This work was supported by National Natural Science Foundation of China (82004031) and Sichuan Science and Technology Program (2022NSFSC1303). We express our great appreciation to Jiayi Sun at Innovative Institute of Chinese Medicine and Pharmacy, Chengdu University of Traditional Chinese Medicine, for the assistance with western blot.

1. Downloading the structures of xanthatin and Keap1
<ol>
	<li>Open the PubChem database (https://pubchem.ncbi.nlm.nih.gov/), input xanthatin (small molecule), then press&#160;Search and click on The First Result. Click on Download and click Save under 2D structure to save the compound in .sdf format.</li>
	<li>Download the crystal structure of the protein.
	<ol>
		<li>Open the UniProt database (https://www.uniprot.org/), input Keap1 and click on&#160;Search. Click Human under popular organisms in the left column, and then click on The First Entry Named Q14145 in the right column.</li>
		<li>Click on Structure under the function in the left column, find the structure with PDB ID: 2FLU, and click on RCSB-PDB. Click on Download Files and select PDB Format to download the crystal structure of Keap1 protein.</li>
	</ol>
	</li>
</ol>
2. Molecular docking
<ol>
	<li>Create a new folder named molecular docking on the desktop and save the downloaded xanthatin (small molecule) and Keap1 (protein) structures in this folder. 
	NOTE: Folder name and path must be in English.</li>
	<li>Open Maestro software, click File, select Change Working Directory, click Desktop, double-click to select the newly created molecular docking folder and click Choose Options.</li>
	<li>Small molecule processing
	<ol>
		<li>Click File and import structures options, click Desktop, double-click on molecular docking folder, click on xanthatin Structure File, and then click Open to import small molecule file.</li>
		<li>Click Tasks in the upper right corner of the software and select LigPrep option. Use structures from the workspace; the rest of the parameters are the default parameters and click Run to perform small molecule processing.</li>
	</ol>
	</li>
	<li>Protein structure processing
	<ol>
		<li>Click the File and Import Structures options, click Desktop, and double-click on the Molecular Docking folder. Click on the Keap1 Protein Structure file, and then click Open to import the protein structure file.</li>
		<li>Click Tasks in the upper right corner of the software and select the Protein Preparation Wizard option. Check the box Before Deleting Waters Beyond 5 &#197; From Het Groups and enter a pH of 7.4+/-0.0. Click Preprocess, click Refine, and then click Optimize &#62; Remove Waters &#62; Minimize.</li>
		<li>Click on the next task when the previous task is completed. Click Tasks and select the Sitemap option, use default settings for all parameters, and click Run.</li>
		<li>Click File and Import Structures options, click Desktop, double-click on the molecular docking folder, and open the folder named sitemap_1. Click on the first file named Sitemap_1_out.maegz, and then click Open.</li>
		<li>Double-click on the dot to select sitemap_1_protein in the workspace, and then click on Sitemap_1_site_1 to select it . Click Tasks and select the Receptor Grid Generation option. 
		NOTE: The protein sites predicted by the sitemap are ranked according to the scoring, with the highest-scoring site in the first place.</li>
		<li>Select the Entry option under receptor, with the remaining parameters unchanged. Click on the Small White Ball in the protein structure to generate a docking box of default size and change the job name to glide-grid_2FLU. Click Run.</li>
	</ol>
	</li>
	<li>Molecular docking
	<ol>
		<li>Click Tasks and select the Ligand Docking option. Select the Receptor Grid As from the file and click Browse. Click Desktop, double click on molecular docking folder, double click on glide-grid_2FLU file, click Glide-grid_2FLU.zip file, and then click Open.</li>
		<li>Click on Use Ligands From Files &#62; Browse &#62; Desktop. Double-click on the molecular docking folder, double-click on the ligprep_1 file, click Ligprep_1-out.maegz file, and then click Open.</li>
		<li>Click Settings and select Precision as XP (extra precision) option, change the job name to glide-dock_XP_2FLU, and click Run.</li>
	</ol>
	</li>
	<li>View docking results
	<ol>
		<li>Click File and Import Structures options, click Desktop, and double-click on the Molecular Docking folder. Double-click on the glide-dock_XP_2FLU file, click the glide-dock_XP_1_pv.maegz file and then click Open.</li>
		<li>Double-click on the dot to select ligand in the workspace, and then click to select sitemap_1_protein. Click Table, slide to the far right, and view the score under docking score.</li>
	</ol>
	</li>
	<li>Visualization of 2D results
	<ol>
		<li>Double click on the dot to select ligand in the workspace, and then click to also select sitemap_1_protein. Click Ligand Interaction &#62; View and check the LID legend box.</li>
		<li>Click File, choose Save Screenshot, input 6000 at the width, uncheck the box for Transparent Background, and click OK. Click on Desktop, name the file as 2D-xanthatin-2FLU, and save the image.</li>
	</ol>
	</li>
	<li>Visualization of 3D results
	<ol>
		<li>Double click on the dot to select ligand in the workspace, and then click to also select sitemap_1_protein.</li>
		<li>Click L in Quick Select to select small molecules, and then click Style. Click on the Second Line in the style to change the size of the small molecule bond and click on the color atoms to change the small molecule color.</li>
		<li>Select the small molecule in the figure, click the right mouse button, select Expand Selection, and choose 4 &#197;. Click Apply Labels in style to display the labels.</li>
		<li>Click on the second line in the style to change the size of amino acid bonds and click on the Color Atoms to change amino acid color.</li>
		<li>Click on Next &#62; Invert, hold down the control key and click on the first eye position under style to show only the amino acids with labels.</li>
		<li>Click on Style &#62; Ribbons to show the protein as a ribbon style and click on Edit Ribbon under ribbons to change the ribbon color.</li>
		<li>Click on Interactions Toggle in the bottom right corner of the screen, click the right mouse button and uncheck contacts/clashes.</li>
		<li>Search for measure in tasks, select Distance, and click on the two atoms connected by the yellow dotted line (hydrogen bond) in the screen to measure hydrogen bonds lengths.</li>
		<li>Press and hold the mouse&#39;s wheel to rotate the protein, adjust the protein to the center of the screen, and ensure that each amino acid label is visible.</li>
		<li>Click Workspace &#62; Save Image as and click on the desktop. Save the image as 600dpi by default, name the file as 3D-xanthatin-2FLU, choose file type as tiff format, and save the image.</li>
	</ol>
	</li>
</ol>
3. Cell culture and CETSA sample preparation
<ol>
	<li>Cell culture
	<ol>
		<li>Add 5 x 106 RAW264.7 cells/mL into a 100 mm culture dish and incubate with 6 mL of DMEM medium with 10% FBS and 1% antibiotic-antimycotic solution at 37 &#176;C, 95% humidity, and 5% CO2. Prepare three dishes and perform the experiment when the cells reach 80% confluency.</li>
	</ol>
	</li>
	<li>CETSA sample preparation
	<ol>
		<li>Aspirate the old medium, add 2 mL of phosphate buffer saline (PBS) warmed to room temperature to each dish, and wash 2x.</li>
		<li>Add 2 mL of PBS to each dish of cells, mix the cells, and collect cells into two 1.5 mL microcentrifuge tubes. Centrifuge at 377 x g for 3 min at room temperature.</li>
		<li>Discard the supernatant and resuspend cells with 2 mL of PBS containing 1% Broad-spectrum protease inhibitor mixtures. Divide into individual microcentrifuge tubes with 1 mL of cell suspension in each.</li>
		<li>Freeze microcentrifuge tubes in liquid nitrogen until white solid, then immediately thaw in a 37 &#176;C water bath and repeat 3x. Centrifuge at 12,000 x g at 4 &#176;C for 10 min.</li>
		<li>Take two microcentrifuge tubes, one with 100 &#181;M xanthatin and one with an equal volume of DMSO, and add 450 &#181;L of centrifuged supernatant to each tube, incubate at 37 &#176;C for 30 min.</li>
		<li>Add the supernatant from the two microcentrifuge tubes to 14 PCR tubes at a volume of 60 &#181;L/tube, and heat tubes simultaneously at varying temperatures (45 &#176;C, 48 &#176;C, 51 &#176;C, 54 &#176;C, 57 &#176;C, 60 &#176;C and 63 &#176;C) for 3 min, with two PCR tubes at each temperature, for the DMSO and xanthatin groups, respectively.</li>
		<li>For the unheated group, take supernatant from each of the two microcentrifuge tubes and add to 14 PCR tubes, 7 for the DMSO group and 7 for the xanthatin group, in a volume of 60 &#181;L each. Do not heat the samples here.</li>
		<li>Cool the tubes at room temperature for the heated group. Centrifuge all the tubes (heated and unheated) at 12,000 x g for 10 min at 4 &#176;C and take 48 &#181;L of supernatant from each tube to a new 1.5 mL microcentrifuge tube.Add 12 &#181;L of SDS-PAGE protein loading buffer to each tube, vortex to mix well, and heat in a water bath at 100 &#176;C for 5 min. 
		NOTE: Prepared CETSA samples must be stored at -80 &#176;C and tested by western blot within 1 week.</li>
	</ol>
	</li>
</ol>
4. Western blot
<ol>
	<li>Preparation of SDS-PAGE gel
	<ol>
		<li>Rinse one long and one short glass plate with ultrapure water. Place the plates into the clamping slot, then on a transparent board. Add ultrapure water and leak check for 10 min.</li>
		<li>Add 8 mL of lower gel solution and 8 mL of lower gel buffer into a conical flask and mix by gently shaking. Add 160 &#181;L of coagulant promoter to the gel solution and mix well. 
		NOTE: The volume of the lower layer of adhesive should not exceed the green line on the clamping slot.</li>
		<li>Drain the ultrapure water from the plates and blot them dry with filter paper. Add the prepared lower gel solution. Add 2 mL of isopropyl alcohol and wait for 20 min for solidification.</li>
		<li>Take 2 mL of upper gel solution and 2 mL of upper gel buffer into a conical flask and mix by gently shaking the flask.</li>
		<li>Remove the isopropyl alcohol from the gel layer and blot the excess isopropyl alcohol with filter paper.</li>
		<li>Add 40 &#181;L of coagulant promoter to the mixed upper gel solution, mix well, then add between the long and short glass plates and insert a 1.5 mm 15-hole comb, wait for 20 min. 
		NOTE: The comb should be inserted perpendicular to the upper layer, with no air bubbles between the comb and the solution.</li>
	</ol>
	</li>
	<li>Electrophoresis
	<ol>
		<li>Add 1 L of ultrapure water to a beaker, add a sachet of SDS-PAGE running buffer powder, stir well with a glass rod, and add the prepared gel to the electrophoresis unit. Place the unit in the box and pull out the comb.</li>
		<li>Thaw marker and CETSA samples at room temperature, vortex, and spin down. Add 2.5 &#181;L of marker to the first well and 20 &#181;L of CETSA samples to each of the remaining wells according to the predetermined sequence. 
		NOTE: CETSA samples were added successively according to temperature treatment described in step 3.2.6 from 45 &#176;C to 63 &#176;C; the DMSO group was added first, then the xanthatin group (Figure 1).</li>
		<li>Add the remaining electrophoresis solution to the electrophoresis unit, close the lid, and insert the electrode, positive pole to positive pole, negative pole to negative pole.</li>
		<li>Turn ON the power switch and adjust the voltage switch to 80 V to start electrophoresis.</li>
	</ol>
	</li>
	<li>Transfer to membrane
	<ol>
		<li>Add 850 mL of ultrapure water, 100 mL of anhydrous ethanol, and 50 mL of rapid transfer buffer to a beaker and stir well. Prepare foam board, plastic knives, tweezers, trays, and membrane transfer devices.</li>
		<li>Cut the PVDF membrane according to the size of the gel, mark it, and soak it in methanol for 30 s to activate it.</li>
		<li>Pour the transfer solution into the tray, place and open the transfer clamps, and place a sponge pad and three layers of transfer filter paper.</li>
		<li>Stop electrophoresis and then open the lid of the electrophoresis box. Take the electrophoresis device, pour out the electrophoresis solution, and remove the plate. Place the plate on the foam board, gently pry it off with a plastic knife, and cut the gel to clearly visualize the protein marker and the target proteins.</li>
		<li>Hold one side of the marker with tweezers, wash it in a tray containing the transmembrane solution, and lay it flat on filter paper. Dip the activated PVDF membrane into the transfer solution, hold the labeled side with tweezers, and cover the gel face-down. 
		NOTE: The PVDF membrane marked side is the front side and pay attention to the fact that no bubbles are generated during the placement process.</li>
		<li>Place two layers of transfer filter paper and one layer of sponge pad on the PVDF membrane, cover and clamp the transfer clips, and put them into the transfer tank. 
		NOTE: Black color of transfer clip to black color of transfer slot.</li>
		<li>Add the remaining transmembrane solution to the transmembrane cassette, cover with the lid, positive to positive, negative to negative.</li>
		<li>Adjust the current to 400 mA, time to 30 min, and then press Run to start transferring the membrane at room temperature.</li>
	</ol>
	</li>
	<li>Blocking
	<ol>
		<li>Add 2 L of ultrapure water to a glass vial, add a sachet of TBS powder, and then add 2 mL of Tween to make TBST solution. Weigh the BSA powder to prepare a 5% BSA solution with TBST and add 6 mL of BSA solution to the incubation box.</li>
		<li>Stop the membrane transfer and then open the transfer clip. Clamp the marker side of the PVDF membrane and place it in the incubation box. Place the incubation box on a shaker and block for 1.5 h at room temperature.</li>
	</ol>
	</li>
	<li>Incubation of primary antibody
	<ol>
		<li>Prepare the primary antibody (see Table of Materials for details) with 5% BSA solution at 1:1000, recycle the BSA solution in the incubator box, and add 6 mL of primary antibody to the incubation box. Place the incubation box in a foam box with ice packs and keep it on a shaker overnight.</li>
	</ol>
	</li>
	<li>Incubation of secondary antibody
	<ol>
		<li>Collect the primary antibody in the incubation box the next day, then add 6 mL of TBST solution and place it on a shaker to wash for 5 min.</li>
		<li>Pour off the washed TBST solution and add fresh for washing; repeat 5x and prepare the secondary antibody with 5% BSA solution at 1:5000.</li>
		<li>Pour off the TBST solution and add 6 mL of secondary antibody to the incubation box. Place the incubation box on a shaker and incubate for 1.5 h at room temperature.</li>
		<li>Collect the secondary antibody (see Table of Materials for details), then add 6 mL of TBST solution and place it on a shaker to wash for 5 min. Pour off the washed TBST solution and add fresh for washing; repeat 5x.</li>
	</ol>
	</li>
	<li>Exposing the strips
	<ol>
		<li>Retain the TBST solution at the end of the last wash. Take equal volumes of chemiluminescent reagents A and B, shake, and mix well. Shield the solution from light.</li>
		<li>Open the gel imaging system, click on Samples, check calibration, wait for calibration to complete, and then click on Marker, check calibration.</li>
		<li>Pick up the marker side of the strip with tweezers and fully immerse in the chemiluminescent reagent solution. Clamp one side of the marker to place the strip face down in the imager, close the lid of the imager, click on Exposure, and set the exposure time to 1 s. 
		NOTE: The developer must completely cover the strip, and the development should be in a dark environment.</li>
		<li>Click Save, name the image, and save it as a .tif file. Analyze the optical density of the western blot. The gray values of each temperature in the DMSO group were compared with the gray values of 45 &#176;C, and the gray values of each temperature in the xanthatin group were compared with the gray values of xanthatin at 45 &#176;C.</li>
	</ol>
	</li>
</ol>

Validating Xanthatin-Keap1 Interaction with Docking and CETSA

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+advances+in+CETSA.">Current advances in CETSA.</a> Front Mol Biosci. 9, 866764 (2022).</li><li>Liu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xanthatin+inhibits+STAT3+and+NF-&#954;B+signalling+by+covalently+binding+to+JAK+and+IKK+kinases.">Xanthatin inhibits STAT3 and NF-&#954;B signalling by covalently binding to JAK and IKK kinases.</a> J Cell Mol Med. 23 (6), 4301-4312 (2019).</li><li>Liu, Y., Chen, W., Zheng, F., Yu, H., Wei, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xanthatin+alleviates+LPS-Induced+inflammatory+response+in+RAW264.7+macrophages+by+Inhibiting+NF-&#954;B,+MAPK+and+STATs+activation.">Xanthatin alleviates LPS-Induced inflammatory response in RAW264.7 macrophages by Inhibiting NF-&#954;B, MAPK and STATs activation.</a> Molecules. 27 (14), 4603 (2022).</li><li>Dinkova-Kostova, A. T., Kostov, R. V., Canning, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keap1,+the+cysteine-based+mammalian+intracellular+sensor+for+electrophiles+and+oxidants.">Keap1, the cysteine-based mammalian intracellular sensor for electrophiles and oxidants.</a> Arch Biochem Biophys. 617, 84-93 (2017).</li><li>Tabana, Y., Babu, D., Fahlman, R., Siraki, A. G., Barakat, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+identification+of+small+molecules:+An+overview+of+the+current+applications+in+drug+discovery.">Target identification of small molecules: An overview of the current applications in drug discovery.</a> BMC Biotechnol. 23 (1), 44 (2023).</li><li>Schenone, M., Dan&#269;&#237;k, V., Wagner, B. K., Clemons, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+identification+and+mechanism+of+action+in+chemical+biology+and+drug+discovery.">Target identification and mechanism of action in chemical biology and drug discovery.</a> Nat Chem Biol. 9 (4), 232-240 (2013).</li><li>Hughes, J. P., Rees, S., Kalindjian, S. B., Philpott, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+early+drug+discovery.">Principles of early drug discovery.</a> Br J Pharmacol. 162 (6), 1239-1249 (2011).</li><li>Chakraborty, 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=SARS-CoV-2+protein+drug+targets+landscape:+A+potential+pharmacological+insight+view+for+the+new+drug+development.">SARS-CoV-2 protein drug targets landscape: A potential pharmacological insight view for the new drug development.</a> Expert Rev Clin Pharmacol. 14 (2), 225-238 (2021).</li><li>Ziegler, S., Pries, V., Hedberg, C., Waldmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+identification+for+small+bioactive+molecules:+Finding+the+needle+in+the+haystack.">Target identification for small bioactive molecules: Finding the needle in the haystack.</a> Angew Chem Int Ed Engl. 52 (10), 2744-2792 (2013).</li><li>Zhang, X., 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=Highly+effective+identification+of+drug+targets+at+the+proteome+level+by+pH-dependent+protein+precipitation.">Highly effective identification of drug targets at the proteome level by pH-dependent protein precipitation.</a> Chem Sci. 13 (42), 12403-12418 (2022).</li><li>Zhang, X., 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=A+simplified+thermal+proteome+profiling+approach+to+screen+protein+targets+of+a+ligand.">A simplified thermal proteome profiling approach to screen protein targets of a ligand.</a> Proteomics. 20, 1900372 (2020).</li><li>Hou, 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=Salidroside+intensifies+mitochondrial+function+of+CoCl2-damaged+HT22+cells+by+stimulating+PI3K-AKT-MAPK+signaling+pathway.">Salidroside intensifies mitochondrial function of CoCl2-damaged HT22 cells by stimulating PI3K-AKT-MAPK signaling pathway.</a> Phytomedicine. 109, 154568 (2022).</li><li>Wang, X., 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=Salidroside,+a+phenyl+ethanol+glycoside+from+Rhodiolacrenulata,+orchestrates+hypoxic+mitochondrial+dynamics+homeostasis+by+stimulating+Sirt1/p53/Drp1+signaling.">Salidroside, a phenyl ethanol glycoside from Rhodiolacrenulata, orchestrates hypoxic mitochondrial dynamics homeostasis by stimulating Sirt1/p53/Drp1 signaling.</a> J Ethnopharmacol. 293, 115278 (2022).</li></ol>

The authors have nothing to disclose.

The identification of disease targets and the discovery and development of drugs are closely interconnected27. By precisely targeting specific targets, drug candidates can be developed to treat particular diseases more effectively while concurrently minimizing the side effects associated with the drugs28,29. The most commonly used targets are protein targets30. However, the identification of special protein targets ...

Proteins are highly important macromolecules in living organisms and possess a diverse range of unique functions within cells, such as membrane composition, cytoskeleton formation, enzyme activity, transportation, cell signaling, and involvement in both intracellular and extracellular mechanisms1,2,3. Proteins manifest their biological functions primarily through specific interactions with a variety of molecules, including other proteins, nucleic acids, small molecule ligands, and metal ions1,4. Ligands are small molecular compounds that specifically bind to proteins in an organism. The interaction between proteins and ligands occurs at specific sites on the protein, called the binding sites, also known as the binding pockets5. In medicinal chemistry research, the focus lies in identifying key proteins that are clearly associated with diseases, which serve as targets for drugs6. Therefore, gaining a deep understanding of the binding sites between proteins and ligands is of utmost importance in promoting drug discovery, design, and research7,8.
Molecular docking is a widely used computational tool for studying protein-ligand binding, which employs the three-dimensional structures of proteins and ligands to explore their primary binding modes and affinities when forming stable complexes9,10,11. The application of molecular docking technology originated in the 1970s. Based on the lock and key pairing principle and utilizing the algorithms of molecular docking software, one can determine the interaction between compounds and molecular targets by analyzing docking results. This approach enables the prediction of active binding sites for both the compound and the target molecule. Consequently, it facilitates the identification of an optimal binding conformation (here called the binding model) for ligand-receptor interactions, which is crucial for understanding the mechanics of these molecular engagements12,13,14,15. While molecular docking provides valuable computer-based predictions of ligand-receptor interactions, it is important to note that these are preliminary findings. Consequently, further experimental verification is essential to confirm these interactions.
The cellular thermal shift assay (CETSA), initially proposed by P&#228;r Nordlund&#39;s research team in 2013, serves as a method for validating drug-target protein interactions. This technique specifically tests the thermal stability of target proteins induced by drug binding, providing a practical approach for confirming molecular interactions16,17,18. This approach is based on the fundamental principle that ligand binding initiates a thermal shift within target proteins and is applicable to a wide array of biological samples, including cell lysates, intact living cells, and tissues19,20. CETSA supports direct target engagement of small molecules in intact cells by detecting thermodynamic stabilization of proteins due to ligand binding and linking the observed phenotypic response to the target compound21,22. Among the various methodologies derived from CETSA, Western Blot-CETSA (WB-CETSA) is considered a classical approach. Following sample preparation using the CETSA method, western blot analysis is utilized to detect alterations in the thermal stability of the target protein. This allows for the precise determination of drug-protein interactions within cellular systems17,23.
Xanthatin is a bioactive compound isolated from the plant Xanthium L. with properties such as anti-inflammatory, which has been used in traditional Chinese medicine to treat diseases like nasal sinusitis and arthritis24,25. The kelch-like ECH-associated protein 1 (Keap1) is a component of the Cullin3-based Cullin-RING E3 ubiquitin ligase multi-subunit protein complex and an important regulator of intracellular redox homeostasis, which influences the intensity and duration of the inflammatory response by modulating the intracellular redox state26. In this study, we first utilized molecular docking to investigate the interaction between xanthatin (small molecule) and Keap1 protein, aiming to predict their binding mode. Subsequently, we employed the CETSA method to validate this interaction by assessing the impact of xanthatin on the thermal stability of the Keap1 protein.

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			<td>0.45 &#956;m Polyvinylidene fluoride membrane</td>
			<td>Millipore</td>
			<td>PR05509</td>
			<td></td>
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		<tr>
			<td>Anhydrous ethanol</td>
			<td>Chron chemicals</td>
			<td>64-17-5</td>
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			<td>Bovine serum albumin</td>
			<td>BioFroxx</td>
			<td>4240GR100</td>
			<td></td>
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			<td>Broad-spectrum protease inhibitor mixtures</td>
			<td>Boster Biological Technology Co., Ltd</td>
			<td>AR1193</td>
			<td></td>
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		<tr>
			<td>DMSO</td>
			<td>Boster Biological Technology Co., Ltd</td>
			<td>PYG0040</td>
			<td></td>
		</tr>
		<tr>
			<td>Enhanced chemiluminescence reagent</td>
			<td>Beyotime Biotechnology Co., Ltd</td>
			<td>P0018S</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH antibody</td>
			<td>ProteinTech Group Co., Ltd</td>
			<td>10494-1-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Imaging Instrument</td>
			<td>E-BLOT</td>
			<td>Touch Imager Pro</td>
			<td></td>
		</tr>
		<tr>
			<td>Gradient PCR instrument</td>
			<td>Biometra TADVANCED</td>
			<td>Biometra Tadvanced 96SG</td>
			<td></td>
		</tr>
		<tr>
			<td>High-speed freezing centrifuge</td>
			<td>Beckman Coulter</td>
			<td>Allegra X-30R</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish peroxidase-conjugated affiniPure goat antibody</td>
			<td>ProteinTech Group Co., Ltd</td>
			<td>SA00001-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl alcohol&#160;</td>
			<td>Chron chemicals</td>
			<td>67-63-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Keap1 antibody</td>
			<td>Zen BioScience Co., Ltd</td>
			<td>R26935</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal bath</td>
			<td>Analytik Jena</td>
			<td>TSC</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Chron chemicals</td>
			<td>67-56-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ncmblot rapid transfer buffer (20&#215;)</td>
			<td>NCM Biotech Co., Ltd</td>
			<td>WB4600</td>
			<td></td>
		</tr>
		<tr>
			<td>Omni-Easy OneStep PAGE gel fast preparation kie</td>
			<td>Epizyme Biotech Co., Ltd</td>
			<td>PG212</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer saline</td>
			<td>Boster Biological Technology Co., Ltd</td>
			<td>PYG0021</td>
			<td></td>
		</tr>
		<tr>
			<td>Prestained Color Protein Marker</td>
			<td>Biosharp&#160;</td>
			<td>BL741A</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein Blotting Electrophoresis System</td>
			<td>Bio-Rad</td>
			<td>MiniPROTEAN&#210;Tetra Cell</td>
			<td></td>
		</tr>
		<tr>
			<td>RAW264.7 cell</td>
			<td>Beyotime Biotechnology Co., Ltd</td>
			<td>C7505</td>
			<td></td>
		</tr>
		<tr>
			<td>RAW264.7 cell-specific medium</td>
			<td>Procell Life Science&#38;Technology Co., Ltd</td>
			<td>CM-0597</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE protein loading buffer</td>
			<td>Boster Biological Technology Co., Ltd</td>
			<td>AR1112-10</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE running buffer powder</td>
			<td>Servicebio</td>
			<td>G2018</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris buffered saline powder</td>
			<td>Servicebio</td>
			<td>G0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>BioFroxx</td>
			<td>1247ML100</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Memmert</td>
			<td>WNE10</td>
			<td></td>
		</tr>
		<tr>
			<td>Water purifier</td>
			<td>Millipore</td>
			<td>Milli- IQ 7005</td>
			<td></td>
		</tr>
		<tr>
			<td>Xanthatin</td>
			<td>ChemConst Biotechnology Co., Ltd</td>
			<td>CONST210706</td>
			<td></td>
		</tr>
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protein target prediction and validation of small molecule compound

Proteins are fundamental to human physiology, with their targets being crucial in research and drug development. The identification and validation of crucial protein targets have become integral to drug development. Molecular docking is a computational tool widely utilized to investigate protein-ligand binding, especially in the context of drug and protein target interactions. For the experimental verification of the binding and to access the binding of the drug and its target directly, the cellular thermal shift assay (CETSA) method is used. This study aimed to integrate molecular docking with CETSA to predict and validate interactions between drugs and vital protein targets. Specifically, we predicted the interaction between xanthatin and Keap1 protein as well as its binding mode through molecular docking analysis, followed by verification of the interaction using the CETSA assay. Our results demonstrated that xanthatin could establish hydrogen bonds with specific amino acid residues of Keap1 protein and reduce the thermostability of Keap1 protein, indicating that xanthatin could directly interact with Keap1 protein.

Molecular docking analysis predicted the interaction between xanthatin and Keap1 protein. Figure 2 demonstrates the formation of hydrogen bonds between xanthatin and amino acid residues Gly-367 and Val-606 of Keap1 protein, with a hydrogen bond length of 2.17 &#197; for Gly-367 and 2.13 &#197; for Val-606. In addition, the calculated docking score of -5.69 kcal/mol signifies a good binding affinity between xanthatin and Keap1 protein.
The CETSA method showed that ...

Watch this Scientific Journal Video about Author Spotlight: Streamlining Protein Target Prediction and Validation via Molecular Docking and CETSA at JoVE.com

Author Spotlight: Streamlining Protein Target Prediction and Validation via Molecular Docking and CETSA

The experiment used here shows a method of molecular docking combined with cellular thermal shift assay to predict and validate the interaction between small molecules and protein targets.

Protein Target Prediction and Validation of Small Molecule Compound

Proteins are fundamental to human physiology, with their targets being crucial in research and drug development. The identification and validation of ...

protein-target-prediction-and-validation-of-small-molecule-compound

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

2.2K Views.  Chengdu University of Traditional Chinese Medicine. The experiment used here shows a method of molecular docking combined with cellular thermal shift assay to predict and validate the interaction between small molecules and protein targets.

Video: Author Spotlight: Streamlining Protein Target Prediction and Validation via Molecular Docking and CETSA

An Experimental Paradigm for the Prediction of Post-Operative Pain (PPOP)

Many women undergo cesarean delivery without problems, however some experience significant pain after cesarean section. Pain is associated with negative short-term and long-term effects on the mother. Prior to women undergoing surgery, can we predict who is at risk for developing significant postoperative pain and potentially prevent or minimize its negative consequences? These are the fundamental questions that a team from the University of Washington, Stanford University, the Catholic University in Brussels, Belgium, Santa Joana Women's Hospital in S&atilde;o Paulo, Brazil, and Rambam Medical Center in Israel is currently evaluating in an international research collaboration. The ultimate goal of this project is to provide optimal pain relief during and after cesarean section by offering individualized anesthetic care to women who appear to be more 'susceptible' to pain after surgery.
A significant number of women experience moderate or severe acute post-partum pain after vaginal and cesarean deliveries. 1 Furthermore, 10-15% of women suffer chronic persistent pain after cesarean section. 2 With constant increase in cesarean rates in the US 3 and the already high rate in Brazil, this is bound to create a significant public health problem. When questioning women's fears and expectations from cesarean section, pain during and after it is their greatest concern. 4 Individual variability in severity of pain after vaginal or operative delivery is influenced by multiple factors including sensitivity to pain, psychological factors, age, and genetics. The unique birth experience leads to unpredictable requirements for analgesics, from 'none at all' to 'very high' doses of pain medication. Pain after cesarean section is an excellent model to study post-operative pain because it is performed on otherwise young and healthy women. Therefore, it is recommended to attenuate the pain during the acute phase because this may lead to chronic pain disorders. The impact of developing persistent pain is immense, since it may impair not only the ability of women to care for their child in the immediate postpartum period, but also their own well being for a long period of time.
In a series of projects, an international research network is currently investigating the effect of pregnancy on pain modulation and ways to predict who will suffer acute severe pain and potentially chronic pain, by using simple pain tests and questionnaires in combination with genetic analysis. A relatively recent approach to investigate pain modulation is via the psychophysical measure of Diffuse Noxious Inhibitory Control (DNIC). This pain-modulating process is the neurophysiological basis for the well-known phenomenon of 'pain inhibits pain' from remote areas of the body. The DNIC paradigm has evolved recently into a clinical tool and simple test and has been shown to be a predictor of post-operative pain.5 Since pregnancy is associated with decreased pain sensitivity and/or enhanced processes of pain modulation, using tests that investigate pain modulation should provide a better understanding of the pathways involved with pregnancy-induced analgesia and may help predict pain outcomes during labor and delivery. For those women delivering by cesarean section, a DNIC test performed prior to surgery along with psychosocial questionnaires and genetic tests should enable one to identify women prone to suffer severe post-cesarean pain and persistent pain. These clinical tests should allow anesthesiologists to offer not only personalized medicine to women with the promise to improve well-being and satisfaction, but also a reduction in the overall cost of perioperative and long term care due to pain and suffering. On a larger scale, these tests that explore pain modulation may become bedside screening tests to predict the development of pain disorders following surgery.

An Experimental Paradigm for the Prediction of Post-Operative Pain PPOP

Diffuse noxious inhibitory control, temporal summation and wound hyperalgesia testing are demonstrated in the obstetric patient. These tests evaluate inhibitory and excitatory mechanisms of pain processing and are here utilized to evaluate endogenous analgesia at different time-points during pregnancy and the peripartum period to help reveal individual s risk for persistent pain.

Many women undergo cesarean delivery without problems, however some experience significant pain after cesarean section. Pain is associated with negative ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Protocol for Long Duration Whole Body Hyperthermia in Mice

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.

This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease

Unbiased discovery proteomics strategies have the potential to identify large numbers of novel biomarkers that can improve diagnostic and prognostic testing in a clinical setting and may help guide therapeutic interventions. When large numbers of candidate proteins are identified, it may be difficult to validate candidate biomarkers in a timely and efficient fashion from patient plasma samples that are event-driven, of finite volume and irreplaceable, such as at the onset of acute graft-versus-host disease (GVHD), a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT).
Here we describe the process of performing commercially available ELISAs for six validated GVHD proteins: IL-2R&alpha;5, TNFR16, HGF7, IL-88, elafin2, and REG3&alpha;3 (also known as PAP1) in a sequential fashion to minimize freeze-thaw cycles, thawed plasma time and plasma usage. For this procedure we perform the ELISAs in sequential order as determined by sample dilution factor as established in our laboratory using manufacturer ELISA kits and protocols with minor adjustments to facilitate optimal sequential ELISA performance. The resulting plasma biomarker concentrations can then be compiled and analyzed for significant findings within a patient cohort. While these biomarkers are currently for research purposes only, their incorporation into clinical care is currently being investigated in clinical trials.
This technique can be applied to perform ELISAs for multiple proteins/cytokines of interest on the same sample(s) provided the samples do not need to be mixed with other reagents. If ELISA kits do not come with pre-coated plates, 96-well half-well plates or 384-well plates can be used to further minimize use of samples/reagents.

High throughput validation of multiple candidate biomarkers can be performed by sequential ELISA in order to minimize freeze/thaw cycles and use of precious plasma samples. Here, we demonstrate how to sequentially perform ELISAs for six different validated plasma biomarkers1-3 of graft-versus-host disease (GVHD)4 on the same plasma sample.

Unbiased discovery proteomics strategies have  the potential to identify large numbers of novel biomarkers that can improve  diagnostic and prognostic ...

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

Cell-based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the prognosis of idiopathic scoliosis in asymptomatic and affected children. The assay consists of the evaluation of the functional status of Gi and Gs proteins in peripheral blood mononuclear cells (PBMCs) by cellular dielectric spectroscopy (CDS), using an automated CDS-based instrument, and the classification of children into three functional groups (FG1, FG2, FG3) with respect to the profile of imbalance between the degree of response to Gi and Gs proteins stimulation. The classification is further confirmed by the differential effect of osteopontin (OPN) on response to Gi stimulation among groups and the severe progression of disease is referenced by FG2. Approximately, a volume of 10 ml of blood is required to extract PBMCs by Ficoll-gradient and cells are then stored in liquid nitrogen. The adequate number of PBMCs to perform the assay is obtained after two days of cell culture. Essentially, cells are first incubated with phytohemmaglutinin (PHA). After 24 hr incubation, medium is replaced by a PHA-free culture medium for an additional 24 hr prior to cell seeding and OPN treatment. Cells are then spectroscopically screened for their responses to somatostatin and isoproterenol, which respectively activate Gi and Gs proteins through their cognate receptors. Both somatostatin and isoproterenol are simultaneously injected with an integrated fluidics system and the cells&#39; responses are monitored for 15 min. The assay can be performed with fresh or frozen PBMCs and the procedure is completed within 4 days.

A structured protocol is presented for a cell-based assay as a functional test to predict the prognosis of idiopathic scoliosis using cellular dielectric spectroscopy (CDS). The assay can be performed with fresh or frozen peripheral blood mononuclear cells (PBMCs) and the procedure is completed within 4 days.

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

Fat-Water Phantoms for Magnetic Resonance Imaging Validation: A Flexible and Scalable Protocol

As new techniques are developed to image adipose tissue, methods to validate such protocols are becoming increasingly important. Phantoms, experimental replicas of a tissue or organ of interest, provide a low cost, flexible solution. However, without access to expensive and specialized equipment, constructing stable phantoms with high fat fractions (e.g., &#62;50% fat fraction levels such as those seen in brown adipose tissue) can be difficult due to the hydrophobic nature of lipids. This work presents a detailed, low cost protocol for creating 5x 100 mL&#160;phantoms with fat fractions of 0%, 25%, 50%, 75%, and 100% using basic lab supplies (hotplate, beakers, etc.) and easily accessible components (distilled water, agar, water-soluble surfactant, sodium benzoate, gadolinium-diethylenetriaminepentacetate (DTPA) contrast agent, peanut oil, and oil-soluble surfactant). The protocol was designed to be flexible; it can be used to create phantoms with different fat fractions and a wide range of volumes. Phantoms created with this technique were evaluated in the feasibility study that compared the fat fraction values from fat-water magnetic resonance imaging to the target values in the constructed phantoms. This study yielded a concordance correlation coefficient of 0.998 (95% confidence interval: 0.972-1.00). In summary, these studies demonstrate the utility of&#160;fat phantoms for validating adipose tissue imaging techniques across a range of clinically relevant tissues and organs.

The purpose of this work is to describe a protocol for creating a practical fat-water phantom that can be customized to produce phantoms with varying fat percentages and volumes.

As new techniques are developed to image adipose tissue, methods to validate such protocols are becoming increasingly important. Phantoms, experimental ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

Virtual Prism Adaptation Therapy: Protocol for Validation in Healthy Adults

Hemispatial neglect is a common impairment after stroke. It is associated with poor functional and social outcomes. Therefore, an adequate intervention is imperative for the successful management of hemispatial neglect. However, the clinical use of various interventions is limited in real clinical practice. Prism adaptation therapy is one of the most evidence-based rehabilitation modalities to treat hemispatial neglect. To overcome any possible shortcoming that may occur with prism therapy, we developed a new system using immersive virtual reality and depth-sensing camera to create a virtual prism adaptation therapy (VPAT). To validate the VPAT system, we designed an experimental protocol investigating the behavioral errors and changes in cortical activation via the VPAT system. Cortical activation was measured by functional near infrared spectroscopy (fNIRS). The experiment consisted of four phases. All four included clicking, pointing or rest applied to right-handed healthy people. Clicking versus pointing was used for investigating the cortical region related with the gross motor task, and pointing with VPAT versus pointing without VPAT was used for investigating the cortical region associated with visuospatial perception. The preliminary results from four healthy participants showed that pointing errors by the VPAT system was similar to the conventional prism adaptation therapy. Further analysis with more participants and fNIRS data, as well as a study in patients with stroke may be required.

This experimental protocol demonstrates the use of virtual prism adaptation therapy (VPAT) in healthy adults and the association between VPAT and functional near infrared spectroscopy to determine the effect of VPAT on cortical activation. Results suggest that VPAT may be feasible and could induce similar behavioral adaptation as conventional prism adaptation therapy.

Hemispatial neglect is a common impairment after stroke. It is associated with poor functional and social outcomes. Therefore, an adequate intervention is ...

Protein Target Prediction and Validation of Small Molecule Compound

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High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Cell-based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Fat-Water Phantoms for Magnetic Resonance Imaging Validation: A Flexible and Scalable Protocol

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Virtual Prism Adaptation Therapy: Protocol for Validation in Healthy Adults