この作品は、A. - LHへネイサンショックセンター（P30 AG13283）と国立老化研究所（NIA）からR01助成金（R01 AG028516）からのパイロット賞によってサポートされていました

<html> 1。細菌の濃度を推定するための検量線の確立このセクションでは、C言語の食事制限の実験のための食料源として使用される任意の細菌株（例えば、OP50、HT115）のための細菌の濃度を推定する方法を説明しますエレガンス 。別々の検量線は、株が使用されている各細菌のために確立されるべきである。 <ol><li> E.のシングルコロニーを植菌大腸菌 OP50細菌は、3 mLの液体LBブロス中のLB寒天培地上の細菌の新鮮な筋から選んだ。 </li><li>場所OP50 37文化℃のシェーカーと細菌が一晩増殖することができます。 </li><li>一晩培養液からの細菌懸濁液（2倍、4倍、8倍、16倍、32倍速）の連続2倍希釈液を調製する。 </li><li>三重での分光光度計により600 nmで、各希釈液の吸光度（光学密度）を測定します。 </li><li>さらにそれぞれの細菌懸濁液100万倍（1000倍希釈倍）希釈する。 </li><li> 100mmのLB寒天プレート上に均等に各希釈液の細菌懸濁液0.4 mLを広げる。 </li><li> 18〜24時間、37℃インキュベーターでOP50のある場所LBプレートを。 </li><li>各プレート上で生育した細菌のコロニーの数を数えます。コロニー数は、各サンプルに対してmL当たりのcfu（コロニー形成単位）の計算に使用されます。 </li><li>測定OD 600の値とOP50細菌濃度との関係は、細菌濃度（CFU / mL）をの関数としてOD 600をプロットし、線形回帰分析（図1）を実行することによって確立することができます。 </li></ol>ソリューション： <table border="1"><tbody><tr><td colspan="2">ルリアブロス（LB）、1L： </td></tr><tr><td > 10グラム</td><td>バクトトリプトン</td></tr><tr><td> 5グラム</td><td>酵母エキス</td></tr><tr><td> 10グラム</td><td> NaClの</td></tr></tbody></table> 1Lに脱イオン水を追加。 オートクレーブ、室温で保管。 2。固体プレートベースの食事制限（SDR）プレートの調製このセクションでは、固体プレートベースの食事制限（SDR）の実験で使用するためのプレートの準備について説明します。通常は、我々は、条件と1この方法のための最適なDRの給餌条件として× 10 8 CFU / mLの送りを自由に摂取として1 × 10 11 CFU / mLの細菌の濃度を考慮してください。しかし、4-6異なる細菌の濃度は、SDRとその応答のための完全な用量依存関係を確立するために必要な場合があります。 <ol><li> E.のシングルコロニーを植菌3 mLの液体LBブロス中の大腸菌 OP50細菌。 </li><li> 37℃のシェーカーでOP50文化を置き、細菌が6〜8時間のために成長することができます。 </li><li> 500mLのLBブロスを含む新しいフラスコにOP50文化を転送し、℃でシェーカー一晩37に戻ってOP50文化を置きます。 </li><li>細菌濃度（濃度は第1部で生成された検量線を用いて計算される）を決定するOP50文化のOD 600を測定する。適切な希釈液は吸光度の正確な測定のために必要になる場合があります。 </li><li> 20分1,500 gで遠心分離によってペレットOP50細菌。 </li><li> 1 × 10 12 CFU / mLの濃度にバクテリアを再懸濁します。 </li><li> 6つの異なる濃度で10 mLの細菌の培養を準備：1 × 10 12、10 11、10 10、10 9、10 8、10 7 CFU / mLの。 </li><li> 60mmの中央に細菌の培養のピペット0.2mLのは、NGMプレートを固めた。表面に打痕が寒天プレートに穴にワームを許可するように、ピペットチップとプレートの表面に触れないようにしてください。 </li><li> 1時間37℃のインキュベータ内の場所のプレート。 </li><li>細菌の増殖を阻止するために、各プレートに100mg/mLカルベニシリン及び50mg/mLのカナマイシン10μLの10μLを適用します。 </li><li> 4の抗生物質 - 死菌の異なる濃度° C今後の使用のために（最大1ヶ月）。と店舗プレート少なくとも1〜2日後に控えて全体の計画的な実験のための十分なプレートを準備して格納することが重要です。全体の実験のための板の同じバッチを使用することが望ましい。 </li><li>細菌の生存性を確認するには、SDRのプレートのいくつかの細菌をこすり取ってカルベニシリンおよびカナマイシンによる空のNGMプレート上に広げる。 6-8時間、37℃のインキュベーターでプレートを配置し、細菌の増殖（ない細菌の増殖が観察されないはず）を確認してください。 </li></ol>ソリューション<table border="1"><tbody><tr><td colspan="2">線虫の増殖培地（NGM）、1L： </td></tr><tr><td > 3グラム</td><td> NaClの</td></tr><tr><td> 2.5グラム</td><td>バクトペプトン</td></tr><tr><td> 20グラム</td><td>寒天</td></tr></tbody></table>ove_contentは&quot;&gt; 40分間オートクレーブし、65に冷却させ° C、その後に追加： <table border="1"><tbody><tr><td > 1 mLの</td><td> 1 M MgSO 4を </td></tr><tr><td> 1 mLの</td><td> 1 M CaCl 2を </td></tr><tr><td> 1 mLの</td><td> 5 mg / mLのコレステロール</td></tr><tr><td> 25 mLの</td><td> 1Mリン酸カリウム、pH6.0の</td></tr></tbody></table>すぐに10mLのアリコートで60ミリメートル× 15ミリメートルのペトリ皿に水などの液体NGMを注ぐ 3。 SDRの実験のセットアップこのような寿命の解析と出生率のプロファイリングとして、SDRに関連する研究、のほとんどは、ワームの年齢同期人口の準備が不可欠です。他の研究では、単に食事制限を開始するために、SDRの板に定期的なOP50プレートからワームを転送する。このセクションでは、SDRの動物のために寿命の解析を設定するプロトコルを例として説明されています。 <ol><li> OP50でいくつかの定期的なNGMプレート上に20〜30生殖アクティブな大人を転送するためにプラチナのワームピッカーを使用してください。実験に必要な卵の数に応じて、より多くの大人が必要になることがあります。 </li><li> 20℃でプレートを残す℃で4時間は、産卵を可能にする。 </li><li>プレートから成人を取り外します。プレートを視覚的に卵のために点検してください。 </li><li> 20℃でプレートを残し° Cと子孫がLate-L4/day大人1名のステージに発展することができます。通常、これは20℃N2野生型のワームのために3日かかる℃に</li><li>食事制限を開始するSDRのプレートに1日成人の適切な数を転送する。 </li><li>寿命の分析については、各SDR条件のプレート当たり10から20までのワームで6〜8枚の合計を設定します。 </li><li>すべてのワームが死亡するまで2日ごとにそれぞれのワームの実行可能性を得点する。それは、ワームは、食品の完全な剥奪を避けるために、すべての12から48時間新鮮なSDRの板に転送することが重要です。 </li></ol> 4。代表的な結果： 図1に示すようにOD 600との関係でOP50濃度の推定のための代表的な検量線のプロットである。ライナーの回帰分析によって生成された方程式は、OP50細菌濃度の将来のすべての計算に使用することができます。図2に示すように、別のワームの株の平均寿命に慢性SDRの治療の効果を測定することの例です。青いラインは、SDRに野生型ワームの通常の用量依存性の応答を表します。 DAF - 16（mu86）は SDR -誘導寿命の延長には何の影響もないまま、赤いラインは、SDRの寿命の効果が部分的にDRR - 2 OEによって抑制されていることを示します。 Cで寿命の解析を行うための詳細の方法虫は 1-3以前の文献に記載されている。 <img src="/files/ftp_upload/2701/2701fig1.jpg" alt="図1" /> 図1 E.の推定のための検量線の代表結果大腸菌 OP50濃度。 OP50濃度は、測定OD 600値の関数としてプロットされます。エラーバー= SD泊細菌の培養は、cfuの測定前に1,000 ×倍の吸光度測定の前に8倍、16倍、32倍、1.5倍2倍、4倍希釈し、そしてさらに希釈した。 <img src="/files/ftp_upload/2701/2701fig2.jpg" alt="図2" /> 図2：C.の代表結果虫の寿命実験では、SDRへの応答の異なる2つの変異体（緑と赤）（チンらから図4）と野生型のN2の動物（青）を比較。エラーバーは= SEM

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食事制限（DR）が寿命を増やすおよび5-7哺乳類を含む種の広い範囲で様々な加齢に伴う病気の発症を遅らせることが知られている。両方の環境と遺伝子操作は、CのモデルDRに使用されている半定義された液体培地8,9、液体培地10-12で細菌の食物源の希釈、およびより遅い速度13で送り、その動作の変異体の使用で培養型ワームなど、 エレガンス、。</e...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>カルベニシリン</td><td> Duchefa Biochemie </td><td> C0109.25 </td><td>水に溶解。 </td></tr><tr><td>カナマイシン</td><td>ロッシュ</td><td> 10106801001 </td><td>水に溶解。 </td></tr><tr><td>バクトペプトン</td><td>ベクトンディッキンソン社</td><td> DF0118072 </td><td></td></tr><tr><td>バクトトリプトン</td><td>ベクトンディッキンソン社</td><td> DF0123075 </td><td></td></tr><tr><td>バクト酵母エキス</td><td>ベクトンディッキンソン社</td><td> DF0127071 </td><td></td></tr><tr><td>寒天</td><td>ベクトンディッキンソン社</td><td> DF00145170 </td><td></td></tr><tr><td>無菌培養管</td><td>米国サイエンティフィック</td><td> 1485-0810 </td><td></td></tr><tr><td>分光光度計</td><td>アマシャムバイオサイエンス</td><td>遺伝子クワントプロ</td><td>アマシャムは現在GEヘルスケアの一部です。 </td></tr><tr><td>解剖顕微鏡</td><td>ツァイス</td><td> STEMI 2000 </td><td></td></tr><tr><td>遠心</td><td>ベックマンコールター</td><td> Accuspin FR </td><td></td></tr></tbody></table>

solid plate-based dietary restriction in caenorhabditis elegans

栄養失調や飢餓のない食物摂取の減少は、寿命を向上させ、哺乳動物を含めた種の広い範囲で様々な加齢に伴う病気の発症を遅らせることが知られている。また、体重と生殖能力の低下だけでなく、血漿グルコース、インスリン、およびこれらの動物におけるIGF - 1の低いレベルになります。この治療はしばしば、食事制限（DR）やカロリー制限（CR）と呼ばれています。線虫Caenorhabditis elegansは老化の生物学を研究するための重要なモデル生物として注目されています。環境と遺伝子操作モデルDRに使用されてきたとCで寿命を延ばすことが示されている両方エレガンス 。しかし、C言語で報告されたDRの研究の多くエレガンスは、加齢分野における遺伝学的研究のほとんどはペトリ皿の標準固形寒天上で行われた一方、液体培地で動物を伝播することによって行われた。ここでは、死菌と標準固体NGM寒天ベースのプレートを使用してDRのプロトコルを提示する。

Watch this Scientific Journal Video about Solid Plate-based Dietary Restriction in Caenorhabditis elegans at JoVE.com

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

ここでは、固体プレートベースの食事制限を行うためのプロトコルを提示<em&gt; C.エレガンス</em&gt;死菌で。

固体プレートベースの食餌制限線虫（Caenorhabditis elegans）</em

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

solid-plate-based-dietary-restriction-in-caenorhabditis-elegans

Research

JoVE Journal

Biology

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

16.5K ビュー.  University of Michigan. ここでは、固体プレートベースの食事制限を行うためのプロトコルを提示 C.エレガンス死菌で。

ビデオ: Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

計測線虫（Caenorhabditis elegans）</em固体培地上>寿命

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode ...

生物学

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 ...

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 ...

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, ...

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, ...

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 ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington&#8217;s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

カエノハブディティス・エレガンスにおける組織特異的プロテオスタティック低下の定量化

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their detrimental effects on the human life quality and the healthcare system, the majority of human neurodegenerative disorders still remain incurable and non-preventable. Therefore, the development of novel therapeutic interventions against such maladies is becoming a pressing urgency. Age-associated deterioration of neuronal circuits and function is evolutionarily conserved in organisms as diverse as the lowly worm Caenorhabditis elegans and humans, signifying similarities in the underlying cellular and molecular mechanisms. C. elegans is a highly malleable genetic model, which offers a well-characterized nervous system, body transparency and a diverse repertoire of genetic and imaging techniques to assess neuronal activity and quality control during ageing. Here, we introduce and describe methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis (e.g., deg-3(d) and mec-4(d)) and protein aggregate (e.g., &#945;-syunclein and poly-glutamate)-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-related neuronal breakdown. A combination of these animal neurodegeneration models, together with genetic and pharmacological screens for cell death modulators will lead to an unprecedented understanding of age-related breakdown of neuronal function and will provide critical insights with broad relevance to human health and quality of life.

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Here, we introduce and describe widely accessible methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis and protein aggregate-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-associated neurodegenerative diseases.

カエノハブディティス・エレガンスにおける年齢関連神経変性疾患のモデル化

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their ...

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

Caenorhabditis elegansにおける凝集定量の自動化

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...