Funding: Novartis FreeNovation, NCCR, and EPFL.

<ol>
	<li>Long, M. J. C., Ly, P., Aye, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+primer+on+harnessing+non-enzymatic+post-translational+modifications+for+drug+design.">A primer on harnessing non-enzymatic post-translational modifications for drug design.</a> RSC Medicinal Chemistry. 12 (11), 1797-1807 (2021).</li><li>Parvez, S., Long, M. J. C., Poganik, J. R., Aye, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redox+signaling+by+reactive+electrophiles+and+oxidants.">Redox signaling by reactive electrophiles and oxidants.</a> Chemical Reviews. 118 (18), 8798-8888 (2018).</li><li>Parvez, S., 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=T-REX+on-demand+redox+targeting+in+live+cells.">T-REX on-demand redox targeting in live cells.</a> Nature Protocols. 11 (12), 2328-2356 (2016).</li><li>Long, M. J. C., 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=Precision+electrophile+tagging+in+Caenorhabditis+elegans.">Precision electrophile tagging in Caenorhabditis elegans.</a> Biochemistry. 57 (2), 216-220 (2018).</li><li>Van Hall-Beauvais, A., Zhao, Y., Urul, D. A., Long, M. J. C., Aye, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-protein-specific+redox+targeting+in+live+mammalian+cells+and+C.+elegans.">Single-protein-specific redox targeting in live mammalian cells and C. elegans.</a> Current Protocols in Chemical Biology. 10 (3), 43 (2018).</li><li>Long, M. J. C., 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=Akt3+is+a+privileged+first+responder+in+isozyme-specific+electrophile+response.">Akt3 is a privileged first responder in isozyme-specific electrophile response.</a> Nature Chemical Biology. 13 (3), 333-338 (2017).</li><li>Poganik, J. R., 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=Wdr1+and+cofilin+are+necessary+mediators+of+immune-cell-specific+apoptosis+triggered+by+Tecfidera.">Wdr1 and cofilin are necessary mediators of immune-cell-specific apoptosis triggered by Tecfidera.</a> Nature Communications. 12 (1), 5736 (2021).</li><li>Lin, H. -. Y., Haegele, J. A., Disare, M. T., Lin, Q., Aye, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+generalizable+platform+for+interrogating+target-+and+signal-specific+consequences+of+electrophilic+modifications+in+redox-dependent+cell+signaling.">A generalizable platform for interrogating target- and signal-specific consequences of electrophilic modifications in redox-dependent cell signaling.</a> Journal of the American Chemical Society. 137 (19), 6232-6244 (2015).</li><li>Schmittgen, T. D., Livak, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+real-time+PCR+data+by+the+comparative+CT+method.">Analyzing real-time PCR data by the comparative CT method.</a> Nature Protocols. 3 (6), 1101-1108 (2008).</li><li>Wakabayashi, N., 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=Keap1-null+mutation+leads+to+postnatal+lethality+due+to+constitutive+Nrf2+activation.">Keap1-null mutation leads to postnatal lethality due to constitutive Nrf2 activation.</a> Nature Genetics. 35 (3), 238-245 (2003).</li><li>Huang, K. -. T., 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=Z-REX:+Shepherding+reactive+electrophiles+to+specific+proteins+expressed+either+tissue-specifically+or+ubiquitously,+and+recording+the+resultant+functional+electrophile-induced+redox+responses+in+larval+fish.">Z-REX: Shepherding reactive electrophiles to specific proteins expressed either tissue-specifically or ubiquitously, and recording the resultant functional electrophile-induced redox responses in larval fish.</a> Nature Protocols. , (2023).</li><li>Long, M. J. C., Assari, M., Aye, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hiding+in+plain+sight:+The+issue+of+hidden+variables.">Hiding in plain sight: The issue of hidden variables.</a> ACS Chemical Biology. 17 (6), 1285-1292 (2022).</li></ol>

Isoform-specific small-molecule kinase inhibitors, the discovery of which were enabled by REX technologies, have been filed for patent application

Z-REX described in this protocol demonstrates a robust strategy for electrophile-target pair investigation and signaling pathway deconvolution in live fish. The proximity-directed delivery enables dosage and spatial control of the electrophilic compound treatment. Unlike conventional bolus dosing methods, in which the supraphysiological concentrations of electrophile deployed often lead to off-target issues, the relatively minor amount of electrophile released to the system renders Z-REX largely noninvasive. We have used 0.1-6 &#181;M Ht-PreHNE(alkyne) in zebrafish embryos, and the results showed the treatment is not detrimental to embryo development11.
The Z-REX procedure is generally longer than T-REX, a technique for screening/studying electrophile-sensing proteins in cultured cells. Suppose the experiment&#39;s purpose is to screen for electrophile-target interactions; we suggest first performing extensive screening by T-REX in cultured cells and using Z-REX for in vivo validation and phenotypic/pathway analysis. Compared to cell culture, requirements for performing Z-REX are basic fish husbandry techniques in addition to biochemical experimental skills required by T-REX. A typical timeframe for Z-REX (from fish crossing to light-inducible electrophile delivery) is 2-3 days, which is no more than 1 day longer than the typical time for a T-REX experiment on transfected live cells. Live imaging for phenotypic analysis can be performed 2-10 h after light illumination; click coupling with biotin-azide for pull-down assay takes 3 days; qRT-PCR for assaying transcriptional response takes 3 days; IF staining takes 5 days. These steps are roughly similar to their cell culture equivalents, although the interpretation of data requires an understanding of fish physiology and reporter strains.
As a multiple variable procedure12, several control groups are necessary for Z-REX to exclude uncertainties in the results (Figure 1D). Common control groups are: (1) DMSO/vehicle treatment only; (2) probe treatment, but without light illumination; (3) light illumination, but without probe treatment; (4) P2A-split construct, in which Halo and the POI are expressed separately, so the proximity delivery is ablated; and (5) hypomorphic mutants, whose electrophile-sensing residue(s) is/are mutated, such as Akt3 (C119S)6 and Keap1 (C151S, C273W, C288E)5, which we have used in previous studies.
If downstream assays involve western blot analysis, deyolking must be performed before harvest. The yolk proteins reduce the fidelity of lysate-concentration assessments and may bind non-specifically to antibodies. When performing live fish imaging or whole-mount IF staining, we have also observed non-specific fluorescent signals in the yolk sac, likely resulting from autofluorescent proteins in the yolk sac, or non-specific binding of the antibodies themselves. If the autofluorescence signal interferes with the signal, we suggest excluding the yolk sac from quantification, or quantifying different regions separately. Dechorination is necessary for live fish imaging and whole-mount IF staining assay. The chorion can interfere with imaging, and later with quantification/cell counting. However, dechorination is only applicable to embryos older than 1 dpf; younger embryos at blastulation/gastrulation/segmentation stages are too fragile to be dechorionated.
The Z-REX protocol described here is based on mRNA-driven ectopic POI expression. The procedure is rapid compared to using/generating transgenic fish lines. mRNA-driven expression is ubiquitous and transient, and lasts for at least 2 days for mRNAs used in this protocol. However, the duration of expression is likely to vary in other cases. Thus, this approach provides a fast, and more global investigative window into the effects of a specific electrophile-labeling event, compatible with several high-throughput/high-content assays. Transgenic lines with stable Halo-POI expression in specific tissues are also compatible with Z-REX11. Such lines are best used when a more precise question needs to be asked, for instance, when a phenotype in a specific organ is predicted from cell-culture data, or when screening from mRNA-injection experiments predicts that a specific organ is sensitive to an electrophile-labeling event. A heart-specific antioxidant response induction through Z-REX was demonstrated using Tg(gstp1:GFP;DsRed-P2A-myl7:Halo-TeV-Keap1) fish in our previous publication11. It may also be possible to perform Z-REX on transgenic fish older than 2 dpf.

A myriad of cellular signaling events involve reactions between small reactive molecules (endogenously produced in the cell or xenobiotics/xenometabolites, such as drugs) and their protein target. In many instances, a substoichiometric level of such covalent binding events can trigger cellular responses, leading to changes in, for instance, development, metabolism, apoptosis, and/or immune response1. However, deconstructing the mode of action (MOA) by linking specific binding events to their phenotypic consequences has proven challenging. Traditional bolus dosing methods that involve the introduction of high concentrations of the reactive species often result in a multitude of proteins being modified, as well as excessive toxicity to the model organism2. Such conditions are far from ideal. A method was developed to resolve these issues in cell culture using precision localized electrophile delivery in a native cellular context, named T-REX (targetable reactive electrophiles and oxidants)3. In the intervening years, the focus has turned to experiments in whole organisms, which allow the opportunity to study proteins in specific cellular contexts in non-transformed cells. Thus, we have extended the technique to be compatible with several models, including Danio rerio embryo models. Herein, we present Z-REX (zebrafish targeting reactive electrophiles and oxidants) (Figure 1).
To understand Z-REX, this article first presents REX technologies and their underlying concepts. At their core, these techniques model endogenous physiological reactive electrophilic species (RES) signaling by mimicking how natural electrophiles are locally produced in vivo with spatiotemporal precision. The protein of interest (POI) is expressed as a fusion construct to Halo; the latter anchors the tissue-permeable and inert probe bearing the photocaged RES in a 1:1 stoichiometry. One such endogenous RES is 4-hydroxynonenal (HNE hereafter), which is photocaged in the probe Ht-PreHNE. In many instances, we use an alkyne-functionalized version of HNE [i.e., HNE(alkyne)], which has essentially identical biological properties to HNE, but can be labeled by click chemistry. The probe, which is also functionalized with a chloroalkane for reactivity with Halo, is referred to as Ht-PreHNE(alkyne). The complex of the Halo-POI fusion and the probe thus formed allows proximal delivery of RES to the fused POI upon irradiation with UV light. If the POI reacts rapidly with the liberated RES, the resulting covalent labeling of the POI with RES allows us to identify kinetically-privileged cysteines.
Z-REX takes the aforementioned advantages of REX technologies and applies them broadly to study specific signaling pathways in live fish. This protocol has been optimized for zebrafish (D. rerio), since they are genetically-tractable vertebrate organisms that are transparent during development, and thus ideal for opto-chemical/-genetic techniques like REX technologies. Nevertheless, a similar strategy is also likely to work well on other genetically-tractable fish species, since the broad applicability of the method is due to the pseudo-intramolecular mechanism of lipid-derived electrophile (LDE) delivery. Indeed, the procedure is highly biocompatible, since fish can be treated with the Z-REX photocaged-electrophile [e.g., Ht-PreHNE(alkyne)] for at least 48 h without any noticeable impacts on development. A similar protocol functions in C. elegans4,5.
The protocol first describes the use of mRNA injection to produce a transient expression of a non-native Halo-POI fusion construct in embryonic zebrafish models, 1-1.5 days post-fertilization (dpf). This results in the expression of the ectopic protein in the majority of cells within the fish (hereafter referred to as &#39;ubiquitous&#39;), rather than in specific tissues or locales; however, the data shows that cell-specific effects can be observed in certain cases. Following injection, the embryos are incubated with a low concentration [0.3-5 &#181;M Ht-PreHNE(alkyne)] of the probe for up to 30.5 h post fertilization (hpf). Then, at a user-prescribed time, delivery of the RES to the POI within fish is achieved by photouncaging for 2-5 min. Following photouncaging of the RES, a variety of downstream phenotypic assays can be performed over the next 2-10 h: 1) live imaging of reporter lines (Figure 2A); 2) target-labeling assessment by western blot analysis (Figure 3); 3) transcriptomic analysis (Figure 4); or 4) whole-mount immunofluorescence (Figure 5).
As an example of live imaging of reporter lines, Z-REX is demonstrated in tandem with live imaging of fish lines, Tg(lyz:TagRFP) and Tg(mpeg1:eGFP), to measure how RES modification of a specific electrophile-sensor POI (namely Keap1) decreases neutrophil and macrophage levels, respectively, with no observable effects on other cells in the fish. However, we have shown previously that the POI-labeling and the consequential pathway signaling from T-REX studies can be reproduced using Z-REX for several proteins: Akt36, Keap17, and Ube2v26. Overall, with Z-REX, scientists can study the consequence(s) of covalent modification of POIs by RES in the context of several complex redox pathways. This technique is primed to pinpoint targets and their functional residues for covalent drug design and novel drug mechanisms in a more contextually-relevant whole animal model.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Mercaptoethanol (BME)</td>
			<td>Sigma-Aldrich</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5-mL microcentrifuge tube</td>
			<td>Axygen</td>
			<td>MCT-150-C-S</td>
			<td></td>
		</tr>
		<tr>
			<td>10-cm Petri dishes</td>
			<td>Celltreat</td>
			<td>229692</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mL microcentrifuge tube</td>
			<td>Axygen</td>
			<td>MCT-200-C-S</td>
			<td></td>
		</tr>
		<tr>
			<td>30% Acrylamide and bis-acrylamide solution</td>
			<td>BioRad</td>
			<td>1610154</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Celltreat</td>
			<td>229506</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Fisher</td>
			<td>A/0600/15</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>GoldBio</td>
			<td>A201-100</td>
			<td></td>
		</tr>
		<tr>
			<td>All Blue Prestained Protein Standards</td>
			<td>BioRad</td>
			<td>1610373</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>Sigma</td>
			<td>A3678</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin-dPEG11-azide</td>
			<td>Quanta Biodesign</td>
			<td>102856-142</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford Dye</td>
			<td>BioRad</td>
			<td>5000205</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Fisher</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Capillary tubes</td>
			<td>VWR</td>
			<td>HARV30-00200</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Supelco, Inc</td>
			<td>1.02445.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>11836170001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cu(TBTA)</td>
			<td>Lumiprobe</td>
			<td>21050</td>
			<td></td>
		</tr>
		<tr>
			<td>CuSO4</td>
			<td>Sigma</td>
			<td>209198</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Fisher</td>
			<td>D128-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-mouse-Alexa Fluor 647</td>
			<td>Abcam</td>
			<td>ab150107</td>
			<td>1:800 (IF)</td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit-Alexa Fluor 647</td>
			<td>Abcam</td>
			<td>ab150075</td>
			<td>1:1000 (IF)</td>
		</tr>
		<tr>
			<td>Donkey anti-rat-AlexaFluor 568</td>
			<td>Abcam</td>
			<td>ab175475</td>
			<td>1:1000 (IF)</td>
		</tr>
		<tr>
			<td>ECL substrate</td>
			<td>Thermo Fisher Scientific</td>
			<td>32106</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL-Plus substrate</td>
			<td>Thermo Fisher Scientific</td>
			<td>32132</td>
			<td></td>
		</tr>
		<tr>
			<td>End-to-end rotator</td>
			<td>FinePCR</td>
			<td>Rotator AG</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher</td>
			<td>E/0650DF/15</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium bromide</td>
			<td>Sigma</td>
			<td>E1510</td>
			<td></td>
		</tr>
		<tr>
			<td>Flaming/Brown Micropipette Puller</td>
			<td>Sutter Instrument Co.</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence stereomicroscope</td>
			<td>Leica</td>
			<td>M165 FC</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps (blunt)</td>
			<td>self made</td>
			<td></td>
			<td>self made by blunting sharp forceps (Fine Science Tools, Dumont #5, 11252-40)</td>
		</tr>
		<tr>
			<td>Forceps (sharp)</td>
			<td>Fine Science Tools</td>
			<td>Dumont #5, 11252-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel loading tip</td>
			<td>Fisher</td>
			<td>02-707-181</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel/blot imager</td>
			<td>Vilber</td>
			<td>Fusion FX imager</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beads</td>
			<td>Sigma</td>
			<td>45-G1145</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass stage micrometer</td>
			<td>Meiji Techno.</td>
			<td>MA285</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat inactivated FBS</td>
			<td>Sigma</td>
			<td>F2442</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated ultrasonic bath</td>
			<td>VWR</td>
			<td>89375-470</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher</td>
			<td>BP310-1</td>
			<td></td>
		</tr>
		<tr>
			<td>High capacity streptavidin agarose</td>
			<td>Thermo Fisher Scientific</td>
			<td>20359</td>
			<td></td>
		</tr>
		<tr>
			<td>Ht-PreHNE alkyne probe</td>
			<td>self-made</td>
			<td>-</td>
			<td>Parvez, S. et al. T-REX on-demand redox targeting in live cells. Nat Protoc. 11 (12), 2328-2356, (2016).</td>
		</tr>
		<tr>
			<td>Imaging plate (10% HBSS buffer, for live embryos)</td>
			<td></td>
			<td></td>
			<td>Made with Petri dish, and 2% agarose in 10% HBSS buffer</td>
		</tr>
		<tr>
			<td>Imaging plate (PBS, for formaldehyde-fixed embryos)</td>
			<td></td>
			<td></td>
			<td>Made with Petri dish, and 2% agarose in PBS</td>
		</tr>
		<tr>
			<td>Injection plate</td>
			<td></td>
			<td></td>
			<td>Made with microinjection mold, Petri dish, and 2% agarose in 10% HBSS buffer</td>
		</tr>
		<tr>
			<td>LDS</td>
			<td>Apollo</td>
			<td>APOBI3331</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>VWR</td>
			<td>20864.32</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjection mold</td>
			<td>Adaptive Science Tools</td>
			<td>TU-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Microloader tips</td>
			<td>Eppendorf</td>
			<td>930001007</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Narishige</td>
			<td>MN-153</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope for micro-injection</td>
			<td>Olympus</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope light source</td>
			<td>Olympus</td>
			<td>&#160;KL 1600 LED</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma</td>
			<td>M3516</td>
			<td></td>
		</tr>
		<tr>
			<td>mMessage mMachine SP6 Transcription Kit</td>
			<td>Ambion</td>
			<td>AM1340</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti- &#946;-actin-HRP</td>
			<td>Sigma</td>
			<td>A3854</td>
			<td>1:20000 (WB)</td>
		</tr>
		<tr>
			<td>Mouse anti-HaloTag</td>
			<td>Promega</td>
			<td>G921A</td>
			<td>1:500 (IF)</td>
		</tr>
		<tr>
			<td>Multi-mode reader</td>
			<td>BioTek Instruments</td>
			<td>Cytation 5</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleic acid agarose gel electrophoresis apparatus</td>
			<td>Biorad</td>
			<td>Mini-Sub Cell GT Systems</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligo(dT)20</td>
			<td>Integrated DNA Technologies</td>
			<td>customized: (dT)20</td>
			<td></td>
		</tr>
		<tr>
			<td>Orange G</td>
			<td>Sigma</td>
			<td>O3756</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>pCS2+8 Halo-2XHA-P2A-TeV-Keap1-2xHA&#160;</td>
			<td>self-cloned</td>
			<td>-</td>
			<td>Available from Prof. Yimon AYE&#39;s group at EPFL</td>
		</tr>
		<tr>
			<td>pCS2+8 Halo-TeV-Keap1-2xHA</td>
			<td>self-cloned</td>
			<td>-</td>
			<td>Available from Prof. Yimon AYE&#39;s group at EPFL</td>
		</tr>
		<tr>
			<td>Pneumatic PicoPump</td>
			<td>WPI</td>
			<td>SYS-PV820</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein electrophoresis equipment and supplies</td>
			<td>Biorad</td>
			<td>Mini-PROTEAN Tetra Vertical Electrophoresis</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-active Caspase-3</td>
			<td>BD Pharmingen&#160;</td>
			<td>559565</td>
			<td>1:800 (IF)</td>
		</tr>
		<tr>
			<td>Rat anti-HA-HRP</td>
			<td>Sigma</td>
			<td>H3663</td>
			<td>1:500 (IF and WB)</td>
		</tr>
		<tr>
			<td>Rat anti-RFP</td>
			<td>ChromoTek</td>
			<td>5F8</td>
			<td>1:800 (IF)</td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge</td>
			<td>Eppendorf</td>
			<td>5417R</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAseOUT recombinant ribonuclease inhibitor</td>
			<td>ThermoFisher Scientific</td>
			<td>10777019</td>
			<td></td>
		</tr>
		<tr>
			<td>RnaseZap RNA decontamination solution</td>
			<td>ThermoFisher Scientific</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr>
			<td>Rocking Shaker</td>
			<td>DLAB</td>
			<td>SK-R1807-S</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Teknova</td>
			<td>S9974</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript III reverse transcriptase</td>
			<td>ThermoFisher Scientific</td>
			<td>18080085</td>
			<td></td>
		</tr>
		<tr>
			<td>t-Butanol</td>
			<td>Sigma</td>
			<td>471712</td>
			<td></td>
		</tr>
		<tr>
			<td>TCEP-HCl</td>
			<td>Gold Biotechnology</td>
			<td>TCEP1</td>
			<td></td>
		</tr>
		<tr>
			<td>TeV protease (S219V)</td>
			<td>self-made</td>
			<td>-</td>
			<td>Parvez, S. et al. T-REX on-demand redox targeting in live cells. Nat Protoc. 11 (12), 2328-2356, (2016).</td>
		</tr>
		<tr>
			<td>Tg(lyz:TagRFP)</td>
			<td>Zebrafish International Resource Center (ZIRC)</td>
			<td>uwm11Tg (ZFIN)</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Tg(mpeg1:eGFP)</td>
			<td>Zebrafish International Resource Center (ZIRC)</td>
			<td>gl22Tg (ZFIN)</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Thermal cycler</td>
			<td>Analytik Jana</td>
			<td>846-x-070-280</td>
			<td></td>
		</tr>
		<tr>
			<td>TMEDA</td>
			<td>Sigma</td>
			<td>T7024</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipets</td>
			<td>Fisher</td>
			<td>13-711-9D</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Apollo</td>
			<td>APOBI2888</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher</td>
			<td>BP151-100</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin inhibitor from Glycine max (soybean)</td>
			<td>Sigma</td>
			<td>T9003</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Fisher</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr>
			<td>UV lamp with 365-nm light</td>
			<td>Spectroline</td>
			<td>ENF 240C</td>
			<td></td>
		</tr>
		<tr>
			<td>UV meter&#160;</td>
			<td>Spectroline</td>
			<td>XS-365</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortexer</td>
			<td>Scientific Industries, Inc.</td>
			<td>Vortex-Genie 2</td>
			<td></td>
		</tr>
		<tr>
			<td>Western Blotting Transfer equipment and supplies</td>
			<td>Biorad</td>
			<td>Mini Trans-Blot or Criterion Blotter&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Zebrafish husbandry and breeding equipment</td>
			<td></td>
			<td></td>
			<td>in house</td>
		</tr>
		<tr>
			<td>Zirconia beads</td>
			<td>BioSpec</td>
			<td>11079107zx</td>
			<td></td>
		</tr>
	</tbody>
</table>

monitoring on-target signaling responses in larval zebrafish - z-rex unmasks precise mechanisms of electrophilic drugs and metabolites

Reactive metabolites and related electrophilic drugs are among the most challenging small molecules to study. Conventional approaches to deconstruct the mode of action (MOA) of such molecules leverage bulk treatment of experimental specimens with an excess of a specific reactive species. In this approach, the high reactivity of electrophiles renders non-discriminate labeling of the proteome in a time- and context-dependent manner; redox-sensitive proteins and processes can also be indirectly and often irreversibly affected. Against such a backdrop of innumerable potential targets and indirect secondary effects, linking phenotype to specific target engagement remains a complex task. Zebrafish targeting reactive electrophiles and oxidants (Z-REX)-an on-demand reactive-electrophile delivery platform adapted for use in larval zebrafish-is designed to deliver electrophiles to a specific protein of interest (POI) in otherwise unperturbed live fish embryos. Key features of this technique include a low level of invasiveness, along with dosage-, chemotype-, and spatiotemporally-controlled precision electrophile delivery. Thus, in conjunction with a unique suite of controls, this technique sidesteps off-target effects and systemic toxicity, otherwise observed following uncontrolled bulk exposure of animals to reactive electrophiles and pleiotropic electrophilic drugs. Leveraging Z-REX, researchers can establish a foothold in the understanding of how individual stress responses and signaling outputs are altered as a result of specific reactive ligand engagement with a specific POI, under near-physiologic conditions in intact living animals.

Live imaging of Z-REX-treated transgenic neutrophil/macrophage reporter fish, Tg(lyz:TagRFP) and Tg(mpeg1:EGFP). Induction of neutrophil/macrophage apoptosis through Keap1 HNEylation. (See also Figure 2). The effect of electrophile labeling of Keap1 on neutrophil and macrophage levels was assessed by injecting heterozygous transgenic embryos derived from Tg(lyz:TagRFP) or Tg(mpeg1:EGFP) with mRNA encoding Halo-Keap1, and then treating with Ht-PreHNE(alkyne). Following the procedures for step 6.1-downstream assay Option 1-HNE(alkyne) was liberated and Keap1 was labeled. Neutrophil and macrophage levels were assessed by live imaging of reporter lines, Tg(lyz:TagRFP) and Tg(mpeg1:eGFP), respectively. The level of both cell types decreased by 30%-40% after Z-REX treatment, in which HNE was delivered to Keap1. On the contrary, no loss of neutrophils or macrophages was seen in Z-REX technical control groups [without light and Ht-PreHNE(alkyne), light alone, or Ht-PreHNE(alkyne) alone] (Figure 1D and Figure 2A-D).
The induction of neutrophil/macrophage apoptosis indicated successful HNE delivery to Keap1 through Z-REX. Details for the pathway analysis and apoptosis mechanism have been published5. To account for off-target effects of HNE(alkyne), several controls were used. (1) Under the same experimental conditions, instead of Halo-TeV-Keap1 mRNA, embryos were injected with Halo-P2A-Keap1 mRNA. P2A linker allowed the Halo and Keap1 proteins to be expressed independently. In this scenario, HNE(alkyne) released from Halo could not label Keap1, as it was no longer proximal to Halo (Figure 1D); hence, the apoptosis signaling pathway was not triggered. No changes in macrophage or neutrophil levels were observed in this group (Figure 2A,B). (2) The same experimental conditions were performed using mRNA encoding Halo-TeV-Keap1(C151S,C273W,C288E), a mutant of Keap1 that does not respond to HNE(alkyne) (Figure 1D). No changes in macrophage or neutrophil levels were observed (Figure 2G,H).
Biotin azide-click coupling and biotin pull-down assay. Target-labeling assessment. (See also Figure 3). The target-labeling assessment was carried out using WT embryos, injected with mRNA encoding either Halo-TeV-Keap1-2xHA (Halo-POI fusion construct) or Halo-2xHA-P2A-Keap1-2xHA (P2A-split construct, in which Halo and Keap1 are not fused; Figure 1D). Labeled Keap1 protein was only pulled down in the group expressing fusion protein and treated with Z-REX (second lane in the top anti-HA blot), but not in other control groups (no mRNA injection, fusion construct without Z-REX, or P2A-split construct). The results indicate the HNE(alkyne) was successfully delivered to Keap1, and the modified Keap1 was subsequently conjugated with biotin through click reaction, and the biotin-labeled Keap1 was pulled down by streptavidin resin.
Transcriptional analysis. RNA-seq and qRT-PCR. (See also Figure 4). The transcriptional change after Z-REX treatment was assessed by RNA-seq and qRT-PCR. In RNA-seq, several immune-related genes were downregulated after Z-REX. In contrast, many antioxidant response(AR)-related genes were upregulated after Z-REX, which resulted from the induction of the Keap1-Nrf2-AR pathway upon HNEylation on&#160;Keap110 (Figure 4A). In qRT-PCR analysis, similar results were found when analyzing three immune-related genes (lyz, mpeg1.1, and coro1a) (Figure 4B). The up- and down-regulation of the respective genes showed the successful induction of pathways mediated by Keap1 HNEylation.
Whole-mount (co-)immunofluorescence staining assay and colocalization analysis. (See also Figure 5). The exogenous Halo-TeV-Keap1-2xHA and Halo-2xHA-P2A-Keap1-2xHA expression were assessed by whole-mount immunofluorescence (IF) staining (Figure 5A,B). The P2A-split-construct had two times the number of HA tags than the TeV-fusion-construct, which corresponds to a twofold higher anti-HA signal in the P2A-split-construct-mRNA-injected group than the other, indicating the expression level of the two constructs were similar (Figure 5C). The expression levels of the Halo-TeV-Keap1 (wt) and Halo-TeV-Keap1(C151S,C273W,C288E) were also found similar when probing with anti-Halo (Figure&#160;5D,E). Colocalization of neutrophils and active caspase 3 in Z-REX-treated Tg(lyz:TagRFP) was observed by co-immunostaining with anti-RFP and anti-active-Caspase 3 (Figure 5F). Active Caspase 3 is an indicator of apoptosis events.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64846/64846fig01.jpg" /> 
Figure 1: Z-REX workflow. (A,B) A 1-4 cell stage zebrafish embryo is injected with (morpholino and) mRNA encoding Halo-POI (e.g., Halo-Keap1). Injected embryos are then treated with a probe composed of a HaloTag ligand and a photocaged electrophile appended with an alkyne functional group, such as Ht-PreHNE(alkyne) in B. After removing the excess amount of probe, the embryo is exposed to light to release the electrophile of interest [e.g., HNE or its analog, HNE(alkyne)]. The downstream analysis is performed at a given/user-defined timepoint. (C) Design and mechanism of the Ht-PreLDE probe, which is applicable to different lipid-derived electrophiles (LDE). (D) Negative/technical control groups for Z-REX. <a href="https://www.jove.com/files/ftp_upload/64846/64846fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64846/64846fig02.jpg" /> 
Figure 2: Live imaging of transgenic neutrophil/macrophage reporter fish subjected to Z-REX. Z-REX-mediated Keap1 HNEylation induces neutrophil/macrophage apoptosis. (A) Representative images of Tg(lyz:TagRFP) fish expressing either Halo-TeV-Keap1 (fusion construct) or Halo-P2A-Keap1 (split construct), and subjected to negative control conditions [no treatment, light alone, or Ht-PreHNE(alkyne) alone or Z-REX]. Embryo age: 36 hpf. (B) Quantitation of neutrophil levels in A. (C) Representative images of Tg(mpeg1:eGFP) fish expressing Halo-TeV-Keap1 with or without Z-REX treatment. Embryo age: 34 hpf. (D) Quantitation of macrophage levels in C. (E,F) Time-course measurement of (E) neutrophil and (F) macrophage levels after Z-REX treatment. (G) Similar experiment as in A in fish expressing either Halo-TeV-Keap1 (WT) or Halo-TeV-Keap1 (C151S, C273W, C288E), a mutant that does not have HNE-sensing capability. (H) Quantitation of neutrophil levels in G. Scale bars: 500 &#181;m. All the graphs are presented with mean &#177; SEM. p values were calculated with one-way ANOVA (blue) and two-tailed Student&#39;s t-test (black). This figure has been modified from Poganik et al.7. <a href="https://www.jove.com/files/ftp_upload/64846/64846fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64846/64846fig03.jpg" /> 
Figure 3: Biotin pull-down assay. WT embryos expressing Halo-TeV-Keap1-2XHA or Halo-2XHA-P2A-Keap1-2XHA were treated with Z-REX or respective negative control conditions (no probe treatment in this case). After harvest, embryos were lysed and treated with TeV protease before the biotin pull-down assay. The results were analyzed by western blotting. This figure has been modified from Huang et al. Z-REX: shepherding reactive electrophiles to specific proteins expressed either tissue-specifically or ubiquitously, and recording the resultant functional electrophile-induced redox responses in larval fish. This figure has been modified from Huang et al.11. <a href="https://www.jove.com/files/ftp_upload/64846/64846fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64846/64846fig04.jpg" /> 
Figure 4: Transcriptional analysis. (A) RNA-seq results of Z-REX-treated versus non-treated embryos. Statistically significant differentially-expressed (SDE) genes are highlighted. Immunity-related SDE genes are colored red. Antioxidant response (AR)-related genes are colored green. Other SDE genes are colored blue. All p values were calculated with CuffDiff. (B-D) Three immunity-related SDE genes from A: (B) lyz, (C) mpeg1.1, and (D) coro1a were further analyzed with qRT-PCR, and only the Z-REX-treated embryos showed the suppression of these transcripts. All the graphs are presented with mean &#177; SEM. p values were calculated with one-way ANOVA (blue) and two-tailed Student&#39;s t-test (black). This figure has been modified from Poganik et al.7. <a href="https://www.jove.com/files/ftp_upload/64846/64846fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64846/64846fig05.jpg" /> 
Figure 5: Whole-mount immunofluorescence staining assay. (A,B) Representative images of embryos expressing either (A) Halo-TeV-Keap1-2xHA or (B) Halo-2xHA-P2A-Keap1-2xHA immunostained with anti-HA and secondary antibody conjugated with AlexaFluor568. mRNA-injected fish were compared to age-matched non-injected fish. (C) Quantification of anti-HA signal in (A,B). (D) Representative images of embryos expressing Halo-TeV-Keap1 (WT) or Halo-TeV-Keap1 (C151S, C273W, C288E) immunostained with anti-Halo and secondary antibody conjugated with AlexaFluor647. mRNA-injected fish were compared to age-matched non-injected fish. (E) Quantification of anti-Halo signal in D. p values were calculated with two-tailed Student&#39;s t-test. (F) Tg(lyz:TagRFP) embryos subjected to Z-REX were co-immunostained with anti-RFP and anti-active Caspase 3, and respective fluorophore-conjugated secondary antibodies. The white box marks the magnified area. White arrows indicate colocalizations of neutrophils and active Caspase 3. Scale bars: 500 &#181;m. All the graphs are presented with mean &#177; SEM. This figure has been modified from Poganik et al.7. and Huang et al.11. <a href="https://www.jove.com/files/ftp_upload/64846/64846fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Table 1: List of buffers used in this study. <a href="https://www.jove.com/files/ftp_upload/64846/Supplementary Table 1.xlsx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Monitoring On-Target Signaling Responses in Larval Zebrafish - Z-REX Unmasks Precise Mechanisms of Electrophilic Drugs and Metabolites at JoVE.com

Monitoring On-Target Signaling Responses in Larval Zebrafish - Z-REX Unmasks Precise Mechanisms of Electrophilic Drugs and Metabolites

Zebrafish targeting reactive electrophiles and oxidants (Z-REX) is a chemical biology-based method for the investigation of reactive small-molecule signaling. This technique can be applied to live fish of different developmental stages. Here, we couple standard assays in zebrafish with Z-REX for signaling pathway analysis.

Reactive metabolites and related electrophilic drugs are among the most challenging small molecules to study. Conventional approaches to deconstruct the ...

monitoring-on-target-signaling-responses-larval-zebrafish-z-rex

Research

JoVE Journal

Biology

700 Views.  Swiss Federal Institute of Technology Lausanne (EPFL). Zebrafish targeting reactive electrophiles and oxidants (Z-REX) is a chemical biology-based method for the investigation of reactive small-molecule signaling. This technique can be applied to live fish of different developmental stages. Here, we couple standard assays in zebrafish with Z-REX for signaling pathway analysis.

Video: Monitoring On-Target Signaling Responses in Larval Zebrafish - Z-REX Unmasks Precise Mechanisms of Electrophilic Drugs and Metabolites

Monitoring Plant Hormones During Stress Responses

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. Among the most severe stresses are insect herbivory, pathogen infection, and drought stress. For each of these stresses a specific set of hormones and/or combinations thereof are known to fine-tune the responses, thereby ensuring the plant's survival. The major hormones involved in the regulation of these responses are jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA). To better understand the role of individual hormones as well as their potential interaction during these responses it is necessary to monitor changes in their abundance in a temporal as well as in a spatial fashion. For the easy, sensitive, and reproducible quantification of these and other signaling compounds we developed a method based on vapor phase extraction and gas chromatography/mass spectrometry (GC/MS) analysis (1, 2, 3, 4). After extracting these compounds from the plant tissue by acidic aqueous 1-propanol mixed with dichloromethane the carboxylic acid-containing compounds are methylated, volatilized under heat, and collected on a polymeric absorbent. After elution into a sample vial the analytes are separated by gas chromatography and detected by chemical ionization mass spectrometry. The use of appropriate internal standards then allows for the simple quantification by relating the peak areas of analyte and internal standard.

A simple method is provided that allows for the rapid extraction and analysis of multiple plant hormones from small tissue samples. The procedure uses vapor phase extraction as the solemn purification step. Samples are analyzed by GC/MS with chemical ionization that produces mainly (M+1)+ ions.

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

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.

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

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 &mu;m) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology.

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies1-3, congenital myopathies4,5, and disruptions in sarcomeric assembly6,7, due to high genomic and structural conservation with mammals8. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral).
Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres9. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres.
The touch-evoked escape assay involves observing an embryo&#39;s swimming behavior in response to tactile stimulation10-12. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance12. The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.

The zebrafish is now an established and powerful tool for modeling muscular dystrophies, congenital myopathies, and related neuromuscular diseases. Birefringence and touch-evoked escape behavior are two common noninvasive assays used to determine the degree of muscular disorganization and locomotive impairment of zebrafish embryos during early development.

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

High-throughput Measurement of Gut Transit Time Using Larval Zebrafish

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and pharmacological similarities to that of the mammals. GI intolerance during clinical testing of drug candidates is common and may pose a serious threat to human health. Testing for GI toxicity in preclinical mammalian models can be expensive in terms of time, test compound, and labor. The high-throughput method presented here may be used to predict GI safety issues. Compared to mammalian models, this method allows for more expedient assessment of test compound effects on GI transit while using low quantities of compound. In this method, larval zebrafish (7 days post fertilization) are fed food containing a fluorescent label. After feeding, each larval fish is placed into a well of a 96-conical-bottom-well plate and dosed with test compound (dissolved in water) or the vehicle. As gut transit occurs, fecal matter accumulates on the bottom of the wells, and the rate at which this happens is monitored by measuring fluorescence from the bottom of the well repeatedly over time using a plate spectrophotometer. The fluorescence from larvae in a given treatment group are averaged and these values are graphed along with standard error, for each measurement time, yielding a curve representing average transit of food over time. Effects on gut transit time are identified by comparing the area under the curve for each treatment group to that of the vehicle-treated group. This method detected changes in zebrafish GI transit time induced by drugs with known clinical GI effects; it can be employed to interrogate dozens of treatments for GI effects per day. As such, safer compounds can be quickly prioritized for mammalian testing, which expedites discovery and proffers 3Rs advancement.

The goal of this protocol is to measure the transit time of fluorescently labeled food through the gut of larval zebrafish in a high throughput fashion.

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...