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Chagas disease is a zoonosis caused by the protozoan parasite Trypanosoma cruzi. Clinical outcomes range from long-term asymptomatic carriage to cardiac, digestive, neurological and composite presentations that can be fatal in both acute and chronic stages of the disease. Studies of T. cruzi in animal models, principally mice, have informed our understanding of the biological basis of this variability and its relationship to infection and host response dynamics. Hamsters have higher translational value for many human infectious diseases, but they have not been well developed as models of Chagas disease. We transposed a real-time bioluminescence imaging system for T. cruzi infection from mice into female Syrian hamsters (Mesocricetus auratus). This enabled us to study chronic tissue pathology in the context of spatiotemporal infection dynamics. Acute infections were widely disseminated, whereas chronic infections were almost entirely restricted to the skin and subcutaneous adipose tissue. Neither cardiac nor digestive tract disease were reproducible features of the model. Skeletal muscle had only sporadic parasitism in the chronic phase, but nevertheless displayed significant inflammation and fibrosis, features also seen in mouse models. Whereas mice had normal locomotion, all chronically infected hamsters developed hindlimb muscle hypertonia and a gait dysfunction resembling spastic diplegia. With further development, this model may therefore prove valuable in studies of peripheral nervous system involvement in Chagas disease.

Benznidazole is the front-line drug used to treat infections with Trypanosoma cruzi, the causative agent of Chagas disease. However, for reasons that are unknown, treatment failures are common. When we examined parasites that survived benznidazole treatment in mice using highly sensitive in vivo and ex vivo bioluminescence imaging, we found that recrudescence is not due to persistence of parasites in a specific organ or tissue that preferentially protects them from drug activity. Surviving parasites are widely distributed and located in host cells where the vast majority contained only one or two amastigotes. Therefore, infection relapse does not arise from a small number of intact large nests. Rather, persisters are either survivors of intracellular populations where co-located parasites have been killed, or amastigotes in single/low-level infected cells exist in a state where they are less susceptible to benznidazole. To better assess the nature of parasite persisters, we exposed infected mammalian cell monolayers to a benznidazole regimen that reduces the intracellular amastigote population to

BackgroundChagas disease is caused by the protozoan parasite Trypanosoma cruzi and is a serious public health problem throughout Latin America. With 6 million people infected, there is a major international effort to develop new drugs. In the chronic phase of the disease, the parasite burden is extremely low, infections are highly focal at a tissue/organ level, and bloodstream parasites are only intermittently detectable. As a result, clinical trials are constrained by difficulties associated with determining parasitological cure. Even highly sensitive PCR methodologies can be unreliable, with a tendency to produce "false-cure" readouts. Improved diagnostic techniques and biomarkers for cure are therefore an important medical need.Methodology/principal findingsUsing an experimental mouse model, we have combined a multiplex assay system and highly sensitive bioluminescence imaging to evaluate serological procedures for diagnosis of T. cruzi infections and confirmation of parasitological cure. We identified a set of three antigens that in the context of the multiplex serology system, provide a rapid, reactive and highly accurate read-out of both acute and chronic T. cruzi infection. In addition, we describe specific antibody responses where down-regulation can be correlated with benznidazole-mediated parasite reduction and others where upregulation is associated with persistent infection. One specific antibody (IBAG39) highly correlated with the bioluminescence flux and represents a promising therapy monitoring biomarker in mice.Conclusions/significanceRobust, high-throughput methodologies for monitoring the efficacy of anti-T. cruzi drug treatment are urgently required. Using our experimental systems, we have identified markers of infection or parasite reduction that merit assessing in a clinical setting for the longitudinal monitoring of drug-treated patients.

Digestive Chagas disease (DCD) is an enteric neuropathy caused by Trypanosoma cruzi infection. The mechanism of pathogenesis is poorly understood and the lack of a robust, predictive animal model has held back research. We screened a series of mouse models using gastrointestinal tracer assays and in vivo infection imaging systems to discover a subset exhibiting chronic digestive transit dysfunction and significant retention of faeces in both sated and fasted conditions. The colon was a specific site of both tissue parasite persistence, delayed transit and dramatic loss of myenteric neurons as revealed by whole-mount immunofluorescence analysis. DCD mice therefore recapitulated key clinical manifestations of human disease. We also exploited dual reporter transgenic parasites to home in on locations of rare chronic infection foci in the colon by ex vivo bioluminescence imaging and then used fluorescence imaging in tissue microdomains to reveal co-localisation of infection and enteric nervous system lesions. This indicates that long-term T. cruzi-host interactions in the colon drive DCD pathogenesis, suggesting that the efficacy of anti-parasitic chemotherapy against chronic disease progression warrants further pre-clinical investigation.

New assay designs are needed to improve the predictive value of the Trypanosoma cruzi in vitro tests used as part of the Chagas' disease drug development pipeline. Here, we employed a green fluorescent protein (eGFP)-expressing parasite line and live high-content imaging to monitor the growth of T. cruzi amastigotes in mouse embryonic fibroblasts. A novel assay design allowed us to follow parasite numbers over 6 days, in four-hour intervals, while occupying the microscope for only 24 hours per biological replicate. Dose-response curves were calculated for each time point after addition of test compounds, revealing how EC50 values first decreased over the time of drug exposure, and then leveled off. However, we observed that parasite numbers could vary, even in the untreated controls, and at different sites in the same well, which caused variability in the EC50 values. To overcome this, we established that fold change in parasite number per hour is a more robust and informative measure of drug activity. This was calculated based on an exponential growth model for every biological sample. The net fold change per hour is the result of parasite replication, differentiation, and death. The calculation of this fold change enabled us to determine the tipping point of drug action, i.e. the time point when the death rate of the parasites exceeded the growth rate and the fold change dropped below 1, depending on the drug concentration and exposure time. This revealed specific pharmacodynamic profiles of the benchmark drugs benznidazole and posaconazole.

BACKGROUND:The long term and complex nature of Chagas disease in humans has restricted studies on vaccine feasibility. Animal models also have limitations due to technical difficulties in monitoring the extremely low parasite burden that is characteristic of chronic stage infections. Advances in imaging technology offer alternative approaches that circumvent these problems. Here, we describe the use of highly sensitive whole body in vivo imaging to assess the efficacy of recombinant viral vector vaccines and benznidazole-cured infections to protect mice from challenge with Trypanosoma cruzi. METHODOLOGY/PRINCIPAL FINDINGS:Mice were infected with T. cruzi strains modified to express a red-shifted luciferase reporter. Using bioluminescence imaging, we assessed the degree of immunity to re-infection conferred after benznidazole-cure. Those infected for 14 days or more, prior to the onset of benznidazole treatment, were highly protected from challenge with both homologous and heterologous strains. There was a >99% reduction in parasite burden, with parasites frequently undetectable after homologous challenge. This level of protection was considerably greater than that achieved with recombinant vaccines. It was also independent of the route of infection or size of the challenge inoculum, and was long-lasting, with no significant diminution in immunity after almost a year. When the primary infection was benznidazole-treated after 4 days (before completion of the first cycle of intracellular infection), the degree of protection was much reduced, an outcome associated with a minimal T. cruzi-specific IFN-γ+ T cell response. CONCLUSIONS/SIGNIFICANCE:Our findings suggest that a protective Chagas disease vaccine must have the ability to eliminate parasites before they reach organs/tissues, such as the GI tract, where once established, they become largely refractory to the induced immune response.

Investigations into intracellular replication and differentiation of Trypanosoma cruzi within the mammalian host have been restricted by limitations in our ability to detect parasitized cells throughout the course of infection. We have overcome this problem by generating genetically modified parasites that express a bioluminescent/fluorescent fusion protein. By combining in vivo imaging and confocal microscopy, this has enabled us to routinely visualise murine infections at the level of individual host cells. These studies reveal that intracellular parasite replication is an asynchronous process, irrespective of tissue location or disease stage. Furthermore, using TUNEL assays and EdU labelling, we demonstrate that within individual infected cells, replication of both mitochondrial (kDNA) and nuclear genomes is not co-ordinated within the parasite population, and that replicating amastigotes and non-replicating trypomastigotes can co-exist in the same cell. Finally, we report the presence of distinct non-canonical morphological forms of T. cruzi in the mammalian host. These appear to represent transitional forms in the amastigote to trypomastigote differentiation process. Therefore, the intracellular life-cycle of T. cruzi in vivo is more complex than previously realised, with potential implications for our understanding of disease pathogenesis, immune evasion and drug development. Dissecting the mechanisms involved will be an important experimental challenge.

El benznidazol y el nifurtimux conpuestos nitroheterocilicos son los medicamentos aprobados para el tratamiento de las infecciones por Trypanosoma cruzi. Ambos son profármacos y no tienen importantes propiedades tripanocidas hasta su activación intraparasitaria. La enzima responsable es una nitroreductasa (TcNTR ), que inicia una cascada reductora que conduce a la generación de los metabolitos tóxicos que matan al parásito. Los procesos que actúan para regular a esta enzima conducen a la resis- tencia cruzada contra ambos fármacos. Estos incluyen la pérdida de uno de los cromosomas que contienen el gen TcNTR o mutaciones puntuales que inactivan la enzima. Los parásitos TcNTR heterocigotos son infecciosos, no muestran un fenotipo nocivo obvio y son hasta 5 veces más resistente a benznidazol y el nifurtimox. Sin embargo, la pérdida completa de la actividad TcNTR hace que T. cruzi no sea infeccioso, lo que sugiere que puede haber un límite para el nivel de resistencia por este mecanismo. En las poblaciones naturales de T. cruzi no se encontraron pruebas de que las amplias variaciones en la sensibilidad al benznidazol estén vinculadas a las mutaciones en TcNTR lo que, junto con la evidencia de que la resistencia a benznidazol y nifurtimox no siempre es conjunta, indica que existen otros mecanismos independientes de TcNTR. Los nuevos avances en tecnología ofrecen la oportunidad de explorar más a fondo esta cuestión.

BACKGROUND:The neglected parasitic infection Chagas disease is rapidly becoming a globalised public health issue due to migration. There are only two anti-parasitic drugs available to treat this disease, benznidazole and nifurtimox. Thus it is important to identify and validate new drug targets in Trypanosoma cruzi, the causative agent. T. cruzi expresses an ER-localised ascorbate-dependent peroxidase (TcAPx). This parasite-specific enzyme has attracted interest from the perspective of targeted chemotherapy. METHODOLOGY/PRINCIPAL FINDINGS:To assess the importance of TcAPx in protecting T. cruzi from oxidative stress and to determine if it is essential for virulence, we generated null mutants by targeted gene disruption. Loss of activity was associated with increased sensitivity to exogenous hydrogen peroxide, but had no effect on susceptibility to the front-line Chagas disease drug benznidazole. This suggests that increased oxidative stress in the ER does not play a significant role in its mechanism of action. Homozygous knockouts could proceed through the entire life-cycle in vitro, although they exhibited a significant decrease in their ability to infect mammalian cells. To investigate virulence, we exploited a highly sensitive bioluminescence imaging system which allows parasites to be monitored in real-time in the chronic stage of murine infections. This showed that depletion of enzyme activity had no effect on T. cruzi replication, dissemination or tissue tropism in vivo. CONCLUSIONS/SIGNIFICANCE:TcAPx is not essential for parasite viability within the mammalian host, does not have a significant role in establishment or maintenance of chronic infections, and should therefore not be considered a priority for drug design.

Human African trypanosomiasis is caused by infection with parasites of the Trypanosoma brucei species complex, and threatens over 70 million people in sub-Saharan Africa. Development of new drugs is hampered by the limitations of current rodent models, particularly for stage II infections, which occur once parasites have accessed the CNS. Bioluminescence imaging of pathogens expressing firefly luciferase (emission maximum 562 nm) has been adopted in a number of in vivo models of disease to monitor dissemination, drug-treatment and the role of immune responses. However, lack of sensitivity in detecting deep tissue bioluminescence at wavelengths below 600 nm has restricted the wide-spread use of in vivo imaging to investigate infections with T. brucei and other trypanosomatids.Here, we report a system that allows the detection of fewer than 100 bioluminescent T. brucei parasites in a murine model. As a reporter, we used a codon-optimised red-shifted Photinus pyralis luciferase (PpyRE9H) with a peak emission of 617 nm. Maximal expression was obtained following targeted integration of the gene, flanked by an upstream 5'-variant surface glycoprotein untranslated region (UTR) and a downstream 3'-tubulin UTR, into a T. brucei ribosomal DNA locus. Expression was stable in the absence of selective drug for at least 3 months and was not associated with detectable phenotypic changes. Parasite dissemination and drug efficacy could be monitored in real time, and brain infections were readily detectable. The level of sensitivity in vivo was significantly greater than achievable with a yellow firefly luciferase reporter.The optimised bioluminescent reporter line described here will significantly enhance the application of in vivo imaging to study stage II African trypanosomiasis in murine models. The greatly increased sensitivity provides a new framework for investigating host-parasite relationships, particularly in the context of CNS infections. It should be ideally suited to drug evaluation programmes.

Patients with single ventricle heart defects requires a series of staged open-heart procedures, termed Fontan palliation. However, while lifesaving, these operations are associated with significant morbidity and early mortality. The attendant complications are thought to arise in response to the abnormal hemodynamics induced by Fontan palliation, although the pathophysiology underlying these physicochemical changes in cardiovascular and other organs remain unknown. Here, we investigated the microRNA (miRNA) content in serum and serum-derived extracellular vesicles (EVs) by sequencing small RNAs from a physiologically relevant sheep model of the Fontan operation. The differential expression analysis identified the enriched miRNA clusters in (1) serum vs. serum-derived EVs and (2) pre-Fontan EVs vs. post-Fontan EVs. Metascape analysis showed that the overexpressed subset of EV miRNAs by Fontan procedure target liver-specific cells, underscoring a potentially important pathway involved in the liver dysfunction that occurs as a consequence of Fontan palliation. We also found that post-Fontan EV miRNAs were associated with senescence and cell death, whereas pre-Fontan EV miRNAs were associated with stem cell maintenance and epithelial-to-mesenchymal transition. This study shows great potential to identify novel circulating EV biomarkers from Fontan sheep serum that may be used for the diagnosis, prognosis, and therapeutics for patients that have undergone Fontan palliation.

Amidinobenzimidazole derivatives connected to 1-aryl-substituted 1,2,3-triazole through phenoxymethylene linkers 7a–7e, 8a–8e, and 9a–9e were designed and synthesised with the aim of evaluating their anti-bacterial and anti-trypanosomal activities and DNA/RNA binding affinity. Results from anti-bacterial evaluations of antibiotic-resistant pathogenic bacteria revealed that both o-chlorophenyl-1,2,3-triazole and N-isopropylamidine moieties in 8c led to strong inhibitory activity against resistant Gram-positive bacteria, particularly the MRSA strain. Furthermore, the non-substituted amidine and phenyl ring in 7a induced a marked anti-bacterial effect, with potency against ESBL-producing Gram-negative E. coli better than those of the antibiotics ceftazidime and ciprofloxacin. UV–Vis and CD spectroscopy, as well as thermal denaturation assays, indicated that compounds 7a and 8c showed also binding affinities towards ctDNA. Anti-trypanosomal evaluations showed that the p-methoxyphenyl-1,2,3-triazole moiety in 7b and 9b enhanced inhibitory activity against T. brucei, with 8b being more potent than nifurtimox, and having minimal toxicity towards mammalian cells.

The protozoan parasite Trypanosoma cruzi causes Chagas disease, an important public health problem throughout Latin America. Current therapeutic options are characterised by limited efficacy, long treatment regimens and frequent toxic side-effects. Advances in this area have been compromised by gaps in our knowledge of disease pathogenesis, parasite biology and drug activity. Nevertheless, several factors have come together to create a more optimistic scenario. Drug-based research has become more systematic, with increased collaborations between the academic and commercial sectors, often within the framework of not-for-profit consortia. High-throughput screening of compound libraries is being widely applied, and new technical advances are helping to streamline the drug development pipeline. In addition, drug repurposing and optimisation of current treatment regimens, informed by laboratory research, are providing a basis for new clinical trials. Here, we will provide an overview of the current status of Chagas disease drug development, highlight those areas where progress can be expected, and describe how fundamental research is helping to underpin the process.

Imidazolium salts are privileged compounds in organic chemistry, and have valuable biological properties. Recent studies show that symmetric imidazolium salts with bulky moieties can display antiparasitic activity against T. cruzi. After developing a facile methodology for the synthesis of tetrasubstituted imidazolium salts from propargylamines and isocyanides, we screened a small library of these adducts against the causative agents of African and American trypanosomiases. These compounds display nanomolar activity against T. brucei and low (or sub) micromolar activity against T. cruzi, with excellent selectivity indexes and favorable molecular properties, thereby emerging as promising hits for the treatment of Chagas disease and sleeping sickness.

Chagas disease is caused by the insect-transmitted protozoan Trypanosoma cruzi, and is the most important parasitic infection in Latin America. [...]

Ever since Freud, the couch has been viewed as an important—some would argue essential—component of psychoanalysis. Although many theoretical papers and case reports have addressed the use of the couch in psychoanalysis, no empirical study has investigated its effect on psychoanalytic process or outcome. After a review of the literature, a number of research designs are proposed that might be used in such an investigation. Finally, preliminary empirical data are presented from archived audiotapes of two psychoanalyses: one in which the patient switched from lying down to sitting up, and one in which the opposite occurred. The aim is to stimulate research-oriented psychoanalysts to undertake empirical investigations of the theoretical concepts underlying use of the couch and, more generally, to present a specific example of research as a paradigm for a broader research agenda for empirical investigation of the key theoretical ideas underlying psychoanalysis. [ABSTRACT FROM PUBLISHER]

Current drug therapies against Trypanosoma cruzi, the causative agent of Chagas disease, have limited effectiveness and are highly toxic. T. cruzi-specific metabolic pathways that utilize trypanothione for the reduction of peroxides are being explored as potential novel therapeutic targets. In the present study we solved the X-ray crystal structure of one of the T. cruzi enzymes involved in peroxide reduction, the glutathione peroxidase-like enzyme TcGPXI (T. cruzi glutathione peroxidase-like enzyme I). We also characterized the wild-type, C48G and C96G variants of TcGPXI by NMR spectroscopy and biochemical assays. Our results show that residues Cys48 and Cys96 are required for catalytic activity. In solution, the TcGPXI molecule readily forms a Cys48–Cys96 disulfide bridge and the polypeptide segment containing Cys96 lacks regular secondary structure. NMR spectra of the reduced TcGPXI are indicative of a protein that undergoes widespread conformational exchange on an intermediate time scale. Despite the absence of the disulfide bond, the active site mutant proteins acquired an oxidized-like conformation as judged from their NMR spectra. The protein that was used for crystallization was pre-oxidized by t-butyl hydroperoxide; however, the electron density maps clearly showed that the active site cysteine residues are in the reduced thiol form, indicative of X-ray-induced reduction. Our crystallographic and solution studies suggest a level of structural plasticity in TcGPXI consistent with the requirement of the atypical two-cysteine (2-Cys) peroxiredoxin-like mechanism implied by the behaviour of the Cys48 and Cys96 mutant proteins. [ABSTRACT FROM AUTHOR]

The microwave (MW)-assisted formation of ZnO micro-javelins from zinc nitrate and urea in aqueous solution is described. The particles (named as ‘micro-javelins’ because of their high aspect ratio and needle-like tips) grow hexagonally with well-defined facets in the 〈0110〉 direction and pointed tips in (0001) direction. Powder X-ray diffraction patterns show the appearance of a strikingly dominant (1000) orientation. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) investigations reveal the morphological evolution of these hexagonal ZnO particles with time. The effect of precursor concentrations, counterion type and MW irradiation power and their consequent influence on pH and Zn2+ion concentration are investigated. A mechanism for the formation of the micro-javelins is postulated. The microwave induced supersaturation of Zn(OH)+species under the weakly basic pH condition and the initial growth through the (0001) direction (oxygen-rich face) are proposed to be the key factors that dictate the formation of these ZnO micro-javelins. The present one-step microwave process is a straightforward and a reproducible method for the bulk synthesis of defect-free ZnO micro-javelins, which would find potential applications in microelectronic devices (e.g.lasers, cantilevers in surface probing equipment, etc.). [ABSTRACT FROM AUTHOR]

The photophysical properties of 5′-guanosine monophosphate (5′-GMP) and polyguanylic acid {poly(G)} in D2O solutions of varying pH have been studied using picosecond transient infrared absorption spectroscopy. Whereas in neutral or weakly alkaline solution only the vibrationally excited electronic ground state of 5′-GMP is observed, in acidic solution the relatively long-lived (229 ± 20 ps) electronic excited state of protonated 5′-GMP, which possesses strong absorptions at 1517 and 1634 cm−1, could be detected. The picosecond transient behaviour of polyguanylic acid in acidic solution is also very different from that of the polynucleotide in neutral solution due not only to the protonation of guanine moieties yielding the protonated excited state but because of the disruption of the guanine stacks which are present in the species in neutral solution. [ABSTRACT FROM AUTHOR]

An industrially viable solution-based processing route using minimal amounts of solvent has been used to prepare bulk quantity nanopowders (average particle size 15 ± 3 nm) for the fabrication of ZnO varistors. The xerogels, calcined powders and sintered materials were fully characterised. The preparation of varistors from nanopowders has been optimised by studying the effect of temperature on grain growth, densification and breakdown voltage. The varistors are prepared by sintering at 1050 °C for 2 hours, a temperature that is significantly lower than that used in the current industrial process. Highly dense varistor discs prepared from the sintered material produce devices, with a breakdown voltage 85% higher than that of commercial varistors, making them suitable for use in miniaturised electronic circuitry. Improved performance of these materials has been attributed to the small grain size and better dispersion of additives on ZnO grains. [ABSTRACT FROM AUTHOR]

There is increasing evidence that Trypanosoma cruzi antioxidant enzymes play a key immune evasion role by protecting the parasite against macrophage-derived reactive oxygen and nitrogen species. Using T. cruzi transformed to overexpress the peroxiredoxins TcCPX (T. cruzi cytosolic tryparedoxin peroxidase) and TcMPX (T. cruzi mitochondrial tryparedoxin peroxidase), we found that both cell lines readily detoxify cytotoxic and diffusible reactive oxygen and nitrogen species generated in vitro or released by activated macrophages. Parasites transformed to overexpress TcAPX (T. cruzi ascorbate-dependent haemoperoxidase) were also more resistant to H2O2 challenge, but unlike TcMPX and TcCPX overexpressing lines, the TcAPX overexpressing parasites were not resistant to peroxynitrite. Whereas isolated tryparedoxin peroxidases react rapidly (k=7.2×105 M−1·s−1) and reduce peroxynitrite to nitrite, our results demonstrate that both TcMPX and TcCPX peroxiredoxins also efficiently decompose exogenous- and endogenously-generated peroxynitrite in intact cells. The degree of protection provided by TcCPX against peroxynitrite challenge results in higher parasite proliferation rates, and is demonstrated by inhibition of intracellular redox-sensitive fluorescence probe oxidation, protein 3-nitrotyrosine and protein–DMPO (5,5-dimethylpyrroline-N-oxide) adduct formation. Additionally, peroxynitrite-mediated over-oxidation of the peroxidatic cysteine residue of peroxiredoxins was greatly decreased in TcCPX overexpressing cells. The protective effects generated by TcCPX and TcMPX after oxidant challenge were lost by mutation of the peroxidatic cysteine residue in both enzymes. We also observed that there is less peroxynitrite-dependent 3-nitrotyrosine formation in infective metacyclic trypomastigotes than in non-infective epimastigotes. Together with recent reports of up-regulation of antioxidant enzymes during metacyclogenesis, our results identify components of the antioxidant enzyme network of T. cruzi as virulence factors of emerging importance. [ABSTRACT FROM AUTHOR]

The photophysical properties of [Re(CO)3(dppz)(py)]+ (dppz = dipyrido-[3,2-a:2′,3′-c] phenazine) (1) and its 11,12 substituted derivatives [Re(CO)3(dppzMe2)(py)]+ (2) and [Re(CO)3(dppzF2)(py)]+ (3) have been examined in organic and aqueous environments using phosphorescence and picosecond transient visible and infrared absorption spectroscopic methods. The roles of the intraligand IL(π–π*) and metal-to-ligand charge transfer MLCT(phz) excited states are evaluated and used to explain the major effect of difluoro-substitution, which is particularly remarkable in water, where the excited state of [Re(CO)3(dppzF2)(py)]+ (3) is strongly quenched. [ABSTRACT FROM AUTHOR]

A rapid, straight-forward method for the preparation of silver colloidal solutions of various colours has been developed. This is based on the reduction of silver ions by ascorbic acid in the presence of citrate-stabilized silver seeds, additional trisodium citrate and a polymer such as polyvinylpyrrolidone. The colour of the sol is controlled by varying the concentration of only one reagent, namely the trisodium citrate. TEM studies show that this effect is due to changes in morphology of the particles. Working at temperatures above 50 °C yields well-defined triangular plates, which are stable in the reaction medium. Samples prepared at lower temperatures and left in the reaction medium experience etching and a consequent change in particle shape. The mechanisms of the initial particle growth and the etching process are discussed. [ABSTRACT FROM AUTHOR]

Trypanosoma cruzi undergo PCD (programmed cell death) under appropriate stimuli, the mechanisms of which remain to be established. In the present study, we show that stimulation of PCD in T. cruzi epimastigotes by FHS (fresh human serum) results in rapid (<1 h) externalization of phosphatidylserine and depletion of the low molecular mass thiols dihydrotrypanothione and glutathione. Concomitantly, enhanced generation of oxidants was established by EPR and immuno-spin trapping of radicals using DMPO (5,5-dimethylpyrroline-N-oxide) and augmentation of the glucose flux through the pentose phosphate pathway. In the early period (<20 min), changes in mitochondrial membrane potential and inhibition of respiration, probably due to the impairment of ADP/ATP exchange with the cytosol, were observed, conditions that favour the generation of O2•−. Accelerated rates of mitochondrial O2•− production were detected by the inactivation of the redox-sensitive mitochondrial aconitase and by oxidation of a mitochondrial-targeted probe (MitoSOX). Importantly, parasites overexpressing mitochondrial FeSOD (iron superoxide dismutase) were more resistant to the PCD stimulus, unambiguously indicating the participation of mitochondrial O2•− in the signalling process. In summary, FHS-induced PCD in T. cruzi involves mitochondrial dysfunction that causes enhanced O2•− formation, which leads to cellular oxidative stress conditions that trigger the initiation of PCD cascades; moreover, overexpression of mitochondrial FeSOD, which is also observed during metacyclogenesis, resulted in cytoprotective effects. [ABSTRACT FROM AUTHOR]

The photophysical properties of stacked G-tetrads in diverse systems, including concentrated solutions of 5′-guanosine monophosphate (5′-GMP), polyguanylic acid (poly(G)) and the G-rich oligodeoxynucleotide sequence characteristic of human telomeric DNA, are probed by ps-TRIR and compared to those of the monomeric 5′-GMP. [ABSTRACT FROM AUTHOR]

Strongly white-emitting (λmax = 495 ± 10 nm) d- and l- penicillamine capped CdS nanoparticles, which show strong circular dichroism in the range 200–390 nm, have been prepared. [ABSTRACT FROM AUTHOR]

A strong infrared band at 1574 cm−1 is observed following 267 nm excitation of 2′-deoxycytidine (τ = 37 ± 4 ps) or 2′-deoxycytidine 5′-monophosphate (τ = 33 ± 4 ps); this band is provisionally attributed to an 1nNπ* state and is absent for cytosine. [ABSTRACT FROM AUTHOR]

Objectives: Our goal was to conduct a hemodynamic analysis of a novel animal model of Fontan physiology. Poor late-term outcomes in Fontan patients are believed to arise from Fontan-induced hemodynamics, but the mechanisms remain poorly understood. Recent advances in surgical experimentation have resulted in the development of a chronic sheep model of Fontan physiology; however, detailed analysis of this model is lacking. Methods: We created a single-stage Fontan model in juvenile sheep with normal biventricular circulation. The superior vena cava was anastomosed to the main pulmonary artery, and the inferior vena cava was connected to the main pulmonary artery using an expanded polytetrafluoroethylene conduit. Longitudinal hemodynamics, including catheterization and magnetic resonance imaging were evaluated. Results: Four out of 12 animals survived, with the longest surviving animal living 3 years after single-stage Fontan. We showed a significant era effect regarding survival (1 out of 8 and subsequently 3 out of 4 animals surviving beyond 2 months) attributed in large part to the procedural learning curve. Key characteristics of Fontan hemodynamics, namely systemic venous hypertension and low normal cardiac output, were observed. However, recapitulation of passive human Fontan hemodynamics is affected by volume loading of the right ventricle given an anatomic difference in sheep azygous venous anatomy draining to the coronary sinus. Conclusions: A significant learning curve exists to ensure long-term survival and future surgical modifications, including banding of the main pulmonary artery and ligation of the azygous to coronary sinus connection are promising strategies to improve the fidelity of model hemodynamics.

Abstract Digestive Chagas disease (DCD) is an enteric neuropathy caused by Trypanosoma cruzi infection. There is a lack of evidence on the mechanism of pathogenesis and rationales for treatment. We used a female C3H/HeN mouse model that recapitulates key clinical manifestations to study how infection dynamics shape DCD pathology and the impact of treatment with the front-line, anti-parasitic drug benznidazole. Curative treatment 6 weeks post-infection resulted in sustained recovery of gastrointestinal transit function, whereas treatment failure led to infection relapse and gradual return of DCD symptoms. Neuro/immune gene expression patterns shifted from chronic inflammation to a tissue repair profile after cure, accompanied by increased cellular proliferation, glial cell marker expression and recovery of neuronal density in the myenteric plexus. Delaying treatment until 24 weeks post-infection led to partial reversal of DCD, suggesting the accumulation of permanent tissue damage over the course of chronic infection. Our study shows that murine DCD pathogenesis is sustained by chronic T. cruzi infection and is not an inevitable consequence of acute stage denervation. The risk of irreversible enteric neuromuscular tissue damage and dysfunction developing highlights the importance of prompt diagnosis and treatment. These findings support the concept of treating asymptomatic, T. cruzi-infected individuals with benznidazole to prevent DCD development.

The repurposing of approved drugs is an appealing method to fast-track the development of novel therapies for neglected diseases. Amiodarone and dronedarone, two approved antiarrhythmic agents, have been reported to have potential for the management of Chagas disease patients displaying symptomatic heart pathology. More recently, it has been suggested that both molecules not only have an antiarrhythmic effect, but also have trypanocidal activity against Trypanosoma cruzi, the causative agent of Chagas disease. In this work, we assessed the in vitro activity of these compounds against T. cruzi, the in vivo pharmacokinetics, and pharmacodynamics, to determine the potential for repurposing these drugs as therapies for Chagas disease. Based on these results, we were unable to reproduce the in vitro potencies of amiodarone and dronedarone described in the literature, and both drugs were found to be inactive or cytotoxic against a variety of different mammalian cell lines. The evaluation of in vivo efficacy in a bioluminescent murine model of T. cruzi did not show antiparasitic activity at the highest tolerated dose tested. While the potential of amiodarone and dronedarone as antiarrhythmic agents in Chagas cardiomyopathic patients cannot be completely excluded, a trypanocidal effect in patients treated with these two drugs appears unlikely.

Chronic Chagas cardiomyopathy (CCC) results from infection with the protozoan parasite Trypanosoma cruzi and is a prevalent cause of heart disease in endemic countries. We previously found that cardiac fibrosis can vary widely in C3H/HeN mice chronically infected with T. cruzi JR strain, mirroring the spectrum of heart disease in humans. In this study, we examined functional cardiac abnormalities in this host:parasite combination to determine its potential as an experimental model for CCC. We utilised electrocardiography (ECG) to monitor T. cruzi-infected mice and determine whether ECG markers could be correlated with cardiac function abnormalities. We found that the C3H/HeN:JR combination frequently displayed early onset CCC indicators, such as sinus bradycardia and right bundle branch block, as well as prolonged PQ, PR, RR, ST, and QT intervals in the acute stage. Our model exhibited high levels of cardiac inflammation and enhanced iNOS expression in the acute stage, but denervation did not appear to have a role in pathology. These results demonstrate the potential of the C3H/HeN:JR host:parasite combination as a model for CCC that could be used for screening new compounds targeted at cardiac remodelling and for examining the potential of antiparasitic drugs to prevent or alleviate CCC development and progression.

Abstract In the field of congenital heart surgery, tissue-engineered vascular grafts (TEVGs) are a promising alternative to traditionally used synthetic grafts. Our group has pioneered the use of TEVGs as a conduit between the inferior vena cava and the pulmonary arteries in the Fontan operation. The natural history of graft remodeling and its effect on hemodynamic performance has not been well characterized. In this study, we provide a detailed analysis of the first U.S. clinical trial evaluating TEVGs in the treatment of congenital heart disease. We show two distinct phases of graft remodeling: an early phase distinguished by rapid changes in graft geometry and a second phase of sustained growth and decreased graft stiffness. Using clinically informed and patient-specific computational fluid dynamics (CFD) simulations, we demonstrate how changes to TEVG geometry, thickness, and stiffness affect patient hemodynamics. We show that metrics of patient hemodynamics remain within normal ranges despite clinically observed levels of graft narrowing. These insights strengthen the continued clinical evaluation of this technology while supporting recent indications that reversible graft narrowing can be well tolerated, thus suggesting caution before intervening clinically.

Flaviviridae infections, such as those caused by hepatitis C (HCV) and dengue viruses (DENVs), represent global health risks. Infected people are in danger of developing chronic liver failure or hemorrhagic fever, both of which can be fatal if not treated. The tropical parasites Trypanosoma brucei and Trypanosoma cruzi cause enormous socioeconomic burdens in Sub-Saharan Africa and Latin America. Anti-HCV chemotherapy has severe adverse effects and is expensive, whereas dengue has no clinically authorized treatment. Antiparasitic medicines are often toxic and difficult to administer, and treatment failures are widely reported. There is an urgent need for new chemotherapies. Based on our previous research, we have undertaken structural modification of lead compound V with the goal of producing derivatives with both antiviral and trypanocidal activity. The novel spirocarbocyclic-substituted hydantoin analogs were designed, synthesized, and tested for antiviral activity against three HCV genotypes (1b, 3a, 4a), DENV, yellow fever virus (YFV), and two trypanosome species (T. brucei, T. cruzi). The optimization was successful and led to compounds with significant antiviral and trypanocidal activity and exceptional selectivity. Several modifications were made to further investigate the structure–activity relationships (SARs) and confirm the critical role of lipophilicity and conformational degrees of freedom.

In this work, we present the synthesis and the anti-trypanosomal activity of the 2-(4-(adamant-1-yl)phenyl)-N-hydroxyarylamides, 1a,b and the 2-(4-(adamant-1-yl)phenoxy)-N-hydroxyacetamide, 1c. The 4-(adamant-1-yl)phenyl- and 4-(adamant-1-yl)phenoxy- moieties, which are endowed with promising drug-like properties, are functionalized at the side chain termini as hydroxamic acids. The phenoxy acetohydroxamic derivative, 1c, shows the most interesting profile in terms of activity and toxicity against trypanosomes and merits further investigation.

A series of 6-amidinobenzothiazoles, linked via phenoxymethylene or directly to the 1,2,3-triazole ring with a p-substituted phenyl or benzyl moiety, were synthesised and evaluated in vitro against four human tumour cell lines and the protozoan parasite Trypanosoma brucei. The influence of the type of amidino substituent and phenoxymethylene linker on antiproliferative and antitrypanosomal activities was observed, showing that the imidazoline moiety had a major impact on both activities. Benzothiazole imidazoline 14a, which was directly connected to N-1-phenyl-1,2,3-triazole, had the most potent growth-inhibitory effect (IC50 = 0.25 µM) on colorectal adenocarcinoma (SW620), while benzothiazole imidazoline 11b, containing a phenoxymethylene linker, exhibited the best antitrypanosomal potency (IC90 = 0.12 µM). DNA binding assays showed a non-covalent interaction of 6-amidinobenzothiazole ligands, indicating both minor groove binding and intercalation modes of DNA interaction. Our findings encourage further development of novel structurally related 6-amidino-2-arylbenzothiazoles to obtain more selective anticancer and anti-HAT agents.

The prospect of eradicating malaria continues to be challenging in the face of increasing parasite resistance to antimalarial drugs so that novel antimalarials active against asexual, sexual, and liver-stage malaria parasites are urgently needed. In addition, new antimalarials need to be affordable and available to those most in need and, bearing in mind climate change, should ideally be sustainable. The West African climbing shrub Cryptolepis sanguinolenta is used traditionally for the treatment of malaria; its principal alkaloid, cryptolepine (1), has been shown to have antimalarial properties, and the synthetic analogue 2,7-dibromocryptolepine (2) is of interest as a lead toward new antimalarial agents. Cryptolepine (1) was isolated using a two-step Soxhlet extraction of C. sanguinolenta roots, followed by crystallization (yield 0.8% calculated as a base with respect to the dried roots). Semi-synthetic 7-bromo- (3), 7, 9-dibromo- (4), 7-iodo- (5), and 7, 9-dibromocryptolepine (6) were obtained in excellent yields by reaction of 1 with N-bromo- or N-iodosuccinimide in trifluoroacetic acid as a solvent. All compounds were active against Plasmodia in vitro, but 6 showed the most selective profile with respect to Hep G2 cells: P. falciparum (chloroquine-resistant strain K1), IC50 = 0.25 µM, SI = 113; late stage, gametocytes, IC50 = 2.2 µM, SI = 13; liver stage, P. berghei sporozoites IC50 = 6.13 µM, SI = 4.6. Compounds 3–6 were also active against the emerging zoonotic species P. knowlesi with 5 being the most potent (IC50 = 0.11 µM). In addition, 3–6 potently inhibited T. brucei in vitro at nM concentrations and good selectivity with 6 again being the most selective (IC50 = 59 nM, SI = 478). These compounds were also cytotoxic to wild-type ovarian cancer cells as well as adriamycin-resistant and, except for 5, cisplatin-resistant ovarian cancer cells. In an acute oral toxicity test in mice, 3–6 did not exhibit toxic effects at doses of up to 100 mg/kg/dose × 3 consecutive days. This study demonstrates that C. sanguinolenta may be utilized as a sustainable source of novel compounds that may lead to the development of novel agents for the treatment of malaria, African trypanosomiasis, and cancer.

Chronic Trypanosoma cruzi infections are typically lifelong, with small numbers of parasites surviving in restricted tissue sites, which include the gastrointestinal tract. There is considerable debate about the replicative status of these persistent parasites and whether there is a role for dormancy in long-term infection. Here, we investigated T. cruzi proliferation in the colon of chronically infected mice using 5-ethynyl-2′deoxyuridine incorporation into DNA to provide ‘snapshots’ of parasite replication status. Highly sensitive imaging of the extremely rare infection foci, at single-cell resolution, revealed that parasites are three times more likely to be in S-phase during the acute stage than during the chronic stage. By implication, chronic infections of the colon are associated with a reduced rate of parasite replication. Despite this, very few host cells survived infection for more than 14 days, suggesting that T. cruzi persistence continues to involve regular cycles of replication, host cell lysis and re-infection. We could find no evidence for wide-spread dormancy in parasites that persist in this tissue reservoir.

ABSTRACT Infections with Trypanosoma cruzi are usually lifelong despite generating a strong adaptive immune response. Identifying the sites of parasite persistence is therefore crucial to understanding how T. cruzi avoids immune-mediated destruction. However, this is a major technical challenge, because the parasite burden during chronic infections is extremely low. Here, we describe an integrated approach involving comprehensive tissue processing, ex vivo imaging, and confocal microscopy, which allowed us to visualize infected host cells in murine tissue with exquisite sensitivity. Using bioluminescence-guided tissue sampling, with a detection level of 200 parasites, which we term mega-nests. In contrast, during the acute stage, when the total parasite burden is considerably higher and many cells are infected, nests containing >50 parasites are rarely found. In C3H/HeN mice, but not BALB/c mice, we identified skeletal muscle as a major site of persistence during the chronic stage, with most parasites being found in large mega-nests within the muscle fibers. Finally, we report that parasites are also frequently found in the skin during chronic murine infections, often in multiple infection foci. In addition to being a site of parasite persistence, this anatomical reservoir could play an important role in insect-mediated transmission and have implications for drug development. IMPORTANCE Trypanosoma cruzi causes Chagas disease, the most important parasitic infection in Latin America. Major pathologies include severe damage to the heart and digestive tract, although symptoms do not usually appear until decades after infection. Research has been hampered by the complex nature of the disease and technical difficulties in locating the extremely low number of parasites. Here, using highly sensitive imaging technology, we reveal the sites of parasite persistence during chronic-stage infections of experimental mice at single-cell resolution. We show that parasites are frequently located in smooth muscle cells in the circular muscle layer of the colon and that skeletal muscle cells and the skin can also be important reservoirs. This information provides a framework for investigating how the parasite is able to survive as a lifelong infection, despite a vigorous immune response. It also informs drug development strategies by identifying tissue sites that must be accessed to achieve a curative outcome.