Name: methods to increase the sensitivity of high resolution melting single nucleotide polymorphism genotyping in malaria
Uploaded: 2015-11-10T13:00:00
Duration: 10 min 27 s
Description: Watch this Scientific Journal Video about Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria at JoVE.com

The authors thank the Bill and Melinda Gates Foundation for its support of technology development and training.

Note: This protocol includes volumes and concentrations for 96-well plate, 8-tube strips, and capillary-based systems (10 &#181;l&#160;reaction volume); however, HRM can also be performed in 384-well plate-based systems with a 5 &#181;l&#160;reaction volume by scaling all volumes correspondingly. The range of detection for most systems is 10 pg to 10 ng template DNA.&#160;
1. Prepare Templates
<ol>
	<li>Obtain Plasmodium falciparum samples directly from blood obtained from patients participating in field site studies and stored as whole blood, pelleted red blood cells, or on filter paper. 
	Note: Samples may also be culture-adapted to grow in vitro; some standard laboratory strains for analysis can be ordered from the Malaria Research and Reference Reagent Resource Center (MR4, Samples may be used directly from pelleted red blood cells or culture or extracted from whole blood, red blood cells, or filter paper.</li>
	<li>DNA extracted from filter paper or culture-adapted strains 
	Note: HRM is sensitive to salt concentration; concentration differences due to variable extraction methods can significantly affect melting temperatures. While specific final buffers are not a significant factor for success, use a standard common elution or resuspension buffer (i.e., water, 1x Te [&#8216;low Te&#8217; or &#8216;DNA suspension buffer&#8217;: 10 mM Tris-Cl, 0.10 mM EDTA] or 1x TE [10 mM Tris-Cl, 1 mM EDTA]) for all samples to avoid anomalous or inconsistent melt curves.
	<ol>
		<li>Prepare template dilutions of 10 pg &#8211;10 ng per &#181;l&#160;in standard Te, TE, or water for each 10 &#181;l&#160;reaction.</li>
	</ol>
	</li>
	<li>Direct amplification from pelleted red blood cells
	<ol>
		<li>Determine parasitemia of red blood cells using thin-smear microscopy 
		Note: Methods for determining parasitemia of infected red blood cells are available from the Centers for Disease Control and Prevention (CDC): http://www.cdc.gov/dpdx/diagnosticProcedures/blood/microexam.html</li>
		<li>Dilute parasitemias above 0.5% 1:400 in PCR-grade water, TE, or Te. Use 3 &#181;l&#160;for each 5&#8211; 10 &#181;l&#160;reaction.</li>
	</ol>
	</li>
	<li>Controls 
	Note: HRM can be used for gene scanning to detect sequence variants in a population12; however, accurate genotyping requires standards sequence-verified to contain the SNP being interrogated. Plasmid constructs or malaria isolates with known genotypes can be used. For most applications, 3D7 (MR4 MRA-151) is used as a wild-type control. Due to between-run variability, always include positive and negative controls.
	<ol>
		<li>Positive controls: Prepare 10 pg &#8211; 10 ng per 1 &#181;l&#160;dilutions of plasmid or standards with sequence-verified SNP genotypes.</li>
		<li>Negative control: Use PCR grade water as a no-template control (NTC) for the amplification reactions.</li>
	</ol>
	</li>
</ol>

2. PCR Amplification
<ol>
	<li>Prepare reaction mixture
	<ol>
		<li>(Optional) Mineral oil overlay. Note: Some HRM systems are standalone melting platforms and do not have a heated lid to prevent condensation and product evaporation during amplification.
		<ol>
			<li>Add 20 &#181;l&#160;light mineral oil to each well plate before loading the reaction mixture.</li>
		</ol>
		</li>
		<li>Prepare 10x working stocks of all assays
		<ol>
			<li>Refer to Table 1 for recommended 10x working stock concentrations as well as final concentrations for blocked-probe-based HRM genotyping.</li>
		</ol>
		</li>
		<li>Prepare reaction mixtures
		<ol>
			<li>To each well in plate or tube, add 1 &#181;l&#160;10x working stock forward and reverse primers and probes, 4.0 &#8211; 5.0 &#181;l&#160;2.5x or 2.0x HRM master mix, 1 &#181;l&#160;template, and PCR grade water for a total reaction volume of 10 &#181;l.</li>
		</ol>
		</li>
		<li>Cover plates or tubes
		<ol>
			<li>Use optical plate seals for plate-based amplification, optical caps for strip-tube-based systems, and plastic caps for capillary-based systems. Be sure coverage is complete and plates and caps are completely sealed and secured to prevent evaporation during amplification.</li>
		</ol>
		</li>
		<li>Spin samples
		<ol>
			<li>Spin plates 3 min at 1,800 x g to remove air bubbles and to separate oil and aqueous phases if mineral oil is used.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Amplification protocol
	<ol>
		<li>Place reaction plates or tubes in thermal cycler. Run standard blocked-probe or MAAB amplification protocols (Tables 2 and 3, respectively). Note the difference in annealing temperature between these methods.</li>
	</ol>
	</li>
</ol>
3. HRM Analysis
<ol>
	<li>Melt
	<ol>
		<li>After PCR amplification, proceed to melting analysis. For standalone melting systems that do not have built-in amplification (e.g., BioFire Defense LightScanner systems), remove amplified plate from thermal cycler and place in HRM instrument. From the system software interface, melt the plate from 40 &#8211; 80 &#176;C to produce both probe and amplicon melting peaks. 
		Note: To save melting and analysis time, the melting window can be shortened to cover temperature ranges for just probe or amplicon melting regions. After melting, plates and tubes can be re-melted if necessary and stored at -20 &#176;C or 4 &#176;C. Store glass tubes only at 4 &#176;C to prevent tubes from cracking.</li>
	</ol>
	</li>
	<li>Melt analysis
	<ol>
		<li>Remove negative samples&#160;
		<ol>
			<li>Within the analysis software, remove from further analysis samples that did not amplify or that have jagged melting peaks. Note: After selecting the mode of analysis for an individual run file, the instrument automatically groups the positive and negative samples. Before genotyping each subset, the user can manually alter the calls.&#160;
			<ol>
				<li>From either the melting curve or melting peak view, right-click on the curves to be removed to select them. After selecting the misclassified samples, go to &#8220;New Call,&#8221; select the appropriate grouping, and click &#8220;Apply Change.&#8221;</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>Normalize
		<ol>
			<li>From the negative derivative of normalized fluorescence with respect to temperature (&#8211;dF/dT) (&#8216;Difference Curves&#8217;) view, move normalization bars to surround the probe melt region. 
			Note: The probe melt region is the lower temperature of the two melting regions. The normalization bars should be at the base of the melt peaks.</li>
		</ol>
		</li>
		<li>Calculate groups&#160;
		<ol>
			<li>After normalization, select &#8216;Calculate groups&#8217; to automatically assign samples to groups. 
			If necessary, manually re-assign samples to specific groups. 
			Note: Some analysis software has high sensitivity thresholds that cannot be changed; in these cases, the software might assign samples to different groups that visually belong to another.</li>
		</ol>
		</li>
		<li>Assign genotypes
		<ol>
			<li>Assign genotypes for each group based on standards (positive controls).
			<ol>
				<li>After the user confirms that the computed groups are correct (if not, that correct changes have been made under the Grouping tab by selecting misclassified samples and selecting the right group from the drop-box beside &#8220;New Call&#8221;), select &#8220;Edit Group Names&#8221; under the &#8220;Grouping&#8221; tab.</li>
				<li>Enter the genotypes of the standards into the corresponding colored box (for example, if standard 3d7 has a known A genotype and is in the red group, the red group has genotype A). Press &#8220;OK&#8221; and save the results.</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>Export results
		<ol>
			<li>Export genotype calls as a spreadsheet for further sample population analysis.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> World Health Organization report 2013. , 1-286 (2013).</li><li>Hayton, K., Su, X. -. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+resistance+and+genetic+mapping+in+Plasmodium+falciparum.">Drug resistance and genetic mapping in Plasmodium falciparum.</a> Current genetics. 54 (5), (2008).</li><li>Neafsey, D. E., 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=Genome-wide+SNP+genotyping+highlights+the+role+of+natural+selection+in+Plasmodium+falciparum+population+divergence.">Genome-wide SNP genotyping highlights the role of natural selection in Plasmodium falciparum population divergence.</a> Genome biology. 9 (12), R171 (2008).</li><li>Volkman, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+map+of+diversity+in+Plasmodium+falciparum.">A genome-wide map of diversity in Plasmodium falciparum.</a> Nature. 39 (1), 113-119 (2007).</li><li>Volkman, S. K., Neafsey, D. E., Schaffner, S. F., Park, D. J., Wirth, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+genomics+and+genome+biology+to+understand+malaria+biology.">Harnessing genomics and genome biology to understand malaria biology.</a> Nature Reviews Genetics. 13 (5), 315-328 (2012).</li><li>Manske, 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=Analysis+of+Plasmodium+falciparum+diversity+in+natural+infections+by+deep+sequencing.">Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing.</a> Nature. 487 (7407), 375-379 (2012).</li><li>Auburn, 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=Characterization+of+within-host+Plasmodium+falciparum+diversity+using+next-generation+sequence+data.">Characterization of within-host Plasmodium falciparum diversity using next-generation sequence data.</a> PLoS ONE. 7 (2), e32891 (2012).</li><li>Daniels, R., 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=A+general+SNP-based+molecular+barcode+for+Plasmodium+falciparum+identification+and+tracking.">A general SNP-based molecular barcode for Plasmodium falciparum identification and tracking.</a> Malaria Journal. 7, 223 (2008).</li><li>Daniels, R., 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=Genetic+surveillance+detects+both+clonal+and+epidemic+transmission+of+malaria+following+enhanced+intervention+in+Senegal.">Genetic surveillance detects both clonal and epidemic transmission of malaria following enhanced intervention in Senegal.</a> PLoS ONE. 8 (4), e60780 (2013).</li><li>Daniels, R. R., 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=field-deployable+method+for+genotyping+and+discovery+of+single-nucleotide+polymorphisms+associated+with+drug+resistance+in+Plasmodium+falciparum.">field-deployable method for genotyping and discovery of single-nucleotide polymorphisms associated with drug resistance in Plasmodium falciparum.</a> Antimicrobial agents and chemotherapy. 56 (6), 2976-2986 (2012).</li><li>Olbadia, N., 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=Clonal+outbreak+of+Plasmodium+falciparum+in+eastern+Panama.">Clonal outbreak of Plasmodium falciparum in eastern Panama.</a> The Journal of infectious diseases. , (2014).</li><li>Montgomery, J., Wittwer, C. T., Palais, R., Zhou, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+mutation+scanning+and+genotyping+by+high-resolution+DNA+melting+analysis.">Simultaneous mutation scanning and genotyping by high-resolution DNA melting analysis.</a> Nature protocols. 2 (1), 59-66 (2007).</li><li>Mharakurwa, S., Daniels, R., Scott, A., Wirth, D. F., Thuma, P., Volkman, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-amplification+methods+for+tracking+low-grade+Plasmodium+falciparum+populations+during+scaled-up+interventions+in+Southern+Zambia.">Pre-amplification methods for tracking low-grade Plasmodium falciparum populations during scaled-up interventions in Southern Zambia.</a> Malaria Journal. 13, 89 (2014).</li></ol>

The authors have nothing to disclose.

&#160;&#160; Continous&#160;HRM is a post-PCR analysis step; therefore, sample and assay setup is similar to standard PCR protocols, with the additional incorporation of a fluorescent intercalating dye used to track the transition from double-stranded to single-stranded DNA during the high-resolution melting step. The single most important factor for successful HRM analysis is a robust PCR product. Assay design is key, and several tools optimized for HRM assay design are available commercially and online13. Amplicon lengths of 80-150 base pairs with accompanying ~20 base pair blocked probes centered over the SNP or SNPs of interest work best to differentiate SNPs as well as haplotypes of multiple SNPs within the probe region. Probes can be blocked using either a 3&#8217; C3 spacer or a 2 base pair mismatch at the 3&#8217; end, which prevent extension during amplification. Probes can be designed to match the Watson or Crick template strand; the choice depends on empirical testing to determine which probe design produces acceptable melting peaks. Excess forward or reverse primers are used to produce single-stranded amplification product that anneals to the blocked probes. If the probe contains the forward strand sequence, then reverse primer is used in excess, typically in a 1:5 ratio, and vice-versa for probes that match the reverse strand.
Similarly, HRM works best with sufficient and robust PCR product. PCR reaction optimization requires gradient PCR and agarose gels or Bioanalyzer analysis to determine optimal annealing temperatures. Template concentration can be increased using methods such as pre-amplification, biased multiplexed amplification of all targets using low primer concentration and cycle numbers to increase the template concentration13.
Only a handful of instruments are compatible with MAAB. The thermal properties of the glass capillaries tubes used in these systems facilitate this method. The small interior diameter of the capillaries as well as the rapid heat transfer through glass offer more stringent temperature control than standard PCR plasticware, which has slower transition rates between annealing and denaturation cycles due to the thermal insulating properties of the plastics. With this method, detection sensitivities can be reduced from 2-5% to less than 1% of a mutant allele in a mixture of wild-type and mutant alleles10.
HRM is a facile, efficient, and economical tool for SNP genotyping in a variety of settings and numerous applications, from infectious disease surveillance to scanning for cancer gene variants. Several other instruments, such as the Roche Nano and LightCycler systems (480 and 96) as well as the Eco Real-time PCR system offer HRM in addition to standard amplification, real-time, and copy-number functionality. Using higher-throughput machines, HRM barcoding costs less than $0.50 per assay in our hands, including all reagents and consumables.
In the context described here, field-based applications allow real-time population surveillance for changes associated with malaria control efforts, including reduced population diversity, detection of imported parasite types, and the appearance and spread of SNPs associated with reduced drug sensitivity. Refinements to the method offer additional sensitivity useful for informing progress towards malaria elimination and eradication.

Despite renewed interest in malaria control and eradication, malaria remains a worldwide burden, with nearly half of the world&#8217;s population at risk of infection and more than 550,000 deaths annually, particularly children in sub-Saharan Africa1.
These new control and eradication programs have been supported by the genomic renaissance, with large numbers of malaria parasites sequenced and analyzed for mutations associated with reduced drug sensitivity, increased virulence, and for population characteristics2,3. Single-nucleotide polymorphisms (SNPs) are among the most commonly identified genetic variants 4-7.
Portable SNP genotyping methods offer on-site and real-time population surveillance and tracking8. In addition to fingerprinting individual parasites, the &#8216;molecular barcode&#8217; is also used to detect dramatic temporal shifts in allele frequency as well as variance effective population size and complexity of infection9.
While this set of informative SNPs is easily adapted to many genotyping platforms, high resolution melting (HRM) analysis is particularly well-suited to field-based studies, where sensitive and simple operation and detection of novel mutations at low costs compared to sequencing and other approaches are attractive in resource-poor settings.
HRM starts with standard polymerase chain reaction (PCR) that incorporates a fluorescent dye. Post-PCR melting analysis determines the peak amplicon melting temperature; a single SNP difference in a short amplicon can result in substantial peak melting temperature (Tm) differences.
Several refinements to this method offer better genotyping resolution to differentiate SNPs including class IV (A-T) SNPs, and detect minor mutant alleles in samples with multiple alleles present (polygenomic infections). First, the assays incorporate short probes centered over the SNP region in addition to forward and reverse primers that are present at different concentrations. These blocked probes are not amplified during PCR, but bind to the strand produced in excess due to asymmetric primer concentrations that increase production of the probe-template product. These separate double-stranded amplicons consisting of probe bound to the excess template strand are ~20-30 bp; their significantly decreased length compared to the entire amplicon (80-150 bp) lead to much larger Tm differences associated with single or multiple base probe-template mismatches10.
Second, mutant allele amplification bias (MAAB) lowers the reaction annealing temperature to bias the reaction towards mutant alleles present at low ratios in polygenomic infections. The annealing temperature is set between the Tms of perfectly matched (wild-type) and mismatched (mutant) probes. At this temperature, the binding of the wild-type allele is stable enough that amplification is hindered compared to its mismatch equivalent, thus biasing the amplification towards the mutant allele when both are present in a single sample10.
With these HRM refinements, this technology has allowed tracking of origins and identity of parasites associated with epidemic infections in South America11 and detection of new mutations, differentiation of multiple SNPs located immediately next to each other in sequence, and mutations present in less than 1% of the alleles in polygenomic samples10.
Increased sensitivity is particularly important in regions approaching pre-elimination status and those at risk for emerging drug resistance. The ability to quickly and easily identify imported parasites and SNPs associated with reduced sensitivity on-site informs surveillance efforts and control programs about the effectiveness of their implementations and identifies hotspots for increased malaria eradication and elimination efforts. This protocol describes the methods for blocked-probe-based and MAAB for increased HRM genotyping sensitivity.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>LightScanner Master Mix</td>
			<td>BioFire Defense</td>
			<td>HRLS-ASY-0003</td>
			<td></td>
		</tr>
		<tr>
			<td>Light mineral oil</td>
			<td>Sigma</td>
			<td>M5904</td>
			<td>for BioFire Defense plate-based HRM systems</td>
		</tr>
		<tr>
			<td>TE</td>
			<td>Teknova</td>
			<td>T0226</td>
			<td></td>
		</tr>
		<tr>
			<td>Te</td>
			<td>Teknova</td>
			<td>T0221</td>
			<td>TE buffer with reduced EDTA</td>
		</tr>
		<tr>
			<td>PCR grade water</td>
			<td>Teknova</td>
			<td>W3330</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmodium falciparum standards</td>
			<td>MR4</td>
			<td>MRA-151, 156, 205, 330</td>
			<td>MR4.org offers free genomic DNA</td>
		</tr>
		<tr>
			<td>Light Scanner Primer Design Software</td>
			<td>BioFire Defense</td>
			<td></td>
			<td>commercial sofware for HRM assay design</td>
		</tr>
		<tr>
			<td>LightCycler 480 High Resolution Melting Master Mix</td>
			<td>Roche</td>
			<td>4909631001</td>
			<td>For use in LightCycler-480, -96, and nano. 2x concentration</td>
		</tr>
		<tr>
			<td>Bioanalyzer</td>
			<td>Agilent</td>
			<td>G2938A</td>
			<td>Agilent 2100 bioanalyzer</td>
		</tr>
		<tr>
			<td>LightCycler 2.0</td>
			<td>Roche</td>
			<td>3531414001</td>
			<td>Capillary-based system for MAAB</td>
		</tr>
		<tr>
			<td>LightCycler Nano</td>
			<td>Roche</td>
			<td>6407773001</td>
			<td>Strip tube-based HRM and amplification</td>
		</tr>
		<tr>
			<td>LightCycler 96</td>
			<td>Roche</td>
			<td>5815916001</td>
			<td>Strip-tube and plate-based HRM and amplification</td>
		</tr>
		<tr>
			<td>LightCycler 480</td>
			<td>Roche</td>
			<td>5015278001</td>
			<td>plate-based HRM (96 and 384-well plates) and amplification</td>
		</tr>
		<tr>
			<td>LightScanner-96</td>
			<td>BioFire Defense</td>
			<td>LSCN-ASY-0011</td>
			<td>plate-based HRM (96-well)&#160;</td>
		</tr>
		<tr>
			<td>LightScanner-384</td>
			<td>BioFire Defense</td>
			<td>LSCN-ASY-0001</td>
			<td>plate-based HRM (96-well)</td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Roche</td>
			<td>4729692001</td>
			<td>96-well plates for HRM (LightCycler</td>
		</tr>
		<tr>
			<td>384-well plates</td>
			<td>Roche</td>
			<td>4729749001</td>
			<td>384-well plates for HRM (LightCycler)</td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Bio-Rad</td>
			<td>HSP-9665&#160;</td>
			<td>96-well plates for HRM (LightScanner)</td>
		</tr>
		<tr>
			<td>384-well plates</td>
			<td>Bio-Rad</td>
			<td>HSP-3865&#160;</td>
			<td>384-well plates for HRM (LightScanner)</td>
		</tr>
		<tr>
			<td>LightCycler 8-Tube Strips (clear)</td>
			<td>Roche</td>
			<td>6327672001</td>
			<td>strip tubes for HRM (Light Cycler 96 and Light Cycler Nano)</td>
		</tr>
		<tr>
			<td>LightCycler Capillaries (20 &#956;l)</td>
			<td>Roche</td>
			<td>4929292001</td>
			<td>capillaries for HRM</td>
		</tr>
		<tr>
			<td>Optical plate seals</td>
			<td>Roche</td>
			<td>4729757001</td>
			<td>other brands also work</td>
		</tr>
	</tbody>
</table>

methods to increase the sensitivity of high resolution melting single nucleotide polymorphism genotyping in malaria

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs.
One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures.
SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status.
While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele.
In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs can inform control programs.
This manuscript describes modifications to high resolution melting technology that further increase its sensitivity to identify polygenomic infections in patient samples.

Data can be visualized in several different ways, depending on the analysis software and instrument. Typically, a plot of the negative derivative of normalized fluorescence with respect to temperature (&#8211;dF/dT) against temperature results is most straightforward for visualizing melting peaks and determining genotypes.
A full melting window (40&#176;C&#8211;80&#176;C) will result in both amplicon and probe melting regions. Figure 1 shows an example of normalized melting peaks for both regions. Setting the analysis window to just the probe region results in clear differentiation of peaks corresponding to specific SNPs (Figure 2).
Probe-based analysis clearly shows the presence of both alleles as individual peaks with melting temperatures that match homozygous SNP peaks (Figure 3).
Figure 4 shows that progressively reducing the annealing temperature during amplification (MAAB) results in a bias towards the mutant allele (left-side peak) (Figure 4A), resulting in increased sensitivity for the mutant allele in a population of mixed alleles (Figure 4B).
<img alt="Figure 1" src="/files/ftp_upload/52839/52839fig1.jpg" />
Figure 1.&#160;Probe and amplicon melting regions. Plotting the negative derivative of the normalized fluorescence with respect to temperature over a wide melting window results in both probe (lower temperature) and amplicon melting peaks that can be analyzed. (Adapted from Daniels et al. DOI: 10.1128/AAC.05737-11) <a href="https://www.jove.com/files/ftp_upload/52839/52839fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52839/52839fig2.jpg" /> 
Figure 2.&#160;Probe melting peaks. Perfect matches (typically wild-type alleles) result in higher melting peaks (red), while SNP mismatches have lower melting temperatures (grey). <a href="https://www.jove.com/files/ftp_upload/52839/52839fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52839/52839fig3.jpg" /> 
Figure 3.&#160;Polygenomic melting peaks. When both alleles are present in a sample, probe-based HRM analysis represents both alleles as two-peak curves (orange) with peaks that match single-allele samples (red and grey). (Adapted from Daniels et al. DOI: 10.1128/AAC.05737-11) <a href="https://www.jove.com/files/ftp_upload/52839/52839fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52839/52839fig4.jpg" /> 
Figure 4.&#160;Mutant allele amplification bias (MAAB). A. Progressively lowering the amplification annealing temperature biases the melting peak reaction towards peak corresponding to the mutant (lower temperature) allele in a polygenomic or polyallelic sample. B. MAAB results in HRM sensitivity to detect minor alleles present at less than 1% in polygenomic or polyallelic samples. (Part B adapted from Daniels et al. DOI: 10.1128/AAC.05737-11) <a href="https://www.jove.com/files/ftp_upload/52839/52839fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1.&#160;Set-up of probe-based high resolution melting amplification reactions.
<table>
	<tbody>
		<tr>
			<td>Component</td>
			<td>Volume per reaction (96-well plate and capillary tube)</td>
			<td>Volume per reaction (384-well plate)</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>HRM Master Mix&#160;</td>
			<td>4&#8211;5 &#181;l</td>
			<td>2-2.5 &#181;l</td>
			<td>1 x</td>
		</tr>
		<tr>
			<td>10 x Excess primer</td>
			<td>1 &#181;l</td>
			<td>0.5 &#181;l</td>
			<td>0.50 &#181;M</td>
		</tr>
		<tr>
			<td>10 x Other primer</td>
			<td>1 &#181;l</td>
			<td>0.5&#181;l</td>
			<td>0.1 &#181;M&#160;&#160;</td>
		</tr>
		<tr>
			<td>10x Probe</td>
			<td>1 &#181;l</td>
			<td>0.5 &#181;l</td>
			<td>0.40 &#181;M</td>
		</tr>
		<tr>
			<td>PCR-grade water</td>
			<td>1&#8211;2 &#181;l</td>
			<td>0.5-1 &#181;l</td>
			<td></td>
		</tr>
		<tr>
			<td>Template DNA&#160;</td>
			<td>1 &#181;l</td>
			<td>0.5 &#181;l</td>
			<td>0.01-10 ng/&#181;l</td>
		</tr>
		<tr>
			<td></td>
			<td>10 &#181;l</td>
			<td>5 &#181;l</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2.&#160;Standard high resolution melting amplification conditions. 
<table>
	<tbody>
		<tr>
			<td>Temperature</td>
			<td>Time</td>
		</tr>
		<tr>
			<td>Hold</td>
			<td>95 &#176;C</td>
			<td>120 sec</td>
		</tr>
		<tr>
			<td rowspan="2">45 cycles</td>
			<td>95 &#176;C</td>
			<td>30 sec</td>
		</tr>
		<tr>
			<td>68 &#176;C*</td>
			<td>30 sec</td>
		</tr>
		<tr>
			<td rowspan="2">1 cycle</td>
			<td>95 &#176;C</td>
			<td>30 sec</td>
		</tr>
		<tr>
			<td>28 &#176;C</td>
			<td>30 sec</td>
		</tr>
	</tbody>
</table>
* this temperature is amplicon-dependent
Table 3.&#160;Amplification conditions for mutant allele amplification bias.
<table>
	<tbody>
		<tr>
			<td>Temperature</td>
			<td>Time</td>
			<td>Cycles</td>
			<td>Ramp rate</td>
			<td>Acquisition mode</td>
			<td>Analysis mode</td>
		</tr>
		<tr>
			<td>Denature</td>
			<td>95 &#176;C</td>
			<td>30 sec</td>
			<td>1</td>
			<td>20</td>
			<td>None</td>
			<td>None</td>
		</tr>
		<tr>
			<td rowspan="2">Cycle</td>
			<td>95 &#176;C</td>
			<td>2 sec</td>
			<td rowspan="2">55</td>
			<td>20</td>
			<td>None</td>
			<td rowspan="2">Quantification</td>
		</tr>
		<tr>
			<td>56 &#176;C*</td>
			<td>15 sec</td>
			<td>20</td>
			<td>Single</td>
		</tr>
		<tr>
			<td rowspan="3">Melt</td>
			<td>40 &#176;C</td>
			<td>0 sec</td>
			<td rowspan="3"></td>
			<td rowspan="3">0.3</td>
			<td>None</td>
			<td>None</td>
		</tr>
		<tr>
			<td>45 &#176;C*</td>
			<td>0 sec</td>
			<td rowspan="2">Continous</td>
			<td rowspan="2">Melt</td>
		</tr>
		<tr>
			<td>90 &#176;C*</td>
			<td>0 sec</td>
		</tr>
	</tbody>
</table>
* this temperature is amplicon-dependent and will be reduced when performing MAAB

Watch this Scientific Journal Video about Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria at JoVE.com

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

While high resolution melting analysis offers the ability to differentiate between single nucleotide polymorphisms in a heterogeneous population, mutant allele amplification bias can increase its ability to detect alleles present at relatively low percentages within a sample. This protocol describes improvements that improve the sensitivity of high resolution melting analysis.

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been ...

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Research

JoVE Journal

Immunology and Infection

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3 and humans4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10.

Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in human non-hepatic and murine cell lines, the efficiency was very low and required long-term selection procedures using HCV replicon constructs expressing dominant antibiotic-selectable markers1-5. HCV in vitro research is therefore limited to human hepatoma cell lines permissive for virus entry and completion of the viral life cycle. Due to HCVs narrow species tropism, there is no immunocompetent small animal model available that sustains the complete HCV replication cycle 6-8. Inefficient replication of HCV in non-human cells e.g. of mouse origin is likely due to lack of genetic incompatibility of essential host dependency factors and/or expression of restriction factors.
We investigated whether HCV propagation is suppressed by dominant restriction factors in either human cell lines derived from non-hepatic tissues or in mouse liver cell lines. To this end, we developed two independent conditional trans-complementation methods relying on somatic cell fusion. In both cases, completion of the viral replication cycle is only possible in the heterokaryons. Consequently, successful trans-complementation, which is determined by measuring de novo production of infectious viral progeny, indicates absence of dominant restrictions.
Specifically, subgenomic HCV replicons carrying a luciferase transgene were transfected into highly permissive human hepatoma cells (Huh-7.5 cells). Subsequently, these cells were co-cultured and fused to various human and murine cells expressing HCV structural proteins core, envelope 1 and 2 (E1, E2) and accessory proteins p7 and NS2. Provided that cell fusion was initiated by treatment with polyethylene-glycol (PEG), the culture released infectious viral particles which infected na&iuml;ve cells in a receptor-dependent fashion.
To assess the influence of dominant restrictions on the complete viral life cycle including cell entry, RNA translation, replication and virus assembly, we took advantage of a human liver cell line (Huh-7 Lunet N cells 9) which lacks endogenous expression of CD81, an essential entry factor of HCV. In the absence of ectopically expressed CD81, these cells are essentially refractory to HCV infection 10 . Importantly, when co-cultured and fused with cells that express human CD81 but lack at least another crucial cell entry factor (i.e. SR-BI, CLDN1, OCLN), only the resulting heterokaryons display the complete set of HCV entry factors requisite for infection. Therefore, to analyze if dominant restriction factors suppress completion of the HCV replication cycle, we fused Lunet N cells with various cells from human and mouse origin which fulfill the above mentioned criteria. When co-cultured cells were transfected with a highly fusogenic viral envelope protein mutant of the prototype foamy virus (PFV11) and subsequently challenged with infectious HCV particles (HCVcc), de novo production of infectious virus was observed. This indicates that HCV successfully completed its replication cycle in heterokaryons thus ruling out expression of dominant restriction factors in these cell lines. These novel conditional trans-complementation methods will be useful to screen a large panel of cell lines and primary cells for expression of HCV-specific dominant restriction factors.

We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in ...

2D and 3D Chromosome Painting in Malaria Mosquitoes

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.

Chromosome painting is a useful method for studying organization of the cell nucleus and evolution of the karyotype. Here, we demonstrate an approach to isolate and amplify specific regions of interest from single polytene chromosomes that are subsequently used for two- and three-dimensional fluorescent in situ hybridization (FISH).

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2. H. pylori is the primary cause of gastric cancer, the second leading cause of cancer-related deaths worldwide3. One virulence factor that has been associated with increased risk of gastric disease is the Cag-pathogenicity island, a 40-kb region within the chromosome of H. pylori that encodes a type IV secretion system and the cognate effector molecule, CagA4,5. The Cag-T4SS is responsible for translocating CagA and peptidoglycan into host epithelial cells5,6. The activity of the Cag-T4SS results in numerous changes in host cell biology including upregulation of cytokine expression, activation of proinflammatory pathways, cytoskeletal remodeling, and induction of oncogenic cell-signaling networks5-8. The Cag-T4SS is a macromolecular machine comprised of sub-assembly components spanning the inner and outer membrane and extending outward from the cell into the extracellular space. The extracellular portion of the Cag-T4SS is referred to as the &#8220;pilus&#8221;5. Numerous studies have demonstrated that the Cag-T4SS pili are formed at the host-pathogen interface9,10. However, the environmental features that regulate the biogenesis of this important organelle remain largely obscure. Recently, we reported that conditions of low iron availability increased the Cag-T4SS activity and pilus biogenesis. Here we present an optimized protocol to grow H. pylori in varying conditions of iron availability prior to co-culture with human gastric epithelial cells. Further, we present the comprehensive protocol for visualization of the hyper-piliated phenotype exhibited in iron restricted conditions by high resolution scanning electron microscopy analyses.

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Here we describe a method to visualize the oncogenic bacterial organelle known as the Cag Type IV Secretion System (Cag-T4SS). We find that the Cag-T4SS is differentially produced on the surface of H. pylori in response to varying conditions of iron availability.

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2.  H. pylori is ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism pathway and contributes to increased oxidative stress resistance. Pyomelanin production in Pseudomonas aeruginosa is reduced in a dose dependent manner through treatment with 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). We describe a titration method using multiple concentrations of NTBC to determine the concentration of drug that will reduce or abolish pyomelanin production in bacteria. The titration method has an easily quantifiable outcome, a visible reduction in pigment production with increasing drug concentrations. We also describe a microtiter plate method to assay antibiotic minimum inhibitory concentration (MIC) in bacteria. This method uses a minimum of resources and can easily be scaled up to test multiple antibiotics in one microtiter plate for one strain of bacteria. The MIC assay can be adapted to test the affects of non-antibiotic compounds on bacterial growth at specific concentrations. Finally, we describe a method for testing bacterial sensitivity to oxidative stress by incorporating H2O2 into agar plates and spotting multiple dilutions of bacteria onto the plates. Sensitivity to oxidative stress is indicated by reductions in colony number and size for the different dilutions on plates containing H2O2 compared to a no H2O2 control. The oxidative stress spot plate assay uses a minimum of resources and low concentrations of H2O2. Importantly, it also has good reproducibility. This spot plate assay could be adapted to test bacterial sensitivity to various compounds by incorporating the compounds in agar plates and characterizing the resulting bacterial growth.

Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...