The authors would like to thank California Department of Food and Agriculture for funding of the project which led to the development of this protocol. CDFA SCB11019.

1. Planting
<ol>
	<li>Fill plug trays (128 cell; 28 x 54 cm with cells 3 cm square and 5 cm deep) with plug soil mix. To aid in uniform filling of cells compress soil in each tray using an empty plug tray.</li>
	<li>Plant lettuce seed &#188; inch in depth 2-3 seeds per cell. Plant all experimental lines in replicated trays to provide both a drought stressed and control trays for the drought trial.</li>
	<li>Place plug trays into a base tray without holes.</li>
	<li>Water trays and cover with inverted tray or plastic dome. Germination normally occurs in 48-72 hr. After germination remove the cover and move trays into greenhouse. 
	NOTE: This protocol could also be performed in growth chambers.</li>
</ol>
2. Growth and Plant Care
<ol>
	<li>Water trays as needed from 1-4 weeks after germination, normally this is 2-3 times weekly, by filling lower tray and allowing soil to become saturated (~1 hr)&#160;rays to sit for extended periods in water as this may negatively impact plant health and root development and may affect the drought screen.</li>
	<li>Fertilize trays once weekly with a commercial soluble 20-20-20 fertilizer mixed at a rate of 1.5 tsp/gal.</li>
	<li>Thin trays to one plant per cell 1-2 weeks post germination.</li>
</ol>
3. Initiating Drought Stress
<ol>
	<li>When the plants are four weeks old separate trays into 2 groups. Subject one group to severe water stress. Use the other trays as well watered controls. Water the control group as in step 2.1 throughout the experiment. At the initiation of the drought stress suspend fertilizing all trays for the remainder of the experiment.</li>
	<li>The experimental drought period occurs over one week. On the initial day of the drought stress trial (Day 0) fill all lower trays with water and allow the soil in the plug trays to become fully saturated then drain water from trays. Saturating the soil immediately before the initiation of the stress period will help to minimize any variation in soil moisture between individual cells in the plug trays.</li>
</ol>
4. Conducting the Basic Drought Stress Screen with Parameters to be Measured
NOTE: The following steps outline how to collect the three physiological measurements that will be recorded over the course of the drought screen while Table 1 provides a schedule of the measurements taken on each day of the drought-stress trial period.
<ol>
	<li>Measure the Leaf Relative Water Content (RWC) on Days 0, 2, 4 of the trial.
	<ol>
		<li>Clip a lettuce plant from the tray and remove 2 leaves.</li>
		<li>Punch 3 discs from each leaf using a #9 cork borer (~ 1.6 cm diameter). Combine leaf punches as one sample.</li>
		<li>Weigh the leaf discs, this is the fresh weight (FW) value used for the RWC calculation, then place the discs in a petri plate.</li>
		<li>Add just enough Distilled water to the plate to allow all leaf discs to float. This is to fully hydrate the leaf discs to gather turgid weights for RWC analysis. Allow the leaf discs to float for 24 hr at RT.</li>
		<li>Remove the now fully hydrated leaf discs from the plates and gently dry the exterior of the leaf discs with paper towels before weighing the discs. Record these weights as turgid weight (TW) for the RWC calculation.</li>
		<li>Place leaf discs on a paper lab wipe or filter paper disc in an open petri dish and dry in incubator at 55 &#176;C for 24 hr. 
		NOTE: The paper prevents the leaf discs from adhering to the petri dish during drying.</li>
		<li>Weigh dry leaf discs and record as dry weights (DW) for RWC calculation.</li>
		<li>Calculate RWC using Weatherley&#8217;s formula as described by Smart and Bingham11,12. 
		&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; RWC=(FW-DW/TW-DW)*100 
		&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; FW=fresh weight, TW=turgid weight, DW=dry weight</li>
		<li>Repeat step 4.1 for each germplasm and treatment in the trial.</li>
	</ol>
	</li>
	<li>Record the plant wilt once daily beginning on day 1 of the drought stress trial and ending when 100% of plants are wilted, normally day 4 or 5.</li>
	<li>Recovery phase and growth differential
	<ol>
		<li>On day 6, conclude the drought-stress period and the stressed plants enter the recovery phase of the trial. Fill the lower trays with water and allow the drought-stressed trays to soak for 24 hr before resuming the standard watering schedule, step 2.1, as the control trays.</li>
		<li>Allow all plants to recover for 10 days.</li>
		<li>At 10 days post drought-stress harvest the entire above ground portion of each plant and record the fresh weight.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Dijk, A. I. J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Millennium+Drought+in+southeast+Australia+(2001&#8211;2009):+Natural+and+human+causes+and+implications+for+water+resources,+ecosystems,+economy,+and+society.">The Millennium Drought in southeast Australia (2001&#8211;2009): Natural and human causes and implications for water resources, ecosystems, economy, and society.</a> Water Resour. Res. 49, (2013).</li><li>Aghakouchak, A., Feldman, D., Stewardson, M. J., Saphores, J., Grant, S., Sanders, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Australia&#8217;s+Drought:+Lessons+for+California.">Australia&#8217;s Drought: Lessons for California.</a> Science. 343, 1430 (2014).</li><li>Tolomeo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=California+Agricultural+Statistics+2012+Crop+Year.">California Agricultural Statistics 2012 Crop Year.</a> USDA National Agricultural Statistics Service Pacific Regional Office-California. , (2013).</li><li>Cavagnaro, T., 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=Climate+Change:+Challenges+and+Solutions+for+California+Agricultural+Landscapes.+White+paper+CEC-500-2005-189-SF.">Climate Change: Challenges and Solutions for California Agricultural Landscapes. White paper CEC-500-2005-189-SF.</a> California Climate Change Center. , (2005).</li><li>Lobell, D. B., Gourdji, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Influence+of+Climate+Change+on+Global+Crop+Productivity.">The Influence of Climate Change on Global Crop Productivity.</a> Plant Physiol. 160, 1686-1697 (2012).</li><li>Jackson, L. E., Stivers, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Root+distribution+of+lettuce+under+commercial+production:+implications+for+crop+uptake+of+nitrogen.">Root distribution of lettuce under commercial production: implications for crop uptake of nitrogen.</a> Biological Agriculture and Horticulture. 9, 273-293 (1993).</li><li>Jackson, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Root+architecture+in+cultivated+and+wild+lettuce+(Lactuca+spp).">Root architecture in cultivated and wild lettuce (Lactuca spp).</a> Plant. Cell and Environment. 18, 885-897 (1995).</li><li>Malcom, S., Marshall, E., Aillery, M., Heisey, P., Livingston, M., Day-Rubenstein, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agricultural+Adaptation+to+a+Changing+Climate:+Economic+and+Environmental+Implications+Vary+by+U.S+Region.+USDA+Economic+Research+Service.">Agricultural Adaptation to a Changing Climate: Economic and Environmental Implications Vary by U.S Region. USDA Economic Research Service.</a> Economic Research Report Number. 136, (2012).</li><li>Chaves, M. M., Maroco, J. P., Pereira, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+plant+responses+to+drought-+from+genes+to+the+whole+plant.">Understanding plant responses to drought- from genes to the whole plant.</a> Funct. Plant Biol. 30, 239-264 (2003).</li><li>Ingram, J., Bartels, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Molecular+Basis+of+Dehydration+Tolerance+in+Plants.">The Molecular Basis of Dehydration Tolerance in Plants.</a> Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 377-403 (1996).</li><li>Weatherley, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+in+the+water+relations+of+the+cotton+plant.+I.+The+field+measurement+of+water+deficits+in+leaves.">Studies in the water relations of the cotton plant. I. The field measurement of water deficits in leaves.</a> New Phlytol. 49, 81-97 (1950).</li><li>Smart, R. E., Bingham, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Estimates+of+Relative+Water+Content.">Rapid Estimates of Relative Water Content.</a> Plant Physiol. 53, 258-260 (1974).</li></ol>

The authors declare that they have no competing financial interests.

Considerations of sample number for screen.
The number of samples needed should be based on the desired use of the data from this screen. If publication quality results are desired it is recommended to harvest 3 individual plants (3 biological replicates) from each line and perform a minimum of 2 experimental replicates to give adequate points for quality statistical analysis. If the desired outcome is simply to quickly narrow a large pool of candidate germplasm in order to pe...

Water availability for irrigation has been a concern across much of the United States and globally for decades, but research into the response to drought-stress, along with other abiotic stresses, has lagged behind work in the areas of disease and insect resistance, at the industrial, academic, and governmental levels largely due to a lack of funding. Water availability for agriculture has historically been only an afterthought at the level of policy makers. Recently, due to several severe droughts in important agricultural production regions in both the United States and Australia1,2 fresh water availability has been thrust into the spotlight at both the national and international levels leading to many more resources being directed towards research into developing drought-tolerant cultivars of the major grain crops. While this shifting focus toward the development of drought-tolerant major crops is beneficial, like many areas of plant research specialty crops have largely been left behind. 
A severe drought is currently limiting vegetable production in California, the largest production region for Lettuce (Lactuca sativa L.) in the United States3. These short-term weather patterns coupled with legislative and judicial action along with long-term climate change4,5 have combined to reduce water available for agriculture in many of the most productive regions of California. Lettuce production in California represents a 1.5 billion dollar industry accounting for nearly 80% of lettuce production in the United States3. Leafy vegetables have high leaf water content and lettuce, in particular, has a shallow root system6,7 which leaves the crop vulnerable to water-stress. In lettuce, as in all crops the development of drought-tolerant varieties will become increasingly important as fresh water supplies for irrigation become more constrained8.
This protocol lays out a method by which an initial screen was performed on a large collection of lettuce germplasm in order to identify a pool of potentially drought-tolerant candidate lines for use in physiological, molecular, and genetic studies to identify specific drought-tolerance traits. These candidates can also be used directly as sources of drought-tolerance in breeding programs to improve water use efficiency in commercial cultivars. This protocol was developed specifically to address the challenges that arise during the course of any screen of an extensive germplasm collection, especially issues of space availability and labor. Also, the protocol as presented was developed for use in lettuce, but has been successfully adapted for use in the screening of a germplasm collection of spinach (Spinacia oleracea) for potential drought-tolerance and could be modified simply to screen any leafy vegetable crop. 
An important consideration before initiating a drought-tolerance screen is to understand what this method is and is not. This protocol is meant to represent a rapid method by which a large germplasm pool can be quickly and efficiently narrowed to a manageable number of candidate germplasm for use in more focused and thorough studies to identify individual tolerance traits. This protocol subjects the plants to a rapidly induced severe water-stress in contrast to a more natural slowly-induced moderate continuous drought-stress that would be observed under field conditions. The type of response induced within the plant during these two types of water-stress events (rapid dehydration versus natural drought) is not identical9 which could lead to the exclusion of some materials from future trials. These two stress events are not without overlap though10 and this protocol should serve as an effective way to identify potentially drought-tolerant germplasm contained within a large collection. This protocol alone should not be considered sufficient to identify with a high degree of certainty germplasm that contains durable drought-tolerance under field water-stress conditions, but it represents a significant step forward in the rapid screening of leafy green vegetables for potential drought-tolerance traits.

<table border="0" cellpadding="0" cellspacing="0" width="791">
	<tbody>
		<tr height="54">
			<td height="54" style="height:54px;width:171px;">Name of Material/ equipment</td>
			<td style="width:135px;">Manufacturer</td>
			<td style="width:121px;">Catalog number</td>
			<td style="width:188px;">Comments</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">plug tray 128</td>
			<td>T.O. Plastics 
			Hummert International</td>
			<td>11-8595-1</td>
			<td style="width:188px;">Any brand plug tray will work, but use the same style of trays for all trials.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">lower tray (Display tray)</td>
			<td>T.O. Plastics 
			Hummert International</td>
			<td>11-3305-1</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:171px;">plug/planting mix (Sunshine Mix #5)</td>
			<td>Sunshine 
			Hummert International</td>
			<td>10-0467-1</td>
			<td style="width:188px;">A different mix may need to be substituted if adapting this protocol to a different crop.&#160; Sunshine mix #4 was used in spinach trials.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">fertilizer (20-20-20)</td>
			<td>Jack&#39;s: Professional water-soluble fertilizer 
			Hummert International</td>
			<td>07-5915-1</td>
			<td style="width:188px;">Any fertilizer can be used, adjust type as needed for adapting this protocol to specific crop needs.</td>
		</tr>
	</tbody>
</table>

semi-high throughput screening for potential drought-tolerance in lettuce (lactuca sativa) germplasm collections

This protocol describes a method by which a large collection of the leafy green vegetable lettuce (Lactuca sativa L.) germplasm was screened for likely drought-tolerance traits. Fresh water availability for agricultural use is a growing concern across the United States as well as many regions of the world. Short-term drought events along with regulatory intervention in the regulation of water availability coupled with the looming threat of long-term climate shifts that may lead to reduced precipitation in many important agricultural regions has increased the need to hasten the development of crops adapted for improved water use efficiency in order to maintain or expand production in the coming years. This protocol is not meant as a step-by-step guide to identifying at either the physiological or molecular level drought-tolerance traits in lettuce, but rather is a method developed and refined through the screening of thousands of different lettuce varieties. The nature of this screen is based in part on the streamlined measurements focusing on only three water-stress indicators: leaf relative water content, wilt, and differential plant growth following drought-stress. The purpose of rapidly screening a large germplasm collection is to narrow the candidate pool to a point in which more intensive physiological, molecular, and genetic methods can be applied to identify specific drought-tolerant traits in either the lab or field. Candidates can also be directly incorporated into breeding programs as a source of drought-tolerance traits.

When performing a large screen in order to segregate a population by the desired experimental trait, in the case of this protocol drought-stress response, the data generated will correspondingly be varied from very stress susceptible to likely drought-tolerant and all points in between. Figure 1 contains graphs representing the type of results that can be expected from this protocol. Representative cultivars of three different lettuce types (romaine (cos), crisphead, and butterhead) are included in the d...

Watch this Scientific Journal Video about Semi-High Throughput Screening for Potential Drought-tolerance in Lettuce Lactuca sativa Germplasm Collections at JoVE.com

Semi-High Throughput Screening for Potential Drought-tolerance in Lettuce Lactuca sativa Germplasm Collections

This protocol was developed to screen a large germplasm collection of the leafy vegetable lettuce (Lactuca sativa L.) for drought-tolerance, in order to identify a small candidate pool of lettuce for use in physiological, molecular, and genetic studies to identify underlying drought-tolerance traits along with breeding programs.

Semi-High Throughput Screening for Potential Drought-tolerance in Lettuce (Lactuca sativa) Germplasm Collections

This protocol describes a method by which a large collection of the leafy green vegetable lettuce (Lactuca sativa L.) germplasm was screened for likely ...

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8.9K Views.  United States Department of Agriculture. This protocol was developed to screen a large germplasm collection of the leafy vegetable lettuce (Lactuca sativa L.) for drought-tolerance, in order to identify a small candidate pool of lettuce for use in physiological, molecular, and genetic studies to identify underlying drought-tolerance traits along with breeding programs.

Video: Semi-High Throughput Screening for Potential Drought-tolerance in Lettuce Lactuca sativa Germplasm Collections

High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities

Microbes in soils and other environments produce extracellular enzymes to depolymerize and hydrolyze organic macromolecules so that they can be assimilated for energy and nutrients. Measuring soil microbial enzyme activity is crucial in understanding soil ecosystem functional dynamics. The general concept of the fluorescence enzyme assay is that synthetic C-, N-, or P-rich substrates bound with a fluorescent dye are added to soil samples. When intact, the labeled substrates do not fluoresce. Enzyme activity is measured as the increase in fluorescence as the fluorescent dyes are cleaved from their substrates, which allows them to fluoresce. Enzyme measurements can be expressed in units of molarity or activity. To perform this assay, soil slurries are prepared by combining soil with a pH buffer. The pH buffer (typically a 50 mM sodium acetate or 50 mM Tris buffer), is chosen for the buffer&#39;s particular acid dissociation constant (pKa) to best match the soil sample pH. The soil slurries are inoculated with a nonlimiting amount of fluorescently labeled (i.e. C-, N-, or P-rich) substrate. Using soil slurries in the assay serves to minimize limitations on enzyme and substrate diffusion. Therefore, this assay controls for differences in substrate limitation, diffusion rates, and soil pH conditions; thus detecting potential enzyme activity rates as a function of the difference in enzyme concentrations (per sample).
Fluorescence enzyme assays are typically more sensitive than spectrophotometric (i.e. colorimetric) assays,&#160;but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light; so caution is required when handling fluorescent substrates. Likewise, this method only assesses potential enzyme activities under laboratory conditions when substrates are not limiting. Caution should be used when interpreting the data representing cross-site comparisons with differing temperatures or soil types, as in situ soil type and temperature can influence enzyme kinetics.

To measure potential rates of soil extracellular enzyme activities, synthetic substrates that are bound to a fluorescent dye are added to soil samples. Enzyme activity is measured as the fluorescent dye is released from the substrate by an enzyme-catalyzed reaction, where higher fluorescence indicates more substrate degradation.

Microbes in soils and other environments produce extracellular enzymes to depolymerize and hydrolyze organic macromolecules so that they can be ...

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3 . Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high&#8208;resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5. Moreover, OpNS can be a high&#8208;throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.

More than half of proteins are small proteins (molecular mass &#60;200 kDa) that are challenging for both electron microscope imaging and three-dimensional reconstructions. Optimized negative staining is a robust and high-throughput protocol to obtain high contrast and relatively high resolution (~1 nm) images of small asymmetric proteins or complexes under different physiological conditions.

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of ...

Screening Foodstuffs for Class 1 Integrons and Gene Cassettes

Antibiotic resistance is one of the greatest threats to health in the 21st century. Acquisition of resistance genes via lateral gene transfer is a major factor in the spread of diverse resistance mechanisms. Amongst the DNA elements facilitating lateral transfer, the class 1 integrons have largely been responsible for spreading antibiotic resistance determinants amongst Gram negative pathogens. In total, these integrons have acquired and disseminated over 130 different antibiotic resistance genes. With continued antibiotic use, class 1 integrons have become ubiquitous in commensals and pathogens of humans and their domesticated animals. As a consequence, they can now be found in all human waste streams, where they continue to acquire new genes, and have the potential to cycle back into humans via the food chain. This protocol details a streamlined approach for detecting class 1 integrons and their associated resistance gene cassettes in foodstuffs, using culturing and PCR. Using this protocol, researchers should be able to: collect and prepare samples to make enriched cultures and screen for class 1 integrons; isolate single bacterial colonies to identify integron-positive isolates; identify bacterial species that contain class 1 integrons; and characterize these integrons and their associated gene cassettes.

This protocol describes the detection of class 1 integrons and their associated gene cassettes in foodstuffs.

Antibiotic resistance is one of the greatest threats to health in the 21st century. Acquisition of resistance genes via lateral gene transfer is a major ...

High-throughput Screening of Recalcitrance Variations in Lignocellulosic Biomass: Total Lignin, Lignin Monomers, and Enzymatic Sugar Release

The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the use of non-renewable energy sources. In order to identify which plants, out of a diverse pool, have the desired chemical traits for downstream applications, attributes, such as cellulose and lignin content, or monomeric sugar release following an enzymatic saccharification, must be compared. The experimental and data analysis protocols of the standard methods of analysis can be time-consuming, thereby limiting the number of samples that can be measured. High-throughput (HTP) methods alleviate the shortcomings of the standard methods, and permit the rapid screening of available samples to isolate those possessing the desired traits. This study illustrates the HTP sugar release and pyrolysis-molecular beam mass spectrometry pipelines employed at the National Renewable Energy Lab. These pipelines have enabled the efficient assessment of thousands of plants while decreasing experimental time and costs through reductions in labor and consumables.

Plant cell wall structure and chemistry traits are evaluated to identify ideal feedstocks for biofuels and bio-materials. Standard methods have limitations when applied to large data sets. These high-throughput pretreatment, enzyme saccharification, and pyrolysis-molecular beam mass spectrometry methods compare large numbers of biomass samples with decreased experimental time and cost.

The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the ...

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been hindered partly due to a lack of standardized methods and availability of robust, user-friendly technology. In this study, commercially available, division-arrested cell lines were employed to quantitatively screen for endocrine activity of chemicals present in water samples of interest to environmental quality professionals. A single, standardized protocol that included comprehensive quality assurance/quality control (QA/QC) checks was developed for Estrogen and Glucocorticoid Receptor activity (ER and GR, respectively) using a cell-based Fluorescence Resonance Energy Transfer (FRET) assay. Samples of treated municipal wastewater effluent and surface water from freshwater systems in California (USA), were extracted using solid phase extraction and analyzed for endocrine activity using the standardized protocol. Background and dose-response for endpoint-specific reference chemicals met QA/QC guidelines deemed necessary for reliable measurement. The bioassay screening response for surface water samples was largely not detectable. In contrast, effluent samples from secondary treatment plants had the highest measurable activity, with estimated bioassay equivalent concentrations (BEQs) up to 392 ng dexamethasone/L for GR and 17 ng 17&#946;-estradiol/L for ER. The bioassay response for a tertiary effluent sample was lower than that measured for secondary effluents, indicating a lower residual of endocrine active chemicals after advanced treatment. This protocol showed that in vitro transactivation bioassays that utilize commercially available, division-arrested cell &#34;kits&#34;, can be adapted to screen for endocrine activity in water.

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

A protocol to screen for endocrine activity in organic extracts of water samples, including treated wastewater effluent and surface (receiving) water, was adapted using commercially available division-arrested ("freeze and thaw") in vitro transactivation bioassays.

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been ...

Basic Research in Plasma Medicine - A Throughput Approach from Liquids to Cells

In plasma medicine, ionized gases with temperatures close to that of vertebrate systems are applied to cells and tissues. Cold plasmas generate reactive species known to redox regulate biological processes in health and disease. Pre-clinical and clinical evidence points to beneficial effects of plasma treatment in the healing of chronic ulcer of the skin. Other emerging topics, such as plasma cancer treatment, are receiving increasing attention. Plasma medical research requires interdisciplinary expertise in physics, chemistry, and biomedicine. One goal of plasma research is to characterize plasma-treated cells in a variety of specific applications. This includes, for example, cell count and viability, cellular oxidation, mitochondrial activity, cytotoxicity and mode of cell death, cell cycle analysis, cell surface marker expression, and cytokine release. This study describes the essential equipment and workflows required for such research in plasma biomedicine. It describes the proper operation of an atmospheric pressure argon plasma jet, specifically monitoring its basic emission spectra and feed gas settings to modulate reactive species output. Using a high-precision xyz-table and computer software, the jet is hovered in millisecond-precision over the cavities of 96-well plates in micrometer-precision for maximal reproducibility. Downstream assays for liquid analysis of redox-active molecules are shown, and target cells are plasma-treated. Specifically, melanoma cells are analyzed in an efficient sequence of different consecutive assays but using the same cells: measurement of metabolic activity, total cell area, and surface marker expression of calreticulin, a molecule important for the immunogenic cell death of cancer cells. These assays retrieve content-rich biological information about plasma effects from a single plate. Altogether, this study describes the essential steps and protocols for plasma medical research.

A high-throughput cold physical plasma treatment protocol of liquids and cells is shown. It involves setting up different feed gas compositions for plasma ignition, measuring the plasma&#39;s emission spectra, and the subsequent analysis of liquids and cellular activity after plasma treatment.

In plasma medicine, ionized gases with temperatures close to that of vertebrate systems are applied to cells and tissues. Cold plasmas generate reactive ...

Evaluating Dryocosmus Kuriphilus-induced Damage on Castanea Sativa

Dryocosmus kuriphilus Yasumatsu has become a major pest for Castaneasativa since its arrival in Europe. Its galling activity results in the formation of different gall types and prevents the development of normal shoots. Repeated and uncontrolled attacks cause, besides the production of galls and the attendant gall-related reduction in leaf area, progressive corruption of the branch architecture, including the death of branch parts, and an increase in dormant bud activation. Thus far, there have been few attempts to quantify branch architecture damage. Further, the different methods for assessing infestation degree (MAID) that have been developed focus only on the galls' presence and abundance.
Using the leaf area to sapwood area relationship as a green biomass indicator, we developed in a previous study a damage composite index (DCI) that takes into account the most important branch architectural features, allowing for realistic damage assessment during the entire epidemic process. 
The aim of this study is to present this novel method and highlight differences in the damage description with respect to other broadly used indices. Results show how the DCI depicts branch damage better, especially during the epidemic peak, compared to MAID, which tend to underestimate it. We conclude by suggesting how to properly evaluate the overall impact of the pest by means of our composite damage index, the infestation degree using classic methods, and crown transparency evaluations.

Evaluating Dryocosmus Kuriphilus-induced Damage on Castanea Sativa

It is common practice to assess the damage caused by Dryocosmus kuriphilus by considering the abundance of galls alone rather than by also taking related branch corruption into consideration. We propose a composite damage index that takes into account the most important branch features, thus enabling more realistic damage assessment.

Dryocosmus kuriphilus Yasumatsu has become a major pest for Castaneasativa since its arrival in Europe. Its galling activity results in the formation of ...

High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize (Zea mays L.)

Maize is an important grain crop in the United States and worldwide. However, maize grain must be processed prior to human consumption. Furthermore, whole grain composition and processing characteristics vary among maize hybrids and can impact the quality of the final processed product. Therefore, in order to produce healthier processed food products from maize, it is necessary to know how to optimize processing parameters for particular sets of germplasm to account for these differences in grain composition and processing characteristics. This includes a better understanding of how current processing techniques impact the nutritional quality of the final processed food product. Here, we describe a microscale protocol that both simulates the processing pipeline to produce cornflakes from large flaking grits and allows for the processing of multiple grain samples simultaneously. The flaking grits, the intermediate processed products, or final processed product, as well as the corn grain itself, can be analyzed for nutritional content as part of a high-throughput analytical pipeline. This procedure was developed specifically for incorporation into a maize breeding research program, and it can be modified for other grain crops. We provide an example of the analysis of insoluble-bound ferulic acid and p-coumaric acid content in maize. Samples were taken at five different processing stages. We demonstrate that sampling can take place at multiple stages during microscale processing, that the processing technique can be utilized in the context of a specialized maize breeding program, and that, in our example, most of the nutritional content was lost during food product processing.

High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize Zea mays L.

Here, we present a microscale protocol for processing grain samples and for incorporating this microscale approach into a high-throughput analytical pipeline. This is a higher throughput adaptation of currently available protocols.

Maize is an important grain crop in the United States and worldwide. However, maize grain must be processed prior to human consumption. Furthermore, whole ...

A 3-dimensional (3D)-printed Template for High Throughput Zebrafish Embryo Arraying

The zebrafish is a globally recognized fresh water organism frequently used in developmental biology, environmental toxicology, and human disease related research fields. Thanks to its unique features, including large fecundity, embryo translucency, rapid and simultaneous development, etc., zebrafish embryos are often used for large scale toxicity assessment of chemicals and drug/compound screening. A typical screening procedure involves adult zebrafish spawning, embryos selection, and arraying the embryos into multi-well plates. From there, embryos are subjected to exposure and the toxicity of chemical, or the effectiveness of the drugs/compounds can be evaluated relatively quickly based on phenotypic observations. Among these processes, embryos arraying is one of the most time-consuming and labor-intensive steps that limits the throughput level. In this protocol, we present an innovative approach that makes use of a 3D-printed arraying template coupled with vacuum manipulation to speed up this laborious step. The protocol herein describes the overall design of the arraying template, a detailed experimental setup and step-by-step procedure, followed by representative results. When implemented, this approach should prove beneficial in a variety of research applications using zebrafish embryos as testing subjects.

A 3-dimensional 3D-printed Template for High Throughput Zebrafish Embryo Arraying

Here, we present a protocol to design and fabricate a zebrafish embryo arraying template, followed by a detailed procedure on the use of such template for high throughput zebrafish embryo arraying into a 96-well plate.

The zebrafish is a globally recognized fresh water organism frequently used in developmental biology, environmental toxicology, and human disease related ...

Choice and No-Choice Bioassays to Study the Pupation Preference and Emergence Success of Ectropis grisescens

Many insects live above the ground as larvae and adults and as pupate below the ground. Compared to the above-ground stages of their life cycles, less attention has been paid on how environmental factors affect these insects when they pupate within the soil. The tea looper, Ectropis grisescens Warren (Lepidoptera: Geometridae), is a severe pest of tea plants and has caused huge economic losses in South China. The protocols described here aim to investigate, through multiple-choice bioassays, whether mature last-instar E. grisescens larvae can discriminate soil variables such as the substrate type and moisture content, and determine, through no-choice bioassays, the impact of the substrate type and moisture content on pupation behaviors and the emergence success of E. grisescens. The results would enhance the understanding of the pupation ecology of E. grisescens and may bring insights into soil-management tactics for suppressing E. grisescens populations. In addition, these bioassays can be modified to study the influences of various factors on the pupation behaviors and survivorship of soil-pupating pests.

Choice and No-Choice Bioassays to Study the Pupation Preference and Emergence Success of Ectropis grisescens

Here, we present a protocol to investigate the pupation preference of mature larvae of Ectropis grisescens in response to soil factors (e.g., substrate type and moisture content) using choice bioassays. We also present a protocol of no-choice bioassays to determine the factors that affect the pupation behaviors and survivorship of E. grisescens.

Many insects live above the ground as larvae and adults and as pupate below the ground. Compared to the above-ground stages of their life cycles, less ...