Name: investigation of xenobiotics metabolism in salix alba leaves via mass spectrometry imaging
Uploaded: 2020-06-15T12:30:00
Description: Watch this Scientific Journal Video about Investigation of Xenobiotics Metabolism In Salix alba Leaves via Mass Spectrometry Imaging at JoVE.com

We thank Charles Pineau, M&#233;lanie Lagarrigue and R&#233;gis Lavigne for their tips and tricks regarding sample preparation for MALDI imaging of plant samples.

1. Sample preparation
<ol>
	<li>Obtain the biological sample and either keep it fresh and intact (e.g., do not force it into a tube) or freeze it. The proposed protocol applies to any type of solid biological sample (i.e., plant, animal, or human tissues) to localize compounds in specific tissues.</li>
	<li>Cool down a cryomicrotome to -20 &#176;C. Keep the sample holder and the blade at the same temperature.</li>
	<li>If necessary, embed the object in M1 embedding medium to preserve it during cutting.
	<ol>
		<li>Pour...

<ol>
	<li>Zhang, D., Gersberg, R. M., Ng, W. J., Tan, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+pharmaceuticals+and+personal+care+products+in+aquatic+plant-based+systems:+A+review.">Removal of pharmaceuticals and personal care products in aquatic plant-based systems: A review.</a> Environmental Pollution. 184, 620-639 (2014).</li><li>Adeel, M., Song, X., Wang, Y., Francis, D., Yang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+impact+of+estrogens+on+human,+animal+and+plant+life:+A+critical+review.">Environmental impact of estrogens on human, animal and plant life: A critical review.</a> Environment International. 99, 107-119 (2017).</li><li>Prosser, R. S., Sibley, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+health+risk+assessment+of+pharmaceuticals+and+personal+care+products+in+plant+tissue+due+to+biosolids+and+manure+amendments,+and+wastewater+irrigation.">Human health risk assessment of pharmaceuticals and personal care products in plant tissue due to biosolids and manure amendments, and wastewater irrigation.</a> Environment International. 75, 223-233 (2015).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+biochar+to+soils+may+result+in+plant+contamination+and+human+cancer+risk+due+to+exposure+of+polycyclic+aromatic+hydrocarbons.">Application of biochar to soils may result in plant contamination and human cancer risk due to exposure of polycyclic aromatic hydrocarbons.</a> Environment International. 121, 169-177 (2018).</li><li>Marsik, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+ibuprofen+in+higher+plants:+A+model+Arabidopsis+thaliana+cell+suspension+culture+system.">Metabolism of ibuprofen in higher plants: A model Arabidopsis thaliana cell suspension culture system.</a> Environmental Pollution. 220, 383-392 (2017).</li><li>He, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+ibuprofen+by+Phragmites+australis:+uptake+and+phytodegradation.">Metabolism of ibuprofen by Phragmites australis: uptake and phytodegradation.</a> Environmental Science and Technology. 51 (8), 4576-4584 (2017).</li><li>Huber, C., Bartha, B., Harpaintner, R., Schr&#246;der, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+acetaminophen+(paracetamol)+in+plants-two+independent+pathways+result+in+the+formation+of+a+glutathione+and+a+glucose+conjugate.">Metabolism of acetaminophen (paracetamol) in plants-two independent pathways result in the formation of a glutathione and a glucose conjugate.</a> Environmental Science and Pollution Research. 16 (2), 206-213 (2009).</li><li>Thomas, F., C&#233;bron, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+rhizosphere+effect+on+available+carbon+sources,+phenanthrene+degradation,+and+active+microbiome+in+an+aged-contaminated+industrial+soil.">Short-term rhizosphere effect on available carbon sources, phenanthrene degradation, and active microbiome in an aged-contaminated industrial soil.</a> Frontiers in Microbiology. 7, 1-15 (2016).</li><li>Villette, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+localization+of+micropollutants+and+associated+stress+response+in+Populus+nigra+leaves.">In situ localization of micropollutants and associated stress response in Populus nigra leaves.</a> Environment International. 126, 523-532 (2019).</li><li>Sandermann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+metabolism+of+organic+xenobiotics.+Status+and+prospects+of+the+'Green+Liver'+concept.">Plant metabolism of organic xenobiotics. Status and prospects of the 'Green Liver' concept.</a> Plant Biotechnology and In Vitro Biology in the 21st Century. , 321-328 (1999).</li><li>Sula, B., Deveci, E., &#214;zevren, H., Ekinci, C., Elbey, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+and+histopathological+changes+in+the+skin+of+rats+after+administration+of+lead+acetate.">Immunohistochemical and histopathological changes in the skin of rats after administration of lead acetate.</a> International Journal of Morphology. 34 (3), 918-922 (2016).</li><li>Villette, C., Maurer, L., Wanko, A., Heintz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenobiotics+metabolization+in+Salix+alba+leaves+uncovered+by+mass+spectrometry+imaging.">Xenobiotics metabolization in Salix alba leaves uncovered by mass spectrometry imaging.</a> Metabolomics. 15, 122 (2019).</li><li>Khatib-Shahidi, S., Andersson, M., Herman, J. L., Gillespie, T. A., Caprioli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Molecular+Analysis+of+Whole-Body+Animal+Tissue+Sections+by+Imaging+MALDI+Mass+Spectrometry.">Direct Molecular Analysis of Whole-Body Animal Tissue Sections by Imaging MALDI Mass Spectrometry.</a> Analytical Chemistry. 78 (18), 6448-6456 (2006).</li></ol>

The critical part of this protocol is the sample preparation: the sample must be soft and intact. Cutting is the most difficult part, as the temperature and thickness of the sample can vary depending on the type of sample studied. Animal tissues are usually homogeneous and easier to cut. Plant samples often incorporate different structures and therefore are more difficult to keep intact as the blade encounters soft, hard, or empty vascular tissues. It is highly recommended to use fresh tissues when working with plant sam...

Xenobiotics are widely distributed around the world due to human activities. Some of these compounds are water-soluble and absorbed by soil1, and enter the food chain when they accumulate in plant tissues2,3,4. The plants are eaten by insects and herbivores, which are prey to other organisms. The intake of some xenobiotics and their impact on a plant&#8217;s health have been described5,6,7,8, but only recently at a tissue level9. Therefore, it is still unclear where or how the metabolism of xenobiotics occurs, or if specific plant metabolites are correlated to xenobiotic accumulation in specific tissues10. Moreover, most research has overlooked the metabolism of xenobiotics and their metabolites in plants, so little is known about these reactions in plant tissues.
Proposed here is a method to investigate enzymatic reactions in biological samples that can be associated to the tissue localization of substrates and products of the reactions. The method can draw the complete metabolic profile of a biological sample in one experiment, as the analysis is non-targeted and can be investigated using custom lists of analytes of interest. Provided is a list of candidates tracked in the original dataset. If one or several analytes of interest are noted in the sample, the specific tissue localization can provide important information on the related biological processes. The analytes of interest can then be modified in silico using relevant biological laws to search for possible products/metabolites. The list of metabolites obtained is then used to analyze the original data by identifying the enzymes involved and localizing the reactions in the tissues, thus helping to understand the occurring metabolic processes. No other method provides information on the types of reactions occurring in the biological samples, the localization of the compounds of interest, and their related metabolites. This method can be used on any type of biological material once fresh and intact tissues are available and the compounds of interest can be ionized. The proposed protocol was published in Villette et al.12 and is detailed here for use by the scientific community.

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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Cover slips</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td align="right" style="width:119px;">267942</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Cryomicrotome</td>
			<td style="width:163px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Excel</td>
			<td style="width:163px;">Microsoft corporation</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">flexImaging</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">ftmsControl</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">GTX primescan</td>
			<td style="width:163px;">GX Microscopes</td>
			<td style="width:119px;"></td>
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			<td height="20" style="height:20px;width:216px;">HCCA MALDI matrix</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td align="right" style="width:119px;">8201344</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">ImagePrep</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td style="width:119px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">ITO-coated slides</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td align="right" style="width:119px;">237001</td>
			<td></td>
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			<td height="20" style="height:20px;width:216px;">M1-embedding matrix</td>
			<td style="width:163px;">ThermoScientific</td>
			<td align="right" style="width:119px;">1310</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Metabolite Predict</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Metaboscape</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Methanol</td>
			<td style="width:163px;">Fisher Chemicals</td>
			<td style="width:119px;"></td>
			<td>No specific reference needed</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">MX 35 Ultra blades</td>
			<td style="width:163px;">Thermo Scientific</td>
			<td align="right" style="width:119px;">15835682</td>
			<td></td>
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			<td height="20" style="height:20px;width:216px;">Plastic molds</td>
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			<td>No specific reference needed</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">SCiLS Lab</td>
			<td style="width:163px;">Bruker Daltonics</td>
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			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">SolariX XR 7Tesla</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td style="width:119px;"></td>
			<td>The method proposed is not limited to this instrument</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Spray sheets for ImagePrep</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td align="right" style="width:119px;">8261614</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">TFA</td>
			<td style="width:163px;">Sigma Aldrich</td>
			<td style="width:119px;"></td>
			<td>No specific reference needed</td>
		</tr>
	</tbody>
</table>

investigation of xenobiotics metabolism in salix alba leaves via mass spectrometry imaging

The method presented uses mass spectrometry imaging (MSI) to establish the metabolic profile of S. alba leaves when exposed to xenobiotics. Using a non-targeted approach, plant metabolites and xenobiotics of interest are identified and localized in plant tissues to uncover&#160;specific distribution patterns. Then, in silico prediction of potential metabolites (i.e., catabolites and conjugates) from the identified xenobiotics is performed. When a xenobiotic metabolite is located in the tissue, the type of enzyme involved in its alteration by the plant is recorded. These results were used to describe different types of biological reactions occurring in S. alba leaves in response to xenobiotic accumulation in the leaves. The metabolites were predicted in two generations, allowing the documentation of successive biological reactions to transform xenobiotics in the leaf tissues.

This protocol was applied to fresh leaves sampled from a S. alba tree exposed to xenobiotics in the environment. The process is depicted in Figure 1. The first step is to prepare thin slices of the sample of interest. Plant samples are often more difficult to cut than animal samples, as the tissues are heterogeneous and can contain water and/or air. This difficulty is handled using embedding medium, which forms a homogeneous block around the sample. The matrix deposition is facilita...

Watch this Scientific Journal Video about Investigation of Xenobiotics Metabolism In Salix alba Leaves via Mass Spectrometry Imaging at JoVE.com

Investigation of Xenobiotics Metabolism In Salix alba Leaves via Mass Spectrometry Imaging

This method uses mass spectrometry imaging (MSI) to understand metabolic processes in S. alba leaves when exposed to xenobiotics. The method allows the spatial localization of compounds of interest and their predicted metabolites within specific, intact tissues.

Investigation of Xenobiotics Metabolism In Salix alba Leaves via Mass Spectrometry Imaging

The method presented uses mass spectrometry imaging (MSI) to establish the metabolic profile of S. alba leaves when exposed to xenobiotics. Using a ...

This protocol allows direct identification of predicted metabolites in situ. It facilitates the determination of the type of tissues or cells that are affected by a specific metabolic process. This method is quick and straightforward.It does not require a complicated sample preparation or compound extraction, and it allows the identification and localization of low abundant compounds. Upon acquisition of the biological sample, cool a cryo microtome sample holder and blade to minus 20 degrees Celsius. If necessary, pour embedding medium into a plastic mold placed within the cryo microtome chamber and quickly add the sample to the medium.Cover the sample with additional embedding medium and adjust the position of the sample to the center of the mold as necessary as the matrix solidifies. When the sample is ready, use embedding medium to mount the tissue onto the cryo microtome holder and use the cooled blade to cut the tissue into appropriate sized sections, modifying the cutting thickness and temperature as necessary to obtain optimal samples. As each section is acquired, use an appropriate tool to carefully move the slice to an indium tin oxide coated glass microscope slide, and place a finger under the slide to warm and dry the sample before placing the slide into the cryo microtome chamber.When all of the slices have been acquired, slowly remove the slides from the chamber and use an appropriate marker to indicate the exact location of each sample on the slides. When all of the slides have been marked, use a high-resolution scanner to scan the samples. After scanning, clean the matrix deposition robot with 100%methanol, and use methanol and a precision wipe to clean each slide, holding the samples without touching them and without removing the marks.Place a clean coverslip over each slide in an area without sample and place the coverslip end of the first slide over the optical detector, then place no more than six milliliters of freshly prepared matrix solution in the matrix deposition reservoir and add two milliliters of 100%methanol to a total volume of eight milliliters. For data acquisition set up, place one microliter of matrix onto a MALDI plate and insert the plate into the source. Click load target in the device software and click on the spot in the image that represents the MALDI plate to select the position of the matrix spot.Indicate the sample name and folder in the sample info tab. Click acquisition to start the acquisition and use the mouse pointer to move the plate slightly so that the laser points at different spots. When the acquisition is finished, open the calibration tab and select HCCA calibration list in the quadratic mode and click automatic.The global calibration result will be indicated in the calibration plot window. If the calibration is in the appropriate range, click accept and save the method. Once the matrix deposition on the samples is finished, place the slides in the slide adapter and use a plastic cover to retrieve the position of the marks.In the imaging software, set up a new imaging run in the first window that opens in the software and name the imaging run. Select the result directory and click next. Indicate the raster size, select the method to be used, and click next.Load the optical image from a scanned slide and click next. The image will open in a wider window and place the plastic cover bearing the marks onto a MALDI plate to recover its position and to facilitate the teaching. Under FTMS control, place the target of the MALDI video window on the exact position of a mark and open flex imaging, then click on the exact same point on the optical image.Use the add measurement regions tools to draw the regions of interest in the samples and save the imaging run. If several samples are to be analyzed, run the auto execute sequence and use auto execute batch runner to launch a sequence. When all of the images have been acquired, use the batch importer tool to select the raw data, indicate the target directory, and click import.Click file and new to select the data set tool to select the type of instrument used for the acquisition and click plus to add the imported dataset. Click and drag to arrange the images and check and modify the mass range settings as necessary. Click next to see the import summary and launch the import.Visualize the master charge ratio in different tissues within a sample or between different samples and click spectra to select the mass-to-charge ratios of interest. Export the mass-to-charge ratios of interest as a CSV file from the object tab and click export. Create a new dataset in an annotation software program and click projects and import CSV project to import the CSV file into the software.Annotate with custom-made analyte lists, which can be derived from publicly available databases. A template is given by the software to create analyte lists. Use the prediction software to perform in silico prediction of the metabolites of the annotated compounds.Recover the list of metabolites, create an analyte list, and use the list in the annotation software to annotate the raw data with predicted metabolites, then right-click on the predicted metabolite of interest to recover the names of the enzymes involved in the metabolic processes in the prediction software. In this example, the drug Telmisartan was determined to be distributed throughout the plant leaf tissue samples. The drug metabolites were predicted and searched for in the raw data, and the annotations revealed that one first-generation metabolite was detected in the internal tissues of the leaves and further degraded into second-generation metabolites that were localized in internal tissues or more generally distributed in all of the leaf tissues.These results suggest an active metabolic reaction in the leaves to degrade Telmisartan. The process was then applied to several compounds of interest annotated in the leaves and the enzymes involved in the reactions were recovered to investigate their role in the plant's response to xenobiotic accumulation. Although this procedure is quite simple, a good sample preparation is key.Be sure to take the time to find the parameters that fit your sample type during tissue sectioning. The sample size can be further used for microscopy observations. It can be stained to obtain a panel of complimentary biological observations.

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Environment

Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil

New techniques and methods are being sought to try to win the battle against mosquitoes. Recent advances in molecular techniques have led to the development of new and innovative methods of mosquito control based around the Sterile Insect Technique (SIT)1-3. A control method known as RIDL&#160;(Release of Insects carrying a Dominant Lethal)4, is based around SIT, but uses genetic methods to remove the need for radiation-sterilization5-8. A RIDL&#160;strain of Ae. aegypti was successfully tested in the field in Grand Cayman9,10; further field use is planned or in progress in other countries around the world.
Mass rearing of insects has been established in several insect species and to levels of billions a week. However, in mosquitoes, rearing has generally been performed on a much smaller scale, with most large scale rearing being performed in the 1970s and 80s. For a RIDL&#160;program it is desirable to release as few females as possible as they bite and transmit disease. In a mass rearing program&#160;there are several stages to produce the males to be released: egg production, rearing eggs until pupation, and then sorting males from females before release. These males are then used for a RIDL&#160;control program,&#160;released as either pupae or adults11,12.
To suppress a mosquito population using RIDL&#160;a large number of high quality male adults need to be reared13,14. The following describes the methods for the mass rearing of OX513A, a RIDL&#160;strain of Ae. aegypti 8, for release and covers the techniques required for the production of eggs and mass rearing RIDL&#160;males for a control program.

Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil

To achieve population suppression of Aedes aegypti using the RIDL&#174; (Release of Insects carrying a Dominant Lethal) system, large numbers of male mosquitoes need to be released. This requires the use of mass rearing techniques and technology to provide reliable systems to obtain the maximum number of high quality male mosquitoes.

New techniques and methods are being sought to try to win the battle against mosquitoes. Recent advances in molecular techniques have led to the ...

Optimize Flue Gas Settings to Promote Microalgae Growth in Photobioreactors via Computer Simulations

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently than plants3, but also synthesize advanced biofuels2-4. Generally, atmospheric CO2 is not a sufficient source for supporting maximal algal growth5. On the other hand, the high concentrations of CO2 in industrial exhaust gases have adverse effects on algal physiology. Consequently, both cultivation conditions (such as nutrients and light) and the control of the flue gas flow into the photo-bioreactors are important to develop an efficient &ldquo;flue gas to algae&rdquo; system. Researchers have proposed different photobioreactor configurations4,6 and cultivation strategies7,8 with flue gas. Here, we present a protocol that demonstrates how to use models to predict the microalgal growth in response to flue gas settings. We perform both experimental illustration and model simulations to determine the favorable conditions for algal growth with flue gas. We develop a Monod-based model coupled with mass transfer and light intensity equations to simulate the microalgal growth in a homogenous photo-bioreactor. The model simulation compares algal growth and flue gas consumptions under different flue-gas settings. The model illustrates: 1) how algal growth is influenced by different volumetric mass transfer coefficients of CO2; 2) how we can find optimal CO2 concentration for algal growth via the dynamic optimization approach (DOA); 3) how we can design a rectangular on-off flue gas pulse to promote algal biomass growth and to reduce the usage of flue gas. On the experimental side, we present a protocol for growing Chlorella under the flue gas (generated by natural gas combustion). The experimental results qualitatively validate the model predictions that the high frequency flue gas pulses can significantly improve algal cultivation.

Flue gas from power plants is a cheap CO2 source for algal growth. We have built prototype &#34;flue gas to algal cultivation&#34; systems and described how to scale up the algal cultivation process. We have demonstrated the use of a mass-transfer bio-reaction model to simulate and to design the optimal operation of flue gas for the growth of Chlorella sp. in algal photo-bioreactors.

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently ...

An Aquatic Microbial Metaproteomics Workflow: From Cells to Tryptic Peptides Suitable for Tandem Mass Spectrometry-based Analysis

Meta-omic technologies such as metagenomics, metatranscriptomics and metaproteomics can aid in the understanding of microbial community structure and metabolism. Although powerful, metagenomics alone can only elucidate functional potential. On the other hand, metaproteomics enables the description of the expressed in situ metabolism and function of a community. Here we describe a protocol for cell lysis, protein and DNA isolation, as well as peptide digestion and extraction from marine microbial cells collected on a cartridge filter unit (such as the Sterivex filter unit) and preserved in an RNA stabilization solution (like RNAlater). In mass spectrometry-based proteomics studies, the identification of peptides and proteins is performed by comparing peptide tandem mass spectra to a database of translated nucleotide sequences. Including the metagenome of a sample in the search database increases the number of peptides and proteins that can be identified from the mass spectra. Hence, in this protocol DNA is isolated from the same filter, which can be used subsequently for metagenomic analysis.

This protocol is for the extraction and concentration of protein and DNA from microbial biomass collected from seawater, followed by the generation of tryptic peptides suitable for tandem mass spectrometry-based proteomic analysis.

Meta-omic technologies such as metagenomics, metatranscriptomics and metaproteomics can aid in the understanding of microbial community structure and ...

Measuring Rates of Herbicide Metabolism in Dicot Weeds with an Excised Leaf Assay

In order to isolate and accurately determine rates of herbicide metabolism in an obligate-outcrossing dicot weed, waterhemp (Amaranthus tuberculatus), we developed an excised leaf assay combined with a vegetative cloning strategy to normalize herbicide uptake and remove translocation as contributing factors in herbicide-resistant (R) and &#8211;sensitive (S) waterhemp populations. Biokinetic analyses of organic pesticides in plants typically include the determination of uptake, translocation (delivery to the target site), metabolic fate, and interactions with the target site. Herbicide metabolism is an important parameter to measure in herbicide-resistant weeds and herbicide-tolerant crops, and is typically accomplished with whole-plant tests using radiolabeled herbicides. However, one difficulty with interpreting biokinetic parameters derived from whole-plant methods is that translocation is often affected by rates of herbicide metabolism, since polar metabolites are usually not mobile within the plant following herbicide detoxification reactions. Advantages of the protocol described in this manuscript include reproducible, accurate, and rapid determination of herbicide degradation rates in R and S populations, a substantial decrease in the amount of radiolabeled herbicide consumed, a large reduction in radiolabeled plant materials requiring further handling and disposal, and the ability to perform radiolabeled herbicide experiments in the lab or growth chamber instead of a greenhouse. As herbicide resistance continues to develop and spread in dicot weed populations worldwide, the excised leaf assay method developed and described herein will provide an invaluable technique for investigating non-target site-based resistance due to enhanced rates of herbicide metabolism and detoxification.

This manuscript describes how herbicide metabolism rates can be effectively quantified with excised leaves from a dicot weed, thereby reducing variability and removing any possible confounding effects of herbicide uptake or translocation typically observed in whole-plant assays.

In order to isolate and accurately determine rates of herbicide metabolism in an obligate-outcrossing dicot weed, waterhemp (Amaranthus tuberculatus), we ...

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic pathways requires a model in which external factors can be well controlled, allowing for reproducible and meaningful results. Many studies employ transformed cellular models for these purposes; however, metabolic reprogramming that occurs in many cancer cell lines may introduce confounding variables. For this reason primary cells are desirable, though attaining adequate biomass for metabolic studies can be challenging. Here we show that human platelets can be utilized as a platform to carry out metabolic studies in combination with liquid chromatography-tandem mass spectrometry analysis. This approach is amenable to relative quantification and isotopic labeling to probe the activity of specific metabolic pathways. Availability of platelets from individual donors or from blood banks makes this model system applicable to clinical studies and feasible to scale up. Here we utilize isolated platelets to confirm previously identified compensatory metabolic shifts in response to the complex I inhibitor rotenone. More specifically, a decrease in glycolysis is accompanied by an increase in fatty acid oxidation to maintain acetyl-CoA levels. Our results show that platelets can be used as an easily accessible and medically relevant model to probe the effects of xenobiotics on cellular metabolism.

Here we show isolated human platelets can be used as an accessible ex vivo model to study metabolic adaptations in response to the complex I inhibitor rotenone. This approach employs isotopic tracing and relative quantification by liquid chromatography-mass spectrometry and can be applied to a variety of study designs.

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic ...

Determination of Inorganic Arsenic in a Wide Range of Food Matrices using Hydride Generation - Atomic Absorption Spectrometry.

The European Food Safety Authority (EFSA) underlined in its Scientific Opinion on Arsenic in Food that in order to support a sound exposure assessment to inorganic arsenic through diet, information about distribution of arsenic species in various food types must be generated. A method, previously validated in a collaborative trial, has been applied to determine inorganic arsenic in a wide variety of food matrices, covering grains, mushrooms and food of marine origin (31 samples in total). The method is based on detection by flow injection-hydride generation-atomic absorption spectrometry of the iAs selectively extracted into chloroform after digestion of the proteins with concentrated HCl. The method is characterized by a limit of quantification of 10 &#181;g/kg dry weight, which allowed quantification of inorganic arsenic in a large amount of food matrices. Information is provided about performance scores given to results obtained with this method and which were reported by different laboratories in several proficiency tests. The percentage of satisfactory results obtained with the discussed method is higher than that of the results obtained with other analytical approaches.

The usefulness of an analytical method to determine inorganic arsenic in a wide range of food matrices is demonstrated. The method consists of selective extraction of inorganic arsenic into chloroform with a final determination by hydride generation-atomic absorption spectrometry.

The European Food Safety Authority (EFSA) underlined in its Scientific Opinion on Arsenic in Food that in order to support a sound exposure assessment to ...

Assessing the Particulate Matter Removal Abilities of Tree Leaves

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on collecting various sized particulate matter (PM) retained on leaf surfaces. We further characterized the retention efficiency of leaves to various sized PM, which will help to assess the abilities of urban trees to remove PM from ambient air quantitatively. 
Taking three broadleaf tree species (Ginkgo biloba, Sophora japonica, and Salix babylonica) and two needleleaf tree species (Pinus tabuliformis and Sabina chinensis) as the research objects, leaf samples were collected 4 days (short PM retention period) and 14 days (long PM retention period) after the latest rainfall. PM retained on the leaf surfaces was collected by means of WC, BC, and UC in sequence. Then, retention efficiencies of leaves (AEleaf) to three types of the various sized PM, including easily removable PM (ERP), difficult-to-remove PM (DRP), and totally removable PM (TRP), were calculated. Only around 23%-45% of the total PM retained on leaves could be cleaned off and collected by WC. When the leaves were cleaned through WC+BC, the underestimation of the PM retention capacity of different tree species was in the range of 29%-46% for various sized PM. Almost all PM retained on leaves could be removed if UC was supplemented to WC+BC. 
In conclusion, if the UC was complemented after the conventional cleaning methods, more PM on leaf surfaces could be eluted and collected. The procedure developed in this study can be used for assessing the PM removal abilities of different tree species.

The ultrasonic cleaning method was applied to elute the particulate matter (PM) retained on leaf surfaces after PM was eluted by the conventional cleaning methods (water cleaning only or water cleaning plus brush cleaning). The methodology can help to improve the estimation accuracy for PM retention capacity of leaves.

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on ...

Identifying Per- and Polyfluorinated Chemical Species with a Combined Targeted and Non-Targeted-Screening High-Resolution Mass Spectrometry Workflow

Historical and emerging per- and polyfluoroalkyl substances (PFASs) have garnered significant interest from the public and government agencies from the local to federal levels. The continuing evolution of PFAS chemistries presents a challenge to the environmental monitoring, where ongoing development of targeted methods necessarily lags the discovery of new chemical compounds. There is a need, therefore, to have forward-looking methodologies that can detect emerging and unexpected compounds, monitor these species over time, and resolve details of their chemical structure to enable future work in human health. To this end, non-targeted analysis by high-resolution mass spectrometry offers a broad base detection approach that can be combined with almost any sample preparation scheme and provides significant capabilities for compound identification after detection. Herein, we describe a solid-phase extraction (SPE) based sample concentration method tuned for shorter chain and more hydrophilic PFAS chemistries, such as per fluorinated ether acids and sulfonates, and describe analysis of samples prepared in this fashion in both targeted and non-targeted modes. Targeted methods provide superior quantification when reference standards are available but are intrinsically limited to expected compounds when performing analysis. In contrast, a non-targeted approach can identify the presence of unexpected compounds and provide some information about their chemical structure. Information about chemical features can be used to correlate compounds across sample locations and track abundance and occurrence over time.

Here, we present a protocol for the sequential targeted quantification and non-targeted analysis of fluorinated compounds in water by mass spectrometry. This methodology provides quantitative levels of known fluorochemical compounds and identifies unknown chemicals in related samples with semi-quantitative estimates of their abundance.

Historical and emerging per- and polyfluoroalkyl substances (PFASs) have garnered significant interest from the public and government agencies from the ...

Investigation of Plant Interactions Across Common Mycorrhizal Networks Using Rotated Cores

Arbuscular mycorrhizal (AM) fungi influence plant mineral nutrient uptake and growth, hence, they have the potential to influence plant interactions. The power of their influence is in extraradical mycelia that spread beyond nutrient depletion zones found near roots to ultimately interconnect individuals within a common mycorrhizal network (CMN). Most experiments, however, have investigated the role of AM fungi in plant interactions by growing plants with versus without mycorrhizal fungi, a method that fails to explicitly address the role of CMNs. Here, we propose a method that manipulates CMNs to investigate their role in plant interactions. Our method uses modified containers with conical bottoms with a nylon mesh and/or hydrophobic material covering slotted openings, 15N fertilizer, and a nutrient-poor interstitial sand. CMNs are left either intact between interacting individuals, severed by rotation of containers, or prevented from forming by a solid barrier. Our findings suggest that rotating containers is sufficient to disrupt CMNs and prevent their effects on plant interactions across CMNs. Our approach is advantageous because it mimics aspects of nature, such as seedlings tapping into already established CMNs and the use of a suite of AM fungi that may provide diverse benefits. Although our experiment is limited to investigating plants at the seedling stage, plant interactions across CMNs can be detected using our approach which therefore can be applied to investigate biological questions about the functioning of CMNs in ecosystems.

Most plants within communities likely are interconnected by arbuscular mycorrhizal (AM) fungi, but mediation of plant interactions by them has been investigated primarily by growing plants with versus without mycorrhizas. We present a method to manipulate common mycorrhizal networks among mycorrhizal plants to investigate their consequences for plant interactions.

Arbuscular mycorrhizal (AM) fungi influence plant mineral nutrient uptake and growth, hence, they have the potential to influence plant interactions. The ...

Mass-Rearing and Molecular Studies in Tortricidae Pest Insects

Tortricidae (Lepidoptera), commonly known as tortrix or leafroller moths, comprises many agricultural and forestry pests, which cause serious agricultural losses. To understand the biology of such pest moths, fundamental techniques have been in high demand. Here, methods for mass-rearing, observations, and molecular studies are developed using two tea tortrix, Homona magnanima and Adoxophyes honmai (Lepidoptera: Tortricidae). Insects were mass-reared with sliced artificial diet and maintained by inbreeding for over 100 generations by considering their biological characteristics. Insects have various sex dimorphisms; hence it is difficult to distinguish the sex during the developing stages, which have prevented subsequent assays. The present work highlighted that the sex of tortricids larvae could be determined by observing testes or lactic-acetic orcein staining to visualize the female-specific W chromosome. Moreover, using the sex determination methods, the present study enabled nucleic acid extractions from sex determined embryos and application toward high throughput sequencing. These tips are applicable for other pest insects and will facilitate further morphological and genetic studies.

The present protocol describes the rearing method of tortricid pest insects in the laboratories. The procedures to distinguish insects' sex and extract nucleic acids for high throughput sequencing are established using two tortricid pests.

Tortricidae (Lepidoptera), commonly known as tortrix or leafroller moths, comprises many agricultural and forestry pests, which cause serious agricultural ...