Intrinsic and Chemotherapeutic Stressors Modulate ABCC-Like Transport in Trypanosoma cruzi

Trypanosoma cruzi is the etiologic agent for Chagas disease, which affects 6–7 million people worldwide. The biological diversity of the parasite reflects on inefficiency of benznidazole, which is a first choice chemotherapy, on chronic patients. ABC transporters that extrude xenobiotics, metabolites, and mediators are overexpressed in resistant cells and contribute to chemotherapy failure. An ABCC-like transport was identified in the Y strain and extrudes thiol-conjugated compounds. As thiols represent a line of defense towards reactive species, we aimed to verify whether ABCC-like transport could participate in the regulation of responses to stressor stimuli. In order to achieve this, ABCC-like activity was measured by flow cytometry using fluorescent substrates. The present study reveals the participation of glutathione and ceramides on ABCC-like transport, which are both implicated in stress. Hemin modulated the ABCC-like efflux which suggests that this protein might be involved in cellular detoxification. Additionally, all strains evaluated exhibited ABCC-like activity, while no ABCB1-like activity was detected. Results suggest that ABCC-like efflux is not associated with natural resistance to benznidazole, since sensitive strains showed higher activity than the resistant ones. Although benznidazole is not a direct substrate, ABCC-like efflux increased after prolonged drug exposure and this indicates that the ABCC-like efflux mediated protection against cell stress depends on the glutathione biosynthesis pathway.     

Quantifying Light and Ultraviolet Radiation in Plant Biology

Abstract— Fluence rate and irradiance, two parameters used to quantify light and ultraviolet radiation, do not have a constant relation under natural light conditions. A method is described for measuring fluence rate, the preferred parameter, using meters intended for irradiance measurements. Values measured under natural light conditions are compared to a daylight model.     

A Model for Inactivation of Microbes Suspended in the Atmosphere by Solar Ultraviolet Radiation

Solar ultraviolet (UV) light within 280–320 nm (UVB) is the primary cause for virus inactivation in the atmosphere. Only the effect of the direct component has been previously evaluated. We developed a simple regression model to estimate the inactivation of a virus due to direct (unscattered), diffuse (scattered) and total (direct + diffuse) components of solar UV (daily integrated irradiances). The model predicts the maximum number of radiation-days a virus will survive at a given altitude above the ground in rural and urban environments under clear skies. We explored the effect of several environmental variables: visibility, altitude and ground reflectivity. We found that the effect of diffuse radiation on virus inactivation was larger than the direct component. The diffuse irradiance increased with ground albedo (mainly due to reflection of the direct attenuated solar off the ground) and decreased with increased visibility (proportional to aerosol loading in the atmosphere). The diffuse component increased with altitude, but the ratio of diffuse to the total decreased with increased altitude, highlighting the importance of the diffuse component of UV near the ground. Our model may help public health studies in predicting and understanding the effect of environmental parameters on the survival of germs.     

Hydrogen Atoms Are Produced When Tryptophan within a Protein Is Irradiated with Ultraviolet Light

Abstract— The UV photolysis of the aromatic amino acid, tryptophan (Trp), in the Ca²⁺-binding protein, cod paralbumin, type III, was studied using electron paramagnetic resonance (EPR) spectroscopy in the temperature range 4–80 K. For the Ca²⁺-bound protein, irradiation with UV light (250–400 nm) resulted in the generation of atomic hydrogen with a hyperfine splitting of 50.9 mT, whereas in the Ca²⁺-free form, where the Trp is exposed to solvent, the trapped atomic hydrogen was not in evidence. In the same spectra, the radical signal in the g = 2.00 region could be detected. The line shape of the Ca²⁺-bound form is similar to the EPR line shape obtained for Trp in micellar systems. In contrast, the EPR line shape for the Ca²⁺-free form is essentially featureless up to 80 K. The EPR spectra of the photoproducts of Trp and the nature of the photoreactions are therefore sensitive to the environment of Trp within the protein.     

Cytotoxicity of Essential Oil Cordia verbenaceae against Leishmania brasiliensis and Trypanosoma cruzi

The species Cordia verbenacea DC (Boraginaceae), known as the whaling herb and camaradinha, is a perennial shrub species native to the Atlantic Forest. Its leaves are used in folk medicine as an anti-inflammatory, analgesic, antiulcerogenic and curative agent, in the form of teas or infusions for internal or topical use. The present study aimed to verify the cytotoxicity of the essential oil and the leishmanicidal and trypanocidal potential of C. verbenacea. The essential oil was characterized by GC-MS. The in vitro biological activity was determined by anti-Leishmania and anti-Trypanosoma assays. The cytotoxixity was determined using mammalian fibroblasts. The C. verbenacea species presented α-pinene (45.71%), β-caryophyllene (18.77%), tricyclo[2,2,1-(2.6)]heptane (12.56%) as their main compounds. The essential oil exhibited strong cytotoxicity at concentrations below 250 μg/mL (LC₅₀ 138.1 μg/mL) in mammalian fibroblasts. The potent anti-trypanosome and anti-promastigote activities occurred from the concentration of 62.5 μg/mL and was considered clinically relevant. The results also demonstrate that at low concentrations (<62.5 μg/mL), the essential oil of C. verbenacea managed to be lethal for these activities. This can be considered an indication of the power used in daily human consumption. Therefore, it can be concluded that the essential oil of C. verbenacea contains a compound with remarkable antiparasitic activities and requires further research.     

Sialic acid C-glycosides with aromatic residues: investigating enzyme binding and inhibition of Trypanosoma cruzi trans-sialidase

Several α-configured C-sialosides were synthesised by cross metathesis and further synthetic derivatisation to obtain ligands for Trypanosoma cruzi trans-sialidase (TcTS), a key enzyme in Chagas disease. Affinities of these compounds to immobilised TcTS were measured by surface plasmon resonance (SPR). The K(D) values thus obtained are in the lower millimolar range and will be discussed. The results show the importance of addressing Tyr(119) and Trp(312) side chains of TcTS in target oriented ligand synthesis, since these amino acids constitute the acceptor binding region in the active site of TcTS. The best ligand showed a significant decrease of TcTS activity in a preliminary NMR based inhibition assay.     

Heteroleptic Oxidovanadium(V) Complexes with Activity against Infective and Non-Infective Stages of Trypanosoma cruzi

Five heteroleptic compounds, [V^VO(IN-2H)(L-H)], where L are 8-hydroxyquinoline derivatives and IN is a Schiff base ligand, were synthesized and characterized in both the solid and solution state. The compounds were evaluated on epimastigotes and trypomastigotes of Trypanosoma cruzi as well as on VERO cells, as a mammalian cell model. Compounds showed activity against trypomastigotes with IC₅₀ values of 0.29–3.02 μM. IN ligand and the new [V^VO₂(IN-H)] complex showed negligible activity. The most active compound [V^VO(IN-2H)(L2-H)], with L2 = 5-chloro-7-iodo-8-hydroxyquinoline, showed good selectivity towards the parasite and was selected to carry out further biological studies. Stability studies suggested a partial decomposition in solution. [V^VO(IN-2H)(L2-H)] affects the infection potential of cell-derived trypomastigotes. Low total vanadium uptake by parasites and preferential accumulation in the soluble proteins fraction were determined. A trypanocide effect was observed when incubating epimastigotes with 10 × IC₅₀ values of [V^VO(IN-2H)(L2-H)] and the generation of ROS after treatments was suggested. Fluorescence competition measurements with DNA:ethidium bromide adduct showed a moderate DNA interaction of the complexes. In vivo toxicity study on C. elegans model showed no toxicity up to a 100 μM concentration of [V^VO(IN-2H)(L2-H)]. This compound could be considered a prospective anti-T. cruzi agent that deserves further research.     

Long-wavelength Ultraviolet A1 and Visible Light Photoprotection: A Multimodality Assessment of Dose and Response

Human skin is exposed to visible light (VL; 400–700 nm) and long-wavelength ultraviolet A1 (UVA1) radiation (370–400 nm) after the application of organic broad-spectrum sunscreens. The biologic effects of these wavelengths have been demonstrated; however, a dose–response has not been investigated. Ten subjects with Fitzpatrick skin phototype IV-VI were enrolled. Subjects were irradiated with 2 light sources (80–480 J cm⁻²): one comprising VL with less than 0.5% UVA1 (VL+UVA1) and the other pure VL. Skin responses were evaluated for 2 weeks using clinical and spectroscopic assessments. 4-mm punch biopsies were obtained from nonirradiated skin and sites irradiated with 480 J cm⁻² of VL+UVA1 and pure VL 24 h after irradiation. Clinical and spectroscopic assessments demonstrated a robust response at VL+UVA1 sites compared with pure VL. Histology findings demonstrated a statistically significant increase in the marker of inflammation (P < 0.05) and proliferation (P < 0.05) at the irradiated sites compared with nonirradiated control. Threshold doses of VL+UVA1 resulting in biologic responses were calculated. Results indicate that approximately 2 h of sun exposure, which equates to VL+UVA1 dose (~400 J cm⁻²), is capable of inducing inflammation, immediate erythema and delayed tanning. These findings reinforce the need of photoprotection beyond the UV range.     

Inactivation by Pulsed Light of Bacillus subtilis Spores with Impaired Protection Factors

The resistance to pulsed light (PL) of spores of Bacillus subtilis strain 168 and of strains with mutations increasing sensitivity to UV‐C or affecting spore structure was evaluated and compared to resistance to continuous UV‐C and moist heat, in order to reveal original mechanisms of inactivation by PL. Spores of B. subtilis strain 168 (1A1) and eight mutant strains (sspA, sspB, sspAB, cotA, gerE, cotE, uvrA and recA) were exposed to PL (up to 1.77 J cm^?2), continuous UV‐C (up to 147 mJ cm^?2) and moist heat at 90°C. Spores of the strains lacking proteins linked to coat formation or structure (cotA, gerE and cotE) were markedly more sensitive to PL than 1A1, while their sensitivity to continuous UV‐C or to moist heat was similar to the one of strain 1A1. Coat proteins had a major contribution to the resistance of B. subtilis spores to PL irradiation characterized by short‐time and high‐energy pulses of white light in the wavelengths 200–1100 nm. In contrast the role of coat proteins to UV‐C or to moist heat resistance was marginal or null.     

Natural products and Chagas' disease: a review of plant compounds studied for activity against Trypanosoma cruzi

Here, we review studies that have investigated the activity of plant-derived compounds against Trypanosoma cruzi, the etiologic agent of Chagas’ disease. In the last decade, more than 300 species belonging to almost 100 families have been evaluated for activity, and here we describe the compounds isolated; 85 references are cited.     

Probing the acceptor substrate binding site of Trypanosoma cruzi trans-sialidase with systematically modified substrates and glycoside libraries

Systematically modified octyl galactosides and octyl N-acetyllactosamines were assessed as inhibitors of, and substrates for, T. cruzi trans-sialidase (TcTS) in the context of exploring its acceptor substrate binding site. These studies show that TcTS, which catalyses the α-(2→3)-sialylation of non-reducing terminal β-galactose residues, is largely intolerant of substitution of the galactose 2 and 4 positions whereas substitution of the galactose 6 position is well tolerated. Further studies show that even the addition of a bulky sugar residue (glucose, galactose) does not impact negatively on TcTS binding and turnover, which highlights the potential of 'internal' 6-substituted galactose residues to serve as TcTS acceptor substrates. Results from screening a 93-membered thiogalactoside library highlight a number of structural features (notably imidazoles and indoles) that are worthy of further investigation in the context of TcTS inhibitor development.     

Chemical and chemoenzymatic synthesis of glycosyl-amino acids and glycopeptides related to Trypanosoma cruzi mucins

This study describes the synthesis of the alpha- and beta-linked N-acetyllactosamine (Galp-beta-1,4-GlcNAc; LacNAc) glycosides of threonine (LacNAc-Thr). LacNAc-a-Thr was prepared by direct chemical coupling of a 2-azido-2-deoxy-lactose disaccharide donor to a suitable partially protected threonine unit. In contrast, stepwise chemical generation of beta-linked N-acetylglucosamine followed by enzymatic galactosylation to give LacNAc-beta-Thr proved effective, whereas use of a 2-azido-2-deoxy-lactose donor in acetonitrile failed to give the desired beta-linked disaccharyl glycoside. This study illustrates that it is possible to overcome the inherent stereoselection for 1,2-trans chemical glycosylation with a GlcNAc donor, and that the well-established preference of bovine beta-1,4-galactosyltransferase for beta-linked acceptor substrates can also be overcome. Using this knowledge, short glycopeptide fragments based on T. cruzi mucin sequences, Thr-Thr-[LacNAcThr]-Thr-Thr-Gly, were synthesised. All LacNAc-based compounds outlined were shown to serve as acceptor substrates for sialylation by T. cruzi trans-sialidase.     

Inactivation of Bacillus subtilis Spores Using Various Combinations of Ultraviolet Treatment with Addition of Hydrogen Peroxide

This study aims at comparing the inactivation of Bacillus subtilis spores by various combinations of UV treatment and hydrogen peroxide (H₂O₂) addition. The combinations included sequential (UV–H₂O₂, H₂O₂–UV) and simultaneous (UV/H₂O₂) processes. Results showed that B. subtilis spores achieved a certain inactivation effect through UV treatment. However, hardly any inactivation effect by H₂O₂ alone was observed. H₂O₂ had a significant synergetic effect when combined with UV treatment, while high irradiance and H₂O₂ concentration both favored the reaction. When treated with 0.60 mm H₂O₂ and 113.0 μW/cm² UV irradiance for 6 min, the simultaneous UV/H₂O₂ treatment showed significantly improved disinfection effect (4.13 log) compared to that of UV–H₂O₂ (3.03 log) and H₂O₂–UV (2.88 log). The relationship between the inactivation effect and the exposure time followed a typical pseudo‐first‐order kinetics model. The pseudo‐first‐order rate constants were 0.478, 0.447 and 0.634 min^?1, for the UV‐H₂O₂, H₂O₂–UV and UV/H₂O₂ processes, respectively, further confirming the optimal disinfection effect of the UV/H₂O₂ process. The disinfection could be ascribed to the OH radicals, as verified by the level of para‐chlorobenzoic acid (pCBA).     

Molecular dynamics study of triosephosphate isomerase from Trypanosoma cruzi in water/decane mixtures

A comprehensive study of the triosephosphate isomerase from the parasite Trypanosoma cruzi (TcTIM) in water, in decane, and in three water/decane mixtures was performed using molecular dynamics (MD) simulations in a time scale of 40 ns. The structure and dynamics of the enzyme, as well as the solvent molecules' distribution and mobility, were analyzed in detail. In the presence of decane, the amplitudes of the most important internal motions of the enzyme backbone were observed to depend on the solvent concentration: the higher the water concentration, the greater the amplitudes. Contrary to this trend, the amplitudes of the TcTIM motions in pure water were similar to those of the simulation with the lowest water concentration. The enzyme was observed to be almost motionless in pure decane due to a sharp increase of the number of intramolecular hydrogen bonds. This caused a contraction of the enzyme structure accompanied by a loss of secondary structure and of a decrease of the hydrophilic solvent accessible surface. A similar behavior, although to a lesser extent, was observed in the simulation at the lowest water concentration. Our results suggest that the presence of decane molecules located at specific sites of the enzyme might accelerate its internal movements, although a minimum number of water molecules is needed for the protein to keep its structure and dynamics. Altogether, this work provides new insight into protein and water behavior in organic solvents as well as into the dynamics of TcTIM itself.     

Inactivation of Photosensitizing Merocyanine Dyes by Plasma, Serum and Serum Components

Abstract— Merocyanine dyes with an oxygen in the electron donor heterocycle were rapidly degraded by plasma, serum and serum components. Replacement of the oxygen by a sulfur or selenium atom rendered the dyes refractory to degradation. The degradation of labile merocyanine dyes was temperature dependent and oxygen independent. The plasma component that was responsible for the degradation of merocyanine dyes was sensitive to heat and detergent, suggesting an enzymatic process. The identification of the structural requirements for sensitivity/resistance to degradation provides the experimenter with a simple means to manipulate the stability of mer-ocyanines in high serum or plasma environments and may expand the clinical utility of merocyanine photosen-sitizers beyond their traditional role in the extracorporeal purging of bone marrow grafts.