The authors thank Dara Frank and Carrie Harwood for their generous contribution of strains. University of Wisconsin Milwaukee Research Foundation holds patent no. 8,354,451; with claims broadly directed to treating or inhibiting the progression of infection of a microorganism in a patient by administering a 4-hydroxyphenylpyruvate dioxygenase-inhibiting compound such as 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). Inventors are Graham Moran and Pang He. This research was supported by the National Institutes of Health (R00-GM083147). The University of Washington P. aeruginosa transposon mutant library is supported by NIH P30 DK089507.

1. Preparation of Culture Media, Antibiotics, and 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC)
<ol>
	<li>Make LB broth (1% tryptone, 0.5% yeast extract, 0.25% NaCl in H2O) and aliquot into appropriate volumes. Sterilize by autoclave. Store at room temperature.</li>
	<li>Make 100 ml LB agar (1% tryptone, 0.5% yeast extract, 0.25% NaCl, 1.5% agar in H2O) in 250 ml flasks. Sterilize by autoclave and store at room temperature. Ensure that the agar is melted before pouring into plates. 
	NOTE: Flasks containing 100 ml of LB agar will yield 4 plates. The amount of LB agar can be altered to correspond to the number of plates needed for the assay.</li>
	<li>Make PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4)16. Sterilize by autoclave or filtration and store at room temperature.</li>
	<li>Prepare the antibiotic stock solutions for gentamicin, kanamycin, and tobramycin.
	<ol>
		<li>Prepare appropriate antibiotic stock concentrations for P. aeruginosa strains containing 100 mg/ml gentamicin, 30 mg/ml kanamycin, and 10 mg/ml tobramycin. Dissolve the antibiotics in water, filter sterilize (0.2 &#181;m), and store at 4 &#176;C. Alter the antibiotics and concentrations depending on the bacterium studied.</li>
	</ol>
	</li>
	<li>Prepare the NTBC stock solutions. Dissolve 10 mg of NTBC in 400 &#181;l of DMSO. This yields a concentration of 75.9 mM NTBC. Store NTBC stock solutions at -20 &#176;C. Thaw solutions at room temperature as needed. 
	NOTE: Different sources of NTBC have differences in solubility. Determine the appropriate vehicle in which to dissolve the NTBC based on the manufacturer&#8217;s recommendations and adjust Step 1.5 accordingly.</li>
</ol>
2. NTBC Titrations of Bacterial Strains
<ol>
	<li>Set up overnight cultures of the strains to be tested. Add 2 ml LB broth to 16 x 150 mm test tubes (one per strain) and inoculate with 1 isolated colony from each strain. Incubate overnight at 37 &#176;C with aeration on a tissue culture rotator in an air incubator.</li>
	<li>The following day, prepare titrations of NTBC in LB broth. Use an initial range from 0 to 900 &#181;M NTBC since different strains have differences in sensitivity to NTBC.
	<ol>
		<li>Add 1 ml LB broth to 4 to 5 test tubes (16 x 150 mm) per strain.</li>
		<li>Add the NTBC stock solution (75.9 mM) to the test tubes (16 x 150 mm) in a range of concentrations. See Table 1 for NTBC concentrations and corresponding stock volumes to add to 1 ml of LB broth.</li>
	</ol>
	</li>
	<li>Measure the OD600 of the overnight cultures. Wash cultures before taking OD600 readings to eliminate pyomelanin present in the media.
	<ol>
		<li>Wash the cultures by centrifuging 1 ml of culture in a microcentrifuge at 16,000 x g for 2 min. Remove the supernatant and any loosely pelleted cells with a micropipettor and resuspend the solid cell pellet in 1 ml LB.</li>
	</ol>
	</li>
	<li>Inoculate titration tubes at OD600 0.05. Calculate the amount of washed culture needed to inoculate the tubes. 
	NOTE: Use the washed cultures for inoculations since pyomelanin should not be present.</li>
	<li>Incubate the titration tubes for approximately 24 hr&#160;at 37 &#176;C with aeration using a tissue culture rotator in an air incubator.</li>
	<li>Photograph the titration tubes and compare pigment production within and between strains to determine the amount of NTBC to use for MIC and oxidative stress assays. Use OD600 readings to determine the amount of pyomelanin in cell free culture supernatant and to determine cell density. 
	NOTE: The OD600 ratio of pyomelanin in culture supernatant to cells can be calculated to quantify differences in pyomelanin production after treatment with NTBC.</li>
</ol>
3. Antibiotic Minimum Inhibitory Concentration (MIC) Assay in 96-well Plates
<ol>
	<li>Set up overnight cultures of the strains to be tested in LB with and without NTBC. 
	NOTE: This protocol is described using the representative level of 300 &#181;M NTBC. The appropriate level of NTBC to be used is determined in Step 2.6.
	<ol>
		<li>Add 300 &#181;M NTBC to 2 ml LB. Add an equivalent volume of vehicle (DMSO) to 2 ml LB for the no NTBC condition.</li>
		<li>Using sterile toothpicks, inoculate tubes with one isolated colony of bacteria. There will be one culture with NTBC and one culture without NTBC for each strain. Incubate overnight at 37 &#176;C with aeration using a tissue culture rotator in an air incubator.</li>
	</ol>
	</li>
	<li>The following day, make LB + NTBC and LB + DMSO master solutions for setting up the MIC assay. Add NTBC at a concentration of 600 &#181;M as this will be diluted two fold when inoculum is added, yielding a final concentration of 300 &#181;M.
	<ol>
		<li>To test one antibiotic for one strain, add 600 &#181;M NTBC to 2 ml LB and mix to make the NTBC master solution. Add an equivalent volume of vehicle (DMSO) to 2 ml LB and mix to make the no NTBC master solution. Use these solutions for creating antibiotic stock solutions as well as for setting up the dilution series in 96-well plates. 
		NOTE: The master solution formulations will yield extra solution to account for pipetting errors. The master solutions can be scaled up or down as required depending on the number of antibiotics and strains tested.</li>
	</ol>
	</li>
	<li>Prepare the antibiotic solutions in the LB + NTBC or LB + DMSO master solutions. 
	NOTE: The antibiotic concentration in these solutions should be double the final desired concentration. Enough solution should be made to transfer 100 &#181;l to four wells in a 96-well plate.
	<ol>
		<li>Prepare gentamicin +/- NTBC stock solution at 64 &#181;g/ml. To make this solution, add 0.288 &#181;l of gentamicin stock (100 mg/ml) to 450 &#181;l LB + NTBC or LB + DMSO master solution. 
		NOTE: The maximum concentration of gentamicin for P. aeruginosa PAO1 is 32 &#181;g/ml.</li>
		<li>Make the kanamycin +/- NTBC stock solution at 256 &#181;g/ml. To make this solution, add 3.84 &#181;l of kanamycin stock (30 mg/ml) to 450 &#181;l LB + NTBC or LB + DMSO master solutions. 
		NOTE: The maximum concentration of kanamycin for P. aeruginosa PAO1 is 128 &#181;g/ml.</li>
		<li>Prepare tobramycin +/- NTBC stock solution at 8 &#181;g/ml. To make this solution, add 0.36 &#181;l of tobramycin stock (10 mg/ml) to 450 &#181;l LB + NTBC or LB + DMSO master solutions. 
		NOTE: For P. aeruginosa PAO1, the maximum concentration of tobramycin is 4 &#181;g/ml. 
		NOTE: The antibiotics and concentrations can be adjusted for the bacteria to be tested.</li>
	</ol>
	</li>
	<li>Add 100 &#181;l of each 2x antibiotic solution to four wells in a 96-well plate. Place these solutions in row A. For example, gentamicin should be placed in A1 through A4, kanamycin should be placed in A5 through A8, and tobramycin should be placed in A9 through A12. See Figure 1A for a diagram of a 96-well plate set up. 
	NOTE: Multiple antibiotics can be tested in one plate, but only one strain should be tested per plate to eliminate the potential for cross-contamination from other strains.</li>
	<li>Add 50 &#181;l of the LB + NTBC or LB + DMSO master solution to rows B through H of the 96-well plate. Ensure that one plate is LB + NTBC and one plate is LB + DMSO. See Figure 1A.
	<ol>
		<li>Use LB + NTBC for the antibiotics in LB + NTBC. Use LB + DMSO for the antibiotics in LB + DMSO.</li>
	</ol>
	</li>
	<li>Using a micropipettor perform two fold serial dilutions of the antibiotics by transferring 50 &#181;l of the solution from row A to row B. Mix the solution, change the pipet tips, and transfer 50 &#181;l of the solution from row B to row C. Repeat for the remaining rows. After diluting row G, remove 50 &#181;l of the solution from that row and discard. Use row H as a no antibiotic control for bacterial growth. See Figure 1B. 
	NOTE: Each well in rows A through G now contains 50 &#181;l of antibiotic in LB + NTBC or LB + DMSO at 2X the final desired concentration. Row H contains LB + NTBC or LB + DMSO with no antibiotics.</li>
	<li>Measure the OD600 of the overnight cultures. Wash all cultures before taking OD600 readings to eliminate pyomelanin present in the media.
	<ol>
		<li>Wash the cultures by centrifuging 1 ml of culture in a microcentrifuge at 16,000 x g for 2 min. Remove the supernatant with a micropipettor and resuspend the cell pellet in 1 ml LB.</li>
	</ol>
	</li>
	<li>Dilute the overnight cultures to 2.75x105 CFU/ml in LB. 
	NOTE: Assume that one OD600 unit is the equivalent of 1x109 CFU/ml for P. aeruginosa. OD to CFU/ml conversions may be different in other bacteria.</li>
	<li>Add 50 &#181;l of the diluted bacterial culture to the appropriate well. 
	NOTE: Cultures grown in NTBC should be added to the wells containing NTBC and cultures grown in DMSO should be added to the wells containing DMSO. See Figure 1B.
	<ol>
		<li>Add bacteria to three wells for each strain and antibiotic concentration. Add 50 &#181;l of LB to the fourth well to act as a control for bacterial contamination. See Figure 1B.</li>
		<li>Use a multi-channel micropipettor to inoculate the wells. Ensure that pipet tips are near the bottom of the wells when adding inoculum to prevent contamination of neighboring wells. 
		NOTE: Adding bacterial culture to the wells will dilute the antibiotic and NTBC concentrations two fold.</li>
	</ol>
	</li>
	<li>Cover the 96-well plates with parafilm and incubate approximately 24 hr&#160;at 37 &#176;C. Incubate 96-well plates statically, in an air incubator.</li>
	<li>Examine the plates for bacterial growth in the wells. The MIC is the lowest concentration of antibiotic in which no bacterial growth is seen for all three replicates of each strain.
	<ol>
		<li>Visually examine the plate for growth or read using a plate reader set to OD600.</li>
	</ol>
	</li>
</ol>
4. Spot Plate Assay for Oxidative Stress Response
<ol>
	<li>Set up overnight cultures of the strains to be tested in LB with and without NTBC as described in step 3.1.</li>
	<li>The next day, prepare LB agar plates containing H2O2 as an oxidative stressor. A range of H2O2 concentrations from 0 to 1 mM is a good starting point.
	<ol>
		<li>Melt the LB agar flasks. Cool media to approximately 50 &#176;C at room temperature.</li>
		<li>Add H2O2 directly to the cooled media at the desired concentrations. Swirl flasks to mix. See Table 2 for concentrations of H2O2 and volumes of concentrated H2O2 to add. These values are based on 100 ml of LB agar.</li>
		<li>Pour plates immediately after adding H2O2 and flame the surface to remove bubbles. The yield is 4 plates per 100 ml of LB agar. Mark the plates with the H2O2 concentration.</li>
		<li>Place the plates uncovered in a biological flow hood with the fan running for 30 min to remove excess moisture from the plates. 
		NOTE: Use the plates the same day they are prepared. Failure to do so may result in inconsistent data. 
		NOTE: Oxidative stressors such as paraquat can be substituted for H2O2 in this assay. The concentrations used for other oxidative stressors may be different than those used for H2O2.</li>
	</ol>
	</li>
	<li>Wash and measure the OD600 of the overnight cultures as described in step 3.7.</li>
	<li>Normalize the OD600 of all the overnight cultures to the lowest value for the set of strains being tested. P. aeruginosa generally has an OD600 of approximately 2.5 when grown overnight in LB + NTBC or LB + DMSO.
	<ol>
		<li>Determine the volume of culture needed to dilute the culture to the lowest OD600 in a total volume of 1 ml. For example, if a culture has an OD600 of 3 and the lowest OD600 for the set of strains is 2.5, perform the following calculation: (2.5)(1 ml) = (3)(x). x = 0.833 ml. 0.833 ml of culture will be placed in a microfuge tube.</li>
		<li>Calculate the amount of LB + NTBC (300 &#181;M) or LB + DMSO needed to bring the culture volume to 1 ml. For the example in step 4.4.1, the amount of LB + NTBC or LB + DMSO added to the culture would be 0.167 ml (1 ml total volume &#8211; 0.833 ml culture). Make stock solutions of LB + NTBC and LB + DMSO to use for these dilutions based on the volume needed for diluting all strains.</li>
		<li>Mix the culture and LB + NTBC or LB + DMSO by vortexing.</li>
	</ol>
	</li>
	<li>To maintain cultures in a constant concentration of NTBC or DMSO, perform ten fold serial dilutions of the normalized overnight cultures in PBS + NTBC or PBS + DMSO.
	<ol>
		<li>Make stock solutions of PBS + NTBC and PBS + DMSO. For one set of dilutions for one strain, mix 300 &#181;M NTBC or an equivalent volume of DMSO with PBS to yield a total volume of 720 &#181;l. Scale these stocks up or down depending on how many strains are tested.</li>
		<li>Label microfuge tubes for 10-1 through 10-7 serial dilutions. Add 90 &#181;l of PBS + NTBC or PBS + DMSO to the appropriate tubes. Use PBS + NTBC for strains grown in LB + NTBC and use PBS + DMSO for strains grown in LB + DMSO.</li>
		<li>Add 10 &#181;l of culture to the appropriate 10-1 dilution tube. Mix by vortexing and transfer 10 &#181;l of the 10-1 dilution to the 10-2 dilution tube. Repeat until all dilutions have been performed. Change pipet tips between dilutions.</li>
	</ol>
	</li>
	<li>Spot 5 &#181;l of the 10-3 through 10-7 dilutions on LB + H2O2 plates in duplicate for each strain. Use one pipet tip if spots are plated from most dilute to least dilute (10-7 to 10-3). Do not tip or tilt the plate until the liquid has dried into the plate.</li>
	<li>Incubate the plates for 24-48 hr&#160;at 37 &#176;C (air incubator), depending on the strain. 
	NOTE: P. aeruginosa PAO1 will have good sized colonies on LB after 24 hr&#160;of incubation. Incubate strains until they have colonies approximately the same size as PAO1.</li>
	<li>Photograph the plates using a CCD camera above a transluminator. Optionally, edit the photos for contrast and crop to the same size. Count the number of colonies in each spot to determine changes in sensitivity to oxidative stress.</li>
</ol>

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The authors declare that they have no competing financial interests.

The NTBC titration method described in this protocol will allow the user to determine if NTBC can reduce or eliminate pyomelanin production in bacteria, and determine the concentration of NTBC required. The most critical step in the NTBC titration assay is determining the range of NTBC concentrations to use in the assay. Different strains of P. aeruginosa have different sensitivities to NTBC, and laboratory strains may be more sensitive to NTBC than clinical isolates12 (Figure 2). The...

Pseudomonas aeruginosa is a Gram negative bacterium that produces a variety of pigments including pyomelanin, a red-brown pigment that helps provide protection from oxidative stress1-4 and binds a variety of compounds, including aminoglycoside antibiotics5-7. Pyomelanin production is caused by a defect in the tyrosine catabolism pathway4,8, either through deletions or mutations of the gene encoding homogentisate 1,2-dioxygenase (HmgA)1,9 or through imbalances in the various enzymes in the pathway10. Homogentisate accumulates due to inactivation of HmgA, and is secreted and oxidized to form pyomelanin11. Production of pyomelanin can be abolished or reduced in a dose dependent manner through treatment with the herbicide 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC)12, which inhibits 4-hydroxyphenylpyruvate dioxygenase (Hpd) in the tyrosine catabolism pathway13. Hpd is required for the formation of homogentisate, and therefore pyomelanin11. 
We describe in detail three techniques that were important in our studies of NTBC treatment of pyomelanin producing strains of P. aeruginosa. These techniques include titration of NTBC to determine the concentrations that will abolish or reduce pyomelanin production in laboratory and clinical pyomelanin producing strains, determination of the minimum inhibitory concentration (MIC) of antibiotics when bacteria are treated with NTBC, and the resulting sensitivity to oxidative stress with NTBC treatment.
The titration assay we developed serves two purposes. First, the assay will allow the user to determine if NTBC can abolish or reduce pyomelanin production in the bacterium being studied and at which concentrations. This will allow the user to determine sensitivity to NTBC, since different strains of bacteria may have different sensitivities to this compound, as observed in P. aeruginosa12. Second, the NTBC titration assay will allow the user to determine the appropriate concentration of NTBC to use in subsequent assays, such as antibiotic MIC and oxidative stress response assays, if the goal is to abolish or reduce pyomelanin production and determine the effects of pigment reduction. 
The titration assay works because a visible difference in pyomelanin production can be seen in strains treated with NTBC and the differences in pyomelanin production are dose dependent12. Additionally, this technique can be applied to the study of other compounds that may eliminate or enhance pigment production in bacteria.
Antibiotic MICs are used to determine the sensitivity of bacteria to antibiotics. There are several methods to determine MICs, including agar dilution plates and broth dilutions14. Broth dilutions can be performed in small test tubes or in a 96-well microtiter plate. The microtiter plate method of MIC determination described herein will allow the user to test a wide range of antibiotics using a minimum of resources. The assay provides reproducibility as well as flexibility in the number of antibiotics and strains tested by this method. Additionally, with the incorporation of NTBC in the assay, the user can determine if elimination or reduction of pyomelanin production alters antibiotic sensitivity in bacteria that produce pyomelanin.
Bacterial response to oxidative stress can be tested in several ways. The most common methods described are either viable counts of bacteria subjected to oxidative stress for a period of time1, or oxidative stress disc diffusion assays15. These methods tend to use high concentrations of oxidative stressors to examine the effects of oxidative stress in bacteria and results can be quite variable between biological replicates. The viable count assay also tends to use more agar plates than the other methods. The spot plate assay we describe uses low concentrations of H2O2 and allows the user to test the oxidative stress response of multiple strains using a minimum of plates. The assay is also consistently reproducible between technical and biological replicates. As pyomelanin is involved in resistance to oxidative stress, the incorporation of NTBC in the assay allows the user to determine the effects of elimination of pyomelanin production on oxidative stress resistance.

<table><tbody><tr><td>2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC)</td><td>Sigma-Aldrich</td><td>SML0269-50mg</td><td>Also called nitisinone.&amp;nbsp; Soluble in DMSO.</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>216763-100ML</td><td>30 wt. % in H&lt;sub&gt;2&lt;/sub&gt;O.&amp;nbsp; Stabilized.</td></tr><tr><td>Gentamicin</td><td>Gold Bio</td><td>G-400-100</td><td>Soluble in H&lt;sub&gt;2&lt;/sub&gt;O.&amp;nbsp; Filter sterilize.</td></tr><tr><td>Kanamycin</td><td>Fisher Scientific</td><td>BP906-5</td><td>Soluble in H&lt;sub&gt;2&lt;/sub&gt;O.&amp;nbsp; Filter sterilize.</td></tr><tr><td>Tobramycin</td><td>Sigma-Aldrich</td><td>T4014-100MG</td><td>Soluble in H&lt;sub&gt;2&lt;/sub&gt;O.&amp;nbsp; Filter sterilize.</td></tr></tbody></table>

methods to inhibit bacterial pyomelanin production and determine the corresponding increase in sensitivity to oxidative stress

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism pathway and contributes to increased oxidative stress resistance. Pyomelanin production in Pseudomonas aeruginosa is reduced in a dose dependent manner through treatment with 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). We describe a titration method using multiple concentrations of NTBC to determine the concentration of drug that will reduce or abolish pyomelanin production in bacteria. The titration method has an easily quantifiable outcome, a visible reduction in pigment production with increasing drug concentrations. We also describe a microtiter plate method to assay antibiotic minimum inhibitory concentration (MIC) in bacteria. This method uses a minimum of resources and can easily be scaled up to test multiple antibiotics in one microtiter plate for one strain of bacteria. The MIC assay can be adapted to test the affects of non-antibiotic compounds on bacterial growth at specific concentrations. Finally, we describe a method for testing bacterial sensitivity to oxidative stress by incorporating H2O2 into agar plates and spotting multiple dilutions of bacteria onto the plates. Sensitivity to oxidative stress is indicated by reductions in colony number and size for the different dilutions on plates containing H2O2 compared to a no H2O2 control. The oxidative stress spot plate assay uses a minimum of resources and low concentrations of H2O2. Importantly, it also has good reproducibility. This spot plate assay could be adapted to test bacterial sensitivity to various compounds by incorporating the compounds in agar plates and characterizing the resulting bacterial growth.

NTBC titrations
The NTBC titrations were used to determine if NTBC was able to reduce pyomelanin production in P. aeruginosa, and also identify the concentration of NTBC that eliminates or reduces pyomelanin production for use in additional assays. There may be variations in the levels of pyomelanin produced in different replications, but general trends remain constant. The NTBC titration assay could also be modified to test other compounds that may affect pigment production in other ...

Watch this Scientific Journal Video about Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress at JoVE.com

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism ...

methods-to-inhibit-bacterial-pyomelanin-production-determine

Research

JoVE Journal

Immunology and Infection

8.1K Views.  University of Wisconsin Milwaukee. Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

Video: Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including destruction of the sample, introduction of potential artifacts, and a lack of specificity for the reactive species involved. Thus, there is a current need in the field of toxicology for non-destructive, sensitive, and specific methods that can be used to observe and quantify intracellular redox perturbations, more commonly referred to as oxidative stress. Here, we present a method for the use of two genetically-encoded fluorogenic sensors, roGFP2 and HyPer, to be used in live-cell imaging studies to observe xenobiotic-induced oxidative responses. roGFP2 equilibrates with the glutathione redox potential (EGSH), while HyPer directly detects hydrogen peroxide (H2O2). Both sensors can be expressed into various cell types via transfection or transduction, and can be targeted to specific cellular compartments. Most importantly, live-cell microscopy using these sensors offers high spatial and temporal resolution that is not possible using conventional methods. Changes in the fluorescence intensity monitored at 510 nm serves as the readout for both genetically-encoded fluorogenic sensors when sequentially excited by 404 nm and 488 nm light. This property makes both sensors ratiometric, eliminating common microscopy artifacts and correcting for differences in sensor expression between cells. This methodology can be applied across a variety of fluorometric platforms capable of exciting and collecting emissions at the prescribed wavelengths, making it suitable for use with confocal imaging systems, conventional wide-field microscopy, and plate readers. Both genetically-encoded fluorogenic sensors have been used in a variety of cell types and toxicological studies to monitor cellular EGSH and H2O2 generation in real-time. Outlined here is a standardized method that is widely adaptable across cell types and fluorometric platforms for the application of roGFP2 and HyPer in live-cell toxicological assessments of oxidative stress.

This manuscript describes the use of genetically-encoded fluorogenic reporters in an application of live-cell imaging for the examination of xenobiotic-induced oxidative stress. This experimental approach offers unparalleled spatiotemporal resolution, sensitivity, and specificity while avoiding many of the shortcomings of conventional methods used for the detection of toxicological oxidative stress.

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including ...

Cancer Research

Quantification of Hypopigmentation Activity In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is synthesized in response to multiple cellular and environmental factors. Melanin protects skin cells from ultraviolet damage, but also has biophysical and biochemical functions. Excessive production or accumulation of melanin in melanocytes can cause dermatological problems, such as freckles, dark spots, melasma, and moles. Therefore, the control of melanogenesis with hypopigmentation agents is important in individuals with clinical or cosmetic needs. Melanin is primarily synthesized in the melanosomes of melanocytes in a complex biochemical process called melanogenesis, which is influenced by extrinsic and intrinsic factors, such as hormones, inflammation, age, and ultraviolet light exposure. We describe three methods to determine the hypopigmentation activity of chemicals or natural substances in melanocytes: measurement of the 1) cellular tyrosinase activity and 2) melanin content, and 3) staining and quantifying cellular melanin with image analysis.
In melanogenesis, tyrosinase catalyzes the rate-limiting step that converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and then into dopaquinone. Therefore, the inhibition of tyrosinase is a primary hypopigmentation mechanism. In cultured melanocytes, tyrosinase activity can be quantified by adding L-DOPA as a substrate and measuring dopaquinone production by spectrophotometry. Melanogenesis can also be measured by quantifying the melanin content. The melanin-containing cellular fraction is extracted with NaOH and melanin is quantified spectrophotometrically. Finally, the melanin content can be quantified by image analysis following Fontana-Masson staining of melanin. Although the results of these in vitro assays may not always be reproduced in human skin, these methods are widely used in melanogenesis research, especially as the initial step to identify potential hypopigmentation activity. These methods can also be used to assess melanocyte activity, growth, and differentiation. Consistent results with the three different methods ensure the validity of the effects.

We describe three experimental methods for evaluating the hypopigmentation activity of chemicals in vitro: quantification of 1) cellular tyrosinase activity and 2) melanin content, and 3) measurement of melanin by cellular melanin staining and image analysis.

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

Biochemistry

Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection 1. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.

Epidermal melanin is induced by topical application of forskolin in a murine model of the fair-skinned UV-sensitive human. Pharmacologic manipulation of cAMP levels in the skin and epidermal darkening strongly protect against UV-mediated inflammation (sunburn) as measured by the minimum erythematous dose (MED) assay.

Fairness of skin, UV sensitivity and skin cancer  risk all correlate with the physiologic function of the melanocortin 1  receptor, a Gs-coupled signaling ...

Medicine

Adsorptive Bioprocess Improves Yield of Melanin from Pseudomonas stutzeri

Melanins are natural pigments, and the presence of indole ring and numerous functional groups makes melanin an ideal choice for many applications such as UV protective agents, skincare, cosmetics etc. A marine Pseudomonas stutzeri produces melanin without the addition of tyrosine. The feedback inhibition was observed by melanin in the culture of a melanin-producing marine bacterium, Pseudomonas stutzeri. Melanin also demonstrated microbial growth inhibition. The Han-Levenspiel model-based analysis identified uncompetitive type product inhibition of melanin on the cell growth. Tyrosinase enzyme, which produces melanin, was inhibited by melanin. The double reciprocal plot of the enzymatic reaction in the presence of different melanin concentrations revealed uncompetitive product inhibition. An adsorbent-based adsorptive bioprocess was developed to reduce the feedback inhibition by melanin. Different adsorbents were screened to select the best adsorbent for melanin adsorption. Dosage amount and time were optimized to develop the adsorptive bioprocess, which resulted in an 8.8-fold enhancement in melanin production by the marine bacteria Pseudomonas stutzeri (153 mg/L to 1349 mg/L) without supplementation of tyrosine and yeast extract.

Adsorptive Bioprocess Improves Yield of Melanin from Pseudomonas stutzeri

Here we present a protocol on adsorptive bioprocess to produce a biological product, especially a pigment that inhibits its own biosynthesis and inhibits the growth of the microorganism that produces it.

Melanins are natural pigments, and the presence of indole ring and numerous functional groups makes melanin an ideal choice for many applications such as ...

Engineering

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5&#8242;-Phosphate

Riboflavin-5&#39;-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can be generated from FMN photolysis upon irradiation with visible light. The ROS generated from FMN photolysis are harmful to microorganisms, including pathogenic bacteria such as Staphylococcus aureus (S. aureus). This article presents a protocol for deactivating S. aureus, as an example, via photochemical reactions involving FMN under visible light irradiation. The superoxide radical anion (<img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01v3.png" style="margin: auto;" />) generated during the FMN photolysis is evaluated via nitro blue tetrazolium (NBT) reduction. The microbial viability of S. aureus that is attributed to reactive <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01v3.png" style="margin: auto;" /> species was used to determine the effectiveness of the process. The bacterial inactivation rate is proportional to FMN concentration. Violet light is more efficient in inactivating S. aureus than blue light irradiation, while the red or green light does not drive FMN photolysis. The present article demonstrates FMN photolysis as a simple and safe method for sanitary processes.

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5&#8242;-Phosphate

Here, we present a protocol to inactivate pathogenic bacteria with reactive oxygen species produced during photolysis of flavin mononucleotide (FMN) under blue and violet light irradiation of low intensity. FMN photolysis is demonstrated to be a simple and safe method for sanitary processes.

Riboflavin-5&#39;-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can ...

Bioengineering

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...

Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In Situ

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient production of ROS in situ efficiently activates cell proliferation in stem cell niches and stimulates tissue regeneration as strongly manifested through the acceleration of burn healing and hair follicle growth processes. The protocol is based on a regulatable photodynamic treatment that treats the tissue with precursors of the endogenous photosensitizer protoporphyrin IX and further irradiates the tissue with red light under tightly controlled physicochemical parameters. Overall, this protocol constitutes an interesting experimental tool to analyze ROS biology.

The aim of this protocol is to induce transient in vivo production of nonlethal levels of reactive oxygen species (ROS) in mouse skin, further promoting physiological responses in the tissue.

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient ...

Biology

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical applications. Isolating primary melanocytes from skin tissues by the conventional method usually takes about 3 to 4 weeks to passage sufficiently. More importantly, the tissues used are usually newborn foreskins and it is still a challenge to efficiently isolate primary melanocytes from adult tissues. We recently developed a new isolation method for melanocytes that adds Y-27632, a Rho kinase inhibitor, to the initial culture medium for 48 h. Compared with the conventional protocol, this new method dramatically increases the yield of melanocytes and shortens the time required to isolate melanocytes from foreskin tissues. We now describe this new method in more detail using adult epidermis to efficiently culture primary melanocytes. Importantly, we show that melanocytes obtained from adult tissues prepared by this new method can function normally. This new protocol will significantly benefit studies of pigmentation defects and melanomas using primary melanocytes prepared from easily accessed adult skin tissues.

This paper reports that the addition of Y-27632 to TIVA medium can significantly increase the yield of melanocytes from adult skin tissues.

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical ...

Minimum Erythematous Dose Assay: A Method for Measuring UV Sensitivity in Mouse Models

Source: Amaro-Ortiz, A. et al. <a href="https://www.jove.com/v/50670"> Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model</a>. J. Vis. Exp. (2013)
This video describes a method to measure UV sensitivity in a humanized mouse model&#8212;a mouse model that mimics human skin. We determine the minimal erythematous dose (MED) of UV radiation that can cause erythema or edema on the skin. The method can help to find out the efficacy of drugs used to reduce UV sensitivity.

Test minimalnej dawki rumieniowej: metoda pomiaru wrażliwości na promieniowanie UV w modelach mysich

Source: Amaro-Ortiz, A. et al.  Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model. J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Skin Cancer

Animal model studies

A Spot Plate Assay for Evaluating Oxidative Stress Responses in Bacterial Strains

Source: Ketelboeter, L. M. et al., <a href="https://app.jove.com/v/53105">Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress.</a> J. Vis. Exp. (2015)
This video demonstrates a spot plate assay tailored to assess the oxidative stress responses of bacterial strains. Some strains produce pyomelanin, an antioxidant pigment. Conversely, strains treated with a test agent, leading to reduced pyomelanin production, exhibit increased susceptibility to oxidative stress, resulting in decreased cell viability compared to pyomelanin-producing strains.

Test punktowy do oceny reakcji na stres oksydacyjny w szczepach bakteryjnych

1. Preparation of Culture Media, Antibiotics, and 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC)

	Make Luria-Bertani (LB) broth (1% ...

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

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Rozdziały w tym wideo

Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors

Quantification of Hypopigmentation Activity In Vitro

Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

Adsorptive Bioprocess Improves Yield of Melanin from Pseudomonas stutzeri

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5′-Phosphate

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In Situ

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

Test minimalnej dawki rumieniowej: metoda pomiaru wrażliwości na promieniowanie UV w modelach mysich

Test punktowy do oceny reakcji na stres oksydacyjny w szczepach bakteryjnych