The authors acknowledge COST Action CM1406 (Epigenetic Chemical Biology). Research reported in this publication was supported by the Pierre Mercier foundation.

1. Cell Culture
NOTE: The following steps are performed in a standard tissue culture hood. Familiarity with sterile technique is expected.
<ol>
	<li>Culture HeLa cells to 80% confluency in a T75 cell culture flask in MEM supplemented with 10% fetal bovine serum, penicillin G (100 U/mL), and streptomycin (100 mg/mL). 
	NOTE: Cell culture, treatment, and lysis steps are conducted under laminar flow, with cell incubation steps at 37 &#176;C performed in a humidified atmosphere of 5% CO2 under sterile conditions.</li>
	<li>Wash the cells twice with 5 mL of DPBS, aspirate the DPBS, and add 1 mL of cell dissociation reagent. Incubate at 37 &#176;C for 5 min to detach the cells.</li>
	<li>Add 9 mL of MEM culture medium, thoroughly resuspend the cell suspension, and count the cells using a hemocytometer.</li>
	<li>Adjust the density of the cell suspension to 6 x 104 cells/mL in MEM.</li>
	<li>Add 100 &#181;L of cell suspension to control and test the wells of a sterile, flat-bottom, tissue culture-treated 96-well plate (Figure 1A).</li>
	<li>Add 100 &#181;L of MEM culture medium to blank wells of the 96-well plate (Figure 1A).</li>
	<li>Incubate the 96-well plate at 37 &#176;C for 24 h. 
	NOTE: A 24 h incubation is a suitable time to test for endogenous HDAC activity in HeLa cells. Different cell types with unknown HDAC activity may need a timecourse evaluation of control cells.</li>
</ol>
2. Cell Treatment with Test Compounds and HDAC Substrates
<ol>
	<li>Aspirate the medium from the wells of the 96-well plate.</li>
	<li>Add 25 &#181;L of 2x test compounds (e.g., trichostatin A, MS275, and tubastatin A) in MEM to test wells. Add 25 &#181;L of MEM to control and blank wells. 
	NOTE: The concentration of test compounds is to be adjusted to provide a range of at least 5 final test concentrations (one concentration per well). The DMSO final concentration in the test compound should not exceed 0.5% and should be the same in each well, including blanks and controls. The final incubation volume is 50 &#181;L per well. Trichostatin A is tested at 5 concentrations ranging from 500 to 1.95 nM (1:4 dilution). MS275 and tubastatin A are tested at 5 concentrations ranging from 8,000 to 6.17 nM (1:6 dilution). The desired final test concentration for each test compound should be determined by each individual user by creating a dose-response curve.</li>
	<li>Add 25 &#181;L of the substrate mixture (42 &#181;M of MAL, 42 &#181;M of MOCPAC, and 42 &#181;M of BATCP in MEM) to control and test wells. Add 25 &#181;L of MEM to blank wells. 
	NOTE: The final concentration of each substrate is 21 &#181;M. MOCPAC and BATCP distinguish between HDAC1 and HDAC6 activity, respectively (Figure 1B). When no activity is observed with those substrates, the use of MAL could potentially lead to the identification of activity against another HDAC isoform.</li>
	<li>Incubate the 96-well plate at 37 &#176;C for 8 h in a humidified 5% CO2 incubator. 
	NOTE: This incubation time is suitable for HeLa cells and allows the endogenous HDAC to interact with the synthetic substrates. Different cell lines may require adjustments in incubation time and/or substrate concentration.</li>
</ol>
3. Cell Lysis and Sample Preparation for UHPLC-ESI-MS/MS
Caution: The sample preparation steps use organic chemicals, which are highly flammable and toxic by ingestion or inhalation. Wear appropriate personal protection, such as gloves and safety glasses.
<ol>
	<li>Add 10 &#181;L of 6x RIPA buffer (diluted from 10x RIPA buffer) supplemented with protease inhibitor to each well of the 96-well plate.</li>
	<li>Stop the reaction by adding 160 &#181;L of cold acetonitrile to each well and mix by pipetting up and down. 
	NOTE: Keep the acetonitrile at -20 &#176;C prior to this step.</li>
	<li>Place the plate in a -80 &#176;C freezer for 10 min.</li>
	<li>Remove the plate from the freezer and transfer 220 &#181;L of the content of each well to a non-sterile, conical-bottom (V-bottom) 96-well plate. Centrifuge the plate at 5,000 x g and 4 &#176;C for 10 min. 
	NOTE: This step aims at removing proteins and salts. If necessary, the solutions can be transferred to individual conical centrifuge tubes prior to centrifugation or filtered through a set of 96-well, 0.65 &#181;m PVDF filter plates. The successful removal of the precipitate is required prior to UHPLC analysis.</li>
	<li>Transfer 200 &#181;L of supernatant (or filtrate) into a 96-well plate compatible with the UHPLC system and seal it with peelable, heat-sealing foil using a plate sealer. 
	NOTE: Avoid pipetting the pellets when removing the supernatants. If the UHPLC analysis cannot be immediately performed, the plate can be stored at 4 &#176;C for a maximum of 24 h until analysis.</li>
</ol>
4. UHPLC/MS-MS Analysis
<ol>
	<li>Prepare the UHPLC system by filling it with the mobile phase system (95% A:5% B): 
	A: H2O, HPLC-grade, containing 0.1% formic acid (prepare 1 L) 
	B: Acetonitrile, HPLC-grade, containing 0.1% formic acid (prepare 1 L). 
	Caution: The mobile phase contains organic chemicals, which are highly flammable and toxic by ingestion or inhalation. Wear appropriate personal protection, such as gloves and safety glasses.</li>
	<li>Place the 96-well plate into the sample manager containing a plate holder. Run the samples according to the UHPLC-ESI-MS/MS conditions provided in Table 1. 
	NOTE: Every column of the analytical 96-well plate should have one control well and one blank well (as depicted in the plate scheme of Figure 1A). Controls are representative of 100% enzymatic activity in the tested cell line, while blanks are there to check whether the MS source is providing consistent background. Unusual MS peaks detected in a given blank should be investigated to prevent MS sensitivity changes.</li>
</ol>
5. Data Analysis
<ol>
	<li>Integrate the peak area of each substrate and its deacetylated product with an appropriate UHPLC integration software. 
	NOTE: Use automatic integration parameters with a smoothing method (Mean, window size 3, and number of smooth 2, for example). Using the chromatographic conditions provided in Table 1, the elution order is dMAL (5.8 min), dBATCP (6.0 min), dMOCPAC (6.2 min), MAL (7.6 min), MOCPAC (8.9 min), and BATCP (9.8 min), as depicted in Figure 1C.</li>
	<li>For each substrate, calculate the peak area ratio (deacetylated/acetylated) in each chromatogram (test and control samples).</li>
	<li>Calculate the percent HDAC inhibition of each test sample: 
	<img alt="Equation" src="/files/ftp_upload/55878/55878eq1.jpg" /> 
	NOTE: HDAC1 inhibition is calculated using the peak area ratio of dMOCPAC/MOCPAC; HDAC6 inhibition is calculated with dBATCP/BATCP. The ratio dMAL/MAL can be used to express general HDAC inhibition.</li>
</ol>

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The authors have nothing to disclose.

HDAC inhibition is a hot topic in drug discovery, with a current focus on HDAC6-selective inhibitors for cancer therapy21. HDAC selectivity is usually assessed by a series of high-throughput, cell-free assays, which intend to determine the inhibitory potency towards individual HDAC isoforms21. However, the selectivity of an inhibitor must be additionally confirmed in living cells by evaluating the acetylation status of endogenous protein substrates, such as histones (for cl...

HDACs belong to a family of enzymes able to deacetylate histones within the chromatin structure. They also have other protein substrates in the cytosol and are located in various cell compartments. A total of 18 HDAC isoforms have been identified so far and have been related to several cell mechanisms, including the regulation of transcription factors and gene expression, as well as cell signaling and transport1,2,3,4,5,6,7. HDAC catalytic inhibitors have emerged as potential therapeutic drugs for cancer therapy. Most HDAC inhibitors currently approved by the FDA are for the treatment of T-cell lymphoma and multiple myeloma, and are non-specific HDAC inhibitors, such as vorinostat (SAHA), belinostat, and panobinostat8,9. However, a series of side effects have been associated with pan-inhibitors, and the search for isoform-specific small molecules is a hot topic in medicinal chemistry and drug discovery. Accordingly, the class I (HDAC1-3 and HDAC8) selective inhibitor romidepsin is an already approved drug10, while HDAC6-specific inhibitors are currently under clinical trials, with increased therapeutic potential in multiple myeloma11,12,13,14,15.
Screening assays to characterize HDAC inhibitors are based on the incubation of an HDAC substrate with an enzymatic source (single isoform, nuclear extract, or cell lysate). The substrate is usually a small peptide sequence containing an acetyl lysine residue coupled to a cleavable fluorophore (e.g., coumarin), such as N-(4-methyl-7-aminocoumarinyl)-N&#945;-(t-butoxycarbonyl)-N&#969;-acetyllysineamide (MAL)16. To distinguish between isoform-specific activities, separate cell-free assays involving each isoform are necessary and might not reflect the real isoform activity in living cells. Isoform-specific substrates are commercially available, such as benzyl (S)-[1-(4-methyl-2-oxo-2H-chromen-7-ylcarbamoyl)-5-propionylaminopentyl]carbamate (MOCPAC, HDAC1 specific substrate) and (S)-[5-acetylamino-1-(2-oxo-4-trifluoromethyl-2H-chromen-7-ylcarbamoyl)pentyl]carbamic acid tert-butyl ester (BATCP, HDAC6 specific substrate) (Figure 1B). However, a multi-substrate mixture containing MAL, MOCPAC, and BATCP given to living cells will not permit the detection of the individual deacetylated products by fluorometric measurement, given that they bear the same cleavable fluorophore.
The method described here allows for the detection and relative quantification of each substrate and its deacetylated product in HeLa cells using a multi-substrate assay followed by UHPLC-ESI-MS/MS analysis17. An HDAC assay is conducted on HeLa cells to enable the direct identification of HDAC inhibitory activity and of the specificity of test compounds on endogenous HDACs. There is a focus on HDAC1 and HDAC6, which are simultaneously evaluated. To achieve these enzymatic measurements in a single incubation assay, a mixture of non-specific and specific HDAC substrates is added to treated and untreated HeLa cells plated on a 96-well plate. Following an incubation step, cells are lysed to release the substrates and their respective reaction products, which are separated and detected using a UHPLC-MS method (Figure 1C). The deacetylated products of the MAL, MOCPAC, and BATCP substrates are the deacetylated MAL (dMAL), deacetylated MOCPAC (dMOCPAC), and deacetylated BATCP (dBATCP), respectively. Dose-response curves can be built with active compounds.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55878/55878fig1.jpg" /> 
Figure 1: General scheme for the cell-based HDAC assay to identify HDAC1- and HDAC6-specific inhibitors by the UHPLC-MS analysis of multiple substrates. (A) Scheme of a typical 96-well plate containing treated (test compounds) and untreated (control) HeLa cells, as well as cell-free blanks. (B) Chemical structure of substrates added as a mixture (21 &#181;M each) to be deacetylated by endogenous HDACs. (C) Typical UHPLC-MS chromatogram showing the peaks of the added substrates (MAL, MOCPAC, and BATCP) and their deacetylated products (dMAL, dMOCPAC, and dBACTP, respectively). <a href="http://cloudflare.jove.com/files/ftp_upload/55878/55878fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0" width="887">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">BATCP</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:152px;">B4061</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MAL</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td>SCP0168</td>
			<td>Synonym: BOC-Ac-Lys-AMC&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">MOCPAC</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td>M2195</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">MS275</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:152px;">EPS002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Trichostatin A</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:152px;">T8552</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Tubastatin A</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:152px;">SML0044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Acetonitrile, HPLC grade</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:152px;">10660131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Formic acid, LC/MS grade</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:152px;">10596814</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:271px;">H2O, HPLC grade</td>
			<td style="width:191px;"></td>
			<td style="width:152px;"></td>
			<td>distilled H2O filtered through a Milli-Q purification system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">HeLa cells</td>
			<td style="width:191px;">ATCC</td>
			<td style="width:152px;">ATCC CRM-CCL-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell dissociation reagent TrypLE Express</td>
			<td style="width:191px;">ThermoFisher Scientific</td>
			<td style="width:152px;">12604013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">DMSO, cell culture grade</td>
			<td style="width:191px;">Applichem</td>
			<td style="width:152px;">146463</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">DPBS</td>
			<td style="width:191px;">ThermoFisher Scientific</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Fetal bovine serum&#160;</td>
			<td style="width:191px;">Biowest</td>
			<td>S1810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">MEM</td>
			<td style="width:191px;">ThermoFisher Scientific</td>
			<td>22561021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin</td>
			<td style="width:191px;">Bioconcept</td>
			<td>4-01F00-H&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10X RIPA buffer</td>
			<td style="width:191px;">Abcam</td>
			<td style="width:152px;">ab156034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">SigmaFast protease inhibitor tablets</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:152px;">S8820</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">96-well plates, sterile, flat-bottom, tissue culture treated Corning</td>
			<td style="width:191px;">VWR</td>
			<td style="width:152px;">29442-058</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">96-well plates, non-sterile, V-bottom Corning</td>
			<td style="width:191px;">VWR</td>
			<td style="width:152px;">29442-404</td>
			<td>used in the centrifugation step</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">96-well plate, conical bottom, Nunc</td>
			<td style="width:191px;">ThermoFisher Scientific</td>
			<td style="width:152px;">249944</td>
			<td>compatible with the Acquity UHPLC system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">T75&#160; cell culture flasks Corning</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:152px;">CLS430641&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Peelable heat sealing foil&#160;</td>
			<td style="width:191px;">Waters</td>
			<td style="width:152px;">186002789</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Acquity UPLC system</td>
			<td style="width:191px;">Waters</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Eppendorf Centrifuge 5810 R</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:152px;">05-413-323</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Integration software: MassLynx V4.1</td>
			<td style="width:191px;">Waters</td>
			<td style="width:152px;"></td>
			<td>Catalog number not available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Combi thermo-sealer SP-0669/240</td>
			<td style="width:191px;">Waters</td>
			<td style="width:152px;"></td>
			<td>Catalog number not available</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Quattro micro API Tandem Quadrupole System</td>
			<td style="width:191px;">Waters</td>
			<td style="width:152px;"></td>
			<td>Catalog number not available</td>
		</tr>
	</tbody>
</table>

simultaneous measurement of hdac1 and hdac6 activity in hela cells using uhplc-ms

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography &#8211; mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.

To demonstrate the application of the method to identifying non-selective and selective inhibitors for HDAC1 (class I) and HDAC6 (class IIb), HeLa cells were treated with known standard compounds: trichostatin A (non-selective between HDAC1 and HDAC6)18, MS275 (HDAC1-selective inhibitor)19, and tubastatin A (HDAC6-selective inhibitor)20. By using the present method (Figure 1), IC50</...

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Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

The present method serves to identify isoform-specific inhibitors of histone deacetylases (HDAC) in HeLa cells by the UHPLC-MS analysis of multiple substrates. This is an antibody-free method developed to reflect HDAC1 and HDAC6 activity in the living cell environment, in contrast to single-isoform cell-free assays.

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since ...

simultaneous-measurement-hdac1-hdac6-activity-hela-cells-using-uhplc

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

8.4K Views.  Universités des Geneva–Lausanne (EPGL). The present method serves to identify isoform-specific inhibitors of histone deacetylases (HDAC) in HeLa cells by the UHPLC-MS analysis of multiple substrates. This is an antibody-free method developed to reflect HDAC1 and HDAC6 activity in the living cell environment, in contrast to single-isoform cell-free assays.

Video: Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, koff). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (koff). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (&lt;1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E&gt;S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.

Time courses for the glycosylase activity of 8-oxoguanine DNA glycosylase are biphasic exhibiting a burst of product formation and a linear steady-state phase. Utilizing quench-flow techniques, the burst and the steady-state rates can be measured, which correspond to excision of 8-oxoguanine and release of the glycosylase from the product DNA, respectively.

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization ...

A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal fermentation processes and screening for new oil-rich strains requires a fast and reliable protocol for determination of intracellular lipid content. Current practices rely largely on gravimetric methods to determine oil content, techniques developed decades ago that are time consuming and require large sample volumes. In this paper, Nile Red, a fluorescent dye that has been used to identify the presence of lipid bodies in numerous types of organisms, is incorporated into a simple, fast, and reliable protocol for measuring the neutral lipid content of Auxenochlorella protothecoides, a green alga. The method uses ethanol, a relatively mild solvent, to permeabilize the cell membrane before staining and a 96 well micro-plate to increase sample capacity during fluorescence intensity measurements. It has been designed with the specific application of monitoring bioprocess performance. Previously dried samples or live samples from a growing culture can be used in the assay.

A simple protocol to determine the neutral lipid content of algal cells using a Nile Red staining procedure is described. This time-saving technique offers an alternative to traditional gravimetric-based lipid quantification protocols. It has been designed for the specific application of monitoring bioprocess performance.

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal ...

HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2&#8217;-deoxyguanosine, in Cultured Cells and Animal Tissues

Oxidative stress is associated with many physiological and pathological processes, as well as xenobiotic metabolism, leading to the oxidation of biomacromolecules, including DNA. Therefore, efficient detection of DNA oxidation is important for a variety of research disciplines, including medicine and toxicology. A common biomarker of oxidatively damaged DNA is 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxo-dGuo; often erroneously referred to as 8-hydroxy-2&#39;-deoxyguanosine (8-OH-dGuo or 8-oxo-dG)). Several protocols for 8-oxo-dGuo measurement by high pressure liquid chromatography with electrochemical detection (HPLC-ED) have been described. However, these were mainly applied to purified DNA treated with pro-oxidants. In addition, due to methodological differences between laboratories, mainly due to differences in analytical equipment, the adoption of published methods for detection of 8-oxo-dGuo by HPLC-ED requires careful optimization by each laboratory. A comprehensive protocol, describing such an optimization process, is lacking. Here, a detailed protocol is described for the detection of 8-oxo-dGuo by HPLC-ED, in DNA from cultured cells or animal tissues. It illustrates how DNA sample preparation can be easily and rapidly optimized to minimize undesirable DNA oxidation that can occur during sample preparation. This protocol shows how to detect 8-oxo-dGuo in cultured human alveolar adenocarcinoma cells (i.e., A549 cells) treated with the oxidizing agent KBrO3, and from the spleen of mice exposed to the polycyclic aromatic hydrocarbon dibenzo(def,p)chrysene (DBC, formerly known as dibenzo(a,l)pyrene, DalP). Overall, this work illustrates how an HPLC-ED methodology can be readily optimized for the detection of 8-oxo-dGuo in biological samples.

The goal of this protocol is the detection of the DNA oxidation marker, 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxo-dGuo) by HPLC-ED, in DNA from cultured cells or animal tissues.

Oxidative stress is associated with many physiological and pathological processes, as well as xenobiotic metabolism, leading to the oxidation of ...

Dynamic Electrochemical Measurement of Chloride Ions

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver chloride electrode is used extensively for potentiometric measurement of chloride ions concentration in electrolyte. In this measurement, long-term and continuous monitoring is limited due to the inherent drift and the requirement of a stable reference electrode. We utilized the chronopotentiometric approach to minimize drift and avoid the use of a conventional reference electrode. A galvanostatic pulse is applied to an Ag/AgCl electrode which initiates a faradic reaction depleting the Cl&#713; ions near the electrode surface. The transition time, which is the time to completely deplete the ions near the electrode surface, is a function of the ion concentration, given by the Nernst equation. The square root of the transition time is in linear relation to the chloride ion concentration. Drift of the response over two weeks is negligible (59 &#181;M/day) when measuring 1 mM [Cl&#713;]using a current pulse of 10 Am-2. This is a dynamic measurement where the moment of transition time determines the response and thus is independent of the absolute potential. Any metal wire can be used as a pseudo-reference electrode, making this approach feasible for long-term measurement inside concrete structures.

Dynamic measurement of chloride ions is presented. Transition time of an Ag/AgCl electrode, during a chronopotentiometric technique, can give the concentration of chloride ions in electrolyte. This method does not require a stable conventional reference electrode.

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Fabrication and Testing of Photonic Thermometers

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being leveraged for developing sophisticated photonic sensors. Silicon photonic sensors seek to exploit the strong confinement of light in nano-waveguides to transduce changes in physical state to changes in resonance frequency. In the case of thermometry, the thermo-optic coefficient, i.e., changes in refractive index due to temperature, causes the resonant frequency of the photonic device such as a Bragg grating to drift with temperature. We are developing a suite of photonic devices that leverage recent advances in telecom compatible light sources to fabricate cost-effective photonic temperature sensors, which can be deployed in a wide variety of settings ranging from controlled laboratory conditions, to the noisy environment of a factory floor or a residence. In this manuscript, we detail our protocol for the fabrication and testing of photonic thermometers.

We describe the process of fabrication and testing of photonic thermometers.

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being ...

Disentangling High Strength Copolymer Aramid Fibers to Enable the Determination of Their Mechanical Properties

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to diversify the fiber choices in the United States body armor market, copolymer fibers based on the combination of 5-amino-2-(p-aminophenyl) benzimidazole (PBIA) and the more conventional PPTA were introduced. Little is known regarding the long-term stability of these fibers, but as condensation polymers, they are expected to have potential sensitivity to moisture and humidity. Therefore, characterizing the strength of the materials and understanding their vulnerability to environmental conditions is important for evaluating their use lifetime in safety applications. Ballistic resistance and other critical structural properties of these fibers are predicated on their strength. To accurately determine the strength of the individual fibers, it is necessary to disentangle them from the yarn without introducing any damage. Three aramid-based copolymer fibers were selected for the study. The fibers were washed with acetone followed by methanol to remove an organic coating that held the individual fibers in each yarn bundle together. This coating makes it difficult to separate single fibers from the yarn bundle for mechanical testing without damaging the fibers and affecting their strength. After washing, fourier transform infrared (FTIR) spectroscopy was performed on both washed and unwashed samples and the results were compared. This experiment has shown that there are no significant variations in the spectra of poly(p-phenylene-benzimidazole-terephthalamide-co-p-phenylene terephthalamide) (PBIA-co-PPTA1) and PBIA-co-PPTA3 after washing, and only a small variation in intensity for PBIA. This indicates that the acetone and methanol rinses are not adversely affecting the fibers and causing chemical degradation. Additionally, single fiber tensile testing was performed on the washed fibers to characterize their initial tensile strength and strain to failure, and compare those to other reported values. Iterative procedural development was necessary to find a successful method for performing tensile testing on these fibers.

The primary goal of the study is to develop a protocol to prepare consistent specimens for accurate mechanical testing of high strength copolymer aramid fibers, by removing a coating and disentangling the individual fiber strands without introducing significant chemical or physical degradation.

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to ...

High Resolution Physical Characterization of Single Metallic Nanoparticles

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single nanometer-scale pore. The signal is characteristic of the molecule&#39;s physicochemical properties and its interactions with the pore. We demonstrate that the nanopore formed by the bacterial protein exotoxin Staphylococcus aureus alpha hemolysin (&#945;HL) can detect polyoxometalates (POMs, anionic metal oxygen clusters), at the single molecule limit. Moreover, multiple degradation products of 12-phosphotungstic acid POM (PTA, H3PW12O40) in solution are simultaneously measured. The single molecule sensitivity of the nanopore method allows for POMs to be characterized at significantly lower concentrations than required for nuclear magnetic resonance (NMR) spectroscopy. This technique could serve as a new tool for chemists to study the molecular properties of polyoxometalates or other metallic clusters, to better understand POM synthetic processes, and possibly improve their yield. Hypothetically, the location of a given atom, or the rotation of a fragment in the molecule, and the metal oxidation state could be investigated with this method. In addition, this new technique has the advantage of allowing the real-time monitoring of molecules in solution.&#160;

Here, we present a protocol to detect discrete metal oxygen clusters, polyoxometalates (POMs), at the single molecule limit using a biological nanopore-based electronic platform. The method provides a complementary approach to traditional analytical chemistry tools used in the study of these molecules.

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single ...

A New Straightforward Method for Lipophilicity (logP) Measurement using 19F NMR Spectroscopy

Fluorination has become an effective tool to optimize physicochemical properties of bioactive compounds. One of the applications of fluorine introduction is to modulate the lipophilicity of the compound. In our group, we are interested in the study of the impact of fluorination on lipophilicity of aliphatic fluorohydrins and fluorinated carbohydrates. These are not UV-active, resulting in a challenging lipophilicity determination. Here, we present a straightforward method for the measurement of lipophilicity of fluorinated compounds by 19F NMR spectroscopy. This method requires no UV-activity. Accurate solute mass, solvent and aliquot volume are also not required to be measured. Using this method, we measured the lipophilicities of a large number of fluorinated alkanols and carbohydrates.

A New Straightforward Method for Lipophilicity logP Measurement using 19F NMR Spectroscopy

A novel and straightforward variation of the shake-flask method was developed for accurate lipophilicity measurement of fluorinated compounds by 19F NMR spectroscopy.

Fluorination has become an effective tool to optimize physicochemical properties of bioactive compounds. One of the applications of fluorine introduction ...

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

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