Susceptibility of Leishmania to novel pentavalent organometallics: Investigating impact on DNA and membrane integrity in antimony(III)-sensitive and -resistant strains.

The aim the present study was to investigate the impact of novel pentavalent organobismuth and organoantimony complexes on membrane integrity and their interaction with DNA, activity against Sb(III)-sensitive and -resistant Leishmania strains and toxicity in mammalian peritoneal macrophages. Ph3M(L)2 type complexes were synthesized, where M = Sb(V) or Bi(V) and L = deprotonated 3-(dimethylamino)benzoic acid or 2-acetylbenzoic acid. Both organobismuth(V) and organoantimony(V) complexes exhibited efficacy at micromolar concentrations against Leishmania amazonensis and L. infantum but only the later ones demonstrated biocompatibility. Ph3Sb(L1)2 and Ph3Bi(L1)2 demonstrated distinct susceptibility profiles compared to inorganic Sb(III)-resistant strains of MRPA-overexpressing L. amazonensis and AQP1-mutated L. guyanensis. These complexes were able to permeate the cell membrane and interact with the Leishmania DNA, suggesting that this effect may contribute to the parasite growth inhibition via apoptosis. Taken altogether, our data substantiate the notion of a distinct mechanism of uptake pathway and action in Leishmania for these organometallic complexes, distinguishing them from the conventional inorganic antimonial drugs.

Next-Generation Leishmanization: Revisiting Molecular Targets for Selecting Genetically Engineered Live-Attenuated Leishmania.

Despite decades of research devoted to finding a vaccine against leishmaniasis, we are still lacking a safe and effective vaccine for humans. Given this scenario, the search for a new prophylaxis alternative for controlling leishmaniasis should be a global priority. Inspired by leishmanization-a first generation vaccine strategy where live L. major parasites are inoculated in the skin to protect against reinfection-live-attenuated Leishmania vaccine candidates are promising alternatives due to their robust elicited protective immune response. In addition, they do not cause disease and could provide long-term protection upon challenge with a virulent strain. The discovery of a precise and easy way to perform CRISPR/Cas-based gene editing allowed the selection of safer null mutant live-attenuated Leishmania parasites obtained by gene disruption. Here, we revisited molecular targets associated with the selection of live-attenuated vaccinal strains, discussing their function, their limiting factors and the ideal candidate for the next generation of genetically engineered live-attenuated Leishmania vaccines to control leishmaniasis.

Exploring direct and indirect targets of current antileishmanial drugs using a novel thermal proteomics profiling approach.

Visceral leishmaniasis (VL), caused by Leishmania infantum, is an oft-fatal neglected tropical disease. In the absence of an effective vaccine, the control of leishmaniasis relies exclusively on chemotherapy. Due to the lack of established molecular/genetic markers denoting parasite resistance, clinical treatment failure is often used as an indicator. Antimony-based drugs have been the standard antileishmanial treatment for more than seven decades, leading to major drug resistance in certain regions. Likewise, drug resistance to miltefosine and amphotericin B continues to spread at alarming rates. In consequence, innovative approaches are needed to accelerate the identification of antimicrobial drug targets and resistance mechanisms. To this end, we have implemented a novel approach based on thermal proteome profiling (TPP) to further characterize the mode of action of antileishmanials antimony, miltefosine and amphotericin B, as well as to better understand the mechanisms of drug resistance deployed by Leishmania. Proteins become more resistant to heat-induced denaturation when complexed with a ligand. In this way, we used multiplexed quantitative mass spectrometry-based proteomics to monitor the melting profile of thousands of expressed soluble proteins in WT, antimony-resistant, miltefosine-resistant, and amphotericin B-resistant L. infantum parasites, in the presence (or absence) of the above-mentioned drugs. Bioinformatics analyses were performed, including data normalization, melting profile fitting, and identification of proteins that underwent changes (fold change > 4) caused by complexation with a drug. With this unique approach, we were able to narrow down the regions of the L. infantum proteome that interact with antimony, miltefosine, and amphotericin B; validating previously-identified and unveiling novel drug targets. Moreover, analyses revealed candidate proteins potentially involved in drug resistance. Interestingly, we detected thermal proximity coaggregation for several proteins belonging to the same metabolic pathway (i.e., tryparedoxin peroxidase and aspartate aminotransferase in proteins exposed to antimony), highlighting the importance of these pathways. Collectively, our results could serve as a jumping-off point for the future development of innovative diagnostic tools for the detection and evaluation of antimicrobial-resistant Leishmania populations, as well as open the door for new on-target therapies.

Leishmania amazonensis from distinct clinical forms/hosts has polymorphisms in Lipophosphoglycans, displays variations in immunomodulatory properties and, susceptibility to antileishmanial drugs.

Lipophosphoglycan (LPG), the major Leishmania glycoconjugate, induces pro-inflammatory/immunosuppressive innate immune responses. Here, we evaluated functional/biochemical LPG properties from six Leishmania amazonensis strains from different hosts/clinical forms. LPGs from three strains (GV02, BA276, and LV79) had higher pro-inflammatory profiles for most of the mediators, including tumor necrosis factor alpha and interleukin 6. For this reason, glycoconjugates from all strains were biochemically characterized and had polymorphisms in their repeat units. They consisted of three types: type I, repeat units devoid of side chains; type II, containing galactosylated side chains; and type III, containing glucosylated side chains. No relationship was observed between LPG type and the pro-inflammatory properties. Finally, to evaluate the susceptibility against antileishmanial agents, two strains with high (GV02, BA276) and one with low (BA336) pro-inflammatory activity were selected for chemotherapeutic tests in THP-1 cells. All analyzed strains were susceptible to amphotericin B (AmB) but displayed various responses against miltefosine (MIL) and glucantime (GLU). The GV02 strain (canine visceral leishmaniasis) had the highest IC50 for MIL (3.34 µM), whereas diffuse leishmaniasis strains (BA276 and BA336) had a higher IC50 for GLU (6.87-12.19 mM). The highest IC50 against MIL shown by the GV02 strain has an impact on clinical management. Miltefosine is the only drug approved for dog treatment in Brazil. Further studies into drug susceptibility of L. amazonensis strains are warranted, especially in areas where dog infection by this species overlaps with those caused by Leishmania infantum.

Validation of a colorimetric LAMP to detect Loxosceles experimental envenomation.

Diagnostic tests for brown spider accidents are unavailable and impact treatment decisions, increasing costs and patient risks. In this work, we used for the first time a fast, simple, and visual method based on the loop-mediated isothermal amplification assay (LAMP) to detect Loxosceles envenomation. Using the DNA from L. similis legs, we observed a high sensitivity using this test since as low as 0.32 pg of DNA could be detected. This pH-dependent colorimetric assay was 64 times more sensitive than PCR to detect spider DNA. The test was specific for Loxosceles once no cross-reaction was observed when testing DNA from different agents that cause similar dermonecrotic injuries. The test allowed the detection of Loxosceles intermedia DNA from hair, serum, and exudate samples obtained from experimentally-envenomed rabbit within 72 h. The method sensitivity varied according to the sample and the collection time, reaching 100% sensitivity in serum and hair, respectively, 1 h and 24 h after the experimental envenomation. Due to its ease of execution, speed, sensitivity, and specificity, LAMP presents an excellent potential for identifying Loxosceles spp. Envenomation. This can reduce the burden on the Health System and the morbidity for the patient by implementing the appropriate therapy immediately.In addition, this work opens up the perspective to other venomous animal accident identification using LAMP.

Sex under pressure: stress facilitates Leishmania in vitro hybridization.

The selection of Leishmania hybrids in axenic culture was considered rare until recently, when Louradour and Ferreira et al., demonstrated that induced DNA damage facilitates genetic exchange, resulting in full genome tetraploid progenies in vitro. Meiosis-related gene homologues HAP2, GEX1, and RAD51 were found to be involved, opening new avenues for functional genomic studies.

Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern.

The coronavirus disease 2019 (COVID-19) pandemic unfolded due to the widespread severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission reinforced the urgent need for affordable molecular diagnostic alternative methods for massive testing screening. We present the clinical validation of a pH-dependent colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) for SARS-CoV-2 detection. The method revealed a limit of detection of 19.3 ± 2.7 viral genomic copies/µL when using RNA extracted samples obtained from nasopharyngeal swabs collected in guanidine-containing viral transport medium. Typical RT-LAMP reactions were performed at 65°C for 30 min. When compared to reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), up to cycle-threshold (Ct) value 32, RT-LAMP presented 98% [95% confidence interval (CI) = 95.3-99.5%] sensitivity and 100% (95% CI = 94.5-100%) specificity for SARS-CoV-2 RNA detection targeting E and N genes. No cross-reactivity was detected when testing other non-SARS-CoV virus, confirming high specificity. The test is compatible with primary RNA extraction-free samples. We also demonstrated that colorimetric RT-LAMP can detect SARS-CoV-2 variants of concern and variants of interest, such as variants occurring in Brazil named gamma (P.1), zeta (P.2), delta (B.1.617.2), B.1.1.374, and B.1.1.371. The method meets point-of-care requirements and can be deployed in the field for high-throughput COVID-19 testing campaigns, especially in countries where COVID-19 testing efforts are far from ideal to tackle the pandemics. Although RT-qPCR is considered the gold standard for SARS-CoV-2 RNA detection, it requires expensive equipment, infrastructure, and highly trained personnel. In contrast, RT-LAMP emerges as an affordable, inexpensive, and simple alternative for SARS-CoV-2 molecular detection that can be applied to massive COVID-19 testing campaigns and save lives.

Network-Based Approaches Reveal Potential Therapeutic Targets for Host-Directed Antileishmanial Therapy Driving Drug Repurposing.

Leishmania parasites are the causal agent of leishmaniasis, an endemic disease in more than 90 countries worldwide. Over the years, traditional approaches focused on the parasite when developing treatments against leishmaniasis. Despite numerous attempts, there is not yet a universal treatment, and those available have allowed for the appearance of resistance. Here, we propose and follow a host-directed approach that aims to overcome the current lack of treatment. Our approach identifies potential therapeutic targets in the host cell and proposes known drug interactions aiming to improve the immune response and to block the host machinery necessary for the survival of the parasite. We started analyzing transcription factor regulatory networks of macrophages infected with Leishmania major. Next, based on the regulatory dynamics of the infection and available gene expression profiles, we selected potential therapeutic target proteins. The function of these proteins was then analyzed following a multilayered network scheme in which we combined information on metabolic pathways with known drugs that have a direct connection with the activity carried out by these proteins. Using our approach, we were able to identify five host protein-coding gene products that are potential therapeutic targets for treating leishmaniasis. Moreover, from the 11 drugs known to interact with the function performed by these proteins, 3 have already been tested against this parasite, verifying in this way our novel methodology. More importantly, the remaining eight drugs previously employed to treat other diseases, remain as promising yet-untested antileishmanial therapies. IMPORTANCE This work opens a new path to fight parasites by targeting host molecular functions by repurposing available and approved drugs. We created a novel approach to identify key proteins involved in any biological process by combining gene regulatory networks and expression profiles. Once proteins have been selected, our approach employs a multilayered network methodology that relates proteins to functions to drugs that alter these functions. By applying our novel approach to macrophages during the Leishmania infection process, we both validated our work and found eight drugs already approved for use in humans that to the best of our knowledge were never employed to treat leishmaniasis, rendering our work as a new tool in the box available to the scientific community fighting parasites.

Three different mutations in the DNA topoisomerase 1B in Leishmania infantum contribute to resistance to antitumor drug topotecan.

BACKGROUND: The evolution of drug resistance is one of the biggest challenges in leishmaniasis and has prompted the need for new antileishmanial drugs. Repurposing of approved drugs is a faster and very attractive strategy that is gaining supporters worldwide. Different anticancer topoisomerase 1B (TOP1B) inhibitors have shown strong antileishmanial activity and promising selective indices, supporting the potential repurposing of these drugs. However, cancer cells and Leishmania share the ability to become rapidly resistant. The aim of this study was to complete a whole-genome exploration of the effects caused by exposure to topotecan in order to highlight the potential mechanisms deployed by Leishmania to favor its survival in the presence of a TOP1B inhibitor. METHODS: We used a combination of stepwise drug resistance selection, whole-genome sequencing, functional validation, and theoretical approaches to explore the propensity of and potential mechanisms deployed by three independent clones of L. infantum to resist the action of TOP1B inhibitor topotecan. RESULTS: We demonstrated that L. infantum is capable of becoming resistant to high concentrations of topotecan without impaired growth ability. No gene deletions or amplifications were identified from the next-generation sequencing data in any of the three resistant lines, ruling out the overexpression of efflux pumps as the preferred mechanism of topotecan resistance. We identified three different mutations in the large subunit of the leishmanial TOP1B (Top1BF187Y, Top1BG191A, and Top1BW232R). Overexpression of these mutated alleles in the wild-type background led to high levels of resistance to topotecan. Computational molecular dynamics simulations, in both covalent and non-covalent complexes, showed that these mutations have an effect on the arrangement of the catalytic pentad and on the interaction of these residues with surrounding amino acids and DNA. This altered architecture of the binding pocket results in decreased persistence of topotecan in the ternary complex. CONCLUSIONS: This work helps elucidate the previously unclear potential mechanisms of topotecan resistance in Leishmania by mutations in the large subunit of TOP1B and provides a valuable clue for the design of improved inhibitors to combat resistance in both leishmaniasis and cancer. Our data highlights the importance of including drug resistance evaluation in drug discovery cascades.

The mutation G133D on Leishmania guyanensis AQP1 is highly destabilizing as revealed by molecular modeling and hypo-osmotic shock assay.

The Leishmania aquaglyceroporin 1 (AQP1) plays an important role in osmoregulation and antimony (Sb) uptake, being determinant for resistance to antimony. We have previously demonstrated that G133D mutation on L. guyanensis AQP1 (LgAQP1) leads to reduced Sb uptake. Here, we investigated the effects of G133D mutation on LgAQP1 structure, associated with Sb uptake and alterations in osmoregulation capacity. High confidence molecular models of wild-type LgAQP1 as well as the LgAQP1::G133D mutant were constructed and optimized via comparative homology modeling. Computational methods from the mCSM platform were used to evaluate the effects on protein stability and on its ability to bind to glycerol. Functional validation of the disruptive effect of the mutation on LgAQP1 was done by challenging the parasites with hypo-osmotic chock. Glycine 133 is on transmembrane helix 3, buried in the membrane in both open and closed conformation. G133D mutation was predicted to be highly destabilizing, as it alters the helical bundling arrangement in order to accommodate the aspartic acid side chain. The shift in helices also resulted in fewer favorable contacts with glycerol in the channel, which would explain the reduced affinity for similar small molecules as SbO3. Under hypo-osmotic condition, L. guyanensis AQP1G133D presented a 3-fold increase in cellular volume and pronounced delay to recover osmosis homeostasis when compared to the wild-type, a profile that was enhanced in LgAQP1-/- mutants. In conclusion, G133D is a highly disruptive mutation that will destabilize the monomer, compromise tetramer formation and alter pore conformation, leading to reduced Sb uptake and deficient osmoregulation.

Clinical validation of colorimetric RT-LAMP, a fast, highly sensitive and specific COVID-19 molecular diagnostic tool that is robust to detect SARS-CoV-2 variants of concern

The COVID-19 pandemics unfolded due to the widespread SARS-CoV-2 transmission reinforced the urgent need for affordable molecular diagnostic alternative methods for massive testing screening. We present the clinical validation of a pH-dependent colorimetric RT-LAMP (reverse transcription loop-mediated isothermal amplification) for SARS-CoV-2 detection. The method revealed a limit of detection of 19.3 {+/-} 2.7 viral genomic copies/L when using RNA extracted samples obtained from nasopharyngeal swabs collected in guanidine-containing viral transport medium. Typical RT-LAMP reactions were performed at 65 {o}C for 30 min. When compared to RT-qPCR, up to Ct value 32, RT-LAMP presented 97% (87.4-99.4% 95% CI) sensitivity and 100% (86.2-100%) specificity for SARS-CoV-2 RNA detection targeting N gene. No cross-reactivity was detected when testing other non-SARS-CoV virus, confirming high specificity. The test is compatible with primary RNA extraction free samples. We also demonstrated that colorimetric RT-LAMP can detect SARS-CoV-2 variants of concern (VOC) and variants of interest (VOI), such as variants occurring in Brazil named P.1, P.2, B.1.1.374 and B.1.1.371. The method meets point-of-care requirements and can be deployed in the field for high-throughput COVID-19 testing campaigns, especially in countries where COVID-19 testing efforts are far from ideal to tackle the pandemics. Although RT-qPCR is considered the gold standard for SARS-CoV-2 RNA detection, it requires expensive equipments, infrastructure and highly trained personnel. In contrast, RT-LAMP emerges as an affordable, inexpensive and simple alternative for SARS-CoV-2 molecular detection that can be applied to massive COVID-19 testing campaigns and save lives.

Impact of Homeopathic Treatment on the Quality of Life of Women with Chronic Diseases: A Randomized Controlled Pragmatic Trial.

BACKGROUND: Despite the increasing demand for complementary and integrative medicine, only a few studies have evaluated the effect of these types of treatments on the quality of life (QoL) of patients with chronic diseases. The objective of this study was to evaluate the QoL of women treated with homeopathy within the Public Health System of Belo Horizonte, Brazil. METHODS: This is a prospective randomized controlled pragmatic trial. The patients were divided into two independent groups, one group underwent homeopathic treatment in the first 6-month period and the other did not receive any homeopathic treatment. In both randomized groups, patients maintained their conventional medical treatment when necessary. The World Health Organization Quality of Life abbreviated questionnaire (WHOQOL-BREF) was used for QoL analysis prior to treatment and 6 months later. RESULTS: Randomization afforded similar baseline results in three domains of QoL analysis for both groups. After 6 months' treatment, there was a statistically significant difference between groups in the physical domain of WHOQOL-BREF: the average score improved to 63.6 ± (SD) 15.8 in the homeopathy group, compared with 53.1 ± (SD) 16.7 in the control group. CONCLUSIONS: Homeopathic treatment showed a positive impact at 6 months on the QoL of women with chronic diseases. Further studies should be performed to determine the long-term effects of homeopathic treatment on QoL and its determinant factors.

Unveiling six potent and highly selective antileishmanial agents via the open source compound collection 'Pathogen Box' against antimony-sensitive and -resistant Leishmania braziliensis.

Despite all efforts to provide new chemical entities to tackle leishmaniases, we are still dependent on a the limited drug arsenal, together with drawbacks like toxicity and drug-resistant parasites. Collaborative drug discovery emerged as an option to speed up the way to find alternative antileishmanial agents. This is the case of Medicines for Malaria Ventures - MMV, that promotes an open source drug discovery initiative to fight diseases worldwide. Here, we screened 400 compounds from 'Pathogen Box' (PBox) collection against Leishmania braziliensis, the main etiological agent of cutaneous leishmaniasis in Brazil. Twenty-three compounds were able to inhibit ≥ 80 % L. braziliensis growth at 5 µM. Six out of the PBox selected 23 compounds were found to be highly selective against L. braziliensis intracellular amastigotes with selectivity index varying from > 104 to > 746 and IC50s ranging from 47 to 480 nM. The compounds were also active against antimony-resistant L. braziliensis isolated from the field or laboratory selected mutants, revealing the potential on treating patients infected with drug resistant parasites. Most of the selected compounds were known to be active against kinetoplastids, however, two compounds (MMV688703 and MMV676477) were part of toxoplasmosis and tuberculosis 'PBox' disease set, reinforcing the potential of phenotyping screening to unveil drug repurposing. Here we applied a computational prediction of pharmacokinetic properties using the ADMET predictor pkCSM (http://biosig.unimelb.edu.au/pkcsm/). The tool offered clues on potential drug development needs and can support further in vivo studies. Molecular docking analysis identified CRK3 (LbrM.35.0660), CYP450 (LbrM.30.3580) and PKA (LbrM.18.1180) as L. braziliensis targets for MMV676604, MMV688372 and MMV688703, respectively. Compounds from 'Pathogen Box' thus represents a new hope for novel (or repurposed) small molecules source to tackle leishmaniases.

Anticancer and antileishmanial in vitro activity of gold(I) complexes with 1,3,4-oxadiazole-2(3H)-thione ligands derived from δ-D-gluconolactone.

Four gold(I) complexes conceived as anticancer agents were synthesized by reacting [Au(PEt3 )Cl] and [Au(PPh3 )Cl] with ligands derived from δ-d-gluconolactone. The ligands' structure was designed to combine desired biological properties previously reported for each group. Ligands were synthesized from δ-d-gluconolactone via ketal protection and hydrazide formation followed by cyclization with CS2 to produce the novel oxadiazolidine-2-thione 7 and 8. Increasing of the ligands' lipophilicity via ketal protection proved useful since all four gold(I) complexes showed anticancer and antileishmanial properties. The IC50 values are at low micromolar range, varying from 2 to 3 µm for the most active compounds. The free D-gluconate 1,3,4 oxadiazole-derived ligands were neither toxic nor presented anticancer or antileishmanial properties. Triethylphosphine-derived compounds 9 and 10 were more selective against B16-F10 melanoma cell line. Although similar in vitro antileishmanial activity was observed for the gold(I) precursors themselves and their derived complexes, the latter were three times less toxic for human THP-1 macrophage cell line; this result is attributed to an isomeric variation of the D-gluconate ligand and the oxadiazole portion, which was one of the key concepts behind this work. These findings should encourage further research on gold(I) complexes to develop novel compounds with potential application in cancer and leishmaniasis chemotherapy.

Of Drugs and Trypanosomatids: New Tools and Knowledge to Reduce Bottlenecks in Drug Discovery.

Leishmaniasis (Leishmania species), sleeping sickness (Trypanosoma brucei), and Chagas disease (Trypanosoma cruzi) are devastating and globally spread diseases caused by trypanosomatid parasites. At present, drugs for treating trypanosomatid diseases are far from ideal due to host toxicity, elevated cost, limited access, and increasing rates of drug resistance. Technological advances in parasitology, chemistry, and genomics have unlocked new possibilities for novel drug concepts and compound screening technologies that were previously inaccessible. In this perspective, we discuss current models used in drug-discovery cascades targeting trypanosomatids (from in vitro to in vivo approaches), their use and limitations in a biological context, as well as different examples of recently discovered lead compounds.

Preclinical Gold Complexes as Oral Drug Candidates to Treat Leishmaniasis Are Potent Trypanothione Reductase Inhibitors.

The drugs currently used to treat leishmaniases have limitations concerning cost, efficacy, and safety, making the search for new therapeutic approaches urgent. We found that the gold(I)-derived complexes were active against L. infantum and L. braziliensis intracellular amastigotes with IC50 values ranging from 0.5 to 5.5 µM. All gold(I) complexes were potent inhibitors of trypanothione reductase (TR), with enzyme IC50 values ranging from 1 to 7.8 µM. Triethylphosphine-derived complexes enhanced reactive oxygen species (ROS) production and decreased mitochondrial respiration after 2 h of exposure, indicating that gold(I) complexes cause oxidative stress by direct ROS production, by causing mitochondrial damage or by impairing TR activity and thus accumulating ROS. There was no cross-resistance to antimony; in fact, SbR (antimony-resistant mutants) strains were hypersensitive to some of the complexes. BALB/c mice infected with luciferase-expressing L. braziliensis or L. amazonensis and treated orally with 12.5 mg/kg/day of AdT Et (3) or AdO Et (4) presented reduced lesion size and parasite burden, as revealed by bioimaging. The combination of (3) and miltefosine allowed for a 50% reduction in miltefosine treatment time. Complexes 3 and 4 presented favorable pharmacokinetic and toxicity profiles that encourage further drug development studies. Gold(I) complexes are promising antileishmanial agents, with a potential for therapeutic use, including in leishmaniasis caused by antimony-resistant parasites.

Decrease in Attentional Performance After Repeated Bouts of High Intensity Exercise in Association-Football Referees and Assistant Referees.

Referees and assistant referees are submitted to high physical stress during matches. Pressure to make decisions in front of large crowds is another potential stressor. These two stressors can impair attention executive control, depending on physical fitness and individual vulnerability or resilience to situational pressure. Error percentage for referees and assistants may reach around 14% during a soccer match. Although previous studies have suggested that soccer referees and assistants should take cognitive assessments, they are only required by Fédération Internationale de Football Association (FIFA) to demonstrate knowledge of the rules and pass annually in a fitness test (FIFA-Test). This study aimed to assess attention performance in referees and assistants before and after the mandatory FIFA-Test. It is hypothesized that the high physical demands associated with the pressure to pass the FIFA-Test would interfere with attention performance. The sample included 33 referees and 20 assistants. The Continuous Visual Attention Test (CVAT) consisted of a 15-min Go/No-go task. Performance in the CVAT is based on four variables: omission and commission errors, reaction time, and variability of reaction time (VRT). Failure in the CVAT was defined by a performance below the 5th percentile of the age- and sex-matched normative data in at least one variable of the CVAT. Before the FIFA-Test all participants performed the CVAT. The second CVAT began 3-7 min directly following completion of the FIFA-test. Considering only the officials who passed both the FIFA-Test and the first CVAT (19 referees and 15 assistants), 44% (9 referees and 6 assistants) exhibited a performance decline in the second CVAT. A significant increase in VRT was found after the high intensity exercise. As increase in VRT is thought to reflect executive dysfunctions and lapses of attention, we concluded that physical fitness alone may not be enough to help officials cope with the physical and contextual stresses associated with the FIFA-Test. These data suggest that over 35% of soccer referees and their assistants who were considered physically able to referee matches may not be mentally prepared for the attentional demands of refereeing soccer matches.

Planarians as models to investigate the bioactivity of gold(I) complexes in vivo.

Gold(I)-containing complexes are used in drug discovery research for rheumatoid arthritis, cancer, and parasitic infections. In this study, we tested the bioactivity of gold(I) complexes in vivo using planarians. The planarian Schmidtea mediterranea possesses orthologues of tumor suppressor genes, such as p53, that, when silenced, cause deregulation of cell proliferation and apoptosis. In this context, we tested two triethylphosphine-gold(I) complexes (AdO and AdT) to determine if they can attenuate phenotypes that result from p53 inhibition. First, we identified the drug concentration that did not affect survival or regeneration and evaluated the drug's effect on cell division and apoptosis. We found that AdT treatment decreased the number of mitotic cells and that all drug treatments increased the number of apoptotic cells. We then performed p53(RNAi) and drug treatments concomitantly and observed the phenotype progression. Drug treatment increased survival three-fold and decreased apoptosis, which resulted in an attenuated phenotype. Our results indicate that planarians can be treated with gold(I) complexes, and that this treatment can diminish the p53(RNAi) phenotype and extend survival. In this work we show that planarians can be used as a model to study the in vivo effect of gold(I) complexes and to further investigate their mechanisms of action.

Intraspecies susceptibility of Leishmania (Viannia) braziliensis to antileishmanial drugs: Antimony resistance in human isolates from atypical lesions.

Leishmania (Viannia) braziliensis is the most common etiological agent of cutaneous and mucocutaneous leishmaniasis (MCL) in Latin America. An interesting aspect of the disease outcome caused by this species is the appearance of non-ulcerated atypical cutaneous leishmaniasis. Atypical (AT) lesions are often associated with therapeutic failure when treated with antimony(Sb)-based drugs. Refractory cases are not necessarily due to intrinsic parasite drug resistance. The status of in vitro drug susceptibility from L. braziliensis field isolates is less assessed than patient treatment outcome. In this work, L. braziliensis isolated from typical CL (6), MCL (1) and AT (3) lesions and vector (1) were tested for their susceptibility to amphotericin B (AmB), miltefosine (MIL), glucantime (GLU) and non-comercial meglumine antimoniate (MA). Overall, intracellular amastigotes of all isolates were sensitive to the tested antileishmanial drugs except AT lesions-derived strains 316, 330 and 340 that presented in vitro resistance against SbV-based drugs. Although susceptible to miltefosine - based on phenotypic screening - intramacrophagic quiescent amastigotes could restore infection. L. braziliensis promastigotes isolated from AT lesions also displayed 29% reduced capacity to infect human monocyte-derived macrophages when compared with parasites obtained from patients with typical lesions, MCL or from sand-fly. These data indicate differences in drug susceptibility and infectiveness among L. braziliensis isolated from patients exhibiting different types of lesions and highlight the importance of its characterization for drug response prediction outcome in clinical practice.