We would like to thank Dr. Vesper Fe Marie Ramos for critical reading of the manuscript. This work was supported by the National Center for Research Resources (P20 RR0115635-6, C06 RR17417-01 and G20RR024001) and the National Institute for Neurological Disorders and Stroke (2R01 NS052609).

1. Preparing the Equipment
<ol>
	<li>Use a Misonix 3000 or Misonix 4000 sonicator (Farmingdale, NY) connected to a Thermo Electron Neslab EX-7 water bath (Newington, NH) to keep a constant temperature of 37 &deg;C. Sonicate the samples in 200 &mu;l thin-walled PCR tube strips with domed caps obtained from Thermo Scientific (Waltham, MA).</li>
	<li>A brand new sonicator requires a "break-in" period of continuous operation9. A two-month break-in period consists of a 40-sec sonication burst and 10-minute incubation cycles with the amplitude level set to 10. One should observe erosion throughout the surface of the titanium microplate horn (Figure 1, panel B). The circulating water will look white turbid due to erosion of the titanium microplate horn. Replace this water daily.</li>
	<li>The amplification of PrPSc will vary throughout the microplate horn. The best PrPSc amplification efficiency can be obtained within a radius of 5 cm from the center of the microplate horn (See Figure 1, panel C).</li>
	<li>One round of PMCA consists 144 cycles. Each cycle consists of 5 sec sonication and 10 min incubation. This will roughly entail to 24 hr per round of PMCA.</li>
	<li>The power output of sonication, displayed on the sonicator's control panel, varies with the number of PCR tubes present in the sonicator's rack and the temperature of the circulating water from the water bath. Ensure that the water is equilibrated at 37 &deg;C, and do not fill more than 30% of the sonicator's tube rack (Figure 1, panel C). Spread the PCR tubes through the microplate horn. Have at least one row of empty space between PCR tube strips and allow no more than four tubes connected together per strip (Figure 1, panel C).</li>
	<li>The strength of sonication is critical for a successful prion strain amplification. While sonicating, adjust the amplitude to have a power output ranging between 155 ~ 170 watts. In our laboratory the power level is set to six and seven for the Misonix 3000 and Misonix 4000, respectively.</li>
	<li>Use distilled water in the water bath to avoid accumulating hard water stains. Replace this water weekly. During the incubation and sonication cycles, water condensation will occur inside the microplate horn lid. To avoid condensation, attach a small aquarium heat pad to the top of the sonicator's enclosure box as shown in Figure 1, panel A.</li>
</ol>
2. Preparing the Samples
<ol>
	<li>All procedures involving animals were approved by the Creighton University Institutional Animal Care and Use Committee and comply with the Guide for the Care and Use of Laboratory Animals. Before any tissue collection, the animal must be anesthetized and transcardially perfused using ice-cold phosphate buffered saline solution with 5 mM EDTA at pH 7.4 to remove blood from the brain and avoid the inhibition of PMCA reaction by blood7. Collect animal tissues rapidly using aseptic technique and have dedicated dissection tools for each prion strain. After dissection, place the tissue in a 1.5 ml microcentrifuge tube, flash freeze it on dry ice, and store it at -80 &deg;C until homogenization.</li>
	<li>To prepare the PMCA substrate (PrPC) solution, homogenize an uninfected hamster brain (UN BH) to a 10% weight/volume (w/v) solution with ice cold conversion buffer (see part 3) using a Tenbroeck tissue grinder. While homogenizing, be careful not to get air inside the grinder to avoid excessive foaming of the solution. Clarify the solution by centrifuging at 500 x g for 30 sec and aliquot the supernatant in 1.5 ml microcentrifuge tubes. Store at -80 &deg;C until further use.</li>
	<li>To prepare the PMCA seed (PrPSc) solution, homogenize an infectious material (brain, spleen, lymph nodes, other tissues or contaminated soil) to a 10% w/v solution with ice cold water and aliquot the solution in 0.6 ml microcentrifuge tubes. Store at -80 &deg;C until further use.</li>
</ol>
3. Preparing Conversion Buffer
<ol>
	<li>Weigh 0.5093 g of Triton X-100 in a 50 ml volumetric flask (result is a 1% final concentration of Triton X-100) (Sigma-Aldrich GmbH; Steinheim, Germany).</li>
	<li>Add one Complete Protease Inhibitor Tablet (Roche Diagnostics GmbH; Mannheim, Germany).</li>
	<li>Add 0.0931 g of EDTA (this will result in a 5 mM final concentration of EDTA) (Mallinckrodt Baker Inc.; Phillipsburg, NJ).</li>
	<li>Adjust the pH to 7.4 with 1N NaOH.</li>
	<li>Adjust volume to 50 ml with Dulbecco's Phosphate Buffered Saline (DPBS) (Mediatech Inc.; Manassas, VA).</li>
	<li>Filter the solution through a 0.45 &mu;m nitrocellulose membrane with a vacuum filtration apparatus. The conversion buffer can be stored at 4 &deg;C for no more than one month.</li>
</ol>
4. Amplification of Prion Strains Using PMCA
<ol>
	<li>Dilute the PMCA seed into the PMCA substrate at a ratio of 1:20 (i.e. mix 5 &mu;l of the PMCA seed and 95 &mu;l of the PMCA substrate) into a PCR tube. Remove and store a 15 &mu;l aliquot at -80 &deg;C for unsonicated control; this unsonicated control will be used to quantify the PrPSc amplification via proteinase K (PK) digestion followed by Western blot (WB) analysis. An additional unsonicated control could be generated by placing a second 15 &mu;l aliquot at 37 &deg;C for the duration of the PMCA reaction. A minimum of three repetitions is required for statistical purposes.</li>
	<li>Subject the remainder of the sample (70 &mu;l) to one round of PMCA as depicted on Figure 1, Panel A. Shake tubes every 5 hr to avoid water condensation on the domed cap of the PCR tube.</li>
	<li>The sample's tubes should be spun down and gently vortexed (being careful that the solution do not go into the domed cap) before opening after any procedure to ensure homogeneity and avoid potential contamination.</li>
	<li>For serial PMCA (sPMCA) of short incubation period strains (i.e. HY TME, HaCWD, or 263K), dilute the sonicated material at a ratio of 1:20 by transferring 5 &mu;l of the sonicated sample from round one into 95 &mu;l of fresh uninfected brain homogenate (Figure 2, Panel B).</li>
	<li>For sPMCA of long incubation period strains (i.e. DY TME, 139H, 22CH, 22AH, or ME7H), dilute the sonicated material at a ratio of 1:1 by transferring 50 &mu;l of the sonicated sample from round one into 50 &mu;l of fresh uninfected brain homogenate (Figure 2, Panel B).</li>
	<li>Remove two 15 &mu;l aliquots from the previously diluted sonicated mixture (from steps 4.4 or 4.5) and store one of them at -80 &deg;C and the other at 37 &deg;C (unsonicated control solutions for PMCA round two). Subject the remnants to a PMCA round two.</li>
	<li>This procedure can be repeated indefinitely. In our laboratory, we have performed up to fifteen PMCA rounds, with the strain properties remaining unchanged.</li>
	<li>PK digest the samples. For a typical WB, mix 5 &mu;l of PK 80 &mu;g/ml to 5 &mu;l of sample (this solution will have a final PK concentration of 40 &mu;g/ml) and incubate at 37 &deg;C for one hour. Add 10 &mu;l of gel loading buffer. Boil this solution for 10 min. Let the solution cool down and load 10 &mu;l into the gel.</li>
	<li>To calculate the success of amplification perform a WB analysis on the PK-digested samples to estimate the amount of amplified PrPSc by comparing them to either, the unsonicated controls20,21 Figure 2, Panel C or to known amounts of PK-undigested recombinant PrP.</li>
</ol>
5. Avoiding Cross-contamination
Critical steps have to be taken to minimize cross-contamination and the occurrence of false positives due to de novo formation of protease resistant products.
<ol>
	<li>Use a dedicated set of dissection tools (i.e. forceps, tweezers, scissors) for sample collection for each prion strain.</li>
	<li>Before homogenizing uninfected brains to be used as a PMCA substrate, wipe the pipettes with 1% bleach. Soak the sonicator's PCR tube rack, tissue homogenizers, and PCR tube storage racks with 1% bleach for 10 min and rinse thoroughly with distilled water.</li>
	<li>Cover the work area with a fresh Versi-Dry Lab Soaker every time a new sample is to be homogenized, a new PMCA experiment is to be prepared, or when aliquoting between sPMCA rounds.</li>
	<li>Use new gloves each time new samples are handled (especially when transferring aliquots between sPMCA rounds).</li>
	<li>Use short sonication bursts for each PMCA cycle. Cycles of 5 sec sonication combined with 10 min incubation is sufficient to amplify prions without generating de novo PrPSc 1,20,21,23,24 . As other laboratories have shown, increasing the sonication time, increasing the sonication amplitude, and extending the number of PMCA cycles increases the probability for de novo PrPSc formation2.</li>
</ol>

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+PrP+properties+suggest+the+molecular+basis+of+strain+variation+in+transmissible+mink+encephalopathy.">Distinct PrP properties suggest the molecular basis of strain variation in transmissible mink encephalopathy.</a> J. Virol. 68, 7859-7868 (1994).</li><li>Bessen, R. A., Marsh, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+two+biologically+distinct+strains+of+transmissible+mink+encephalopathy+in+hamsters.">Identification of two biologically distinct strains of transmissible mink encephalopathy in hamsters.</a> J. Gen. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Competition+between+different+scrapie+agents+in+mice.">Competition between different scrapie agents in mice.</a> Nat. New Biol. 237, 244-245 (1972).</li><li>Gonzalez-Romero, D., Barria, M. A., Leon, P., Morales, R., Soto, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+infectious+prions+in+urine.">Detection of infectious prions in urine.</a> FEBS Lett. 582, 3161-3166 (2008).</li><li>Green, K. M., Castilla, J., Seward, T. S., Napier, D. L., Jewell, J. E., Soto, C., Telling, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+high+fidelity+prion+amplification+within+and+across+prion+species+barriers.">Accelerated high fidelity prion amplification within and across prion species barriers.</a> PLoS Pathog. 4, e1000139 (2008).</li><li>Hadlow, W. J., Race, R. E., Kennedy, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+distribution+of+transmissible+mink+encephalopathy+virus+in+mink+inoculated+subcutaneously.">Temporal distribution of transmissible mink encephalopathy virus in mink inoculated subcutaneously.</a> J. Virol. 61, 3235-3240 (1987).</li><li>Kocisko, D. A., Come, J. H., Priola, S. A., Chesebro, B., Raymond, G. J., Lansbury, P. 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No conflicts of interest declared.

Challenges of amplifying infectious prion proteins are the long incubation periods and the expenses of in vivo experiments. The PMCA technique is a cost effective means to amplify infectious prion agents. Several laboratories have confirmed the ability of PMCA to accurately amplify prion strains in vitro 7, 9, 12, 14, 19,24 .
Prion diseases can be transmitted between species. Bessen and Marsh have effectively inoculated hamsters with transmissible mink encephalopat...

<table border="1">
 <tbody>
 <tr>
 <td>Reagent / Equipment</td>
 <td>Manufacturer</td>
 <td>Cat. Number</td>
 </tr>
 <tr>
 <td>Misonix 3000</td>
 <td>Misonix</td>
 <td>S-3000</td>
 </tr>
 <tr>
 <td>Misonix 4000</td>
 <td>Misonix</td>
 <td>S-4000</td>
 </tr>
 <tr>
 <td>Tenbroeck Tissue Grinder</td>
 <td>Kontes</td>
 <td>885000-0007</td>
 </tr>
 <tr>
 <td>Neslab EX-7 Water Bath</td>
 <td>Thermo Electron</td>
 <td>Neslab EX-7</td>
 </tr>
 <tr>
 <td>0.2 ml PCR Tube Strips</td>
 <td>Thermo Scientific</td>
 <td>AB-0451</td>
 </tr>
 <tr>
 <td>Triton X-100</td>
 <td>Sigma Aldrich</td>
 <td>T9284-100ML</td>
 </tr>
 <tr>
 <td>Complete Protease Inhibitor</td>
 <td>Roche</td>
 <td>11 697 498 001</td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>J.T. Baker</td>
 <td>4040-00</td>
 </tr>
 <tr>
 <td>DPBS</td>
 <td>Mallinckrodt Baker Mediatech</td>
 <td>21-031-CV</td>
 </tr>
 <tr>
 <td>Versi-Dry Lab Soakers</td>
 <td>Fisher Scientific</td>
 <td>14 206 28</td>
 </tr>
 <tr>
 <td>Repti Therm Heater</td>
 <td>Zoo Med Laboratories, Inc.</td>
 <td>RH-4</td>
 </tr>
 </tbody>
</table>

protein misfolding cyclic amplification of prions

Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans9,18. The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrPC) and the infectious protein (PrPSc), an abnormally-folded isoform of PrPC 8.
One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation13. This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrPSc are poorly characterized due to their unusual conformation and aggregation states.
PrPSc can seed the conversion of PrPC to PrPSc in vitro14. PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrPSc, at an accelerated rate, from a system containing excess amounts of PrPC and minute amounts of the PrPSc seed19. This technique has proven to effectively recapitulate the species and strain specificity of PrPSc conversion from PrPC, to emulate prion strain interference, and to amplify very low levels of PrPSc from infected tissues, fluids, and environmental samples6,7,16,23 .
This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.

Protein misfolding cyclic amplification (PMCA) is used to amplify PrPSc in vitro7, 12, 14, 19, 24. A successful PrPSc amplification is shown by an increase in band intensity on Western blots of the PK-resistant prion protein (migrating between 19 and 30 kDa for hamster-derived prion strains) as shown in Figure 3. The increase in its band intensity after PMCA indicates amplification of the PK-resistant PrPSc material. Successful amplification of the ham...

Watch this Scientific Journal Video about Protein Misfolding Cyclic Amplification of Prions at JoVE.com

Protein Misfolding Cyclic Amplification of Prions

Protein misfolding cyclic amplification (PMCA) is an in vitro assay for the study of prion conversion and strain and species barriers. It can also be used as a prion detection assay.

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

protein-misfolding-cyclic-amplification-of-prions

Research

JoVE Journal

Immunology and Infection

19.4K ビュー.  University of Nebraska at Lincoln. タンパク質のミスフォールディングサイクリック増幅（PMCA）はプリオン変換とひずみと種の壁の研究のためのin vitroアッセイである。また、プリオン検出アッセイとして使用することができます。

ビデオ: Protein Misfolding Cyclic Amplification of Prions

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant prion protein (PrP) substrate using prion seeds as a template for the conversion. RT-QuIC is a novel high-throughput technique which is analogous to real-time polymerase chain reaction (PCR). Detection of amyloid fibril growth is based on the dye Thioflavin T, which fluoresces upon specific interaction with &#7526;-sheet rich proteins. Thus, amyloid formation can be detected in real time. We attempted to develop a reliable non-invasive screening test to detect chronic wasting disease (CWD) prions in fecal extract. Here, we have specifically adapted the RT-QuIC technique to reveal PrPSc seeding activity in feces of CWD infected cervids. Initially, the seeding activity of the fecal extracts we prepared was relatively low in RT-QuIC, possibly due to potential assay inhibitors in the fecal material. To improve seeding activity of feces extracts and remove potential assay inhibitors, we homogenized the fecal samples in a buffer containing detergents and protease inhibitors. We also submitted the samples to different methodologies to concentrate PrPSc on the basis of protein precipitation using sodium phosphotungstic acid, and centrifugal force. Finally, the feces extracts were tested by optimized RT-QuIC which included substrate replacement in the protocol to improve the sensitivity of detection. Thus, we established a protocol for sensitive detection of CWD prion seeding activity in feces of pre-clinical and clinical cervids by RT-QuIC, which can be a practical tool for non-invasive CWD diagnosis.

Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC) method.

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant ...

Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast cancers. However, a minority of cases displays a heterogeneous distribution of HER2-positive cells, which generates major clinical challenges. To date, no reliable and standardized protocols for the characterization and quantification of HER2 heterogeneous gene amplification in large cohorts have been proposed. Here, we present a high-throughput methodology to simultaneously assess the HER2 status across different topographic areas of multiple breast cancers. In particular, we illustrate the laboratory procedure to construct enhanced tissue microarrays (TMAs) incorporating a targeted mapping of the tumors. All TMA parameters have been specifically optimized for the silver in situ hybridization (SISH) of formalin-fixed paraffin-embedded (FFPE) breast tissues. Immunohistochemical analysis of the prognostic and predictive biomarkers (i.e., ER, PR, Ki67, and HER2) should be performed using automated procedures. A customized SISH protocol has been implemented to allow a high-quality molecular analysis across multiple tissues that underwent different fixation, processing, and storage procedures. In this study, we provide a proof-of-principle that specific DNA sequences could be localized simultaneously in distinct topographic areas of multiple and heterogeneously processed breast cancers using an efficient and cost-effective method.

Heterogeneous distribution of HER2-positive cells can be observed in a subset of breast cancers and generates clinical dilemmas. Here, we introduce a reliable and cost-effective protocol to define, quantify, and compare HER2 intra-tumor genetic heterogeneity in a large series of heterogeneously processed breast cancers.

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Specific and Rapid Detection of Tilapia Lake Virus

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the aquaculture industry that has resulted in the mass morbidity and mortality of tilapia in many parts of the world. An effective, rapid, and accurate diagnostic assay for TiLV infection is therefore necessary to detect the initial infection and to prevent the spread of the disease in aquaculture farming. In this study, a highly sensitive and practical reverse transcription loop-mediated isothermal amplification (RT-LAMP) method is presented to detect tilapia lake virus in fish tissue. A comparison of the RT-qPCR and RT-LAMP assays of infected samples revealed positive results in 63 (100%) and 51 (80.95%) samples, respectively. Moreover, an analysis of uninfected samples showed that all 63 uninfected tissues yielded negative results for both the RT-qPCR and RT-LAMP assays. The cross-reactivity with five pathogens in tilapia was evaluated using RT-LAMP, and all the tests showed negative results. Both the liver and mucus samples obtained from infected fish showed comparable results using the RT-LAMP method, suggesting that mucus can be used in RT-LAMP as a nonlethal assay to avoid killing fish. In conclusion, the results demonstrated that the presented RT-LAMP assay provides an effective method for TiLV detection in tilapia tissue within 1 h. The method is therefore recommended as a screening tool on farms for the rapid diagnosis of TiLV.

Reverse Transcription Loop-Mediated Isothermal Amplification RT-LAMP Assay for the Specific and Rapid Detection of Tilapia Lake Virus

We present an RT-LAMP assay for the detection of TiLV in tilapia fish using simple instruments over a relatively short period of time compared to conventional RT-PCR techniques. This protocol may help control the epidemic spread of TiLVD, especially in developing countries.