Die Arbeit im Labor Duennwald wird durch Zuschüsse aus dem American Federation für Alternsforschung (Afar), der Erbkrankheit Stiftung (HDF) und der William Wood-Stiftung unterstützt.

1. Expression von toxischen PolyQ-Erweiterung Proteine ​​in Hefe Eine systematische Analyse der genauen Aminosäuresequenz eines Proteins Polyglutamindomänen-Erweiterung, die erforderlich ist, um Toxizität in Hefe 7 erzeugen hergestellt wird. Diese toxische Polyglutamindomänen-Expansion-Protein enthält eine Amino-terminalen FLAG-Tag durch 17 Aminosäuren von der ursprünglichen Sequenz des Huntingtin-Protein, einem Polyglutamindomänen Region und eine Carboxy-terminale Fusion mit einem fluoreszierenden Protein (GFP oder entweder GFP gefolgt, siehe Abbildung 1 a). Die Expression von Proteinen mit Polyglutamindomänen Erweiterungen von 46 Glutaminen oder mehr (z. B. 72 und 103 Glutaminen) erzeugt Toxizität in Hefe, wenn sie unter der Kontrolle des induzierbaren und relativ starke GAL1-Promotor 7, 9 ausgedrückt. Wie zuvor beschrieben, ist Polyglutamindomänen Toxizität in Hefe nur scheinbar in Zellen, die das Protein in seiner Durchführung Rnq1p Prion-Konformation, [RnQ +], zB die Hefe-Stamm W303 9. Die toxische Polyglutamindomänen-Erweiterung Protein recapitulates zentrale Aspekte der Polyglutamindomänen-Biologie in Hefe, wie Polyglutamindomänen Länge-abhängige Toxizität (siehe unten) und Aggregation (Abbildung 1 b). Bemerkenswert ist, müssen toxische Proteine ​​Polyglutamindomänen-Erweiterung nicht töten Hefezellen sofort, sondern eher beeinträchtigen oder die Festnahme des Zellzyklus und der Zell-Devision, was zu einer Verlangsamung oder Hemmung des Wachstums von Hefe-Kolonien auf Platten oder flüssigen Kulturen (eigene unveröffentlichte Daten). 2. Potenzielle Probleme mit Hefezellen, Toxic PolyQ-Erweiterung Proteine Hefezellen, die sonst giftige Polyglutamindomänen Expansion Proteine ​​dürfen keine Fehler Wachstum 9. Die genetische Beschaffenheit dieser Suppressoren Polyglutamindomänen-Toxizität scheint nicht auf einfachen Mendelschen Mutationen beruhen und somit mit relativ hoher Frequenz (eigene unveröffentlichte Ergebnisse) auftreten. Wir spekulieren, dass diese spontanen Unterdrücker durch die Härtung von nicht identifizierten Prionen, die ähnlich wie in [RnQ +] bei der Bestimmung Polyglutamindomänen toxicity.Thes funktionieren verursacht werdene spontane Suppressoren Polyglutamindomänen Toxizität kann gefährden den erfolgreichen Ausgang eines Experiments, das zu charakterisieren Polyglutamindomänen Toxizität oder zur Identifizierung und Charakterisierung von Modifikatoren Polyglutamindomänen Toxizität zielt. Um das häufige Vorkommen dieser spontanen Unterdrücker zu vermeiden, befolgen Sie die unten aufgeführten Vorsichtsmaßnahmen, die sich als sehr wirksam sind: <ol><li> Verwenden Sie frische Hefe-Zellen für Experimente Toxizität. Lagern Hefe für längere Zeiträume. Häufig abrufen frische Hefe-Kolonien aus gefrorenen Beständen. </li><li> Häufig beobachten die Expression und Aggregation von toxischen Proteinen Polyglutamindomänen-Expansion durch Fluoreszenzmikroskopie. </li><li> Halten der Hefezellen in Medien, die die Expression des toxischen Polyglutamindomänen Toxizität zu allen Zeiten (dh in selektiven Medien mit Glucose als einziger Kohlenstoffquelle) unterdrücken, bevor keine Toxizität Messungen. </li><li> Verwenden Sie mindestens drei unabhängigen Transformanten für jedes Polyglutamindomänen-Toxizität Experiment. &lt;/ Li&gt; </ol> 3. Wachstums-Assays <ol><li> Für jede experimentelle Bedingung, eine Kolonie beimpft jeweils aus drei unabhängigen Transformanten von Hefe-Zellen, die toxische Polyglutamindomänen-Expansion Proteins in 3 ml Hefe-selektive Medien mit Glucose als einziger Kohlenstoffquelle. </li><li> Inkubieren Sie diese Kulturen über Nacht bei 30 ° C. Unter diesen Bedingungen werden die Zellen nicht exprimiert polyglutamine-Expansion Protein, da die Glucose im Medium verdrängt ihre Expression. Lassen Sie sich nicht diese Kulturen überwuchern, halten Sie die OD 600 (Absorption von Licht der Wellenlänge 600 nm) der Übernacht-Kulturen unter 1. </li><li> Wir verwenden routinemäßig drei verschiedenen Assays, um die Mängel Wachstum von Hefe-Zellen, die das toxische Protein Polyglutamindomänen-Erweiterung zu überwachen: </li></ol> 3,1 Wachstum auf Platten <ol><li> Verdünnen Sie die Nacht Hefekulturen (aufgewachsen mit 220 rpm geschüttelt) auf eine OD 600 von 0,0005 (dh eine 1:1000 Verdünnung eines OD600 = 0,5 Kultur) in selektiver michØ 7 mit Glucose. </li><li> Gleichmäßig verteilt 50 ul (was ca.. 700 Kolonien pro Platte) der verdünnten Kultur auf einer Platte (10 cm Durchmesser) mit selektiven Medium, das Glucose als einzige Kohlenstoffquelle und eine Platte mit selektiven Medium, das Galactose als einziger Kohlenstoffquelle. </li><li> Die Inkubation erfolgt während drei bis vier Tage bei 30 ° C Nach der Inkubation, um Aufnahmen von jeder Platte und die Anzahl der Kolonien auf dem Glucose und die Anzahl der Kolonien auf den Platten Galactose. Unter idealen Bedingungen, sollte es keine oder nur sehr wenige Kolonien auf der Platte sein Galactose bei der Verwendung von Hefe-Zellen, eine hochgiftige Polyglutamindomänen-Erweiterung Protein (103q, Abbildung 2a). </li></ol> 3,2 Spotting-Assays Dieser Assay ist quantitativ als die Plattierung Assay oben beschrieben, und kann somit offenbaren auch feine Unterschiede in Polyglutamindomänen Toxizität mit dem gleichen Experiment auf der gleichen Platte. <ol><li> Verdünnen Sie die ÜNight Kulturen in Medium mit Glukose zu einer OD 600 von 0,1 gezüchtet. </li><li> Je 200 ul dieser verdünnten Kulturen in sterile 96-well Platten und bereiten fünf Fünffache serielle Verdünnungen in sterilem Wasser unter Verwendung einer Mehrkanal-Pipette. </li><li> Verwendung eines frogger, (auch als ein Spotter, mit 8 x 6 Pins für die Übertragung von Zellsuspensionen) übertragen Zellsuspensionen auf Platten, die selektive Medien mit Glucose als einziger Kohlenstoffquelle und Platten, die selektive Medien mit Galactose als einziger Kohlenstoffquelle. </li><li> Die Platten vor Inkubation bei 30 ° C für drei bis vier Tage trocknen. </li><li> Nach der Inkubationszeit, machen Fotos von jeder Platte (Abb. 2b). </li></ol> 3.3. Die Überwachung Polyglutamindomänen Toxizität in Hefe durch das Wachstum der Flüssigkulturen Dieses Protokoll ist die quantitative (OD600-Nummern) der drei beschriebenen Tests hier und kann sogar erkennen, sehr feine Unterschiede in Polyglutamindomänen Toxizität. Die vorgenannten ocCurrence spontane Suppressoren kann jedoch potenziell irreführende Ergebnisse zur Folge. Ich empfehlen daher die Kombination dieser Assay mit mindestens einer der beiden oben beschriebenen Assays Plattieren. Wir schlagen vor, den BioscreenC Instrument für diese Experimente zu verwenden. Die BioscreenC ist ein Instrument, misst automatisch die optische Dichte von Hefezellen in 100-Well-Platten, während bei bestimmten Temperaturen mit definierten Bewegung inkubiert. Andere Verfahren zum Züchten von Hefezellen und Messung ihrer optischen Dichte kann ebenfalls Anwendung finden. <ol><li> Waschen der Hefezellen aus Minimalmedium mit Glucose als einziger Kohlenstoffquelle dreimal in 3 ml sterilem Wasser. </li><li> Verdünnen Sie die Übernacht-Kulturen in Medium mit Galactose zu einer OD 600 von 0,1 gezüchtet. </li><li> Füllen Sie jede Position auf der 100-Well-Platte BioscreenC mit 300 ul der Hefekulturen. </li><li> Öffnen Sie das Programm einfach Bioscreen Experiment. Bestimmen Sie die Anzahl der Proben Sie überwachen möchten (einschließlich Leerzeichen und nur mittel-Kontrollen), die Temperatur auf 30 ° C, muss die Länge der Versuche zu 3 Tage, setzen Sie die Mess-Intervalle bis 15 Minuten, setzen Sie den Filter bis 600 nm / Brown, und stellen Sie den Modus Schütteln bis 15 Sekunden vor jeder Messung an mittlerer Stärke. </li><li> Die BioscreenC Instrument und der beigefügten Software produziert Excel-Tabellen von jedem Datenpunkt während des Experiments übernommen. </li><li> Bereiten Wachstumskurven mit Excel für jede Probe und vergleichen Wachstum von verschiedenen Proben (Abbildung 2c). Eine detaillierte Beschreibung der Analyse von Daten, BioscreenC Experimenten hergestellt wurde vor dem 10. vorgesehen. </li></ol> 4. Repräsentative Ergebnisse <img alt="1" src="/files/ftp_upload/3461/3461fig1.jpg" /> Abbildung 1. Die Hefe Polyglutamindomänen Modell. a) Schematische Darstellung des toxischen Polyglutamindomänen-Expansion-Protein. b) Fluoreszenzmikroskopie zeigt Hefe-Zellen, die eine kurze, nicht-toxische PolyglutamindomänenExpansion Protein (25Q, linkes Bild) und Hefe-Zellen, die eine lange, giftige Polyglutamindomänen-Erweiterung Protein (103q, rechtes Bild). <img alt="2" src="/files/ftp_upload/3461/3461fig2.jpg" /> Abbildung 2. Repräsentative Ergebnisse der Wachstums-Assays von Hefezellen, die Polyglutamindomänen-Expansion Proteine. a) Plating-Assay. Rund 700 Hefezellen wurden auf Platten verteilt und für drei Tage bei 30 ° C. Das obere Feld zeigt eine Platte mit Glucose-Medium, dh die Expression des toxischen Polyglutamindomänen-Expansion Protein (103q) nicht induziert. Das untere Feld zeigt eine Hefe Platte mit Galactose Medium, dh die Expression des toxischen Polyglutamindomänen-Expansion Protein induziert wird. Beachten Sie, dass in dem Experiment hier dargestellt, traten keine spontane Suppressor. B) Spotting-Assay. Fünf serielle fünf Verdünnungen von Hefe-Zellen, die entweder ein nicht-toxisches Protein Polyglutamindomänen (25Q) oder eine toxische Polyglutamindomänen-Expansion Protein (103q) wurden auf Glucose-Platten, die die Expression der Proteine ​​(linkes Feld) oder auf Galactose Platten, die ihre Expression zu induzieren unterdrücken entdeckt. Die Platten wurden dann für drei Tage bei 30 ° C inkubiert c) BioscreenC Experiment. Das Wachstum von Hefe-Zellen, die entweder ein nicht-toxisches Protein Polyglutamindomänen (25Q) oder eine toxische Polyglutamindomänen-Expansion Protein (103q) durch die überwacht wurden, BioscreenC Instrument. Die experimentellen Bedingungen und die Analyse der BioscreenC Experimente sind in den Haupttext beschrieben.

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Dieses Manuskript beschreibt drei sich ergänzende experimentelle Ansätze zu Polyglutamindomänen-Toxizität in der Modell-Organismus Hefe zu vermindertem Wachstum von Hefe-Zellen, die toxische Polyglutamindomänen-Erweiterung der Basis von Proteinen zu messen. Work in Hefe hat tiefe Einblicke in die grundlegenden zellulären und molekularen Mechanismen der Fehlfaltung und dessen entsprechend Toxizität, einschließlich der Fehlfaltung und Toxizität von Polyglutamindomänen-Erweiterung Proteine ​​9,11,12</sup...

<table border="1"><tbody><tr><td> Name des Instruments </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> Frogger (6x8 Pins) </td><td> V &amp; P Scientific, San Diego </td><td> VP 407 AH </td></tr><tr><td> BioscreenC </td><td> Growthcurves USA </td><td> 5101370 </td></tr><tr><td> 100-Well-Wabenplatten </td><td> Growthcurves USA </td><td> 9602550 </td></tr></tbody></table>

growth assays to assess polyglutamine toxicity in yeast

Fehlfaltung mit vielen Krankheiten, insbesondere neurodegenerativer Erkrankungen, wie Alzheimer-Krankheit, Parkinson-Krankheit und Huntington-Krankheit 1 zugeordnet ist. Huntington-Krankheit (HD) durch die anormalen Entwicklung eines Polyglutamin (Polyglutamindomänen) Bereich innerhalb des Proteins Huntingtin verursacht. Die Polyglutamindomänen-expanded Huntingtin-Protein erreicht eine anomale Konformation (dh es misfolds) und verursacht zelluläre Toxizität 2. Mindestens acht weiteren neurodegenerativen Krankheiten werden durch Polyglutamindomänen-Erweiterungen, einschließlich der Spinozerebelläre Ataxien und Kennedy-Syndrom 3 verursacht. Der Modellorganismus Hefe hat wesentliche Einblicke in die zellulären und molekularen Grundlagen der Polyglutamindomänen-Toxizität erleichtert, einschließlich der Auswirkungen von intra-und inter-molekularen Faktoren der Polyglutamindomänen-Toxizität und die Identifizierung von zellulären Signalwegen, die in Zellen beeinträchtigt sind Ausdruck Polyglutamindomänen-Expansion Proteine ​​8.3. Wichtigly, wurden viele Aspekte der Polyglutamindomänen-Toxizität, die in der Hefe gefunden wurden, in anderen experimentellen Systemen und in gewissem Maße in Proben von HD-Patienten reproduziert und bewiesen damit, die Bedeutung des Hefe-Modell für die Entdeckung der grundlegenden Mechanismen zugrunde Polyglutamindomänen-Toxizität. Eine direkte und relativ einfachen Weg, um Polyglutamindomänen-Toxizität in Hefe zu bestimmen ist, um das Wachstum von Hefe-Defekte Zellen, die Proteine ​​Polyglutamindomänen-Expansion zu messen. Dieses Manuskript beschreibt drei komplementäre experimentelle Ansätze zur Polyglutamindomänen-Toxizität in Hefe durch Messung bestimmen das Wachstum von Hefe-Zellen, die Proteine ​​Polyglutamindomänen-Expansion. Die ersten zwei experimentelle Ansätze verfolgen das Wachstum der Hefe auf Tellern, überwacht der dritte Ansatz, das Wachstum von flüssigen Hefekulturen mit dem BioscreenC Instrument. Darüber hinaus beschreibt diese Handschrift experimentellen Schwierigkeiten, die auftreten beim Umgang mit Hefe Polyglutamindomänen Modelle können und skizziert Strategien, die helfen, oder zu vermeiden wirddiese Schwierigkeiten zu minimieren. Die Protokolle können die hier beschriebenen zu identifizieren und zu genetischen Wegen und kleinen Molekülen, die Polyglutamindomänen-Toxizität modulieren zu charakterisieren. Darüber hinaus können die beschriebenen Assays als Template für eine genaue Analyse der Toxizität von anderen Krankheit assoziierte fehlgefaltete Proteine ​​in Hefe verursacht Modelle dienen.

Watch this Scientific Journal Video about Growth Assays to Assess Polyglutamine Toxicity in Yeast at JoVE.com

Growth Assays to Assess Polyglutamine Toxicity in Yeast

Dieses Manuskript beschreibt drei sich ergänzenden Protokolle zur Beurteilung der Toxizität von Polyglutamin (Polyglutamindomänen)-Expansion Proteinen in der Hefe<em&gt; Saccharomyces cerevisiae</em&gt;. Diese Protokolle können einfach modifiziert, um die Toxizität von anderen fehlgefaltete Proteine ​​in Hefe zu überwachen.

Wachstums-Assays zur Beurteilung Polyglutamin Toxizität in Hefe

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer&#x2019;s disease, Parkinson's ...

growth-assays-to-assess-polyglutamine-toxicity-in-yeast

Research

JoVE Journal

Biology

13.5K Aufrufe.  Boston Biomedical Research Institute. Dieses Manuskript beschreibt drei sich ergänzenden Protokolle zur Beurteilung der Toxizität von Polyglutamin (Polyglutamindomänen)-Expansion Proteinen in der Hefe Saccharomyces cerevisiae. Diese Protokolle können einfach modifiziert, um die Toxizität von anderen fehlgefaltete Proteine ​​in Hefe zu überwachen.

Serie: Growth Assays to Assess Polyglutamine Toxicity in Yeast

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

Identification of Growth Inhibition Phänotypen durch Expression von bakteriellen Typ III Effektoren in Hefe Induced

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

Forschung

Biologie

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

Modifizierten Hefe-Zwei-Hybrid-System, um Proteine ​​interagieren mit dem Growth Factor Progranulin Identifizieren

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Langfristige letale Toxizität Test mit dem Krustentier Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

Methoden zur subzellulärer Kompartimente der Muskel in Beurteilen<em&gt; C. elegans</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 and minimum levels of 0.03 ng ml&#8722;1) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.

The proposed work assesses the diagnostic potential of direct and amplified surface plasmon resonance imaging (SPRi) assays, particularly for the detection of recombinant human growth hormone in spiked human serum, by comparing SPRi results directly with commercially available enzyme-linked immunosorbent assay (ELISA) kit.

Vergleichende Analyse von Human Growth Hormone im Serum Mit SPRi, Nano-SPRi und ELISA-Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 ...

ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, log-phase, and stationary-phase. Each growth phase can reveal different aspects of fitness that are related to various environmental and genetic conditions. High-resolution and quantitative measurements of all 3 phases of growth are generally difficult to obtain. Here we present a detailed method to characterize all 3 growth phases on solid media using an assay called One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). ODELAY quantifies growth phenotypes of individual cells growing into colonies on solid media using time-lapse microscopy. This method can directly observe population heterogeneity with each growth parameter in genetically identical cells growing into colonies. This population heterogeneity offers a unique perspective for understanding genetic and epigenetic regulation, and responses to genetic and environmental perturbations. While the ODELAY method is demonstrated using yeast, it can be utilized on any colony forming microorganism that is visible by bright field microscopy.

We present a method for quantifying growth phenotypes of individual yeast cells as they grow into colonies on solid media using time-lapse microscopy termed, One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). Population heterogeneity of genetically identical cells growing into colonies can be directly observed and quantified.

ODELAY: Eine groß angelegte Methode für die Mehrparameter-Quantifizierung des Hefewachstums

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

Wirkung von fluoreszierenden Proteinen auf Fusion Partner mit Trinukleotiderkrankungen Toxizität Assays in Hefe

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm cell to penetrate the zona pellucida and fertilize the egg. It is therefore of great interest to test compounds (e.g., environmental chemicals or drug candidates) for their effect on Ca2+-signaling and acrosome reaction in human sperm either to examine the potential adverse effects on human sperm cell function or to investigate a possible role as a contraceptive. Here, two medium-throughput assays are described: 1) a fluorescence-based assay for assessment of effects on Ca2+-signaling in human sperm, and 2) an image cytometric assay for assessment of acrosome reaction in human sperm. These assays can be used to screen a large number of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm. Furthermore, the assays can be used to generate highly specific dose-response curves of individual compounds, determine potential additivity/synergism for two or more compounds, and to study the pharmacological mode of action through competitive inhibition experiments with CatSper inhibitors.

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Here, two medium-throughput assays for assessment of effects on Ca2+-signaling and acrosome reaction in human sperm are described. These assays can be used to quickly and easily screen large amounts of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm.

Medium-Throughput Screening Tests zur Beurteilung der Auswirkungen auf Ca2 +-Signalisierung und Akrosom Reaktion in menschliches Sperma

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm ...

Measuring RAN Peptide Toxicity in C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington&#8217;s disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

Measuring RAN Peptide Toxicity in C. elegans

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

Messung der RAN-Peptidtoxizität in C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis ...

Characterizing Mechanical Properties of Primary Cell Wall in Living Plant Organs Using Atomic Force Microscopy

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the plant. Many sophisticated techniques have been developed to measure these properties; however, atomic force microscopy (AFM) remains the most convenient for studying cell wall elasticity at the cellular level. One of the most important limitations of this technique has been that only superficial or isolated living cells can be studied. Here, the use of atomic force microscopy to investigate the mechanical properties of primary cell walls belonging to the internal tissues of a plant body is presented. This protocol describes measurements of the apparent Young&#39;s modulus of cell walls in roots, but the method can also be applied to other plant organs. The measurements are performed on vibratome-derived sections of plant material in a liquid cell, which allows (i) avoiding the use of plasmolyzing solutions or sample impregnation with wax or resin, (ii) making the experiments fast, and (iii) preventing dehydration of the sample. Both anticlinal and periclinal cell walls can be studied, depending on how the specimen was sectioned. Differences in the mechanical properties of different tissues can be investigated in a single section. The protocol describes the principles of study planning, issues with specimen preparation and measurements, as well as the method of selecting force-deformation curves to avoid the influence of topography on the obtained values of elastic modulus. The method is not limited by sample size but is sensitive to cell size (i.e., cells with a large lumen are difficult to examine).

Studies of cell wall biomechanics are essential for understanding plant growth and morphogenesis. The following protocol is proposed to investigate thin primary cell walls in the internal tissues of young plant organs using atomic force microscopy.

Charakterisierung mechanischer Eigenschaften der primären Zellwand in lebenden Pflanzenorganen mittels Rasterkraftmikroskopie

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the ...