A Screening of 10,240 NatureBank Fractions Identifies Nematicidal Activity in Agelasine-Containing Extracts from Sponges.

Nematode infections affect a fifth of the human population, livestock, and crops worldwide, imposing a burden to global public health and economies, particularly in developing nations. Resistance to commercial anthelmintics has increased over the years in livestock infections and driven the pursuit for new drugs. We herein present a rapid, cost-effective, and automated assay for nematicide discovery using the free-living nematode Caenorhabditis elegans to screen a highly diverse natural product library enriched in bioactive molecules. Screening of 10,240 fractions obtained from extracts of various biological sources allowed the identification of 7 promising hit fractions, all from marine sponges. These fractions were further assayed for nematicidal activity against the sheep nematode parasite Haemonchus contortus and for innocuity in zebrafish. The most active extracts against parasites and innocuous toward vertebrates belong to two chemotypes. High-performance liquid chromatography (HPLC) coupled with nuclear magnetic resonance (NMR) revealed that the most abundant compound in one chemotype is halaminol A, an aminoalcohol previously identified in a small screen against H. contortus. Terpene-nucleotide hybrids known as agelasines predominate in the other chemotype. This study reinforces the power of C. elegans for nematicide discovery from large collections and the potential of the chemical diversity derived from marine invertebrate biota.

Glueball Dark Matter Revisited.

We revisit the possibility that dark matter is composed of stable scalar glueballs of a confining dark SU(3) gauge theory coupled only to gravity. The relic abundance of dark glueballs is studied for the first time in a thermal effective theory accounting for strong-coupling dynamics. An important ingredient of our analysis is the use of an effective potential for glueballs that is fitted by lattice simulations. We predict the relic abundance to be in the range 0.12ζ_{T}^{-3}Λ/(137.9 eV)≲Ωh^{2}≲0.12ζ_{T}^{-3}Λ/(82.7 eV), with Λ being the confinement scale, ζ_{T} the visible-to-dark sector temperature ratio, and the uncertainty is coming from the fit to lattice data. This prediction is an order of magnitude smaller than the existing glueball abundance results in the literature. Our framework can be easily generalized to different gauge groups and modified cosmological histories paving the way toward consistent exploration of strongly coupled dark sectors and their cosmological implications.

Structure-Based Bioisosterism Design, Synthesis, Biological Evaluation and In Silico Studies of Benzamide Analogs as Potential Anthelmintics.

A recent screen of 67,012 compounds identified a new family of compounds with excellent nematicidal activity: the ortho-substituted benzamide families Wact-11 and Wact-12. These compounds are active against Caenorhabditis elegans and parasitic nematodes by selectively inhibiting nematode complex II, and they display low toxicity in mammalian cells and vertebrate organisms. Although a big number of benzamides were tested against C. elegans in high-throughput screens, bioisosteres of the amide moiety were not represented in the chemical space examined. We thus identified an opportunity for the design, synthesis and evaluation of novel compounds, using bioisosteric replacements of the amide group present in benzamides. The compound Wact-11 was used as the reference scaffold to prepare a set of bioisosteres to be evaluated against C. elegans. Eight types of amide replacement were selected, including ester, thioamide, selenoamide, sulfonamide, alkyl thio- and oxo-amides, urea and triazole. The results allowed us to perform a structure-activity relationship, highlighting the relevance of the amide group for nematicide activity. Experimental evidence was complemented with in silico structural studies over a C. elegans complex II model as a molecular target of benzamides. Importantly, compound Wact-11 was active against the flatworm Echinococcus granulosus, suggesting a previously unreported pan-anthelmintic potential for benzamides.

Normal transaminases in obese patients with metabolic associated steatohepatitis (MASH): a cohort of Peruvian patients.

INTRODUCTION: Metabolic associated steatohepatitis (MASH) is one of the most frequent causes of chronic liver disease. Liver transaminases are important biomarkers to measure liver injury, however, a proportion of patients with MASH may present with normal levels of transaminases. The levels of serum transaminases may not correlate with the severity of histopathological changes. OBJECTIVE: We aimed to identify the frequency of normal transaminases in obese patients with MASH, as well as to describe the clinical, biochemical and histological characteristics in this specific group of patients. MATERIALS AND METHODS: A retrospective cross-sectional study was conducted in the bariatric surgery service of a private clinic. Obese patients older than 18 years with a body mass index (BMI) >30Kg/m2 and 2 co-morbidities undergoing a gastric sleeve surgery were included. Measurement of biochemical routine laboratory exams was performed. Insulin resistance was calculated using the homeostasis evaluation model (HOMA-IR). All patients underwent liver biopsies prior to surgery and the diagnosis of MASH was based on the Brunt criteria. RESULTS: 159 obese patients with MASH were included, of which 47.2% had normal transaminases and 52.8% elevated transaminases. Factors associated with alteration in transaminases were: being male OR=4.02 (95% CI: 2.03- 7.96; p<0.01), diagnosis of type 2 diabetes mellitus OR=4.86 (95% CI: 1.97- 11.95; p<0.01) and levels of GGT >50 IU/L OR=7.50 (95% CI: 3.40-16.56; p<0.01). The values of HOMA-IR and GGT were significantly higher in the group of high transaminases (p<0.01). Differences in the degree of fibrosis were not associated with transaminases levels. CONCLUSION: In conclusion we found that the frequency of normal transaminases was 47.2% in obese patients with MASH. Factors associated with elevation in liver enzymes were being male, diagnosis of diabetes mellitus and elevation in GGT levels. The degree of fibrosis was not associated with elevations in liver transaminases. These findings suggest that transaminases levels alone are not accurate markers to assess liver injury, as they do not necessarily correlate with histological liver damage.

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

Five heteroleptic compounds, [VVO(IN-2H)(L-H)], where L are 8-hydroxyquinoline derivatives and IN is a Schiff base ligand, were synthesized and characterized in both the solid and solution state. The compounds were evaluated on epimastigotes and trypomastigotes of Trypanosoma cruzi as well as on VERO cells, as a mammalian cell model. Compounds showed activity against trypomastigotes with IC50 values of 0.29-3.02 µM. IN ligand and the new [VVO2(IN-H)] complex showed negligible activity. The most active compound [VVO(IN-2H)(L2-H)], with L2 = 5-chloro-7-iodo-8-hydroxyquinoline, showed good selectivity towards the parasite and was selected to carry out further biological studies. Stability studies suggested a partial decomposition in solution. [VVO(IN-2H)(L2-H)] affects the infection potential of cell-derived trypomastigotes. Low total vanadium uptake by parasites and preferential accumulation in the soluble proteins fraction were determined. A trypanocide effect was observed when incubating epimastigotes with 10 × IC50 values of [VVO(IN-2H)(L2-H)] and the generation of ROS after treatments was suggested. Fluorescence competition measurements with DNA:ethidium bromide adduct showed a moderate DNA interaction of the complexes. In vivo toxicity study on C. elegans model showed no toxicity up to a 100 µM concentration of [VVO(IN-2H)(L2-H)]. This compound could be considered a prospective anti-T. cruzi agent that deserves further research.

The kynurenine pathway is essential for rhodoquinone biosynthesis in Caenorhabditis elegans.

A key metabolic adaptation of some species that face hypoxia as part of their life cycle involves an alternative electron transport chain in which rhodoquinone (RQ) is required for fumarate reduction and ATP production. RQ biosynthesis in bacteria and protists requires ubiquinone (Q) as a precursor. In contrast, Q is not a precursor for RQ biosynthesis in animals such as parasitic helminths, and most details of this pathway have remained elusive. Here, we used Caenorhabditis elegans as a model animal to elucidate key steps in RQ biosynthesis. Using RNAi and a series of C. elegans mutants, we found that arylamine metabolites from the kynurenine pathway are essential precursors for RQ biosynthesis de novo Deletion of kynu-1, encoding a kynureninase that converts l-kynurenine (KYN) to anthranilic acid (AA) and 3-hydroxykynurenine (3HKYN) to 3-hydroxyanthranilic acid (3HAA), completely abolished RQ biosynthesis but did not affect Q levels. Deletion of kmo-1, which encodes a kynurenine 3-monooxygenase that converts KYN to 3HKYN, drastically reduced RQ but not Q levels. Knockdown of the Q biosynthetic genes coq-5 and coq-6 affected both Q and RQ levels, indicating that both biosynthetic pathways share common enzymes. Our study reveals that two pathways for RQ biosynthesis have independently evolved. Unlike in bacteria, where amination is the last step in RQ biosynthesis, in worms the pathway begins with the arylamine precursor AA or 3HAA. Because RQ is absent in mammalian hosts of helminths, inhibition of RQ biosynthesis may have potential utility for targeting parasitic infections that cause important neglected tropical diseases.

An isomerase completes the circuit for a redox switch.

The activity of human transglutaminase 2 (TG2), which forms protein cross-links between glutamine and lysine residues, is controlled by an allosteric disulfide bond. However, the mechanism by which this bond is formed, like many systems regulated by oxidative cysteine modifications, was not clear. A new study from Khosla and colleagues shows that TG2 is oxidatively inactivated by the protein disulfide isomerase ERp57, providing the first example of a defined and reversible protein-controlled redox switch and pointing to new strategies to inhibit undesirable TG2 activity in pathological states.

The genomes of four tapeworm species reveal adaptations to parasitism.

Tapeworms (Cestoda) cause neglected diseases that can be fatal and are difficult to treat, owing to inefficient drugs. Here we present an analysis of tapeworm genome sequences using the human-infective species Echinococcus multilocularis, E. granulosus, Taenia solium and the laboratory model Hymenolepis microstoma as examples. The 115- to 141-megabase genomes offer insights into the evolution of parasitism. Synteny is maintained with distantly related blood flukes but we find extreme losses of genes and pathways that are ubiquitous in other animals, including 34 homeobox families and several determinants of stem cell fate. Tapeworms have specialized detoxification pathways, metabolism that is finely tuned to rely on nutrients scavenged from their hosts, and species-specific expansions of non-canonical heat shock proteins and families of known antigens. We identify new potential drug targets, including some on which existing pharmaceuticals may act. The genomes provide a rich resource to underpin the development of urgently needed treatments and control.

New heteroleptic oxidovanadium(V) complexes: synthesis, characterization and biological evaluation as potential agents against Trypanosoma cruzi.

Searching for prospective vanadium-based agents against Trypanosoma cruzi, the parasite causing Chagas disease, four new [VVO(8HQ-H)(L-2H)] compounds, where 8HQ is 8-hydroxyquinoline and L are tridentate salicylaldehyde semicarbazone derivatives L1-L4, were synthesized and characterized in the solid state and in solution. The compounds were evaluated on T. cruzi epimastigotes (CL Brener) as well as on VERO cells, as mammalian cell model. Compounds showed activity against T. cruzi (IC50 6.2-10.5 µM) of the same order than Nifurtimox and 8HQ, and a four- to sevenfold activity increase with respect to the free semicarbazones. For comparison, [VVO2(L-H)] series was prepared and the new [VVO2(L3-H)] was fully characterized. They showed negligible activity and low selectivity towards the parasite. The inclusion of 8HQ as ligand in [VVO(8HQ-H)(L-2H)] compounds led to good activities and increased selectivity towards the parasite with respect to 8HQ. 51V NMR experiments, performed to get insight into the nature of the active species, suggested partial decomposition of the compounds in solution to [VVO2(L-H)] and 8HQ. Depending on the dose, the compounds act as trypanocide or trypanostatic. A high uptake of vanadium in the parasites (58.51-88.9% depending on dose) and a preferential accumulation in the soluble protein fraction of the parasite was determined. Treated parasites do not seem to show a late apoptotic/necrotic phenotype suggesting a different cell death mechanism. In vivo toxicity study on zebrafish model showed no toxicity up to a 25 µM concentration of [VVO(8HQ-H)(L1-2H)]. These compounds could be considered prospective anti-T. cruzi agents that deserve further research.

Caenorhabditis elegans: nature and nurture gift to nematode parasitologists.

The free-living nematode Caenorhabditis elegans is the simplest animal model organism to work with. Substantial knowledge and tools have accumulated over 50 years of C. elegans research. The use of C. elegans relating to parasitic nematodes from a basic biology standpoint or an applied perspective has increased in recent years. The wealth of information gained on the model organism, the use of the powerful approaches and technologies that have advanced C. elegans research to parasitic nematodes and the enormous success of the omics fields have contributed to bridge the divide between C. elegans and parasite nematode researchers. We review key fields, such as genomics, drug discovery and genetics, where C. elegans and nematode parasite research have convened. We advocate the use of C. elegans as a model to study helminth metabolism, a neglected area ready to advance. How emerging technologies being used in C. elegans can pave the way for parasitic nematode research is discussed.

Selenoprotein Gene Nomenclature.

The human genome contains 25 genes coding for selenocysteine-containing proteins (selenoproteins). These proteins are involved in a variety of functions, most notably redox homeostasis. Selenoprotein enzymes with known functions are designated according to these functions: TXNRD1, TXNRD2, and TXNRD3 (thioredoxin reductases), GPX1, GPX2, GPX3, GPX4, and GPX6 (glutathione peroxidases), DIO1, DIO2, and DIO3 (iodothyronine deiodinases), MSRB1 (methionine sulfoxide reductase B1), and SEPHS2 (selenophosphate synthetase 2). Selenoproteins without known functions have traditionally been denoted by SEL or SEP symbols. However, these symbols are sometimes ambiguous and conflict with the approved nomenclature for several other genes. Therefore, there is a need to implement a rational and coherent nomenclature system for selenoprotein-encoding genes. Our solution is to use the root symbol SELENO followed by a letter. This nomenclature applies to SELENOF (selenoprotein F, the 15-kDa selenoprotein, SEP15), SELENOH (selenoprotein H, SELH, C11orf31), SELENOI (selenoprotein I, SELI, EPT1), SELENOK (selenoprotein K, SELK), SELENOM (selenoprotein M, SELM), SELENON (selenoprotein N, SEPN1, SELN), SELENOO (selenoprotein O, SELO), SELENOP (selenoprotein P, SeP, SEPP1, SELP), SELENOS (selenoprotein S, SELS, SEPS1, VIMP), SELENOT (selenoprotein T, SELT), SELENOV (selenoprotein V, SELV), and SELENOW (selenoprotein W, SELW, SEPW1). This system, approved by the HUGO Gene Nomenclature Committee, also resolves conflicting, missing, and ambiguous designations for selenoprotein genes and is applicable to selenoproteins across vertebrates.

Del Nido cardioplegia in low risk adults undergoing first time coronary artery bypass surgery.

OBJECTIVES: Single-dose del Nido cardioplegia has been used in the pediatric population for many years. Only a small amount of data exists about its use in adult cardiac surgery. We sought to compare the outcomes of all patients undergoing coronary artery bypass, using our 4:1 blood cardioplegia versus single-dose 1:4 del Nido cardioplegia, at our institution. METHODS: Data were retrospectively reviewed from all patients during 2 consecutive years (2013-2014). We switched our cardioplegia protocol from 4:1 blood cardioplegia to exclusively 1:4 single-dose del Nido cardioplegia in early 2014. A total of 408 patients were evaluated. Two hundred and forty-nine consecutive patients underwent coronary artery bypass using blood cardioplegia and 159 using del Nido Cardioplegia. RESULTS: Cardiopulmonary bypass time, cross-clamp time, in-hospital mortality and length of stay were similar (p>0.05): 63 ± 23 vs. 65 ± 21 min, 50 ± 20 vs. 52 ± 20 min, 0.8% vs. 0.6% and 6.4 ± 3 vs. 5.8 ± 3 days, respectively. For secondary outcomes: patients requiring defibrillation was 105/249 (42%) vs. 13/159 (8%) (p<0.0001), blood transfusion was required in 96/249 (38%) vs. 48/159 (30%) (p<0.085), total volume administered was 1139mL vs. 813 mL per case (p<0.0001), hematocrit change was 11.6% vs. 10.9% (p<0.04) and the mean cost per dose was $157.54 vs $5.74. CONCLUSIONS: Single-dose del Nido cardioplegia is an effective and economic cardioplegia and can be used with good outcomes in coronary surgery. Most patients have spontaneous return of sinus rhythm and there is a trend towards decreased transfusion rate.

Adjustments, extinction, and remains of selenocysteine incorporation machinery in the nematode lineage.

Selenocysteine (Sec) is encoded by an UGA codon with the help of a SECIS element present in selenoprotein mRNAs. SECIS-binding protein (SBP2/SCBP-2) mediates Sec insertion, but the roles of its domains and the impact of its deficiency on Sec insertion are not fully understood. We used Caenorhabditis elegans to examine SBP2 function since it possesses a single selenoprotein, thioredoxin reductase-1 (TRXR-1). All SBP2 described so far have an RNA-binding domain (RBD) and a Sec-incorporation domain (SID). Surprisingly, C. elegans SBP2 lacks SID and consists only of an RBD. An sbp2 deletion mutant strain ablated Sec incorporation demonstrating SBP2 essentiality for Sec incorporation. Further in silico analyses of nematode genomes revealed conservation of SBP2 lacking SID and maintenance of Sec incorporation linked to TRXR-1. Remarkably, parasitic plant nematodes lost the ability to incorporate Sec, but retained SecP43, a gene associated with Sec incorporation. Interestingly, both selenophosphate synthetase (SPS) genes are absent in plant parasitic nematodes, while only Cys-containing SPS2 is present in Sec-incorporating nematodes. Our results indicate that C. elegans and the nematode lineage provide key insights into Sec incorporation and the evolution of Sec utilization trait, selenoproteomes, selenoproteins, and Sec residues. Finally, our study provides evidence of noncanonical translation initiation in C. elegans, not previously known for this well-established animal model.

Inhibition of Tapeworm Thioredoxin and Glutathione Pathways by an Oxadiazole N-Oxide Leads to Reduced Mesocestoides vogae Infection Burden in Mice.

Parasitic flatworms cause serious infectious diseases that affect humans and livestock in vast regions of the world, yet there are few effective drugs to treat them. Thioredoxin glutathione reductase (TGR) is an essential enzyme for redox homeostasis in flatworm parasites and a promising pharmacological target. We purified to homogeneity and characterized the TGR from the tapeworm Mesocestoides vogae (syn. M. corti). This purification revealed absence of conventional TR and GR. The glutathione reductase activity of the purified TGR exhibits a hysteretic behavior typical of flatworm TGRs. Consistently, M. vogae genome analysis revealed the presence of a selenocysteine-containing TGR and absence of conventional TR and GR. M. vogae thioredoxin and glutathione reductase activities were inhibited by 3,4-bis(phenylsulfonyl)-1,2,5-oxadiazole N2-oxide (VL16E), an oxadiazole N-oxide previously identified as an inhibitor of fluke and tapeworm TGRs. Finally, we show that mice experimentally infected with M. vogae tetrathyridia and treated with either praziquantel, the reference drug for flatworm infections, or VL16E exhibited a 28% reduction of intraperitoneal larvae numbers compared to vehicle treated mice. Our results show that oxadiazole N-oxide is a promising chemotype in vivo and highlights the convenience of M. vogae as a model for rapid assessment of tapeworm infections in vivo.

Discovering Echinococcus granulosus thioredoxin glutathione reductase inhibitors through site-specific dynamic combinatorial chemistry.

In this study, we report a strategy using dynamic combinatorial chemistry for targeting the thioredoxin (Trx)-reductase catalytic site on Trx glutathione reductase (TGR), a pyridine nucleotide thiol-disulfide oxido-reductase. We chose Echinococcus granulosus TGR since it is a bottleneck enzyme of platyhelminth parasites and a validated pharmacological target. A dynamic combinatorial library (DCL) was constructed based on thiol-disulfide reversible exchange. We demonstrate the use of 5-thio-2-nitrobenzoic acid (TNB) as a non-covalent anchor fragment in a DCL templated by E. granulosus TGR. The heterodimer of TNB and bisthiazolidine (2af) was identified, upon library analysis by HPLC (IC50 = 24 µM). Furthermore, 14 analogs were synthetically prepared and evaluated against TGR. This allowed the study of a structure-activity relationship and the identification of a disulfide TNB-tricyclic bisthiazolidine (2aj) as the best enzyme inhibitor in these series, with an IC50 = 24 µM. Thus, our results validate the use of DCL for targeting thiol-disulfide oxido-reductases.

Antifungal and Antiparasitic Activities of Metallocene-Containing Fluconazole Derivatives.

The search for new anti-infectives based on metal complexes is gaining momentum. Among the different options taken by researchers, the one involving the use of organometallic complexes is probably the most successful one with a compound, namely, ferroquine, already in clinical trials against malaria. In this study, we describe the preparation and in-depth characterization of 10 new (organometallic) derivatives of the approved antifungal drug fluconazole. Our rationale is that the sterol 14α-demethylase is an enzyme part of the ergosterol biosynthesis route in Trypanosoma and is similar to the one in pathogenic fungi. To demonstrate our postulate, docking experiments to assess the binding of our compounds with the enzyme were also performed. Our compounds were then tested on a range of fungal strains and parasitic organisms, including the protozoan parasite Trypanosoma cruzi (T. cruzi) responsible for Chagas disease, an endemic disease in Latin America that ranks among some of the most prevalent parasitic diseases worldwide. Of high interest, the two most potent compounds of the study on T. cruzi that contain a ferrocene or cobaltocenium were found to be harmless for an invertebrate animal model, namely, Caenorhabditis elegans (C. elegans), without affecting motility, viability, or development.

A Minimal Kynurenine Pathway Was Preserved for Rhodoquinone but Not for De Novo NAD+ Biosynthesis in Parasitic Worms: The Essential Role of NAD+ Rescue Pathways.

Aims: To determine the role of the kynurenine (KYN) pathway in rhodoquinone (RQ) and de novo NAD+ biosynthesis and whether NAD+ rescue pathways are essential in parasitic worms (helminths). Results: We demonstrate that RQ, the key electron transporter used by helminths under hypoxia, derives from the tryptophan (Trp) catabolism even in the presence of a minimal KYN pathway. We show that of the KYN pathway genes only the kynureninase and tryptophan/indoleamine dioxygenases are essential for RQ biosynthesis. Metabolic labeling with Trp revealed that the lack of the formamidase and kynurenine monooxygenase genes did not preclude RQ biosynthesis in the flatworm Mesocestoides corti. In contrast, a minimal KYN pathway prevented de novo NAD+ biosynthesis, as revealed by metabolic labeling in M. corti, which also lacks the 3-hydroxyanthranilate 3,4-dioxygenase gene. Our results indicate that most helminths depend solely on NAD+ rescue pathways, and some lineages rely exclusively on the nicotinamide salvage pathway. Importantly, the inhibition of the NAD+ recycling enzyme nicotinamide phosphoribosyltransferase with FK866 led cultured M. corti to death. Innovation: We use comparative genomics of more than 100 hundred helminth genomes, metabolic labeling, HPLC-mass spectrometry targeted metabolomics, and enzyme inhibitors to define pathways that lead to RQ and NAD+ biosynthesis in helminths. We identified the essential enzymes of these pathways in helminth lineages, revealing new potential pharmacological targets for helminthiasis. Conclusion: Our results demonstrate that a minimal KYN pathway was evolutionary maintained for RQ and not for de novo NAD+ biosynthesis in helminths and shed light on the essentiality of NAD+ rescue pathways in helminths.

Linked thioredoxin-glutathione systems in platyhelminth parasites: alternative pathways for glutathione reduction and deglutathionylation.

In most organisms, thioredoxin (Trx) and/or glutathione (GSH) systems are essential for redox homeostasis and deoxyribonucleotide synthesis. Platyhelminth parasites have a unique and simplified thiol-based redox system, in which the selenoprotein thioredoxin-glutathione reductase (TGR), a fusion of a glutaredoxin (Grx) domain to canonical thioredoxin reductase domains, is the sole enzyme supplying electrons to oxidized glutathione (GSSG) and Trx. This enzyme has recently been validated as a key drug target for flatworm infections. In this study, we show that TGR possesses GSH-independent deglutathionylase activity on a glutathionylated peptide. Furthermore, we demonstrate that deglutathionylation and GSSG reduction are mediated by the Grx domain by a monothiolic mechanism and that the glutathionylated TGR intermediate is resolved by selenocysteine. Deglutathionylation and GSSG reduction via Grx domain, but not Trx reduction, are inhibited at high [GSSG]/[GSH] ratios. We found that Trxs (cytosolic and mitochondrial) provide alternative pathways for deglutathionylation and GSSG reduction. These pathways are operative at high [GSSG]/[GSH] and function in a complementary manner to the Grx domain-dependent one. Despite the existence of alternative pathways, the thioredoxin reductase domains of TGR are an obligate electron route for both the Grx domain- and the Trx-dependent pathways. Overall, our results provide an explanation for the unique array of thiol-dependent redox pathways present in parasitic platyhelminths. Finally, we found that TGR is inhibited by 1-hydroxy-2-oxo-3-(N-3-methyl-aminopropyl)-3-methyl-1-triazene (NOC-7), giving further evidence for NO donation as a mechanism of action for oxadiazole N-oxide TGR inhibitors. Thus, NO donors aimed at TGR could disrupt the entire redox homeostasis of parasitic flatworms.

Phagocyte-specific S100 proteins in the local response to the Echinococcus granulosus larva.

Infection by larval Echinococcus granulosus is usually characterized by tight inflammatory control. However, various degrees of chronic granulomatous inflammation are also observed, reaching a high point in infection of cattle by the most prevalent parasite strain worldwide, which is not well adapted to this host species. In this context, epithelioid and multinucleated giant macrophages surround the parasite, and the secreted products of these cells often associate with the larval wall. The phagocyte-specific S100 proteins, S100A8, S100A9 and S100A12, are important non-conventionally secreted amplifiers of inflammatory responses. We have analysed by proteomics and immunohistochemistry the presence of these proteins at the E. granulosus larva-host interface. We found that, in the context of inflammatory control as observed in human infections, the S100 proteins are not abundant, but S100A9 and S100A8 can be expressed by eosinophils distal to the parasite. In the granulomatous inflammation context as observed in cattle infections, we found that S100A12 is one of the most abundant host-derived, parasite-associated proteins, while S100A9 and S100A8 are not present at similarly high levels. As expected, S100A12 derives mostly from the epithelioid and multinucleated giant cells. S100A12, as well as cathepsin K and matrix metalloproteinase-9, also expressed by E. granulosus-elicited epithelioid cells, are connected to the Th17 arm of immunity, which may therefore be involved in this granulomatous response.

Caenorhabditis elegans as a valuable model for the study of anthelmintic pharmacodynamics and drug-drug interactions: The case of ivermectin and eprinomectin.

Caenorhabditis elegans is a free-living nematode that has been validated for anthelmintic drug screening. However, this model has not been used to address anthelmintic dose-response-time and drug-drug interactions through matrix array methodology. Eprinomectin (EPM) and Ivermectin (IVM) are macrocyclic lactones widely used as anthelmintics. Despite being very similar, EPM and IVM are combined in commercial formulations or mixed by farmers, under the assumption that the combination would increase their efficacy. However, there is no data reported on the pharmacological evaluation of the combination of both drugs. In this study, we assessed the pharmacodynamics and drug-drug interactions of these two anthelmintic drugs. Since the action of these drugs causes worm paralysis, we used an infrared motility assay to measure EPM and IVM effects on worm movement over time. The results showed that EPM was slightly more potent than IVM, that drug potency increased with drug time exposure, and that once paralyzed, worms did not recover. Different EPM/IVM concentration ratios were used and synergy and combination sensitivity scores were determined at different exposure times, applying Highest Single Agent (HSA), Loewe additivity, Bliss and Zero Interaction Potency (ZIP) models. The results clearly indicate that there is neither synergy nor antagonism between both macrocyclic lactones. This study shows that it is more relevant to prioritize the exposure time of each individual drug than to combine them to improve their effects. The results highlight the utility of C. elegans to address pharmacodynamics studies, particularly for drug-drug interactions. Models in vitro can be integrated to facilitate preclinical and clinical translational studies and help researchers to understand drug-drug interactions and achieve rational therapeutic regimes.