Name: a high-throughput, high-content, liquid-based c. elegans pathosystem
Uploaded: 2018-07-01T15:00:00.000+00:00
Duration: 9 min 44 s
Description: Watch this Scientific Journal Video about A High-throughput, High-content, Liquid-based C. elegans Pathosystem at JoVE.com

Questo studio è stato sostenuto dal Research Institute del Texas (CPRIT) premio RR150044, Welch Foundation Research Grant C-1930 e la prevenzione di cancro e dell'istituti nazionali di salute K22 AI110552 assegnato a NVK. I finanziatori non avevano alcun ruolo nel disegno dello studio, raccolta dati e analisi, decisione di pubblicare o preparazione del manoscritto.

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+and+novel+screening+model+for+assessing+the+bioactivity+of+extracts+against+multidrug-resistant+Pseudomonas+aeruginosa+using+Caenorhabditis+elegans.">An efficient and novel screening model for assessing the bioactivity of extracts against multidrug-resistant Pseudomonas aeruginosa using Caenorhabditis elegans.</a> Bioscience, Biotechnology, and Biochemistry. 75 (9), 1746-1751 (2011).</li><li>Leung, C. K., Deonarine, A., Strange, K., Choe, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+and+biosensing+with+fluorescent+C.+elegans+strains.">High-throughput screening and biosensing with fluorescent C. elegans strains.</a> Journal of Visual Experiments. (51), (2011).</li><li>Kirienko, N. V., Ausubel, F. M., Ruvkun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitophagy+confers+resistance+to+siderophore-mediated+killing+by+Pseudomonas+aeruginosa.">Mitophagy confers resistance to siderophore-mediated killing by Pseudomonas aeruginosa.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (6), 1821-1826 (2015).</li><li>Kirienko, N. V., Cezairliyan, B. O., Ausubel, F. M., Powell, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+PA14+pathogenesis+in+Caenorhabditis+elegans.">Pseudomonas aeruginosa PA14 pathogenesis in Caenorhabditis elegans.</a> Methods in Molecular Biology. 1149, 653-669 (2014).</li><li>Kang, D., Kirienko, D. R., Webster, P., Fisher, A. L., Kirienko, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyoverdine,+a+siderophore+from+Pseudomonas+aeruginosa,+translocates+into+C.+elegans,+removes+iron,+and+activates+a+distinct+host+response.">Pyoverdine, a siderophore from Pseudomonas aeruginosa, translocates into C. elegans, removes iron, and activates a distinct host response.</a> Virulence. , 1-41 (2018).</li><li>Garsin, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-lived+C.+elegans+daf-2+mutants+are+resistant+to+bacterial+pathogens.">Long-lived C. elegans daf-2 mutants are resistant to bacterial pathogens.</a> Science. 300 (5627), 1921 (2003).</li><li>Estes, K. A., Dunbar, T. L., Powell, J. R., Ausubel, F. M., Troemel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=bZIP+transcription+factor+zip-2+mediates+an+early+response+to+Pseudomonas+aeruginosa+infection+in+Caenorhabditis+elegans.">bZIP transcription factor zip-2 mediates an early response to Pseudomonas aeruginosa infection in Caenorhabditis elegans.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (5), 2153-2158 (2010).</li><li>Tjahjono, E., Kirienko, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+mitochondrial+surveillance+pathway+is+required+for+defense+against+Pseudomonas+aeruginosa.">A conserved mitochondrial surveillance pathway is required for defense against Pseudomonas aeruginosa.</a> PLoS Genetics. 13 (6), e1006876 (2017).</li><li>Moy, T. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+novel+antimicrobials+using+a+live-animal+infection+model.">Identification of novel antimicrobials using a live-animal infection model.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (27), 10414-10419 (2006).</li><li>Henderson, S. T., Bonafe, M., Johnson, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=daf-16+protects+the+nematode+Caenorhabditis+elegans+during+food+deprivation.">daf-16 protects the nematode Caenorhabditis elegans during food deprivation.</a> The Journals of Gerontology. Series A, Biological Sciences and Medical Sciences. 61 (5), 444-460 (2006).</li><li>Beanan, M. 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Gli autori dichiarano di non avere conflitti di interesse a divulgare.

Questo saggio (o simili saggi dove altri agenti patogeni vengono sostituiti con p. aeruginosa o E. faecalis) sono utile per una varietà di scopi, tra cui la scoperta di nuovi farmaci. È anche utile per affrontare questioni biologiche fondamentali, come identificare i fattori di virulenza, la delucidazione dei meccanismi di difesa ospite e determinare il regolamentazione macchinari coinvolti nell'interazione ospite-patogeno.
Anche se il dosaggio di uccisione liquido p. a...

L'identificazione e lo sviluppo di efficaci antibiotici ad ampio spettro, ora quasi un secolo fa, ha portato a un momento di svolta nella sanità pubblica dove c'era una credenza diffusa che infettiva malattia sarebbe un flagello del passato. Entro pochi decenni, questo ottimismo iniziò a scemare, come agente patogeno dopo agente patogeno ha sviluppato meccanismi di resistenza che limitato questi trattamenti una volta miracolosi. Per qualche tempo, la corsa agli armamenti tra azioni di individuazione di droga e gli agenti patogeni sembrava equilibrata. Tuttavia, l'uso improprio degli antimicrobici è recentemente culminato nell'emergere di ceppi di Klebsiella pneumoniae, Acinetobacter baumanii, Serratia marcescense p. aeruginosa1, pan-farmaco resistenti 2,3,4.
P. aeruginosa è un patogeno di negativo, multi-host di grammo di opportunisti, che è una grave minaccia ai pazienti con gravi ustioni, coloro che sono immunocompromessi, o hanno la fibrosi cistica. È inoltre sempre più identificato come un agente eziologico nelle infezioni nosocomial severa, particolarmente dovuto la sua acquisizione in corso della resistenza antimicrobica. Per cominciare ad affrontare questa minaccia, abbiamo usato la ben documentata elegans del c.-p. aeruginosa infezione sistema5. Il nostro laboratorio ha sfruttato questo sistema per sviluppare una piattaforma di base liquida, ad alta velocità, ad alto contenuto di screening per identificare nuove molecole che limitano la capacità del patogeno di uccidere l' host6. Curiosamente, questi composti sembrano appartenere ad almeno tre categorie generali, compresi gli antimicrobici7 e virulenza inibitori8. Altri saggi di scoperta di droga ad alto contenuto in c. elegans sono stati segnalati per del micobatterio tuberculosum, Chlamydia trachomatis, pestis di Yersinia, Listeria monocytogenes, Francisella tularensis, Staphylococcus aureus, Candida albicans, e Enterococcus faecalis, tra gli altri9,10,11,12,13,14,15,16. Questi tipi di analisi presentano parecchi vantaggi ben riconosciuti, ad esempio limitando falsi positivi successi che possono essere tossici per sia l'host e l'agente patogeno, probabilità aumentata della biodisponibilità rispetto a uno schermo di chimico e la capacità di identificare successi di là semplicemente limitando la crescita microbica, come anti-virulents, stimolatore immunitario molecole o composti che altrimenti inclinare la bilancia dell'interazione ospite-patogeno in favore della ex. Inoltre, i composti scoperti in queste schermate sono spesso efficaci in mammiferi padroni di casa.
Vale la pena notare che almeno due altri saggi17,18 sono disponibili per realizzare schermi di alto-rendimento in c. elegans in liquido. Tuttavia, ciascuno di questi test è una modifica che permette il dosaggio di colonizzazione intestinale prototipo, conosciuto come lento-uccisione, da eseguirsi in liquido, aumentando la produttività e consentendo di composti più facilmente essere schermato. Un'attenta caratterizzazione ha dimostrato conclusivamente che i meccanismi di virulenza batterica sono diversi tra questi saggi e nostra base liquida schermo7. Poiché entrambi i tipi di virulenza sono osservati nei sistemi mammiferi, è importante considerare quali determinanti di virulenza sono più rilevante per gli interessi dello sperimentatore prima della selezione di test.
Qui dimostriamo una versione ottimizzata del liquido basato su analisi di c. elegans-P. aeruginosa . Segnaliamo anche l'adattamento del nostro metodo di analisi liquido-basata per ospitare l'agente patogeno batterico gram-positivo Enterococcus faecalis. Come p. aeruginosa, E. faecalis è sempre più identificato come una minaccia seria nosocomial con un armamento crescente di resistenza antimicrobica vie1. Anche se esiste un metodo precedente per high throughput screening di E. faecalis 14, richiede preinfection con l'agente patogeno, che complica la procedura e aumenta la probabilità di contaminazione di attrezzature come il FlowSort COPAS. Il nostro protocollo Elimina la necessità di pre-infezione, miglioramento del profilo di sicurezza. Infine, segnaliamo un mezzo mediante il quale uno di questi test possono essere combinato con alimentazione di RNAi, permettendo all'utente di cercare per fattori ospite che giocano un ruolo nella creazione di, o resistenza a, infezione.

<table><tbody><tr><td>COPAS FP BioSorter</td><td>Union Biometrica</td><td></td><td>Large object flow cytometer/worm sorter</td></tr><tr><td>Cytation 5</td><td>BioTek</td><td></td><td></td></tr><tr><td>EL406 Washer Dispenser</td><td>BioTek</td><td></td><td></td></tr><tr><td>Multitron Pro</td><td>Infors HT</td><td></td><td></td></tr><tr><td>24 Deep-Well RB Block</td><td>Thermo Fisher Scientific</td><td>CS15124</td><td></td></tr><tr><td>384-Well plate</td><td>Greiner Bio-One</td><td>MPG-781091</td><td></td></tr><tr><td>Nematode Growth Media (NGM)</td><td></td><td></td><td>Amount per liter: 18 grams agar, 3 grams NaCl, 2.5 grams Peptone, 1 mL CaCl&lt;sub&gt;2&lt;/sub&gt; (1 M), 1 mL MgSO&lt;sub&gt;4&lt;/sub&gt; (1 M), 25 mL Phospate buffer, and 973 mL of milli-Q water</td></tr><tr><td>Slow Killing (SK) plates</td><td></td><td></td><td>Amount per liter: 18 grams agar, 3 grams NaCl, 3.5 grams Peptone, 1 mL CaCl&lt;sub&gt;2&lt;/sub&gt; (1 M), 1 mL MgSO&lt;sub&gt;4&lt;/sub&gt; (1 M), 25 mL Phospate buffer, and 973 mL of milli-Q water</td></tr><tr><td>Slow Killing (SK) media</td><td></td><td></td><td>Amount per liter:&amp;nbsp; 3 grams NaCl, 3.5 grams Peptone, 1 mL CaCl&lt;sub&gt;2 &lt;/sub&gt;(1 M), 1 mL MgSO&lt;sub&gt;4&lt;/sub&gt; (1 M), 25 mL Phosphate buffer, and 973 mL of milli-Q water</td></tr><tr><td>Lysogeny Broth (LB)</td><td>USBiological Life Sciences</td><td>L1520</td><td></td></tr><tr><td>Brian Heart Infusion broth (BHI)</td><td>Research Products International Corp</td><td>50-488-526</td><td></td></tr><tr><td>Worm Bleach Solution</td><td></td><td></td><td>Amount per 100 mL: 10 mL of 5 M NaOH solution, 20 mL of 5% Sodium Hypochlorite Solution, and 70 mL of sterile water</td></tr><tr><td>S Basal</td><td></td><td></td><td>Amount per liter: 5.85 grams NaCl, 6 grams KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 1 gram K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, and 1 Liter of milli-Q water</td></tr><tr><td>Agar</td><td>USBiological Life Sciences</td><td>A0930</td><td></td></tr><tr><td>NaCl</td><td>USBiological Life Sciences</td><td>S5000</td><td></td></tr><tr><td>Peptone</td><td>USBiological Life Sciences</td><td>P3300</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>USBiological Life Sciences</td><td></td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>M63-500</td><td></td></tr><tr><td>Phospate buffer</td><td></td><td></td><td>amount per liter: 132 mL of K2HPO4 (1M) and 868 mL of KH2PO4 (1M)</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Acros Organics</td><td>7778-77-0</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>USBiological Life Sciences</td><td>P5100</td><td></td></tr><tr><td>5% Sodium Hypochlorite Solution</td><td>BICCA</td><td>7495.5-32</td><td></td></tr><tr><td>NaOH solution</td><td>Fisher Scientific</td><td>SS255-1</td><td></td></tr><tr><td>Breathe-easy</td><td>Diversified Biotech</td><td>BEM-1</td><td></td></tr><tr><td>SYTOX Orange Nucleic Acid Stain</td><td>Fisher Scientific</td><td>S11368</td><td></td></tr><tr><td>&lt;strong&gt;Bacterial Strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;P. aeruginosa &lt;/em&gt;(PA14)</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;E. faecalis&lt;/em&gt;(OG1RF)</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;E. coli &lt;/em&gt;superfood (OP50)</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;E. coli &lt;/em&gt;RNAi expressing bacteria (HT115)</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Worm Strains &lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;glp-4&lt;/em&gt;(&lt;em&gt;bn2&lt;/em&gt;) (Beanan and Strome, 1992, PMID: 1289064)</td><td></td><td></td><td></td></tr><tr><td>PINK-1::GFP reporter (Kang et al., 2018, PMID: 29532717)</td><td></td><td></td><td></td></tr></tbody></table>

a high-throughput, high-content, liquid-based c. elegans pathosystem

Il numero di nuovi farmaci identificati da tradizionale, schermi in vitro è scemato, riducendo il successo di questo approccio nella ricerca di nuove armi combattere la Resistenza multifarmaco. Questo ha portato alla conclusione che i ricercatori non solo la necessità di trovare nuovi farmaci, ma anche bisogno di sviluppare nuovi modi di trovare loro. Tra i candidati più promettenti metodi sono intero-organismo, in vivo dosaggi che uso alto-rendimento, fenotipica letture e ospita che vanno da Caenorhabditis elegans di Danio rerio. Questi host presentano parecchi vantaggi potenti, tra cui le riduzioni drammatiche in falsi positivi colpisce, come composti che sono tossici per l'ospite e/o biounavailable vengono in genere eliminati nella schermata iniziale, prima di seguire costosi fino.
Qui vi mostriamo come nostra analisi sono stato utilizzato per interrogare variazione host la ben documentata di c. elegans— patosistema come contributo liquido uccisione diPseudomonas aeruginosa . Inoltre dimostriamo diverse estensioni di questa tecnica ben lavorata fuori. Per esempio, siamo in grado di svolgere high throughput genetiche schermate mediante RNAi in 24 o 96 pozzetti formati piastra di fattori dell'ospite query in questa interazione ospite-patogeno. Usando questa analisi, schermi intero genoma possono essere completati in pochi mesi, che possono di semplificare notevolmente il compito di individuare bersagli farmacologici, potenzialmente senza la necessità di approcci biochimici laboriosa purificazione.
Inoltre segnaliamo qui una variazione del nostro metodo che sostituisce il batterio gram-positivo Enterococcus faecalis per l'agente patogeno gram-negativo p. aeruginosa. Molto come è il caso per p. aeruginosa, uccisione di E. faecalis è dipendente dal tempo. A differenza del precedente c. elegans— saggi diE. faecalis , nostra analisi per E. faecalis non richiede preinfection, migliorando il suo profilo di sicurezza e riducendo le possibilità di contaminazione liquido-per la movimentazione. Il dosaggio è estremamente robusto, mostrando ~ 95% tassi di mortalità 96h post infezione.

Parametri importanti per le prestazioni
Una corretta comprensione della biologia alla base di questa analisi è necessaria per risolvere i problemi e ottimizzare il dosaggio. A tal fine, ci riferiamo in primo luogo a parecchie carte chiave delucidamento dei meccanismi di patogenesi di p. aeruginosa-mediata uccidendo in liquido7,20. Condizione che i pass...

Watch this Scientific Journal Video about A High-throughput, High-content, Liquid-based C. elegans Pathosystem at JoVE.com

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Qui descriviamo un protocollo che è un host adattabile, intero, strumento di screening ad alto contenuto che possa essere utilizzata per studiare le interazioni ospite-patogeno ed essere utilizzato per la scoperta di nuovi farmaci.

Un ad alta velocità, ad alto contenuto, liquido a base di c. elegans patosistema come contributo

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...

a-high-throughput-high-content-liquid-based-c-elegans-pathosystem

Research

JoVE Journal

Immunology and Infection

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

14.4K Views.  Rice University. Qui descriviamo un protocollo che è un host adattabile, intero, strumento di screening ad alto contenuto che possa essere utilizzata per studiare le interazioni ospite-patogeno ed essere utilizzato per la scoperta di nuovi farmaci.

Video: A High-throughput, High-content, Liquid-based C. elegans Pathosystem

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Un metodo semplice per High Throughput Screening chimico in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Ricerca

Biochimica

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection of the neural circuits underlying these behaviors, the development of robust and reproducible behavioral assays is necessary. Previous C. elegans behavioral studies have used variations of a "drop test", a "chemotaxis assay", and a "retention assay" to investigate the response of C. elegans to soluble compounds. The method described in this article seeks to combine the complementary strengths of the three aforementioned assays. Briefly, a small circle in the middle of each assay plate is divided into four quadrants with the control and experimental solutions alternately placed. After the addition of the worms, the assay plates are loaded into a behavior chamber where microscope cameras record the worms' encounters with the treated regions. Automated video analysis is then performed and a preference index (PI) value for each video is generated. The video acquisition and automated analysis features of this method minimizes the experimenter's involvement and any associated errors. Furthermore, minute amounts of the experimental compound are used per assay and the behavior chamber's multi-camera setup increases experimental throughput. This method is particularly useful for conducting behavioral screens of genetic mutants and novel chemical compounds. However, this method is not appropriate for studying stimulus gradient navigation due to the close proximity of the control and experimental solution regions. It should also not be used when only a small population of worms is available. While suitable for assaying responses only to soluble compounds in its current form, this method can be easily modified to accommodate multimodal sensory interaction and optogenetic studies. This method can also be adapted to assay the chemosensory responses of other nematode species.

We present a behavior recording setup and protocol that enables automated analysis of the nematode, Caenorhabditis elegans' preference for soluble compounds in a population-based assay. This article describes the construction of a behavior chamber, the behavioral assay protocol, and video analysis software usage.

Analisi automatizzata di un esame preferenziale di Chemosensory basato sulla popolazione di Nematode

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection ...

Comportamento

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and maintenance issues. On the contrary, the nematode Caenorhabditis elegans (C. elegans) has advantages to make it an ideal choice for toxicity testing: a short lifespan, easy cultivation, and efficient reproduction. Here, we describe a protocol for the automatic phenotypic profiling of C. elegans in a 384-well plate. The nematode worms are cultured in a 384-well plate with liquid medium and chemical treatment, and videos are taken of each well to quantify the chemical influence on 33 worm features. Experimental results demonstrate that the quantified phenotype features can classify and predict the acute toxicity for different chemical compounds and establish a priority list for further traditional chemical toxicity assessment tests in a rodent model.

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

A quantitative method has been developed to identify and predict the acute toxicity of chemicals by automatically analyzing the phenotypic profiling of Caenorhabditis elegans. This protocol describes how to treat worms with chemicals in a 384-well plate, capture videos, and quantify toxicological related phenotypes.

Un saggio di High-throughput per la previsione di tossicità chimica di Automated fenotipica profilatura di Caenorhabditis elegans

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and ...

Ambiente

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The large body of work on C. elegans makes it an ideal candidate to integrate into large-population, whole-animal studies to dissect the complex biological components and their relationships with another organism. In order to use C. elegans in collaborative -omics research, a method is needed to generate large populations of animals where a single sample can be split and assayed across diverse platforms for comparative analyses.
Here, a method to culture and collect an abundant mixed-stage C. elegans population on a large-scale culture plate (LSCP) and subsequent phenotypic data is presented. This pipeline yields sufficient numbers of animals to collect phenotypic and population data, along with any data needed for -omics experiments (i.e., genomics, transcriptomics, proteomics, and metabolomics). In addition, the LSCP method requires minimal manipulation to the animals themselves, less user preparation time, provides tight environmental control, and ensures that handling of each sample is consistent throughout the study for overall reproducibility. Lastly, methods to document population size and population distribution of C. elegans life stages in a given LSCP are presented.

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

To use Caenorhabditis elegans (C. elegans) in omics research, a method is needed to generate large populations of worms where a single sample can be measured across platforms for comparative analyses. Here, a method to culture C. elegans populations on large-scale culture plates (LSCPs) and to document population growth is presented.

Cultura e dosaggio delle popolazioni di caenorhabditis elegans a grande scala

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The ...

Genetica

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

A<em&gt; Caenorhabditis elegans</em&gt; Sistema modello per Amylopathy Studio

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

Neuroscienze

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

High-throughput screening e biopercezione con fluorescente C. elegans Ceppi

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

Biologia

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

Quantitative e automatica High-throughput Genome-wide Schermi RNAi in C. elegans</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

Analisi del comportamento di grande c. elegans popolazioni utilizzando un ampio campo di vista Monitoraggio piattaforma automatizzata

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...

Immunologia e infezioni

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

C. elegans Chemiotassi Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on host physiology. It also fluoresces in blue when dying, providing a convenient means of pinpointing death. This property has been exploited to develop high-throughput label-free C. elegans survival assays (LFASS). These involve time-lapse fluorescence recording of worm populations set in multiwell plates, from which population median time of death can be derived. The present study adopts the LFASS approach to screen multiple microbial isolates at once for the effects on C. elegans susceptibility to severe heat and oxidative stresses. Such microbial screening pipeline, which can notably be used to prescreen probiotics, using severe stress resistance as a proxy for host health is reported here. The protocol describes how to grow both C. elegans gut microbiota isolate collections and synchronous worm populations in multiwell arrays before combining them for the assays. The example provided covers the testing of 47 bacterial isolates and one control strain on two worm strains, in two stress assays in parallel. However, the approach pipeline is readily scalable and applicable to the screening of many other modalities. Thus, it provides a versatile setup to rapidly survey a multiparametric landscape of biological and biochemical conditions that impact C. elegans health.

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

Gut microbes may positively or negatively impact the health of their host via specific or conserved mechanisms. Caenorhabditis elegans is a convenient platform to screen for such microbes. The present protocol describes high-throughput screening of 48 bacterial isolates for impact on nematode stress resistance, used as a proxy for worm health.

Screening ad alta produttività di isolati microbici con impatto sulla salute di Caenorhabditis elegans

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on ...

Un ad alta velocità, ad alto contenuto, liquido a base di c. elegans patosistema come contributo

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