Name: in vitro drug screening against all life cycle stages of trypanosoma cruzi using parasites expressing &#946;-galactosidase
Uploaded: 2021-11-05T13:00:00
Description: Watch this Scientific Journal Video about In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing ÃŽÂ²-galactosidase at JoVE.com

We thank Dr. Buckner for kindly providing the pLacZ plasmid. This work was supported by Agencia Nacional de Promoci&#243;n Cient&#237;fica y Tecnol&#243;gica, Ministerio de Ciencia e Innovaci&#243;n Productiva from Argentina (PICT2016-0439, PICT2019-0526, PICT2019-4212), and Research Council United Kingdom [MR/P027989/1]. Servier Medical Art was used to produce Figure 1 (https://smart.servier.com).

NOTE: An overview of the entire experimental design is depicted in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63210/63210fig01.jpg" /> 
Figure 1: Overview of the in vitro screening assay of Trypanosoma cruzi Dm28c/pLacZ line using CPRG as a substrate for the colorimetric reaction. The assay consists of seeding the parasites (1), incubati...

<ol>
	<li>Rassi, A., Rassi, A., Rassi, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predictors+of+mortality+in+chronic+Chagas+disease:+a+systematic+review+of+observational+studies.">Predictors of mortality in chronic Chagas disease: a systematic review of observational studies.</a> Circulation. 115 (9), 1101-1108 (2007).</li><li>P&#233;rez-Molina, J. A., Molina, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chagas+disease.">Chagas disease.</a> The Lancet. 391 (10115), 82-94 (2018).</li><li>Messenger, L. A., Miles, M. A., Bern, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Between+a+bug+and+a+hard+place:+Trypanosoma+cruzi+genetic+diversity+and+the+clinical+outcomes+of+Chagas+disease.">Between a bug and a hard place: Trypanosoma cruzi genetic diversity and the clinical outcomes of Chagas disease.</a> Expert Review of Anti-infective Therapy. 13 (8), 995-1029 (2015).</li><li>Steverding, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+history+of+Chagas+disease.">The history of Chagas disease.</a> Parasites &amp; Vectors. 7, 317 (2014).</li><li>Viotti, 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=Towards+a+paradigm+shift+in+the+treatment+of+chronic+Chagas+disease.">Towards a paradigm shift in the treatment of chronic Chagas disease.</a> Antimicrobial Agents and Chemotherapy. 58 (2), 635-639 (2014).</li><li>Bern, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chagas’+Disease.">Chagas’ Disease.</a> The New England Journal of Medicine. 373 (19), 1882 (2015).</li><li>Bustamante, J. M., Tarleton, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodological+advances+in+drug+discovery+for+Chagas+disease.">Methodological advances in drug discovery for Chagas disease.</a> Expert Opinion on Drug Discovery. 6 (6), 653-661 (2011).</li><li>Buckner, F. S., Verlinde, C. L., La Flamme, A. C., Van Voorhis, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+technique+for+screening+drugs+for+activity+against+Trypanosoma+cruzi+using+parasites+expressing+beta-galactosidase.">Efficient technique for screening drugs for activity against Trypanosoma cruzi using parasites expressing beta-galactosidase.</a> Antimicrobial Agents and Chemotherapy. 40 (11), 2592-2597 (1996).</li><li>Vega, C., Rol&#243;n, M., Mart&#237;nez-Fern&#225;ndez, A. R., Escario, J. A., G&#243;mez-Barrio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+pharmacological+screening+assay+with+Trypanosoma+cruzi+epimastigotes+expressing+beta-galactosidase.">A new pharmacological screening assay with Trypanosoma cruzi epimastigotes expressing beta-galactosidase.</a> Parasitology Research. 95 (4), 296-298 (2005).</li><li>Bettiol, 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=Identification+of+three+classes+of+heteroaromatic+compounds+with+activity+against+intracellular+Trypanosoma+cruzi+by+chemical+library+screening.">Identification of three classes of heteroaromatic compounds with activity against intracellular Trypanosoma cruzi by chemical library screening.</a> PLoS Neglected Tropical Diseases. 3 (2), 384 (2009).</li><li>Gulin, J. E. 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=Optimization+and+biological+validation+of+an+in+vitro+assay+using+the+transfected+Dm28c/pLacZ+Trypanosoma+cruzi+strain.">Optimization and biological validation of an in vitro assay using the transfected Dm28c/pLacZ Trypanosoma cruzi strain.</a> Biology Methods and Protocols. 6 (1), 004 (2021).</li><li>da Silva Santos, A. C., Moura, D. M. N., Dos Santos, T. A. R., de Melo Neto, O. P., Pereira, V. R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+Leishmania+cell+lines+expressing+high+levels+of+beta-galactosidase+as+alternative+tools+for+the+evaluation+of+anti-leishmanial+drug+activity.">Assessment of Leishmania cell lines expressing high levels of beta-galactosidase as alternative tools for the evaluation of anti-leishmanial drug activity.</a> Journal of Microbiological Methods. 166, 105732 (2019).</li><li>McFadden, D. C., Seeber, F., Boothroyd, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Toxoplasma+gondii+expressing+beta-galactosidase+for+colorimetric+assessment+of+drug+activity+in+vitro.">Use of Toxoplasma gondii expressing beta-galactosidase for colorimetric assessment of drug activity in vitro.</a> Antimicrobial Agents and Chemotherapy. 41 (9), 1849-1853 (1997).</li><li>Moreno-Viguri, 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=In+vitro+and+in+vivo+anti-Trypanosoma+cruzi+activity+of+new+arylamine+Mannich+base-type+derivatives.">In vitro and in vivo anti-Trypanosoma cruzi activity of new arylamine Mannich base-type derivatives.</a> Journal of Medicinal Chemistry. 59 (24), 10929-10945 (2016).</li><li>Garc&#237;a, P., Alonso, V. L., Serra, E., Escalante, A. M., Furlan, R. L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+a+biologically+active+bromodomain+inhibitor+by+target-directed+dynamic+combinatorial+chemistry.">Discovery of a biologically active bromodomain inhibitor by target-directed dynamic combinatorial chemistry.</a> ACS Medicinal Chemistry Letters. 9 (10), 1002-1006 (2018).</li><li>Vela, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+susceptibility+of+Trypanosoma+cruzi+discrete+typing+units+(DTUs)+to+benznidazole:+A+systematic+review+and+meta-analysis.">In vitro susceptibility of Trypanosoma cruzi discrete typing units (DTUs) to benznidazole: A systematic review and meta-analysis.</a> PLoS Neglected Tropical Diseases. 15 (3), 0009269 (2021).</li><li>Alonso-Padilla, J., Rodr&#237;guez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+screening+for+anti-Trypanosoma+cruzi+drug+discovery.">High throughput screening for anti-Trypanosoma cruzi drug discovery.</a> PLoS Neglected Tropical Diseases. 8 (12), 3259 (2014).</li><li>Martinez-Peinado, 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=Amaryllidaceae+alkaloids+with+anti-Trypanosoma+cruzi+activity.">Amaryllidaceae alkaloids with anti-Trypanosoma cruzi activity.</a> Parasites &amp; Vectors. 13 (1), 299 (2020).</li><li>Puente, V., Demaria, A., Frank, F. M., Batlle, A., Lombardo, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-parasitic+effect+of+vitamin+C+alone+and+in+combination+with+benznidazole+against+Trypanosoma+cruzi.">Anti-parasitic effect of vitamin C alone and in combination with benznidazole against Trypanosoma cruzi.</a> PLoS Neglected Tropical Diseases. 12 (9), 0006764 (2018).</li><li>Muelas-Serrano, S., Nogal-Ruiz, J. J., G&#243;mez-Barrio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Setting+of+a+colorimetric+method+to+determine+the+viability+of+Trypanosoma+cruzi+epimastigotes.">Setting of a colorimetric method to determine the viability of Trypanosoma cruzi epimastigotes.</a> Parasitology Research. 86 (12), 999-1002 (2000).</li></ol>

This paper describes an assay based on determining the cytoplasmic &#946;-galactosidase activity released due to membrane lysis of T. cruzi epimastigotes, trypomastigotes, or infected cells with amastigotes in the presence of the substrate CPRG. We used T. cruzi Dm28c/pLacZ parasites, a stable parasite strain obtained after transfection with a &#946;-galactosidase-bearing plasmid constructed by Buckner and co-authors10. This assay has been used to search for antitrypanocidal comp...

Chagas disease (ChD), or American trypanosomiasis, is a parasitic disease caused by the flagellated protozoan, Trypanosoma cruzi (T. cruzi). ChD begins with an asymptomatic or oligosymptomatic acute phase that is usually undiagnosed, followed by a lifelong chronic phase. In the chronicity, ~30% of patients manifest-decades after the infection-a variety of debilitating conditions, including myocardiopathy, mega-digestive syndromes, or both, with a mortality rate ranging from 0.2% to 20%1,2,3. Asymptomatic chronic patients may have no clinical signs but remain seropositive throughout their life.
Estimations suggest that ~7 million people are infected worldwide, mostly from Latin America, where ChD is endemic. In these countries, T. cruzi is mainly transmitted through infected blood-sucking triatomine bugs (vector-borne transmission) and less frequently by oral transmission through the ingestion of food contaminated with triatomine feces containing the parasites2. Additionally, the parasite can be transmitted via the placenta from chagasic mothers to newborns, through blood transfusions, or during organ transplantation. These vector-independent ways of acquiring the infection and human migration have contributed to the worldwide spread of the disease, evidenced by an increasing number of cases in North America, Europe, and some African, Eastern Mediterranean, and Western Pacific countries4. ChD is considered a neglected disease as vector-borne transmission is closely associated with poverty and is a leading public health issue, especially in Latin American low-income countries. Although there are available treatments, mortality due to ChD in Latin America is the highest among parasitic diseases, including malaria2.
There are two registered drugs for ChD treatment introduced in the late 1960s and early 1970s: nifurtimox and benznidazole5. Both drugs are effective in the acute phase of the disease in adults, children, and congenitally infected newborns, as well as in children with chronic infection, where cure is usually achieved. However, only a few people are diagnosed early enough to be treated in time. According to the latest clinical trials, both drugs have important limitations in adults and were ineffective in reducing symptoms in people with chronic disease; hence, their use in this stage is controversial. Other drawbacks are the prolonged treatment periods required (60-90 days) and the frequent, severe adverse effects observed, which lead to discontinuation of therapy in a proportion of infected people6,7. It is estimated that fewer than 10% of the people with ChD have been diagnosed, and even fewer have access to treatment, as many affected individuals live in rural areas with no or scarce access to healthcare8. These facts highlight the urgent need to find new drugs against T. cruzi to allow for more efficient, safe, and applicable-to-the-field treatments, especially for the chronic phase. In this regard, another challenge in the development of more efficacious compounds is the limitation of systems for assessing drug efficacy in vitro and in vivo9.
Although chemical biology and genomic approaches for the identification of potential drug targets have been used in kinetoplastid parasites, the available genomic tools in T. cruzi are limited in contrast to T. brucei or Leishmania. Thus, the screening of compounds with trypanocidal activity is still the most used approach in the search for new chemotherapeutic drug candidates against ChD. Usually, drug discovery in T. cruzi must start with testing the effects of a new drug in an in vitro assay against the epimastigote stage. For decades, the only way for measuring the inhibitory effects of candidate compounds on T. cruzi was manual microscopic counting, which is laborious, time-consuming, and operator-dependent. Moreover, this approach is suitable for assaying a small number of compounds but is unacceptable for high-throughput screening of large compound libraries. Nowadays, many investigations begin with the analysis of a vast number of compounds from different origins that are assayed in vitro, testing their capacity for inhibiting parasite growth. Both colorimetric and fluorometric methods have been developed to increase throughput in these assays, improving the objectivity of the screening and making the whole process less tedious9.
One of the most widely used colorimetric methods is based on the &#946;-galactosidase activity of transfected parasites first described by Bucknet and collaborators10. The &#946;-galactosidase enzyme expressed by the recombinant parasites hydrolyzes the chromogenic substrate, chlorophenol red &#946;-D-galactopyranoside (CPRG), to chlorophenol red, which can be easily measured colorimetrically using a microplate spectrophotometer. Thus, parasite growth in the presence of a variety of compounds can be simultaneously evaluated and quantitated in microtiter plates. This method has been applied to test drugs in epimastigote forms (present in the insect vector), trypomastigotes, and intracellular amastigotes, the mammalian stages of the parasite. Further, several recombinant T. cruzi strains transfected with the pBS:CL-Neo-01/BC-X-10 plasmid (pLacZ)10 to express the Escherichia coli &#946;-galactosidase enzyme are already available (and new ones can be constructed), which allows the evaluation of parasites from different discrete typing units (DTUs) that may not behave equally toward the same compounds10,11,12,13. This method has already been successfully used to evaluate compounds for activity against T. cruzi in low- and high-throughput screening12,13. Similar approaches have also been used in other protozoan parasites, including Toxoplasma gondii and Leishmania mexicana14,15.
This paper describes and shows a detailed method for an in vitro drug screening against all life cycle stages of T. cruzi using parasites expressing &#946;-galactosidase. The assays presented here have been performed with a &#946;-galactosidase-expressing T. cruzi line obtained by transfection of T. cruzi Dm28c strain from DTU I13 with pLacZ plasmid (Dm28c/pLacZ). Additionally, the same protocol could be easily adapted to other strains to compare the performance between compounds and between T. cruzi strains or DTUs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 L beaker</td>
			<td>Schott Duran</td>
			<td>10005227</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL serological pipette sterile</td>
			<td>Jet Biofil</td>
			<td>GSP211010</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL serological pipette sterile</td>
			<td>Jet Biofil</td>
			<td>GSP010005</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Corning</td>
			<td>3599</td>
			<td></td>
		</tr>
		<tr>
			<td>Benznidazole</td>
			<td>Sigma Aldrich</td>
			<td>419656</td>
			<td>N-Benzyl-2-nitro-1H-imidazole-1-acetamide</td>
		</tr>
		<tr>
			<td>Biosafty Cabinet</td>
			<td>Telstar</td>
			<td>Bio II A/P</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tube 15 mL conical bottom sterile</td>
			<td>Tarson</td>
			<td>546021</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tube 50 mL conical bottom sterile</td>
			<td>Tarson</td>
			<td>546041</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Sanyo</td>
			<td>MCO-15A</td>
			<td></td>
		</tr>
		<tr>
			<td>CPRG</td>
			<td>Roche</td>
			<td>10 884308001</td>
			<td>Chlorophenol Red-&#946;-D-galactopyranoside</td>
		</tr>
		<tr>
			<td>DMEM, High Glucose</td>
			<td>Thermo Fisher Cientific</td>
			<td>12100046</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sintorgan</td>
			<td>SIN-061</td>
			<td>Dimethylsulfoxid</td>
		</tr>
		<tr>
			<td>Fetal Calf Serum</td>
			<td>Internegocios SA</td>
			<td>FCS FRA 500</td>
			<td>Sterile and heat-inactivated</td>
		</tr>
		<tr>
			<td>G418 disulphate salt solution</td>
			<td>Roche</td>
			<td>G418-RO</td>
			<td>stock concentration: 50 mg/mL</td>
		</tr>
		<tr>
			<td>Glucose D(+)</td>
			<td>Cicarelli</td>
			<td>716214</td>
			<td></td>
		</tr>
		<tr>
			<td>Graduated cylinder</td>
			<td>Nalgene</td>
			<td>3663-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemin</td>
			<td>Frontier Scientific</td>
			<td>H651-9</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Cicarelli</td>
			<td>867212</td>
			<td></td>
		</tr>
		<tr>
			<td>Liver Infusion</td>
			<td>Difco</td>
			<td>226920</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tube 1.5 mL</td>
			<td>Tarson</td>
			<td>500010-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate Spectrophotometer</td>
			<td>Biotek</td>
			<td>Synergy HTX</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Cicarelli</td>
			<td>834214</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Cicarelli</td>
			<td>750214</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer chamber</td>
			<td>Boeco</td>
			<td>BOE 01</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonidet P-40</td>
			<td>Antrace</td>
			<td>NIDP40</td>
			<td>2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol</td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>Graphpad</td>
			<td></td>
			<td>Statistical Analysis software</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Cicarelli</td>
			<td>929211</td>
			<td>NaHCO3</td>
		</tr>
		<tr>
			<td>Sorvall ST 16 Centrifuge</td>
			<td>Thermo Fisher Cientific</td>
			<td>75004380</td>
			<td></td>
		</tr>
		<tr>
			<td>T-25 flasks</td>
			<td>Corning</td>
			<td>430639</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptose</td>
			<td>Merck</td>
			<td>1106760500</td>
			<td></td>
		</tr>
		<tr>
			<td>Vero cells</td>
			<td>ATCC</td>
			<td>CRL-1587</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro drug screening against all life cycle stages of trypanosoma cruzi using parasites expressing &#946;-galactosidase

Trypanosoma cruzi is the causative agent of Chagas disease (ChD), an endemic disease of public health importance in Latin America that also affects many non-endemic countries due to the increase in migration. This disease affects nearly 8 million people, with new cases estimated at 50,000 per year. In the 1960s and 70s, two drugs for ChD treatment were introduced: nifurtimox and benznidazole (BZN). Both are effective in newborns and during the acute phase of the disease but not in the chronic phase, and their use is associated with important side effects. These facts underscore the urgent need to intensify the search for new drugs against T. cruzi.
T. cruzi is transmitted through hematophagous insect vectors of the Reduviidae and Hemiptera families. Once in the mammalian host, it multiplies intracellularly as the non-flagellated amastigote form and differentiates into the trypomastigote, the bloodstream non-replicative infective form. Inside the insect vector, trypomastigotes transform into the epimastigote stage and multiply through binary fission.
This paper describes an assay based on measuring the activity of the cytoplasmic &#946;-galactosidase released into the culture due to parasites lysis by using the substrate, chlorophenol red &#946;-D-galactopyranoside (CPRG). For this, the T. cruzi Dm28c strain was transfected with a &#946;-galactosidase-overexpressing plasmid and used for in vitro pharmacological screening in epimastigote, trypomastigote, and amastigote stages. This paper also describes how to measure the enzymatic activity in cultured epimastigotes, infected Vero cells with amastigotes, and trypomastigotes released from the cultured cells using the reference drug, benznidazole, as an example. This colorimetric assay is easily performed and can be scaled to a high-throughput format and applied to other T. cruzi strains.

Following the protocol described above, &#946;-galactosidase-expressing Dm28c epimastigotes were incubated with 6 concentrations of BZN (2.5, 5, 10, 20, 40, 80 &#181;M) (or compounds of interest) for 72 h. After this period, CPRG reagent was added along with detergent, which lyses the cells and releases &#946;-galactosidase. CPRG is cleaved by the &#946;-galactosidase to produce chlorophenol red, leading to a change in color from yellow to reddish (Figure 2A). Chlorophenol red was measured b...

Watch this Scientific Journal Video about In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing ÃŽÂ²-galactosidase at JoVE.com

In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing ÃƒÅ½Ã‚Â²-galactosidase

We describe a high-throughput colorimetric assay measuring &#946;-galactosidase activity in three life cycle stages of Trypanosoma cruzi, the causative agent of Chagas disease. This assay can be used to identify trypanocidal compounds in an easy, fast, and reproducible manner.

In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing &#946;-galactosidase

Trypanosoma cruzi is the causative agent of Chagas disease (ChD), an endemic disease of public health importance in Latin America that also affects many ...

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Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave ...

Screening for Phytoestrogens using a Cell-based Estrogen Receptor  &#946; Reporter Assay

Screening for Phytoestrogens using a Cell-based Estrogen Receptor  ÃƒÅ½Ã‚Â² Reporter Assay

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in ...

Derivatization of Protein Crystals with I3C using Random Microseed Matrix Screening

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction ...