1. Preparing samples
<ol>
	<li>1,000 mL of 0.1 M sodium acetate buffer at pH 4.6
	<ol>
		<li>Measure 800 mL of distilled water in a 1,000 mL graduated beaker.</li>
		<li>Weigh 8.2 g of anhydrous sodium acetate and add it to the beaker.</li>
		<li>Place the beaker on a stir plate, place a stir rod into the beaker, turn on the stir plate and stir until completely dissolved.</li>
		<li>When the anhydrous sodium acetate is completely dissolved, measure the pH of the solution with a calibrated pH meter.</li>
		<li>Add 1 M HCl or NaOH accordingly to obtain the desired pH 4.6.</li>
		<li>Add distilled water until the total volume is 1,000 mL.</li>
		<li>Store at room temperature until use.</li>
	</ol>
	</li>
	<li>Enzyme solution
	<ol>
		<li>Prepare 10 mL of the enzyme solution within the 10-30 mg/mL range by first measuring 8 mL of the 0.1 M sodium acetate buffer pH 4.6 in a 15 mL graduated cylinder.&#160;</li>
		<li>Add the buffer solution into a 15 mL conical tube with enzyme and shake vigorously until the enzyme has dissolved.</li>
		<li>Add more buffer solution until the total volume is 10 mL.</li>
		<li>Store the enzyme solution at 4 &#176;C until use.</li>
	</ol>
	</li>
	<li>Substrate solution
	<ol>
		<li>To prepare a substrate solution within the 300-600 mM range, calculate the amount of substrate needed in grams to make the desired concentration.</li>
		<li>Weigh out the substrate and place into a 100 mL glass beaker</li>
		<li>Measure 20 mL of the buffer solution using a 25 mL graduated cylinder and then add it to the glass beaker.</li>
		<li>Place the beaker on a stir plate and place a magnetic stir rod into the beaker. Turn on the heat and adjust the stirring speed accordingly.</li>
		<li>Allow stirring to continue until the substrate has dissolved.</li>
		<li>Pour the substrate solution into a 50 mL conical tube and add 0.1 M sodium acetate buffer pH 4.6 until the total volume is 45 mL. Mix by shaking.</li>
		<li>Store the substrate solution at room temperature until use.</li>
	</ol>
	</li>
</ol>
2. Performing the experiment
<ol>
	<li>Preparing the ITC instrument&#160;
	<ol>
		<li>Ensure that the reference cell is loaded with 350 &#181;L of distilled water. Before loading the enzyme into the sample cell, verify that the sample cell has been cleaned.</li>
		<li>Cleaning protocol- Fill the loading syringe with 500 &#181;L of 2% cleaning solution (Table of Materials), carefully insert the needle into the sample cell, fill the cell and slowly remove the liquid using the same syringe. Dispose the liquid into a beaker. Repeat this step twice with 2% cleaning solution (Table of Materials), three times with 70% ethanol and then wash ten times with distilled water.</li>
		<li>Fill the loading syringe with 450 &#181;L of enzyme solution, carefully insert the needle all the way to the bottom of the sample cell and press the plunger down to the 100 &#181;L line slowly to prevent formation of air bubbles.</li>
		<li>Wash the 50 &#181;L titration syringe with distilled water three times by placing the needle tip into water then slowly taking up the water into the syringe, then dispensing the water into a waste container.</li>
		<li>Remove residual water by rinsing with substrate solution three times.</li>
		<li>Fill the titration syringe with substrate solution by drawing the solution up until the syringe is full without any air bubbles.&#160;</li>
		<li>With the syringe still in the substrate solution, remove the plunger and allow approximately 2 &#181;L of air to enter the top of the syringe and reinsert the plunger.</li>
		<li>Remove the buret handle of the ITC, place the syringe inside the buret handle and screw until tight.&#160;</li>
		<li>Wipe the tip of the stirrer with a lint-free tissue, then carefully place the buret handle into the ITC instrument and lock it in place.</li>
	</ol>
	</li>
</ol>
3. Setting up ITCrun&#160;
<ol>
	<li>On the computer, open ITCrun and click Set up.</li>
	<li>Click Stirring rate and set to 350 RPM. Check the syringe size (&#181;L) and ensure it is at 50 &#181;L.</li>
	<li>Set the temperature and press Update. It is recommended that this step be performed at least 1 h before preparing the ITC. This allows enough time for the instrument to heat up or cool down as needed.</li>
	<li>For the experiment setup, select incremental titration.</li>
	<li>Click Insert to setup the injections. Adjust the injection interval to 5,400 s, injection volume (&#181;L) to 4 and number of injections to 4. Press OK to confirm settings.</li>
	<li>In the Equilibration box, select Auto-equilibrate and Large expected heats. (If the expected heats are small, one can select Small under expected heats; however, this will increase the equilibration time.)</li>
	<li>Set the initial baseline to 300 s.</li>
	<li>To start the run, click the Start symbol next to Stirring rate and then click Start which is located next to the wrench symbol.</li>
	<li>Save the file and allow the instrument to run.</li>
</ol>
4. Analyzing data
<ol>
	<li>Open the file in NanoAnalyze. Click Data and select Data columns.</li>
	<li>Select all data, copy and then paste the data into Microsoft Excel.</li>
	<li>Adjust zero baseline by adding the value required at 300 s to make it zero. Apply this correction to the entire column of heat rate values.&#160;</li>
	<li>Find the minimum or maximum value of the heat rate for each injection by using the equation: =MIN(cell:cell) or MAX(cell:cell). Each data point represents the peak enzymatic activity of the enzyme at each injection.</li>
	<li>Plot graphs of the MIN or MAX values against the time at which the value occurred during the titration.</li>
</ol>

<ol>
	<li>Anastas, P., Eghbali, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+chemistry:+principles+and+practice.">Green chemistry: principles and practice.</a> Chemical Society Reviews. 39 (1), 301-312 (2010).</li><li>Jin, L. Q., 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=Immobilization+of+Recombinant+Glucose+Isomerase+for+Efficient+Production+of+High+Fructose+Corn+Syrup.">Immobilization of Recombinant Glucose Isomerase for Efficient Production of High Fructose Corn Syrup.</a> Applied Biochemistry Biotechnoiogy. 183 (1), 293-306 (2017).</li><li>Budak, &. #. 3. 5. 0. ;. &. #. 2. 1. 4. ;., Ko&#231;ak, C., Bron, P. A., de Vries, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Microbial Cultures and Enzymes Dairy Technology. , 182-203 (2018).</li><li>van Donkelaar, L. H. G., Mostert, J., Zisopoulos, F. K., Boom, R. M., van der Goot, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+enzymes+for+beer+brewing:+Thermodynamic+comparison+on+resource+use.">The use of enzymes for beer brewing: Thermodynamic comparison on resource use.</a> Energy. 115, 519-527 (2016).</li><li>Rodriguez-Colinas, B., Fernandez-Arrojo, L., Ballesteros, A. O., Plou, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galactooligosaccharides+formation+during+enzymatic+hydrolysis+of+lactose:+Towards+a+prebiotic-enriched+milk.">Galactooligosaccharides formation during enzymatic hydrolysis of lactose: Towards a prebiotic-enriched milk.</a> Food Chemistry. 145, 388-394 (2014).</li><li>Lawrence, P. B., Price, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+PEGylation+influences+protein+conformational+stability.">How PEGylation influences protein conformational stability.</a> Current Opinions in Chemical Biology. 34, 88-94 (2016).</li><li>Bernal, C., Rodriguez, K., Martinez, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+enzyme+immobilization+and+protein+engineering:+An+alternative+path+for+the+development+of+novel+and+improved+industrial+biocatalysts.">Integrating enzyme immobilization and protein engineering: An alternative path for the development of novel and improved industrial biocatalysts.</a> Biotechnology Advances. 36 (5), 1470-1480 (2018).</li><li>Rigoldi, F., Donini, S., Redaelli, A., Parisini, E., Gautieri, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+Engineering+of+thermostable+enzymes+for+industrial+applications.">Review: Engineering of thermostable enzymes for industrial applications.</a> Applied Bioengeneering. 2 (1), 011501 (2018).</li><li>Johnson, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+scanning+calorimetry+as+a+tool+for+protein+folding+and+stability.">Differential scanning calorimetry as a tool for protein folding and stability.</a> Archives of Biochemistry and Biophysics. 531 (1), 100-109 (2013).</li><li>Chen, N. G., Gregory, K., Sun, Y., Golovlev, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+model+of+thermal+deactivation+of+enzymes.">Transient model of thermal deactivation of enzymes.</a> Biochimica et biophysica acta. 1814 (10), 1318-1324 (2011).</li><li>Mason, 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=Calorimetric+Methods+for+Measuring+Stability+and+Reusability+of+Membrane+Immobilized+Enzymes.">Calorimetric Methods for Measuring Stability and Reusability of Membrane Immobilized Enzymes.</a> Jounal of Food Science. , (2017).</li><li>Leksmono, C. S., 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=Measuring+Lactase+Enzymatic+Activity+in+the+Teaching+Lab.">Measuring Lactase Enzymatic Activity in the Teaching Lab.</a> Journal of visualized experiments : Journal of Visual Experiments. (138), e54377 (2018).</li></ol>

A major advantage of the ITC enzyme stability assay described here is automation. Once all the appropriate buffers and solutions are made, the set-up time for each assay is approximately 15 min for the person doing the assay. In contrast, the conventional assays for invertase and lactase activity require about 2 h with continual involvement of the person doing the assay and many enzymatic activity assays take considerably more person-hours. In a previous publication, we have demonstrated how data from the ITC method comp...

Enzymes are proteins capable of catalyzing a wide array of organic reactions. Most enzymes function in aqueous solution at near neutral pH thus avoiding the use of harsh solvents. Because of their high selectivity, enzyme catalyzed reactions produce fewer (in some cases no byproducts) byproducts than non-selective catalysts such as acids and bases1. This is especially relevant in food manufacturing where all chemical reactions must be done so the final product is safe for human consumption. Currently, enzymes are used to produce high fructose corn syrup2, cheese3, beer4, lactose-free milk5, and other important food products. While this paper focuses on enzyme use in the food industry, there are many other uses for enzymes including in green chemistry and drug synthesis.&#160;
The utility of enzymes is limited by the stability of enzyme activity, which depends on maintaining the three-dimensional structure of the enzyme. The enzyme structure can be stabilized by modifications such as PEGylation6, immobilization on a solid support7, genetic modifications8, and formulations. Currently, enzyme stability is typically measured by differential scanning calorimetry (DSC) and endpoint enzyme activity assays9. DSC measures the temperature at which an enzyme unfolds; the higher the temperature, the more stable the structure. However, loss of activity often occurs at a lower temperature than required to unfold the enzyme or domains within the enzyme10. Therefore, DSC is not sufficient to determine whether an enzyme modification increases the stability of enzyme activity. Endpoint enzyme assays are usually time intensive, require multiple samples, and often involve a coupled colorimetric reaction that is not applicable to highly colored or opaque solutions or suspensions.
This work demonstrates a method for direct measurement of the stability of enzyme activity by isothermal titration calorimetry (ITC). ITC measures the rate of heat released or absorbed during the course of a reaction. Since nearly all reactions produce or absorb heat, ITC can be used for most enzyme-catalyzed reactions, including reactions that do not have a coupled reaction or occur in opaque media such as milk. ITC has been used for many decades to measure chemical kinetic parameters for many kinds of reactions, but the protocol presented here focuses on using ITC to measure the peak heat rate of enzyme-catalyzed reactions and demonstrates that enzyme activity is linearly correlated with the peak heat rate. ITC measurements of peak heat rates are mostly autonomous and require very little personnel time to setup and analyze.&#160;

<table border="0" cellpadding="0" cellspacing="0" style="width:600px;" width="601">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">a-Lactose</td>
			<td style="width:99px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">unknown (too old)</td>
			<td style="width:167px;">500g</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium Acetate, Anhydrous 99% min</td>
			<td style="width:99px;">Alfa Aesar</td>
			<td style="width:119px;">A13184-30</td>
			<td>250g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lactase&#160;</td>
			<td style="width:99px;">MP Bio</td>
			<td align="right" style="width:119px;">100780</td>
			<td>5g</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hydrocholric Acid Solution, 1N&#160;</td>
			<td style="width:99px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">SA48-500</td>
			<td>500mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Benchtop Meter- pH</td>
			<td style="width:99px;">VWR</td>
			<td style="width:119px;">89231-622</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethanol 70%</td>
			<td style="width:99px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">BP8231GAL</td>
			<td>1gallon</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Micro-90</td>
			<td style="width:99px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">NC024628</td>
			<td>1L (cleaning solution)</td>
		</tr>
	</tbody>
</table>

measuring enzymatic stability by isothermal titration calorimetry

This work demonstrates a new method for measuring the stability of enzyme activity by isothermal titration calorimetry (ITC). The peak heat rate observed after a single injection of the substrate solution into an enzyme solution is correlated with enzyme activity. Multiple injections of the substrate into the same enzyme solution over time show the loss of enzyme activity. The assay is autonomous, requiring very little personnel time, and is applicable to most media and enzymes.&#160; &#160;

The representative results in Figure 1 and Figure 5 show data from two enzymes, lactase and invertase. Lactase and invertase catalyze the hydrolysis of a disaccharide into two monosaccharides, endothermically and exothermically, respectively. Both enzymatic reactions were run at concentrations that precluded saturation of the enzyme.&#160;
The lactase data demonstrate how ITC data can be used to estimate enzyme stability. Four sequent...

Watch this Scientific Journal Video about Measuring Enzymatic Stability by Isothermal Titration Calorimetry at JoVE.com

Measuring Enzymatic Stability by Isothermal Titration Calorimetry

The thermal stability of enzyme activity is readily measured by isothermal titration calorimetry (ITC). Most protein stability assays currently used measure protein unfolding, but do not provide information about enzymatic activity. ITC enables direct determination of the effect of enzyme modifications on the stability of enzyme activity.

This work demonstrates a new method for measuring the stability of enzyme activity by isothermal titration calorimetry (ITC). The peak heat rate observed ...

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13.0K Views.  Brigham Young University. The thermal stability of enzyme activity is readily measured by isothermal titration calorimetry (ITC). Most protein stability assays currently used measure protein unfolding, but do not provide information about enzymatic activity. ITC enables direct determination of the effect of enzyme modifications on the stability of enzyme activity.

Video: Measuring Enzymatic Stability by Isothermal Titration Calorimetry

Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. Radioisotope tracing is a major technique used to measure such fluxes, both within plants, and between plants and their environments. Flux data obtained with radiotracer protocols can help elucidate the capacity, mechanism, regulation, and energetics of transport systems for specific mineral nutrients or toxicants, and can provide insight into compartmentation and turnover rates of subcellular mineral and metabolite pools. Here, we describe two major radioisotope protocols used in plant biology: direct influx (DI) and compartmental analysis by tracer efflux (CATE). We focus on flux measurement of potassium (K+) as a nutrient, and ammonia/ammonium (NH3/NH4+) as a toxicant, in intact seedlings of the model species barley (Hordeum vulgare L.). These protocols can be readily adapted to other experimental systems (e.g., different species, excised plant material, and other nutrients/toxicants). Advantages and limitations of these protocols are discussed.

In planta measurement of nutrient and toxicant fluxes is essential to the study of plant nutrition and toxicity. Here, we cover radiotracer protocols for influx and efflux determination in intact plant roots, using potassium (K+) and ammonia/ammonium (NH3/NH4+) fluxes as examples. Advantages and limitations of such techniques are discussed.

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. ...

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 ...

Measuring Carbon-based Contaminant Mineralization Using Combined CO2 Flux and Radiocarbon Analyses

A method is described which uses the absence of radiocarbon in industrial chemicals and fuels made from petroleum feedstocks which frequently contaminate the environment. This radiocarbon signal &#8212; or rather the absence of signal &#8212; is evenly distributed throughout a contaminant source pool (unlike an added tracer) and is not impacted by biological, chemical or physical processes (e.g., the 14C radioactive decay rate is immutable). If the fossil-derived contaminant is fully degraded to CO2, a harmless end-product, that CO2 will contain no radiocarbon. CO2 derived from natural organic matter (NOM) degradation will reflect the NOM radiocarbon content (usually &#60;30,000 years old). Given a known radiocarbon content for NOM (a site background), a two end-member mixing model can be used to determine the CO2 derived from a fossil source in a given soil gas or groundwater sample. Coupling the percent CO2 derived from the contaminant with the CO2 respiration rate provides an estimate for the total amount of contaminant degraded per unit time. Finally, determining a zone of influence (ZOI) representing the volume from which site CO2 is collected allows determining the contaminant degradation per unit time and volume. Along with estimates for total contaminant mass, this can ultimately be used to calculate time-to-remediate or otherwise used by site managers for decision-making.

Measuring Carbon-based Contaminant Mineralization Using Combined CO2 Flux and Radiocarbon Analyses

A protocol is described wherein CO2 mineralized from organic contaminant (derived from petroleum feedstocks) biodegradation is trapped, quantified, and analyzed for 14C content. A model is developed to determine CO2 capture zone's spatial extent. Spatial and temporal measurements allow integrating contaminant mineralization rates for predicting remediation extent and time.

A method is described which uses the absence of radiocarbon in industrial chemicals and fuels made from petroleum feedstocks which frequently contaminate ...

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 ...

Protocol for Measuring the Thermal Properties of a Supercooled Synthetic Sand-water-gas-methane Hydrate Sample

Methane hydrates (MHs) are present in large amounts in the ocean floor and permafrost regions. Methane and hydrogen hydrates are being studied as future energy resources and energy storage media. To develop a method for gas production from natural MH-bearing sediments and hydrate-based technologies, it is imperative to understand the thermal properties of gas hydrates.
The thermal properties' measurements of samples comprising sand, water, methane, and MH are difficult because the melting heat of MH may affect the measurements. To solve this problem, we performed thermal properties' measurements at supercooled conditions during MH formation. The measurement protocol, calculation method of the saturation change, and tips for thermal constants' analysis of the sample using transient plane source techniques are described here.
The effect of the formation heat of MH on measurement is very small because the gas hydrate formation rate is very slow. This measurement method can be applied to the thermal properties of the gas hydrate-water-guest gas system, which contains hydrogen, CO2, and ozone hydrates, because the characteristic low formation rate of gas hydrate is not unique to MH. The key point of this method is the low rate of phase transition of the target material. Hence, this method may be applied to other materials having low phase-transition rates.

We present a protocol for measuring the thermal properties of synthetic hydrate-bearing sediment samples comprising sand, water, methane, and methane hydrate.

Methane hydrates (MHs) are present in large amounts in the ocean floor and permafrost regions. Methane and hydrogen hydrates are being studied as future ...

Spotting Cheetahs: Identifying Individuals by Their Footprints

The cheetah (Acinonyx jubatus) is Africa's most endangered large felid and listed as Vulnerable with a declining population trend by the IUCN1. It ranges widely over sub-Saharan Africa and in parts of the Middle East. Cheetah conservationists face two major challenges, conflict with landowners over the killing of domestic livestock, and concern over range contraction. Understanding of the latter remains particularly poor2. Namibia is believed to support the largest number of cheetahs of any range country, around 30%, but estimates range from 2,9053 to 13,5204. The disparity is likely a result of the different techniques used in monitoring.
Current techniques, including invasive tagging with VHF or satellite/GPS collars, can be costly and unreliable. The footprint identification technique5 is a new tool accessible to both field scientists and also citizens with smartphones, who could potentially augment data collection. The footprint identification technique analyzes digital images of footprints captured according to a standardized protocol. Images are optimized and measured in data visualization software. Measurements of distances, angles, and areas of the footprint images are analyzed using a robust cross-validated pairwise discriminant analysis based on a customized model. The final output is in the form of a Ward's cluster dendrogram. A user-friendly graphic user interface (GUI) allows the user immediate access and clear interpretation of classification results.
The footprint identification technique algorithms are species specific because each species has a unique anatomy. The technique runs in a data visualization software, using its own scripting language (jsl) that can be customized for the footprint anatomy of any species. An initial classification algorithm is built from a training database of footprints from that species, collected from individuals of known identity. An algorithm derived from a cheetah of known identity is then able to classify free-ranging cheetahs of unknown identity. The footprint identification technique predicts individual cheetah identity with an accuracy of &gt;90%.

The cheetah (Acinonyx jubatus) is an iconic, endangered species, but conservation efforts are challenged by habitat shrinkage and conflict with commercial farmers. The footprint identification technique, a robust, accurate and cost-effective image classification system, is a new approach to monitoring cheetahs.

The cheetah (Acinonyx jubatus) is Africa's most endangered large felid and listed as Vulnerable with a declining population trend by the IUCN1. It ranges ...

Measuring Stolons and Rhizomes of Turfgrasses Using a Digital Image Analysis System

Length and diameter of stolons or rhizomes are usually measured using simple rulers and calipers. This procedure is slow and laborious, so it is often used on a limited number of stolons or rhizomes. For this reason, these traits are limited in their use for morphological characterization of plants. The use of digital image analysis software technology may overcome measurement errors due to human mistakes, which tend to increase as the number and size of samples also increase. The protocol can be used for any kind of crop but is particularly suitable for forage or grasses, where plants are small and numerous. Turf samples consist of aboveground biomass and an upper soil layer to the depth of maximum rhizome development, depending on the species of interest. In studies, samples are washed from the soil, and stolons/rhizomes are cleaned by hand before analysis by digital image analysis software. The samples are further dried in a laboratory heating oven to measure dry weight; therefore, for each sample, the resultant data are total length, total dry weight, and average diameter. Scanned images can be corrected before analysis by excluding visible extraneous parts, such as remaining roots or leaves not removed with the cleaning process. Indeed, these fragments normally have much smaller diameters than stolons or rhizomes, so they can be easily excluded from analysis by fixing the minimum diameter below which objects are not considered. Stolon or rhizome density per unit area can then be calculated based on sample size. The advantage of this method is quick and efficient measurement of the length and average diameter of large sample numbers of stolons or rhizomes.

A software-based image analysis system provides an alternative method to study the morphology of stoloniferous and rhizomatous species. This protocol permits measurement of the length and diameter of stolons and rhizomes and can be applied to samples with a large amount of biomass and to a wide variety of species.

Length and diameter of stolons or rhizomes are usually measured using simple rulers and calipers. This procedure is slow and laborious, so it is often ...

Measuring Hypopharyngeal Gland Acinus Size in Honey Bee (Apis mellifera) Workers

The nurse hypopharyngeal glands produce the protein fraction of the worker and royal jelly that is fed to developing larvae and queens. These paired glands that are located in the head of the bee are highly sensitive to the quantity and quality of pollen and pollen substitutes that the nurse bee consumes. The glands get smaller when nurses are fed deficient diets and are large when they are fed complete diets. Because nurse hypopharyngeal gland size is a robust indicator of nurse nutrition, it is essential that those studying honey bee nutrition know how to measure these glands. Here, we provide detailed methods for dissecting, staining, imaging, and measuring nurse bee hypopharyngeal glands. We present comparisons of unstained and stained tissue and data that were used to study the impact of pollen on gland size. This method has been used to test how diet impacts hypopharyngeal gland size but has further use for understanding the role of these glands in hive health.

Measuring Hypopharyngeal Gland Acinus Size in Honey Bee Apis mellifera Workers

Hypopharyngeal gland acinus size is a robust measure of nurse honey bee nutrition. Here, we provide a detailed protocol for dissecting, staining, imaging, and measuring nurse bee hypopharyngeal gland acini.

The nurse hypopharyngeal glands produce the protein fraction of the worker and royal jelly that is fed to developing larvae and queens. These paired ...

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many countries around the world. Severe yield losses are caused by direct feeding, and even more importantly, also by the transmission of more than 100 harmful plant pathogenic viruses. As for other invasive pests, increased international trade facilitates the dispersal of B. tabaci to areas beyond its native range. Inspections of plant import products at points of entry such as seaports and airports are, therefore, seen as an important prevention measure. However, this last line of defense against pest invasions is only effective if rapid identification methods for suspicious insect specimens are readily available. Because the morphological differentiation between the regulated B. tabaci and close relatives without quarantine status is difficult for non-taxonomists, a rapid molecular identification assay based on the loop-mediated isothermal amplification (LAMP) technology has been developed. This publication reports the detailed protocol of the novel assay describing rapid DNA extraction, set-up of the LAMP reaction, as well as interpretation of its read-out, which allows identifying B. tabaci specimens within one hour. Compared to existing protocols for the detection of specific B. tabaci biotypes, the developed method targets the whole B. tabaci species complex in one assay. Moreover the assay is designed to be applied on-site by plant health inspectors with minimal laboratory training directly at points of entry. Thorough validation performed under laboratory and on-site conditions demonstrates that the reported LAMP assay is a rapid and reliable identification tool, improving the management of B. tabaci.

A Loop-mediated Isothermal Amplification LAMP Assay for Rapid Identification of Bemisia tabaci

This paper reports the protocol for a rapid identification assay for Bemisia tabaci based on loop-mediated isothermal amplification (LAMP) technology. The protocol requires minimal laboratory training and can, therefore, be implemented on-site at points of entry for plant imports such as seaports and airports.

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many ...

Measuring Phosphorus Release in Laboratory Microcosms for Water Quality Assessment

Phosphorus (P) is a critical limiting nutrient in agroecosystems requiring careful management to reduce transport risk to aquatic environments. Routine laboratory measures of P bioavailability are based on chemical extractions performed on dried samples under oxidizing conditions. While useful, these tests are limited with respect to characterizing P release under prolonged water saturation. Labile orthophosphate bound to oxidized iron and other metals can rapidly desorb to solution in reducing environments, increasing P mobilization risk to surface runoff and groundwater. To better quantify P desorption potential and mobility during extended saturation, a laboratory microcosm method was developed based on repeated sampling of porewater and overlying floodwater over time. The method is useful for quantifying P release potential from soils and sediments varying in physicochemical properties and can improve site-specific P mitigation efforts by better characterizing P release risk in hydrologically active areas. Advantages of the method include its ability to simulate in situ dynamics, simplicity, low cost, and flexibility.

Accurate quantification of phosphorus (P) desorption potential in saturated soils and sediments is important for P modeling and transport mitigation efforts. To better account for in situ soil-water redox dynamics and P mobilization under prolonged saturation, a simple approach was developed based on repeated sampling of laboratory microcosms.

Phosphorus (P) is a critical limiting nutrient in agroecosystems requiring careful management to reduce transport risk to aquatic environments. Routine ...