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>

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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. 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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 inc...

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.
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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

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 ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

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 ...

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 ...