Name: catalytic scavenging of plant reactive oxygen species in vivo by anionic cerium oxide nanoparticles
Uploaded: 2018-08-26T15:00:00.000+00:00
Duration: 9 min 46 s
Description: Watch this Scientific Journal Video about Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles at JoVE.com

This work was supported by the University of California, Riverside and USDA National Institute of Food and Agriculture, Hatch project 1009710 to J.P.G. This material is based upon work supported by the National Science Foundation under Grant No. 1817363 to J.P.G.

1. Growing A. thaliana Plants
<ol>
	<li>Sow A. thaliana seeds in 5 cm x 5 cm disposable pots filled with standard soil mix. Put 32 of these pots into a plastic tray filled with water (~ 0.5 cm depth) and transfer the plastic tray with the plants into a plant growth chamber.
	<ol>
		<li>Set the growth chamber settings as follows: 200 &#181;mol/ms photosynthetic active radiation (PAR), 24 &#177; 1 &#176;C day and 21 &#177; 1 &#176;C night, 60% humidity, and 14/10 h day/night light regime, respectively.</li>
	</ol>
	</li>
	<li>Thin each pot to leave only one individual plant after one week of germination. Take note to keep the seedlings with similar size in each pot.</li>
	<li>Water the pots by pouring tap water directly on the plastic tray once every two days. Grow the plants for four weeks. A. thaliana plants are ready for further use.</li>
</ol>
2. Synthesis and Characterization of PNC
<ol>
	<li>Weigh 1.08 g of cerium (III) nitrate and dissolve it in 2.5 mL of molecular biology grade water in a 50 mL conical tube.</li>
	<li>Weigh 4.5 g of poly (acrylic) acid&#160;and dissolve it in 5 mL of molecular biology grade water in a 50 mL conical tube.</li>
	<li>Mix these two solutions thoroughly at 2,000 rpm for 15 min using a digital vortex mixer.</li>
	<li>Transfer 15 mL of ammonium hydroxide solution (7.2 M) to a 50 mL glass beaker.</li>
	<li>While stirring at 500 rpm, add the mixture from Step 2.3 dropwise to the ammonium hydroxide solution and stir at 500 rpm at room temperature for 24 hr in a fume hood.</li>
	<li>Cover the beaker with a piece of paper to avoid the substantial loss of solution during the overnight reaction.</li>
	<li>After 24 h, transfer the resulting solution to a 50 mL conical tube and centrifuge it at 3,900 x g for 1 h to remove any possible debris and large agglomerates.</li>
	<li>Transfer this 22.5 mL of supernatant solution into three 15 mL 10 kDa filters and fill the remainder of the filter with molecular grade water to make a total dilution of 45 mL.</li>
	<li>Purify the supernatant solution from free polymers and other reagents with a benchtop centrifuge by adding the supernatant to a 15 mL 10 kDa filter and centrifuge at 3,900 x g for 15 min. Repeat this step at least six times.</li>
	<li>Measure the absorbance of the eluent in each cycle with a UV-VIS spectrophotometer from 220-700 nm to ensure no free polymers and other reagents are present in the final PNC solution.</li>
	<li>Take the collected PNC solution into the 5 mL syringe and filter it against a 20 nm pore size syringe filter. Collect the filtered PNC solution in a 50 mL conical tube.</li>
	<li>Take a diluted final PNC solution in a plastic cuvette and measure its absorbance with the UV-VIS spectrophotometer from 220-700 nm. PNC absorbance peak is at 271 nm.</li>
	<li>Calculate its concentration by using Beer-Lambert&#39;s law: A = &#949;CL. A is the absorbance of the peak value for a given sample, &#603; is the molar absorption coefficient of PNC (cm-1 M-1), L is the optical path length (cuvette width, 1 cm in this method), and C is the molar concentration of measured nanoparticles.</li>
	<li>Measure the hydrodynamic diameter and zeta potential of the synthesized PNC using a particle size and zeta potential analyzer (Figure 1).</li>
	<li>Store the final PNC solution in a refrigerator (4 &#176;C) until further use. 
	NOTE: Please refer to Wu et al.8 for more protocol details about PNC characterization.</li>
</ol>
3. Labeling PNC with DiI Fluorescent Dye
<ol>
	<li>Mix 0.4 mL of 5 mM (58 mg/L) PNC with 3.6 mL of molecular biology grade water in a 20 mL glass vial and stir at 500 rpm.</li>
	<li>Add 24 &#181;L 1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate dye solution (DiI, 2.5 mg/mL; dilute in DMSO) into 176 &#181;L of DMSO (dimethyl sulfoxide) to make the DiI dye solution.</li>
	<li>Add the DiI dye dropwise to the PNC solution, stirring at 1,000 rpm for 1 min at ambient temperature.</li>
	<li>Transfer this resulting mixture into a 15 mL 10 kDa filter and fill the tube to the top with molecular biology grade water to make the total dilution 15 mL.</li>
	<li>Purify the DiI labeled PNC (DiI-PNC) solution from DMSO and any possible free DiI dye by a benchtop centrifugation with the 15 mL 10 kDa filter at 3,900 x g for 5 min.
	<ol>
		<li>Repeat Step 3.5 at least five times.</li>
	</ol>
	</li>
	<li>Filter the final DiI-PNC solutions through a 20 nm pore size syringe filter.</li>
	<li>Measure the absorbance of final DiI-PNC by UV-VIS spectrophotometry and calculate its concentration according to Beer-Lambert&#39;s law (Figure 2). See Step 2.13 for more details.</li>
	<li>Store it in a refrigerator at 4 &#176;C for further use.</li>
</ol>
4. Infiltration of Plant Leaves with PNC
<ol>
	<li>Add 0.1 mL of infiltration buffer (100 mM TES, 100 mM MgCl2, pH 7.5, adjusted by HCl) into 0.9 mL of 0.5 mM PNC or DiI-PNC solution and vortex it. Use a solution of 10 mM TES infiltration buffer as a negative control.</li>
	<li>Transfer 0.2 mL of the PNC or DiI-PNC infiltration solution to a 1 mL sterile needleless syringe. Tap to remove any possible air bubbles.</li>
	<li>Retrieve the plant from the growth chamber just before infiltration with nanoparticles to avoid possible stomata closure under room light conditions.</li>
	<li>Before infiltration, measure the chlorophyll content from A. thaliana leaves with similar size using a chlorophyll meter. Measure each leaf with three replicates (each replicate consisting of at least three measurements)14. Choose the A. thaliana leaves with similar chlorophyll content for the infiltration experiment.</li>
	<li>Infiltrate the leaves slowly with the recently prepared PNC or DiI-PNC solution by gently pressing the tip of the needleless syringe against the bottom of the leaf lamina (abaxial side) and depress the plunger (Figure 3A).</li>
	<li>Gently wipe off the excess solution that remains on the surface of leaf lamina (Figure 3B) using a delicate task wiper (Figure 3C) and label the plant. Use new delicate task wipes for each group of leaves.</li>
	<li>Keep the infiltrated A. thaliana plants on the bench for leaf adaptation and incubation with PNC or DiI-PNC for 3 h. 
	NOTE: Infiltrated A. thaliana plants are then ready for further use (Figure 3D).</li>
</ol>
5. Preparation of Leaf Samples for Confocal Microscopy
<ol>
	<li>Roll a pea-size amount of observation gel to about a 1 cm radius (Figure 4A) and then spread it out until it is 1 mm thin on a glass slide (Figure 4B).</li>
	<li>Use a cork borer (diameter 0.3 cm) to cut out a circular section at the center of the observation gel on the glass slide (Figure 4C).</li>
	<li>Fill the cut well entirely with perfluorodecalin (PFD) for deeper and better confocal imaging resolution in leaf tissues.</li>
	<li>Use a cork borer (diameter 0.2 cm) to collect leaf discs from the adapted DiI-PNC infiltrated A. thaliana plants (Figure 4D).</li>
	<li>Mount the leaf disc in the PFD filled well; face the infiltrated (abaxial) side of the leaf up.</li>
	<li>Put a square coverslip on top of the leaf disc and gently press on the slide coverslip evenly to seal it with the well of observation gel and ensure no air bubbles remain trapped (Figure 4E).</li>
</ol>
6. Imaging DiI-PNC in Leaf Tissues by Confocal Microscopy
<ol>
	<li>Use a 40X objective lens in an inverted laser scanning confocal microscope.</li>
	<li>Drop two to three drops of ddH2O on the top of the 40X objective lens.</li>
	<li>Place the prepared DiI-PNC infiltrated leaf sample slide on top of the inverted 40X objective lens.
	<ol>
		<li>Make sure the coverslip side but not the glass slide contact directly with the ddH2O on the lens.</li>
	</ol>
	</li>
	<li>Find a region of interest in the sample under the microscope with either laser light or bright field.</li>
	<li>Start the microscope software and turn on the Argon laser (set at 20%).</li>
	<li>Set the pinhole to collect an optical slice less than 2 &#181;m and a line average of 4.</li>
	<li>Image the sample with confocal microscope settings: 514 nm laser excitation (30 %); Z-Stack section thickness: 2 &#181;m; PMT1: 550-615 nm (for DiI-PNC imaging); PMT2: 700-800 nm (for chloroplast imaging).</li>
	<li>Take representative confocal images of leaf samples from different individuals, a minimum of three biological replicates.</li>
</ol>
7. Imaging PNC in vivo ROS Scavenging by Confocal Microscopy
<ol>
	<li>Prepare 25 &#181;M 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (H2DCFDA, a dye for indicating a general ROS) and 10 &#181;M dihydroethidium (DHE, a dye for indicating superoxide anion) dyes in TES infiltration buffer (pH 7.5) in 1.5 mL microcentrifuge tubes, separately.</li>
	<li>Use a cork borer (diameter 0.2 cm) to collect leaf discs from the adapted PNC infiltrated A. thaliana plants.
	<ol>
		<li>Use the sharp tip of the forceps to make three to four holes on the leaf discs to accelerate dye loading process.</li>
	</ol>
	</li>
	<li>Transfer the leaf discs to microcentrifuge tubes with H2DCFDA and DHE separately and incubate for 30 min under darkness.</li>
	<li>After incubation, rinse the leaf discs with ddH2O three times and mount it into the glass slide with observation gel (see Protocol Section 5).</li>
	<li>Put the slide on the confocal microscope and manually focus to a region of leaf mesophyll cells. See Protocol Section 6 for details.</li>
	<li>Expose the leaf discs to the UV-A (405 nm) laser for 3 min to generate ROS and record the ROS signal intensity change in time-series (&#34;xyt&#34;) per leaf disc.</li>
	<li>Image the leaf disc with confocal microscope settings: 40X water objective; 496 nm laser excitation; PMT1: 500-600 nm (for DHE and DCFDA dye detection); PMT2: 700-800 nm (for chloroplasts detection). Use a plant infiltrated with only infiltration buffer solution as the negative control.</li>
</ol>
8. PNC Scavenging of H2O2 in vitro
<ol>
	<li>Conduct the CAT (catalase) mimetic activity of the synthesized PNC in vitro by following the methods in previous publications3,8,15</li>
	<li>Add 45.4 &#181;L of 1x TES infiltration buffer (10 mM TES, 10 mM MgCl2, pH 7.5, adjusted by HCl), PNC (60 nM, 3 &#181;L), and H2O2 (2 &#181;M, 1 &#181;L) into a well (white round bottom 96 well plate), and gently mix it by pipetting.</li>
	<li>Add 10-acetyl-3,7-dihydroxyphenoxazine (working concentration 100 &#181;M, 0.5 &#181;L) and horseradish peroxidase (HRP; working concentration 0.2 U/mL, 0.1 &#181;L) into the well, gently mix it by pipetting, and incubate it for 30 min. 10-acetyl-3,7-dihydroxyphenoxazine reacts with H2O2 and is converted into resorufin in the presence of HRP.
	<ol>
		<li>Wrap the plate with aluminum foil to avoid light during the incubation.</li>
		<li>Prepare a negative control by using reaction buffer or water to replace H2O2.</li>
		<li>Except for the stock solution, prepare all other solutions at ambient temperature.</li>
	</ol>
	</li>
	<li>After the incubation, with a plate reader, monitor the absorbance at 560 nm to use resorufin for indicating the level of H2O2. Set time regime at 0, 2, 5, 10, 20, and 30 min.</li>
</ol>

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The authors have nothing to disclose.

In this protocol, we describe PNC synthesis, characterization, fluorescent dye labeling, and confocal imaging of the nanoparticles within plant mesophyll cells to exhibit their in vivo ROS scavenging activity. PNC are synthesized from a mixture of cerium nitrate and PAA solution in ammonium hydroxide. PNC are characterized by absorption spectrophotomery and the concentration determined using Beer-Lamberts law. Zeta potential measurements confirmed the negatively charged surface of PNC for enhancing delivery to chloroplasts8. Labeling of PNC with a fluorescent DiI dye enables in vivo imaging by confocal microscopy within leaf mesophyll cells where the nanoparticles show high levels of colocalization with chloroplasts. Using DHE and DCF fluorescent dyes, we confirmed that PNC act as a potent scavenger of superoxide anion and ROS in vivo.
The method for synthesizing PNC is a simple step-wise procedure that generates cerium oxide nanoparticles with controlled size, negative charge, and ROS scavenging capabilities16. Other methods, such as thermal hydrolysis, require high temperatures and expensive chemistry equipment21,22. The synthesis and characterization of PNC is a low-cost method performed with common laboratory equipment. It does not require a steep learning curve compared with molecular methods in plants based on overexpression of an antioxidant enzyme, e.g., SOD, APX, and CAT, for scavenging ROS species in model systems23. PNC is a robust, water-soluble ROS catalytic scavenger that will not require laborious cloning and transformation methods that are dependent on the plant&#39;s genetic tractability and available molecular toolkit.
A critical step in this protocol is the syringe-based infiltration of leaf mesophyll cells with PNC. Infiltration of nanoparticles into live plants should be done gently to avoid physical damage to the leaf24. Thus, as in Protocol Step 4 of the methods, gently push against the leaf surface with the syringe to avoid tearing or puncturing the abaxial leaf surface. It is better to infiltrate from the abaxial surface of A. thaliana leaf since it has higher stomatal density than the adaxial surface25,26. Furthermore, a buffered solution of PNC or DiI-PNC within the physiological pH range (~ pH 7.5) should be used during leaf lamina infiltration. Another critical step in this protocol is to apply the appropriate concentration of PNC to the studied plant tissue. In this protocol, 50 mg/L of PNC was not toxic to A. thaliana leaves while enabling catalytic PNC ROS scavenging. Some limitations of the current nanoparticle delivery method are 1) not applicable to plant species having thick and waxy cuticles or low stomatal densities, 2) not scalable for applications in the field, 3) the high cost of a confocal microscopy system to monitor in vivo nanoparticle distribution and ROS scavenging.
This protocol demonstrates the application of ROS scavenging PNC for studying and improving abiotic stress tolerance in plants through a facile method of leaf lamina infiltration. ROS accumulation is accompanied by abiotic stresses in plants, which in turn reduces plant photosynthesis, growth, and yield8,27,28. Plant genetic modifications methods for ROS manipulation in vivo are often limited to plant model species while PNC ROS-scavenging has the potential to be applied to diverse wild type plant species. Leaf lamina infiltration is a practical research method to increase ROS scavenging in leaf tissues for understanding and engineering plant abiotic stress tolerance.

Cerium oxide nanoparticles (nanoceria) are widely used in living organisms, from basic research to bioengineering, due to their distinct catalytic reactive oxygen species (ROS) scavenging ability1,2,3. Nanoceria have ROS scavenging abilities due to a large number of surface oxygen vacancies that alternate between two oxidation states (Ce3+ and Ce4+) 4,5,6. The Ce3+ dangling bonds effectively scavenge ROS while the lattice strains at the nanoscale promote the regeneration of these defect sites via redox cycling reactions7. Nanoceria have also been recently used for studying and engineering plant function8,9. Plants under abiotic stress experience accumulation of ROS, causing oxidative damage to lipids, proteins, and DNA10. In A. thaliana plants,&#160;nanoceria catalytic scavenging of ROS in vivo leads to improved plant photosynthesis under high light, heat, and chilling stresses8. Applying nanoceria to soil also increases shoot biomass and grain yield of wheat (Triticum aestivum)11; canola (Brassica napus) plants treated with nanoceria have higher plant biomass under salt stress12.
Nanoceria offer&#160;bioengineers and plant biologists a nanotechnology-based tool to understand abiotic stress responses and enhance plant abiotic stress tolerance. Nanoceria&#39;s&#160;in vivo ROS&#160;scavenging capabilities are independent of plant species, and the facile delivery into plant tissues has the potential to enable broad application outside of model organisms. Unlike other genetically-based methods, nanoceria do&#160;not require generating plant lines with the overexpression of antioxidant enzymes for higher ROS scavenging ability13. Leaf lamina infiltration of nanoceria to plants is a&#160;practical approach&#160;for lab-based research.
The overall goal of this protocol is to describe 1) the synthesis and characterization of negatively charged poly (acrylic) acid nanoceria (PNC), 2) the delivery and tracking of PNC throughout leaf cells, and 3) the monitoring of PNC-enabled ROS scavenging in vivo. In this protocol, negatively charged poly (acrylic) acid&#160;nanoceria (PNC) are synthesized and characterized by their absorption spectrum, hydrodynamic diameter, and zeta potential. We describe a simple leaf lamina infiltration method to deliver PNC into plant leaf tissues. For in vivo imaging of nanoparticle distribution within mesophyll cells, a fluorescent dye (DiI) was used to label PNC (DiI-PNC) and observe the nanoparticles via confocal fluorescence microscopy. Finally, we explain how to monitor in vivo PNC ROS scavenging through confocal microscopy.

<table><tbody><tr><td>Cerium (III) nitrate hexahydrate</td><td>Sigma-Aldrich</td><td>238538-100G</td><td></td></tr><tr><td>Molecular Biology Grade Water, Corning</td><td>VWR</td><td>45001-044&amp;nbsp;</td><td></td></tr><tr><td>Falcon 50 mL Conical Centrifuge Tubes</td><td>VWR</td><td>14-959-49A</td><td></td></tr><tr><td>Poly (acrylic acid) 1,800 Mw</td><td>Sigma-Aldrich</td><td>323667-100G</td><td></td></tr><tr><td>Fisherbrand Digital Vortex Mixer</td><td>Fisher Scientific</td><td>02-215-370</td><td></td></tr><tr><td>Fisherbrand Digital Vortex Mixer Accessory, Insert Retainer</td><td>Fisher Scientific</td><td>02-215-391</td><td></td></tr><tr><td>Fisherbrand Digital Vortex Mixer Accessories: Foam Insert Set</td><td>Fisher Scientific</td><td>02-215-395</td><td></td></tr><tr><td>Ammonium hydroxide solution</td><td>Sigma-Aldrich</td><td>05002-1L</td><td></td></tr><tr><td>PYREX Griffin Beakers, Graduated, Corning</td><td>VWR</td><td>13912-149&amp;nbsp;</td><td></td></tr><tr><td>RCT basic</td><td>IKA</td><td>3810001</td><td></td></tr><tr><td>Eppendorf Microcentrifuge 5424</td><td>VWR</td><td>80094-126</td><td></td></tr><tr><td>Amicon Ultra-15 Centrifugal Filter Units</td><td>Millipore-Sigma</td><td>UFC901024</td><td></td></tr><tr><td>Allegra X-30 Series Benchtop Centrifuge</td><td>Beckman Coulter</td><td>B06314</td><td></td></tr><tr><td>UV-2600 Sptecrophotometer</td><td>Shimadzu</td><td>UV-2600 120V</td><td></td></tr><tr><td>Whatman Anotop 10 syringe filter</td><td>Sigma-Aldrich</td><td>WHA68091102</td><td></td></tr><tr><td>BD Disposable Syringes with Luer-Lok Tips</td><td>Fisher Scientific</td><td>14-829-45</td><td></td></tr><tr><td>Zetasizer Nano S</td><td>Malvern Panalytical</td><td>Zen 1600</td><td></td></tr><tr><td>1,1&amp;prime;-Dioctadecyl-3,3,3&amp;prime;,3&amp;prime;-tetramethylindocarbocyanine perchlorate</td><td>Sigma-Aldrich</td><td>42364-100MG</td><td></td></tr><tr><td>Dimethyl Sulfoxide, ACS</td><td>VWR</td><td>BDH1115-1LP</td><td></td></tr><tr><td>Sunshine Mix #1 LC1</td><td>Green Island Distributors, Inc</td><td>5212601.CFL080P</td><td></td></tr><tr><td>Adaptis 1000</td><td>Conviron</td><td>A1000</td><td></td></tr><tr><td>TES, &gt;99% (titration</td><td>Sigma-Aldrich</td><td>T1375-100G</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>M8266-1KG</td><td></td></tr><tr><td>Air-Tite All-Plastic Norm-Ject Syringe</td><td>Fisher Scientific</td><td>14-817-25</td><td></td></tr><tr><td>Kimberly-Clark Professional Kimtech Science Kimwipes Delicate Task Wipers</td><td>Fisher Scientific</td><td>06-666A</td><td></td></tr><tr><td>Carolina Observation Gel</td><td>Carolina</td><td>132700</td><td></td></tr><tr><td>Corning microscope slides, frosted one side, one end</td><td>Sigma-Aldrich</td><td>CLS294875X25-72EA</td><td></td></tr><tr><td>Cork Borer Sets with Handles</td><td>Fisher Scientific</td><td>S50166A</td><td></td></tr><tr><td>Perfluorodecalin</td><td>Sigma-Aldrich</td><td>P9900-25G</td><td></td></tr><tr><td>Micro Cover Glasses, Square, No. 1</td><td>VWR</td><td>48366-045</td><td></td></tr><tr><td>Leica Laser Scanning Confocal Microscope TCS SP5</td><td>Leica Microsystems</td><td>TCS SP5</td><td></td></tr><tr><td>2&amp;prime;,7&amp;prime;-Dichlorofluorescin diacetate</td><td>Sigma-Aldrich</td><td>D6883-250MG</td><td></td></tr><tr><td>Dihydroethidium</td><td>Sigma-Aldrich</td><td>D7008-10MG</td><td></td></tr><tr><td>Fisherbrand Premium Microcentrifuge Tubes: 1.5 mL</td><td>Fisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>Eppendorf Uvette cuvettes</td><td>Sigma-Aldrich</td><td>Z605050-80EA</td><td></td></tr><tr><td>Chlorophyll meter&amp;nbsp;</td><td>Konica Minolta</td><td>SPAD-502</td><td></td></tr></tbody></table>

catalytic scavenging of plant reactive oxygen species in vivo by anionic cerium oxide nanoparticles

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling molecules at low levels and damaging molecules at high levels. Accumulation of ROS in stressed plants can damage metabolites, enzymes, lipids, and DNA, causing a reduction of plant growth and yield. The ability of cerium oxide nanoparticles (nanoceria) to catalytically scavenge ROS in vivo provides a unique tool to understand and bioengineer plant abiotic stress tolerance. Here, we present a protocol to synthesize and characterize poly (acrylic) acid coated nanoceria (PNC), interface the nanoparticles with plants via leaf lamina infiltration, and monitor their distribution and ROS scavenging in vivo using confocal microscopy. Current molecular tools for manipulating ROS accumulation in plants are limited to model species and require laborious transformation methods. This protocol for in vivo ROS scavenging has the potential to be applied to wild type plants with broad leaves and leaf structure like Arabidopsis thaliana.

PNC synthesis and characterization. 
PNC were synthesized, purified and characterized following the method described in Protocol Section 2. Figure 1A shows the coloration of the solutions of cerium nitrate, PAA, the mixture of cerium nitrate and PAA, and PNC. A color change from white to light yellow is seen after PNC is synthesized. After purification with a 10 kDa filter, PNC were characterized with a UV-VIS spectrophotometer. A peak of absorbance for PNC was observed at 271 nm (Figure 1B). The final eluent was also measured with UV-VIS to confirm that the non-reacted chemicals were washed during the purification. The hydrodynamic diameter and zeta potential of the synthesized PNC were measured with a particle sizer and zeta potential analyzer (Figure 1C).
PNC labeling with DiI dye. 
To determine the distribution of the nanoparticles in vivo, PNC were labeled with a fluorescent DiI dye following the method described in Protocol Section 3. DiI dye embeds within the PNC coating spontaneously since it can encapsulate into the hydrophobic domains inside the polymer coatings of nanoceria16. After adding DiI dye to the PNC solution, a rapid color change to pink was observed (Figure 2A). The DiI labeled PNC were then purified with a 10 kDa filter and characterized by a UV-VIS spectrophotometry. Three clear peaks of absorbance for the DiI labeled PNC were observed (Figure 2B). The final eluent was measured by UV-VIS spectrophotometry to confirm that the non-reacted chemicals were washed out during the purification.
Leaf Lamina infiltration. 
PNC or DiI-PNC were delivered into A. thaliana leaf via leaf lamina infiltration method as described in Protocol Section 4. Leaf was infiltrated at four different spots to ensure the full leaf area was perfused with PNC solution (Figure 3A). Any remaining solution was removed from&#160;the leaf surface (Figure 3B and 3C). The leaf color changed during infiltration from green to darker green (Figure 3D). The syringe was gently pressed against the leaf to avoid any physical damage.
Leaf sample preparation for fluorescence microscopy. 
Leaf samples were mounted on glass slides within an observation gel made well filled with PFD. After rolling the pea size observation gel on the slide (Figure 4A and 4B), a well was made in the middle of the flat gel (Figure 4C). Then, the freshly prepared leaf disc was transferred to the well previously filled with PFD solution (Figure 4D). A coverslip was used to immobilize the leaf sample on the slide (Figure 4E).
Confocal imaging of DiI-PNC in vivo. 
DiI-PNC infiltrated A. thaliana leaves were used for determining the distribution of DiI-PNC in leaf mesophyll cells via confocal imaging (Figure 5A and 5B). To visualize colocalization between the DiI-PNC and chloroplasts, DiI-PNC infiltrated leaf samples were excited with a 514 nm laser. The emission of DiI-PNC was set at 550-615 nm to avoid the possible interference of chloroplast pigments signals after ~650 nm17. The chlorophyll auto-fluorescence from chloroplasts were detected from 700-800 nm. The confocal imaging settings were set (laser power and gain) to make sure no DiI dye signals were detected in the control leaf sample (infiltrated with only buffer) (Figure 5C). The colocalization of DiI-PNC with chloroplasts in leaf mesophyll cells can be observed by the overlay image of detected DiI-PNC and chloroplast pigment autofluorescence (Figure 5D).
Confocal imaging of PNC ROS scavenging in vivo. 
PNC in 10 mM TES buffer solution were delivered into A. thaliana leaves via the leaf lamina infiltration method as described in Protocol Section 7. DHE (dihydroethidium) and H2DCFDA (2&#8242;,7&#8242;-dichlorodihydrofluorescein diacetate) fluorescent dyes are used to visualize ROS in plant tissues8,18. H2DCFDA is known to be converted to fluorescent DCF (2&#8242;,7&#8242;-dichlorofluorescein, an indicator of the degree of general oxidative stress) due to the cleavage of the acetate groups by ROS19. DHE is a more specific dye for superoxide anion having its fluorescent product (2-hydroxyethidium) increase upon reaction with a superoxide anion20. In vivo ROS scavenging enabled by PNC was monitored in leaf discs measuring DHE and DCF dye fluorescence intensity changes (Figures 6A and 6B). PNC infiltrated leaf samples were excited with 496 nm laser. The emission of DHE and DCF dye was set at 500-600 nm to avoid the possible interference with chloroplast auto-fluorescence signals. Pigment auto-fluorescence from chloroplasts were detected from 700-800 nm. After 3 minutes of UV stress, the ROS dye signals in PNC and buffer- infiltrated leaf samples were monitored separately. Compared to the no-nanoparticle buffer control (NNP), PNC infiltrated leaves showed significantly less ROS-activated fluorescent DCF dye signal (Figure 6A). Similar results were also found with the superoxide anion-activated DHE dye, where PNC infiltrated leaves had significantly less DHE dye intensity than buffer infiltrated control leaves (Figure 6B).
PNC CAT mimetic activity assay. Assay of PNC scavenging of H2O2 was described in Protocol Section 8. A decrease of resorufin which indicates H2O2 level in the reaction mixture containing PNC was observed (Figure 7), confirming the CAT mimetic activity of the synthesized PNC.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58373/58373fig1.jpg" /> 
Figure 1: Synthesis and characterization of PNC. A. Coloration of cerium nitrate, poly acrylic acid (PAA), mixture of cerium nitrate and PAA, and the synthesized PNC (PAA coated cerium oxide nanoparticles, light yellow). B. Absorbance spectrum of PNC measured by UV-VIS spectrophotometry. C. Hydrodynamic diameter and zeta potential of the synthesized PNC. Mean &#177; standard error (n = 4). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58373/58373fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58373/58373fig2.jpg" /> 
Figure 2: PNC labelling with DiI fluorescent dye. A. PNC (light yellow) and DiI dye labelled PNC solution (DiI-PNC, pink). B. Absorbance spectrum of DiI-PNC solution. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58373/58373fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58373/58373fig3.jpg" /> 
Figure 3: Leaf lamina infiltration of PNC or DiI-PNC. A. A. thaliana leaf before infiltration. The solution inside the syringe is DiI-PNC. B. Leaf infiltrated with DiI-PNC. C. Cleaning the remaining solution from the leaf surface with delicate task wipes. D. Cleaned A. thaliana leaf infiltrated with DiI-PNC. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58373/58373fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58373/58373fig4.jpg" /> 
Figure 4: Preparation of leaf sample slides. A. Microscopy glass slide with pea size observation gels. B. Slide with flat observation gels. C. Slide with flat observation gel having a well at the middle. D. Slide with leaf discs in the observation gel well filled with perfluorodecalin (PFD) solution. E. Slide with leaf discs immobilized by cover slip. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58373/58373fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58373/58373fig5.jpg" /> 
Figure 5: Imaging DiI-PNC in leaf mesophyll cells via confocal microscopy. A. Leaf sample is mounted on an inverted confocal microscope having a 40X water immersion lens. B. A confocal microscope is used for imaging DiI-PNC and chloroplasts. C. Chloroplast auto-fluorescence is recorded in buffer infiltrated leaf samples without nanoparticles (NNP). D. DiI-PNC signal and chloroplast auto-fluorescence is imaged in DiI-PNC infiltrated leaf samples. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58373/58373fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58373/58373fig6.jpg" /> 
Figure 6: Monitoring PNC ROS scavenging in planta via confocal microscopy. A. DCF fluorescent dye (for monitoring general ROS signal) is significantly lower in mesophyll cells of PNC infiltrated plants than buffer control (no nanoparticles, NNP). B. Reduced DHE fluorescent dye (for monitoring superoxide anion) in mesophyll cells of PNC infiltrated plants relative to that of buffer control (NNP). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58373/58373fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58373/58373fig7.jpg" /> 
Figure 7: Catalase (CAT) mimetic activity of PNC. In the presence of horseradish peroxidase, the fluorescent probe reacts with hydrogen peroxide and is converted to resorufin (absorbance 560 nm). Absorbance of resorufin, which is indicative of hydrogen peroxide levels, was monitored at 560 nm. PNC showed a CAT mimetic activity. Mean &#177; SE (standard error) (n = 4). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58373/58373fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles at JoVE.com

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Here, we present a protocol for the synthesis and characterization of cerium oxide nanoparticles (nanoceria) for ROS (reactive oxygen species) scavenging in vivo, nanoceria imaging in plant tissues by confocal microscopy, and in vivo monitoring of nanoceria ROS scavenging by confocal microscopy.

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling ...

catalytic-scavenging-plant-reactive-oxygen-species-vivo-anionic

Nanyang Technological University

Research

JoVE Journal

Bioengineering

8.8K Views.  University of California. Hier presenteren we een protocol voor de synthese en de karakterisering van nanodeeltjes (nanoceria) oxide cerium voor ROS (reactieve zuurstof soorten) opruiming in vivo, nanoceria in plantaardige weefsels door confocale microscopie en in vivo imaging monitoring van nanoceria ROS opruiming door confocale microscopie.

Video: Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted through ion exchange. The resultant production bleed water (PBW) contains contaminants such as arsenic and other heavy metals. Samples of PBW from an active ISR uranium facility were treated with cupric oxide nanoparticles (CuO-NPs). CuO-NP treatment of PBW reduced priority contaminants, including arsenic, selenium, uranium, and vanadium. Untreated and CuO-NP treated PBW was used as the liquid component of the cell growth media and changes in viability were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in human embryonic kidney (HEK 293) and human hepatocellular carcinoma (Hep G2) cells. CuO-NP treatment was associated with improved HEK and HEP cell viability. Limitations of this method include dilution of the PBW by growth media components and during osmolality adjustment as well as necessary pH adjustment. This method is limited in its wider context due to dilution effects and changes in the pH of the PBW which is traditionally slightly acidic&#160;however; this method could have a broader use assessing CuO-NP treatment in more neutral waters.

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

Production bleed water (PBW) was treated with cupric oxide nanoparticles (CuO-NPs) and cellular toxicity was assessed in cultured human cells. The goal of this protocol was to integrate the native environmental sample into a cell culture format assessing the changes in toxicity due to CuO-NP treatment.

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted ...

Environment

A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12. Although tobacco tars and related products in the particle phase of cigarette smoke are major causes of carcinogenic and mutagenic related diseases, cigarette smoke contains significant amounts of free radicals that are also considered as an important group of carcinogens9,10. Free radicals attack cell constituents by damaging protein structure, lipids and DNA sequences and increase the risks of developing various types of cancers. Inhaled radicals produce adducts that contribute to many of the negative health effects of tobacco smoke in the lung3. Studies have been conducted to reduce free radicals in cigarette smoke to decrease risks of the smoking-induced damage. It has been reported that haemoglobin and heme-containing compounds could partially scavenge nitric oxide, reactive oxidants and carcinogenic volatile nitrosocompounds of cigarette smoke4. A 'bio-filter' consisted of haemoglobin and activated carbon was used to scavenge the free radicals and to remove up to 90% of the free radicals from cigarette smoke14. However, due to the cost-ineffectiveness, it has not been successfully commercialized. Another study showed good scavenging efficiency of shikonin, a component of Chinese herbal medicine8. In the present study, we report a protocol for introducing common natural antioxidant extracts into the cigarette filter for scavenging gas phase free radicals in cigarette smoke and measurement of the scavenge effect on gas phase free radicals in mainstream cigarette smoke (MCS) using spin-trapping Electron Spin Resonance (ESR) Spectroscopy1,2,14. We showed high scavenging capacity of lycopene and grape seed extract which could point to their future application in cigarette filters. An important advantage of these prospective scavengers is that they can be obtained in large quantities from byproducts of tomato or wine industry respectively11,13

Spin-trapping ESR spectroscopy was used to study the effect of plant antioxidants lycopene, pycnogenol and grape seed extract on scavenging gas-phase free radicals in cigarette smoke.

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12.  ...

Photochemical Oxidative Growth of Iridium Oxide Nanoparticles on CdSe@CdS Nanorods

We demonstrate a procedure for the photochemical oxidative growth of iridium oxide catalysts on the surface of seeded cadmium selenide-cadmium sulfide (CdSe@CdS) nanorod photocatalysts. Seeded rods are grown using a colloidal hot-injection method and then moved to an aqueous medium by ligand exchange. CdSe@CdS nanorods, an iridium precursor and other salts are mixed and illuminated. The deposition process is initiated by absorption of photons by the semiconductor particle, which results with formation of charge carriers that are used to promote redox reactions. To insure photochemical oxidative growth we used an electron scavenger. The photogenerated holes oxidize the iridium precursor, apparently in a mediated oxidative pathway. This results in the growth of high quality crystalline iridium oxide particles, ranging from 0.5 nm to about 3 nm, along the surface of the rod. Iridium oxide grown on CdSe@CdS heterostructures was studied by a variety of characterization methods, in order to evaluate its characteristics and quality. We explored means for control over particle size, crystallinity, deposition location on the CdS rod, and composition. Illumination time and excitation wavelength were found to be key parameters for such control. The influence of different growth conditions and the characterization of these heterostructures are described alongside a detailed description of their synthesis. Of significance is the fact that the addition of iridium oxide afforded the rods astounding photochemical stability under prolonged illumination in pure water (alleviating the requirement for hole scavengers).

A protocol for the photochemical oxidative growth of small crystalline iridium oxide nanoparticles on the surface of CdSe@CdS seeded rod nanoparticles is presented.

We demonstrate a procedure for the photochemical oxidative growth of iridium oxide catalysts on the surface of seeded cadmium selenide-cadmium sulfide ...

Chemistry

Use of Electron Paramagnetic Resonance in Biological Samples at Ambient Temperature and 77 K

The accurate and specific detection of reactive oxygen species (ROS) in different cellular and tissue compartments is essential to the study of redox-regulated signaling in biological settings. Electron paramagnetic resonance spectroscopy (EPR) is the only direct method to assess free radicals unambiguously. Its advantage is that it detects physiologic levels of specific species with a high specificity, but it does require specialized technology, careful sample preparation, and appropriate controls to ensure accurate interpretation of the data. Cyclic hydroxylamine spin probes react selectively with superoxide or other radicals to generate a nitroxide signal that can be quantified by EPR spectroscopy. Cell-permeable spin probes and spin probes designed to accumulate rapidly in the mitochondria allow for the determination of superoxide concentration in different cellular compartments.
In cultured cells, the use of cell permeable 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) along with and without cell-impermeable superoxide dismutase (SOD) pretreatment, or use of cell-permeable PEG-SOD,&#160;allows for the differentiation of extracellular from cytosolic superoxide. The mitochondrial 1-hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethyl-piperidine,1-hydroxy-2,2,6,6-tetramethyl-4-[2-(triphenylphosphonio)acetamido] piperidinium dichloride (mito-TEMPO-H) allows for measurement of mitochondrial ROS (predominantly superoxide).
Spin probes and EPR spectroscopy can also be applied to in vivo models. Superoxide can be detected in extracellular fluids such as blood and alveolar fluid, as well as tissues such as lung tissue. Several methods are presented to process and store tissue for EPR measurements and deliver intravenous 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH) spin probe in vivo. While measurements can be performed at room temperature, samples obtained from in vitro and in vivo models can also be stored at -80 &#176;C and analyzed by EPR at 77 K. The samples can be stored in specialized tubing stable at -80 &#176;C and run at 77 K to enable a practical, efficient, and reproducible method that facilitates storing and transferring samples.

Electron paramagnetic resonance (EPR) spectroscopy is an unambiguous method to measure free radicals. The use of selective spin probes allows for detection of free radicals in different cellular compartments. We present a practical, efficient method to collect biological samples that facilitate treating, storing, and transferring samples for EPR measurements.

The accurate and specific detection of reactive oxygen species (ROS) in different cellular and tissue compartments is essential to the study of ...

Biochemistry

The Effect of Interfacial Chemical Bonding in TiO2-SiO2 Composites on Their Photocatalytic NOx Abatement Performance

The chemical bonding of particulate photocatalysts to supporting material surfaces is of great importance in engineering more efficient and practical photocatalytic structures. However, the influence of such chemical bonding on the optical and surface properties of the photocatalyst and thus its photocatalytic activity/reaction selectivity behavior has not been systematically studied. In this investigation, TiO2 has been supported on the surface of SiO2 by means of two different methods: (i) by the in situ formation of TiO2 in the presence of sand quartz via a sol-gel method employing tetrabutyl orthotitanium (TBOT); and (ii) by binding the commercial TiO2 powder to quartz on a surface silica gel layer formed from the reaction of quartz with tetraethylorthosilicate (TEOS). For comparison, TiO2 nanoparticles were also deposited on the surfaces of a more reactive SiO2 prepared by a hydrolysis-controlled sol-gel technique as well as through a sol-gel route from TiO2 and SiO2 precursors. The combination of TiO2 and SiO2, through interfacial Ti-O-Si bonds, was confirmed by FTIR spectroscopy and the photocatalytic activities of the obtained composites were tested for photocatalytic degradation of NO according to the ISO standard method (ISO 22197&#8722;1). The electron microscope images of the obtained materials showed that variable photocatalyst coverage of the support surface can successfully be achieved but the photocatalytic activity towards NO removal was found to be affected by the preparation method and the nitrate selectivity is adversely affected by Ti-O-Si bonding.

The Effect of Interfacial Chemical Bonding in TiO2-SiO2 Composites on Their Photocatalytic NOx Abatement Performance

The focus of the present work is to establish means to generate and quantify levels of Ti-O-Si linkages and to correlate these with the photocatalytic properties of the supported TiO2.

The chemical bonding of particulate photocatalysts to supporting material surfaces is of great importance in engineering more efficient and practical ...

Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, fluorophores are prone to loss of signal via photobleaching by dissolved oxygen (O2). To prevent photobleaching and extend the fluorophore lifetime, oxygen scavenging systems (OSS) are employed to reduce O2. Commercially available OSS may be contaminated by nucleases that damage or degrade nucleic acids, confounding interpretation of experimental results. Here we detail a protocol for the expression and purification of highly active Pseudomonas putida protocatechuate-3,4-dioxygenase (PCD) with no detectable nuclease contamination. PCD can efficiently remove reactive O2 species by conversion of the substrate protocatechuic acid (PCA) to 3-carboxy-cis,cis-muconic acid. This method can be used in any aqueous system where O2 plays a detrimental role in data acquisition. This method is effective in producing highly active, nuclease free PCD in comparison with commercially available PCD.

Protocatechuate 3,4-dioxygenase (PCD) can enzymatically remove free diatomic oxygen from an aqueous system using its substrate protocatechuic acid (PCA). This protocol describes the expression, purification, and activity analysis of this oxygen scavenging enzyme.

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, ...

A Complete Method for Evaluating the Performance of Photocatalysts for the Degradation of Antibiotics in Environmental Remediation

Various antibiotics such as tetracycline, aureomycin, amoxicillin, and levofloxacin are found in large quantities in groundwater and soil systems, potentially leading to the development of resistant and multi-drug resistant bacteria, posing a threat to humans, animals, and environmental systems. Photocatalytic technology has attracted keen interest due to its rapid and stable treatment and direct use of solar energy. However, most studies evaluating the performance of semiconductor catalysts for the photocatalytic degradation of organic pollutants in water are currently incomplete. In this paper, a complete experimental protocol is designed to comprehensively evaluate the photocatalytic performance of semiconductor catalysts. Herein, rhombic dodecahedral silver phosphate was prepared by a simple solvent phase synthesis method at room temperature and atmospheric pressure. BrSubphthalocyanine/Ag3PO4 heterojunction materials were prepared by the solvothermal method. The catalytic performance of as-prepared materials for the degradation of tetracycline was evaluated by studying different influencing factors such as catalyst dosage, temperature, pH, and anions at atmospheric pressure using a 300 W xenon lamp as a simulated solar light source and a light intensity of 350 mW/cm2. Compared with the first cycle, the constructed BrSubphthalocyanine/Ag3PO4 maintained 82.0% of the original photocatalytic activity after five photocatalytic cycles, while the pristine Ag3PO4 maintained only 28.6%. The stability of silver phosphate samples was further tested by a five-cycle experiment. This paper provides a complete process for evaluating the catalytic performance of semiconductor catalysts in the laboratory for the development of semiconductor catalysts with potential for practical applications.

Presented here is a protocol to explore a universal set of experimental procedures for comprehensive laboratory evaluation of photocatalysts in the field of environmental purification, using the example of photocatalytic removal of antibiotic organic pollutant molecules from water by phthalocyanine sensitized silver phosphate composites.

Various antibiotics such as tetracycline, aureomycin, amoxicillin, and levofloxacin are found in large quantities in groundwater and soil systems, ...

A Continuous-flow Photocatalytic Reactor for the Precisely Controlled Deposition of Metallic Nanoparticles

In this work, a novel photocatalytic reactor for the pulsed and controlled excitation of the photocatalyst and the precise deposition of metallic nanoparticles is developed. Guidelines for the replication of the reactor and its operation are provided in detail. Three different composite systems (Pt/graphene, Pt/TiO2, and Au/TiO2) with monodisperse and uniformly distributed particles are produced by this reactor, and the photodeposition mechanism, as well as the synthesis optimization strategy, are discussed. The synthesis methods and their technical aspects are described comprehensively. The role of the ultraviolet (UV) dose (in each excitation pulse) on the photodeposition process is investigated and the optimum values for each composite system are provided.

For a continuous and scalable synthesis of noble-metal-based nanocomposites, a novel photocatalytic reactor is developed and its structure, operation principles, and product quality optimization strategies are described.

In this work, a novel photocatalytic reactor for the pulsed and controlled excitation of the photocatalyst and the precise deposition of metallic ...

Cercosporin-Photocatalyzed [4+1]- and [4+2]-Annulations of Azoalkenes Under Mild Conditions

The interest on nitrogen-containing heterocycles has expanded rapidly in the synthetic community since they are important motifs for new drugs. Traditionally, they were synthesized through thermal cycloaddition reactions, whereas today, photocatalysis is preferred due to the mild and efficient conditions. With this focus, a new photocatalytic method for the synthesis of nitrogen-containing heterocycles is highly desired. Here, we report a protocol for the biosynthesis of cercosporin, which could function as a metal-free photocatalyst. We then illustrate cercosporin-photocatalyzed protocols for the synthesis of nitrogen-containing heterocycles 1,2,3-thiadiazoles through annulation of azoalkenes with KSCN, and synthesis of 1,4,5,6-tetrahydropyridazines [4+2] through cyclodimerization of azoalkenes under mild conditions, respectively. As a result, there is a new bridge between the microbial fermentation method and organic synthesis in a mild, cost-effective, environmentally friendly and sustainable manner.

New routes for the synthesis of nitrogen-containing heterocycles utilizing cercosporin as a metal-free photocatalyst were developed.

The interest on nitrogen-containing heterocycles has expanded rapidly in the synthetic community since they are important motifs for new drugs. ...

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

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