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Nearly all expression vectors currently available for Trypanosoma cruzi were conceived to produce a single primary transcript containing the genes of interest along with those that confer antibiotic resistance. However, since each messenger RNA (mRNA) matures separately, drug selection will only guarantee the expression of those derived from the selectable marker. Therefore, commonly a considerable fraction of the cells recovered after selection with these expression vectors, although resistant do not express the protein of interest. Consequently, in order to counteract this disadvantage, we developed vectors with an alternative arrangement in which the gene of interest and antibiotic resistance are fused sharing the same mRNA. To test this configuration, we included the coding sequence for the green fluorescent protein (mEGFP) linked to the one conferring neomycin resistance (Neo). Additionally, to allow for the production of two independent proteins the sequence for a Thosea asigna virus self-cleaving 2A peptide (T2A) was inserted in-between. Cells obtained with these vectors displayed higher mEGFP expression levels with more homogeneous transgenic parasite populations than those transfected with more conventional independent mRNA-based alternatives. Moreover, as determined by Western blot, 2A mediated fusion protein dissociation occurred with high efficiency in all parasite stages. In addition, these vectors could easily be transformed into endogenous tagging constructs that allowed the insertion, by ends-in homologous recombination, of a hemagglutinin tag (HA) fused to the actin gene. The use of 2A self-cleaving peptides in the context of single mRNA vectors represents an interesting strategy capable of improving ectopic transgene expression in T. cruzi as well as providing a simple alternative to more sophisticated methods, such as the one based on CRISPR/Cas9, for the endogenous labeling of genes., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Background: TolT was originally described as a Trypanosoma cruzi molecule that accumulated on the trypomastigote flagellum bearing similarity to bacterial TolA colicins receptors. Preliminary biochemical studies indicated that TolT resolved in SDS-PAGE as ~3-5 different bands with sizes between 34 and 45 kDa, and that this heterogeneity could be ascribed to differences in polypeptide glycosylation. However, the recurrent identification of TolT-deduced peptides, and variations thereof, in trypomastigote proteomic surveys suggested an intrinsic TolT complexity, and prompted us to undertake a thorough reassessment of this antigen., Methods/principle Findings: Genome mining exercises showed that TolT constitutes a larger-than-expected family of genes, with at least 12 polymorphic members in the T. cruzi CL Brener reference strain and homologs in different trypanosomes. According to structural features, TolT deduced proteins could be split into three robust groups, termed TolT-A, TolT-B, and TolT-C, all of them showing marginal sequence similarity to bacterial TolA proteins and canonical signatures of surface localization/membrane association, most of which were herein experimentally validated. Further biochemical and microscopy-based characterizations indicated that this grouping may have a functional correlate, as TolT-A, TolT-B and TolT-C molecules showed differences in their expression profile, sub-cellular distribution, post-translational modification(s) and antigenic structure. We finally used a recently developed fluorescence magnetic beads immunoassay to validate a recombinant protein spanning the central and mature region of a TolT-B deduced molecule for Chagas disease serodiagnosis., Conclusion/significance: This study unveiled an unexpected genetic and biochemical complexity within the TolT family, which could be exploited for the development of novel T. cruzi biomarkers with diagnostic/therapeutic applications., Competing Interests: The authors have declared that no competing interests exist.

Trypanosoma cruzi, the protozoan agent of Chagas disease, has evolved an innovative metabolic pathway by which protective sialic acid (SA) residues are scavenged from host sialylglycoconjugates and transferred onto parasite surface mucin-like molecules (or surface glycoconjugates from host target cells) by means of a unique trans-sialidase (TS) enzyme. TS-induced changes in the glycoprotein sialylation profile of both parasite and host cells are crucial for the establishment of a persistent T. cruzi infection and for the development of Chagas disease-associated pathogenesis. In this chapter, we describe a novel metabolic labeling method developed in our labs that enables straightforward identification and molecular characterization of SA acceptors of the TS-catalyzed reaction.

Trypanosoma cruzi, the flagellate protozoan agent of Chagas disease or American trypanosomiasis, is unable to synthesize sialic acids de novo. Mucins and trans-sialidase (TS) are substrate and enzyme, respectively, of the glycobiological system that scavenges sialic acid from the host in a crucial interplay for T. cruzi life cycle. The acquisition of the sialyl residue allows the parasite to avoid lysis by serum factors and to interact with the host cell. A major drawback to studying the sialylation kinetics and turnover of the trypomastigote glycoconjugates is the difficulty to identify and follow the recently acquired sialyl residues. To tackle this issue, we followed an unnatural sugar approach as bioorthogonal chemical reporters, where the use of azidosialyl residues allowed identifying the acquired sugar. Advanced microscopy techniques, together with biochemical methods, were used to study the trypomastigote membrane from its glycobiological perspective. Main sialyl acceptors were identified as mucins by biochemical procedures and protein markers. Together with determining their shedding and turnover rates, we also report that several membrane proteins, including TS and its substrates, both glycosylphosphatidylinositol-anchored proteins, are separately distributed on parasite surface and contained in different and highly stable membrane microdomains. Notably, labeling for α(1,3)Galactosyl residues only partially colocalize with sialylated mucins, indicating that two species of glycosylated mucins do exist, which are segregated at the parasite surface. Moreover, sialylated mucins were included in lipid-raft-domains, whereas TS molecules are not. The location of the surface-anchored TS resulted too far off as to be capable to sialylate mucins, a role played by the shed TS instead. Phosphatidylinositol-phospholipase-C activity is actually not present in trypomastigotes. Therefore, shedding of TS occurs via microvesicles instead of as a fully soluble form.

Background: A study was carried out to elucidate the anatomical and functional outcome after surgical excision of subfoveal choroidal neovascular membranes in high myopia., Methods: Sixty-five patients with high myopia (> or =6 diopters), well-defined subfoveal neovascular membranes on fluorescein angiography and preoperative visual acuity < or =20/100 were selected for surgery. A standardized surgical technique was used in all cases, by a single surgeon. The main outcomes assessed were Snellen visual acuity, surgical retinal pigment epithelium defect and postoperative perfusion of the choriocapillaris. Multifactor analysis of variance and chi-square/Fisher's exact test statistics were used to assess the association between patients' pre- and postoperative characteristics and outcome measures., Results: Follow-up ranged from 6 to 48 months (mean 16 months). Mean postoperative visual acuity (0.18) was significantly better than mean preoperative visual acuity (0.09). Visual acuity improved by at least two lines in 29 eyes (45%) and was unchanged in 24 (37%). Overall, 43 eyes (66%) had visual acuity of 20/200 or better and 15 (23%), 20/60 or better. Predictive factors with a significant effect on final visual acuity were mean visual acuity, preoperative status of retinal pigment epithelium and postoperative perfusion of the choriocapillaris. Postoperative perfusion was detected in 31 (48%) of the total 65 eyes and in 12 (67%) of the 18 eyes with normal retinal pigment epithelium at baseline. The mean postoperative retinal pigment epithelium defect was 4.6 times larger than the original neovascular membrane. In selected patients, SLO macular scotometry showed areas of retained retinal sensitivity within the atrophic scar., Conclusion: The natural history of subfoveal neovascularization in high myopia is rarely visually restorative. By contrast, surgical excision of the membranes is feasible and may restore visual acuity in selected patients. This therapeutic approach merits a formal multicenter clinical trial.

Background: We investigated the functional outcomes of macular surgery for idiopathic, myopic and age-related subfoveal neovascular membranes and looked for preoperative features associated with better final visual acuity (VA)., Methods: We retrospectively studied 61 patients who had undergone macular surgery in our department between October 1992 and September 1994. Follow-up ranged from 5 to 27 months. Of 61 eyes with subfoveal neovascularizations, 6 were idiopathic, 21 had high myopia (-13 D median) and 34 displayed age-related macular degeneration (AMD). Median preoperative VA was 5/200 in AMD, 20/300 in myopia and 20/200 in idiopathic cases., Results: Four of the six eyes with idiopathic subfoveal neovascularization had VA 20/60 or better after a median follow-up of 18 months. Of the 21 eyes with high myopia, 10 (48%) improved and 13 (62%) were 20/200 or greater after a median follow-up of 12 months. Among the 34 eyes with AMD, VA improved in 7 (21%), but only 6 (18%) were 20/200 or better after a median follow-up of 7 months. Overall, complications included five retinal detachments, ten cataracts and an increase in size of the retinal pigment epithelium defect over the neovascular membrane of 2.3 +/- 0.8 times (mean +/- SD) in idiopathic eyes. 5.9 +/- 3.6 times in myopia and 19.5 +/- 12.2 times in AMD. Recurrence rates for idiopathic, myopic and age-related neovascular membranes were 33%, 19% and 18% respectively. Etiology (P = 0.035), initial VA in myopic eyes (P = 0.026) and initial size of the neovascular membranes in AMD (P = 0.025) were preoperative factors with a significant effect on final visual outcome., Conclusions: Surgical excision of subfoveal neovascular membranes yields different functional results depending on the underlying disease. Severe alteration of the retinal pigment epithelium-Bruch's membrane complex may be responsible for the poor visual outcomes in AMD.

Monoamine oxidase (MAO, E.C. 1.4.3.4) activity was determined by spectrophotofluorimetric assay of the reaction product of the artificial substrate kynuramine in platelets from venous blood samples from 50 outpatients with glaucomas and 47 sex- and age-matched controls. No significant differences were found between MAO activity in control subjects and that in subjects with glaucomas grouped according to diagnostic subcategories. Irrespective of ocular disease, women had significantly higher enzyme activity than men. Age-related changes in platelet MAO were inconsistent.