Delayed Activation of T Cells at the Site of Infection Facilitates the Establishment of Trypanosoma cruzi in Both Naive and Immune Hosts.

Although parasite entry through breaks in the skin or mucosa is one of the main routes of natural transmission of Trypanosoma cruzi, little is known about the host cell types initially invaded nor the ability of those host cells to initiate immune responses at the site of infection. To gain insights into these early events, we studied the fate of fluorescently tagged T. cruzi delivered subcutaneously in mouse footpads or ears. We demonstrate that the majority of parasites introduced into the skin initially proliferate there until 8 to 10 days postinfection, when the parasite load decreases. This decline in parasite numbers is dependent on the presence of an intact T cell compartment and on the ability of hosts to produce gamma interferon (IFN-γ). Many of the parasite-containing cells at the initial infection site display a macrophage/monocyte phenotype but with low expression of activation markers, suggesting that these cells provide an early niche for T. cruzi proliferation, rather than being active in parasite control. It is only after the first round of T. cruzi replication and release from host cells that signs of immune activation and control of parasites become apparent. The delay in the activation and failure to rapidly control parasite replication are observed even when T. cruzi-primed T cells are present, such as in chronically infected mice. This failure of a primed immune system to recognize and react prior to extensive parasite expansion at the infection site likely poses a significant challenge for the development of vaccines aiming to prevent T. cruzi infection. IMPORTANCE Trypanosoma cruzi, the parasite causing Chagas disease, usually infects through the mucosa or breaks in the skin, but little is known about the parasite's fate at the site of entry or the early events involving immune control there. Here, we track the local proliferation and subsequent dissemination of fluorescently tagged T. cruzi and the initial immune response at the point of entry. We show that T. cruzi preferentially infects innate immune cells in the skin and that the stimulation of an adaptive T cell response does not occur until after the release of parasites from this first round of infected host cells. This first immunologically "silent" proliferation occurs even in the presence of a strong immune T cell memory generated by previous infection. This capacity of T. cruzi to establish infections while avoiding initial immune recognition has important implications for the potential to develop vaccines to prevent T. cruzi infection.

Medical cost of acute diarrhea in children in ambulatory care.

OBJECTIVE: The aim of this study was to estimate the direct medical cost per episode and the annual cost for acute diarrhea (AD) in children under five years of age in Ambulatory Care Centers of the Ministry of Public Health (MOPH) of Ecuador. METHODS: A cost of illness study with a provider perspective was carried out through a micro-costing of health resources and valuated in international dollars. Medical consultations and laboratory tests were valued using the tariff framework of services for the National Health System and for the prescribed medications, a reported cost registry of pharmacy purchases made in the year of study was used. RESULTS: A total of 332 electronic health records of children under five years of age were included in the analysis. Laboratory tests were performed on 37.95% (126/332), medications were prescribed to 93.67% (311/332) of the children, and antimicrobials were prescribed to 37.35% (124/332) of the children, representing an antibiotic prescription rate of 26.51% (88/332) and an antiparasitic prescription rate of 10.84% (36/332). The mean cost of the MOPH per child per episode of AD was US$45.24 (2019 dollars) (95% CI:43.71 to 46.76). CONCLUSION: The total estimated cost of AD in children under five years of age for the MOPH in 2019 was about US$6,645,167.88 million (2019 dollars) (95% CI: 6,420,430.77 to 6,868,436.12). A high proportion of the direct medical cost of AD in children under five years of age in outpatient settings is due to unnecessary laboratory tests.

Prophylactic low-dose, bi-weekly benznidazole treatment fails to prevent Trypanosoma cruzi infection in dogs under intense transmission pressure.

Trypanosoma cruzi naturally infects a wide variety of wild and domesticated mammals, in addition to humans. Depending on the infection dose and other factors, the acute infection can be life-threatening, and in all cases, the risk of chagasic heart disease is high in persistently infected hosts. Domestic, working, and semi-feral dogs in the Americas are at significant risk of T. cruzi infection and in certain settings in the southern United States, the risk of new infections can exceed 30% per year, even with the use of vector control protocols. In this study, we explored whether intermittent low-dose treatment with the trypanocidal compound benznidazole (BNZ) during the transmission season, could alter the number of new infections in dogs in an area of known, intense transmission pressure. Preliminary studies in mice suggested that twice-weekly administration of BNZ could prevent or truncate infections when parasites were delivered at the mid-point between BNZ doses. Pre-transmission season screening of 126 dogs identified 53 dogs (42.1%) as T. cruzi infection positive, based upon blood PCR and Luminex-based serology. Serial monitoring of the 67 uninfected dogs during the high transmission season (May to October) revealed 15 (22.4%) new infections, 6 in the untreated control group and 9 in the group receiving BNZ prophylaxis, indicating no impact of this prophylaxis regimen on the incidence of new infections. Although these studies suggest that rigorously timed and more potent dosing regimen may be needed to achieve an immediate benefit of prophylaxis, additional studies would be needed to determine if drug prophylaxis reduced disease severity despite this failure to prevent new infections.

Discovery of an orally active benzoxaborole prodrug effective in the treatment of Chagas disease in non-human primates.

Trypanosoma cruzi, the agent of Chagas disease, probably infects tens of millions of people, primarily in Latin America, causing morbidity and mortality. The options for treatment and prevention of Chagas disease are limited and underutilized. Here we describe the discovery of a series of benzoxaborole compounds with nanomolar activity against extra- and intracellular stages of T. cruzi. Leveraging both ongoing drug discovery efforts in related kinetoplastids, and the exceptional models for rapid drug screening and optimization in T. cruzi, we have identified the prodrug AN15368 that is activated by parasite carboxypeptidases to yield a compound that targets the messenger RNA processing pathway in T. cruzi. AN15368 was found to be active in vitro and in vivo against a range of genetically distinct T. cruzi lineages and was uniformly curative in non-human primates (NHPs) with long-term naturally acquired infections. Treatment in NHPs also revealed no detectable acute toxicity or long-term health or reproductive impact. Thus, AN15368 is an extensively validated and apparently safe, clinically ready candidate with promising potential for prevention and treatment of Chagas disease.

Design and evaluation of neutralizing and fusion inhibitory peptides to Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever (CCHF) is a medically relevant tick-borne viral disease caused by the Bunyavirus, Crimean-Congo hemorrhagic fever virus (CCHFV). CCHFV is endemic to Asia, the Middle East, South-eastern Europe, and Africa and is transmitted in enzootic cycles among ticks, mammals, and birds. Human infections are mostly subclinical or limited to mild febrile illness. Severe disease may develop, resulting in multi-organ failure, hemorrhagic manifestations, and case-fatality rates up to 30%. Despite the widespread distribution and life-threatening potential, no treatments have been approved for CCHF. Antiviral inhibitory peptides, which antagonize viral entry, are licensed for clinical use in certain viral infections and have been experimentally designed against human pathogenic bunyaviruses, with in vitro and in vivo efficacies. We designed inhibitory peptides against CCHFV with and without conjugation to various polyethylene glycol and sterol groups. These additions have been shown to enhance both cellular uptake and antiviral activity. Peptides were evaluated against pseudotyped and wild-type CCHFV via neutralization tests, Nairovirus fusion assays, and cytotoxicity profiling. Four peptides neutralized CCHFV with two of these peptides shown to inhibit viral fusion. This work represents the development of experimental countermeasures for CCHF, describes a nairovirus immunofluorescence fusion assay, and illustrates the utility of pseudotyped CCHFV for the screening of entry antagonists at low containment settings for CCHF.

Impact of Cattle Feeding Strategy on the Beef Metabolome.

The present study explored changes in the meat metabolome of animals subjected to different finishing systems and growth rates. Thirty-six Angus × Nellore crossbred steers were used in a completely randomized design with four treatments: (1) feedlot system with high average daily gain (ADG; FH); (2) feedlot system with low ADG (FL); (3) pasture system with high ADG (PH); and (4) pasture system with low ADG (PL). After harvest and chilling, Longissimus thoracis (LT) muscle samples were taken for metabolite profile analysis using nuclear magnetic resonance. Spectrum was analyzed using chenomx software, and multi- and mega-variate data analyses were performed. The PLS-DA showed clear separation between FH and PL groups and overlap among treatments with different finishing systems but similar for matching ADG (FL and PH) treatments. Using a VIP cut-off of around 1.0, ATP and fumarate were shown to be greater in meat from PL cattle, while succinate, leucine, AMP, glutamate, carnosine, inosine, methionine, G1P, and choline were greater in meat from FH. Comparing FL and PH treatments, glutamine, carnosine, urea, NAD+, malonate, lactate, isoleucine, and alanine were greater in the meat of PH cattle, while G6P and betaine were elevated in that of FL cattle. Relevant pathways were also identified by differences in growth rate (FH versus PL) and finishing system were also noted. Growth rate caused a clear difference in meat metabolism that was highlighted by energy metabolism and associated pathways, while the feeding system tended to alter protein and lipid metabolism.

Quantitative 3D Imaging of Trypanosoma cruzi-Infected Cells, Dormant Amastigotes, and T Cells in Intact Clarified Organs.

Chagas disease is a neglected pathology that affects millions of people worldwide, mainly in Latin America. The Chagas disease agent, Trypanosoma cruzi (T. cruzi), is an obligate intracellular parasite with a diverse biology that infects several mammalian species, including humans, causing cardiac and digestive pathologies. Reliable detection of T. cruzi in vivo infections has long been needed to understand Chagas disease's complex biology and accurately evaluate the outcome of treatment regimens. The current protocol demonstrates an integrated pipeline for automated quantification of T. cruzi-infected cells in 3D-reconstructed, cleared organs. Light-sheet fluorescent microscopy allows for accurately visualizing and quantifying of actively proliferating and dormant T. cruzi parasites and immune effector cells in whole organs or tissues. Also, the CUBIC-HistoVision pipeline to obtain uniform labeling of cleared organs with antibodies and nuclear stains was successfully adopted. Tissue clearing coupled with 3D immunostaining provides an unbiased approach to comprehensively evaluate drug treatment protocols, improve the understanding of the cellular organization of T. cruzi-infected tissues, and is expected to advance discoveries related to anti-T. cruzi immune responses, tissue damage, and repair in Chagas disease.

Metabolome fingerprints, performance and carcass quality of beef calves supplemented with antibiotic free additive.

The aim of this study was to investigate the effects of probiotics on pre-weaning performance, muscle, and fat deposition and serum metabolite profiles in male and female Senepol calves. Thirty new-born Senepol calves, 15 males and 15 females, were randomly allocated to the following treatments: CON a control group that received the basal creep feeding diet and PRO animals that received the basal diet with addition of 2 g/100 kg of body weight (BW) of probiotic. PRO supplementation did not change the DMI but increased average BW, final BW, ADG relative to animals fed CON. Additionally, PRO improved LMA and marbling. Regardless of the serum metabolite profile, the important metabolites for discriminating PRO and CON were glutamine, leucine, creatine, acetate, creatinine, arginine, glutamate, hippurate, glycerol, carnitine, lactate, carnosine, myo-inositol and histidine. According to gender, males had an overabundance of glutamine, glycerol, isoleucine, creatinine and glucose, whereas females had an overabundance of acetyl carnitine, glutamate and carnitine. In conclusion, the addition of PRO in the pre-weaning diet of calves increases performance, weight at weaning and muscle and fat deposition on the carcass, improving proteins and fatty acid metabolism, the immune system response and rumen development.

Metabolomic signature of genetic potential for muscularity in beef cattle.

The aim of this study was to investigate the serum and meat metabolomic changes according to the genetic potential for muscularity of non-castrated Nellore males and its association with phenotypic traits. Forty-eight non-castrated Nellore males were separated into two groups based on their genetic potential for post-weaning muscularity: high (HM) and low (LM). Selection for muscularity did not cause noticeable differences in the traits evaluated during the finishing phase and after slaughter. However, several metabolites in meat and serum, have changed according to the muscularity group. HM animals presented an over-abundance of glycerol, glutamine, choline, methylhistidine, betaine, creatinine and methionine in serum, compared with their LM counterparts. Similarly, the meat samples of HM animals were rich in glucose-6-phosphate, lactate, pyruvate, creatinine, betaine, choline, glycerol and arginine relative to LM bulls. Inosine monophosphate was the only metabolite over-abundant in LM animals. In conclusion, the genetic potential for post-weaning muscularity did not affect performance during the finishing phase, carcass traits and meat quality. However, multivariate analysis shows that the genetic potential of muscularity can be correlated with serum lipid and protein metabolites, and with energy metabolism in meat, providing a footprint of cattle muscularity metabolism.

Using TD-NMR relaxometry and 1D 1H NMR spectroscopy to evaluate aging of Nellore beef.

The aim of this study was to explore the use of TD-NMR relaxometry and 1H NMR spectroscopy-based for detecting differences in meat quality attributes. There was limited association between various TD-NMR signals and any physicochemical parameters of fresh and aged meat differing in tenderness ratings. Samples were then divided into three groups based on statistical changes in metabolite concentration. Group A samples possessed near linear increases in metabolite concentration over aging time; whereas samples assigned to Groups B and C were characterized by increases in metabolites that peaked between 7 and 14 days, and up to 14 days aging, respectively. 1H NMR spectroscopy discriminated meat quality using changes in metabolites reflective of glycolysis, the citric acid cycle, protein degradation, amino acid generation and purine metabolisms. These data suggest segregation of meat quality is possible using both NMR technologies but additional work is necessary to understand fully their utility in a commercial industry setting.

High variation in immune responses and parasite phenotypes in naturally acquired Trypanosoma cruzi infection in a captive non-human primate breeding colony in Texas, USA.

Trypanosoma cruzi, the causative agent of human Chagas disease, is endemic to the southern region of the United States where it routinely infects many host species. The indoor/outdoor housing configuration used in many non-human primate research and breeding facilities in the southern of the USA provides the opportunity for infection by T. cruzi and thus provides source material for in-depth investigation of host and parasite dynamics in a natural host species under highly controlled and restricted conditions. For cynomolgus macaques housed at such a facility, we used a combination of serial blood quantitative PCR (qPCR) and hemoculture to confirm infection in >92% of seropositive animals, although each method alone failed to detect infection in >20% of cases. Parasite isolates obtained from 43 of the 64 seropositive macaques were of 2 broad genetic types (discrete typing units, (DTU's) I and IV); both within and between these DTU groupings, isolates displayed a wide variation in growth characteristics and virulence, elicited host immune responses, and susceptibility to drug treatment in a mouse model. Likewise, the macaques displayed a diversity in T cell and antibody response profiles that rarely correlated with parasite DTU type, minimum length of infection, or age of the primate. This study reveals the complexity of infection dynamics, parasite phenotypes, and immune response patterns that can occur in a primate group, despite being housed in a uniform environment at a single location, and the limited time period over which the T. cruzi infections were established.

Preliminary study on the characterization of Longissimus lumborum dark cutting meat in Angus × Nellore crossbreed cattle using NMR-based metabolomics.

The aim of this preliminary study was to evaluate meat quality properties, muscle metabolite profile and metabolic pathways associated with the occurrence of dark cutting meat in Angus x Nellore crossbreed cattle. After 14 days' ageing, dark cutting meat presented a higher pH, lower cooking loss and colour parameters, and greater tenderness compared with normal meat. Dark cutting meat had a higher ATP level and lower concentrations of glucose-6-phosphate, lactate, glucose, serine, threonine, creatine phosphate, inosine, leucine, methionine, succinate and glucose-1-phosphate compared to normal meat. In dark cutting samples, the ultimate pH was positively correlated with carnitine and negatively correlated with glucose-6-phosphate. However, in normal meat, the ultimate pH presented a positive correlation with arginine, leucine, methionine, proline, threonine, tyrosine and valine. Pathway analysis showed that differentiation of the groups was linked to energetic pathways such as starch and sucrose metabolism, the pentose phosphate pathway, amino sugar, nucleotide sugar metabolism, and glycolysis or gluconeogenesis. In conclusion, the occurrence of dark cutting meat has a notable impact on meat quality attributes and concentrations of post-mortem glycolytic metabolites, appears to be correlated with mitochondrial activity and affects energetic metabolic pathways.

Characterization of chicken muscle disorders through metabolomics, pathway analysis, and water relaxometry: a pilot study.

Metabolite profiles of chicken breast extracts and water mobility in breasts were studied using proton nuclear magnetic resonance (1H-NMR) spectroscopy and time-domain NMR (TD-NMR) relaxometry, respectively, using normal breast (NB), and wooden breast (WB) and white striping (WS) myopathies in broilers. One thousand eight hundred sixty broilers were raised to commercial standards, receiving the same diets that were formulated as per the different growth stages. At 49 D of age, 200 animals were slaughtered following routine commercial procedures, and at 4 h postmortem, the whole breast (pectoralis major muscle) was removed and visually inspected by an experienced meat inspector who selected NB (without myopathies) and samples with the presence of WS and WB myopathies. Fifteen breasts (5 each of NB, WS, and WB) were analyzed through TD-NMR relaxometry, and samples of approximately 20 g were taken from each breast and frozen at -80°C for metabolite profiling through 1H-NMR spectroscopy. Multivariate statistical analysis was used to evaluate the effect on water relaxometry and metabolite profile in accordance with the presence and type of myopathy in the breast. 1H-NMR data showed that the metabolite profiles in WS and WB breasts were different from each other and from NB. This pilot study shows that myopathies appear to be related to hypoxia, connective tissue deposition, lower mitochondrial function, and greater oxidative stress compared with NB. The longitudinal and transverse relaxation time of the breasts determined by TD-NMR relaxometry was shorter for NB than that for WS and WB, indicating greater water mobility in breasts affected by myopathies. 1H-NMR spectroscopy can be used to differentiate the metabolism of WS, WB, and NB, and TD-NMR has the potential to be a fast, simple, and noninvasive method to distinguish NB from WB and WS. As a practical application, the metabolomic profile as per the occurrence of breast myopathies may be used for a better understanding of these issues, which opens a gap to mitigate the incidence and severity of WS and WB. In addition, the present study brings an opportunity for the development of a new and objective tool to classify the incidence of breast myopathies through TD-NMR relaxometry.

A modified drug regimen clears active and dormant trypanosomes in mouse models of Chagas disease.

A major contributor to treatment failure in Chagas disease, caused by infection with the protozoan parasite Trypanosoma cruzi, is that current treatment regimens do not address the drug insensitivity of transiently dormant T. cruzi amastigotes. Here, we demonstrated that use of a currently available drug in a modified treatment regimen of higher individual doses, given less frequently over an extended treatment period, could consistently extinguish T. cruzi infection in three mouse models of Chagas disease. Once per week administration of benznidazole at a dose 2.5 to 5 times the standard daily dose rapidly eliminated actively replicating parasites and ultimately eradicated the residual, transiently dormant parasite population in mice. This outcome was initially confirmed in "difficult to cure" mouse infection models using immunological, parasitological, and molecular biological approaches and ultimately corroborated by whole organ analysis of optically clarified tissues using light sheet fluorescence microscopy (LSFM). This tool was effective for monitoring pathogen load in intact organs, including detection of individual dormant parasites, and for assessing treatment outcomes. LSFM-based analysis also suggested that dormant amastigotes of T. cruzi may not be fully resistant to trypanocidal compounds such as benznidazole. Collectively, these studies provide important information on the phenomenon of dormancy in T. cruzi infection in mice, demonstrate methods to therapeutically override dormancy using a currently available drug, and provide methods to monitor alternative therapeutic approaches for this, and possibly other, low-density infectious agents.

A straightforward catalytic approach to obtain deuterated chloroform at room temperature.

We report the catalytic activity for the complexes-cis-[RuCl2 (dppb)(bipy)] (A), and [η6 -(p-cymene)Ru (dppb)Cl]PF6 (B), wherein dppb = 1,4-bis(diphenylphosphine)butane, and bipy = 2,2'-bipyridine-for the synthesis of CDCl3 from CHCl3 using D2 O as deuterium source. H/D exchange reactions were performed using a chloroform/D2 O, 1:2 molar ratio, vigorously stirred, at room temperature. One mole of KOH was dissolved in D2 O fraction and catalytic complexes from 0.002 to 0.05 mmol were dissolved in chloroform. The H/D exchange reactions were monitored using 13 C nuclear magnetic resonance sequences without proton decoupling. The reaction using 0.01 mmol of compound A reached approximately 55% of H/D conversion in 1 h. In the same time, the reactions with 0.002 mmol of compound A and without catalyst show approximately 28% and 3% H/D exchange, respectively. Without the catalysts, the H/D exchange was only 12.0% in 5 h. For compound B, 55% H/D conversion was observed in 1 h, only when 0.05 mmol was used, which is much higher catalyst concentration. After the isolation of the chloroform fraction and two more addition of D2 O, it was possible to obtain 95.0% H/D exchange in approximately 3 h, using 0.01 mmol of the compound A. Therefore, compound A is an efficient catalyst for a rapid and straightforward synthesis of CDCl3 from CHCl3 at room temperature and using D2 O as deuterium source.

Selection for Growth and Precocity Alters Muscle Metabolism in Nellore Cattle.

To clarify the relationship between beef genetic selection for growth and precocity with muscle metabolism and metabolites, we performed metabolomic analysis using Longissimus lumborum (LL) muscle from Nellore cattle with divergent selection for these traits (high growth, HG; low growth, LG; high precocity, HP; low precocity, LP). Genetic potential for growth affected muscle protein and energetic metabolism. HG animals had a high concentration of arginine, carnosine, and leucine compared to LG animals. HP animals presented a high concentration of glutamine, betaine, creatinine, isoleucine, carnitine, acetyl carnitine, and lower levels of glucose compared to LP animals, affecting protein and fatty acid metabolism. Intensity of selection (high or low) was correlated with changes in protein metabolism, and the type of selection (growth or precocity) affected fat metabolism. In conclusion, both HG and HP appear to be correlated with a high concentration of protein metabolites and changes in protein metabolic pathways, while selection for precocity is more correlated with changes in fat metabolism compared to animals selected for growth.

Chagas Disease Drug Discovery: Multiparametric Lead Optimization against Trypanosoma cruzi in Acylaminobenzothiazole Series.

Acylaminobenzothiazole hits were identified as potential inhibitors of Trypanosoma cruzi replication, a parasite responsible for Chagas disease. We selected compound 1 for lead optimization, aiming to improve in parallel its anti-T. cruzi activity (IC50 = 0.63 µM) and its human metabolic stability (human clearance = 9.57 mL/min/g). A total of 39 analogues of 1 were synthesized and tested in vitro. We established a multiparametric structure-activity relationship, allowing optimization of antiparasite activity, physicochemical parameters, and ADME properties. We identified compound 50 as an advanced lead with an improved anti-T. cruzi activity in vitro (IC50 = 0.079 µM) and an enhanced metabolic stability (human clearance = 0.41 mL/min/g) and opportunity for the oral route of administration. After tolerability assessment, 50 demonstrated a promising in vivo efficacy.

In Situ Detection of Dormant Trypanosoma cruzi Amastigotes Using Bioluminescent-Fluorescent Reporters.

Chagas disease agent, Trypanosoma cruzi, is capable to persist after prolonged drug treatment using effective drugs. The reason of treatment failure is not known, but recent development of highly sensible bioluminescence imaging coupled to tissue clarification techniques has made possible the detection of individual amastigotes within chronically infected murine tissues and the study of their replicative status. In this chapter, we provide a step-by-step explanation for these protocols that allowed the visualization of nonproliferating amastigotes in tissues of chronically infected mice for the first time.

Magnetic resonance studies of copper (II) sorbitol complex, in solution, reveal a supramolecular structure compatible to the crystal structure.

Although the Cu2+ -sorbitol complex [Cu2+ -Sorb] structure in crystalline state has been determined by X rays, it is not known in solution, where most studies of this complex are performed. Therefore, the goal of this work was to obtain information about the structure of this complex in aqueous solution using nuclear magnetic resonance and electron paramagnetic resonance spectroscopies. The magnetic resonance results indicate that the complex is formed at approximately pH 12. In this pH the sorbitol 1 H relaxation times were so short (broad line) that was not possible to use standard nuclear magnetic resonance parameters (nuclear Overhauser effect and spin-spin coupling constants values) to solve the three-dimensional structure. However, valuable structural information about the complex in solution was obtained. The relaxation results indicate that the Cu2+ ions are buried in the structure and not accessible to solvent; the 1 H and 13 C spectra shows strong paramagnetic shift effect indicating short distance between these nuclei and Cu2+ in the structure. No electron paramagnetic resonance signal was observed in pH 12 indicating strong Cu2+ - Cu2+ dipolar interaction, compatible to Cu2+ -Cu2+ distances measured in crystal, from 1.148 to 1.393 Angstroms. The complex self-diffusion coefficient (D) of 1.58 × 10-10  m2 /s value, determined by Diffusion-Ordered Spectroscopy, is compatible to a molecular weight of 3-6 KDa. Therefore, these results corroborate that the [Cu2+ -Sorb] complex is assembled in solution, at pH 12, with several structural parameters compatible to the toroidal hexadecacuprate supramolecular structure determined in solid state.

Spontaneous dormancy protects Trypanosoma cruzi during extended drug exposure.

The ability of the Chagas disease agent Trypanosoma cruzi to resist extended in vivo exposure to highly effective trypanocidal compounds prompted us to explore the potential for dormancy and its contribution to failed drug treatments in this infection. We document the development of non-proliferating intracellular amastigotes in vivo and in vitro in the absence of drug treatment. Non-proliferative amastigotes ultimately converted to trypomastigotes and established infections in new host cells. Most significantly, dormant amastigotes were uniquely resistant to extended drug treatment in vivo and in vitro and could re-establish a flourishing infection after as many as 30 days of drug exposure. These results demonstrate a dormancy state in T. cruzi that accounts for the failure of highly cytotoxic compounds to completely resolve the infection. The ability of T. cruzi to establish dormancy throws into question current methods for identifying curative drugs but also suggests alternative therapeutic approaches.