Research in NC lab is supported by a National Institutes of Health (NIH) grant R01AI24499 and a New Jersey Health Foundation (NJHF) grant, #PC40-18.

NOTE: The detailed stepwise protocol of the assay is described below, and the schematic protocol is shown in Figure 1.
1. C. albicans strains and growth conditions
<ol>
	<li>Grow the C. albicans strains&#160;in liquid Yeast Extract-Peptone-Dextrose (YPD) medium at 30 &#176;C in an incubator shaker overnight. 
	NOTE: Maintain Candida strains as frozen stocks and grow on YPD agar (1 % yeast extract, 2% peptone, 2% dextrose, and 2% agar).</li>
</ol>
2. Preparation of reagents
<ol>
	<li>Prepare the assay medium as follows:
	<ol>
		<li>For mitochondrial function assay, dissolve 1.04 g Roswell Park Memorial Institute (RPMI) 1640 powder and 2 g glucose (2%) in 90 mL sterile water and warm the media to 37 &#176;C. Adjust the pH to 7.4 using 5 M NaOH and make up the volume to 100 mL with sterile water.</li>
		<li>For glycolytic stress assay, dissolve 1.04 g RPMI 1640 powder alone in 90 mL sterile water, warm the media to 37 &#176;C and adjust the pH to 7.4. Make up the volume to 100 mL with sterile water. RPMI 1640 powder has no bicarbonate, which is critical to monitor the pH change as a measure of glycolysis during the assay.</li>
	</ol>
	</li>
	<li>Injection compounds.
	<ol>
		<li>Prepare 1 M glucose stock in sterile water and store at -20 &#176;C.</li>
		<li>Prepare 100 mM oligomycin stock in dimethyl sulfoxide (DMSO), aliquot in small volumes and store at -20 &#176;C.</li>
		<li>Prepare 100 mM antimycin A stock in DMSO, aliquot in small volumes and store at -20 &#176;C.</li>
		<li>Prepare 100 mM SHAM (salicylhydroxamic acid) stock in ethanol on the day of assay.</li>
		<li>Prepare 1 M KCN in the sterile water on the day of the assay.</li>
	</ol>
	</li>
</ol>
3. Coating of the assay plate with Poly-D-Lysine (PDL)
NOTE: Perform all the below steps in a laminar hood.
<ol>
	<li>Dissolve Poly-D Lysine in tissue culture grade water to make 50 &#181;g/mL final concentration. Mix it well and aliquot into a 1.5 mL microcentrifuge tubes and store at -20 &#176;C for the long term. 
	NOTE: 50 &#181;L per well is needed, and for 24 wells, 1.2 mL is required. Therefore, aliquot at least 1.3 mL per microcentrifuge tube.</li>
	<li>Add 50 &#181;L per well and incubate at room temperature with the lid covered for 1-2 h.</li>
	<li>Aspirate the solution and rinse one time with 500 &#181;L sterile tissue culture grade water.</li>
	<li>Open the lid and allow the wells to air dry. Use the plate on the same day or store at 4 &#176;C for a maximum of 2-3 days.</li>
</ol>
4. Hydration of sensor cartridge
NOTE: Perform this step one day before the experiment.
<ol>
	<li>Open the extra flux Assay Kit and remove the contents. Place the sensor cartridge upside down next to the utility plate (Figure 2).</li>
	<li>Fill each well of the utility plate with 1 mL of calibrant and place the sensor cartridge back. Make sure the sensors which contain fluorophores (to measure the oxygen and pH) are submerged in the calibrant.</li>
	<li>Incubate the sensor cartridge overnight in a non-CO2 incubator at 37 &#176;C.</li>
</ol>
5. Growing and seeding cells in the PDL-coated plates
<ol>
	<li>Inoculate C. albicans in the YPD broth and grow overnight at 30 &#176;C in a shaker at 200 rpm. 
	NOTE: Based on the assay design and the interest, C. albicans can also be grown in YPG or minimal medium.</li>
	<li>On the day of assay, dilute an appropriate number of cells in the assay medium to yield a final concentration of 100,000 cells per 100 &#181;L.</li>
	<li>Add 100 &#181;L of the diluted cells into each well of the assay plate except wells A1, B4, C3, and D6, in which add only 100 &#181;L of the assay medium for background correction (Figure 3).</li>
	<li>Transfer the plate to a non-CO2 incubator at 37 &#176;C and incubate for 60 min, which will let the cells adhere to the plate surface.</li>
</ol>
6. Assay Protocol
NOTE: The protocol outlined here is for the 24-well format of the instrument. Volumes will need to be adjusted if another format is used.
<ol>
	<li>Mitochondrial function assay
	<ol>
		<li>Preparation of compounds
		<ol>
			<li>Prepare compounds at 10x concentration for mitochondrial function assay: Prepare 20 mM SHAM, 100 &#181;M Oligomycin, 100 mM KCN, and 20 &#181;M Antimycin A in the corresponding assay medium.</li>
			<li>Add 50 &#181;L SHAM into the port A, 55 &#181;L Oligomycin into port B, 62 &#181;L KCN into port C and 68 &#181;L Antimycin A into port D (Figure 4).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Glycolytic stress assay
	<ol>
		<li>Preparation of compounds
		<ol>
			<li>Prepare compounds at 10x concentration for glycolytic stress assay. Prepare 100 mM glucose, 100 &#181;M Oligomycin, 500 mM 2-Deoxy Glucose (2DG) and 20 &#181;M Antimycin A in the corresponding assay medium.</li>
			<li>Add 50 &#181;L glucose into port A, 55 &#181;L Oligomycin into port B, 62 &#181;L 2-DG into port C and 68 &#181;L Antimycin A into port D.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Employ extra flux analyzer, which measures oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of live cells in a 24-well plate format. Set up the assay protocol in advance.</li>
	<li>Open the extra flux analyzer and set up the assay template by using the assay wizard tab and follow step by step instruction to fill out all the information that pops out during the setup. Generate the group layout as similar to shown in Figure 5. Set up the protocol as shown in Table 1. Set up these layouts well ahead before assay and save in the computer. At the time of assay, restore the saved protocol by opening the corresponding file in the open file option in the assay wizard tab (Figure 5).</li>
	<li>Load the 10x compounds in the respective ports of the hydrated sensor cartridge containing the calibrant and load in the carrier tray of the extra flux analyzer. Start the calibration by pressing the Start button on the screen.</li>
	<li>Add 350 &#181;L of the assay medium gently to the cell plate along the side of the wells to minimize cell disturbance to bring the final volume to 450 &#181;L.</li>
	<li>Replace the utility plate containing the calibrant with the assay plate and continue.</li>
	<li>Remove the sensor cartridge and the plate once the assay is completed. Save the file in the appropriate destination folder.</li>
</ol>
7. Data Analysis
<ol>
	<li>Use the area under the curve- analysis of variance (AUC-ANOVA) analysis tab in the software to calculate the significant difference between the groups by selecting the respective parameters (OCR or ECAR) as shown in Figure 6.</li>
	<li>Select the groups that need to be compared.</li>
	<li>Add to the analysis groups for ANOVA and click Ok. 
	NOTE: This AUC ANOVA analysis will add a new sheet to the file in which the AUC is calculated for each group and compared between them by ANOVA. This will give a table of p values to show the significance.</li>
</ol>

<ol>
	<li>Brown, G. D., 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=Hidden+killers:+human+fungal+infections.">Hidden killers: human fungal infections.</a> Science Translational Medicine. 4 (165), (2012).</li><li>Wisplinghoff, H., 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=Nosocomial+bloodstream+infections+in+US+hospitals:+analysis+of+24,179+cases+from+a+prospective+nationwide+surveillance+study.">Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study.</a> Clinical Infectious Diseases. 39 (3), 309-317 (2004).</li><li>Ascioglu, S., 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=Defining+opportunistic+invasive+fungal+infections+in+immunocompromised+patients+with+cancer+and+hematopoietic+stem+cell+transplants:+an+international+consensus.">Defining opportunistic invasive fungal infections in immunocompromised patients with cancer and hematopoietic stem cell transplants: an international consensus.</a> Clinical Infectious Diseases. 34 (1), 7-14 (2002).</li><li>Stover, B. H., 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=Nosocomial+infection+rates+in+US+children's+hospitals'+neonatal+and+pediatric+intensive+care+units.">Nosocomial infection rates in US children's hospitals' neonatal and pediatric intensive care units.</a> American Journal of Infection Control. 29 (3), 152-157 (2001).</li><li>Wilson, L. S., 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+direct+cost+and+incidence+of+systemic+fungal+infections.">The direct cost and incidence of systemic fungal infections.</a> Value in Health. 5 (1), 26-34 (2002).</li><li>Wenzel, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nosocomial+candidemia:+risk+factors+and+attributable+mortality.">Nosocomial candidemia: risk factors and attributable mortality.</a> Clinical Infectious Diseases. 20 (6), 1531-1534 (1995).</li><li>Wisplinghoff, H., 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=Nosocomial+bloodstream+infections+in+pediatric+patients+in+United+States+hospitals:+epidemiology,+clinical+features+and+susceptibilities.">Nosocomial bloodstream infections in pediatric patients in United States hospitals: epidemiology, clinical features and susceptibilities.</a> Pediatric Infectious Disease Journal. 22 (8), 686-691 (2003).</li><li>Cheng, W. C., Leach, K. M., Hardwick, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+death+pathways+in+yeast+and+mammalian+cells.">Mitochondrial death pathways in yeast and mammalian cells.</a> Biochimica et Biophysica Acta. 1783 (7), 1272-1279 (2008).</li><li>Shingu-Vazquez, M., Traven, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+and+fungal+pathogenesis:+drug+tolerance,+virulence,+and+potential+for+antifungal+therapy.">Mitochondria and fungal pathogenesis: drug tolerance, virulence, and potential for antifungal therapy.</a> Eukaryotic Cell. 10 (11), 1376-1383 (2011).</li><li>Brown, A. J., Brown, G. D., Netea, M. G., Gow, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+impacts+upon+Candida+immunogenicity+and+pathogenicity+at+multiple+levels.">Metabolism impacts upon Candida immunogenicity and pathogenicity at multiple levels.</a> Trends in Microbiology. 22 (11), 614-622 (2014).</li><li>Tucey, T. 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=Glucose+Homeostasis+Is+Important+for+Immune+Cell+Viability+during+Candida+Challenge+and+Host+Survival+of+Systemic+Fungal+Infection.">Glucose Homeostasis Is Important for Immune Cell Viability during Candida Challenge and Host Survival of Systemic Fungal Infection.</a> Cell Metabolism. 27 (5), 988-1006 (2018).</li><li>Barelle, C. 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=Niche-specific+regulation+of+central+metabolic+pathways+in+a+fungal+pathogen.">Niche-specific regulation of central metabolic pathways in a fungal pathogen.</a> Cellular Microbiology. 8 (6), 961-971 (2006).</li><li>Carman, A. J., Vylkova, S., Lorenz, M. C. <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+acetyl+coenzyme+A+synthesis+and+breakdown+in+alternative+carbon+source+utilization+in+Candida+albicans.">Role of acetyl coenzyme A synthesis and breakdown in alternative carbon source utilization in Candida albicans.</a> Eukaryotic Cell. 7 (10), 1733-1741 (2008).</li><li>Fradin, 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=Granulocytes+govern+the+transcriptional+response,+morphology+and+proliferation+of+Candida+albicans+in+human+blood.">Granulocytes govern the transcriptional response, morphology and proliferation of Candida albicans in human blood.</a> Molecular Microbiology. 56 (2), 397-415 (2005).</li><li>Lorenz, M. C., Bender, J. A., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+response+of+Candida+albicans+upon+internalization+by+macrophages.">Transcriptional response of Candida albicans upon internalization by macrophages.</a> Eukaryotic Cell. 3 (5), 1076-1087 (2004).</li><li>Ramirez, M. A., Lorenz, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+alternative+carbon+utilization+pathways+in+Candida+albicans+attenuate+virulence+and+confer+pleiotropic+phenotypes.">Mutations in alternative carbon utilization pathways in Candida albicans attenuate virulence and confer pleiotropic phenotypes.</a> Eukaryotic Cell. 6 (2), 280-290 (2007).</li><li>Bambach, 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=Goa1p+of+Candida+albicans+localizes+to+the+mitochondria+during+stress+and+is+required+for+mitochondrial+function+and+virulence.">Goa1p of Candida albicans localizes to the mitochondria during stress and is required for mitochondrial function and virulence.</a> Eukaryotic Cell. 8 (11), 1706-1720 (2009).</li><li>Li, D., 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=Enzymatic+dysfunction+of+mitochondrial+complex+I+of+the+Candida+albicans+goa1+mutant+is+associated+with+increased+reactive+oxidants+and+cell+death.">Enzymatic dysfunction of mitochondrial complex I of the Candida albicans goa1 mutant is associated with increased reactive oxidants and cell death.</a> Eukaryotic Cell. 10 (5), 672-682 (2011).</li><li>Desai, C., Mavrianos, J., Chauhan, N. <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+SRR1,+a+putative+two-component+response+regulator+gene,+is+required+for+stress+adaptation,+morphogenesis,+and+virulence.">Candida albicans SRR1, a putative two-component response regulator gene, is required for stress adaptation, morphogenesis, and virulence.</a> Eukaryotic Cell. 10 (10), 1370-1374 (2011).</li><li>Mavrianos, 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=Mitochondrial+two-component+signaling+systems+in+Candida+albicans.">Mitochondrial two-component signaling systems in Candida albicans.</a> Eukaryotic Cell. 12 (6), 913-922 (2013).</li><li>Koch, B., 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+Mitochondrial+GTPase+Gem1+Contributes+to+the+Cell+Wall+Stress+Response+and+Invasive+Growth+of+Candida+albicans.">The Mitochondrial GTPase Gem1 Contributes to the Cell Wall Stress Response and Invasive Growth of Candida albicans.</a> Frontiers in Microbiology. 8, 2555 (2017).</li><li>Qu, Y., 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=Mitochondrial+sorting+and+assembly+machinery+subunit+Sam37+in+Candida+albicans:+insight+into+the+roles+of+mitochondria+in+fitness,+cell+wall+integrity,+and+virulence.">Mitochondrial sorting and assembly machinery subunit Sam37 in Candida albicans: insight into the roles of mitochondria in fitness, cell wall integrity, and virulence.</a> Eukaryotic Cell. 11 (4), 532-544 (2012).</li><li>Brandt, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Candida and Candidiasis. , (2002).</li><li>Huh, W. K., Kang, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+and+functional+expression+of+alternative+oxidase+from+Candida+albicans.">Molecular cloning and functional expression of alternative oxidase from Candida albicans.</a> Journal of Bacteriology. 181 (13), 4098-4102 (1999).</li><li>Yan, 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=The+alternative+oxidase+of+Candida+albicans+causes+reduced+fluconazole+susceptibility.">The alternative oxidase of Candida albicans causes reduced fluconazole susceptibility.</a> Journal of Antimicrobial Chemotherapy. 64 (4), 764-773 (2009).</li><li>de Moura, M. B., Van Houten, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioenergetic+analysis+of+intact+mammalian+cells+using+the+Seahorse+XF24+Extracellular+Flux+analyzer+and+a+luciferase+ATP+assay.">Bioenergetic analysis of intact mammalian cells using the Seahorse XF24 Extracellular Flux analyzer and a luciferase ATP assay.</a> Methods in Molecular Biology. 1105, 589-602 (2014).</li></ol>

The authors have nothing to disclose

The bioenergetics extra flux assay serves as an excellent tool to read out the mitochondrial function by measuring oxidative phosphorylation (OXPHOS)-dependent oxygen consumption in real-time. In addition, a glycolytic function which is measured as an extracellular acidification rate (change in extracellular pH) can also be investigated at the same time in real-time analysis.
Successful plating of C. albicans in the assay plate is one of the critical steps in the assay because the incubation of the cells in the PDL coated plates allow the cells to adhere to the assay plate. Visualization of adhering C. albicans cells is accomplished by light microscopy. Improper cell adherence may lead to cells floating in the wells, which will affect the assay outcome with inconsistent readings between replicates. Similarly, it is important to perform the cell number titration as the first step in this assay to obtain an OCR between 100-300 pmol/min, which is an optimal range. Also, titrating the compounds to obtain the optimal concentration is also critical to achieving the optimal response. This may be performed whenever there is a change in the basal medium composition.
DMEM is the most commonly used basal medium in the extra flux assay for mammalian cells. However, in our hands, RPMI 1640 medium gave the optimal results with C. albicans. This was primarily due to the efficacy of mitochondrial electron transport chain complex inhibitors that worked better in RPMI 1640 medium compared to DMEM.
In this assay, wild type, mam33&#916;/&#916;, and mam33&#916;/&#916;::MAM33 strains of C. albicans were used to assess the mitochondrial respiration and glycolytic efficiency. The results clearly show that the deletion of MAM33 gene reduced the ability of the C. albicans to shift to glycolysis under stress conditions such as inhibition of C-IV (Figure 7C and 7E). Similarly, when cells were subjected to stress condition like glucose starvation, both mitochondrial respiration and glycolytic efficiency is significantly compromised in the mam33&#916;/&#916; mutant but not in the wild type and complement strains (Figure 8). Therefore, depending on experiments, carbon sources in the RPMI 1640 may also be modified to glycerol or galactose, which forces the cells to use OXPHOS rather than glycolysis.
The significance of using the extra flux assay is that by using various substrates and compounds that perturb mitochondrial function, the cells ability to utilize OXPHOS versus glycolysis can be inferred. This may be used as an informative tool to understand the effect of any treatments or gene on the mitochondrial function and glycolysis in Candida albicans without isolating mitochondria. The assay measures basal oxygen consumption rate (OCR), which is a measure of mitochondrial respiration, and extracellular acidification rate (ECAR), which is a measure of glycolytic function in both the presence of glucose and after glucose starvation. The extra flux cellular assay is well employed in a variety of mammalian models26. Despite the medical importance of C. albicans, research into fungal mitochondria has been somewhat hampered due to the lack of standardized experimental procedures to study mitochondrial function and glycolysis in real time.
This method provides an easy and convenient way of investigating the mitochondrial respiration and glycolytic efficiency by using real-time extracellular flux assay in C. albicans. In future, this assay may also be applied to study the drug-efficacy testing using commonly employed antifungals such as fluconazole. Additionally, another potential future application of this method is in drug discovery, especially for screening compounds targeting mitochondrial function of C. albicans.

Invasive fungal infections kill over 1.5 million people a year worldwide. This number is on the rise due to an increase in the numbers of people living with compromised immunity, including the elderly, premature infants, transplant recipients, and cancer patients1. C. albicans is an opportunistic human fungal pathogen that is a part of the human microflora. It also inhabits mucosal surfaces and the gastrointestinal tract as a commensal organism. C. albicans produces serious systemic disease in people who have immune deficiencies, who have undergone surgery, or who have been treated with long courses of antibiotics. The Candida species rank among the top three to four causes of nosocomial infectious diseases (NID) in humans2,3,4,5,6,7. The annual global number of Candida bloodstream infections is estimated to be ~400,000 cases, with associated mortalities of 46-75%1. The annual mortality because of candidiasis is roughly 10,000 in the United States alone. The extent of NID caused by fungi is also reflected in astronomical patient expenses5. In the United States, the yearly expense for the treatment of invasive fungal infections surpasses $2 billion, adding a huge strain to already overburdened health care system. Currently, available standard antifungal therapies are limited because of toxicity, increasingly prevalent drug resistance, and drug-drug interactions. Therefore, there is an urgent need to identify new antifungal drug targets that will result in better treatment options for high-risk patients. However, the discovery of new drugs acting on fungal targets is complicated because fungi are eukaryotes. This greatly limits the number of fungal-specific drug targets.
Recent studies have indicated that mitochondria are a critical contributor to the fungal virulence and tolerance to antifungal drugs since mitochondria are important for cellular respiration, oxidative metabolism, signal transduction, and apoptosis8,9,10,11. Both glycolytic and non-glycolytic metabolism are essential for the survival of C. albicans in the mammalian host12,13,14,15,16. Furthermore, several C. albicans mutants lacking mitochondrial proteins, such as Goa1, Srr1, Gem1, Sam37 etc. have been shown to be defective in filamentation, an important virulence factor of C. albicans17,18,19,20,21,22. In addition, these mutants were also shown to be attenuated for virulence in a mouse model of disseminated candidiasis17,18,19,20,21,22. Thus, fungal mitochondria represent an attractive target for drug discovery. However, the study of mitochondrial function in C. albicans is challenging because C. albicans is petite negative23, which means that it cannot survive without the mitochondrial genome.
Here, we describe a protocol that can be used to investigate mitochondrial and glycolytic function in C. albicans without the need to purify mitochondria. This method can also be optimized to investigate the effect of the genetic manipulation or chemical modulators on mitochondrial and glycolytic pathways in C. albicans.

<table border="0" cellpadding="0" cellspacing="0" style="width:664px;" width="664">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">RPMI 1640</td>
			<td style="width:84px;">Corning</td>
			<td style="width:119px;">MT50020PB</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Antimycin A</td>
			<td style="width:84px;">Sigma</td>
			<td>A8674</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCN</td>
			<td style="width:84px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Mito stress kit</td>
			<td style="width:84px;">Agilent</td>
			<td>103015-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Oligomycin</td>
			<td style="width:84px;">Calbiochem</td>
			<td>495455</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">pH meter</td>
			<td style="width:84px;">Accumet</td>
			<td>AR20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Phenol red</td>
			<td style="width:84px;">Sigma</td>
			<td>P5530</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Poly-D lysine</td>
			<td style="width:84px;">Sigma</td>
			<td>P6407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Rotenone</td>
			<td style="width:84px;">Santa cruz</td>
			<td>203242</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Seahorse XF24 FluxPak</td>
			<td style="width:84px;">Agilent</td>
			<td>100850-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SHAM</td>
			<td style="width:84px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Sodium Chloride</td>
			<td style="width:84px;">Amresco&#160;</td>
			<td>241</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Sodium hydroxie pellets</td>
			<td style="width:84px;">J.T Baker</td>
			<td>3722</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Tissue culture grade water</td>
			<td style="width:84px;">Gibco</td>
			<td>1523-0147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">XF assay calibrant solution</td>
			<td style="width:84px;">Agilent</td>
			<td>100840-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Yeast extract Peptone Dextrose</td>
			<td>Fisher scientific,</td>
			<td>BP2469</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Yeast extract Peptone Dextrose Agar</td>
			<td style="width:84px;">Sigma</td>
			<td>A1296</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:295px;">Yeast extract Peptone Glycerol</td>
			<td style="width:84px;">Sigma</td>
			<td>G2025</td>
			<td></td>
		</tr>
	</tbody>
</table>

bio-energetics investigation of candida albicans using real-time extracellular flux analysis

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular respiration, oxidative metabolism, signal transduction, and apoptosis. Consequently, mitochondrial dysfunction is reported to play an important role in the antifungal drug tolerance and virulence of pathogenic fungi. Recent data have also led to the recognition of the importance of the mitochondria as an important contributor to fungal pathogenesis. Despite the importance of the mitochondria in fungal biology, standardized methods to understand its function are poorly developed. Here, we present a procedure to study the basal oxygen consumption rate (OCR), a measure of mitochondrial respiration, and extracellular acidification rates (ECAR), a measure of glycolytic function in C. albicans strains. The method described herein can be applied to any Candidaspp. strains without the need to purify mitochondria from the intact fungal cells. Furthermore, this protocol can also be customized to screen for inhibitors of mitochondrial function in C. albicans strains.

The focus of this protocol is to determine the bioenergetic functions of C. albicans assessed by extra flux analyzer. A C. albicans mutant lacking mitochondrial protein Mam33 is also included along with its complement strain, mam33&#916;/&#916;::MAM33 to study the effects of the deletion of a mitochondrial protein on OCR and ECAR. MAM33 encodes for a putative mitochondrial acidic matrix protein and its function in Candida is not known.
Cell numbers for extra flux assay 
We employed 1x 105&#160;C. albicans wild type cells per well in the extra flux assay, which showed an OCR of 145 pmol/min (Figure 7), which is within the optimal range of 100-300 pmol/min for any extracellular flux assay. It is also important to titrate the cell number to obtain an optimal OCR before the assay or when there is a change in growth medium composition between different assays.
Analysis of data 
The area under the curve- analysis of variance (AUC-ANOVA) analysis tab in the software was used to calculate the significant difference between the groups by selecting the respective parameters (OCR or ECAR) as shown in Figure 7. This analysis added a new sheet to the file in which the AUC was calculated for each group and compared between them by ANOVA automatically, which gave a table of p values.
Mitochondrial function assay 
In the mitochondrial stress test, the assay starts with measuring the basal oxygen consumption rate (OCR) followed by injection of inhibitors of an alternate oxidase (AOX), ATP synthase, and electron transport chain complexes, IV and III. In addition to the classical respiratory chain, C. albicans also utilizes the AOX pathway for energy generation24. Therefore, we investigated the effect of AOX system on OCR by inhibiting its activity by using salicylhydroxamic acid (SHAM)25. The mitochondrial function, which was measured as an oxygen consumption rate (OCR) in pmol/min at basal and after injecting respective inhibitors is shown in Figure 7. The basal OCR of wild type, mam33&#916;/&#916;, and mam33&#916;/&#916;::MAM33 strains showed no differences (Figure 7A and B). Similarly, no significant difference in the basal ECAR was observed between these strains (Figure 7C and D). However, by inhibiting complex IV by KCN, wild type and mam33&#916;/&#916;::MAM33 showed a substantial shift toward the glycolysis in contrast to mam33&#916;/&#916;, which significantly failed to show a compensatory glycolytic shift (Figure 7E), suggesting that C-IV dependent glycolytic pathway is impaired in the mam33&#916;/&#916; mutant compared to wild type and mam33&#916;/&#916;::MAM33 strains.
Glycolytic stress assay 
In the glycolytic stress test, cells were starved for glucose for 1 h and the basal OCR and ECAR was measured. Upon starvation, mam33&#916;/&#916; showed significantly lower OCR (Figure 8A and B) and ECAR (Figure 8C and D) compared to the wild type and mam33&#916;/&#916;::MAM33 strains suggesting an impaired utilization of glucose for both respiration and glycolysis when cells are forced to the starvation condition. After injecting glucose, all strains showed stimulation of both OCR and ECAR. Although oligomycin has less effect on both OCR and ECAR, 2-DG, which is a competitive inhibitor of glycolysis, inhibits both OCR and ECAR. Specifically, 2-DG partially inhibits OCR, which is further inhibited by Antimycin A. In contrast, 2-DG almost completely inhibits ECAR.
<img alt="Table 1" src="/files/ftp_upload/58913/58913table1.jpg" /> 
Table 1. Protocol commands for the assay
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58913/58913fig1.jpg" /> 
Figure 1. Schematic representation of the experiment <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58913/58913fig1large.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/58913/58913fig2.jpg" /> 
Figure 2. Sensor cartridge (green) placed upside down near the utility plate before adding the calibrant. Fluorophores are indicated by arrows. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58913/58913fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58913/58913fig3.jpg" /> 
Figure 3. Cell culture plate showing background wells A1, B4, C3, and D6. The blue mark shows the notch, oriented to the lower left. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58913/58913fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58913/58913fig4.jpg" /> 
Figure 4. The top portion of the sensor cartridge showing ports A, B, C and D. The arrow mark shows the notch, oriented to the lower left. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58913/58913fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58913/58913fig5.jpg" /> 
Figure 5. The layout of wells and group assignment as visualized in the software. WT- wild type; MT- mam33&#916;/&#916; mutant; COMP- mam33&#916;/&#916;::MAM33 complement strain. Arrow mark shows the open file tab. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58913/58913fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58913/58913fig6.jpg" /> 
Figure 6. The area under the curve (AUC) ANOVA analysis tab to calculate the significance between the groups. Select the groups that need to be compared and add to the analysis groups for ANOVA and click ok. This AUC ANOVA analysis will add a new sheet to the file in which the AUC is calculated for each group and compared between them by ANOVA. This will give a table of p values to show the significance. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58913/58913fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58913/58913fig7.jpg" /> 
Figure 7.&#160;Mitochondrial function assay in C. albicans. Oxygen consumption and extracellular acidification rates were measured in wild type, mam33 &#916;/&#916;, and mam33 &#916;/&#916;::MAM33 strains of C. albicans. A) A real-time graph of oxygen consumption rate (OCR) at basal respiration followed by sequential injection of SHAM (2 mM), Oligomycin (10 &#181;M), KCN (10 mM), and Antimycin A (2 &#181;M). B) Comparison of area under the curve (AUC) basal OCR between wild type, mam33&#916;/&#916;, and mam33&#916;/&#916;::MAM33 strains of C. albicans show no significant difference. C) A real-time graph of extracellular acidification rate (ECAR) at basal glycolysis followed by sequential injection of SHAM (2 mM), Oligomycin (10 &#181;M), KCN (10 mM), and Antimycin A (2 &#181;M). D) Comparison of area under the curve (AUC) basal ECAR between wild type, mam33&#916;/&#916;, and mam33&#916;/&#916;::MAM33 strains of C. albicans show no significant difference. E) Comparison of area under curve (AUC) ECAR between wild type, mam33&#916;/&#916; and mam33 &#916;/&#916;::MAM33 strains of C. albicans after the injection of C-IV inhibitor KCN show significant decrease in glycolytic shift in mam33&#916;/&#916; mutant compared to wild type and complementary strains Values are expressed as average &#177; standard error mean. * p&#60;0.05, ** p&#60;0.001 is considered as significant by one-way ANOVA. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58913/58913fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/58913/58913fig8.jpg" /> 
Figure 8. Glycolytic stress assay in C. albicans. Oxygen consumption and extracellular acidification were measured in wild type, mam33&#916;/&#916; mutant after they were starved for glucose followed by acute injection of saturated glucose concentration. A) A real-time graph of oxygen consumption rate after cells were subjected to basal ECAR followed by sequential injection of glucose (10 mM), Oligomycin (1 &#181;M), 2-DG (40 mM), and Antimycin A (2 &#181;M). B) Bar graph showing the area under the curve ECAR of basal glycolysis between wild type, mam33&#916;/&#916;, and mam33&#916;/&#916;::MAM33 strains of C. albicans. C) A real-time graph of the glycolytic function after cells were subjected to basal ECAR followed by sequential injection of glucose (10 mM), Oligomycin (1 &#181;M), 2-DG (40mM), and Antimycin A (2 &#181;M). D) Bar graph showing the area under the curve ECAR of basal glycolysis between wild type, mam33&#916;/&#916;, and mam33&#916;/&#916;::MAM33 strains of C. albicans Values are expressed as average &#177; standard error mean. * p&#60;0.05, *** p&#60;0.0005, **** p&#60;0.0001 is considered as significant by one-way ANOVA. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58913/58913fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis at JoVE.com

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Here, we present a stepwise protocol to investigate the mitochondrial respiration and glycolytic function in Candida Albicans using an extra flux analyzer.

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular ...

bio-energetics-investigation-candida-albicans-using-real-time

Nanyang Technological University

Research

JoVE Journal

Medicine

6.9K Views.  The State University of New Jersey. Here, we present a stepwise protocol to investigate the mitochondrial respiration and glycolytic function in Candida Albicans using an extra flux analyzer.

Video: Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Real-time fMRI Biofeedback Targeting the Orbitofrontal Cortex for Contamination Anxiety

We present a method for training subjects to control activity in a region of their orbitofrontal cortex associated with contamination anxiety using biofeedback of real-time functional magnetic resonance imaging (rt-fMRI) data. Increased activity of this region is seen in relationship with contamination anxiety both in control subjects1 and in individuals with obsessive-compulsive disorder (OCD),2 a relatively common and often debilitating psychiatric disorder involving contamination anxiety. Although many brain regions have been implicated in OCD, abnormality in the orbitofrontal cortex (OFC) is one of the most consistent findings.3, 4 Furthermore, hyperactivity in the OFC has been found to correlate with OCD symptom severity5 and decreases in hyperactivity in this region have been reported to correlate with decreased symptom severity.6 Therefore, the ability to control this brain area may translate into clinical improvements in obsessive-compulsive symptoms including contamination anxiety. Biofeedback of rt-fMRI data is a new technique in which the temporal pattern of activity in a specific region (or associated with a specific distributed pattern of brain activity) in a subject's brain is provided as a feedback signal to the subject. Recent reports indicate that people are able to develop control over the activity of specific brain areas when provided with rt-fMRI biofeedback.7-12 In particular, several studies using this technique to target brain areas involved in emotion processing have reported success in training subjects to control these regions.13-18 In several cases, rt-fMRI biofeedback training has been reported to induce cognitive, emotional, or clinical changes in subjects.8, 9, 13, 19 Here we illustrate this technique as applied to the treatment of contamination anxiety in healthy subjects. This biofeedback intervention will be a valuable basic research tool: it allows researchers to perturb brain function, measure the resulting changes in brain dynamics and relate those to changes in contamination anxiety or other behavioral measures. In addition, the establishment of this method serves as a first step towards the investigation of fMRI-based biofeedback as a therapeutic intervention for OCD. Given that approximately a quarter of patients with OCD receive little benefit from the currently available forms of treatment,20-22 and that those who do benefit rarely recover completely, new approaches for treating this population are urgently needed.

Here we present a method for training people to control a brain area involved in contamination anxiety and for probing the relationship between contamination anxiety and brain connectivity patterns.

We present a method for training subjects to control activity in a region of their orbitofrontal cortex associated with contamination anxiety using ...

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Movement Retraining using Real-time Feedback of Performance

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular patterns responsible for governing the movement performance. This motor learning can be enhanced through a number of methods that are utilized in research and clinical settings alike. In general, verbal feedback of performance in real-time or knowledge of results following movement is commonly used clinically as a preliminary means of instilling motor learning. Depending on patient preference and learning style, visual feedback (e.g. through use of a mirror or different types of video) or proprioceptive guidance utilizing therapist touch, are used to supplement verbal instructions from the therapist. Indeed, a combination of these forms of feedback is commonplace in the clinical setting to facilitate motor learning and optimize outcomes.
Laboratory-based, quantitative motion analysis has been a mainstay in research settings to provide accurate and objective analysis of a variety of movements in healthy and injured populations. While the actual mechanisms of capturing the movements may differ, all current motion analysis systems rely on the ability to track the movement of body segments and joints and to use established equations of motion to quantify key movement patterns. Due to limitations in acquisition and processing speed, analysis and description of the movements has traditionally occurred offline after completion of a given testing session.
This paper will highlight a new supplement to standard motion analysis techniques that relies on the near instantaneous assessment and quantification of movement patterns and the display of specific movement characteristics to the patient during a movement analysis session. As a result, this novel technique can provide a new method of feedback delivery that has advantages over currently used feedback methods.

Retraining abnormal movement patterns following injury or disease is a key component of physical rehabilitation. Recent advances in technology have permitted accurate assessment of movement during a variety of tasks, with near instantaneous quantification of results. This provides new opportunities for modification of faulty movement patterns in real time.

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans ...

Real-time Pressure-volume Analysis of Acute Myocardial Infarction in Mice

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any potential therapeutic approach directed against acute left ventricular (LV) dysfunction. Current imaging modalities (e.g., echocardiography and magnetic resonance imaging) have several limitations since data on LV pressure cannot directly be measured. LV catheterization in mice undergoing coronary artery occlusion could serve as a novel method for a real-time evaluation of LV function.
At the beginning of the procedure, mice were anesthetized followed by endotracheal intubation. For LV catheterization, the right carotid artery was exposed via middle-neck incision. The catheter was introduced and placed into the LV cavity. Left thoracotomy was conducted and the left main coronary artery (LCA) was ligated. To induce reperfusion, the suture was released after 45 min. Pressure-volume data was recorded at all times.
Ligation of the LCA caused a decrease in LV systolic function as evidenced by a 30% reduction in stroke volume, LV ejection fraction (EF), and cardiac output. Maximum dP/dt as a parameter for LV contractility was also significantly reduced and diastolic function was severely impaired (minimum dP/dt -40%). Reperfusion over a period of 20 min did not lead to a complete recovery of LV function.
Real-time pressure-volume analysis served as a valid procedure for monitoring cardiac function during acute myocardial infarction in mice. Maintaining stable anesthesia and a standardized surgical approach was crucial to ensure valid results. As the early phase of acute myocardial infarction is critical for morbidity and mortality, the delineated method could be beneficial for preclinical evaluation of new strategies for cardioprotection.

Acute myocardial infarction in mice induces acute but incompletely characterized changes in left ventricular (LV) function. LV catheterization in mice undergoing coronary artery occlusion serves as a novel method for a real-time evaluation of LV function.

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any ...

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