The authors thank Dr. Alexander Gottschalk for ZX1659, the RCaMP and channelrhodopsin containing worm strain, Dr. Hongkyun Kim for the slo-1(eg142) worm strain, and the National Bioresource Project for the sca-1(tm5339) worm strain.

1. Microscope setup
<ol>
	<li>Use a compound microscope with fluorescence capabilities. For this study, data were collected on an upright microscope (Table of Materials) fitted with LEDs for excitation.</li>
	<li>In order to properly visualize fluorescence changes in body wall muscles, use a high magnification objective.
	<ol>
		<li>For dissected preparations, use a 60x NA 1.0 water immersion objective (Table of Materials).</li>
		<li>For preparations using nanobeads, use a 60x NA 1.4 oil immersion objective (Table of Materials). This magnification ensures sufficient resolution of muscles onto the camera sensor.</li>
	</ol>
	</li>
	<li>Use a high sensitivity camera attached to the microscope to capture images at a high frame rate in order to track rapid changes in calcium levels. For this study, image with an sCMOS system (Table of Materials) capable of full frame imaging at 100 Hz, as rise times for the Ca2+ signals can be as rapid as tens of milliseconds.</li>
	<li>Control camera acquisition and LED fluorescence excitation with a micro-manager software running in ImageJ10 according to the manufacturer&#39;s instructions (Table of Materials).</li>
	<li>Use an open-source electronic platform plugin, which connects an external open-source microcontroller board (Table of Materials), to manage the external timing pulses to control the fluorescence excitation. 
	NOTE: Digital outs are available from digital pins 8 to 13 providing output bits 0 to 5. These can be addressed as base 2 values of 1, 10, 100, etc. Serial port settings are indicated in Table 1 and available from the micro-manager website (Table of Materials). Firmware for the microcontroller board is similarly available at this website.</li>
	<li>To control the timing logic, activate the micromanager acquisition protocol in the software according to the manufacturer&#39;s instructions (Table of Materials). 
	NOTE: This simultaneously initiates a preset camera frame duration and the number of frames to be activated, as well as the logical shutter and preset amber LED for fluorescence excitation. In this case, the amber LED solid state switch is controlled by microcontroller bit 1 output through pin 9. The camera frames out TTL (back plate out 3 connector) triggers a stimulator. This precisely times the activation of the blue light LED for the activation of channelrhodopsin at a set time following the activation of the imaging sequence.</li>
	<li>To stimulate channelrhodopsin with blue light and record RCaMP changes, use two LEDs. Activate channelrhodopsin with an LED with a peak emission wavelength of 470 nm and a bandpass filter (455 to 490 nm) and excite RCaMP with an LED with a peak emission wavelength of 594 nm and a bandpass filter (570 to 600 nm).</li>
	<li>In order to simultaneously activate channelrhodopsin and capture changes in RCaMP fluorescent levels, co-illuminate both LEDs and transmit the light to the same optical path using a dichroic beam combiner.</li>
	<li>Control the timing of LED illumination with solid state switches (Table of Materials) controlled by TTL signals (maximum rated turn-on time, 1 ms, turn-off time 0.3 ms: 0 to 90% turn-on and turn off time of &#60; 100 &#181;s).</li>
	<li>Set the LED intensity with the current controlled low noise linear power supplies (Table of Materials).</li>
	<li>To ensure the precise timing of the blue light LED, activate it directly by TTL pulse to the solid-state relay from a stimulator. Program the blue light stimulation protocol into a stimulator (Table of Materials), which acts as a precise timer from the start of image acquisition to blue LED illumination. In this experiment, turn on blue light stimulation after 2 s of capturing RCaMP fluorescence only and use a train of 5, 2 ms blue light pulses with 50 ms interpulse intervals to activate channelrhodopsin. 
	NOTE: The delay before blue light pulses, the duration of the blue light pulses, the time between pulses, and the number of pulses in the train can all be set at this point and should reflect the specific parameters of experimental interest.</li>
</ol>
2. C. elegans sample preparation and data acquisition
<ol>
	<li>To optically stimulate presynaptic neurons, obtain animals expressing channelrhodopsin in excitatory cholinergic neurons, driven with the unc-17 promoter region, and RCaMP expressed in all body wall muscles driven with the myo-3 promoter region2. 
	NOTE: Only the use of animals with integrated transgenes and robust levels of fluorescence are recommended as the variable expression in the corresponding cell types may affect the reliability of data acquisition.</li>
	<li>Make a working stock of all-trans retinal by diluting the retinal powder (Table of Materials) in ethanol to create a final concentration of 100 mM and store at -20 &#176;C. This working stock will be stable for approximately one year.</li>
	<li>Create a stock of OP50 E. coli, grown in LB media, supplemented with all-trans retinal from the working stock made in step 2.2, at a final concentration of 80 &#181;M. The volume used for the OP50+retinal stock will be dependent on the number of plates necessary for the experiment.</li>
	<li>Seed nematode growth media (NGM) plates with approximately 300 &#181;L of the OP50+retinal stock and allow plates to dry overnight at room temperature in the dark.</li>
	<li>Grow C. elegans strains to the desired age in the dark on OP50+retinal NGM plates at 20 &#176;C. For this experiment, use adult worms.
	<ol>
		<li>For the experiment using the dissected preparation, only use gravid adult worms as performing the dissection on younger, smaller animals is extremely challenging. Leave the animals on the OP50-retinal plates for a minimum of 3 days for an effective channelrhodopsin activation.</li>
	</ol>
	</li>
	<li>If using the dissected preparation8,11 (Figure 1A), perform dissections in low light.
	<ol>
		<li>Place animals in a dissecting dish with a silicone-coated coverslip base that is filled with a 1 mM Ca2+ extracellular solution composed of 150 mM NaCl, 5 mM KCl, 1 mM CaCl2, 4 mM MgCl2, 10 mM glucose, 5 mM sucrose, and 15 mM HEPES (pH 7.3, -340 Osm).</li>
		<li>Glue down animals using the liquid topical skin adhesive with blue coloring along the dorsal side of the worm and make a lateral cuticle incision along the glue/worm interface using glass needles.</li>
		<li>Use a mouth pipette to clear the internal viscera from the worm cavity.</li>
		<li>Glue down the cuticle flap of the animal to expose the ventral medial body wall muscles for imaging.</li>
	</ol>
	</li>
	<li>If using the nanobead preparation (Figure 1B)
	<ol>
		<li>Make a molten 5% agarose solution using ddH2O to a final volume of 100 mL.</li>
		<li>Using a Pasteur pipette, place a drop of molten agarose solution onto a glass slide and immediately place a second glass slide over the top, perpendicular to the first, using gentle pressure to create an even agarose pad. Remove the top slide before use.</li>
		<li>Add approximately 4 &#181;L of polystyrene nanobeads (Table of Materials) to the middle of the agarose pad.</li>
		<li>In the low light, pick 4-6 C. elegans into the nanobead solution, making sure animals do not lay on top of each other, and carefully place a coverslip on the top.</li>
	</ol>
	</li>
	<li>Place the prepared slide or dissection dish onto the microscope, and find and focus on a worm using 10x magnification and dim bright field illumination.</li>
	<li>Switch to 60x magnification and RCaMP fluorescence excitation to identify a ventromedial body wall muscle that is anterior to the vulva and in the correct focal plane. 
	NOTE: Muscles anterior to the vulva are selected as they reflect muscles commonly stimulated in electrophysiology experiments.</li>
	<li>Change the image pathway from the eyepiece to the camera by pulling out the toggle and clicking Live within the data acquisition software (Table of Materials). 
	NOTE: Make sure the blue light stimulation pathway is turned off at this point to ensure that the channelrhodopsin does not get activated before capturing images.</li>
	<li>Focus the image within the data acquisition software using the microscope fine focus.</li>
	<li>Once the muscle is clearly in focus, turn off the live image by unclicking the Live button.</li>
	<li>Click the ROI button in the data acquisition software and create a box around the muscle being focused on.</li>
	<li>On the stimulator, switch on the blue light stimulation pathway that has been previously programmed in step 1.10.</li>
	<li>Click Acquire in the imaging software to capture the image through the CMOS camera. To do this, set the exposure time to 10 s with 1,000 frames and 2x binning.</li>
</ol>
3. Data analysis
<ol>
	<li>Open the data file in the imaging software (Table of Materials) and, using the Polygon Tool, outline the muscle of interest. This is the ROI.</li>
	<li>Go to Image | Stacks | Plot Z-axis profile and export the resulting data points into the spreadsheet software (Table of Materials). This function plots the ROI selection mean value versus the time point.</li>
	<li>Move the muscle ROI created with the Polygon tool outside of the animal to get a background fluorescence measurement using the steps outlined in 3.2. Export the resulting data into the spreadsheet workbook.</li>
	<li>In the spreadsheet workbook, subtract the background fluorescence values from the muscle fluorescence values at each time point. This provides the background subtracted fluorescent signal.</li>
	<li>Average the background subtracted fluorescence for the first 2 s of data points. This will give the baseline fluorescence measurement, F.</li>
	<li>Use the baseline fluorescence measurement to calculate the normalized fluorescence level at each time point. To do this, use the equation (&#916;F/F)+1. &#916;F represents (F(t)-F), where F(t) is the fluorescence measurement at any given time point and F is the baseline value. The +1 is added as a y-axis offset.</li>
	<li>Repeat steps 3.2-3.6 for each muscle image that is collected. Using single or multiple muscle cells per image is at the discretion of the researcher. The n of the experiment can be determined by performing a power analysis.</li>
	<li>Use the processed data from steps 3.2-3.6 to make a trace of the fluorescent values in graphing software according to the manufacturer&#39;s instructions (Table of Materials).</li>
	<li>From this trace, measure the kinetics of the calcium transient, such as rise to peak time and half decay time, using the tools provided in the graphing software as per the manufacturer&#39;s instructions.</li>
</ol>

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The authors have nothing to disclose.

GECIs are a powerful tool in C. elegans neurobiology. Previous work has utilized calcium imaging techniques to examine a wide variety of functions in both neurons and muscle cells, including sensory and behavioral responses, with varied methods of stimulation. Some studies have used chemical stimuli to trigger calcium transients in sensory ASH neurons22,23 or to induce calcium waves in pharyngeal muscles24. Another group utilized ...

This paper presents methods for in vivo calcium imaging of C. elegans body wall muscles using optogenetic neuronal stimulation. Pairing a muscle expressed genetically encoded calcium indicator (GECI) with blue light triggered neuronal depolarization and provides a system to clearly observe the evoked postsynaptic calcium transients. This avoids the use of electrical stimulation, allowing a non-invasive analysis of mutants affecting the postsynaptic calcium dynamics.
Single-fluorophore GECIs, such as GCaMP, uses a single fluorescent protein molecule fused to the M13 domain of myosin light chain kinase at its N-terminal end and calmodulin (CaM) at the C-terminus. Upon calcium binding, the CaM domain, which has a high affinity for calcium, undergoes a conformational change inducing a subsequent conformational change in the fluorescent protein leading to an increase in fluorescence intensity1. GCaMP fluorescence is excited at 488 nm, making it unsuitable to be used in conjunction with channelrhodopsin, which has a similar excitation wavelength of 473 nm. Thus, in order to couple calcium measurements with channelrhodopsin stimulation, the green fluorescent protein of GCaMP needs to be replaced with a red fluorescent protein, mRuby (RCaMP). Using the muscle expressed RCaMP, in combination with the cholinergic motor neuron expression of channelrhodopsin, permits studies at the worm neuromuscular junction (NMJ) with simultaneous use of optogenetics and functional imaging within the same animal2.
The use of channelrhodopsin bypasses the need for the electrical stimulation to depolarize the neuromuscular junctions of C. elegans, which can only be achieved in dissected preparations, thus making this technique both easier to employ and more precise when targeting specific tissues. For example, channelrhodopsin has been previously used in C. elegans to reversibly activate specific neurons, leading to either robust activation of excitatory or inhibitory neurons3,4. The use of light-stimulated depolarization also circumvents the issue of neuronal damage due to the direct electrical stimulation. This provides an opportunity to examine the effects of many different stimulation protocols, including sustained and repeated stimulations, on postsynaptic calcium dynamics4.
The transparent nature of C. elegans makes it ideal for the fluorescent imaging functional analysis. However, when stimulating the excitatory acetylcholine neurons at the NMJ, animals are expected to respond with an immediate muscle contraction4, making the immobilization of the worms critical in visualizing discrete calcium changes. Traditionally, pharmacological agents have been used to paralyze the animals. One such drug used is levamisole, a cholinergic acetylcholine receptor agonist5,6,7. Since levamisole leads to the persistent activation of a subtype of excitatory muscle receptors, this reagent is unsuitable for the study of the muscle calcium dynamics. The action of levamisole induces postsynaptic depolarization, elevating the cytosolic calcium, and occluding observations following presynaptic stimulation. To avoid the use of paralyzing drugs, we examined two alternative methods to immobilize C. elegans. Animals were either glued down and then dissected open to expose the body wall muscles, similar to the existing C. elegans NMJ electrophysiology method8, or nanobeads were used to immobilize intact animals9. Both procedures allowed for the reproducible measurements of the resting and evoked muscle calcium transients that were easily quantifiable.
The methods in this paper can be used to measure the baseline cytosolic calcium levels and transients in postsynaptic body wall muscle cells in C. elegans. Examples of data employing the two different immobilization techniques are given. Both techniques take advantage of optogenetics to depolarize the muscle cells without the use of electrical stimulation. These examples demonstrate the feasibility of this method in evaluating mutations that affect postsynaptic calcium handling in the worms and point out the pros and cons of the two immobilization approaches.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>all-trans retinal</td>
			<td>Sigma-Aldrich</td>
			<td>R2500</td>
			<td>Necessary for excitation of channel rhodopsin</td>
		</tr>
		<tr>
			<td>Amber LED</td>
			<td></td>
			<td></td>
			<td>RCaMP illumination</td>
		</tr>
		<tr>
			<td>Arduino UNO</td>
			<td>Mouser</td>
			<td>782-A000066</td>
			<td>Controls fluorescence illumination</td>
		</tr>
		<tr>
			<td>Blue LED</td>
			<td></td>
			<td></td>
			<td>channelrhodopsin illumination</td>
		</tr>
		<tr>
			<td>BX51WI microscope</td>
			<td>Olympus</td>
			<td></td>
			<td>Fixed state compound microscope</td>
		</tr>
		<tr>
			<td>Current controlled low noise linear power supply</td>
			<td>Ametek</td>
			<td>Sorenson</td>
			<td>Controls LED intensity</td>
		</tr>
		<tr>
			<td>Igor Pro</td>
			<td>Wavemetrics</td>
			<td>Wavemetrics.com</td>
			<td>Graphing software</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td>imagej.nih.gov</td>
			<td>Image processing software</td>
		</tr>
		<tr>
			<td>LUMFLN 60x water NA 1.4</td>
			<td>Olympus</td>
			<td></td>
			<td>Water immersion objective for dissected preparation</td>
		</tr>
		<tr>
			<td>Master-8 Stimulator</td>
			<td>A.M.P.I</td>
			<td></td>
			<td>Master timer for image acquisition and LED illumination</td>
		</tr>
		<tr>
			<td>Micro-Manager</td>
			<td></td>
			<td>micro-manager.org</td>
			<td>Controls camera acquisition and LED excitiation</td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td>Spreadsheet software</td>
		</tr>
		<tr>
			<td>pco.edge 4.2 CMOS camera</td>
			<td>pco.</td>
			<td>4.2</td>
			<td>High-speed camera</td>
		</tr>
		<tr>
			<td>PlanApo N 60x oil NA 1.4</td>
			<td>Olympus</td>
			<td></td>
			<td>Oil immersion objective for nanobead preparation</td>
		</tr>
		<tr>
			<td>Polybead microspheres</td>
			<td>Polysciences, Inc.</td>
			<td>00876-15</td>
			<td>For worm immobilization</td>
		</tr>
		<tr>
			<td>solid state switches</td>
			<td>Sensata Technologies</td>
			<td>Crydom CMX100D6</td>
			<td>Controls timing of LED illumination</td>
		</tr>
		<tr>
			<td>Transgenic strain, sca-1(tm5339); [zxIs6{Punc17::chop-2 
			(h134R)::yfp,lin-15(+)}; 
			Pmyo3::RCaMP35]</td>
			<td>Richmond Lab</td>
			<td>SY1627</td>
			<td>Excitatory neuronal channelrhodopsin and body wall muscle RCaMP expressing worm line with SERCA mutant allele</td>
		</tr>
		<tr>
			<td>Transgenic strain, slo-1 (eg142); [zxIs6{Punc17::chop-2 
			(h134R)::yfp,lin-15(+)}; 
			Pmyo3::RCaMP35]</td>
			<td>Richmond Lab</td>
			<td></td>
			<td>Excitatory neuronal channelrhodopsin and body wall muscle RCaMP expressing worm line with calcium-activated BK potassium channel mutant allele</td>
		</tr>
		<tr>
			<td>Transgeneic strain, [zxIs6{Punc17::chop-2 
			(h134R)::yfp,lin-15(+)}; 
			Pmyo3::RCaMP35]</td>
			<td>Gottschalk Lab</td>
			<td>ZX1659</td>
			<td>Excitatory neuronal channelrhodopsin and body wall muscle RCaMP expressing worm line</td>
		</tr>
	</tbody>
</table>

in vivo calcium imaging in c. elegans body wall muscles

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for the targeted expression of genetically encoded calcium sensors. This protocol outlines the use of these sensors for the in vivo imaging of calcium dynamics in targeted cells, specifically the body wall muscles of the worms. By utilizing the co-expression of presynaptic channelrhodopsin, stimulation of acetylcholine release from excitatory motor neurons can be induced using blue light pulses, resulting in muscle depolarization and reproducible changes in cytoplasmic calcium levels. Two worm immobilization techniques are discussed with varying levels of difficulty. Comparison of these techniques demonstrates that both approaches preserve the physiology of the neuromuscular junction and allow for the reproducible quantification of calcium transients. By pairing optogenetics and functional calcium imaging, changes in postsynaptic calcium handling and homeostasis can be evaluated in a variety of mutant backgrounds. Data presented validates both immobilization techniques and specifically examines the roles of the C. elegans sarco(endo)plasmic reticular calcium ATPase and the calcium-activated BK potassium channel in the body wall muscle calcium regulation.

This technique evaluated changes in mutants thought to affect the calcium handling or muscle depolarization. Baseline fluorescence levels and fluorescent transients were visualized and resting cytosolic calcium and calcium kinetics within the muscle were evaluated. It is important that the animals were grown on all-trans retinal for at least three days to ensure the successful incorporation of retinal, thereby subsequently activating the channelrhodopsin (Figure 2</st...

Watch this Scientific Journal Video about In Vivo Calcium Imaging in C. elegans Body Wall Muscles at JoVE.com

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

This method provides a way to couple optogenetics and genetically encoded calcium sensors to image baseline cytosolic calcium levels and changes in evoked calcium transients in the body wall muscles of the model organism C. elegans.

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

in-vivo-calcium-imaging-in-c-elegans-body-wall-muscles

Research

JoVE Journal

Biochemistry

7.6K Views.  University of Illinois at Chicago. This method provides a way to couple optogenetics and genetically encoded calcium sensors to image baseline cytosolic calcium levels and changes in evoked calcium transients in the body wall muscles of the model organism C. elegans.

Video: In Vivo Calcium Imaging in C. elegans Body Wall Muscles

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

Combining Raman Imaging and Multivariate Analysis to Visualize Lignin, Cellulose, and Hemicellulose in the Plant Cell Wall

The application of Raman imaging to plant biomass is increasing because it can offer spatial and compositional information on aqueous solutions. The analysis does not usually require extensive sample preparation; structural and chemical information can be obtained without labeling. However, each Raman image contains thousands of spectra; this raises difficulties when extracting hidden information, especially for components with similar chemical structures. This work introduces a multivariate analysis to address this issue. The protocol establishes a general method to visualize the main components, including lignin, cellulose, and hemicellulose within the plant cell wall. In this protocol, procedures for sample preparation, spectral acquisition, and data processing are described. It is highly dependent upon operator skill at sample preparation and data analysis. By using this approach, a Raman investigation can be performed by a non-specialist user to acquire high-quality data and meaningful results for plant cell wall analysis.

This protocol aims to present a general method to visualize lignin, cellulose, and hemicellulose in plant cell walls using Raman imaging and multivariate analysis.

The application of Raman imaging to plant biomass is increasing because it can offer spatial and compositional information on aqueous solutions. The ...

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for skin-care cosmetics. The goals of the protocol are: (1) to process chicken feathers into KH by alkaline-enzymatic hydrolysis and purify it by dialysis, and (2) to test if adding KH into an ointment base (OB) increases hydration of the skin and improves skin barrier function by diminishing transepidermal water loss (TEWL). During alkaline-enzymatic hydrolysis feathers are first incubated at a higher temperature in an alkaline environment and then, under mild conditions, hydrolyzed with proteolytic enzyme. The solution of KH is dialyzed, vacuum dried, and milled to a fine powder. Cosmetic formulations comprising from oil in water emulsion (O/W) containing 2, 4, and 6 weight% of KH (based on the weight of the OB) are prepared. Testing the moisturizing properties of KH is carried out on 10 men and 10 women at time intervals of 1, 2, 3, 4, 24, and 48 h. Tested formulations are spread at degreased volar forearm sites. The skin hydration of stratum corneum (SC) is assessed by measuring capacitance of the skin, which is one of the most world-wide used and simple methods. TEWL is based on measuring the quantity of water transported per a defined area and period of time from the skin. Both methods are fully non-invasive. KH makes for an excellent occlusive; depending on the addition of KH into OB, it brings about a 30% reduction in TEWL after application. KH also functions as a humectant, as it binds water from the lower layers of the epidermis to the SC; at the optimum KH addition in the OB, up to 19% rise in hydration in men and 22% rise in women occurs.

The goal of the protocol is to prepare keratin hydrolysate from chicken feathers by alkaline-enzymatic hydrolysis and to test whether adding keratin hydrolysate into a cosmetics ointment base improves skin barrier function (heightening hydration and diminishing transepidermal water loss). Tests are conducted on men and woman volunteers.

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for ...

Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts of purified material. Here, we describe a simple method for gaining detailed polysaccharide structural information, including resolution of structural isomers. For polysaccharide analysis by gel electrophoresis (PACE), plant cell wall material is hydrolyzed with glycosyl hydrolases specific to the polysaccharide of interest (e.g., mannanases for mannan). Large format polyacrylamide gels are then used to separate the released oligosaccharides, which have been fluorescently labeled. Gels can be visualized with a modified gel imaging system (see Table of Materials). The resulting oligosaccharide fingerprint can either be compared qualitatively or, with replication, quantitatively. Linkage and branching information can be established using additional glycosyl hydrolases (e.g., mannosidases and galactosidases). Whilst this protocol describes a method for analyzing glucomannan structure, it can be applied to any polysaccharide for which characterized glycosyl hydrolases exist. Alternatively, it can be used to characterize novel glycosyl hydrolases using defined polysaccharide substrates.

A protocol for the structural analysis of polysaccharides by gel electrophoresis (PACE), using mannan as an example, is described.

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts ...

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to precisely maneuver the nucleation and crystal growth towards desired sizes of protein crystals. The controlled crystallization is based on sample investigation with in situ Dynamic Light Scattering (DLS) while all visual changes in the droplet are monitored online with the help of a microscope coupled to a CCD camera, thus enabling a full investigation of the protein droplet during all stages of crystallization. The use of in situ DLS measurements throughout the entire experiment allows a precise identification of the highly supersaturated protein solution transitioning to a new phase &#8211; the formation of crystal nuclei. By identifying the protein nucleation stage, the crystallization can be optimized from large protein crystals to the production of protein microcrystals. The experimental protocol shows an interactive crystallization approach based on precise automated steps such as precipitant addition, water evaporation for inducing high supersaturation, and sample dilution for slowing induced homogeneous nucleation or reversing phase transitions.

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Here we present a protocol for controlled production of protein microcrystals. The process uses an automated device allowing controlled manipulation of several crystallization parameters. The protein crystallization is carried-out by controlled and automated addition of crystallization solutions while monitoring and investigating the radius distribution of particles in the crystallization droplet.

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom body (MB) is a higher brain center proposed to be the neural substrate of complex honeybee behaviors. Although previous studies identified genes and proteins that are differentially expressed in the MBs and other brain regions, the activities of the proteins in each region are not yet fully understood. To reveal the functions of these proteins in the brain, pharmacologic analysis is a feasible approach, but it is first necessary to confirm that pharmacologic manipulations indeed alter the protein activity in these brain regions.
We previously identified a higher expression of genes encoding phospholipase C (PLC) in the MBs than in other brain regions, and pharmacologically assessed the involvement of PLC in honeybee behavior. In that study, we biochemically tested two pharmacologic agents and confirmed that they decreased PLC activity in the MBs and other brain regions. Here, we present a detailed description of how to detect PLC activity in honeybee brain homogenate. In this assay system, homogenates derived from different brain regions are reacted with a synthetic fluorogenic substrate, and fluorescence resulting from PLC activity is quantified and compared between brain regions. We also describe our evaluation of the inhibitory effects of certain drugs on PLC activity using the same system. Although this system is likely affected by other endogenous fluorescence compounds and/or the absorbance of the assay components and tissues, the measurement of PLC activity using this system is safer and easier than that using the traditional assay, which requires radiolabeled substrates. The simple procedure and manipulations allow us to examine PLC activity in the brains and other tissues of honeybees involved in different social tasks.

To test the inhibitory effects of pharmacologic agents on phospholipase C (PLC) in different regions of the honeybee brain, we present a biochemical assay to measure PLC activity in those regions. This assay could be useful for comparing PLC activity among tissues, as well as among bees exhibiting different behaviors.

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom ...

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, and protein-protein interactions. FPOP utilizes a KrF excimer laser at 248 nm for photolysis of hydrogen peroxide to generate hydroxyl radicals which in turn oxidatively modify solvent-accessible amino acid side chains. Recently, we expanded the use of FPOP of in vivo oxidative labeling in Caenorhabditis elegans (C. elegans), entitled IV-FPOP. The transparent nematodes have been used as model systems for many human diseases. Structural studies in C. elegans by IV-FPOP is feasible because of the animal&#8217;s ability to uptake hydrogen peroxide, their transparency to laser irradiation at 248 nm, and the irreversible nature of the modification. The assembly of a microfluidic flow system for IV-FPOP labeling, IV-FPOP parameters, protein extraction, and LC-MS/MS optimized parameters are described herein.

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

In vivo fast photochemical oxidation of proteins (IV-FPOP) is a hydroxyl radical protein footprinting technique that allows for mapping of protein structure in their native environment. This protocol describes the assembly and set-up of the IV-FPOP microfluidic flow system.

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, ...

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These amyloid fibrils can sequester endogenous metastable proteins as well as components of the proteostasis network (PN) and thereby exacerbate protein misfolding in the cell. There are a limited number of tools available to assess the aggregation process of amyloid proteins within an animal. We present a protocol for fluorescence lifetime microscopy (FLIM) that allows monitoring as well as quantification of the amyloid fibrilization in specific cells, such as neurons, in a noninvasive manner and with the progression of aging and upon perturbation of the PN. FLIM is independent of the expression levels of the fluorophore and enables an analysis of the aggregation process without any further staining or bleaching. Fluorophores are quenched when they are in close vicinity of amyloid structures, which results in a decrease of the fluorescence lifetime. The quenching directly correlates with the aggregation of the amyloid protein. FLIM is a versatile technique that can be applied to compare the fibrilization process of different amyloid proteins, environmental stimuli, or genetic backgrounds in vivo in a non-invasive manner.

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Fluorescence lifetime imaging monitors, quantifies and distinguishes the aggregation tendencies of proteins in living, aging, and stressed C. elegans disease models.

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These ...