This work was supported by the National Institutes of Health (R03DE029270, T32AI089621B), the National Science Foundation (CBET 1511718), the Department of Education (GAANN-P200A180093), and a University of Maryland Cross-Campus Seed Grant.

Approval was obtained from the University of Maryland, College Park, Institutional Biosafety Committee (IBC) for the work with C. albicans in this protocol (PN 274). The C. albicans strain SC5314 (see Table of Materials) was used in the present study; however, any other strain may also be used.
1. Preparation of the buffer, sterile water, and culture medium
<ol>
	<li>Prepare sterile 0.1 M sodium phosphate buffer (NaPB)26 at pH 7.4, and dilute to 2 mM and 1 mM with sterile water. Preparing 100 mL each of 2 mM NaPB and 1 mM NaPB will be more than sufficient for most applications of this protocol.</li>
	<li>Prepare sterile liquid yeast-peptone-dextrose (YPD, see Table of Materials) medium (10 g/L yeast extract, &#160;&#160;20 g/L dextrose, 20 g/L peptone). Preparing 100 mL of YPD will be more than sufficient for most applications of this protocol.</li>
	<li>Prepare sterile ultrapure water. Preparing 100 mL of water will be more than sufficient for most applications of this protocol. 
	&#8203;NOTE: The buffers, media, and water are sterilized by autoclaving at 121 &#176;C on a liquid cycle for an appropriate time based on the volume in the containers being autoclaved27. For example, for bottles containing 500 mL, the exposure time should be 40 min.</li>
</ol>
2. Inoculation, culturing, and subculturing of C.&#160;albicans
CAUTION: Follow all institutional and governmental regulations for working with C. albicans, which is often classified by academic research institutions as a biosafety level (BSL) 2 organism regardless of drug resistance and by the American Type Culture Collection (ATCC) as a BSL1 or BSL2 organism, depending on the drug resistance of the strain28.
NOTE: Perform the inoculation and culturing portions of this step (steps 2.1-2.2) on the day before beginning the testing for antifungal activity. If possible, perform all the protocol steps with the cells (and the solutions that will be incubated with the cells) in a biosafety cabinet.
<ol>
	<li>Inoculate the desired C. albicans strain&#160;into 10 mL of YPD medium in a culture tube. 
	NOTE: The culture can be inoculated from a freezer stock or from an agar plate, but a consistent source must be used for all data that will be compared.</li>
	<li>Grow the culture overnight (~12-16 h) at 30 &#176;C and 230 rpm on a rotary shaker.</li>
	<li>Subculture the overnight culture of C. albicans, and grow to an OD600 of ~1.0-1.2.
	<ol>
		<li>Measure the OD600 of the overnight culture using a UV spectrophotometer (see Table of Materials).</li>
		<li>Based on the OD600 of the overnight culture, use the overnight culture to inoculate a subculture at an OD600 of 0.1 in 10 mL of YPD.</li>
		<li>Grow the subculture at 30 &#176;C on a rotary shaker at 230 rpm until the OD600 reaches ~1.0-1.2, which is likely to take 4-6 h. Complete step 3 (preparation of peptide solutions)&#160;while the subculture is growing; the subculture will be diluted for use in the assay in step 4.</li>
	</ol>
	</li>
</ol>
3. Preparation of the peptide solutions in a 96-well plate
NOTE: This step may be done ahead of time,&#160;if the peptide stock solutions are stable during storage. Typically, the peptide solutions are stored at &#8722;20 &#176;C until use. They can be thawed in a room temperature water bath before proceeding to the next step.
<ol>
	<li>Determine the highest concentration of each peptide to be tested in the assay. This will vary based on the peptides selected. For the data presented in the representative results section, histatin 5 and engineered analogs22 were tested, and the highest concentration tested was 50 &#181;M. 
	NOTE: The highest concentration to be tested is determined using data reported in the literature or by performing preliminary experiments over a broad range of peptide concentrations. When possible, the highest concentration should be selected to observe a plateau for a complete reduction in viability at the highest concentrations tested and a plateau for no reduction in viability at the lowest concentrations tested, allowing the quantification of the full range of peptide activity.</li>
	<li>Dissolve each peptide in sterile water at twice the desired highest concentration. For the data presented in the representative results section, 150 &#181;L of 100 &#181;M solutions of histatin 5 and engineered analogs were prepared in sterile water. 
	NOTE: If a peptide is not soluble in water, solubilization in another solvent may be necessary prior to this step. Dimethyl sulfoxide (DMSO) is commonly used to solubilize peptides at a high concentration before diluting to the working concentration. Ensure that the final concentration of the DMSO is 1% (v/v) or lower29 and that alternative solvents do not affect the growth of the cells. Also, ensure that the solvent concentration is kept constant in all the wells in steps 3.4-3.5.</li>
	<li>Add 40 &#181;L of the desired peptide stock solutions to the first well (Column 1) of each row in a 96-well, round-bottomed culture plate (see Table of Materials). This plate is Plate 1 (Figure 1). 
	NOTE: Each plate has eight rows that are used for testing peptides. These rows can be used for different peptides or for replicates of the same peptides. Include only a single peptide in each row.</li>
	<li>Add 20 &#181;L of sterile ultrapure water to Column 2 to Column 12 of the rows containing peptide (Figure 1). 
	NOTE: If the peptide stocks were prepared with a solvent other than pure water in step 3.2, the sterile water in this step should be replaced with the solvent present in the peptide stock added to the first column. For example, if the peptide was solubilized in DMSO and the peptide stock contains 1% (v/v) DMSO in water, water containing 1% DMSO must be added to Column 2 to Column 12.</li>
	<li>Serially dilute the peptide stock solutions across the plate to Column 10 (Figure 1). 
	NOTE: Rather than performing the serial dilutions in the plate as described below, the dilutions could also be performed in microcentrifuge tubes and transferred to the 96-well plate.
	<ol>
		<li>Remove 20 &#181;L from Column 1, transfer it to Column 2, and mix by pipetting up and down. Column 2 now contains 40 &#181;L of peptide solution at a 1:2 dilution of the concentration in Column 1.</li>
		<li>Remove 20 &#181;L from Column 2, transfer it to Column 3, and mix by pipetting up and down, so Column 3 now contains 40 &#181;L of peptide solution at a 1:4 dilution of the concentration in Column 1.</li>
		<li>Repeat this process until Column 10 contains 40 &#181;L of peptide solution at a 1:512 dilution of the concentration in Column 1.</li>
		<li>Remove 20 &#181;L of peptide solution from Column 10 and discard it. Each column now contains 20 &#181;L of peptide solution (Columns 1-10) or water (Column 11 and Column 12). 
		&#8203;NOTE: The wells in Column 11 serve as sterility controls, and the wells in Column 12 serve as controls containing no peptide.</li>
	</ol>
	</li>
</ol>
4. Dilution of the C. albicans subculture
NOTE: Begin this step after the subculture has reached an OD600 of ~1.0-1.2 (step 2.3.3).
<ol>
	<li>Transfer the subculture to a 15 mL centrifuge tube, and centrifuge at 3,900 x g for 3 min at room temperature to pellet the cells. Remove the supernatant by pipetting or decanting.</li>
	<li>Resuspend the pellet with 1 mL of 2 mM NaPB (step 1.1), and transfer the suspension to a 1.7 mL centrifuge tube.</li>
	<li>Pellet the cells (as in step 4.1), discard the supernatant, and again resuspend the pellet in 1 mL of 2 mM NaPB.</li>
	<li>Repeat step 4.3 two additional times to leave the washed cells in 1 mL of 2 mM NaPB.</li>
	<li>Determine the cell density of the washed suspension, and calculate the dilution factor needed to obtain a cell density of 5 x 105 cells/mL. This concentration will ultimately lead to adding 1 x 104 cells to each well of the 96-well plate. 
	NOTE: The cell density of the suspension may be determined using a number of methods, including a hemocytometer or automated cell counter. A standard curve of cell density versus OD600 for the strain of interest grown under relevant conditions was used for the present study. This curve was prepared using a hemocytometer (see Table of Materials) to determine the cell density of suspensions with varying OD600 values.</li>
	<li>Dilute the cell suspension to 5 x 105 cells/mL in 2 mM NaPB. Preparing 10 mL of this diluted suspension will be more than sufficient for completing this study.</li>
</ol>
5. Incubation of C. albicans with peptide solutions and preparation of the cells for the quantification of viability
<ol>
	<li>Add 20 &#181;L of the diluted C. albicans suspension (step 4.6) to Columns 1-10 and Column 12 of each row (Figure 1). 
	NOTE: The final peptide concentration in the first column is now half of the peptide stock concentration. For the present study, the final peptide concentration was 50 &#181;M at this point. The final cell suspension in each well contains 2.5 x 105 cells/mL. Columns 1-10 serve as experimental wells to evaluate the antifungal activity at different peptide concentrations, and Column 12 serves as a control for growth with no peptide.</li>
	<li>Add 20 &#181;L of 2 mM NaPB to Column 11. All the wells now have a final concentration of 1 mM NaPB. Column 11 serves as a sterility control.</li>
	<li>Cover Plate 1 (containing both cells and peptide), and incubate it at 30 &#176;C for 30 min. 
	NOTE: An incubation time of 30 min is sufficient for many peptides17,21,22,30, but the incubation time can be increased to 60 min if desired to account for peptides that may require a longer time to exert their antifungal activity25,31,32.</li>
	<li>Prepare a new 96-well culture plate (Plate 2) for use in quantifying the viability (Figure 2). 
	NOTE: The culture plate should be prepared during the incubation of Plate 1.
	<ol>
		<li>Add 100 &#181;L of YPD (step 1.2) to all the wells of the plate.</li>
		<li>Add 100 &#181;L of 2 mM NaPB (Step 1.1) to all the wells of the plate.</li>
	</ol>
	</li>
	<li>Dilute the samples in Plate 1 (containing cells and peptides), and transfer to Plate 2 (containing medium and buffer).
	<ol>
		<li>Retrieve Plate 1 from the incubator following the 30 min of incubation.</li>
		<li>Dilute each well of Plate 1 by adding 280 &#181;L of 1 mM NaPB (step 1.1) for a total volume of 320 &#181;L in each well (Figure 2).</li>
		<li>Mix all the wells containing cells and peptides by pipetting up and down to ensure all the cells are resuspended and no settled cells are visible at the bottom of the wells.</li>
		<li>Transfer 8 &#181;L from each well in Plate 1 to the corresponding well of Plate 2. This volume transfers approximately 250 cells from each well of Plate 1 into Plate 2 (Figure 2).</li>
	</ol>
	</li>
	<li>Cover Plate 2 and incubate on a microtiter plate shaker (see Table of Materials) at 350 rpm for 17 h at 30 &#176;C.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64416/64416fig01.jpg" /> 
Figure 1: Preparation of Plate 1 for the incubation of peptide serial dilutions with cells. Serial dilutions of the peptide are made in water, and C. albicans cells are added to Plate 1. A blue color indicates that peptide is present in the well, and gray indicates a well with water and no peptide. Wells containing cells are indicated with a pattern of black dots. After these steps, the plate is incubated to allow the peptide to exert its antifungal activity. <a href="https://www.jove.com/files/ftp_upload/64416/64416fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64416/64416fig2v2.jpg" /> 
Figure 2: Preparation of Plate 2 for quantifying the reduction in viability due to the peptide. YPD medium and NaPB are added to Plate 2. After diluting the contents of Plate 1, an aliquot from each well is transferred from Plate 1 to Plate 2. Plate 2 is then incubated to allow any viable cells to grow for quantification by measuring the OD600. For Plate 2, wells containing only YPD and NaPB are shown in orange. Wells containing aliquots from Plate 1 mixed with the YPD and NaPB are shown in green. See the Figure 1 legend for the description of the colors on Plate 1. <a href="https://www.jove.com/files/ftp_upload/64416/64416fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
6. Determination of antifungal activity
<ol>
	<li>Obtain OD600 readings for each well in Plate 2.
	<ol>
		<li>Remove Plate 2 from the incubator after 17 h of incubation.</li>
		<li>Mix all the wells in Plate 2 by pipetting up and down to ensure all the cells are resuspended and no settled cells are visible at the bottom of the wells. 
		NOTE: Avoid generating bubbles during this step, as they may interfere with the OD600 readings. If bubbles form, they can often be popped with a dry pipet tip or removed by using two pipet tips to lift them out of the well.</li>
		<li>Use an absorbance plate reader (see Table of Materials) at a wavelength of 600 nm to obtain the OD600 for each well.</li>
	</ol>
	</li>
	<li>Calculate the inhibition of growth (as a percentage), and plot it to determine the effect of the peptides on the growth of C. albicans.
	<ol>
		<li>For each well, calculate the reduction in viability compared to the control (as a percentage) using the following equation17,22,23: 
		<img alt="Equation 1" src="/files/ftp_upload/64416/64416eq1.png" style="margin: auto;" /> 
		where ODwith peptide&#160;is the OD600 of a well containing a given concentration of peptide, ODbackground is the OD600 of Column 11 in the same row (control containing no cells), and 
		ODno peptide&#160;is the OD600 of Column 12 in the same row (control containing cells and no peptide).</li>
		<li>For example, use the following expression to calculate the reduction in viability in Well B5 from the OD600 values for Row B: 
		<img alt="Equation 2" src="/files/ftp_upload/64416/64416eq2.png" style="margin: auto;" /></li>
	</ol>
	</li>
	<li>Plot the reduction in viability as a function of the peptide concentration.</li>
</ol>

<ol>
	<li>Gulati, M., Nobile, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+albicans+biofilms:+Development,+regulation,+and+molecular+mechanisms.">Candida albicans biofilms: Development, regulation, and molecular mechanisms.</a> Microbes and Infection. 18 (5), 310-321 (2016).</li><li>Arya, N. R., Rafiq, N. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candidiasis.">Candidiasis.</a> StatPearls. , (2021).</li><li>de Oliveira Santos, G. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+infections+and+therapeutic+strategies:+Mechanisms+of+action+for+traditional+and+alternative+agents.">Candida infections and therapeutic strategies: Mechanisms of action for traditional and alternative agents.</a> Frontiers in Microbiology. 9, 1351 (2018).</li><li>Espinel-Ingroff, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+resistance+to+antifungal+agents:+Yeasts+and+filamentous+fungi.">Mechanisms of resistance to antifungal agents: Yeasts and filamentous fungi.</a> Revista Iberoamericana de Micolog&#237;a. 25 (2), 101-106 (2008).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+strategies+of+amphotericin+B+for+invasive+fungal+infections.">Delivery strategies of amphotericin B for invasive fungal infections.</a> Acta Pharmaceutica Sinica B. 11 (8), 2585-2604 (2021).</li><li>Struyfs, C., Cammue, B. P. A., Thevissen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-interacting+antifungal+peptides.">Membrane-interacting antifungal peptides.</a> Frontiers in Cell and Developmental Biology. 9, 649875 (2021).</li><li>Huan, Y., Kong, Q., Mou, H., Yi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+peptides:+Classification,+design,+application+and+research+progress+in+multiple+fields.">Antimicrobial peptides: Classification, design, application and research progress in multiple fields.</a> Frontiers in Microbiology. 11, 582779 (2020).</li><li>Sarkar, T., Chetia, M., Chatterjee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+peptides+and+proteins:+From+nature's+reservoir+to+the+laboratory+and+beyond.">Antimicrobial peptides and proteins: From nature's reservoir to the laboratory and beyond.</a> Frontiers in Chemistry. 9, 691532 (2021).</li><li>Mahlapuu, M., Bjorn, C., Ekblom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+peptides+as+therapeutic+agents:+Opportunities+and+challenges.">Antimicrobial peptides as therapeutic agents: Opportunities and challenges.</a> Critical Reviews in Biotechnology. 40 (7), 978-992 (2020).</li><li>Lei, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antimicrobial+peptides+and+their+potential+clinical+applications.">The antimicrobial peptides and their potential clinical applications.</a> American Journal of Translational Research. 11 (7), 3919-3931 (2019).</li><li>Mercer, D. K., O'Neil, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+inspiration:+Antifungal+peptides+and+other+immunotherapeutics+from+the+host+immune+response.">Innate inspiration: Antifungal peptides and other immunotherapeutics from the host immune response.</a> Frontiers in Immunology. 11, 2177 (2020).</li><li>Bin Hafeez, A., Jiang, X., Bergen, P. J., Zhu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+peptides:+An+update+on+classifications+and+databases.">Antimicrobial peptides: An update on classifications and databases.</a> International Journal of Molecular Sciences. 22 (21), 11691 (2021).</li><li>Xu, T., Levitz, S. M., Diamond, R. D., Oppenheim, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anticandidal+activity+of+major+human+salivary+histatins.">Anticandidal activity of major human salivary histatins.</a> Infection and Immunity. 59 (8), 2549-2554 (1991).</li><li>Helmerhorst, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphotericin+B-+and+fluconazole-resistant+Candida+spp.,+Aspergillus+fumigatus,+and+other+newly+emerging+pathogenic+fungi+are+susceptible+to+basic+antifungal+peptides.">Amphotericin B- and fluconazole-resistant Candida spp., Aspergillus fumigatus, and other newly emerging pathogenic fungi are susceptible to basic antifungal peptides.</a> Antimicrobial Agents and Chemotherapy. 43 (3), 702-704 (1999).</li><li>Andra, J., Berninghausen, O., Leippe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cecropins,+antibacterial+peptides+from+insects+and+mammals,+are+potently+fungicidal+against+Candida+albicans.">Cecropins, antibacterial peptides from insects and mammals, are potently fungicidal against Candida albicans.</a> Medical Microbiology and Immunology. 189, 169-173 (2001).</li><li>do Nascimento Dias, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+action+of+antimicrobial+peptides+ToAP2+and+NDBP-5.7+against+Candida+albicans+planktonic+and+biofilm+cells.">Mechanisms of action of antimicrobial peptides ToAP2 and NDBP-5.7 against Candida albicans planktonic and biofilm cells.</a> Scientific Reports. 10, 10327 (2020).</li><li>Ikonomova, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+histatin+5+modifications+on+antifungal+activity+and+kinetics+of+proteolysis.">Effects of histatin 5 modifications on antifungal activity and kinetics of proteolysis.</a> Protein Science. 29, 480-493 (2020).</li><li>Clinical Laboratory Standards Institute. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> M27-A3. Reference method for broth dilution antifungal susceptibility testing of yeasts; Approved standard - Third edition. , (2008).</li><li>Lupetti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candidacidal+activities+of+human+lactoferrin+peptides+derived+from+the+N+terminus.">Candidacidal activities of human lactoferrin peptides derived from the N terminus.</a> Antimicrobial Agents and Chemotherapy. 44 (12), 3257-3263 (2000).</li><li>Han, J., Jyoti, M. A., Song, H. Y., Jang, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+activity+and+action+mechanism+of+histatin+5-halocidin+hybrid+peptides+against+Candida+ssp.">Antifungal activity and action mechanism of histatin 5-halocidin hybrid peptides against Candida ssp.</a> PLoS One. 11 (2), 0150196 (2016).</li><li>den Hertog, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candidacidal+effects+of+two+antimicrobial+peptides:+histatin+5+causes+small+membrane+defects,+but+LL-37+causes+massive+disruption+of+the+cell+membrane.">Candidacidal effects of two antimicrobial peptides: histatin 5 causes small membrane defects, but LL-37 causes massive disruption of the cell membrane.</a> Biochemical Journal. 388, 689-695 (2005).</li><li>Ikonomova, S. P., Moghaddam-Taaheri, P., Jabra-Rizk, M. A., Wang, Y., Karlsson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+improved+variants+of+the+antifungal+peptide+histatin+5+with+reduced+susceptibility+to+Candida+albicans+secreted+aspartic+proteases+and+enhanced+antimicrobial+potency.">Engineering improved variants of the antifungal peptide histatin 5 with reduced susceptibility to Candida albicans secreted aspartic proteases and enhanced antimicrobial potency.</a> The FEBS Journal. 285 (1), 146-159 (2018).</li><li>Moghaddam-Taaheri, P., Leissa, J. A., Eppler, H. B., Jewell, C. M., Karlsson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histatin+5+variant+reduces+Candida+albicans+biofilm+viability+and+inhibits+biofilm+formation.">Histatin 5 variant reduces Candida albicans biofilm viability and inhibits biofilm formation.</a> Fungal Genetics and Biology. 149, 103529 (2021).</li><li>Gong, Z., Doolin, M. T., Adhikari, S., Stroka, K. M., Karlsson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+charge+and+hydrophobicity+in+translocation+of+cell-penetrating+peptides+into+Candida+albicans+cells.">Role of charge and hydrophobicity in translocation of cell-penetrating peptides into Candida albicans cells.</a> AIChE Journal. 65 (12), 16768 (2019).</li><li>Gong, Z., Karlsson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translocation+of+cell-penetrating+peptides+into+Candida+fungal+pathogens.">Translocation of cell-penetrating peptides into Candida fungal pathogens.</a> Protein Science. 26 (9), 1714-1725 (2017).</li><li>Green, M. R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A Laboratory Manual. Fourth edition. 3, (2012).</li><li>Consolidated Sterilizer Systems. Laboratory and Research Autoclaves Available from: <a target="_blank" href="https://consteril.com/wp-content/uploads/2020/12/CSS-Product-Brochure.pdf">https://consteril.com/wp-content/uploads/2020/12/CSS-Product-Brochure.pdf</a> (2022)</li><li>Rodriguez-Tudela, J. L., Cuenca-Estrella, M., Diaz-Guerra, T. M., Mellado, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization+of+antifungal+susceptibility+variables+for+a+semiautomated+methodology.">Standardization of antifungal susceptibility variables for a semiautomated methodology.</a> Journal of Clinical Microbiology. 39 (7), 2513-2517 (2001).</li><li>Mbuayama, K. R., Taute, H., Strmstedt, A. A., Bester, M. J., Gaspar, A. R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+activity+and+mode+of+action+of+synthetic+peptides+derived+from+the+tick+OsDef2+defensin.">Antifungal activity and mode of action of synthetic peptides derived from the tick OsDef2 defensin.</a> Journal of Peptide Science. 28 (5), 3383 (2022).</li><li>Rossignol, T., Kelly, B., Dobson, C., d'Enfert, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytosis-mediated+vacuolar+accumulation+of+the+human+ApoE+apolipoprotein-derived+ApoEdpL-W+antimicrobial+peptide+contributes+to+its+antifungal+activity+in+Candida+albicans.">Endocytosis-mediated vacuolar accumulation of the human ApoE apolipoprotein-derived ApoEdpL-W antimicrobial peptide contributes to its antifungal activity in Candida albicans.</a> Antimicrobial Agents and Chemotherapy. 55 (10), 4670-4681 (2011).</li><li>Helmerhorst, E. J., Reijnders, I. M., van't Hof, W., Veerman, E. C., Nieuw Amerongen, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+comparison+of+the+hemolytic+and+fungicidal+activities+of+cationic+antimicrobial+peptides.">A critical comparison of the hemolytic and fungicidal activities of cationic antimicrobial peptides.</a> FEBS Letters. 449 (2-3), 105-110 (1999).</li><li>Kerenga, B. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salt-tolerant+antifungal+and+antibacterial+activities+of+the+corn+defensin+ZmD32.">Salt-tolerant antifungal and antibacterial activities of the corn defensin ZmD32.</a> Frontiers in Microbiology. 10, 795 (2019).</li><li>Lee, I. H., Cho, Y., Lehrer, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+pH+and+salinity+on+the+antimicrobial+properties+of+clavanins.">Effects of pH and salinity on the antimicrobial properties of clavanins.</a> Infection and Immunity. 65 (7), 2898-2903 (1997).</li><li>Li, X. S., Reddy, M. S., Baev, D., Edgerton, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+albicans+Ssa1/2p+is+the+cell+envelope+binding+protein+for+human+salivary+histatin+5.">Candida albicans Ssa1/2p is the cell envelope binding protein for human salivary histatin 5.</a> Journal of Biological Chemistry. 278 (31), 28553-28561 (2003).</li><li>Rothstein, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anticandida+activity+is+retained+in+P-113,+a+12-amino-acid+fragment+of+histatin+5.">Anticandida activity is retained in P-113, a 12-amino-acid fragment of histatin 5.</a> Antimicrobial Agents and Chemotherapy. 45 (5), 1367-1373 (2001).</li><li>Sanders, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aseptic+laboratory+techniques:+Volume+transfers+with+serological+pipettes+and+micropipettors.">Aseptic laboratory techniques: Volume transfers with serological pipettes and micropipettors.</a> Journal of Visualized Experiments. (63), e2754 (2012).</li><li>Mansoury, M., Hamed, M., Karmustaji, R., Al Hannan, F., Safrany, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+edge+effect:+A+global+problem.+The+trouble+with+culturing+cells+in+96-well+plates.">The edge effect: A global problem. The trouble with culturing cells in 96-well plates.</a> Biochemistry and Biophysics Report. 26, 100987 (2021).</li><li>Goughenour, K. D., Balada-Llasat, J. M., Rappleye, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+microplate-based+growth+assay+for+determination+of+antifungal+susceptibility+of+Histoplasma+capsulatum+yeasts.">Quantitative microplate-based growth assay for determination of antifungal susceptibility of Histoplasma capsulatum yeasts.</a> Journal of Clinical Microbiology. 53 (10), 3286-3295 (2015).</li></ol>

The authors declare that they have no competing financial interests.

This protocol describes an efficient approach for obtaining quantitative data on the antifungal activity of AMPs against the fungal pathogen C. albicans. One common alternative approach to testing peptides and other antifungal agents is the broth microdilution described in the Clinical Laboratory Standards Institute&#39;s (CLSI) standard M2718, but this standard focuses on obtaining qualitative visual results instead of quantitative results. Another alternative approach is to use a method...

Candida albicans is a member of the human microbiota that colonizes numerous locations, including the oral cavity, skin, gastrointestinal tract, and vagina1. For patients that are immunocompromised due to diseases such as human immunodeficiency virus (HIV) and immunosuppressive treatments, the colonization of C. albicans may lead to local or systemic candidiasis2,3. The use of currently available small-molecule antifungal therapeutics, such as amphotericin B, azoles, or echinocandins, can be complicated by solubility and toxicity issues and by the resistance of infections to the therapeutics4,5. Due to the limitations of current antifungal agents, researchers are continually searching for new antifungal molecules with activity against C. albicans.
Antimicrobial peptides (AMPs) are a potential alternative to the current small-molecule antifungal agents6,7,8 and are proposed to be less susceptible to the development of resistance compared to small-molecule drugs9. AMPs are a diverse set of peptides, but they are often cationic, with a broad spectrum of activity10,11,12. AMPs with activity against C. albicans include well-known peptides from the histatin and cecropin families13,14,15, along with more recently described peptides like ToAP2, NDBP-5.7, and the histatin 5 variant K11R-K17R16,17. Due to their potential for treating Candida infections, identifying and designing new AMPs that target C. albicans is an important goal for many research groups.
As part of the process to develop effective AMPs (and other antifungal agents) that target C. albicans, in vitro testing is commonly used to identify promising peptides. Methods to test antifungal activity against C. albicans typically involve incubating cells with serial dilutions of the AMPs (in buffer or medium) in 96-well plates. Several methods are available to assess the antifungal activity following incubation. A technique described by the Clinical Laboratory Standards Institute uses a purely visual assessment of the turbidity of the wells to determine the minimum concentration (MIC) for the complete inhibition of growth (at least 50% inhibition for selected antifungal agents, like azoles and echinocandins) and provides no quantification of growth at sub-MIC concentrations18. Another commonly used approach involves quantifying the viability following incubation with AMPs by plating the contents of the wells on agar plates, incubating the plates, and then counting the number of colony-forming units (CFUs) on the plate. This method has been used for evaluating a number of peptides, including histatin 5-based peptides, LL-37, and human lactoferrin19,20,21. This technique requires a relatively large volume of agar and a high number of plates and involves the tedious counting of CFUs on the plates. To obtain more quantitative data while generating less plastic waste and avoiding counting CFUs, the contents of the wells can be used to inoculate fresh medium in another 96-well plate. After incubating the newly inoculated plate, the growth can be quantified by measuring the optical density at 600 nm (OD600) on an absorbance plate reader. This method has been used to determine the antifungal activity of histatin 5 and its degradation fragments and cell-penetrating peptides17,22,23,24,25.
This protocol describes how to test the antifungal activity of peptides and uses the OD600 method to quantify the reduction in viability of C. albicans due to peptides.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well plates (round bottom)</td>
			<td>VWR</td>
			<td>10062-902</td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbance microplate reader</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Any available microplate reader is sufficient</td>
		</tr>
		<tr>
			<td>C. albicans strain SC5314</td>
			<td>ATCC&#160;</td>
			<td>MYA-2876</td>
			<td>Other C. albicans may also be used</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Can be used to make a standard curve relating cell number to OD600</td>
		</tr>
		<tr>
			<td>Microplate shaker</td>
			<td>VWR</td>
			<td>2620-926</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptide(s)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Peptides can be commercially synthesized by any reliable vendor; a purity of &#8805;95% and trifluoroacetic acid salt removal to hydrochloride salt are recommended</td>
		</tr>
		<tr>
			<td>Reagent reservoirs for multichannel pipettors</td>
			<td>VWR</td>
			<td>18900-320</td>
			<td>Simplifies pipetting into multiwell plates with multichannel pipettor</td>
		</tr>
		<tr>
			<td>Sodium phosphate, dibasic</td>
			<td>Fisher Scientific</td>
			<td>BP332-500</td>
			<td>For making NaPB</td>
		</tr>
		<tr>
			<td>Sodium phosphate, monobasic</td>
			<td>Fisher Scientific</td>
			<td>BP329-500</td>
			<td>For making NaPB</td>
		</tr>
		<tr>
			<td>UV spectrophotometer</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Any available UV spectrophotometer is sufficient</td>
		</tr>
		<tr>
			<td>YPD medium powder</td>
			<td>BD Life Sciences</td>
			<td>242820</td>
			<td>May also be made from yeast extract, peptone, and dextrose</td>
		</tr>
	</tbody>
</table>

quantifying the antifungal activity of peptides against candida albicans

Traditional methods for performing antifungal susceptibility testing for Candida albicans are time-consuming and lack quantitative results. For example, a common approach relies on plating cells treated with different concentrations of antifungal molecules on agar plates and then counting the colonies to determine the relationship between molecule concentration and growth inhibition. This method requires many plates and substantial time to count the colonies. Another common approach eliminates the plates and counting of colonies by visually inspecting cultures treated with antifungal agents to identify the minimum concentration required to inhibit growth; however, visual inspection produces only qualitative results, and information on growth at subinhibitory concentrations is lost. This protocol describes a method for measuring the susceptibility of C. albicans to antifungal peptides. By relying on optical density measurements of cultures, the method reduces the time and materials needed to obtain quantitative results on culture growth at different peptide concentrations. The incubation of the fungus with peptides is performed in a 96-well plate using an appropriate buffer, with controls representing no growth inhibition and complete growth inhibition. Following the incubation with the peptide, the resulting cell suspensions are diluted to reduce peptide activity and then grown overnight. After overnight growth, the optical density of each well is measured and compared to the positive and negative controls to calculate the resulting growth inhibition at each peptide concentration. The results using this assay are comparable to the results using the traditional method of plating the cultures on agar plates, but this protocol reduces plastic waste and the time spent on counting colonies. Although the applications of this protocol have focused on antifungal peptides, the method will also be applicable to testing other molecules with known or suspected antifungal activity.

Using OD600 measurements to quantify the reduction in growth due to antifungal peptides saves substantial time compared to plating samples and counting CFUs. The method described in this protocol requires completing the steps on three different days. On the first day, approximately 1 h is needed to prepare the buffers and media (not including the sterilization time) and inoculate the starting culture of C. albicans for overnight incubation. On the second day, the steps require 5-6 h (including subcult...

Watch this Scientific Journal Video about Quantifying the Antifungal Activity of Peptides Against Candida albicans at JoVE.com

Quantifying the Antifungal Activity of Peptides Against Candida albicans

This protocol describes a method for obtaining quantitative data on the antifungal activity of peptides and other compounds, such as small-molecule antifungal agents, against Candida albicans. Its use of optical density rather than counting colony-forming units to quantify growth inhibition saves time and resources.

Quantifying the Antifungal Activity of Peptides Against Candida albicans

Traditional methods for performing antifungal susceptibility testing for Candida albicans are time-consuming and lack quantitative results. For example, a ...

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2.1K Views.  University of Maryland. This protocol describes a method for obtaining quantitative data on the antifungal activity of peptides and other compounds, such as small-molecule antifungal agents, against Candida albicans. Its use of optical density rather than counting colony-forming units to quantify growth inhibition saves time and resources.

Video: Quantifying the Antifungal Activity of Peptides Against Candida albicans

Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, Parkinson's, and Huntington's diseases, hereditary amyotrophic lateral sclerosis, and type 2 diabetes. Owing to the metastable nature of these protein assemblies, it is difficult to assess their oligomer size distribution quantitatively using classical methods, such as electrophoresis, chromatography, fluorescence, or dynamic light scattering. Oligomers of amyloidogenic proteins exist as metastable mixtures, in which the oligomers dissociate into monomers and associate into larger assemblies simultaneously. PICUP stabilizes oligomer populations by covalent cross-linking and when combined with fractionation methods, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or size-exclusion chromatography (SEC), PICUP provides snapshots of the oligomer size distributions that existed before cross-linking. Hence, PICUP enables visualization and quantitative analysis of metastable protein populations and can be used to monitor assembly and decipher relationships between sequence modifications and oligomerization1. Mechanistically, PICUP involves photo-oxidation of Ru2+ in a tris(bipyridyl)Ru(II) complex (RuBpy) to Ru3+ by irradiation with visible light in the presence of an electron acceptor. Ru3+ is a strong one-electron oxidizer capable of abstracting an electron from a neighboring protein molecule, generating a protein radical1,2. Radicals are unstable, highly-reactive species and therefore disappear rapidly through a variety of intra- and intermolecular reactions. A radical may utilize the high energy of an unpaired electron to react with another protein monomer forming a dimeric radical, which subsequently loses a hydrogen atom and forms a stable, covalently-linked dimer. The dimer may then react further through a similar mechanism with monomers or other dimers to form higher-order oligomers. Advantages of PICUP relative to other photo- or chemical cross-linking methods3,4 include short (&#8804;1 s) exposure to non-destructive visible light, no need for pre facto modification of the native sequence, and zero-length covalent cross-linking. In addition, PICUP enables cross-linking of proteins within wide pH and temperature ranges, including physiologic parameters. Here, we demonstrate application of PICUP to cross-linking of three amyloidogenic proteins the 40- and 42-residue amyloid &beta;-protein variants (A&beta;40 and A&beta;42), and calcitonin, and a control protein, growth-hormone releasing factor (GRF).

Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides

Photo-induced cross-linking of unmodified proteins (PICUP) allows characterization of oligomer size distribution in metastable protein mixtures. We demonstrate application of PICUP to three representative amyloidogenic peptides the 40- and 42-residue forms of amyloid &beta;-protein, and calcitonin, and a control peptide growth-hormone releasing factor.

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, ...

Testing Protozoacidal Activity of Ligand-lytic Peptides Against Termite Gut Protozoa in vitro (Protozoa Culture) and in vivo (Microinjection into Termite Hindgut)

We are developing a novel approach to subterranean termite control that would lead to reduced reliance on the use of chemical pesticides. Subterranean termites are dependent on protozoa in the hindguts of workers to efficiently digest wood. Lytic peptides have been shown to kill a variety of protozoan parasites (Mutwiri et al. 2000) and also protozoa in the gut of the Formosan subterranean termite, Coptotermes formosanus (Husseneder and Collier 2009). Lytic peptides are part of the nonspecific immune system of eukaryotes, and destroy the membranes of microorganisms (Leuschner and Hansel 2004). Most lytic peptides are not likely to harm higher eukaryotes, because they do not affect the electrically neutral cholesterol-containing cell membranes of higher eukaryotes (Javadpour et al. 1996). Lytic peptide action can be targeted to specific cell types by the addition of a ligand. For example, Hansel et al. (2007) reported that lytic peptides conjugated with cancer cell membrane receptor ligands could be used to destroy breast cancer cells, while lytic peptides alone or conjugated with non-specific peptides were not effective. Lytic peptides also have been conjugated to human hormones that bind to receptors on tumor cells for targeted destruction of prostate and testicular cancer cells (Leuschner and Hansel 2004).
In this article we present techniques used to demonstrate the protozoacidal activity of a lytic peptide (Hecate) coupled to a heptapeptide ligand that binds to the surface membrane of protozoa from the gut of the Formosan subterranean termite. These techniques include extirpation of the gut from termite workers, anaerobic culture of gut protozoa (Pseudotrichonympha grassii, Holomastigotoides hartmanni, Spirotrichonympha leidyi), microscopic confirmation that the ligand marked with a fluorescent dye binds to the termite gut protozoa and other free-living protozoa but not to bacteria or gut tissue. We also demonstrate that the same ligand coupled to a lytic peptide efficiently kills termite gut protozoa in vitro (protozoa culture) and in vivo (microinjection into hindgut of workers), but is less bacteriacidal than the lytic peptide alone. The loss of protozoa leads to the death of the termites in less than two weeks.
In the future, we will genetically engineer microorganisms that can survive in the termite hindgut and spread through a termite colony as "Trojan Horses" to express ligand-lytic peptides that would kill the protozoa in the termite gut and subsequently kill the termites in the colony. Ligand-lytic peptides also could be useful for drug development against protozoan parasites.

Testing Protozoacidal Activity of Ligand-lytic Peptides Against Termite Gut Protozoa in vitro Protozoa Culture and in vivo Microinjection into Termite Hindgut

We present procedures for demonstrating that ligands bind to the surface membrane of the cellulose-digesting protozoa in the gut of Formosan subterranean termites using fluorescent microscopy and that ligands coupled with lytic peptides kill these protozoa in vitro (anaerobic protozoa culture) and in vivo (injection into the termite hindgut).

We are developing a novel approach to subterranean termite control that would lead to reduced reliance on the use of chemical pesticides. Subterranean ...

Quantifying Agonist Activity at G Protein-coupled Receptors

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors.
Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (Kb) is much greater than that for the inactive state (Ka). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (Kobs), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (&#949;) (Figure 2).
Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the Kobs and relative efficacy of an agonist 1,2. In this report, we show how to modify this analysis to estimate the agonist Kb value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate Kb in absolute units of M-1.
Our method of analyzing agonist concentration-response curves 3,4 consists of global nonlinear regression using the operational model 5. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of Kobs and a parameter proportional to efficacy (&tau;). The estimate of &tau;Kobs of one agonist, divided by that of another, is a relative measure of Kb (RAi) 6. For any receptor exhibiting constitutive activity, it is possible to estimate a parameter proportional to the efficacy of the free receptor complex (&tau;sys). In this case, the Kb value of an agonist is equivalent to &tau;Kobs/&tau;sys 3.
Our method is useful for determining the selectivity of an agonist for receptor subtypes and for quantifying agonist-receptor signaling through different G proteins.

A method for estimating the affinity constant of an agonist for the active state (Kb) of a G protein-coupled receptor is described. The analysis provides absolute or relative measures of Kb depending on whether constitutive receptor activation is measurable. Our method applies to various responses downstream from receptor activation.

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell ...

Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya6. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila7-14. An acetate kinase which can only utilize PPi but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus15,16.
In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann17-20, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD+ by the enzymes pyruvate kinase and lactate dehydrogenase21,22, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine23. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP+ to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase24.
Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PPi.

A method for the determination of acetate kinase activity is described. This assay utilizes a direct reaction for determining enzyme activity and kinetics of acetate kinase in the acetate-forming direction with different phosphoryl acceptors. Furthermore, this method can be utilized for assaying other acetyl phosphate or acetyl-CoA utilizing enzymes.

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

Measuring Lactase Enzymatic Activity in the Teaching Lab

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and biomedical sciences. This easy to follow protocol was developed for first year undergraduate pharmacy students and provides an entry-level introduction to enzyme reactions and analytical procedures for enzyme analysis. The enzyme of choice is lactase, as this represents an example of a commercially available enzyme relevant to human disease/pharmaceutical practice. Lactase is extracted from dietary supplement tablets, and assessed using a colorimetric assay based upon hydrolysis of an artificial substrate for lactase (ortho-nitrophenol-beta-D-galactopyranoside, ONPG). Release of ortho-nitrophenol following the hydrolytic cleavage of ONPG by lactase is measured by a change in absorbance at 420 nm, and the effect of the temperature on the enzymatic reaction is evaluated by carrying out the reaction on ice, at room temperature and at 37 &#176;C. More advanced analysis can be implemented using this protocol by assessing the enzyme activity under different conditions and using different reagents.

The enzymatic activity of lactase is essential for the catabolic processing of the disaccharide lactose. Here, the activity of lactase found in dietary supplements is assayed using a colorimetric assay. This provides students with an experimental platform for understanding the activity of lactase and enzyme kinetics.

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and ...

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this scenario, the search for new antifungal drugs is necessary. The protocol reported here details a method to screen peptides for their antifungal properties. It is based on the broth microdilution susceptibility test from the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines with modifications to suit the research of antimicrobial peptides as potential new antifungals. This protocol describes a functional assay to evaluate the activity of antifungal compounds and may be easily modified to suit any particular class of molecules under investigation. Since the assays are performed in 96-well plates using small volumes, a large-scale screening can be completed in a short amount of time, especially if carried out in an automation setting. This procedure illustrates how a standardized and adjustable clinical protocol can help the bench-work pursuit of new molecules to improve the therapy of fungal diseases.

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

An easy and adaptable broth microdilution method for screening antifungal compounds and extracts.

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this ...

Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis

The successful biochemical and biophysical characterization of ABC transporters depends heavily on the choice of the heterologous expression system. Over the past two decades, we have developed a yeast membrane protein expression platform that has been used to study many important fungal membrane proteins. The expression host Saccharomyces cerevisiae AD&#916;&#916; is deleted in seven major endogenous ABC transporters and it contains the transcription factor Pdr1-3 with a gain-of-function mutation that enables the constitutive overexpression of heterologous membrane protein genes stably integrated as single copies at the genomic PDR5 locus. The creation of versatile plasmid vectors and the optimization of one-step cloning strategies enables the rapid and accurate cloning, mutagenesis, and expression of heterologous ABC transporters. Here, we describe the development and use of a novel protease-cleavable mGFPHis double tag (i.e., the monomeric yeast enhanced green fluorescent protein yEGFP3 fused to a six-histidine affinity purification tag) that was designed to avoid possible interference of the tag with the protein of interest and to increase the binding efficiency of the His tag to nickel-affinity resins. The fusion of mGFPHis to the membrane protein ORF (open reading frame) enables easy quantification of the protein by inspection of polyacrylamide gels and detection of degradation products retaining the mGFPHis tag. We demonstrate how this feature facilitates detergent screening for membrane protein solubilization. A protocol for the efficient, fast, and reliable isolation of the small-scale plasma membrane preparations of the C-terminally tagged Candida albicans multidrug efflux transporter Cdr1 overexpressed in S. cerevisiae AD&#916;&#916;, is presented. This small-scale plasma membrane isolation protocol generates high-quality plasma membranes within a single working day. The plasma membrane preparations can be used to determine the enzyme activities of Cdr1 and Cdr1 mutant variants.

Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis

This article presents a small-scale plasma membrane isolation protocol for the characterization of Candida albicans ABC (ATP-binding cassette) protein Cdr1, overexpressed in Saccharomyces cerevisiae. A protease-cleavable C-terminal mGFPHis double tag with a 16-residue linker between Cdr1 and the tag was designed to facilitate the purification and detergent-screening of Cdr1.

The successful biochemical and biophysical characterization of ABC transporters depends heavily on the choice of the heterologous expression system. Over ...