我々は彼の貴重な支援に感謝博士アントニオミランダ- Vizueteしたいと思います。我々はまた、貴重な技術支援のためにサルマBoulayouneとイザベルキャバに私たちの感謝の意を表したいと。この作品は、アンダルシア州政府（BIO - 272）からの助成金によって賄われた。この研究は、ヨーロッパでは遺伝子実験を規制する現行の法律との合意で行われている。

1：浸透回避アッセイ。 <ol><li>約16-24時間アッセイの前に、OP50 E.を含む新鮮なNGMプレートに各遺伝子型のL4幼虫期の動物を選ぶ大腸菌は、20℃で、それをandincubate翌日、若年成人での実験を開始します。 </li><li>それは、アッセイ&quot;盲目的に&quot;行うことをお勧めします。アッセイすべき各菌株でプレートを実験が終了したときにマスク解除されるrelabelledプレートを用いてアッセイを行うこと、第二実験者によってrelabelledされるべきである。 </li><li>検定の日、1％コンゴーレッド溶液とフルクトースの4Mストック溶液を調製し、室温で完全に溶解する。色素が時間と共に沈殿する可能性があるため私たちは、各アッセイの前に解決策を確認することをお勧めします。 </li><li> NGMプレート上に環状のリング（直径1cm）が赤4Mのフルクトースの溶液15μlを、固体培地の中心に概説されています。フルクトース溶液は寒天に浸透させ、これは通常、2〜5分かかります。 </li><li>リング内の各菌株の個々の若年成人の動物を配置し、浸透障害への応答を決定するために次の10分をオーバー従ってください。行の6倍以上のリングを回避する動物は、通常のように分類され、以下の6回でリングを終了するものは、浸透圧感受性に欠陥とみなされます。 注 ：コントロール株がそれぞれのアッセイで使用されている必要があります。彼らは断固としてリングの障壁を避けるようにN2若い成人の動物は、ポジティブコントロールとして使用されています。以前に、飢えDauer幼虫を通過または乾燥しすぎているプレートから来ているされている動物は使用しないでください。冒頭で、対照動物は、アッセイプレートと解決策が正しいことを確認するために、重複して評価されるべきである。 </li></ol> 2：RNAiの給餌によるノックダウンのワームの発生。 <ol><li> RNAiのプレート：NGM RNAiの餌皿は、リットル当たりが含まれています：17 gの寒天、2,5グラムペプトン、3.0グラムのNaCl、5 mgのml -1のコレステロールを1mlのH 2 Oおよびオートクレーブで1リットルフラスコを埋める。寒天は、約65に冷却した後° C、1 M KPO 4、pH6.0で、1 M CaCl 2を 、1M MgSO 4を 、0,5 mlカルベニシリン（50 mgをml -1）を、1を1 ml 1 mlの25 mlを追加するミリリットル1M IPTG。準備された固体NGMで開始している場合は、電子レンジで固形培地を溶融し、その後冷却してから、以前に示されているKPO 4、pH6.0で、のCaCl 2、MgSO 4を 、カルベニシリンおよびIPTGを追加するには、ベンチに液体NGMを置きます。 それぞれ60ミリメートルシャーレにNGMの10mlを追加します。冷却を可能にし、使用前に室温で一晩、それらを維持するプレートを反転させる。プレートは、約4〜5日間4℃でビニール袋に格納することができます。 </li><li>細菌の準備と誘導：ルリア-ベルターニ（LB）寒天プレート（17 gの寒天、10gトリプトン、5gの上に、標的遺伝子に対応する断片を含むL4440ベクターで形質転換大腸菌 HT115（DE3）株のコロニーを、分離酵母エキス、1リットル当たり10グラムのNaCl）アンピシリン（50μg/ mlの）とテトラサイクリン（15μg/ ml）を含む。 細菌のコロニーをピックアップし、50μg/ mlのアンピシリンを含むLBに接種し、37℃で振盪しながら6〜8時間のために栽培℃、準備NGMプレート上にシードこの文化のドロップをし、一晩インキュベートされる前に徹底的にプレートを乾燥させる（細菌が成長すると誘導を開始できるように室温で12 24時間）。テトラサイクリンは光に敏感であり、それはプレート間でのRNAiアッセイの変動性に影響を与える可能性があるため、インキュベーションは暗闇の中だった。 それは正とRNAiの給餌実験のネガティブコントロールを使用する必要があります。陽性対照は、UNC - 22遺伝子の配列を含むL4440ベクターで形質転換された大腸菌 HT115セルです。 UNC - 22遺伝子のノックダウンは、&quot;けいれん表現型&quot;を生成します。ネガティブコントロールは、空のL4440ベクターで形質転換した大腸菌 HT115セルです。 </li><li>ワームの取り扱いとスコアリング：初日、20℃細菌とインキュベートすることなく、NGMプレート上にOP50を接種したNGMプレートから場所L4段階のワーム℃で12時間（空腹時）。 次の日、特定の標的遺伝子のRNAiを発現している細菌でseeddedプレートの上に若い絶食大人の雌雄同体を転送する。 F1子孫を生成するために20℃40〜48時間° Cにしておきます。 その後、同じ細菌を接種した別のプレートの上にいくつかのF1の成虫を置きます。 24〜48時間後、選択してF2子孫、若年成人（少数の卵に完全に形成された突出外陰部）と表現型のスコアから隔離する。 注：。神経細胞におけるRNAiの誘導が原因C elegansの神経系の耐火特性のRNAiにいくつかの制限があります。ニューロンにおけるこの非効率性の問題を克服するためには、RRF - 3株、ニューロン10のRNAiの影響に過敏な背景を使用することをお勧めします。我々の実験がないdifferencでESは遺伝子のためにブリストルN2とRRF - 3株の間に発見し、表現型が分析された。 </li></ol> 3：使用される菌株。 C.本研究で使用した線虫株を表1に示す。変異株は、N2野生に対して外交配- タイプの少なくとも4倍、突然変異誘発のプロトコル中に生成されている可能性が不要なランダムな突然変異を削除する。 。。OP50 E coli株は、プラスミドNRX - 1を運ぶpL4440（JA：C29A12.5）と線虫遺伝子センター、ミネソタ大学、米国大腸菌 HT115（DE3）でsuppliiedしたとNLG - 1（JA：C40C9.5 ）遺伝子断片は、ピーターAskjaer、セントロアンダルシアデBiologíaデル開発省（CABD）、CSIC大学パブロオラビデ、セビリア、スペインで提供されていました。 4：代表的な結果。 例示的な結果は図1と図2で表されます。各菌株三レプリカ実験の最低の10匹を行った。 <img alt="図1" src="/files/ftp_upload/1616/1616Fig1.jpg"/> 図1。浸透回避行動の実験。 対照株とNRX - 1 V欠損mutantwormsは、彼らが浸透バリア（4Mフルクトース）を発生したときに逆方向に反転することで対応（ok1649とtm1961）。NLG - 1（ok259とtm474）X変異体はこのバリアーを検出に失敗する。 NLG - 1とNRX - 1の欠損二重変異体は、菌株CRR21（ok1649、ok259）VXとCRR23（tm1961、ok259）VXは、野生型の表現型を回復した。 *は、t -学生の試験によるブリストルN2野生型に各菌株の応答では、T -生徒のテストで有意差（P≤0.001）を示しています<img alt="図2" src="/files/ftp_upload/1616/1616Fig2.jpg"/> 図2。 RNAiの給餌によるノックダウンのワームを用いた実験。 大腸菌 HT115（DE3）空のpL4440ベクターで形質転換または標的遺伝子NRX - 1またはNLG - 1に対応するフラグメントを含むが飼育異なるワームの菌株に使用されていました。 *は、空のpL4440ベクトルが供給N2株と比較して、NLG - 1遺伝子を標的断片でpL4440ベクターを保持する細菌が供給N2株の応答では、T -生徒のテストで有意差（P≤0.001）を示しています。

<ol>
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Neurexinsとneuroliginsはシナプス伝達11とシナプス結合12の分化に重要な役割を行う。両分子は、自閉症13,14のための候補遺伝子として同定されている。 このビデオでは、私たちはCの浸透回避反応に影響を及ぼす遺伝子の影響を研究することができるシンプルな方法を示してエレガンス 。ニューロリギン欠損変異体では、浸透力を検出す...

表1。C.虫系統。 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> ひずみ </td><td> 遺伝子 </td><td> 対立遺伝子 </td><td> 元 </td></tr><tr><td>ブリストルN2 </td><td> - </td><td> - </td><td> aCGC </td></tr><tr><td> VC228 </td><td> NLG - 1 </td><td> ok259 </td><td> CGC </td></tr><tr><td> FX00474 </td><td> NLG - 1 </td><td> tm474 </td><td> bNBP - 日本</td></tr><tr><td> VC1416 </td><td> NRX - 1 </td><td> ok1649 </td><td> CGC </td></tr><tr><td> FX1961 </td><td> NRX - 1 </td><td> tm1961 </td><td> NBP -ジャパン</td></tr><tr><td> NL2099 </td><td> RRF - 3 </td><td> pk1436 </td><td> CGC </td></tr><tr><td> CRR21 </td><td> NRX - 1、NLG - 1 </td><td> ok1649; ok259 </td><td>この作業</td></tr><tr><td> CRR22 </td><td> NRX - 1、NLG - 1 </td><td> tm1961; ok259 </td><td>この作業</td></tr></tbody></table> A. 線虫遺伝子センター、ミネソタ大学、米国。 B.実験動物&quot;線虫C. elegans&quot;のためのナショナルバイオリソースプロジェクト。東京女子医大、 日本。

osmotic avoidance in caenorhabditis elegans: synaptic function of two genes, orthologues of human nrxn1 and nlgn1, as candidates for autism

Neurexinsとneuroliginsは、興奮性と抑制性シナプスに存在する細胞接着分子であり、それらは正しいニューロンのネットワーク機能1に必要です。これらのタンパク質は、シナプス前及びシナプス後膜2で見られる。マウスの研究では、neurexinsとneurologinsがシナプス伝達1の本質的な役割を持っていることを示している。最近の報告では、人間の神経系の開発中に変更された神経細胞の接続は、自閉症スペクトラム障害3の多数の例病因の基礎を構成することができることが示されている。 線虫（Caenorhabditis elegans）があるため実験室での実験のためのそのシンプルさ、シナプス成分の機能の研究を促進するために実験的なツールとして使用し、その神経系、シナプスの配線が完全に特徴付けされていることを与えることができます。 Cで。 エレガンスNRX - 1とNLG - 1遺伝子は、それぞれα-ニューレキシン- 1、ニューロリギン- 1タンパク質を、エンコード人間NRXN1とNLGN1遺伝子にオルソロガスです。ヒトと線虫では、neurexinsとneuroliginsの組織は、機能ドメインの点で似ています。 線虫の頭部はamphid、浸透力を含め様々な刺激に応答を媒介する線虫の感覚器官が含まれています。 amphidは繊毛状突起一シナプス前終末軸索4の12の感覚バイポーラの神経細胞で作られています。 ASHRとASHLという名前のこれらのニューロンの二つは、高い浸透力5で水溶性の忌避剤を検出し、浸透圧感覚機能に特に重要です。これら2つのニューロンの樹状突起は、口の先端に長くし、軸索は、彼らが行動反応6を決定する他のニューロンとのシナプス結合を作る神経リング、に拡張します。 高い浸透力の回避にニューレキシンとニューロリギンの影響を評価するために、我々は、4Mフルクトースリング7に基づいてメソッドを使って、NRX - 1とNLG - 1遺伝子に欠陥のある線虫変異株の異なる応答を示しています。行動の表現型が特異的RNAiのクローン8を用いて確認された。 C.で虫は 、RNAiを引き起こすために必要なdsRNAは、9を供給することにより投与することができます。食品を介してdsRNAの配信は、このように遺伝的要素とのネットワーク経路の同定を可能にする目的の遺伝子のRNAiの干渉を誘導する。

Watch this Scientific Journal Video about Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism at JoVE.com

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

Neurexinsとneuroliginsはシナプス分化と伝達に重要な役割を実行する膜ニューロンの接着タンパク質です。のニューロリギン欠損変異体<em&gt; C.エレガンス</em&gt;浸透圧強度を検出するのに欠陥がある、しかし彼らはまた、ニューレキシンをコードする遺伝子に変異が含まれている場合、彼らは野生型の表現型を回復する。

の浸透圧の回避線虫（Caenorhabditis elegans）：二つの遺伝子のシナプス機能、ヒトのオルソログ NRXN1と NLGN1</em自閉症の候補として>、

Neurexins and neuroligins are cell adhesion molecules present in excitatory and inhibitory synapses, and they are required for correct neuron network ...

osmotic-avoidance-caenorhabditis-elegans-synaptic-function-two-genes

Research

JoVE Journal

Biology

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

11.7K ビュー.  Facultad de Ciencias, Universidad de Córdoba. Neurexinsとneuroliginsはシナプス分化と伝達に重要な役割を実行する膜ニューロンの接着タンパク質です。のニューロリギン欠損変異体 C.エレガンス浸透圧強度を検出するのに欠陥がある、しかし彼らはまた、ニューレキシンをコードする遺伝子に変異が含まれている場合、彼らは野生型の表現型を回復する。

ビデオ: Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

アイソレーションと試験管内で</emの>活性化線虫（Caenorhabditis elegans）精子

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

生物学

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

の初期発生のタイムラプス顕微鏡線虫（Caenorhabditis elegans）

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

基本的な<em&gt;線虫Caenorhabditis elegans</em&gt;方法：同期との観測

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

内脂肪量の生化学的および高スループット微視的評価<em&gt;線虫（Caenorhabditis elegans）</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

におけるプロスタグランジンの抽出と分析<em&gt;線虫（Caenorhabditis elegans）</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Caenorhabditis elegans is a useful model to explore the functions of polyunsaturated fatty acids in development and physiology. This protocol describes an efficient method of supplementing the C. elegans diet with polyunsaturated fatty acids.

多価不飽和脂肪酸の食事補給<em&gt;線虫（Caenorhabditis elegans）</em

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

の栽培<em&gt;線虫（Caenorhabditis elegans）</em&gt;ラボにおける三次元で

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

サンプル準備および透過電子顕微鏡によるエクソソームのイメージ投射

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

そのまま線虫の酸素消費量の測定

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...

の浸透圧の回避線虫（Caenorhabditis elegans）：二つの遺伝子のシナプス機能、ヒトのオルソログ NRXN1と NLGN1、