Authors are thankful to K. P. Madhu, Nita Beliappa and staff of the Media Centre of Indian Institute of Science Education and Research, Pune for their help in the video production. The work was supported by funding under the Centre of Excellence program of Department of Biotechnology, Government of India to SG and postdoctoral fellowship by Department of Science and Technology, Government of India to RK.

1. Hydra Cultura e misura della risposta alimentazione <ol><li> Mantenere polipi idra in coltura somministrando loro giornalmente con artemia e tenerli in un mezzo (buffer 1 mM Tris-HCl, pH 7,6, 1 mM NaCl, 1 mM CaCl 2, 0.1 mM KCl, e 0,1 mM MgSO4) contenuta in un ciotola di vetro a 18 ° C in 12 hr luce-12 hr cicli scuri come descritto in precedenza 12. </li><li> Per misurare la risposta di alimentazione, trasferire matura polipo Hydra avere da 5 a 6 tentacoli di un unico pozzetto di una piastra da 24 pozzetti. Rimuovere la quantità residua dal pozzo inclinandolo, e quindi aggiungere immediatamente 500 ml di terreno fresco. </li><li> Preparare la soluzione glutatione 9 micron di media idra. Poiché la soluzione glutatione è incline all&#39;ossidazione, utilizzare sempre soluzione glutatione appena preparata per ciascun esperimento. </li><li> Trasferire la piastra alla piattaforma di imaging di un microscopio avente disposizioni per la registrazione di immagini. Utilizzare uno sfondo scuro in modo tale che il comportamento of polipo Hydra è chiaramente ripreso sullo sfondo contrastante. </li><li> La sala utilizzata per l&#39;osservazione e l&#39;imaging del comportamento di idra privo di luci di intensità fluttuante, correnti d&#39;aria e rumore. Tali disturbi possono anche causare il polipo idra per mostrare la contrazione dei tentacoli - anche in assenza di glutatione. </li><li> Lasciare che il polipo per rilassarsi per 5 minuti. </li><li> Assicurarsi che il polipo si trova lungo la regione centrale del pozzo in modo che il comportamento può essere ripreso in modo chiaro. Se il polipo è al bordo del pozzo, portarlo al centro irrigando il mezzo con una pipetta e ancora permettergli di rilassarsi. </li><li> Scattare una foto di idra nello stato di rilassamento. Questo sarà il punto di osservazione zero tempo. </li><li> Aggiungere rapidamente 9 mM soluzione glutatione per raggiungere una concentrazione finale di 3 micron nel pozzo. A seconda dello scopo dell&#39;esperimento e la risposta dimostrato da idra, testare una gamma di diverse concentrazioni di glutatione e chooSE la concentrazione appropriata richiesta. </li><li> Avviare il timer immediatamente dopo l&#39;aggiunta di soluzione di glutatione e catturare immagini dopo ogni 15-30 secondi per 4-5 min. Non modificare le impostazioni di ingrandimento durante il time-lapse imaging. </li><li> Aggiungere la soluzione glutatione delicatamente e con un flusso uniforme tale che la posizione dell&#39;animale nel pozzo sarebbe minimamente disturbato nel campo visivo del microscopio. Tuttavia, se il polipo muove ampiamente dopo l&#39;aggiunta di soluzione di glutatione, spostare la piastra delicatamente per portare il polipo nel campo di vista per la cattura delle immagini. </li><li> Nell&#39;esperimento di controllo, utilizzare mezzo privo glutatione mantenendo tutti gli altri parametri identici. </li><li> Assicurarsi di eseguire tutte le fasi sperimentali di cui sopra durante la prima metà della giornata - prima di 1:00 per evitare il possibile effetto del ritmo circadiano del grado di risposta di alimentazione. </li><li> Aprire ciascuna delle immagini acquisite utilizzando il GNU Image Manipulation Program (GIMP). </li&gt; <li> Utilizzare lo strumento &quot;Misura&quot; disponibile dal Menu&gt; Strumenti&gt; Misura per determinare la distanza tra l&#39;estremità apicale di ciascuno dei tentacoli e ipostoma. Se l&#39;apertura della bocca è osservata in qualsiasi delle immagini, determinare la distanza tra il centro della bocca aperta e l&#39;estremità apicale del tentacolo. Fare riferimento a questa distanza come la diffusione tentacolo. </li><li> Calcolare lo spread medio tentacolo per ogni polipo prima e dopo l&#39;esposizione glutatione. Calcolare il rapporto di spread medio tentacolo al punto zero tempo a quella in ciascuno dei successivi punti temporali. Questo rapporto si chiamerà relativa diffusione tentacolo. </li><li> Ripetere le misurazioni per almeno 20 polipi. </li></ol><img alt="Equazione 1" fo:content-width="3.5in" src="/files/ftp_upload/52178/52178eq1.jpg" width="400" /> <img alt="Equazione 2" fo:content-width="4in" src="/files/ftp_upload/52178/52178eq2.jpg" width="450" /> 2. Metodo di convalida utilizzando ilLa fame Modello <ol><li> Per fame, trasferire alcuni polipi Hydra in una ciotola di vetro separata e non dar loro da mangiare per 5 giorni. Inserire il gruppo di controllo di alcuni polipi al giorno con Artemia in una ciotola di dimensioni simili. Modificare il supporto sia sperimentale ciotole giorno per evitare la crescita di funghi nel mezzo. </li><li> Il giorno dell&#39;esperimento, alimentare il gruppo di controllo di idra con artemia per 1 ora e utilizzare questi hydra per gli esperimenti successivi dopo aver rimosso tutti artemia non consumati e morti dal mezzo. </li><li> Misurare la risposta alimentazione del idra fame in confronto con l&#39;idra dal gruppo di controllo con il metodo descritto in precedenza al punto 1. Per evitare qualsiasi distorsione dovuta al tempo di osservazione, alternare le misurazioni di ciascuno degli fame e controllare polipi idra. </li></ol>

<ol>
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Pharmacological and biochemical observations.</a> Comp. Biochem. Phys. C. 102, 39-43 (1992).</li><li>Kass-Simon, G., Scappaticci, A. 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J. Neurosci. 20, 2598-2604 (2004).</li><li>Alzugaray, M. E., Adami, M. L., Diambra, L. A., Hernandez-Martinez, S., Damborenea, C., Noriega, F. G., Ronderos, 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=Allatotropin:+An+ancestral+myotropic+neuropeptide+involved+in+feeding.">Allatotropin: An ancestral myotropic neuropeptide involved in feeding.</a> PLoS ONE. 8, (2013).</li><li>Bellis, S. L., Laux, D. C., Rhoads, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+purification+of+Hydra+glutathione+binding+proteins.">Affinity purification of Hydra glutathione binding proteins.</a> FEBS Lett. 354, 320-324 (1994).</li><li>Lenhoff, H. M., Shaw, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+discovery+of+the+GSH+receptor+in+Hydra+and+its+evolutionary+significance.">The discovery of the GSH receptor in Hydra and its evolutionary significance.</a> Glutathione in the Nervous System. , 25-43 (1998).</li><li>Venturini, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hydra+GSH+receptor.+Pharmacological+and+radioligand+binding+studies.">The hydra GSH receptor. Pharmacological and radioligand binding studies.</a> Comp. Biochem. Phys. C. 87, 321-324 (1987).</li><li>Reddy, P. C., Barve, A., Ghaskadbi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Description+and+phylogenetic+characterization+of+common+hydra+from+India.">Description and phylogenetic characterization of common hydra from India.</a> Curr. Sci. 101, 736-738 (2011).</li><li>Horibata, Y., 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=Unique+catabolic+pathway+of+glycosphingolipids+in+a+hydrozoan,+Hydra+magnipapillata.+Involving+endoglycoceramidase.">Unique catabolic pathway of glycosphingolipids in a hydrozoan, Hydra magnipapillata. Involving endoglycoceramidase.</a> J. Biol. Chem. 279, 33379-33389 (2004).</li><li>Koizumi, O., Maeda, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rise+of+feeding+threshold+in+satiated+Hydra.">Rise of feeding threshold in satiated Hydra.</a> J. Comp. Physiol. 142, 75-80 (1981).</li><li>Bellis, S. L., Kass-Simon, G., Rhoads, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+characterization+and+detergent+solubilization+of+the+putative+glutathione+chemoreceptor+from+hydra.">Partial characterization and detergent solubilization of the putative glutathione chemoreceptor from hydra.</a> Biochemistry. 31, 9838-9843 (1992).</li><li>Morita, H., Hanai, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taste+receptor+proteins+in+invertebrates+-+with+special+reference+to+glutathione+receptor+of+hydra.">Taste receptor proteins in invertebrates - with special reference to glutathione receptor of hydra.</a> Chem. Senses. 12, 245-250 (1987).</li><li>Colasanti, M., Venturini, G., Merante, A., Musci, G., Lauro, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+involvement+in+Hydra+vulgaris+very+primitive+olfactory-+like+system.">Nitric oxide involvement in Hydra vulgaris very primitive olfactory- like system.</a> Journal of Neurosci. 17, 493-499 (1997).</li><li>Kuhn, A., Tsiairis, C. D., Williamson, M., Kalbacher, H., Grimmelikhuijzen, C. J., Holstein, T. W., Gr&#252;nder, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+homologous+subunits+form+a+high+affinity+peptide-gated+ion+channel+in+Hydra.">Three homologous subunits form a high affinity peptide-gated ion channel in Hydra.</a> J. Biol. Chem. 285, 11958-11965 (2010).</li><li>Wang, M., Yao, Y., Kuang, D., Hampson, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+family+C+G-protein-coupled+receptors+by+the+tripeptide+glutathione.">Activation of family C G-protein-coupled receptors by the tripeptide glutathione.</a> J. Biol. Chem. 281, 8864-8870 (2006).</li><li>Ruggieri, R. D., Pierobon, P., Kass-Simon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pacemaker+activity+in+hydra+is+modulated+by+glycine+receptor+ligands.">Pacemaker activity in hydra is modulated by glycine receptor ligands.</a> Comp. Biochem. Phys. C. 138, 193-202 (2004).</li><li>Ramazani, R. B., Krishnan, H. R., Bergeson, S. E., Atkinson, N. 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The authors declare no competing financial interests.

Comportamento alimentare in idra rappresenta uno dei sistemi chemosensoriali più ancestrali dei metazoi. Sebbene la presenza di GSH nel fluido crostaceo rilasciato dopo la cattura la preda nematocisti-assistita è stato rilevato da tempo 1, né la proteina GSHR né le putativo gene codifica / s sono stati caratterizzati da idra ad oggi. Alcuni tentativi sono stati fatti per caratterizzare GSH proteine ​​leganti in idra 8, 14, 15. Tuttavia, l&#39;identità di queste proteine ​​recettori put...

Hydra is the most primitive organism possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey1. It feeds on a variety of animals such as nematode, crustacea, insect larvae, tadpoles and newly hatched fish1. The movement of these prey organisms causes mechanosensory discharge of the stinging capsules called nematocysts from hydra, which are inserted into the prey2. GSH present in the fluid released from the injured prey subsequently activates the feeding response in hydra which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth, and consequent engulfing of the prey. Multiple molecules, such as dopamine3, glutamate4, GABA, glycine5, NMDA receptors6, and allatotropin7, have been shown to be involved in the feeding response in hydra. It has also been shown that the chemosensory response induced by GSH is modulated by the feeding status of the animal such that starved hydra exhibited enhanced feeding response1. Such an increase in the GSH sensitivity is biologically relevant since under starvation hydra need to find its prey at higher sensitivity. 
Although the feeding response induced by GSH can be clearly observed under microscope, the methods typically used for measuring the feeding response observations are non-quantitative. In most of the cases, the time during which the mouth of the hydra remains open was taken as a measure of the feeding response8,9; whereas in another case, quantitation was based on the number of hydra out of a population showing the feeding response10. However, observing the mouth opening time of the hydra polyps is cumbersome and subject to variation induced by uncontrollable parameters such as the direction of the mouth orientation during observations. Similarly, since the feeding response is a quantitative parameter, population-based approaches are subject to variations/errors caused by the opinion or observational bias of the individual observer. To circumvent these issues we have developed a method for the relative quantification of the feeding response in hydra (Hydra vulgaris Ind-Pune11) based on the distance of the apical end of the tentacle from the mouth of the hydra polyp.

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measuring glutathione-induced feeding response in hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Glutatione provoca idra esporre arricciamento dei tentacoli verso la bocca al fine di inghiotte la preda. Tale arricciatura di tentacoli porta estremità apicale dei tentacoli più vicino alla ipostoma. Ciò comporta la riduzione dello spread tentacolo, o la distanza lineare tra l&#39;estremità apicale del tentacolo e ipostoma (Figura 1). La diffusione tentacolo relativa, o il rapporto tra tentacolo media si sviluppa prima e dopo l&#39;aggiunta di glutatione, la media su più polipi riduce nel tempo. L...

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Measuring Glutathione-induced Feeding Response in Hydra

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

Misurazione glutatione indotta risposta alimentazione a Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

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11.2K Views.  India Institute of Science Education and Research, Pune. Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

Video: Measuring Glutathione-induced Feeding Response in Hydra

Proboscis Extension Response (PER) Assay in Drosophila

Proboscis extension response (PER) is a taste behavior assay that has been used in flies as well as in honeybees.
On the surface of the fly's mouth (labellum), there are hair-like structures called sensilla which houses taste neurons. When an attractive substance makes contact to the labellum, the fly extends its proboscis to consume the material. Proboscis Extension Response (PER) assay measures this taste behavior response, and it is a useful method to learn about food preferences in a single fly. Solutions of various sugars, such as sucrose, glucose and fructose, are very attractive to the fly. The effect of aversive substances can also be tested as reduction of PER when mixed in a sweet solution.Despite the simplicity of the basic procedure, there are many things that can prevent it from working. One of the factors that requires attention is the fly's responsive state. The required starvation time to bring the fly to the proper responsive state varies drastically from 36 to 72 hours. We established a series of controls to evaluate the fly's state and which allows screening out of non-responsive or hyper-responsive individual animals. Another important factor is the impact level and the position of the contact to the labellum, which would be difficult to describe by words. This video presentation demonstrates all these together with several other improvements that would increase the reproducibility of this method.

Proboscis Extension Response PER Assay in Drosophila

Proboscis extension response or PER is a taste behavior assay that has been used in flies as well as in honeybees. When the proboscis makes contact with an attractive substance, the fly extends its proboscis to consume the substance. Solutions of various sugars are very attractive to the fly.

Proboscide Estensione di risposta (PER) Assay in Drosophila

Proboscis extension response (PER) is a taste behavior assay that has been used in flies as well as in honeybees.
On the surface of the fly's mouth ...

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Biologia

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

Quantificazione dei γH2AX Foci in risposta alle radiazioni ionizzanti

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

Misurare flussi di calcio veloce in cardiomiociti

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

Generation of Transgenic Hydra by Embryo Microinjection

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology1. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

Generation of Transgenic Hydra by Embryo Microinjection

Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.

Generazione di transgenici<em&gt; Hydra</em&gt; Da Embryo microiniezione

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Misurare la rigidità<em&gt; Ex Vivo</em&gt; Mouse aorte Utilizzando microscopia a forza atomica

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

High-fat Feeding Paradigm for Larval Zebrafish: Feeding, Live Imaging, and Quantification of Food Intake

Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipid-rich meal, which consists of an emulsion of chicken egg yolk liposomes created by sonicating egg yolk in embryo media. Detailed instructions are provided to screen larvae for egg yolk consumption so that larvae that fail to feed will not confound experimental results. The chicken egg yolk liposomes can be spiked with fluorescent lipid analogs, including fatty acids and cholesterol, enabling both systemic and subcellular visualization of dietary lipid processing. Several methods are described to mount larvae that are conducive to short- and long-term live imaging with both upright and inverted objectives at high and low magnification. Additionally presented is an assay to quantify larval food intake by extracting the lipids of larvae fed fluorescent lipid analogs, spotting the lipids on a thin layer chromatography plate, and quantifying the fluorescence. Finally, critical aspects of the procedures, important controls, options for modifying the protocols to address specific experimental questions, and potential limitations are discussed. These techniques can be applied not only to focused, hypothesis driven inquiries, but also to a variety of screens and live imaging techniques to study dietary lipid metabolism and the control of food intake.

Zebrafish are emerging as a valuable model of dietary lipid processing and metabolic disease. Described are protocols of lipid-rich larval feeds, live imaging of dietary fluorescent lipid analogs, and quantification of food intake. These techniques can be applied to a variety of screening, imaging, and hypothesis driven inquiry techniques.

Alto contenuto di grassi alimentazione paradigma per larvale Zebrafish: Alimentazione, l&#39;imaging dal vivo, e la quantificazione del cibo

Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipid-rich ...

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.

Misurazione della tossicità del Peptide RAN in C. elegans

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

Measuring Mitochondrial Substrate Flux in Recombinant Perfringolysin O-Permeabilized Cells

Mitochondrial substrate flux is a distinguishing characteristic of each cell type, and changes in its components such as transporters, channels, or enzymes are involved in the pathogenesis of several diseases. Mitochondrial substrate flux can be studied using intact cells, permeabilized cells, or isolated mitochondria. Investigating intact cells encounters several problems due to simultaneous oxidation of different substrates. Besides, several cell types contain internal stores of different substrates that complicate results interpretation. Methods such as mitochondrial isolation or using permeabilizing agents are not easily reproducible. Isolating pure mitochondria with intact membranes in sufficient amounts from small samples is problematic. Using non-selective permeabilizers causes various degrees of unavoidable mitochondrial membrane damage. Recombinant perfringolysin O (rPFO) was offered as a more appropriate permeabilizer, thanks to its ability to selectively permeabilize plasma membrane without affecting mitochondrial integrity. When used in combination with microplate respirometry, it allows testing the flux of several mitochondrial substrates with enough replicates within one experiment while using a minimal number of cells. In this work, the protocol describes a method to compare mitochondrial substrate flux of two different cellular phenotypes or genotypes and can be customized to test various mitochondrial substrates or inhibitors.

In this work, we describe a modified protocol to test mitochondrial respiratory substrate flux using recombinant perfringolysin O in combination with microplate-based respirometry. With this protocol, we show how metformin affects mitochondrial respiration of two different tumor cell lines.

Misurazione del flusso del substrato mitocondriale in cellule ricombinanti perfringolisina O-permeabilizzate

Mitochondrial substrate flux is a distinguishing characteristic of each cell type, and changes in its components such as transporters, channels, or ...

Tick Artificial Membrane Feeding for Ixodes scapularis

Ticks and their associated diseases are an important topic of study due to their public health and veterinary burden. However, the feeding requirements of ticks during both study and rearing can limit experimental questions or the ability of labs to research ticks and their associated pathogens. An artificial membrane feeding system can reduce these problems and open up new avenues of research that may not have been possible with traditional animal feeding systems. This study describes an artificial membrane feeding system that has been refined for feeding and engorgement success for all Ixodes scapularis life stages. Moreover, the artificial membrane feeding system described in this study can be modified for use with other tick species through simple refinement of the desired membrane thickness. The benefits of an artificial membrane feeding system are counterbalanced by the labor intensiveness of the system, the additional environmental factors that may impact feeding success, and the need to refine the technique for each new species and life stage of ticks.

Tick Artificial Membrane Feeding for Ixodes scapularis

Presented here is a method to blood feed ticks in vitro via an artificial membrane system to allow for partial or full engorgement of a variety of tick life stages.

Alimentazione a membrana artificiale di zecche per Ixodes scapularis

Ticks and their associated diseases are an important topic of study due to their public health and veterinary burden. However, the feeding requirements of ...

Manipulation of Rhythmic Food Intake in Mice Using a Custom-Made Feeding System

Rhythmic gene expression is a hallmark of the circadian rhythm and is essential for driving the rhythmicity of biological functions at the appropriate time of day. Studies over the last few decades have shown that rhythmic food intake (i.e., the time at which organisms eat food during the 24 h day), significantly contributes to the rhythmic regulation of gene expression in various organs and tissues throughout the body. The effects of rhythmic food intake on health and physiology have been widely studied ever since and have revealed that restricting food intake for 8 h during the active phase attenuates metabolic diseases arising from a variety of obesogenic diets. These studies often require the use of controlled methods for timing the delivery of food to animals. This manuscript describes the design and use of a low-cost and efficient system, built in-house for measuring daily food consumption as well as manipulating rhythmic food intake in mice. This system entails the use of affordable raw materials to build cages suited for food delivery, following a user-friendly handling procedure. This system can be used efficiently to feed mice on different feeding regimens such as ad libitum, time-restricted, or arrhythmic schedules, and can incorporate a high-fat diet to study its effect on behavior, physiology, and obesity. A description of how wild-type (WT) mice adapt to the different feeding regimens is provided.

Restricting the timing of food intake has emerged as a promising intervention to attenuate diet-induced metabolic diseases. This manuscript details the construction and use of an efficient system built in-house for measuring and manipulating rhythmic food intake in mice.

Manipolazione dell'assunzione ritmica di cibo nei topi utilizzando un sistema di alimentazione personalizzato

Rhythmic gene expression is a hallmark of the circadian rhythm and is essential for driving the rhythmicity of biological functions at the appropriate ...