Molecular phylogenetic relationship between Philometroides tahieli (Nematoda, Philometridae) and other philometrids from South America.

In South America, the family Philometridae is represented by several genera and species. In particular, Philometra and Philometroides are the most speciose genera. In Argentina, only Philometroides tahieli has been reported so far. The main objective of this study was to analyze the phylogenetic relationship between Ps. tahieli and other South American philometrids using molecular data. The molecular analysis was performed using a female specimen of Ps. tahieli found on the opercular muscle of a juvenile Micropogonias furnieri from brackish waters in Argentina. Phylogenetic relationships were studied based on partial sequences of the 18S rDNA and 28S rDNA genes. In the 18S rDNA tree, Ps. tahieli was mainly grouped with other Philometroides from freshwater hosts from China, sharing the site of infection (head tissues or muscles). In the 28S rDNA tree, obtained with fewer sequences, Ps. tahieli is related species from subcutaneous tissues of head. This study contributes with information on philometrids and confirms the presence of different lineages among South American species, with Ps. tahieli representing a new one. Further studies on South American species using more molecular markers and new morphological characters will improve our knowledge of philometrid biodiversity and phylogeny.

Broadening the spectrum of ivermectin: Its effect on Trypanosoma cruzi and related trypanosomatids.

Chagas disease is an endemic American parasitosis, caused by Trypanosoma cruzi. The current therapies, benznidazole (BZN) and nifurtimox (NFX), show limited efficacy and multiple side effects. Thus, there is a need to develop new trypanocidal strategies. Ivermectin (IVM) is a broad-spectrum antiparasitic drug with low human and veterinary toxicity with effects against T. brucei and Leishmania spp. Considering this and its relatively low cost, we evaluate IVM as a potential repurposed trypanocidal drug on T. cruzi and other trypanosomatids. We found that IVM affected, in a dose-dependent manner, the proliferation of T. cruzi epimastigotes as well as the amastigotes and trypomastigotes survival. The Selectivity Index for the amastigote stage with respect to Vero cells was 12. The IVM effect was also observed in Phytomonas jma 066 and Leishmania mexicana proliferation but not in Crithidia fasciculata. On the epimastigote stage, the IVM effect was trypanostatic at 50 µM but trypanocidal at 100 µM. The assays of the drug combinations of IVM with BNZ or NFX showed mainly additive effects among combinations. In silico studies showed that classical structures belonging to glutamate-gated Cl channels, the most common IVM target, are absent in kinetoplastids. However, we found in the studied trypanosomatid genomes one copy for putative IMPα and IMPß, potential targets for IVM. The putative IMPα genes (with 76% similarity) showed conserved Armadillo domains but lacked the canonical IMPß binding sequence. These results allowed us to propose a novel molecular target in T. cruzi and suggest IVM as a good candidate for drug repurposing in the Chagas disease context.

A new wild strain of Caenorhabditis elegans associated with Allograpta exotica (Syrphidae) in Argentina: an update of its ecological niche and worldwide distribution.

Caenorhabditis elegans is a free-living nematode, belonging to the bacterivorous trophic group. Although it was cited in several countries, in different types of ecosystems and in associations with other organisms, the wild habitats of this nematode have not yet been precisely defined. In Argentina, C. elegans was recently isolated from the hoverfly Allograpta exotica, a voracious predator with potential biological control against aphids in horticultural crops. In this frame, the objectives of this study were (i) to characterize it molecularly and morphologically (ii) to report a wild strain of C. elegans for the first time from Argentina, (iii) to present a new ecological niche by associating it with A. exotica and (iv) to evaluate the pathogenicity against these insects. The results of the morphological and molecular analyses made it possible to determine that the isolated nematode was C. elegans, thus establishing the ARGLP1900 wild strain as the first record of this nematode for Argentina. A new association was described, since there are no records of interaction between C. elegans and A. exotica, providing information on a new ecological niche. The new wild strain found in this work, could be appropriate for comparative genomic studies with other C. elegans strains.

Molecular evidence supports the validity of three Parabrachiella species infecting mugilids.

The parasitic copepod genus Parabrachiella is composed of 70 species, 14 of which are found in South America. The finding of new specimens of Parabrachiella mugilis from Turkey allowed us to compare the nucleotide sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene of this species with those of the South American Parabrachiella exilis and Parabrachiella platensis; all these species are parasites of mugilids. In addition, specimens of Parabrachiella fasciata, Parabrachiella oralis and Parabrachiella dispar from Chile, and Parabrachiella chevreuxi from Argentina were included in the comparison. Our results confirmed that the three Parabrachiella species parasitizing mugilids, which had been identified by morphology, are valid entities. However, P. exilis was recently synonymized with P. mugilis. The latter species showed a great genetic distance from P. exilis (16%) and was closer to Parabrachiella fasciata (13%) and to species with long posterior processes. Parabrachiella exilis and P. platensis (parasite on Mugil cephalus and Mugil liza, respectively) had a low genetic distance (9%) and Parabrachiella kabatai (parasite of Isacia conceptionis) had a low genetic distance (12-13%) from P. fasciata, P. platensis and P. exilis. In addition, the three parasitic copepods from South America have short and round posterior processes compared to other species, which have long posterior processes. Most species with long posterior processes are clustered together in a Pacific Ocean clade (P. hugu from the North Pacific Ocean), with the exception of P. chevreuxi, which has been found in the South Atlantic Ocean. This study adds seven new sequences, making a total of nine sequenced South American species of Parabrachiella.

Detection of Flaviviral-Like DNA Sequences in Aedes aegypti (Diptera: Culicidae) Collected From Argentina.

Diseases caused by flaviviruses are a major public health burden across the world. In the past decades, South America has suffered dengue epidemics, the re-emergence of yellow fever and St. Louis encephalitis viruses, and the introduction of West Nile and Zika viruses. Many insect-specific flaviviruses (ISFs) that cannot replicate in vertebrate cells have recently been described. In this study, we analyzed field-collected mosquito samples from six different ecoregions of Argentina to detect flaviviruses. We did not find any RNA belonging to pathogenic flaviviruses or ISFs in adults or immature stages. However, flaviviral-like DNA similar to flavivirus NS5 region was detected in 83-100% of Aedes aegypti (L.). Despite being previously described as an ancient element in the Ae. aegypti genome, the flaviviral-like DNA sequence was not detected in all Ae. aegypti samples and sequences obtained did not form a monophyletic group, possibly reflecting the genetic diversity of mosquito populations in Argentina.

Smoking flies: testing the effect of tobacco cigarettes on heart function of Drosophila melanogaster.

Studies about the relationship between substances consumed by humans and their impact on health, in animal models, have been a challenge due to differences between species in the animal kingdom. However, the homology of certain genes has allowed extrapolation of certain knowledge obtained in animals. Drosophila melanogaster, studied for decades, has been widely used as model for human diseases as well as to study responses associated with the consumption of several substances. In the present work we explore the impact of tobacco consumption on a model of 'smoking flies'. Throughout these experiments, we aim to provide information about the effects of tobacco consumption on cardiac physiology. We assessed intracellular calcium handling, a phenomenon underlying cardiac contraction and relaxation. Flies chronically exposed to tobacco smoke exhibited an increased heart rate and alterations in the dynamics of the transient increase of intracellular calcium in myocardial cells. These effects were also evident under acute exposure to nicotine of the heart, in a semi-intact preparation. Moreover, the alpha 1 and 7 subunits of the nicotinic receptors are involved in the heart response to tobacco and nicotine under chronic (in the intact fly) as well as acute exposure (in the semi-intact preparation). The present data elucidate the implication of the intracellular cardiac pathways affected by nicotine on the heart tissue. Based on the probed genetic and physiological similarity between the fly and human heart, cardiac effects exerted by tobacco smoke in Drosophila advances our understanding of the impact of it in the human heart. Additionally, it may also provide information on how nicotine-like substances, e.g. neonicotinoids used as insecticides, affect cardiac function.This article has an associated First Person interview with the first author of the paper.

Amphimermis enzoni n. sp. (Nematoda: Mermithidae) parasitizing damselflies and dragonflies in Argentina.

A mermithid nematode was found parasitizing nymphs of dragonflies and damselflies. The host specimens were collected from the stream Cajaravilla, Magdalena, Buenos Aires state, Argentina. In this work, we described Amphimermis enzoni n. sp., a nematode new to science. Nematodes were identified through morphological and molecular methods. The combination of the following characters separates A. enzoni n. sp. from other members of the genus Amphimermis Steiner: long and S-shaped vagina, twisted spicules for proximal 34% of their length, untwisted for 12%, again twisted for 30%, and untwisted for the last 24%; genital papillae arranged in three rows, medial row marginally longer than sub-medial rows; medial row bifurcated immediately anterior and posterior to cloaca, with 111 genital papillae (73 pre-anals and 38 post-anals). The sequences of 18S rDNA regions from A. enzoni formed a well-supported monophyletic clade with two GenBank sequences of Amphimermis spp. (EF617354 and EF617355) with 0.63 to 1.26% divergence and two Mermithidae spp. (LC512371 and LC512370) with 0.63 to 1.1% divergence, respectively. To our knowledge, this is the first example of mermithid infection in nymphs of dragonflies and damselflies for South America.A mermithid nematode was found parasitizing nymphs of dragonflies and damselflies. The host specimens were collected from the stream Cajaravilla, Magdalena, Buenos Aires state, Argentina. In this work, we described Amphimermis enzoni n. sp., a nematode new to science. Nematodes were identified through morphological and molecular methods. The combination of the following characters separates A. enzoni n. sp. from other members of the genus Amphimermis Steiner: long and S-shaped vagina, twisted spicules for proximal 34% of their length, untwisted for 12%, again twisted for 30%, and untwisted for the last 24%; genital papillae arranged in three rows, medial row marginally longer than sub-medial rows; medial row bifurcated immediately anterior and posterior to cloaca, with 111 genital papillae (73 pre-anals and 38 post-anals). The sequences of 18S rDNA regions from A. enzoni formed a well-supported monophyletic clade with two GenBank sequences of Amphimermis spp. (EF617354 and EF617355) with 0.63 to 1.26% divergence and two Mermithidae spp. (LC512371 and LC512370) with 0.63 to 1.1% divergence, respectively. To our knowledge, this is the first example of mermithid infection in nymphs of dragonflies and damselflies for South America.

SERCA is critical to control the Bowditch effect in the heart.

The Bowditch effect or staircase phenomenon is the increment or reduction of contractile force when heart rate increases, defined as either a positive or negative staircase. The healthy and failing human heart both show positive or negative staircase, respectively, but the causes of these distinct cardiac responses are unclear. Different experimental approaches indicate that while the level of Ca2+ in the sarcoplasmic reticulum is critical, the molecular mechanisms are unclear. Here, we demonstrate that Drosophila melanogaster shows a negative staircase which is associated to a slight but significant frequency-dependent acceleration of relaxation (FDAR) at the highest stimulation frequencies tested. We further showed that the type of staircase is oppositely modified by two distinct SERCA mutations. The dominant conditional mutation SERCAA617T induced positive staircase and arrhythmia, while SERCAE442K accentuated the negative staircase of wild type. At the stimulation frequencies tested, no significant FDAR could be appreciated in mutant flies. The present results provide evidence that two individual mutations directly modify the type of staircase occurring within the heart and suggest an important role of SERCA in regulating the Bowditch effect.

The regulation of autophagy differentially affects Trypanosoma cruzi metacyclogenesis.

Autophagy is a cellular process required for the removal of aged organelles and cytosolic components through lysosomal degradation. All types of eukaryotic cells from yeasts to mammalian cells have the machinery to activate autophagy as a result of many physiological and pathological situations. The most frequent stimulus of autophagy is starvation and the result, in this case, is the fast generation of utilizable food (e.g. amino acids and basic nutrients) to maintain the vital biological processes. In some organisms, starvation also triggers other associated processes such as differentiation. The protozoan parasite Trypanosoma cruzi undergoes a series of differentiation processes throughout its complex life cycle. Although not all autophagic genes have been identified in the T. cruzi genome, previous works have demonstrated the presence of essential autophagic-related proteins. Under starvation conditions, TcAtg8, which is the parasite homolog of Atg8/LC3 in other organisms, is located in autophagosome-like vesicles. In this work, we have characterized the autophagic pathway during T. cruzi differentiation from the epimastigote to metacyclic trypomastigote form, a process called metacyclogenesis. We demonstrated that autophagy is stimulated during metacyclogenesis and that the induction of autophagy promotes this process. Moreover, with exception of bafilomycin, other classical autophagy modulators have similar effects on T. cruzi autophagy. We also showed that spermidine and related polyamines can positively regulate parasite autophagy and differentiation. We concluded that both polyamine metabolism and autophagy are key processes during T. cruzi metacyclogenesis that could be exploited as drug targets to avoid the parasite cycle progression.

The superfamily keeps growing: Identification in trypanosomatids of RibJ, the first riboflavin transporter family in protists.

BACKGROUND: Trypanosomatid parasites represent a major health issue affecting hundreds of million people worldwide, with clinical treatments that are partially effective and/or very toxic. They are responsible for serious human and plant diseases including Trypanosoma cruzi (Chagas disease), Trypanosoma brucei (Sleeping sickness), Leishmania spp. (Leishmaniasis), and Phytomonas spp. (phytoparasites). Both, animals and trypanosomatids lack the biosynthetic riboflavin (vitamin B2) pathway, the vital precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) cofactors. While metazoans obtain riboflavin from the diet through RFVT/SLC52 transporters, the riboflavin transport mechanisms in trypanosomatids still remain unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that riboflavin is imported with high affinity in Trypanosoma cruzi, Trypanosoma brucei, Leishmania (Leishmania) mexicana, Crithidia fasciculata and Phytomonas Jma using radiolabeled riboflavin transport assays. The vitamin is incorporated through a saturable carrier-mediated process. Effective competitive uptake occurs with riboflavin analogs roseoflavin, lumiflavin and lumichrome, and co-factor derivatives FMN and FAD. Moreover, important biological processes evaluated in T. cruzi (i.e. proliferation, metacyclogenesis and amastigote replication) are dependent on riboflavin availability. In addition, the riboflavin competitive analogs were found to interfere with parasite physiology on riboflavin-dependent processes. By means of bioinformatics analyses we identified a novel family of riboflavin transporters (RibJ) in trypanosomatids. Two RibJ members, TcRibJ and TbRibJ from T. cruzi and T. brucei respectively, were functionally characterized using homologous and/or heterologous expression systems. CONCLUSIONS/SIGNIFICANCE: The RibJ family represents the first riboflavin transporters found in protists and the third eukaryotic family known to date. The essentiality of riboflavin for trypanosomatids, and the structural/biochemical differences that RFVT/SLC52 and RibJ present, make the riboflavin transporter -and its downstream metabolism- a potential trypanocidal drug target.

Novel 2-arylazoimidazole derivatives as inhibitors of Trypanosoma cruzi proliferation: Synthesis and evaluation of their biological activity.

In this work, the synthesis of a series of 2-arylazoimidazole derivatives 6-20 has been achieved through the reaction of imidazole with aryldiazonium salts, followed by ultrasound-assisted alkylation. This approach has important advantages including higher yield, shorter reaction times and milder reaction conditions. The structures of the compounds obtained were determined by MS, IR; and 1H and 13C NMR. The anti-Trypanosoma cruzi activity of the 15 compounds obtained was evaluated. Two compounds with piperidino substituents in the carboxamide moiety proved to be effective inhibitors of epimastigote proliferation, obtaining inhibition values comparable to those achieved with the reference drug Benznidazole. Besides, these compounds displayed low cytotoxicity on mammalian cells. In vivo, both compounds protected mice against a challenge with a lethal Trypanosoma cruzi strain. These results allow us to propose 2-arylazoimidazoles as lead compounds for the design of novel drugs to treat Chagas' disease.

Trypanosoma cruzi Polyamine Transporter: Its Role on Parasite Growth and Survival Under Stress Conditions.

Trypanosoma cruzi is the etiological agent of Chagas disease, a major health problem in Latin America. Polyamines are polycationic compounds that play a critical role as regulators of cell growth and differentiation. In contrast with other protozoa, T. cruzi is auxotrophic for polyamines because of its inability to synthesize putrescine due to the lack of both, arginine and ornithine decarboxylase; therefore, the intracellular availability of polyamines depends exclusively on transport processes. In this work, the polyamine transporter TcPAT12 was overexpressed in T. cruzi epimastigotes demonstrating that growth rates at different concentrations of polyamines strongly depend on the regulation of the polyamine transport. In addition, parasites overexpressing TcPAT12 showed a highly increased resistance to hydrogen peroxide and the trypanocidal drugs nifurtimox and benznidazole, which act by oxidative stress and interfering the synthesis of polyamine derivatives, respectively. Finally, the presence of putative polyamine transporters was analyzed in T. cruzi, Trypanosoma brucei, and Leishmania major genomes identifying 3-6 genes in these trypanosomatids.

Discovery of novel polyamine analogs with anti-protozoal activity by computer guided drug repositioning.

Chagas disease is a parasitic infection caused by the protozoa Trypanosoma cruzi that affects about 6 million people in Latin America. Despite its sanitary importance, there are currently only two drugs available for treatment: benznidazole and nifurtimox, both exhibiting serious adverse effects and limited efficacy in the chronic stage of the disease. Polyamines are ubiquitous to all living organisms where they participate in multiple basic functions such as biosynthesis of nucleic acids and proteins, proliferation and cell differentiation. T. cruzi is auxotroph for polyamines, which are taken up from the extracellular medium by efficient transporters and, to a large extent, incorporated into trypanothione (bis-glutathionylspermidine), the major redox cosubstrate of trypanosomatids. From a 268-compound database containing polyamine analogs with and without inhibitory effect on T. cruzi we have inferred classificatory models that were later applied in a virtual screening campaign to identify anti-trypanosomal compounds among drugs already used for other therapeutic indications (i.e. computer-guided drug repositioning) compiled in the DrugBank and Sweetlead databases. Five of the candidates identified with this strategy were evaluated in cellular models from different pathogenic trypanosomatids (T. cruzi wt, T. cruzi PAT12, T. brucei and Leishmania infantum), and in vitro models of aminoacid/polyamine transport assays and trypanothione synthetase inhibition assay. Triclabendazole, sertaconazole and paroxetine displayed inhibitory effects on the proliferation of T. cruzi (epimastigotes) and the uptake of putrescine by the parasite. They also interfered with the uptake of others aminoacids and the proliferation of infective T. brucei and L. infantum (promastigotes). Trypanothione synthetase was ruled out as molecular target for the anti-parasitic activity of these compounds.

High-throughput drug repositioning for the discovery of new treatments for Chagas disease.

Despite affecting around 8 million people worldwide and representing an economic burden above $7 billion/ year, currently approved medications to treat Chagas disease are still limited to two drugs, nifurtimox and benznidazole, which were developed more than 40 years ago and present important efficacy and safety limitations. Drug repositioning (i.e. finding second or further therapeutic indications for known drugs) has raised considerable interest within the international drug development community. There are many explanations to the current interest on drug repositioning including the possibility to partially circumvent clinical trials and the consequent saving in time and resources. It has been suggested as a particular attractive approach for the development of novel therapeutics for neglected diseases, which are usually driven by public or non-profit organizations. Here we review current computer-guided approaches to drug repositioning and reports on drug repositioning stories oriented to Chagas disease, with a focus on computer-guided drug repositioning campaigns.

Computer-guided drug repurposing: identification of trypanocidal activity of clofazimine, benidipine and saquinavir.

In spite of remarkable advances in the knowledge on Trypanosoma cruzi biology, no medications to treat Chagas disease have been approved in the last 40 years and almost 8 million people remain infected. Since the public sector and non-profit organizations play a significant role in the research efforts on Chagas disease, it is important to implement research strategies that promote translation of basic research into the clinical practice. Recent international public-private initiatives address the potential of drug repositioning (i.e. finding second or further medical uses for known-medications) which can substantially improve the success at clinical trials and the innovation in the pharmaceutical field. In this work, we present the computer-aided identification of approved drugs clofazimine, benidipine and saquinavir as potential trypanocidal compounds and test their effects at biochemical as much as cellular level on different parasite stages. According to the obtained results, we discuss biopharmaceutical, toxicological and physiopathological criteria applied to decide to move clofazimine and benidipine into preclinical phase, in an acute model of infection. The article illustrates the potential of computer-guided drug repositioning to integrate and optimize drug discovery and preclinical development; it also proposes rational rules to select which among repositioned candidates should advance to investigational drug status and offers a new insight on clofazimine and benidipine as candidate treatments for Chagas disease. One Sentence Summary: We present the computer-guided drug repositioning of three approved drugs as potential new treatments for Chagas disease, integrating computer-aided drug screening and biochemical, cellular and preclinical tests.

Identification of levothyroxine antichagasic activity through computer-aided drug repurposing.

Cruzipain (Cz) is the major cysteine protease of the protozoan Trypanosoma cruzi, etiological agent of Chagas disease. A conformation-independent classifier capable of identifying Cz inhibitors was derived from a 163-compound dataset and later applied in a virtual screening campaign on the DrugBank database, which compiles FDA-approved and investigational drugs. 54 approved drugs were selected as candidates, 3 of which were acquired and tested on Cz and T. cruzi epimastigotes proliferation. Among them, levothyroxine, traditionally used in hormone replacement therapy in patients with hypothyroidism, showed dose-dependent inhibition of Cz and antiproliferative activity on the parasite.

Application of computer-aided drug repurposing in the search of new cruzipain inhibitors: discovery of amiodarone and bromocriptine inhibitory effects.

Cruzipain (Cz) is the major cystein protease of the protozoan Trypanosoma cruzi , etiological agent of Chagas disease. From a 163 compound data set, a 2D-classifier capable of identifying Cz inhibitors was obtained and applied in a virtual screening campaign on the DrugBank database, which compiles FDA-approved and investigational drugs. Fifty-four approved drugs were selected as candidates, four of which were acquired and tested on Cz and T. cruzi epimastigotes. Among them, the antiparkinsonian and antidiabetic drug bromocriptine and the antiarrhythmic amiodarone showed dose-dependent inhibition of Cz and antiproliferative activity on the parasite.