The authors thank members of the Geddes-McAlister Lab for their valuable support throughout this investigation and their manuscript feedback. The authors acknowledge the funding support from the Ontario Graduate Scholarship and International Graduate Research Award - University of Guelph to D. G.-G and from the Canadian Foundation of Innovation (JELF 38798) and Ontario Ministry of Colleges and Universities - Early Researcher Award for J. G.-M.

1. Protein extraction from mollusks
<ol>
	<li>Collect mollusks from a designated and approved natural area (e.g., Speed River, Guelph, Ontario). For this study, both native and invasive species were selected to assess a broad range of potential antifungal effects.</li>
	<li>Gently break the shell of the mollusks (e.g., Cepaea nemoralis, Planorbella pilsbryi, and Cipangopaludina chinensis) using a pestle and mortar, and remove the solid pieces with a pair of tweezers. Generally, 10 mollusks are pooled for use throughout the protocol.</li>
	<li>Collect and weigh the organs. Approximately 15-20 g of sample is optimal. Use scissors to cut the organs into small pieces approximately 0.5-1 cm in size.</li>
	<li>Flash-freeze the dissected and cut organ samples with liquid nitrogen. Then, grind the organ samples into a fine powder using a pestle and mortar.</li>
	<li>Add the organ sample powder (approximately 15 g) into 30 mL of cold distilled water (4 &#176;C) to achieve a 1:2 ratio (w/v).</li>
	<li>Pour 2 mL of the sample into high-impact 2 mL tubes. Add a scoopful of 3 mm stainless-steel beads (approximately 500 &#181;g) to each tube, and homogenize using a blender (see Table of Materials) for 3 min at 1,200 rpm and 4 &#176;C.</li>
	<li>Centrifuge at 12,000 &#215; g for 20 min at 4 &#176;C. Collect all the supernatants with a pipette in a fresh 50 mL tube, discarding the pellet.</li>
	<li>Filter-sterilize the supernatant using a 0.22 &#181;m membrane. Store the samples on ice, and proceed to the clarification protocol (section 2), as applicable. 
	&#8203;NOTE: The samples can be flash-frozen in liquid nitrogen and stored at &#8722;20 &#176;C to be used as crude extracts.</li>
</ol>
2. Clarification of the mollusk extract
<ol>
	<li>Place the supernatant sample from step 1.8 in a thermal bath at 60 &#176;C for 30 min. Then, cool the samples quickly by transferring them to a bucket with ice for 20 min.</li>
	<li>Centrifuge the samples at 15,000 &#215; g for 45 min at 4 &#176;C. Collect the supernatant in a fresh 50 mL tube, and discard the pellet. Filter-sterilize the samples using a 0.22 &#181;m membrane filter, and then store them on ice.</li>
	<li>Determine the protein concentration in the samples from step 1.8 (i.e., the crude extract) and step 2.2 (i.e., the clarified extract) using a quantification assay (e.g., bicinchoninic acid assay17). The optimal protein concentration range is 4-8 mg/mL.</li>
	<li>Store the samples on ice for up to 1 h or flash-freeze in liquid nitrogen, and store them at &#8722;20 &#176;C until needed.</li>
</ol>
3. Inhibitory activity assay
<ol>
	<li>Measure the inhibitory activity of the crude and clarified extracts using enzymatic assays in technical triplicate. 
	NOTE: Each enzymatic assay consists of an enzyme (e.g., subtilisin A, which is involved in fungal virulence16,17,18; usually around 1 x 10&#8722;9 mol/L for chromogenic-based assays), a buffer, and a substrate. The reaction starts when the substrate encounters the enzyme. The enzymatic activity is proportional to the rate of substrate conversion into the product.</li>
	<li>Assess the effect of enzyme concentration (EC) over enzymatic activity (EA) until linear dependence is obtained between both parameters. For the next steps, use an enzyme concentration within the measured linear range. 
	NOTE: The enzymatic assay for the peptidase subtilisin A is detailed in the following steps.</li>
	<li>Incubate the crude (step 1.11) or clarified (step 2.5) mollusk protein extracts (e.g., 10 &#181;L containing 4 mg/mL protein) with 10 &#181;L of subtilisin A (concentration determined from step 3.2), and 220 &#181;L of 100 mM Tris-HCl at pH 8.6 (for the control, add 230 &#181;L of Tris buffer without adding the extract) for 10 min at 25 &#176;C in a 96-well flat-bottom plate.</li>
	<li>Add 10 &#181;L of N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, the substrate for subtilisin A, at a final concentration of 1 Km (Michaelis-Menten constant; 0.2 mM for this substrate with subtilisin A) for a final reaction volume of 250 &#181;L.</li>
	<li>Measure the enzymatic activity by monitoring the appearance of the product over time by reading the optical density (OD) at 405 nm every 15 s for 3 min.</li>
	<li>Determine the residual enzymatic activity by calculating the ratio of the enzymatic activity in the presence of the mollusk extract to that in the absence of the extract.</li>
	<li>If inhibitory activity is observed (i.e., residual activity below 1), measure the inhibitory concentration 50 (IC50)&#160;value using a range of concentrations of the extracts (e.g., a twofold dilution series from 200 &#181;g/mL to 1 &#181;g/mL) following standard methods19.</li>
</ol>
4. Effect of mollusk extracts on C. neoformans growth
<ol>
	<li>Use a 10 &#181;L pipette tip to introduce a single colony of C. neoformans var. grubii H99 wild-type (WT) into 5 mL of yeast extract-peptone-dextrose (YPD) medium (10 g/L yeast extract, 20 g/L peptone, and 20 g/L dextrose, pH 6.5), and incubate for 16-18 h at 30 &#176;C and 200 rpm. Perform the experiment in biological triplicate and technical duplicate.</li>
	<li>Collect 500 &#181;L of the culture in a cuvette, and measure the growth by reading the OD at 600 nm. Dilute the culture with yeast nitrogen base (YNB) medium to a final OD600 of 0.02.</li>
	<li>Co-culture C. neoformans cells with different concentrations (e.g., a threefold dilution series from 440 &#181;g/mL to 15 &#181;g/mL protein) of the extracts (i.e., crude and clarified). To do this, mix 10 &#181;L of the extracts with 190 &#181;L of the diluted C. neoformans culture (step 4.2) in a 96-well plate.</li>
	<li>Measure the growth by reading the OD600 using a plate reader every 15 min to 1 h for 72 h at 200 rpm and 37 &#176;C.</li>
</ol>
5. Effect of mollusk extracts on C. neoformans melanin production
<ol>
	<li>Prepare L-3,4-dihydroxyphenylalanine (L-DOPA) plates.
	<ol>
		<li>Autoclave 230 mL of 14 g/L agar, and let it cool until it reaches 50-60 &#176;C.</li>
		<li>Add 3.25 mL of 1 M glycine, 7.35 mL of 1 M KH2PO4, 2.5 mL of 1 M MgSO4.7H2O, 0.6 mL of 40% glucose, 70 &#181;L of 10 mM thiamine, and 2.5 mL of 100 mM L-DOPA (filter-sterilized) to the liquid agar.</li>
		<li>Pour 15 mL of the mixture into Petri plates, let them dry for approximately 1 h in the dark, and store at 2-4 &#176;C for up to 1 week. Ensure the lids are partially lifted for the agar to properly dry.</li>
	</ol>
	</li>
	<li>Inoculate 5 mL of YNB medium with 50 &#181;L of C. neoformans culture from step 4.1. Incubate for 16-18 h at 30 &#176;C and 200 rpm.</li>
	<li>Spin down the cells at 1,000 &#215; g for 5 min at 4 &#730;C. Wash the cells 2x with sterile phosphate-buffered saline (PBS) at pH 7.4.</li>
	<li>Count the cells using a hemocytometer. Resuspend the cells in 1 mL of PBS to reach a final concentration of 106 cells/mL.</li>
	<li>Prepare a dilution series (e.g., twofold) of the crude and clarified mollusk extracts. Similarly, prepare a 10-fold dilution series of C. neoformans cells (step 5.4) from 1 x 106 cells/mL to 1 x 101 cells/mL using sterile PBS in 96-well plates.</li>
	<li>Spread 200 &#181;L of the extracts onto Petri plates containing L-DOPA agar using a swab, and let them dry for 15 min.</li>
	<li>Spot 5 &#181;L of culture from each well onto the L-DOPA plate, leaving 1 cm between drops. Let it dry in the dark for 15 min.</li>
	<li>Incubate the plates at 30 &#176;C and 37 &#176;C in a static incubator for 3-5 days, capturing images every 24 h with a camera or plate imager (e.g., scanner). 
	&#8203;NOTE: Two temperature conditions are used here to explore the influence of temperature on the growth inhibitory effects of the mollusk extracts, with 30 &#176;C representing environmental and 37 &#730;C representing human physiological temperatures.</li>
</ol>
6. Effect of mollusk extracts on C. neoformans polysaccharide capsule production
<ol>
	<li>Prepare low iron medium (LIM) as described below.
	<ol>
		<li>Dissolve 5 g of chelating resin (see Table of Materials) in 60 mL of ultrapure water, and pack into a glass column (2 cm diameter x 25 cm length). Wash the column with 100 mL of water, and discard the collected water.</li>
		<li>Prepare low iron water (LI-water) by passing 2 L of sterile ultrapure water through the chelating column. Store the LI-water at 4 &#730;C for up to 3 months.</li>
		<li>Dissolve 5 g of glucose in 100 mL of LI-water. Dissolve 5 g of L-asparagine in 200 mL of LI-water in a separate beaker.</li>
		<li>Add the following to 500 mL of LI-water in order: 4.78 g of HEPES, 0.4 g of K2HPO4, 0.08 g of MgSO4.7H2O, 1.85 g of NaHCO3, and 0.25 g of CaCl2.2H2O.</li>
		<li>Combine the solutions from step 6.1.3 and step 6.1.4. Add 1 mL of 100 mM bathophenanthrolinedisulfonic acid sodium salt (BPS) to a final concentration of 100 &#181;M.</li>
		<li>Adjust the pH to 7.2 with 1 M LI-MOPS. Add LI-water to a final volume of 1 L, and filter- sterilize the medium by passing it through a 0.22 &#181;m membrane. Add 100 &#181;L of sterile thiamine (4 mg/mL) to the medium.</li>
	</ol>
	</li>
	<li>Inoculate 5 mL of YNB medium with 50 &#181;L of the C. neoformans culture from step 4.1. Incubate for 16-18 h at 30 &#176;C and 200 rpm. Use this culture to inoculate 5 mL of LIM to a final concentration of 105 cells/mL.</li>
	<li>Add appropriate volumes of the mollusk extracts (e.g., a twofold dilution or the volume determined from previous virulence assays) to reach the desired concentrations. Incubate for 72 h at 37 &#176;C and 200 rpm with loosened caps. After incubation, collect 1 mL of cells, and wash 2x with sterile PBS, pH 7.4, at 1,500 &#215; g for 2 min at 4 &#730;C.</li>
	<li>Gently resuspend the cells in 1 mL of PBS. Mix the cells with India Ink at 1:1 ratio (e.g., 4 &#181;L of India Ink with 4 &#181;L of cell suspension) over a glass microscope slide. Place a coverslip on top of the slide.</li>
	<li>Visualize the samples using a differential interference contrast (DIC) microscope with a 63x oil objective (see Table of Materials). Set the contrast to automatic, and focus manually using the microscope dial. Select all the pictures, and export them in TIFF format. No filters need to be applied.</li>
	<li>Using the ruler tool in ImageJ20, quantify the ratio of the total cell size (including the capsule) to the cell body size.</li>
</ol>
7. Effect of mollusk extracts on C. neoformans biofilm production
<ol>
	<li>Inoculate 5 mL of YNB medium with 50 &#181;L of C. neoformans culture from step 4.1. Incubate for 16-18 h at 30 &#176;C and 200 rpm.</li>
	<li>Harvest cells from the 5 mL culture. Wash the cells with sterile PBS, pH 7.4, at 1,500 &#215; g for 2 min at 4 &#730;C.</li>
	<li>Count the cells using a hemocytometer. Resuspend the cells in 3 mL of Dulbecco&#39;s Modified Eagle Medium (DMEM) to a final concentration of 107 cells/mL.</li>
	<li>Transfer 300 &#181;L of the cell suspension into the individual wells of a sterile, polystyrene, flat-bottom 24-well plate. Use wells with media only as a control.</li>
	<li>Add appropriate volumes of the mollusk extracts (e.g., a twofold dilution or the volume determined from previous virulence assays) to reach a final concentration of 10-20 &#181;g/mL protein. Ensure that the extract volume is not greater than 10% of the total volume.</li>
	<li>Wrap the plates with aluminum foil (to avoid media evaporation), and incubate for 48 h using a static incubator at 30 &#176;C and 37 &#176;C.</li>
	<li>Using a bottle or dispenser wash the wells 2x with sterile water, and let them air-dry for 10-15 min at room temperature. Then, add 100 &#181;L of 0.3% crystal violet solution to each well (including the medium-only control wells), and incubate at room temperature for 10 min.</li>
	<li>Wash the wells thoroughly 3x with sterile water (the biofilms will not be disrupted during washing). Add 200 &#181;L of 100% ethanol, and incubate for 10 min at RT.</li>
	<li>Transfer 75 &#181;L to a new 96-well flat-bottom plate, and read the OD550. Quantify the biofilm formation as (OD550nm of sample - OD550nm of blank).</li>
</ol>

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The authors declare no conflicts of interest.

The extraction protocol described here outlines the isolation of compounds from mollusks collected from Ontario, Canada, and demonstrates a novel investigation of using mollusk extracts against the human fungal pathogen, C. neoformans. This protocol adds to a growing body of research investigating peptidase inhibitor activity from invertebrates13. During the extraction, some extract samples were difficult to filter-sterilize, possibly due to the presence of soluble polysaccharides and/or ...

Cryptococcus neoformans is a human fungal pathogen that produces severe disease in immunocompromised hosts, such as individuals living with HIV/AIDS1, and leads to approximately 19% of AIDS-related deaths2. The fungus is susceptible to several classes of antifungals, including azoles, polyenes, and flucytosine, which exert fungicidal and fungistatic activity using distinct mechanisms3,4. However, the extensive use of antifungals in clinical and agricultural settings combined with strain hybridization have amplified the evolution of resistance in multiple fungal species, including C. neoformans5.
To overcome the challenges of antifungal resistance and reduce the prevalence of fungal infections on a global scale, a promising approach is to use the virulence factors of Cryptococcus spp. (e.g., temperature adaptability, polysaccharide capsule, melanin,&#160;and extracellular enzymes) as potential therapeutic targets4,6. This approach has several advantages, as these virulence factors are well-characterized in the literature, and targeting these factors could potentially reduce the rates of antifungal resistance by imposing a weaker selective pressure through impairing virulence rather than targeting cell growth6. In this context, numerous studies have assessed the possibility of targeting extracellular enzymes (e.g., proteases, peptidases) to reduce or inhibit the virulence of Cryptococcus spp.7,8,9. 
Organisms like invertebrates and plants do not possess an adaptive immune system to protect themselves from pathogens. However, they rely on a strong innate immune system with an immense array of chemical compounds to deal with microorganisms and predators10. These molecules include peptidase inhibitors, which play important roles in many biological systems, including the cellular processes of invertebrate immunity, such as the coagulation of hemolymph, the synthesis of cytokines and antimicrobial peptides, and the protection of hosts by directly inactivating the proteases of pathogens11. Thus, peptidase inhibitors from invertebrates such as mollusks possess potential biomedical applications, but many remain uncharacterized10,12,13. In this context, there are approximately 34 species of terrestrial mollusks in Ontario and 180 freshwater mollusks in Canada14. However, their in-depth profiling and characterization are still limited15. These organisms present an opportunity for the identification of new compounds with potential anti-fungal activity10.
In this protocol, methods to isolate and clarify extracts from invertebrates (e.g., mollusks) (Figure 1) followed by measuring the putative peptidase inhibitory activity are described. The antifungal properties of these extracts are then assessed by measuring their impact on C. neoformans virulence factor production using phenotypic assays (Figure 2). It is important to note that differences in antifungal properties between crude and clarified extracts may be indicative of microbial factors (e.g., secondary metabolites or toxins produced by the host microbiome) of the mollusk, which may influence experimental observations. Such findings support the need for this protocol to assess both crude and clarified extracts independently to unravel the modes of action. Additionally, the extraction process is unbiased and may enable the detection of antimicrobial properties against a plethora of fungal and bacterial pathogens. Therefore, this protocol provides an initiation point for the prioritization of mollusk species with antifungal properties against C. neoformans and an opportunity to evaluate the connections between enzymatic activity and virulence factor production through putative inhibitory mechanisms.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 &#956;m Filters</td>
			<td>VWR</td>
			<td>28145-477 (North America)</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Tubes (Safe-Lock)</td>
			<td>Eppendorf</td>
			<td>0030120086</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL Tubes (Safe-Lock)</td>
			<td>Eppendorf</td>
			<td>0030120094</td>
			<td></td>
		</tr>
		<tr>
			<td>3,4-Dihydroxy-L-phenylalanine (L-DOPA)</td>
			<td>Sigma-Aldrich</td>
			<td>D9628-5G</td>
			<td>CAS #: 59-92-7</td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Costar (Corning)</td>
			<td>3370</td>
			<td></td>
		</tr>
		<tr>
			<td>Bullet Blender Storm 24</td>
			<td>NEXT ADVANCE</td>
			<td>BBY24M</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5430R</td>
			<td>Eppendorf</td>
			<td>5428000010</td>
			<td></td>
		</tr>
		<tr>
			<td>Chelex 100 Resin</td>
			<td>BioRad</td>
			<td>142-1253</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator (Static)</td>
			<td>SANYO</td>
			<td>Not available</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryptococcus neoformans H99</td>
			<td>ATCC</td>
			<td>208821</td>
			<td></td>
		</tr>
		<tr>
			<td>DIC Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DIC Microscope software</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose (D-Glucose, Anhydrous, Reagent Grade)</td>
			<td>BioShop</td>
			<td>GLU501</td>
			<td>CAS #: 50-99-7</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Fisher Chemical</td>
			<td>G46-1</td>
			<td>CAS #: 56-40-6</td>
		</tr>
		<tr>
			<td>GraphPad Prism 9</td>
			<td>Dotmatics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>VWR</td>
			<td>15170-208</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate (MgSO4.7 H2O)</td>
			<td>Honeywell</td>
			<td>M1880-500G</td>
			<td>CAS #: 10034-99-8&#160;</td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>BioShop</td>
			<td>PEP403</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosohate buffer salt pH 7.4</td>
			<td>BioShop</td>
			<td>PBS408</td>
			<td>SKU: PBS408.500</td>
		</tr>
		<tr>
			<td>Plate reader (Synergy-H1)</td>
			<td>BioTek (Agilent)</td>
			<td>Not available</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic (KH2PO4)</td>
			<td>Fisher Chemical</td>
			<td>P285-500</td>
			<td>CAS #: 7778-77-0</td>
		</tr>
		<tr>
			<td>Subtilisin A</td>
			<td>Sigma-Aldrich</td>
			<td>P4860</td>
			<td>CAS #: 9014-01-01</td>
		</tr>
		<tr>
			<td>Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide</td>
			<td>Sigma-Aldrich</td>
			<td>573462</td>
			<td>CAS #: 70967-97-4</td>
		</tr>
		<tr>
			<td>Thermal bath</td>
			<td>VWR</td>
			<td>76308-834</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiamine Hydrochloride</td>
			<td>Fisher-Bioreagents</td>
			<td>BP892-100</td>
			<td>CAS #: 67-03-8</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>BioShop</td>
			<td>YEX401</td>
			<td>CAS #: 8013-01-2</td>
		</tr>
		<tr>
			<td>Yeast nitrogen base (with Amino Acids)</td>
			<td>Sigma-Aldrich</td>
			<td>Y1250-250G</td>
			<td>YNB&#160;</td>
		</tr>
	</tbody>
</table>

assessing the putative anticryptococcal properties of crude and clarified extracts from mollusks

Cryptococcus neoformans is an encapsulated human fungal pathogen with a global distribution that primarily infects immunocompromised individuals. The widespread use of antifungals in clinical settings, their use in agriculture, and strain hybridization have led to increased evolution of resistance. This rising rate of resistance against antifungals is a growing concern among clinicians and scientists worldwide, and there is heightened urgency to develop novel antifungal therapies. For instance, C. neoformans produces several virulence factors, including intra- and extra-cellular enzymes (e.g., peptidases) with roles in tissue degradation, cellular regulation, and nutrient acquisition. The disruption of such peptidase activity by inhibitors perturbs fungal growth and proliferation, suggesting this may be an important strategy for combating the pathogen. Importantly, invertebrates such as mollusks produce peptidase inhibitors with biomedical applications and anti-microbial activity, but they are underexplored in terms of their usage against fungal pathogens. In this protocol, a global extraction from mollusks was performed to isolate potential peptidase inhibitors in crude and clarified extracts, and their effects against classical cryptococcal virulence factors were assessed. This method supports the prioritization of mollusks with antifungal properties and provides opportunities for the discovery of anti-virulence agents by harnessing the natural inhibitors found in mollusks.

The workflow described herein enables the isolation of proteins and peptides from mollusks with potential anti-virulence properties against C. neoformans. Similarly, assessing different forms of extracts (i.e., crude and clarified) allows for the semi-purification of the potential active compounds and supports downstream assessment (e.g., mass spectrometry-based proteomics). Typically, the protein extraction workflow produces homogenized solutions with protein concentrations of 4-8 mg/mL. Here, the representativ...

Watch this Scientific Journal Video about Assessing the Putative Anticryptococcal Properties of Crude and Clarified Extracts from Mollusks at JoVE.com

Assessing the Putative Anticryptococcal Properties of Crude and Clarified Extracts from Mollusks

The human fungal pathogen Cryptococcus neoformans produces a variety of virulence factors (e.g., peptidases) to promote its survival within the host. Environmental niches represent a promising source of novel natural peptidase inhibitors. This protocol outlines the preparation of extracts from mollusks and the assessment of their effect on fungal virulence factor production.

Cryptococcus neoformans is an encapsulated human fungal pathogen with a global distribution that primarily infects immunocompromised individuals. The ...

assessing-putative-anticryptococcal-properties-crude-clarified

Research

JoVE Journal

Biology

1.0K Views.  University of Guelph. The human fungal pathogen Cryptococcus neoformans produces a variety of virulence factors (e.g., peptidases) to promote its survival within the host. Environmental niches represent a promising source of novel natural peptidase inhibitors. This protocol outlines the preparation of extracts from mollusks and the assessment of their effect on fungal virulence factor production.

Video: Assessing the Putative Anticryptococcal Properties of Crude and Clarified Extracts from Mollusks

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue interactions that regulate patterning and morphogenesis of vertebrates (e.g., 1, 2, 3, 4). Here, we illustrate a method that exploits this accessibility to test the signaling and patterning properties of ectodermal tissues during facial development. In these experiments, we create quail-chick 5 or mouse-chick 6 chimeras by transplanting the surface cephalic ectoderm that covers the upper jaw from quail or mouse onto either the same region or an ectopic region of chick embryos. The use of quail as donor tissue for transplantation into chicks was developed to take advantage of a nucleolar marker present in quail but not chick cells, thus allowing investigators to distinguish host and donor tissues 7. Similarly, a repetitive element is present in the mouse genome and is expressed ubiquitously, which allows us to distinguish host and donor tissues in mouse-chick chimeras 8. The use of mouse ectoderm as donor tissue will greatly extend our understanding of these tissue interactions, because this will allow us to test the signaling properties of ectoderm derived from various mutant embryos.

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of surface cephalic ectoderm during craniofacial development.

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue ...

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop.
Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group1. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle.
The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress 2. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures 3. DNA damage can be induced by ultraviolet (UV) irradiation, &gamma;-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin3, 4. MMS is an alkylating agent that inhibits DNA replication and activates the ATR/Chk1-mediated DNA damage checkpoint 4-7. UV irradiation also triggers the ATR/Chk1-dependent DNA damage checkpoint 8. The DNA damage-mimicking structure AT70 is an annealed complex of two oligonucleotides poly-(dA)70 and poly-(dT)70. The AT70 system was developed in Bill Dunphy's laboratory and is widely used to induce ATR/Chk1 checkpoint signaling 9-12.
Here, we describe protocols (1) to prepare cell-free egg extracts (LSE), (2) to treat Xenopus sperm chromatin with two different DNA damaging approaches (MMS and UV), (3) to prepare the DNA damage-mimicking structure AT70, and (4) to trigger the ATR/Chk1-mediated DNA damage checkpoint in LSE with damaged sperm chromatin or a DNA damage-mimicking structure.

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

Xenopus egg extract is a useful model system to investigate the DNA damage checkpoint. This protocol is for the preparation of Xenopus egg extracts and DNA damage checkpoint inducing reagents. These techniques are adaptable to a variety of DNA damaging approaches in the study of the DNA damage checkpoint signaling.

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used as manufacturing platforms for added-value products such as biopharmaceuticals. In both cases, liquid plant extracts may need to be clarified to remove particulates. Optimal clarification reduces the costs of filtration and centrifugation by increasing capacity and longevity. This can be achieved by introducing charged polymers known as flocculants, which cross-link dispersed particles to facilitate solid-liquid separation. There are no mechanistic flocculation models for complex mixtures such as plant extracts so empirical models are used instead. Here a design-of-experiments procedure is described that allows the rapid screening of different flocculants, optimizing the clarification of plant extracts and significantly reducing turbidity. The resulting predictive models allow the identification of robust process conditions and sets of polymers with complementary properties, e.g. effective flocculation in extracts with specific conductivities. The results presented for tobacco leaf extracts can easily be adapted to other plant species or tissues and will thus facilitate the development of more cost-effective downstream processes for commodities and plant-derived pharmaceuticals.

The design-of-experiments procedure presented here allows the evaluation of different flocculants in terms of their ability to aggregate dispersed particles in plant extracts, thus reducing turbidity and the costs of downstream processing.

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...

Systematic Approach to Identify Novel Antimicrobial and Antibiofilm Molecules from Plants' Extracts and Fractions to Prevent Dental Caries

Natural products provide structurally different substances, with a myriad of biological activities. However, the identification and isolation of active compounds from plants are challenging because of the complex plant matrix and time-consuming isolation and identification procedures. Therefore, a stepwise approach for screening natural compounds from plants, including the isolation and identification of potentially active molecules, is presented. It includes the collection of the plant material; preparation and fractionation of crude extracts; chromatography and spectrometry (UHPLC-DAD-HRMS and NMR) approaches for analysis and compounds identification; bioassays (antimicrobial and antibiofilm activities; bacterial &#34;adhesion strength&#34; to the salivary pellicle and initial glucan matrix treated with selected treatments); and data analysis. The model is simple, reproducible, and allows high-throughput screening of multiple compounds, concentrations, and treatment steps can be consistently controlled. The data obtained provide the foundation for future studies, including formulations with the most active extracts and/or fractions, isolation of molecules, modeling molecules to specific targets in microbial cells and biofilms. For example, one target to control cariogenic biofilm is to inhibit the activity of Streptococcus mutans glucosyltransferases that synthesize the extracellular matrix&#8217; glucans. The inhibition of those enzymes prevents the biofilm build-up, decreasing its virulence.

Natural products represent promising starting points for the development of new drugs and therapeutic agents. However, due to the high chemical diversity, finding new therapeutic compounds from plants is a challenging and time-consuming task. We describe a simplified approach to identify antimicrobial and antibiofilm molecules from plant extracts and fractions.

Natural products provide structurally different substances, with a myriad of biological activities. However, the identification and isolation of active ...

Assessing Energy Substrate Oxidation In Vitro with 14CO2 Trapping

Mitochondria host the machinery for the tricarboxylic acid (TCA) cycle and electron transport&#160;chain (ETC), which generate adenosine triphosphate (ATP) to maintain energy homeostasis. Glucose, fatty acids, and amino acids are the major energy substrates fueling mitochondrial respiration in most somatic cells. Evidence shows that different cell types may have a distinct preference for certain substrates. However, substrate utilization by various cells in the skeleton has not been studied in detail. Moreover, as cellular metabolism is attuned to physiological and pathophysiological changes, direct assessments of substrate dependence in skeletal cells may provide important insights into the pathogenesis of bone diseases.
The following protocol is based on the principle of carbon dioxide release from substrate molecules following oxidative phosphorylation. By using substrates containing radioactively labeled carbon atoms (14C), the method provides a sensitive and easy-to-use assay for the rate of substrate oxidation in cell culture. A case study with primary calvarial preosteoblasts versus bone marrow-derived macrophages (BMMs) demonstrates different utilization of the main substrates between the two cell types.

Assessing Energy Substrate Oxidation In Vitro with 14CO2 Trapping

This protocol describes an easy-to-use method to examine substrate oxidation by tracking 14CO2&#160;production in vitro.

Mitochondria host the machinery for the tricarboxylic acid (TCA) cycle and electron transport&#160;chain (ETC), which generate adenosine triphosphate ...