruption of overlapping transcripts in the ROSA βgeo 26 gene trap strain leads to widespread expression of β-galactosidase in mouse embryos and hematopoietic cells. Brian P. Zambrowicz, Akira Imamoto, Steve Fiering, Leonard A. Herzenberg, William G. Kerr, and Philippe Soriano. PNAS April 15, 1997. 94 (8) 3789-3794; https://doi.org/10.1073/pnas.94.8.3789
Green fluorescent protein – calmodulin protein – (GCaMP)-type GECI are based on a circularly permutated EGFP molecule (cpEGFP) flanked at the N and C termini by the smooth muscle myosin light chain kinase derived calmodulin binding peptide (RS20) and calmodulin (CaM), respectively. (Helassa et al., Scientific Reports volume 5, Article number: 15978 (2015))
In our laboratory, we have focused our efforts to improve genetically encoded calcium indicator proteins (GECIs), specifically the green fluorescent protein (GFP)-Calmodulin fusion protein (GCaMP).(Badura et al., 2014 doi: 10.1117/1.NPh.1.2.025008)
Here we report the development of a high-affinity Ca(2+) probe composed of a single GFP (named G-CaMP). (Nakai et al., Nat Biotechnol. 2001 Feb;19(2):137-41.)
Generation and Imaging of Transgenic Mice that Express G-CaMP7 under a Tetracycline Response Element. (Sato et al., 2015 PLoS One 10(5):e0125354)
Designed and random alterations in the previously described, circularly permutated eGFP-based, Ca2+-sensing molecule, which we now term GCaMP1 (11), were undertaken to improve brightness and stability. (Imaging cellular signals in the heart in vivo: Cardiac expression of the high-signal Ca2+ indicator GCaMP2. Yvonne N. Tallini, Masamichi Ohkura, Bum-Rak Choi, Guangju Ji, Keiji Imoto, Robert Doran, Jane Lee, Patricia Plan, Jason Wilson, Hong-Bo Xin, Atsushi Sanbe, James Gulick, John Mathai, Jeffrey Robbins, Guy Salama, Junichi Nakai, and Michael I. Kotlikoff. PNAS March 21, 2006. 103 (12) 4753-4758)
Howard Hughes Medical Institute/Janelia Research Campusの研究者らはGCaMPとハイフンを入れない表記をしています。
Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Tian L, Hires SA, Mao T, Huber D, Chiappe ME, Chalasani SH, Petreanu L, Akerboom J, McKinney SA, Schreiter ER, Bargmann CI, Jayaraman V, Svoboda K, Looger LL. Nat Methods. 2009 Dec;6(12):875-81.
PNAS : Calcium waves occur as Drosophila oocytes activate
（In vivo imaging at dissection scope resolution of Ca2+ flux in activating oocytes from transgenic flies expressing GCaMP3 in their oocytes. See text and the legend to Fig. 1A for details. 論文 https://doi.org/10.1073/pnas.1420589112）
Ca2+ dynamics in body wall muscles of wild-type C. elegans
（Genetic crosses were made to transfer the genetically encoded Ca2+ indicator GCaMP3.35 (Schwarz et al., 2011) into the vps-36 (gk427) background. 論文リンク http://jcs.biologists.org/content/129/7/1490）
Neuronal Activity during Visual Perception 脳に映る視覚世界をリアルタイムで観察
(The gSA2AzGFF49A;UAS:GCaMP7a double-transgenic zebrafish used for imaging were also homozygous for the nacre pigmentation mutation [24] to eliminate melanophores. 論文リンク https://doi.org/10.1016/j.cub.2012.12.040）
（Here, we report a method for long-term imaging of a GECI, GCaMP6f, expressed from adeno-associated virus vectors in cortical neurons of the adult common marmoset (Callithrix jacchus), a small New World primate. 論文リンク https://doi.org/10.1016/j.celrep.2015.10.050）
High-performance GFP-based calcium indicators for imaging activity in neuronal populations and microcompartments.HodDana, YiSun, BoazMohar, BradHulse, Jeremy PHasseman, GetahunTsegaye, ArthurTsang, AllanWong, RonakPatel, John JMacklin, YangChen, ArthurKonnerth, VivekJayaraman, Loren LLooger, Eric RSchreiter, KarelSvoboda, Douglas SKim https://www.biorxiv.org/content/early/2018/10/03/434589
Using structure-based mutagenesis and neuron-based screening, we developed a family of ultrasensitive protein calcium sensors (GCaMP6) that outperformed other sensors in cultured neurons and in zebrafish, flies and mice in vivo.
Based on screening in cultured neurons (Fig. 1), we chose three ultrasensitive GCaMP6 sensors (GCaMP6s, 6m, 6f; for slow, medium and fast kinetics, respectively) for characterization in vivo. These sensors vary in kinetics, with the more sensitive sensors having slower kinetics. (Chen et al., 2013 Nature 499, 295-300)
G-CaMP6, G-CaMP7, G-CaMP8およびそのヴァリアント (中井博士らのグループ）
The dynamic range of G-CaMP7 (Fmax/Fmin = 36.6±4.10, n = 3) was ∼3-fold greater than that of G-CaMP6, even though this variant showed a lower Ca2+ affinity (Kd = 243±14 nM, n = 3) than G-CaMP6 (Fig. 1B and C). By performing further random mutagenesis on G-CaMP7, we obtained a more sensitive variant of G-CaMP7 termed G-CaMP8, (Ohkura et al., 2012 PLOS ONE https://doi.org/10.1371/journal.pone.0051286)
we introduced amino acid substitutions into GCaMP-HS, tested their activities, and developed a new version, which we named GCaMP7a (Muto, Ohkura et al., 2013. Curr Biol. 23(4):307-311)
【ジャネリア GCaMP6シリーズ】Ultrasensitive fluorescent proteins for imaging neuronal activity. Chen et al.,2013. Nature 499:295–300. “we developed a family of ultrasensitive protein calcium sensors (GCaMP6) that outperformed other sensors in cultured neurons and in zebrafish, flies and mice in vivo.” “Based on screening in cultured neurons (Fig. 1), we chose three ultrasensitive GCaMP6 sensors (GCaMP6s, 6m, 6f; for slow, medium and fast kinetics, respectively) for characterization in vivo.”
【中井博士ら GCaMP7a】Real-Time Visualization of Neuronal Activity during Perception. Muto, Ohkura et al., 2013.Curr Biol. 23(4):307-311. “we introduced amino acid substitutions into GCaMP-HS, tested their activities, and developed a new version, which we named GCaMP7a”
【中井博士ら G-CaMP6, G-CaMP7, G-CaMP8】Genetically Encoded Green Fluorescent Ca2+ Indicators with Improved Detectability for Neuronal Ca2+ Signals. Ohkura et al., 2012. PLOS ONE 7(12): e51286. “As expected, G-CaMP6, a variant of G-CaMP5.09 bearing an M36L substitution in the CaM domain (Fig. 1A), showed a higher Ca2+ affinity (Kd = 158±4.0 nM, n = 3) than G-CaMP5.09 or the previously reported G-CaMP2 variants G-CaMP-HS [17] and G-CaMP3 [4] (Fig. 1B and C).” ” Next we performed random mutagenesis on G-CaMP6 by using an error-prone PCR [15] and were able to screen a highly responsive variant termed G-CaMP7, which differs from G-CaMP6 by a deletion of histidine (ΔH) in the RSET domain and an S205N mutation in the circularly permutated EGFP domain (Fig. 1A). The dynamic range of G-CaMP7 (Fmax/Fmin = 36.6±4.10, n = 3) was ∼3-fold greater than that of G-CaMP6, even though this variant showed a lower Ca2+ affinity (Kd = 243±14 nM, n = 3) than G-CaMP6 (Fig. 1B and C). By performing further random mutagenesis on G-CaMP7, we obtained a more sensitive variant of G-CaMP7 termed G-CaMP8,”
【ジャネリア GCaMP5シリーズ】Optimization of a GCaMP Calcium Indicator for Neural Activity Imaging. Akerboom et al., 2012.J Neurosci 32 (40) 13819-13840 “Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of “GCaMP5” sensors.” “Consequently, the Asp380Tyr mutation raises the brightness of the calcium-bound state of GCaMP3 for both GCaMP5A and GCaMP5G; in addition, calcium affinity and cooperativity (Hill coefficient) are increased by ∼25% for GCaMP5A (Table 1).”
【中井博士ら G-CaMP 4.1】Tissue-Tissue Interaction-Triggered Calcium Elevation Is Required for Cell Polarization during Xenopus Gastrulation. Shindo et al., 2010. PLoS ONE 5(2): e8897. “G-CaMP 4.1, which is an improved form of a previously reported calcium indicator,”
【中井博士ら GCaMP-HS】Genetic visualization with an improved GCaMP calcium indicator reveals spatiotemporal activation of the spinal motor neurons in zebrafish. Muto, Ohkura et al., 2011. PNAS 108 (13) 5425-5430. “we developed GCaMP-HS (GCaMP-hyper sensitive), an improved version of the genetically encoded calcium indicator GCaMP,” “Kd and Hill coefficient of GCaMP-HS were 102 nM and 5.0, respectively (Fig. 1D). Kd and Hill coefficient of GCaMP2 were 146 nM and 3.8, respectively, indicating that GCaMP-HS has a higher affinity to Ca2+ ions and a higher cooperativity”
【ジャネリア GCaMP3】Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Tian et al., 2009. Nat Methods. 6(12):875-81.”We developed a single-wavelength GCaMP2-based GECI (GCaMP3), with increased baseline fluorescence (3-fold), increased dynamic range (3-fold) and higher affinity for calcium (1.3-fold). We detected GCaMP3 fluorescence changes triggered by single action potentials”
【ジャネリア GCaMP2結晶構造解析】Crystal Structures of the GCaMP Calcium Sensor Reveal the Mechanism of Fluorescence Signal Change and Aid Rational Design. Akerboom et al., 2009. J Biol Chem. 284, 6455-6464
【ジャネリア＆中井博士ら GCaMP2】Imaging cellular signals in the heart in vivo: Cardiac expression of the high-signal Ca2+ indicator GCaMP2.
Tallini, Ohkura, et al., 2006. PNAS 103 (12) 4753-4758. “previously described sensors have proved to be of limited use to report cell signaling in vivo in mammals. Here, we describe an improved Ca2+ sensor, GCaMP2,”
【中井博士ら 元祖G-CaMP】A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein. Nakai et al., 2001. Nat Biotech. 19:137–141.
中教審は「英語教育の早期化」という諮問を慎重に検討せよ Tweet ThisSend to Facebook | by suzumura 2014/11/25 政策の決定に携わる者の狭隘な視野が前途有為な児童や生徒の将来に悪影響を及ぼすことは避けられなければなりませんし、英語の教育を早期から行うという計画が新たな利権を生み出すための措置となることも、断固として排除されなければなりません。その意味でも、下村文科相による中教審への諮問は実態を伴わないものであるばかりでなく、英語を含む今後の日本の教育界に禍根を残すことになりかねないといえます。
Getting your research into an influential journal is certain to give a healthy boost to both academic standing and future career prospects. (How to get published in high-impact journals: Big research and better writing. Posted by Julie Gould, Nature Jobs Blog 03 Nov 2014)
１ 本件対象文書について不開示とした理由について
本件対象文書は「「インターネット上で指摘のあった論文の画像データに係る調査結果について」として調査結果が平成２７年７月３１日付けで公表されている，研究行動規範委員会の調査に関わる資料の一切。具体的には，調査にかかる会議に提出された資料，会議の議事次第，調査委員が示された資料，会議の議事録，調査報告書とその案，調査対象者から提出された実験データなどの資料，その他調査に使われた資料，調査で行われた関係者のヒアリングの記録，外部機関に調査や分析を行っていればその報告書。加えて，調査にかかった費用とその使途がわかる資料（外部調査委員への支払なども含む）」である。本学では，研究不正の事案については，科学研究行動規範委員会において調査を行っているが，請求にかかる文書は以下の５つの理由に該当する部分について不開示とする決定を行った。
① 個人名その他個人を識別できる情報であって法５条１号ただし書イ，ロ，ハのいずれにも該当しないものが記されている部分を不開示とする。
② 審議，検討又は協議に関する情報であって，公にすることにより，率直な意見の交換若しくは意志決定の中立性が不当に損なわれるおそれがあるものについては，法５条３号に該当するため不開示とする。
③ 公にすることにより，当該事務又は事業の適正な遂行に支障を及ぼすおそれがあるものについては，法５条４号柱書きに該当するため不開示
とする。
④ 公にすることにより，正確な事実の把握を困難にするおそれ，若しくはその発見を困難にするおそれがあるものについては，法５条４号ハに該当するため不開示とする。
⑤ 公にすることにより，人事管理に係る事務に関し，その公正かつ円滑な人事の確保に支障を及ぼすおそれがある部分については，法５条４号
ヘに該当するため不開示とする。よって，本件対象文書を平成２６年度～２７年度科学研究行動規範委員会資料並びに支給調書として，部分開示決定を行ったものである。これについて，審査請求人は，平成２９年４月４日受付の審査請求書のなかで，原処分の取消しを求めている。
私：「来週のラボミーティングは自分の番なんですが、データがないんですけど。」
ボス：「それはあなたの問題ね。」
Me: “I’m supposed to present at lab meeting next week, but I have no data.”
Advisor: “That’s your problem.” @sianbehr 14:48 – 2014年2月21日
大学院で最初のラボミーティングだけどデータが無いから休日の写真を1時間見せていよう First lab meeting presentation today as a PhD, no data, just going to show my holiday photos for an hour @Sarah_Bethy_ 0:22 – 2016年2月26日
1. 失敗の過程を話して、ラボからの助言を仰ぐ
2.古いデータを見直してみる 定量的な解析を行うことにより、新しい見方ができるかも
3．解析ソフトウェアを使いこなす バイオインフォマティクスツールなどの利用で新しい発見があるかも
（4.論文紹介の時間にする）
5. 新しいモデル、模式図をつくってみる 手持ちのデータや文献からの知見をあわせて新たなモデルがつくれるかも
6．備えあれば憂いなし 日頃からサ イドプロジェクトを走らせておいて、何もニューデータがないという状態をつくらないようにする （How to present during lab meeting without new data May 25, 2017 BY Brittany Carson）
“I have not failed 10,000 times. I have not failed once. I have succeeded in proving that those 10,000 ways will not work. When I have eliminated the ways that will not work, I will find the way that will work.” (Thomas Edison) (How Failure Taught Edison to Repeatedly Innovate. By Nathan Furr, forbes.com Jun 9, 2011)
Bacon notes that studies can be enjoyable, and they can also be useful (whether for developing critical thinking skills or learning something for one’s profession). But he also notes that if done too much, studying could lead to “sloth”— laziness (in other words, if all you do is study, you won’t make the time to apply what you’ve learned and use your knowledge in a positive way). Bacon also believed that certain subjects could enhance different abilities— for example, mathematics can improve concentration, poetry can enhance one’s “wit” (what we today would call a sense of humor), and studying history can expand one’s wisdom.
Liumbruno et al., How to write a scientific manuscript for publication. Blood Transfus. 2013 Apr; 11(2): 217–226. doi: 10.2450/2012.0247-12 PMCID: PMC3626472
Tips for Academic Writing and Other Formal WritingWrite what you mean, mean what you write For instance, we often speak informally of “going the extra mile”, “at the end of the day”, “hard facts”, things being “crystal clear” … if there was no crystal, do not write about its clarity.
(7:15-) Now, when I show my students the mystery box, they say now show us what’s inside. But, I will never do that. Of course, it’s sealed. Just like science, the mystery box isn’t about the answer. We don’t have access to some sort of universal truth. All we have are the questions. I tell the students, they can go home and make their own mystery box. And, you can do that, too. And, if your mystery box works the same as mine, congratulations, you are successful. But I will never show you the inside of my mystery box. That’s the science demonstration, right?
Teaching science: we’re doing it wrong | Danny Doucette | TEDxRiga
International English Language Testing System(IELTS)
International English Language Testing System（IELTS：アイエルツ）は、海外留学や研修のために英語力を証明する必要のある方、およびイギリス、オーストラリア、カナダなどへの海外移住申請に最適なテストです。イギリス、オーストラリア、カナダ、ニュージーランドのほぼ全ての高等教育機関で認められており、アメリカでも TOEFLに代わる試験として入学審査の際に採用する教育機関が3,000を超え、英語力証明のグローバルスタンダードテストとして世界中で受験者が増え続けています。
International English Language Testing System(IELTS)
Cambridge Assessment English（ケンブリッジ大学英語検定機構）
ケンブリッジ英語検定 [C2 Proficiency, C1 Advanced, B2 First for Schools, B2 First, B1 Preliminary for Schools, B1 Preliminary, A2 Key for Schools, A2 Key]、
Cambridge Englishは、学習者が実生活のさまざまな状況でのコミュニケーションのために英語をどのように使うことができるかを評価できるように設計されています。
Cambridge English はすべて、言語能力評価の国際指標であるヨーロッパ共通参照枠 Common European Framework of Reference for Languages (CEFR) に準拠しています。
世界中の20,000以上の大学、企業 (雇用者)、行政機関が、Cambridge English を認定しています。
Cambridge English の試験の多くが、 ビザや入学申請 のため次の国々で利用できます: 英国, オーストラリア, 米国およびカナダ。 IELTS (Cambridge English が提供) も英国ビザや入国申請に使うことができます。（引用元：http://www.cambridgeenglish.org/jp/why-cambridge-english/）
We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5′ exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular engineering tool. (Gibson et al., Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods volume 6, pages 343–345 (2009) doi:10.1038/nmeth.1318)
Daniel G Gibson, Lei Young, Ray-Yuan Chuang, J Craig Venter, Clyde A Hutchison III & Hamilton O Smith. Enzymatic assembly of DNA molecules up to several hundred kilobases.Nature Methods volume 6, pages 343–345 (2009) doi:10.1038/nmeth.1318
I’m having a lot of troubles with this reaction. Supposedly, it’s pretty quick and easy… but I’ve been working in this construction for months. (Asked 5 years ago
Gabriela Chavez-Calvillo, ResearchGate)
I recently Gibsoned a 9kb insert into a 5kb vector at a 1:1 molar ratio.(Christopher Duran, Colorado State University, 5 years ago ResearchGate)
NEW ENGLAND BioLabs (NEB) 製品情報 NEBuilder HiFi DNA アッセンブリー 製品名：NEBuilder HiFi DNA Assembly Master Mix, 製品番号：E2621S, 容量：10 reactions, 希望小売価格：￥18,000
Gibson Assembly® Cloning Kit: Have you tried NEBuilder HiFi DNA Assembly? NEBuilder HiFi offers several advantages over NEB Gibson Assembly. For more information, visit NEBuilderHiFi.com.
an In-Fusion™ enzyme reaction can join any two pieces of DNA that have 15 bp of identity at their ends. … The In-Fusion mechanism is ligation-independent and while proprietary, likely uses the unique properties of the 3′–5′ exonuclease activity of poxvirus DNA polymerase. When incubated with linear duplex DNAs with homologous ends in the presence of Mg2+ and low concentrations of dNTP, the 3′–5′ proofreading activity of poxvirus DNA polymerase progressively removes nucleotides from the 3′ end. This exposes complementary regions on substrate DNAs that can then spontaneously anneal through base pairing, resulting in joined molecules containing a hybrid region flanked by nicks, 1–5 nucleotide gaps, or short overhangs. The annealed structures are metastable because the poxvirus DNA polymerase has a lower affinity for nicked or gapped DNA ends than for duplex ends. Introduction into Escherichia coli repairs any single-stranded gaps. (Zhu et al., BioTechniques, Vol. 43, No. 3, September 2007, pp. 354–359)
we developed a new restriction site independent cloning method that does not leave any unwanted sequences at the junction sites (seamless) and is based on in vitro recombination between short regions of homologies (15–52 bp) in bacterial cell extracts termed SLiCE (Seamless Ligation Cloning Extract). (Zhang, Werling and Edelmann. Nucleic Acids Res. 2012 Apr; 40(8): e55. Published online 2012 Jan 11. doi: 10.1093/nar/gkr1288)
SLiCE from Escherichia coli laboratory strains（本橋研究室）：seamless cloningに関連する試薬は、Gibson assembly kitやIn-Fusion kitなどの名称で様々なメーカーから商品化されていますが、その多くが高価で、資金の乏しい研究室では購入を躊躇しているかもしれません。そのような方に、ぜひ試していただきたいのがこのSLiCE法です。
Homologous DNA ends are efficiently fused together and produce vector clones with great accuracy. (Cold Fusion Cloning Kit with Competent Cells. How It Works. SBI System Biosciences))
1) gibson leaves no nick on the product, while infusion does.
2) gibson is more robust, it tolerate 3´- & 5´-end mismatch, while infusion doesn’t. … most false positive colonies i got when using gibson were actually caused by primer dimer or unspecific pcr products (they have the same overlap ends as the designed products)
3) there is ligase in gibson. so if you use only one restriction enzyme to linearize your plasmid backbone and use it in the assembly. The ligase may repair the backbone back to empty plasmid.
(Xinglin Jiang, Technical University of Denmark. What‘s the differences between the gibson assembly(NEB) and in-fusion clone(clontech)? ResearchGate)
A major limitation to SLIC/Gibson/CPEC/SLiCE assembly is that the termini of the DNA sequence fragments to be assembled should not have stable single stranded DNA secondary structure, such as a hairpin or a stem loop (as might be anticipated to occur within a terminator sequence), as this would directly compete with the required single-stranded annealing/priming of neighboring assembly fragments. (The SLIC, Gibson, CPEC and SLiCE assembly methods (and GeneArt® Seamless, In-Fusion® Cloning. j5.jbei.org)
Over the past decade, scientists have developed and fine tuned many different ways to clone DNA fragments which have provided appealing alternatives to restriction enzyme cloning. These newer technologies have become more and more common, and for good reason. They offer many advantages over the traditional restriction enzyme cloning we once relied exclusively on. (Addgene’s Blog. Posted by Brook Pyhtila on Mar 1, 2016 太字強調当サイト)
It’s been more than four decades since researchers launched the molecular-biology revolution with the invention of DNA cloning. In that time, the tools of the trade have changed relatively little, and for many researchers, DNA cloning still means restriction enzyme digestion and DNA ligation. … Today, thanks largely to the needs and creativity of the synthetic-biology community, alternative, “seamless” cloning strategies have been developed. Here are some of the more popular options. (Say Goodbye to Genetic Scars with These Seamless Cloning Kits
Posted: May 29, 2014 Jeffrey M. Perkel 太字強調は当サイト)
参考
Gap-Repair Cloning：GRCがinvitrogenからkit化されて発売されています。GeneArt?® Seamless Cloning & Assemblyという名前です。酵母のコンピテントセルやトランスフォーメーション試薬、大腸菌のコンピテントセル等もすべて含まれたキットです HM’s Home page
相同組換え方法およびクローニング方法並びにキット Inventor 正治 磯部 信幸 黒澤 Original Assignee 国立大学法人富山大学 Priority date 2008-03-07
Homologous recombination-based DNA cloning compositions. US8501454B2 Inventor:Weiqiang Liu, Ping Yang, Tao Wang, Zhuying Wang, Wenzhu Chen, Fang Liang Zhang, Original Assignee:Nanjingjinsirui Science and Tech Biology Corp, Priority date 2008-09-10
Cloning Without Restriction (TheScientist September 12, 2005) Key to Invitrogen’s Gateway technology is the “entry clone,” which contains a fragment of interest in a recombinase-ready plasmid. Researchers can quickly and efficiently move between expression systems without subcloning by swapping the insert from vector to vector.
カリフォルニア大学雇用者の給与(Compensation at the University of California) University of California Employee Pay As part of its commitment to transparency and public accountability, each year the University of California publicly reports employee pay data. The report covers UC’s career faculty and staff employees, as well as part-time, temporary and student employees.
被告発人らは、非常勤講師らがこれを知ることのできない状態において、本件就業規則作成の意見を聴く「労働者の過半数を代表する者」の選出のための立候補者を出すため、各事業所の常勤教員に要請して立候補させ、その選出に反対の者のみ不信任の旨の投票をし、その不信任投票をしなかった者は賛成したものとして取り扱う、というやり方をした。（引用元：2013年4月8日 告発状 PDF 太字強調は当サイト)
私はかつて「過半数代表と労働者代表委員会」という小論（日本労働協会雑誌356号〈1989年〉）において，現行の過半数代表制に重大な欠陥があるとして，常設の労働者代表委員会の設置を提案した。その後ほぼ四半世紀が経過したが，この間，過半数代表の機能が一層肥大化する一方，代表者の選出に関する一定の規定（労基法施行規則6条の2）を除いて，制度改革がまったくなされていないために，事態は一層深刻化している。第1の問題は，過半数代表者のあり方である。労働組合の組織率の低下とともに，過半数組合が存在しない事業場が増えているが，そこでは，個々の問題ごとに従業員の意見を代表する者という法律のタテマエとは異なり，通常は一人の労働者がすべての問題に関する代表となっている。しかし，一人の労働者が，すべての問題について「代表」としての役割を果たすことが不可能なのは明らかである。過半数代表制は，少なくとも過半数組合が存在しない場合には虚構と化しているのである。第2の問題は，この間の非正規労働者の急増（20％から35％へ）にもかかわらず，非正規労働者の声を反映させる仕組みが形成されていないことである。過半数組合が存在する場合でも，多くは正社員しか組織していない。パート就業規則を作成するのに正社員組合の意見を聴取すればよい（パート法7条にパート代表の意見聴取に関する努力義務規定はあるが）というのは明らかに奇妙な事態である。以上いずれの面からみても，過半数代表制はきわめて深刻な矛盾をかかえるに至っている。法律のタテマエと現実との乖離は，もはや許容しうる範囲を大きく越えているといわねばならない（提 言労働者代表制度の早急な法制化を■西谷 敏 PDF 太字強調は当サイト）
