We would like to acknowledge Dr. Ali and the Plant Molecular Diagnostic Laboratory as well as Dr. Pingsheng Ji at the University of Georgia, whose leadership and support helped establish our Fon program.

The authors declare that they have no competing financial interests.

Three methods of race typing have been presented. Each of these methods is best suited to particular questions and experimental conditions. The infested kernel inoculation method (soil infestation) is perhaps simpler and more straightforward, making it especially useful for the assessment of pathogenicity30. Using this method for simple race-typing is highly effective. However, applying the method to determine the resistance of a specific cultivar could be challenging, given that each plant may no...

The soilborne fungi that make up the Fusarium oxysporum species complex (FOSC) are impactful hemibiotrophic plant pathogens that can cause serious disease and yield loss in a diverse range of crops1. Fusarium wilt of watermelon, caused by F. oxysporum f. sp. niveum (Fon), has been increasing in scope, incidence, and severity across the world in the last several decades2,3. In seedlings, the symptoms of Fusarium wilt often resemble damping-off. In older plants, the foliage becomes gray, chlorotic, and necrotic. Eventually, wilting of the plants progresses to full plant collapse and death4. Direct yield loss occurs due to the symptoms and plant death, while indirect yield loss can occur due to sun damage caused by the elimination of the foliar canopy5. Sexual reproduction and associated reproductive structures have never been observed in F. oxysporum. However, the pathogen produces two types of asexual spores, micro- and macroconidia, as well as larger, long-term survival structures called chlamydospores, which can survive in the soil for many years6.
The FOSC is classified into formae speciales based on observed host ranges, usually limited to one or a few host species1. Although recent research has indicated that this species complex may be a composite of 15 different species, the particular species that infect watermelon are currently unknown7. F. oxysporum f. sp. niveum (Fon) is the name for the groups of strains that exclusively infect Citrullus lanatus or the domesticated watermelon8,9. F. oxysporum strains within most pathogenic formae speciales display certain levels of diversity with regard to their genetic components and virulence toward a host species. For instance, one strain may infect all cultivars of a host, whereas another may only infect the more susceptible cultivars. To account for such variation, these groups are informally classified into races based on evolutionary relationships or common phenotypic characteristics. Within Fon, four races (0, 1, 2, and 3) have been characterized based on their pathogenicity against a set of select watermelon cultivars, with the discovery of race 3 occurring recently10.
Despite this apparent diversity, the morphologies of spores or hyphae are not distinguishable between the races of Fon races, meaning that molecular or phenotypic assays are needed to identify an isolate&#39;s unique race11. Molecular research has identified some genetic differences. For example, the role of Secreted in Xylem (SIX) effectors has been studied for years in F. oxysporum, and some of these effectors have been located on the chromosomes exchanged during horizontal gene transfer12. For example, SIX6 is found in Fon races 0 and 1 but not in race 213. SIX effectors have been implicated in the pathogenicity of F. oxysporum f. sp. lycopersici and F. oxysporum f. sp. cubense, which cause Fusarium wilt on tomato and banana, respectively14,15,16,17. The analysis of SIX effector profiles among strains of F. oxysporum f. sp. spiniciae, the Fusarium wilt pathogen on spinach, has enabled classification that accurately reflects genetic and phenotypic diversity18. However, the differences between virulence mechanisms of Fon races are currently not entirely understood, and molecular assays developed upon their use have shown inconsistent and inaccurate results19. Therefore, phenotypic results from infection assays are currently the best way to classify isolates.
F. oxysporum initially infects hosts through the roots before making its way up the xylem20. This makes direct inoculation of the roots of a given host cultivar an effective way to perform race-typing and is the basis of the root-dip and tray-dip inoculation methods21. When not infecting a host, F. oxysporum resides in the soil and can remain dormant for years. Growing susceptible watermelon cultivars in soil from a field of interest is one way to test for the presence of Fon. Expanding this method to include cultivars of different known levels of resistance in soil that is deliberately infested with Fon is also a good way to perform race-typing (Table 1) and is the basis of the infested kernel seeding method. The modified tray-dip method is a variation of the original tray-dip method that allows for a high-throughput race-typing where many plants and field isolates can be investigated rapidly22. Important factors of a quick and successful race-typing bioassay include using cultivars that have documented differences in resistance to the different pathogen races, ensuring that the inoculum is both biologically active and abundant during infection, maintaining an environment that is both conducive for the pathogen and host, and using a consistent rating system for severity or incidence of disease. This paper describes the root-dip23,24, infested kernel seeding25,26, and modified tray-dip22 methods for phenotypic race-typing based on the principles described above.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100% Fuller&#8217;s Earth</td>
			<td>Sigma-Aldrich</td>
			<td>F200-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>1 L glass Erlenmeyer Flask</td>
			<td>PYREX</td>
			<td>4980-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL falcon tubes</td>
			<td>Fisher Scientific</td>
			<td>14-959-49B</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL graduated cylinder</td>
			<td>Lab Safety Supply</td>
			<td>41121805</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Eppendorf Conical Tubes</td>
			<td>Fisher Scientific</td>
			<td>05-413-921</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil wrap</td>
			<td>Reynolds Wrap</td>
			<td>720</td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>Walmart</td>
			<td>587192290</td>
			<td></td>
		</tr>
		<tr>
			<td>Bunsen burner</td>
			<td>Fisher Scientific</td>
			<td>03-391-301</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCO3</td>
			<td>sigma-Aldrich</td>
			<td>239216</td>
			<td></td>
		</tr>
		<tr>
			<td>cell spreaders</td>
			<td>Fisher Scientific</td>
			<td>08-100-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Cheesecloth</td>
			<td>Lions Services, Inc</td>
			<td>8305-01-125-0725</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear plastic dishes</td>
			<td>Visions Wave</td>
			<td>999RP6CLSS</td>
			<td>~15 cm diameter</td>
		</tr>
		<tr>
			<td>Clear vinyl tubing for mushroom bag clamps</td>
			<td>Shroom Supply</td>
			<td></td>
			<td>6&#34; for small bag, 8&#34; for medium bag, 10&#34; for large bag</td>
		</tr>
		<tr>
			<td>Cotton Balls</td>
			<td>Fisherbrand</td>
			<td>22-456-885</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Chemical</td>
			<td>A4094</td>
			<td>100%, then combine with water to make 70% for use</td>
		</tr>
		<tr>
			<td>Flourescent Tube Lights</td>
			<td>MaxLume</td>
			<td>Model T5</td>
			<td>2800 K Color Temperature, 24&#39;&#39; or 48&#39;&#39; long</td>
		</tr>
		<tr>
			<td>granulated agar</td>
			<td>VWR International</td>
			<td>90000-786</td>
			<td></td>
		</tr>
		<tr>
			<td>Hand-held Spray Bottle</td>
			<td>Ability One</td>
			<td>24122002</td>
			<td>~0.95 L</td>
		</tr>
		<tr>
			<td>hemacytometer</td>
			<td>Fisher Scientific</td>
			<td>02-671-55A</td>
			<td>Two chamber hemacytometer</td>
		</tr>
		<tr>
			<td>Lab trays</td>
			<td>Fisher Scientific</td>
			<td>15-236-2A</td>
			<td></td>
		</tr>
		<tr>
			<td>Large, sealable plastic bags</td>
			<td>Ziploc</td>
			<td>430805</td>
			<td>38 cm x 38 cm</td>
		</tr>
		<tr>
			<td>Mister / watering can</td>
			<td>Bar5F</td>
			<td>B10H22</td>
			<td></td>
		</tr>
		<tr>
			<td>Mushroom Bag Clamp</td>
			<td>Shroom Supply</td>
			<td></td>
			<td>6&#34; for small bag, 8&#34; for medium bag, 10&#34; for large bag</td>
		</tr>
		<tr>
			<td>Nitrile Gloves</td>
			<td>Fisher Scientific</td>
			<td>19-130-1597D</td>
			<td></td>
		</tr>
		<tr>
			<td>Organic Rye Berries</td>
			<td>Shroom Supply</td>
			<td></td>
			<td>0.5 gallon or 25 lb bags</td>
		</tr>
		<tr>
			<td>P1000 pipette and tips</td>
			<td>Fisher Scientific</td>
			<td>14-388-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Fisherbrand</td>
			<td>FB0875713</td>
			<td>Round, 100 mm diameter, 15 mm height</td>
		</tr>
		<tr>
			<td>Planting media</td>
			<td>Jolly Gardener</td>
			<td>Pro-Line C/B</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Pitcher</td>
			<td>BrandTech</td>
			<td>UX0600850</td>
			<td>1 L or larger</td>
		</tr>
		<tr>
			<td>Plastic planting pots</td>
			<td>Neo/SCI</td>
			<td>01-1177</td>
			<td>~15 cm diameter and ~10 cm height</td>
		</tr>
		<tr>
			<td>Plastic, autoclave-safe bin</td>
			<td>Thermo Scientific</td>
			<td>UX0601022</td>
			<td>3 L</td>
		</tr>
		<tr>
			<td>Quarter-strength potato dextrose agar media</td>
			<td>Cole-Parmer</td>
			<td>UX1420028</td>
			<td>Use powder in combination with recipe for QPDA</td>
		</tr>
		<tr>
			<td>Scientific Balance Scale, measuring in g</td>
			<td>Ohaus</td>
			<td>30208458</td>
			<td>Any precise scale that can hold and measure 200g will work</td>
		</tr>
		<tr>
			<td>Size #4 cork bore</td>
			<td>Cole-Parmer</td>
			<td>NC9585352</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Mushroom grow bag</td>
			<td>Shroom Supply</td>
			<td></td>
			<td>0.5 micron filter, also comes in medium and large sizes</td>
		</tr>
		<tr>
			<td>Soil trowel</td>
			<td>Walmart</td>
			<td>563876946</td>
			<td></td>
		</tr>
		<tr>
			<td>Styrofoam flats (6 x 12 cells)</td>
			<td>Speedling</td>
			<td>Model TR72A</td>
			<td></td>
		</tr>
		<tr>
			<td>Styrofoam flats (8 x 16 cells)</td>
			<td>Speedling</td>
			<td>Model TR128A</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe (5 or 10 mL)</td>
			<td>fisher Scientific</td>
			<td>14-829-19C</td>
			<td></td>
		</tr>
		<tr>
			<td>Timer</td>
			<td>Walmart</td>
			<td>TM-01</td>
			<td></td>
		</tr>
		<tr>
			<td>V8 Original 100% Vegetable Juice</td>
			<td>Walmart</td>
			<td>564638212</td>
			<td></td>
		</tr>
		<tr>
			<td>vortex</td>
			<td>Fisher Scientific</td>
			<td>02-215-418</td>
			<td></td>
		</tr>
		<tr>
			<td>Watermelon Seed - Black Diamond</td>
			<td>Willhite Seed Inc</td>
			<td>17</td>
			<td></td>
		</tr>
		<tr>
			<td>Watermelon Seed - Calhoun Gray</td>
			<td>Holmes Seed Company</td>
			<td>4440</td>
			<td></td>
		</tr>
		<tr>
			<td>Watermelon Seed - Charleston Gray</td>
			<td>Bonnie Plants</td>
			<td>7.15339E+11</td>
			<td></td>
		</tr>
		<tr>
			<td>Watermelon Seed - PI 296341-FR</td>
			<td>Contact authors</td>
			<td>Contact authors</td>
			<td></td>
		</tr>
		<tr>
			<td>Wheat Kernels (Maxie var.) (optional)</td>
			<td>Alachua County Feed &#38; Seed</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a contrast of three inoculation techniques used to determine the race of unknown fusarium oxysporum f.sp. niveum isolates

Fusarium wilt of watermelon (Citrullus lanatus), caused by Fusarium oxysporum f. sp. niveum (Fon), has reemerged as a major production constraint in the southeastern USA, especially in Florida. Deployment of integrated pest management strategies, such as race-specific resistant cultivars, requires information on the diversity and population density of the pathogen in growers' fields. Despite some progress in developing molecular diagnostic tools to identify pathogen isolates, race determination often requires bioassay approaches.
Race typing was conducted by root-dip inoculation, infested kernel seeding method, and the modified tray-dip method with each of the four watermelon differentials (Black Diamond, Charleston Grey, Calhoun Grey, Plant Introduction 296341-FR). Isolates are assigned a race designation by calculation of disease incidence five weeks after inoculation. If less than 33% of the plants for a particular cultivar were symptomatic, they were categorized as resistant. Those cultivars with incidence greater than 33% were regarded as susceptible. This paper describes three different methods of inoculation to ascertain race, root-dip, infested kernel, and modified tray-dip inoculation, whose applications vary according to the experimental design.

These experiments help define the relative resistance of commonly grown cultivars (Table 1). This information can then be used to guide management recommendations based on local Fon populations. In other words, if race 0 or 1 is known to be present in a commercial field, then the farmer may be inclined to grow a &#34;resistant&#34; variety such as Calhoun Gray, Sunsugar, or equivalent. The results of the bioassays using all methods show that when the seedlings were infected with a Race 1 isolate, the Bla...

Watch this Scientific Journal Video about A Contrast of Three Inoculation Techniques used to Determine the Race of Unknown Fusarium oxysporum f.sp. niveum Isolates at JoVE.com

A Contrast of Three Inoculation Techniques used to Determine the Race of Unknown Fusarium oxysporum f.sp. niveum Isolates

Managing Fusarium wilt of watermelon requires knowledge of the pathogen races present. Here, we describe the root-dip, infested kernel seeding, and modified tray-dip inoculation methods to demonstrate their efficacy in race-typing of the pathogenic fungus Fusarium oxysporum f. sp niveum (Fon).

A Contrast of Three Inoculation Techniques used to Determine the Race of Unknown Fusarium oxysporum f.sp. niveum Isolates

Fusarium wilt of watermelon (Citrullus lanatus), caused by Fusarium oxysporum f. sp. niveum (Fon), has reemerged as a major production constraint in the ...

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3.1K Views.  University of Florida. Managing Fusarium wilt of watermelon requires knowledge of the pathogen races present. Here, we describe the root-dip, infested kernel seeding, and modified tray-dip inoculation methods to demonstrate their efficacy in race-typing of the pathogenic fungus Fusarium oxysporum f. sp niveum (Fon).

Video: A Contrast of Three Inoculation Techniques used to Determine the Race of Unknown Fusarium oxysporum f.sp. niveum Isolates

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.
Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured.
The described kit procedure detects the trypsin protease control at a concentration of approximately 0.5 &mu;g/ml (5 ng of trypsin added to the assay). This sensitivity can be increased with a longer incubation time, up to 24 hours. The assay is performed in microcentrifuge tubes and procedures are provided for fluorescence detection using either cuvettes or multiwell plates.

The Protease Fluorescent Detection Kit is designed for the measurement of protease activity using fluorometry. It is also suitable for detection of trace amounts of protease contamination. The method is based on the proteolytic hydroysis of a proprietary formulation of a FITC-labeled casein substrate.

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host macromolecule. ITC has some advantages over common spectroscopic approaches for studying host/ligand interactions. For example, the heat released or absorbed when the two components interact is directly measured and does not require any exogenous reporters. Thus the binding enthalpy and the association constant (Ka) are directly obtained from ITC data, and can be used to compute the entropic contribution. Moreover, the shape of the isotherm is dependent on the c-value and the mechanistic model involved. The c-value is defined as c = n[P]tKa, where [P]t is the protein concentration, and n is the number of ligand binding sites within the host. In many cases, multiple binding sites for a given ligand are non-equivalent and ITC allows the characterization of the thermodynamic binding parameters for each individual binding site. This however requires that the correct binding model be used. This choice can be problematic if different models can fit the same experimental data. We have previously shown that this problem can be circumvented by performing experiments at several c-values. The multiple isotherms obtained at different c-values are fit simultaneously to separate models. The correct model is next identified based on the goodness of fit across the entire variable-c dataset. This process is applied here to the aminoglycoside resistance-causing enzyme aminoglycoside N-6'-acetyltransferase-Ii (AAC(6')-Ii). Although our methodology is applicable to any system, the necessity of this strategy is better demonstrated with a macromolecule-ligand system showing allostery or cooperativity, and when different binding models provide essentially identical fits to the same data. To our knowledge, there are no such systems commercially available. AAC(6')-Ii, is a homo-dimer containing two active sites, showing cooperativity between the two subunits. However ITC data obtained at a single c-value can be fit equally well to at least two different models a two-sets-of-sites independent model and a two-site sequential (cooperative) model. Through varying the c-value as explained above, it was established that the correct binding model for AAC(6')-Ii is a two-site sequential binding model. Herein, we describe the steps that must be taken when performing ITC experiments in order to obtain datasets suitable for variable-c analyses.

ITC is a powerful tool for studying the binding of a ligand to its host. In complex systems however, several models may fit the data equally well. The method described here provides a means to elucidate the appropriate binding model for complex systems and extract the corresponding thermodynamic parameters.

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host ...

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

Random microseed matrix screening (rMMS) is a protein crystallization technique in which seed crystals are added to random screens. By increasing the likelihood that crystals will grow in the metastable zone of a protein's phase diagram, extra crystallization leads are often obtained, the quality of crystals produced may be increased, and a good supply of crystals for data collection and soaking experiments is provided. Here we describe a general method for rMMS that may be applied to either sitting drop or hanging drop vapor diffusion experiments, established either by hand or using liquid handling robotics, in 96-well or 24-well tray format.

Here we describe a general method for random microseed matrix screening. This technique is shown to significantly increase the success rate of protein crystallization screening experiments, reduce the need for optimization, and provide a reliable supply of crystals for data collection and ligand-soaking experiments.

Random microseed matrix screening  (rMMS) is a protein crystallization technique in which seed crystals are added  to random screens. By increasing the ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

Fluorescent labeling is a well-established process for examining the fate of labeled molecules under a variety of experimental conditions both in vitro and in vivo. Fluorescent probes are particularly useful in determining the bio-distribution of administered large molecules, where the addition of a small-molecule fluorescent label is unlikely to affect the kinetics or bio-distribution of the compound. A variety of methods exist to examine bio-distribution that vary significantly in the amount of effort required and whether the resulting measurements are fully quantitative, but using multiple methods in conjunction can provide a rapid and effective system for analyzing bio-distributions.
Ex vivo whole-organ imaging is a method that can be used to quickly compare the relative concentrations of fluorescent molecules within tissues and between multiple types of tissues or treatment groups. Using an imaging platform designed for live-animal or whole-organ imaging, fluorescence within intact tissues can be determined without further processing, saving time and labor while providing an accurate picture of the overall bio-distribution. This process is ideal in experiments attempting to determine the tissue specificity of a compound or for the comparison of multiple different compounds. Quantitative tissue histology on the other hand requires extensive further processing of tissues in order to create a quantitative measure of the labeled compounds. To accurately assess bio-distribution, all tissues of interest must be sliced, scanned, and analyzed relative to standard curves in order to make comparisons between tissues or groups. Quantitative tissue histology is the gold standard for determining absolute compound concentrations within tissues.
Here, we describe how both methods can be used together effectively to assess the ability of different administration methods and compound modifications to target and deliver fluorescently labeled molecules to the central nervous system1.

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

Ex vivo whole-organ imaging is a rapid method for determining the relative concentrations of fluorescently labeled compounds within and between tissues or treatment groups. Conversely, quantitative fluorescence histology, while more labor intensive, allows for the quantification of the absolute tissue levels of labeled molecules.

Fluorescent labeling is a well-established process for examining the fate of labeled molecules under a variety of experimental conditions both in vitro ...

Simple and Effective Administration and Visualization of Microparticles in the Circulatory System of Small Fishes Using Kidney Injection

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, implantation of transgenic cells and tiny optical sensors. However, intravenous microinjections into small animals, which are mostly used in biological and veterinary laboratories, are very difficult and require trained personnel. Herein, we demonstrate a robust and efficient method for the introduction of microparticles into the circulatory system of adult zebrafish (Danio rerio) by injection into the fish kidney. To visualize the introduced microparticles in the vasculature, we propose a simple intravital imaging technique in fish gills. In vivo monitoring of the zebrafish blood pH was accomplished using an injected microencapsulated fluorescent probe, SNARF-1, to demonstrate one of the possible applications of the described technique. This article provides a detailed description of the encapsulation of pH-sensitive dye and demonstrates the principles of the quick injection and visualization of the obtained microcapsules for in vivo recording of the fluorescent signal. The proposed method of injection is characterized by a low mortality rate (0-20%) and high efficiency (70-90% success), and it is easy to institute using commonly available equipment. All described procedures can be performed on other small fish species, such as guppies and medaka.

This article demonstrates the principles of a quick, minimally invasive injection of fluorescent microparticles into the circulatory system of small fishes and the in vivo visualization of the microparticles in fish blood.

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, ...