eoe sub articles

EoE Sub Articles

1. Solutions and Media
<ol>
	<li>Prepare 2X Roswell Park Memorial Institute (RPMI) 1640 medium, phosphate-buffered saline (PBS), Sabouraud dextrose broth, and Sabouraud dextrose agar as per Table 1.</li>
</ol>
2. Fungal Inoculum Growth Conditions
<ol>
	<li>Store all fungal strains as frozen stocks in 35% glycerol at -80 &#176;C, until needed.</li>
	<li>Perform the following steps before each experiment.
	<ol>
		<li>For Candida albicans strains:
		<ol>
			<li>Thaw a stock vial and transfer 200 &#181;L to 10 mL of Sabouraud dextrose broth in a sterile 50 mL polypropylene tube with a cap and culture overnight at 30 &#176;C with agitation (200 rpm). Remember to incline the tube and leave the cap slightly open for better aeration of the culture.</li>
		</ol>
		</li>
		<li>For Cryptococcus neoformans strains:
		<ol>
			<li>Scrape the frozen surface of a stock vial. Plate the cells onto Sabouraud dextrose agar plates and incubate for 48 h at 30 &#176;C. After visible growth of isolated colonies, keep the plates in the refrigerator (4 &#176;C) sealed with paraffin film for up to 15 days.</li>
			<li>Collect a medium-sized isolated colony from the plate using a sterile toothpick or sterile inoculating loop and inoculate 10 mL of Sabouraud dextrose broth in a sterile 50 mL conical tube. Incubate for approximately 24 h at 30 &#176;C under agitation (200 rpm).</li>
			<li>Do not exceed the 24 h incubation. Remember to incline the tube and leave the cap slightly open for better aeration. It is always good to standardize the growth stage of the cells before each test, since this is an important factor affecting antimicrobial resistance. 
			NOTE: Both fungi were grown at 30 &#176;C for rapid propagation in our experiments, but the temperature can be changed to reflect the aims of the study, for example clinical treatment (37 &#176;C). Most importantly, this initial incubation time should be established beforehand for each fungal isolate and maintained throughout all the tests to ensure reproducibility.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>After fungal growth, collect the cells by centrifuging the conical tubes at 1,200 x g for 5 min at room temperature. Discard the supernatant and add 10 mL of PBS.</li>
	<li>Resuspend the cells and centrifuge again at 1,200 x g for 5 min at room temperature.</li>
	<li>Repeat the PBS wash and centrifugation twice more. After the third wash, resuspend the cells in 5 mL (according to the pellet size) of 2X RPMI-1640 medium.</li>
	<li>Prepare 1 mL of a 1:100 or 1:1,000 dilution in a microcentrifuge tube (depending on the turbidity of the cell suspension).</li>
	<li>Aliquot 10 &#181;L of this dilution, place it in a hemocytometer chamber and count the total number of cells in the four corner quadrants under the microscope. Calculate the concentration by the formula: (total cell number/4) x dilution factor x 104 (chamber dilution constant).
	<ol>
		<li>Consider a 100% viability if viability counts and growth conditions have been systematically correlated for the isolate being studied. 
		NOTE: The standardization and quality control of viability counts can be done by back-plating in accordance to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) antifungal Minimum Inhibitory Concentration (MIC) method for yeasts.</li>
		<li>If the growth/viability correlation has not been established for the isolate under study, measure the fungal viability by counting live cells in a hemocytometer with the aid of dyes that selectively color dead cells, such as phloxine B11, trypan blue, or Janus Green. Use the fungal cells for this protocol only if the viability of the population at this point is 90% or above. 
		&#8203;NOTE: Dead/alive dyes may not work well with the fungus of choice. Please test them before use. For example, the trypan blue dye does not work well with C. neoformans.</li>
	</ol>
	</li>
	<li>After that, prepare the cell suspensions in 2X RPMI-1640 medium (2X adjusted inoculum in RPMI-1640 medium). For 96-well plates consider a volume of 5 mL for each plate.
	<ol>
		<li>For all C. albicans strains, prepare a stock cell suspension of 4 x 103 cells/mL. This concentration is 2X of the final cell concentration in each well (2 x 103 cells/mL).</li>
		<li>For C. neoformans strains, prepare a stock culture of cells 2 x 104 cells/mL. This concentration is twice the final cell concentration in each well (1 x 104 cells/mL).</li>
		<li>For other fungi, test with a known antifungal which concentration will be ideal for the particular study in relation to the incubation time. Take metabolism and duplication time of the fungus into consideration.</li>
	</ol>
	</li>
</ol>
3. Peptides (Unknown Agent)
<ol>
	<li>Store the lyophilized peptides at -20 &#176;C, and dissolve them in deionized water before each experiment. The maximum storage time once dissolved in water will depend on the nature of each peptide.</li>
	<li>Prepare aliquots of twice the highest final concentration tested in the assay (2X). Ideally, prepare a small number of aliquots with enough peptide for one time use to avoid freeze-thaw cycles. 
	NOTE: The choice of concentration should be based on literature and the properties of the peptide. It is recommended to start the serial dilutions with approximately 100 &#181;M of the peptide, and then decrease or increase this concentration range depending on the obtained results.</li>
</ol>
4. Reference Antifungals (Positive Controls)
<ol>
	<li>For those diluted in water: prepare a 2X solution of the highest concentration of the analysis (see Section 5: Antifungal Assay for the dilution steps).
	<ol>
		<li>For C. albicans strains, prepare a solution of 128 &#181;g/mL of fluconazole or 128 &#181;g/mL of caspofungin. The plate concentration for both will be 64 &#181;g/mL.</li>
		<li>For C. neoformans strains, prepare a solution of 32 &#181;g/mL of amphotericin B (water-soluble solution). The plate concentration will be 16 &#181;g/mL. 
		&#8203;NOTE: Normally amphotericin B is diluted in dimethyl sulfoxide (DMSO), since this anti-fungal is poorly soluble in water. However, there are water-soluble amphotericin B preparations commercially available.</li>
	</ol>
	</li>
	<li>For antifungals diluted in an organic solvent: prepare a 100X stock solution in DMSO, then dilute it to 10X in water for use. 
	NOTE: Thus, in the well, the final concentration of DMSO will not exceed 1%. A well where the fungus will grow in media containing 1% DMSO as control is required given that some fungi do not tolerate well this concentration of the solvent. Remember that DMSO is photosensitive, so cover the plate with foil or place it in a dark chamber for the duration of the incubation period.</li>
</ol>
5. Antifungal Assay
NOTE: In vitro antifungal assays are performed based on the broth microdilution susceptibility test from Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines with some modifications.
<ol>
	<li>Prepare a two-fold serial dilution of each peptide and control antifungal in 96-well polystyrene microplates to a final volume of 50 &#181;L.
	<ol>
		<li>Using a pipette add 100 &#181;L of the antifungal/peptide in the 2X concentration of the highest desired final concentration in columns 1-3, in row A.</li>
		<li>Using a multichannel pipette, add 50 &#181;L of sterile water into the other wells, in rows B to H.</li>
		<li>Remove 50 &#181;L of the wells with the highest concentration (row A), transfer to the next well of the next concentration (row B) and homogenize.</li>
		<li>Repeat the above steps until the well with the lowest concentration and discard 50 &#181;L of this well (row H). Leave Columns 11 and 12 (rows A, B, C) with only water for either blank control or negative/growth control.</li>
		<li>Add 50 &#181;L of the 2X adjusted inoculum in RPMI-1640 medium to each well (columns 1-3 plus column 11 (negative/growth control)); the final concentration for C. albicans will be 2 x 103 cells/mL, and the final concentration for C. neoformans strains will be 104 cells/mL.</li>
		<li>Prepare blank controls (50 &#181;L water + 50 &#181;L 2X RPMI-1640 medium without cells) and negative/growth control (50 &#181;L water + 50 &#181;L adjusted inoculum 2X RPMI-1640 medium without antifungal), and load into the well plate as described above.</li>
	</ol>
	</li>
	<li>Repeat the procedure for each compound to be tested and the chosen antifungal control (columns 4-10). 
	NOTE: The serial dilutions can be done vertically as the experiment below, or horizontally in the plate (i.e., if the number of dilutions of the given compound/extract that needs to be tested is eight or more).
	<ol>
		<li>If the compounds, reference drugs, or any of its components are photosensitive, perform the assay in reduced light, cover the plates with foil, or place in a dark chamber during incubations.</li>
	</ol>
	</li>
	<li>Consider the following optional recommendations.
	<ol>
		<li>Seal the plate with a clear cover plate that permits gas exchange, as this helps reduce evaporation.</li>
		<li>Place the plate in a humid chamber; it will also help reduce evaporation.</li>
	</ol>
	</li>
	<li>Incubate the plates at 37 &#176;C for 24 h or 48 h for all C. albicans strains, and for 48 h with 200 rpm shaking for C. neoformans. The lower final volume (100 &#181;L) ensures that no spillover occurs. 
	NOTE: No significant difference was observed between MIC readings at 24 h and 48 h for C. albicans strains. Nonetheless, readings at 48 h are easier to visualize.</li>
	<li>Observe all wells, with the assistance of an inverted optical microscope, before the incubation period and after to verify changes in morphology as well as to check for signs of contamination. 
	NOTE: Readings can be done visually and photographed at the end of the experiment. Before each reading, homogenize slightly the plate. The reads can also be performed by measuring its OD at 600 nm, if cell clumps or filamentation are not observed.</li>
	<li>Perform the experiments at least three times on separate dates.</li>
</ol>
Table 1. Media and reagent preparation.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Medium</td>
			<td>Preparation</td>
		</tr>
		<tr>
			<td rowspan="4">2X Roswell Park Memorial Institute (RPMI) 1640 medium &#8211; 500 mL</td>
			<td>10.4 g RPMI-1640 (supplemented with L-glutamine and phenol red; without bicarbonate)</td>
		</tr>
		<tr>
			<td>330 mM 3-(N-morpholino) propane sulfonic acid (MOPS)</td>
		</tr>
		<tr>
			<td>Adjust to pH 7.0 with NaOH</td>
		</tr>
		<tr>
			<td>Sterilize by filtration (0.22 &#181;M filter)</td>
		</tr>
		<tr>
			<td rowspan="5">Phosphate buffered saline (PBS)</td>
			<td>137 mM NaCl</td>
		</tr>
		<tr>
			<td>2.7 mM KCl</td>
		</tr>
		<tr>
			<td>10 mM Na2HPO4</td>
		</tr>
		<tr>
			<td>2 mM KH2PO4</td>
		</tr>
		<tr>
			<td>Autoclave at 121&#176;C for 15 minutes.</td>
		</tr>
		<tr>
			<td rowspan="3">Sabouraud dextrose broth</td>
			<td>15 g of the powder in 500 mL of distilled water.</td>
		</tr>
		<tr>
			<td>Adjust to pH 7.0 with NaOH</td>
		</tr>
		<tr>
			<td>Autoclave at 121&#176;C for 15 minutes.</td>
		</tr>
		<tr>
			<td rowspan="3">Sabouraud Dextrose Agar</td>
			<td>32.5 g of the powder in 500 mL of distilled water.</td>
		</tr>
		<tr>
			<td>Adjust to pH 7.0 with NaOH</td>
		</tr>
		<tr>
			<td>Autoclave at 121&#176;C for 15 minutes.</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Media and Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium with l-glutamine, without sodium bicarbonate</td>
			<td>Thermo Fisher</td>
			<td>31800-022</td>
			<td></td>
		</tr>
		<tr>
			<td>3-(N-morpholino) propane sulfonic acid (MOPS)</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>Use to buffer 2X RPMI medium</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Din&#226;mica</td>
			<td>1528-1</td>
			<td>137 mM for Phosphate buffered saline (PBS)</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>J.T.Baker</td>
			<td>3040-01</td>
			<td>2.7 mM for Phosphate buffered saline (PBS)</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic (Na2HPO4)</td>
			<td>Sigma-Aldrich</td>
			<td>V000129</td>
			<td>10 mM for Phosphate buffered saline (PBS)</td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate (KH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>60230</td>
			<td>2 mM for Phosphate buffered saline (PBS)</td>
		</tr>
		<tr>
			<td>BD Difco Sabouraud dextrose broth</td>
			<td>BD</td>
			<td>238230</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Difco Sabouraud Dextrose Agar</td>
			<td>BD</td>
			<td>210950</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>V000123</td>
			<td>35%</td>
		</tr>
		<tr>
			<td>Sterile water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antifungal drugs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma-Aldrich</td>
			<td>A2942</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluconazole</td>
			<td>Sigma-Aldrich</td>
			<td>F8929</td>
			<td></td>
		</tr>
		<tr>
			<td>Caspofungin</td>
			<td>Sigma-Aldrich</td>
			<td>PHR1160</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastics</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tube</td>
			<td>Sarstedt</td>
			<td>62.547.254</td>
			<td></td>
		</tr>
		<tr>
			<td>Dish petri</td>
			<td>J.Prolab</td>
			<td>0304-5</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plate</td>
			<td>Corning</td>
			<td>3595</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Solution Reservoir</td>
			<td>KASVI</td>
			<td>K30-208</td>
			<td>Use to pippet the solutions using the multichannel pippet</td>
		</tr>
		<tr>
			<td>Equipment and other materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optical microscope</td>
			<td>Nikon</td>
			<td>E200MV</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifugue</td>
			<td>Thermo Fisher</td>
			<td>MegaFuge 16R</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Ethik Technology</td>
			<td>403-3D</td>
			<td>Set to 37&#176; C</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>New Brunswick Scientific</td>
			<td>Excella E25</td>
			<td>Set to 37&#176; C, 200 RPM</td>
		</tr>
		<tr>
			<td>Cell counting chamber, Neubauer</td>
			<td>BOECO Germany</td>
			<td>BOE 13</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette</td>
			<td>HTL</td>
			<td>5123</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro broth microdilution assay for the screening of antifungal compounds

Source: de-Souza-Silva, C. M., et al. <a href="https://app.jove.com/v/57127">Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds.</a> J. Vis. Exp. (2018).
This video demonstrates an in vitro assay to screen antifungal compounds using a broth microdilution assay. Test compounds are serially diluted in a microtiter plate and incubated with a fungal inoculum. Post-incubation, the plate is examined for wells without visible fungal growth to assess the antifungal potential of the test compounds and their minimum inhibitory concentration (MIC).

An In Vitro Broth Microdilution Assay for the Screening of Antifungal Compounds

1. Solutions and Media

	Prepare 2X Roswell Park Memorial Institute (RPMI) 1640 medium, phosphate-buffered saline (PBS), Sabouraud dextrose broth, and ...

Take a microtiter plate, and add serial dilutions of novel antimicrobial peptides or AMPs &#8212; the test compounds.
Designate a subset of wells as growth controls, and add sterile water without the AMPs.
Add a pathogenic fungus suspended in growth media into selected wells.
Add only the media, devoid of the fungus, in a subset of wells designated as blank controls.
Seal the plate and incubate under humid conditions conducive to fungal growth.
Post-incubation, observe the wells under a microscope.
The growth controls appear turbid due to fungal growth in the absence of AMPs. Turbidity across all dilutions of an AMP indicates its ineffectiveness against the fungus.
The blank controls, devoid of fungal growth, appear clear. Compare test wells with the blank controls to confirm the absence of growth, indicating the antifungal activity of AMPs.
The lowest concentration of an AMP that inhibits fungal growth is termed the minimum inhibitory concentration, or MIC.

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Immunology

Immunodiagnostics

In Vitro and In Vivo Models

182 Views. Source: de-Souza-Silva, C. M., et al. Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds. J. Vis. Exp. (2018). This video demonstrates an in vitro assay to screen antifungal compounds using a broth microdilution assay. Test compounds are serially diluted in a microtiter plate and incubated with a fungal inoculum. Post-incubation, the plate is examined for wells without visible fungal growth to assess the antifungal potential of the test compounds ...

Video: An In Vitro Broth Microdilution Assay for the Screening of Antifungal Compounds - Concept

High-throughput Screening of Chemical Compounds to Elucidate Their Effects on Bacterial Persistence

Bacterial persisters are defined as a small subpopulation of phenotypic variants with the capability of tolerating high concentrations of antibiotics. They are an important health concern as they have been associated with recurrent chronic infections. Although stochastic and deterministic dynamics of stress-related mechanisms are known to play a significant role in persistence, mechanisms underlying the phenotypic switch to/from the persistence state are not completely understood. While persistence factors triggered by environmental signals (e.g., depletion of carbon, nitrogen and oxygen sources) have been extensively studied, the impacts of osmolytes on persistence are yet to be determined. Using microarrays (i.e., 96 well plates containing various chemicals), we have designed an approach to elucidate the effects of various osmolytes on Escherichia coli persistence in a high throughput manner. This approach is transformative as it can be readily adapted for other screening arrays, such as drug panels and gene knockout libraries.

In this method paper, we present a high-throughput screening strategy to identify chemical compounds, such as osmolytes, that have a significant impact on bacterial persistence.

Bacterial persisters are defined as a small subpopulation of phenotypic variants with the capability of tolerating high concentrations of antibiotics. ...

JoVE Journal

Immunology and Infection

High-throughput Antiviral Assays to Screen for Inhibitors of Zika Virus Replication

Antiviral drug discovery requires the development of reliable biochemical and cellular assays that can be performed in high-throughput screening (HTS) formats. The flavivirus non-structural (NS) proteins are thought to co-translationally assemble on the endoplasmic reticulum (ER) membranes, forming the replication complex (RC). The NS3 and NS5 are the most studied enzymes of the RC and constitute the main targets for drug development due to their crucial roles in viral genome replication. NS3 protease domain, which requires NS2B as its cofactor, is responsible for the cleavage of the immature viral polyprotein into the mature NS proteins, whereas NS5 RdRp domain is responsible for the RNA replication. Herein, we describe in detail the protocols used in replicon-based screenings and enzymatic assays to test large compound libraries for inhibitors of the Zika virus (ZIKV) replication. Replicons are self-replicating subgenomic systems expressed in mammalian cells, in which the viral structural genes are replaced by a reporter gene. The inhibitory effects of compounds on viral RNA replication can be easily evaluated by measuring the reduction in the reporter protein activity. The replicon-based screenings were performed using a BHK-21 ZIKV replicon cell line expressing Renilla luciferase as a reporter gene. To characterize the specific targets of identified compounds, we established in-vitro fluorescence-based assays for recombinantly expressed NS3 protease and NS5 RdRp. The proteolytic activity of the viral protease was measured by using the fluorogenic peptide substrate Bz-nKRR-AMC, while the NS5 RdRp elongation activity was directly detected by the increase of the fluorescent signal of SYBR Green I during RNA elongation, using the synthetic biotinylated self-priming template 3&#8242;UTR-U30 (5&#39;-biotin-U30-ACUGGAGAUCGAUCUCCAGU-3&#39;).

In this work, we describe the protocols used in replicon-based and viral enzyme-based assays to screen for inhibitors of Zika virus replication in a high-throughput screening format.

Antiviral drug discovery requires the development of reliable biochemical and cellular assays that can be performed in high-throughput screening (HTS) ...

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 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 ...

An In Vitro Broth Microdilution Assay for the Screening of Antifungal Compounds

Transcript

An In Vitro Broth Microdilution Assay for the Screening of Antifungal Compounds

High-throughput Screening of Chemical Compounds to Elucidate Their Effects on Bacterial Persistence

High-throughput Antiviral Assays to Screen for Inhibitors of Zika Virus Replication

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