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Parasitology International. 66 (3), 236-239 (2017).","journal-title":"Parasitology International"},{"issue":"16","key":"ref27","doi-asserted-by":"crossref","first-page":"12051","DOI":"10.1074\/jbc.275.16.12051","type":"journal-article","article-title":"Amastin mRNA abundance in Trypanosoma cruzi is controlled by a 3\u2019-untranslated region position-dependent cis-element and an untranslated region-binding protein","volume":"275","author":"Coughlin","year":"2000","unstructured":"Coughlin, B.C., Teixeira, S.M., Kirchhoff, L. V, Donelson, J.E. Amastin mRNA abundance in Trypanosoma cruzi is controlled by a 3\u2019-untranslated region position-dependent cis-element and an untranslated region-binding protein. The Journal of Biological Chemistry. 275 (16), 12051-60, at <http:\/\/www.ncbi.nlm.nih.gov\/pubmed\/10766837> (2000).","journal-title":"The Journal of Biological Chemistry"},{"issue":"2","key":"ref28","doi-asserted-by":"crossref","DOI":"10.1128\/mSphere.00051-16","type":"journal-article","article-title":"Mitochondrial Gene Expression Is Responsive to Starvation Stress and Developmental Transition in Trypanosoma cruzi","volume":"1","author":"Shaw","year":"2016","unstructured":"Shaw, A.K., Kalem, M.C., Zimmer, S.L. Mitochondrial Gene Expression Is Responsive to Starvation Stress and Developmental Transition in Trypanosoma cruzi. mSphere. 1 (2), 1-16. (2016).","journal-title":"mSphere"},{"issue":"3","key":"ref29","first-page":"146","type":"journal-article","article-title":"Comparative studies of the isolation of metacyclic trypomastigotes of Trypanosoma cruzi by DEAE ion exchange chromatography","volume":"17","author":"Chao","year":"1984","unstructured":"Chao, D., Dusanic, D.G. 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