The authors thank the &#34;Vicerrector&#237;a de Investigaci&#243;n y Extensi&#243;n VIE&#34; of the Universidad Industrial de Santander for financing the grant number 3739 corresponding to the project &#34;Formulation of an ixodicide and ixorepellent designed in a laboratory and validated in the field to prevent tick infestation in cattle.&#34;

All methods described in this protocol have been approved by the Ethics Committee &#34;Comit&#233; de &#233;tica en investigaci&#243;n cient&#237;fica CEINCI&#34;: Scientific Research Ethics Committee of the Industrial University of Santander. Furthermore, these assays complied with the Arrive guidelines and followed the 1989 Colombian Law 84 (Chapter IV, Art. 23-26) and Resolution 8430 (1993, Title IV, Art. 83-93) that regulates animal research in Colombia.
NOTE: It is strongly recommended to read the oxidative phosphorylation chapter of any biochemistry book before starting this protocol.
1. Reagents and solutions
<ol>
	<li>To isolate rat liver mitochondria, prepare 250 mL of mitochondria isolation buffer: 250 mM D-mannitol, 10 mM HEPES pH 7.2, and 1 mM EGTA. Take out 50 mL of isolation buffer (this will be called isolation buffer without BSA) and then add 0.2 g of BSA to the other 200 mL (this will be called isolation buffer with BSA).</li>
	<li>To homogenize Aedes aegypti larvae, prepare 100 mL of buffer: 250 mM sucrose, 10 mM HEPES, 1 mM EGTA pH 7.2.</li>
	<li>Prepare reaction buffer: 80 mM Sodium Phosphate Buffer solution, pH 7.4, 50 &#181;M EDTA.</li>
	<li>Prepare a stock of reagents used for enzymatic activities at pH 7.2 as described below: 25 mM NADH (freshly prepared for each assay), 2 M sodium succinate, 4 mM cytochrome c (freshly prepared for each assay), 200 &#181;M rotenone, 0.5 mM KCN, 6 mM 2,6-dichlorophenolindophenol (DCPIP), 100 mM phenazine methosulfate (PMS), and 60 mM potassium ferricyanide. 
	NOTE: Ultrapure water with a resistivity of 18.2 M&#937;&#183;cm at 25 &#176;C was used for all experiments and reagents preparation. This water was generated using a Milli-Q purification system, ensuring the removal of organic, inorganic, and microbial contaminants to levels below detectable limits, making it suitable for high-sensitivity biochemical and molecular biology applications.</li>
</ol>
2. Liver dissection, homogenization, and mitochondria isolation from rat liver
<ol>
	<li>Before starting, place all materials and buffers on ice, ensuring all materials are ice-cold during the entire assay to avoid mitochondrial damage.</li>
	<li>Place 10 mL of mitochondria isolation buffer in an ice-cold conic tube.</li>
	<li>Euthanize the male Wistar rat by cervical decapitation, avoiding CO2 use that can interfere with respiration assays. For this, the Committee on Ethics in Scientific Research of the Industrial University of Santander (CEINCI) approved the methods on resolution 13 of 2022. 
	NOTE: Rats should weigh no more than 170 g; fat can also affect respiration analyses.</li>
	<li>Make an incision (approx 4-5 cm) with a sterile scalpel through the abdominal rat section.</li>
	<li>Dissect the liver completely once the abdominal area is visible. The rat liver is a multilobulated organ, dark red approx 10 cm x 5 cm.</li>
	<li>Carefully put the liver within the ice-cold isolation buffer and rinse twice with this medium.</li>
	<li>Finely mince the liver (size less than 1 cm) with sharp scissors in a Petri dish with 10 mL of isolation medium.</li>
	<li>Put the solution into the Potter Elvehjem tissue homogenizer tube and carefully homogenize at least ten times, low velocity (~390 rpm).</li>
	<li>Transfer the solution into a conical tube and centrifuge at 600 x g for 10 min at 4 &#176;C.</li>
	<li>After centrifugation, carefully pour the supernatant into a new conical tube. Discard the pellet with part of the tissue, blood, and intact cellular components.</li>
	<li>Fill the tube with more isolation buffer (~35 mL) and centrifuge at 6000 x g for 10 min at 4 &#176;C.</li>
	<li>After centrifugation, discard the supernatant and carefully resuspend the pellet (mitochondria) in a new isolation buffer without BSA. 
	NOTE: For mitochondrial resuspension, it&#39;s recommended to use a soft brush.</li>
	<li>Then, centrifuge at 7000 x g for 10 min at 4 &#176;C, discard the supernatant and resuspend the isolated mitochondria in 2 mL of isolation buffer without BSA.</li>
	<li>Determine protein concentration using the Bradford method or similar14.</li>
</ol>
3. Aedes aegypti colony and larvae homogenization.
NOTE: For this procedure Ae. aegypti colony was kept inside breeding cages (40 cm &#215; 40 cm &#215; 40 cm) under insectary conditions like humidity (70% &#177; 5%), photoperiod (12:12), and temperature (27 &#177; 5 &#176;C). The eggs were collected on filter paper.
<ol>
	<li>Ensure that the eggs are hatched in trays with filtered and dechlorinated water one week before the experiment. Identify the instar by the molting process in the larvae cycle. Observe each molting process to verify its status L1, L2, and L3. When the larva enters L3, wait a maximum of 12 h to collect the material.</li>
	<li>Collect at least 100 Ae. aegypti larvae at instar L3-L4 in chlorine-free water (~30 mL of water). Take the larvae with a Pasteur pipette.</li>
	<li>Filter the larvae using filter paper, rinse the collected larvae, and resuspend them in the Aedes aegypti homogenization buffer (~2 mL).</li>
	<li>Transfer the solution with larvae into the Potter Elvehjem tissue homogenizer tube and carefully homogenize at least ten times at ~390 rpm.</li>
	<li>Transfer the homogenized tissue and filter it through a glass wool syringe to remove large exoskeleton residues.</li>
	<li>Determine the protein concentration by using the Bradford method or similar15. 
	NOTE: It is not necessary to isolate mitochondria from mosquito larvae; it is possible to evaluate the effect of xenobiotics using the protein extract obtained in this step.</li>
</ol>
4. Determination of xenobiotics effects on mitochondrial electron transport chain
NOTE: This protocol was designed to evaluate the effect of xenobiotics on mitochondrial bioenergetics on isolated mitochondria from rat liver and insect tissue homogenized.
The assays are divided into two: polarographic (NADH and Succinate oxidase) and spectrophotometric (NADH dehydrogenase - complex I, succinate dehydrogenase - complex II, cytochrome c reductase - complex III and cytochrome c oxidase - complex IV).
<ol>
	<li>Polarographic assays: For this, use high-resolution respirometry to measure the complete oxidation of NADH and succinate by O2 consumption, and report results as NADH and succinate oxidase.
	<ol>
		<li>Calibrate the instrument every day before starting assays. This step may vary depending on the oxygraph used. Follow the manufacturer&#39;s instructions.
		<ol>
			<li>For Oroboros calibration, add reaction medium into the chambers with an excess of at least 0.2 mL above the experimental chamber volume (2 mL), to fill the chamber and injection capillary with medium.</li>
		</ol>
		</li>
		<li>Switch on the stirrers after medium addition.</li>
		<li>Wait until the oxygen concentration signal stabilizes and record it.</li>
		<li>For zero oxygen calibration, add mitochondria, cell suspensions, baker&#39;s yeast, or dithionite (Baker&#39;s yeast is recommended) that allow the oxygen depletion, and record it. Wait for the signal to reach background levels (typically &#177; 2 pmol/mL/s for the O2k system).</li>
		<li>Perform NADH and succinate oxidase activities by monitoring the oxygen consumption when adding reaction buffer, 0.1 mg of protein (mitochondrial or larvae homogenized), and the substrate (0.17 mM NADH or 10 mM sodium succinate) to the chamber.</li>
		<li>Use electron transport protein inhibitors to confirm that the observed oxygen consumption is due to the mitochondrial respiratory chain. The common ones are rotenone 1 &#181;M and KCN 1mM16. In the oxygraph cell, add the reagents according to Table 1&#160;(the sequence is important).</li>
	</ol>
	</li>
	<li>Spectrophotometric assays 
	NOTE: To assess electron transport through the mitochondrial complexes, enzymatic assays were conducted and measured using a spectrophotometer and artificial electron acceptors such as PMS, DCPIP, and cytochrome c. It is recommended to measure one enzyme per day. Prepare buffers and reagents at least one day before the assays. Some compounds are photosensible; see Table 2 for more recommendations.
	<ol>
		<li>For NADH dehydrogenase activity, the reaction systems contain 50 mM phosphate buffer solution pH 7.4, 2 mM EDTA, 0.1 mg/mL mitochondrial protein, 1 &#181;M rotenone, and 1 mM KCN. In the spectrophotometer cell, add the reagents according to Table 2&#160;(the sequence is important)17. The recommended steps are as follows:
		<ol>
			<li>Prepare 100 mL of buffer phosphate mixed with the EDTA, and double-check that the pH does not change. Keep the buffer in the refrigerator, but it is mandatory to keep it at room temperature (RT; approximately 25 &#176;C) during assays.</li>
			<li>Prepare fresh stock solutions of rotenone, KCN, NADH, and potassium ferricyanide (stable until 4 h). The suggested volume is 1 mL of concentrated solutions (see the first column of the table).</li>
			<li>If the tested compound has low solubility in water, include an additional control for enzyme activity in the experimental design using just the solvent of the compound.</li>
			<li>First, monitor the NADH dehydrogenase activity by the reduction of potassium ferricyanide at 420 nm. Calculate its concentration by its molar extinction coefficient of 1.04 mM-1&#183;cm-1. The spectrophotometer has an option to calculate the maximum activity in a reaction; check the user manual. Subsequently, divide the reported activity by the concentration of protein used to obtain the specific activity of the enzyme.</li>
		</ol>
		</li>
		<li>Evaluate succinate dehydrogenase activity by adding 20 mM sodium succinate and 1 mM phenazine methosulphate (PMS) with 60 &#181;M 2,6-dichlorophenolindophenol (DCPIP) as artificial electron acceptors. In the spectrophotometer cell, add the reagents according to Table 3&#160;(the sequence is important)17. The recommendations are as follows:
		<ol>
			<li>Prepare the same buffer as used for NADH dehydrogenase. Maintain it in the same conditions.</li>
			<li>Prepare fresh stock solutions of Succinate, PMS, and DCPIP. The suggested volume is 1 mL of concentrated solutions. Prepare PMS and DCPIP solutions on the same day of the assay.</li>
			<li>If the tested compound has low solubility in water, include an additional control for enzyme activity in the experimental design.</li>
			<li>Monitor the succinate oxidation by the reduction of DCPIP at 600 nm. Calculate its concentration by its molar extinction coefficient of 21.0 mM&#8722;1&#183;cm&#8722;1, applying Beer&#39;s law. Subsequently, divide the reported activity by the concentration of protein used to obtain the specific activity of the enzyme.</li>
		</ol>
		</li>
		<li>Measure the Cytochrome c reductase (complex III) activity using either NADH or succinate as oxidizable substrates. With this enzyme, the electron transport from complex I (using NADH) or complex II (using succinate) passes to ubiquinone and then to complex III, which passes the electrons to cytochrome c. This reaction is followed by the reduction of cytochrome c. Prepare the reaction system containing 50 mM phosphate buffer solution pH 7.4, 2 mM EDTA, 0.1 mg of mitochondrial protein, 50 &#181;M NADH, and 0.1 mM KCN. In the spectrophotometer cell, add the reagents according to Table 4&#160;(the sequence is important)18. The recommendations are as follows:
		<ol>
			<li>Prepare the same buffer as used for NADH dehydrogenase. Maintain it in the same conditions.</li>
			<li>Use the same NADH and KCN from NADH dehydrogenase.</li>
			<li>If the tested compound has low solubility in water, include an additional control for enzyme activity in the experimental design.</li>
			<li>Monitor the cytochrome c reduction at 550 nm and calculate its concentration by its molar extinction coefficient of 19.8 mM&#8722;1&#183;cm&#8722;1, applying Beer&#39;s law. Subsequently, divide the reported activity by the concentration of protein used to obtain the specific activity of the enzyme.</li>
		</ol>
		</li>
		<li>Measure cytochrome c oxidase (complex IV) expressed as cytochrome c oxidase activity. For this, prepare a system reaction containing 50 mM phosphate buffer solution, pH 7.4, 2 mM EDTA, and 0.1 mg of mitochondrial protein (Table 5). For these enzymatic assays, reduce cytochrome c by mixing equimolar amounts of cytochrome c and sodium dithionite, ensuring complete reduction of the cytochrome c.</li>
		<li>Then, pass the resulting mixture through a cross-linked dextran gel column using the same phosphate-buffered solution as the mobile phase. Subsequently, quantify the collected fractions using a spectrophotometer at 550 nm19. The recommendations are as follows:
		<ol>
			<li>Use the same buffer used for NADH dehydrogenase. Maintain it in the same conditions.</li>
			<li>If the tested compound has low solubility in water, an additional control for enzyme activity should be included in the experimental design.</li>
			<li>Monitor the cytochrome c oxidation at 550 nm and calculate its concentration by its molar extinction coefficient of 19.8 mM&#8722;1&#183;cm&#8722;1, applying Beer&#39;s law. Subsequently, divide the reported activity by the concentration of protein used to obtain the specific activity of the enzyme. 
			NOTE: A spreadsheet file showing the process of calculating the specific activity of enzymes, measured by oxygen consumption and spectrophotometry, is included as Supplementary File 1.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

Changing Mitochondrial Function for Therapeutic and Bioinsecticide Applications

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+uncouplers+act+synergistically+with+the+fumigant+phosphine+to+disrupt+mitochondrial+membrane+potential+and+cause+cell+death.">Mitochondrial uncouplers act synergistically with the fumigant phosphine to disrupt mitochondrial membrane potential and cause cell death.</a> Toxicology. 252 (1-3), 33-39 (2008).</li><li>Sloci&#324;ska, M., Rosinski, G., Jarmuszkiewicz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+mitochondrial+uncoupling+protein+4+and+ATP-sensitive+potassium+channel+cumulatively+decreases+superoxide+production+in+insect+mitochondria.">Activation of mitochondrial uncoupling protein 4 and ATP-sensitive potassium channel cumulatively decreases superoxide production in insect mitochondria.</a> Protein Pept Lett. 23 (1), 63-68 (2016).</li><li>Borrero Landazabal, M. A., Carre&#241;o Otero, A. L., Kouznetsov, V. V., Duque Luna, J. E., Mendez-Sanchez, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+of+mitochondrial+electron+transport+chain+and+oxidative+stress+induced+by+alkaloid-like+&#945;-aminonitriles+on+Aedes+aegypti+larvae.">Alterations of mitochondrial electron transport chain and oxidative stress induced by alkaloid-like &#945;-aminonitriles on Aedes aegypti larvae.</a> Pestic Biochem Physiol. 144, 64-70 (2018).</li><li>Castillo-Morales, R. M., Carre&#241;o Otero, A. L., Mendez-Sanchez, S. C., Da Silva, M. A. N., Stashenko, E. E., Duque, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+affectation,+DNA+damage+and+AChE+inhibition+induced+by+Salvia+officinalis+essential+oil+on+Aedes+aegypti+larvae.">Mitochondrial affectation, DNA damage and AChE inhibition induced by Salvia officinalis essential oil on Aedes aegypti larvae.</a> Comp Biochem Physiol C Toxicol Pharmacol. 221, 29-37 (2019).</li><li>Montagut, D. C., Bueno, Y., Vesga, L. C., Stashenko, E. E., Mendez-Sanchez, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cymbopogon+flexuosus+(nees+ex+steud.)+w.+watson+essential+oil+effect+on+mitochondrial+bioenergetics.">Cymbopogon flexuosus (nees ex steud.) w. watson essential oil effect on mitochondrial bioenergetics.</a> J Essent Oil Res. 34 (3), 233-239 (2022).</li></ol>

All authors declare that they have no conflicts of interest.

A spectrophotometric and polarographic method with high-resolution O2 was employed to investigate the effects of xenobiotics on mitochondrial function. The study specifically assessed the impact of Cymbopogon flexuosus essential oil and carvacrol on NADH and succinate oxidation, as well as electron transport through the mitochondrial electron transport chain, using two different biological models: isolated mitochondria from rat liver and homogenized Aedes aegypti larvae. Additionally, potenti...

Mitochondria have always been called the power plant of cells due to their chemical energy generation during the oxidative phosphorylation process1,2. Beyond their main core metabolic functions, mitochondria are also implicated in many other cellular processes, ranging from intracellular Ca2+ regulation levels, inflammatory processes, and reactive oxygen species production to cell death induction processes3. Mitochondrial bioenergetics and their dysregulation have been associated with intracellular processes that affect cellular function and overall health. This has sparked a growing interest in researching the pharmaceutical potential of targeting mitochondrial function to develop innovative therapies and safe bioproducts4,5,6. Additionally, understanding the intricate interplay of mitochondrial processes and their impact on cellular function is crucial for developing targeted therapies and presenting opportunities for designing bioproducts to control vector-borne diseases and pest infestations7,8.
Targeting specific components of the electron transport chain with xenobiotics has emerged as a strategy to modulate mitochondrial function for biopesticide generation. Natural and synthetic compounds included within pesticide formulations, such as nanoparticle-based mitochondria-targeted pesticides, have been shown to affect mitochondrial function by decreasing mitochondrial potential and ATP synthesis in S. frugiperda9.
Additionally, most of the carbamates and pyrethroid-based products have been reported as complex I inhibitors in mosquitoes; however, their extended exposure has created some pyrethroid-resistant populations in Anopheles gambiae, often related to elevated levels of P450s that can metabolize these substrates10,11. Similarly, plants and their essential oils, extracts, and secondary metabolites have been documented as potential insecticides having a wide range of mortality in different insect stages12,13. The insecticide activity of these natural compounds may be related to the electron transport inhibition in a specific point in the mitochondrial respiratory chain by modulating the reactive oxygen species production, mitochondrial potential &#916;&#936;m, and ATP synthesis that can trigger cell death processes6,8.
Natural compounds, as sources for pesticide formulation, and their effects on mitochondria play a crucial role in the design of new and safe biopesticides. In this context, this manuscript describes a methodology to identify the capacity of xenobiotics to activate or inhibit electron transport through mitochondrial complexes. This comprehensive approach allows precise measurement of xenobiotics&#39; effects on mitochondrial function using a variety of substrates, employing both respirometry and spectrophotometry methods. This methodology has been applied to analyze the mitochondrial function of key vector species, including Aedes aegypti, leading to theorizing the presence of uncoupler proteins and distinct oxidizable substrate preferences specific to each species.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2,6-dichlorophenolindophenol</td>
			<td>Merck</td>
			<td>119814</td>
			<td>Prepare on the same day of use.</td>
		</tr>
		<tr>
			<td>Bradford reagent</td>
			<td>Merck</td>
			<td>B6916</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Merck</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>Carvacrol</td>
			<td>Merck</td>
			<td>W224511</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytochrome c</td>
			<td>Merck</td>
			<td>C2506</td>
			<td>Photosensitive conver cover it with aluminum foil. In solution it should be stored in the freezer.</td>
		</tr>
		<tr>
			<td>D-mannitol</td>
			<td>Merck</td>
			<td>M4125</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Merck</td>
			<td>&#62;E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid tetrasodium salt dihydrate</td>
			<td>Merck</td>
			<td>E6511</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Merck</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>KCN</td>
			<td>Merck</td>
			<td>60178</td>
			<td>In solution works weel for 2 weeks.</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Merck</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiskan FC Microplate Photometer</td>
			<td>Thermo Scientific&#160;</td>
			<td>51119100</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Merck</td>
			<td>567550</td>
			<td></td>
		</tr>
		<tr>
			<td>Oroboros O2k&#160;</td>
			<td>Oroboros Instruments</td>
			<td>10203-03</td>
			<td>https://www.oroboros.at/index.php/product/o2k-startup/</td>
		</tr>
		<tr>
			<td>Phenazine methosulphate&#160;</td>
			<td>Merck</td>
			<td>P9625</td>
			<td>Prepare on the same day of use.</td>
		</tr>
		<tr>
			<td>Potassium ferricyanide&#160;</td>
			<td>Merck</td>
			<td>244023</td>
			<td>Prepare on the same day of use.</td>
		</tr>
		<tr>
			<td>Potter Elvehjem tissue homogenizer tube</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Merck</td>
			<td>557368</td>
			<td>Soluble in ethanol. Photosensitive conver cover it with aluminum foil. In solution it should be stored in the freezer.</td>
		</tr>
		<tr>
			<td>Sodium succinate</td>
			<td>Merck</td>
			<td>S9637</td>
			<td>In solution it should be stored in the freezer.</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Merck</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Nicotinamide adenine dinucleotide, reduced Disodium salt hydrate</td>
			<td>Merck</td>
			<td>N8129</td>
			<td>Photosensitive conver cover it with aluminum foil. In solution it should be stored in the freezer.</td>
		</tr>
	</tbody>
</table>

unveiling xenobiotic transport and effects in isolated mitochondria: insights from respirometric and enzymatic assays

Mitochondria are often referred to as the cell's powerhouse due to their crucial role in energy production through the electron transport chain (ETC). However, their significance extends far beyond energy production. Dysregulation of mitochondrial bioenergetics can trigger intracellular cascades that impact health and cellular function. Given their physiological importance, there is growing interest in exploring the pharmacological potential of mitochondrial function for developing new therapies and bioproducts. Modulating mitochondrial bioenergetics could provide innovative approaches to address challenges such as neurodegenerative disorders, metabolic diseases, and cancer, as well as to develop bioproducts for controlling pests and vector-borne diseases.
This work presents a method for assessing the effects of xenobiotics on the electron transport chain (ETC) using various substrates, including glutamate and NADH for complex I, succinate for complex II, and cytochrome c (both oxidized and reduced) for complexes III and IV. This methodology allows the activation or inhibition of electron transport through mitochondrial complexes to be evaluated using a respirometer and spectrophotometer. The mitochondria can be sourced from isolated mitochondria, fragmented cells, or homogenized tissue from various species.
In the laboratory, mitochondrial function has been analyzed in Aedes aegypti and Wistar rats, but this method is also applicable to other species, such as Rhipicephalus microplus and Rhodnius prolixus. This approach provides the basis for theorizing about the existence of uncoupler proteins and species-specific oxidizable substrate preferences influenced by their unique energetic demands. Recent findings offer valuable insights into innovative bioinsecticide design strategies that target mitochondrial function, holding significant potential for effectively controlling vector-borne diseases and pest infestations.

Before analyzing the results obtained with this protocol, it is essential to remember a little about the biochemistry of the electron transport chain in the mitochondria. First, NADH supplies a pair of electrons to the oxidoreduction centers located in complex I. There, it begins the transport of electrons that pass from complex I to ubiquinone, subsequently to complex III, which donates them to cytochrome c, and finally to complex IV, where oxygen is the final electron acceptor, reducing it to water. On the other hand, ...

Unveiling Xenobiotic Transport and Effects in Isolated Mitochondria: Insights from Respirometric and Enzymatic Assays

This study introduces a methodology for studying xenobiotic effects on mitochondrial function via respirometric and enzymatic assays. It examines electron transport chain modulation in various species, offering insights for targeted biopesticide development against insect vector-borne diseases.

Mitochondria are often referred to as the cell's powerhouse due to their crucial role in energy production through the electron transport chain (ETC). ...

unveiling-xenobiotic-transport-effects-isolated-mitochondria-insights

Research

JoVE Journal

Biochemistry

436 Views.  Universidad Industrial de Santander. Onze onderzoeksgroep gebruikt mitochondriën als farmacologisch doelwit voor de ontwikkeling van verschillende producten zoals chemotheraputes, insecticiden, onder andere. Met deze video willen we de methodologie beschrijven om enzymen van de mitochondriale ademhaling op een meer toegankelijke, begrijpelijke en bruikbare manier te evalueren. In ons laboratorium hebben we een protocol opgesteld om de verschillende effecten van xenobiotica op mitochondriën bij...

Video: Onthulling van xenobiotisch transport en effecten in geïsoleerde mitochondriën: inzichten uit respirometrische en enzymatische tests

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the cell, ATP. The ETC is composed of 5 massive protein complexes, which also assemble into supercomplexes called respirasomes (C-I, C-III, and C-IV) and synthasomes (C-V) that increase the efficiency of electron transport and ATP production. Various methods have been used for over 50 years to measure ETC function, but these protocols do not provide information on the assembly of individual complexes and supercomplexes. This protocol describes the technique of native gel polyacrylamide gel electrophoresis (PAGE), a method that was modified more than 20 years ago to study ETC complex structure. Native electrophoresis permits the separation of ETC complexes into their active forms, and these complexes can then be studied using immunoblotting, in-gel assays (IGA), and purification by electroelution. By combining the results of native gel PAGE with those of other mitochondrial assays, it is possible to obtain a completer picture of ETC activity, its dynamic assembly and disassembly, and how this regulates mitochondrial structure and function. This work will also discuss limitations of these techniques. In summary, the technique of native PAGE, followed by immunoblotting, IGA, and electroelution, presented below, is a powerful way to investigate the functionality and composition of mitochondrial ETC supercomplexes.

This protocol describes the separation of functional mitochondrial electron transport chain complexes (Cx) I-V and supercomplexes thereof using native electrophoresis to reveal information about their assembly and structure. The native gel can be subjected to immunoblotting, in-gel assays, and purification by electroelution to further characterize individual complexes.

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the ...

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for skin-care cosmetics. The goals of the protocol are: (1) to process chicken feathers into KH by alkaline-enzymatic hydrolysis and purify it by dialysis, and (2) to test if adding KH into an ointment base (OB) increases hydration of the skin and improves skin barrier function by diminishing transepidermal water loss (TEWL). During alkaline-enzymatic hydrolysis feathers are first incubated at a higher temperature in an alkaline environment and then, under mild conditions, hydrolyzed with proteolytic enzyme. The solution of KH is dialyzed, vacuum dried, and milled to a fine powder. Cosmetic formulations comprising from oil in water emulsion (O/W) containing 2, 4, and 6 weight% of KH (based on the weight of the OB) are prepared. Testing the moisturizing properties of KH is carried out on 10 men and 10 women at time intervals of 1, 2, 3, 4, 24, and 48 h. Tested formulations are spread at degreased volar forearm sites. The skin hydration of stratum corneum (SC) is assessed by measuring capacitance of the skin, which is one of the most world-wide used and simple methods. TEWL is based on measuring the quantity of water transported per a defined area and period of time from the skin. Both methods are fully non-invasive. KH makes for an excellent occlusive; depending on the addition of KH into OB, it brings about a 30% reduction in TEWL after application. KH also functions as a humectant, as it binds water from the lower layers of the epidermis to the SC; at the optimum KH addition in the OB, up to 19% rise in hydration in men and 22% rise in women occurs.

The goal of the protocol is to prepare keratin hydrolysate from chicken feathers by alkaline-enzymatic hydrolysis and to test whether adding keratin hydrolysate into a cosmetics ointment base improves skin barrier function (heightening hydration and diminishing transepidermal water loss). Tests are conducted on men and woman volunteers.

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for ...

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca2+ from mitochondria is important for determining the role of mitochondrial Ca2+ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca2+ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca2+ uptake using the Ca2+ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca2+ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca2+ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca2+ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca2+ in permeabilized cells using the Ca2+ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca2+. Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca2+ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca2+ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

Here, we present two protocols for the measurement of mitochondrial Ca2+ influx in isolated mitochondria and cultured cells. For isolated mitochondria, we detail a plate reader-based Ca2+ import assay using the Ca2+ sensitive dye calcium green-5N. For cultured cells, we describe a confocal microscopy method using the Ca2+ dye Rhod-2/AM.

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis&#8212;in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before&#8212;and provide some representative results.

Here we present a protocol to perform polysome profiling on the isolated perfused mouse heart. We describe methods for heart perfusion, polysome profiling, and analysis of the polysome fractions with respect to mRNAs, miRNAs, and the polysome proteome.

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

Caffeine Extraction, Enzymatic Activity and Gene Expression of Caffeine Synthase from Plant Cell Suspensions

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a chemical defense because it has antimicrobial activity and is considered a natural insecticide. Caffeine can also produce negative allelopathic effects that prevent the growth of surrounding plants. In addition, people around the world consume caffeine for its analgesic and stimulatory effects. Due to interest in the technological applications of caffeine, research on the biosynthetic pathway of this compound has grown. These studies have primarily focused on understanding the biochemical and molecular mechanisms that regulate the biosynthesis of caffeine. In vitro tissue culture has become a useful system for studying this biosynthetic pathway. This article will describe a step-by-step protocol for the quantification of caffeine and for measuring the transcript levels of the gene (CCS1) encoding caffeine synthase (CS) in cell suspensions of C. arabica L. as well as its activity.

This protocol describes an efficient methodology for the extraction and quantification of caffeine in cell suspensions of C. arabica L. and an experimental process for evaluating the enzymatic activity of caffeine synthase with the expression level of the gene that encodes this enzyme.

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a ...

Isolation of Physiologically Active Thylakoids and Their Use in Energy-Dependent Protein Transport Assays

Chloroplasts are the organelles in green plants responsible for carrying out numerous essential metabolic pathways, most notably photosynthesis. Within the chloroplasts, the thylakoid membrane system houses all the photosynthetic pigments, reaction center complexes, and most of the electron carriers, and is responsible for light-dependent ATP synthesis. Over 90% of chloroplast proteins are encoded in the nucleus, translated in the cytosol, and subsequently imported into the chloroplast. Further protein transport into or across the thylakoid membrane utilizes one of four translocation pathways. Here, we describe a high-yield method for isolation of transport-competent thylakoids from peas (Pisum sativum), along with transport assays through the three energy-dependent cpTat, cpSec1, and cpSRP-mediated pathways. These methods enable experiments relating to thylakoid protein localization, transport energetics, and the mechanisms of protein translocation across biological membranes.

We present protocols herein for high-yield isolation of physiologically active thylakoids and protein transport assays for the chloroplast twin arginine translocation (cpTat), secretory (cpSec1), and signal recognition particle (cpSRP) pathways.

Chloroplasts are the organelles in green plants responsible for carrying out numerous essential metabolic pathways, most notably photosynthesis. Within ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination of chromatin function and DNA-associated processes. This modification involves a sequential action of several enzymes including E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligase and is reversed by deubiquitinases (DUBs). Ubiquitination induces degradation of proteins or alteration of protein function including modulation of enzymatic activity, protein-protein interaction and subcellular localization. A critical step in demonstrating protein ubiquitination or deubiquitination is to perform in vitro reactions with purified components. Effective ubiquitination and deubiquitination reactions could be greatly impacted by the different components used, enzyme co-factors, buffer conditions, and the nature of the substrate.&#160; Here, we provide step-by-step protocols for conducting ubiquitination and deubiquitination reactions. We illustrate these reactions using minimal components of the mouse Polycomb Repressive Complex 1 (PRC1), BMI1, and RING1B, an E3 ubiquitin ligase that monoubiquitinates histone H2A on lysine 119. Deubiquitination of nucleosomal H2A is performed using a minimal Polycomb Repressive Deubiquitinase (PR-DUB) complex formed by the human deubiquitinase BAP1 and the DEUBiquitinase ADaptor (DEUBAD) domain of its co-factor ASXL2. These ubiquitination/deubiquitination assays can be conducted in the context of either recombinant nucleosomes reconstituted with bacteria-purified proteins or native nucleosomes purified from mammalian cells. We highlight the intricacies that can have a significant impact on these reactions and we propose that the general principles of these protocols can be swiftly adapted to other E3 ubiquitin ligases and deubiquitinases.

Ubiquitination is a post-translational modification that plays important roles in cellular processes and is tightly coordinated by deubiquitination. Defects in both reactions underlie human pathologies. We provide protocols for conducting ubiquitination and deubiquitination reaction in vitro using purified components.

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination ...

Mitochondria and Endoplasmic Reticulum Imaging by Correlative Light and Volume Electron Microscopy

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved structures are folded into various shapes to form a dynamic network depending on the cellular conditions. Visualization of this network between mitochondria and ER has been attempted using super-resolution fluorescence imaging and light microscopy; however, the limited resolution is insufficient to observe the membranes between the mitochondria and ER in detail. Transmission electron microscopy provides good membrane contrast and nanometer-scale resolution for the observation of cellular organelles; however, it is exceptionally time-consuming when assessing the three-dimensional (3D) structure of highly curved organelles. Therefore, we observed the morphology of mitochondria and ER via correlative light-electron microscopy (CLEM) and volume electron microscopy techniques using enhanced ascorbate peroxidase 2 and horseradish peroxidase staining. An en bloc staining method, ultrathin serial sectioning (array tomography), and volume electron microscopy were applied to observe the 3D structure. In this protocol, we suggest a combination of CLEM and 3D electron microscopy to perform detailed structural studies of mitochondria and ER.

We present a protocol to study the distribution of mitochondria and endoplasmic reticulum in whole cells after genetic modification using correlative light and volume electron microscopy including ascorbate peroxidase 2 and horseradish peroxidase staining, serial sectioning of cells with and without the target gene in the same section, and serial imaging via electron microscopy.

Unveiling Xenobiotic Transport and Effects in Isolated Mitochondria: Insights from Respirometric and Enzymatic Assays

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