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      Development of a motion-based cell-counting system for Trypanosoma parasite using a pattern recognition approach

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          Abstract

          Automated cell counters that utilize still images of sample cells are widely used. However, they are not well suited to counting slender, aggregate-prone microorganisms such as Trypanosoma cruzi. Here, we developed a motion-based cell-counting system, using an image-recognition method based on a cubic higher-order local auto-correlation feature. The software successfully estimated the cell density of dispersed, aggregated, as well as fluorescent parasites by motion pattern recognition. Loss of parasites activeness due to drug treatment could also be detected as a reduction in apparent cell count, which potentially increases the sensitivity of drug screening assays. Moreover, the motion-based approach enabled estimation of the number of parasites in a co-culture with host mammalian cells, by disregarding the presence of the host cells as a static background.

          Most cited references8

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          CRISPR/Cas9-Induced Disruption of Paraflagellar Rod Protein 1 and 2 Genes in Trypanosoma cruzi Reveals Their Role in Flagellar Attachment

          ABSTRACT Trypanosoma cruzi is the etiologic agent of Chagas disease, and current methods for its genetic manipulation have been highly inefficient. We report here the use of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system for disrupting genes in the parasite by three different strategies. The utility of the method was established by silencing genes encoding the GP72 protein, which is required for flagellar attachment, and paraflagellar rod proteins 1 and 2 (PFR1, PFR2), key components of the parasite flagellum. We used either vectors containing single guide RNA (sgRNA) and Cas9, separately or together, or one vector containing sgRNA and Cas9 plus donor DNA for homologous recombination to rapidly generate mutant cell lines in which the PFR1, PFR2, and GP72 genes have been disrupted. We demonstrate that genome editing of these endogenous genes in T. cruzi is successful without detectable toxicity of Cas9. Our results indicate that PFR1, PFR2, and GP72 contribute to flagellar attachment to the cell body and motility of the parasites. Therefore, CRISPR/Cas9 allows efficient gene disruption in an almost genetically intractable parasite and suggest that this method will improve the functional analyses of its genome.
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            Chagas disease 101.

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              Functional analysis of the intergenic regions of TcP2beta gene loci allowed the construction of an improved Trypanosoma cruzi expression vector.

              TcP2beta ribosomal protein genes in Trypanosoma cruzi are encoded by four different loci, H6.4, H1.8, H1.5 and H1.3. All loci have a similar organization, except for H1.8 that harbors two TcP2beta genes arranged in tandem and separated by a short repetitive sequence, named SIRE (short interspersed repetitive element), which is also found upstream of the first gene of the tandem and downstream of the second. In this locus the trans-splicing signal of TcP2beta is located within the SIRE element, while in the other loci it is positioned within the first 50bases upstream of the AUG with an AG acceptor site at position -12 respective to the initiation codon. Transient transfection experiments were used to evaluate the efficiency of these two different trans-splicing regions to drive CAT activity. The region named HX1 located upstream the TcP2beta H1. 8 gene was clearly more efficient than the SIRE sequence contained in the region named HX2. Therefore, we decided to use the HX1 region to ameliorate the performance of the cryptic trans-splicing signal present in the T. cruzi expression vector pRIBOTEX (Martinez-Calvillo, S., López, I., Hernandez, H., 1997. pRIBOTEX expression vector: a pTEX derivative for a rapid selection of Trypanosoma cruzi transfectants. Gene 199, 71-76). By insertion of the region HX1 downstream of the ribosomal promoter of pRIBOTEX, we constructed pRHX1CAT40 that, in stable transfected cells, was able to drive CAT activity 2760 times more efficiently than the control plasmids. Based on this, a novel plasmid vector was conceived, named pTREX-n, which retains the neo gene of pRIBOTEX as a positive selectable marker and replaces the CAT-SV40 cassette in pRHX1CAT40 by a multiple cloning site.
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                Author and article information

                Journal
                BTN
                BioTechniques
                BioTechniques
                BioTechniques
                Future Science Ltd (London, UK )
                0736-6205
                1940-9818
                April 2019
                13 December 2018
                : 66
                : 4
                : 179-185
                Affiliations
                [1] 1Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan
                [2] 2Artificial Intelligence Research Center, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan
                Author notes
                *Author for correspondence: yuko-takagi@ 123456aist.go.jp
                Article
                10.2144/btn-2018-0163
                31a135af-b202-4617-b30f-dcdb036721bc
                © 2019 Yuko Takagi

                This work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License

                History
                : 29 October 2018
                : 22 November 2018
                : 13 December 2018
                Categories
                Reports

                General life sciences,Cell biology,Molecular biology,Biotechnology,Genetics,Life sciences
                Chagas disease,cell count,image analysis,protozoan parasite,pattern recognition, Trypanosoma cruzi ,microscopy,machine learning

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