Cryptosporidium life cycle small molecule probing implicates translational repression and an Apetala 2 transcription factor in macrogamont differentiation.

The apicomplexan parasite Cryptosporidium is a leading cause of childhood diarrhea in developing countries. Current treatment options are inadequate and multiple preclinical compounds are being actively pursued as potential drugs for cryptosporidiosis. Unlike most apicomplexans, Cryptosporidium spp. sequentially replicate asexually and then sexually within a single host to complete their lifecycles. Anti-cryptosporidial compounds are generally identified or tested through in vitro phenotypic assays that only assess the asexual stages. Therefore, compounds that specifically target the sexual stages remain unexplored. In this study, we leveraged the ReFRAME drug repurposing library against a newly devised multi-readout imaging assay to identify small-molecule compounds that modulate macrogamont differentiation and maturation. RNA-seq studies confirmed selective modulation of macrogamont differentiation for 10 identified compounds (9 inhibitors and 1 accelerator). The collective transcriptomic profiles of these compounds indicates that translational repression accompanies Cryptosporidium sexual differentiation, which we validated experimentally. Additionally, cross comparison of the RNA-seq data with promoter sequence analysis for stage-specific genes converged on a key role for an Apetala 2 (AP2) transcription factor (cgd2_3490) in differentiation into macrogamonts. Finally, drug annotation for the ReFRAME hits indicates that an elevated supply of energy equivalence in the host cell is critical for macrogamont formation.

Modeling temperature-dependent life-cycle toxicity of thiamethoxam in Chironomus riparius using a DEB-TKTD model.

The neonicotinoid insecticide thiamethoxam (TMX) is widely used to protect crops against insect pests. Despite some desirable properties such as its low toxicity to birds and mammals, concerns have been raised about its toxicity to non-target arthropods, including freshwater insects like chironomids. Whereas multiple studies have investigated chronic effects of neonicotinoids in chironomid larvae at standardized laboratory conditions, a better understanding of their chronic toxicity under variable temperatures and exposure is needed for coherent extrapolation from the laboratory to the field. Here, we developed a quantitative mechanistic effect model for Chironomus riparius, to simulate the species' life history under dynamic temperatures and exposure concentrations of TMX. Laboratory experiments at four different temperatures (12, 15, 20, 23 °C) and TMX concentrations between 4 and 51 µg/L were used to calibrate the model. Observed concentration-dependent effects of TMX in C. riparius included slower growth, later emergence, and higher mortality rates with increasing concentrations. Furthermore, besides a typical accelerating effect on the organisms' growth and development, higher temperatures further increased the effects associated with TMX. With some data-informed modeling decisions, most prominently the inclusion of a size dependence that makes larger animals more sensitive to TMX, the model was parametrized to convincingly reproduce the data. Experiments at both a constant (20 °C) and a dynamically increasing temperature (15-23 °C) with pulsed exposure were used to validate the model. Finally, the model was used to simulate realistic exposure conditions using two reference exposure scenarios measured in Missouri and Nebraska, utilizing a moving time window (MTW) and either a constant temperature (20 °C) or the measured temperature profiles belonging to each respective scenario. Minimum exposure multiplication factors leading to a 10% effect (EP10) in the survival at pupation, i.e., the most sensitive endpoint found in this study, were 25.67 and 21.87 for the Missouri scenario and 38.58 and 44.64 for the Nebraska scenario, when using the respective temperature assumptions. While the results illustrate that the use of real temperature scenarios does not systematically modify the EPx in the same direction (making it either more or less conservative when used as a risk indicator), the advantage of this approach is that it increases the realism and thus reduces the uncertainty associated with the model predictions.

Chronic Effects of an Insect Growth Regulator (teflubenzuron) on the Life Cycle and Population Growth Rate of Folsomia candida.

Current standard toxicity tests on nontarget soil invertebrates mainly focus on the endpoints survival and reproduction. Such results are likely insufficient to predict effects at higher organizational levels, for example, the population level. We assessed the effects of exposure to the pesticide teflubenzuron on the collembolan Folsomia candida, by performing a full life-cycle experiment exposing single individuals via contaminated food (uncontaminated control and 0.2, 0.32, 0.48, 0.72, 1.08, and 1.6 mg/kg dry yeast). Several life-history traits were considered by following the growth and development of newly hatched individuals over a period of 65 days. We assessed survival, body length, time to first oviposition, cumulative egg production, and hatchability of eggs. A two-stage model was applied to calculate the population growth rate (λ) combined with elasticity analysis to reveal the relative sensitivity of λ to the effects of teflubenzuron on each life-history parameter. Body length was the least sensitive life-history parameter (median effective concentration = 1.10 mg teflubenzuron/kg dry yeast) followed by time to first oviposition (0.96 mg/kg), survival (median lethal concentration = 0.87 mg/kg), cumulative egg production (0.32 mg/kg), and egg hatchability (0.27 mg/kg). Population growth decreased with increasing concentrations of teflubenzuron (λ = 1.162/day in control to 1.005/day in 0.72 mg/kg dry yeast, with populations going extinct at 1.08 and 1.6 mg/kg dry yeast). Elasticity analysis showed that changes in juvenile survival had a greater impact on the population growth rate compared with the other life-history traits. Our study provides a comprehensive overview of individual-level effects of long-term exposure to teflubenzuron and integrates these effects to assess the potential risk to collembolan populations. Environ Toxicol Chem 2024;43:1173-1183. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Bioretention filtration prevents acute mortality and reduces chronic toxicity for early life stage coho salmon (Oncorhynchus kisutch) episodically exposed to urban stormwater runoff.

As the human population of western North America continues to expand, widespread patterns of urban growth pose increasingly existential threats to certain wild stocks of Pacific salmon and steelhead (Oncorhynchus sp.). Rainfall previously absorbed into the soils of forests and grasslands falls instead on pavement and other hardened surfaces. This creates stormwater runoff that carries toxic metals, oil, and many other contaminants into salmon-bearing habitats. These include freshwater streams where coho salmon (O. kisutch) spawn in gravel beds. Coho salmon embryos develop within a thick eggshell (chorion) for weeks to months before hatching as alevins and ultimately emerging from the gravel as fry. Untreated urban runoff is highly toxic to older coho salmon (freshwater-resident juveniles and adult spawners), but the vulnerability of the earliest life stages remains poorly understood. To address this uncertainty, we fertilized eggs and raised them under an episodic stormwater exposure regimen, using runoff collected from a high-traffic arterial roadway from 15 discrete storm events. We monitored survival and morphological development, as well as molecular markers for contaminant exposure and cardiovascular stress. We also evaluated the benefit of treating runoff with green infrastructure (bioretention filtration) on coho salmon health and survival. Untreated runoff caused subtle sublethal toxicity in pre-hatch embryos with no mortality, followed by high rates of mortality from exposure at hatch. Bioretention filtration removed most measured contaminants (bacteria, dissolved metals, and polycyclic aromatic hydrocarbons), and the treated effluent was considerably less toxic - notably preventing mortality at the alevin stage. Our findings indicate that untreated urban runoff poses an important threat to early life stage coho salmon, in terms of both acute and delayed-in-time mortality. Moreover, while inexpensive management strategies involving bioinfiltration are promising, future green infrastructure effectiveness research should emphasize sublethal metrics for contaminant exposure and adverse health outcomes in salmonids.

Apical and mechanistic effects of 6PPD-quinone on different life-stages of the fathead minnow (Pimephales promelas).

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-quinone) is an emerging contaminant of concern that is generated through the environmental oxidation of the rubber tire anti-degradant 6PPD. Since the initial report of 6PPD-quinone being the cause of urban runoff mortality syndrome of Coho salmon, numerous species have been identified as either sensitive or insensitive to acute lethality caused by 6PPD-quinone. In sensitive species, acute lethality might be caused by uncoupling of mitochondrial respiration in gills. However, little is known about effects of 6PPD-quinone on insensitive species. Here we demonstrate that embryos of fathead minnows (Pimephales promelas) are insensitive to exposure to concentrations as great as 39.97 µg/L for 168 h, and adult fathead minnows are insensitive to exposure to concentrations as great as 9.4 µg/L for 96 h. A multi-omics approach using a targeted transcriptomics array, (EcoToxChips), and proton nuclear magnetic resonance (1H NMR) was used to assess responses of the transcriptomes and metabolomes of gills and livers from adult fathead minnows exposed to 6PPD-quinone for 96 h to begin to identify sublethal effects of 6PPD-quinone. There was little agreement between results of the EcoToxChip and metabolomics analyses, likely because genes present on the EcoToxChip were not representative of pathways suggested to be perturbed by metabolomic analysis. Changes in abundances of transcripts and metabolites in livers and gills suggest that disruption of one­carbon metabolism and induction of oxidative stress might be occurring in gills and livers, but that tissues differ in their sensitivity or responsiveness to 6PPD-quinone. Overall, several pathways impacted by 6PPD-quinone were identified as candidates for future studies of potential sublethal effects of this chemical.

Acute Toxicity of 6PPD-Quinone to Early Life Stage Juvenile Chinook (Oncorhynchus tshawytscha) and Coho (Oncorhynchus kisutch) Salmon.

The breakdown product of the rubber tire antioxidant N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD)-6-PPD-quinone has been strongly implicated in toxic injury and death in coho salmon (Oncorhynchus kisutch) in urban waterways. Whereas recent studies have reported a wide range of sensitivity to 6PPD-quinone in several fish species, little is known about the risks to Chinook salmon (Oncorhynchus tshawytscha), the primary prey of endangered Southern Resident killer whales (Orcinus orca) and the subject of much concern. Chinook face numerous conservation threats in Canada and the United States, with many populations assessed as either endangered or threatened. We evaluated the acute toxicity of 6PPD-quinone to newly feeding (~3 weeks post swim-up) juvenile Chinook and coho. Juvenile Chinook and coho were exposed for 24 h under static conditions to five concentrations of 6PPD-quinone. Juvenile coho were 3 orders of magnitude more sensitive to 6PPD-quinone compared with juvenile Chinook, with 24-h median lethal concentration (LC50) estimates of 41.0 and more than 67 307 ng/L, respectively. The coho LC50 was 2.3-fold lower than what was previously reported for 1+-year-old coho (95 ng/L), highlighting the value of evaluating age-related differences in sensitivity to this toxic tire-related chemical. Both fish species exhibited typical 6PPD-quinone symptomology (gasping, increased ventilation, loss of equilibrium, erratic swimming), with fish that were symptomatic generally exhibiting mortality. The LC50 values derived from our study for coho are below concentrations that have been measured in salmon-bearing waterways, suggesting the potential for population-level consequences in urban waters. The higher relative LC50 values for Chinook compared with coho merits further investigation, including for the potential for population-relevant sublethal effects. Environ Toxicol Chem 2023;42:815-822. © 2023 His Majesty the King in Right of Canada and The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. Reproduced with the permission of the Minister of Fisheries and Oceans Canada.

New method for screening anti-Leishmania compounds in plants extracts by HPTLC-bioautography.

Leishmania genus is responsible for leishmaniasis, a group of diseases affecting 12 million people in the tropical and subtropical zone. Currently, the few drugs that are available to treat this disease are expensive and cause many side effects. Searching for new therapeutics from plant species seems to be a promising path. This work proposes an original HPTLC test against parasites, in particular on Leishmania infantum, to screen new molecules from plant extracts. The technique uses protozoa transformed to express the luciferase gene to observe the bioautogram in bioluminescence. We have developed two different test protocols based on the two dimorphic stages of the parasite. The free promastigote stage, and an intracellular stage parasitizing macrophage cells called the amastigote stage. These two stages only survive under extremely different conditions which required the development of two very different test protocols. For the promastigote free stage of the protozoa, the direct bioautography technique was chosen while for the intracellular amastigote stage, bioautography by immersion (agar overlay) was required. Amphotericine B was chosen as the reference compound for this assay. The development of each of these two tests made it possible to clearly detect areas of activity on the bioautogram, allowing a rapid and inexpensive screening of the antiparasitic properties of molecules in natural extracts.

Artemisinin-independent inhibitory activity of Artemisia sp. infusions against different Plasmodium stages including relapse-causing hypnozoites.

Artemisinin-based combination therapies (ACT) are the frontline treatments against malaria worldwide. Recently the use of traditional infusions from Artemisia annua (from which artemisinin is obtained) or Artemisia afra (lacking artemisinin) has been controversially advocated. Such unregulated plant-based remedies are strongly discouraged as they might constitute sub-optimal therapies and promote drug resistance. Here, we conducted the first comparative study of the anti-malarial effects of both plant infusions in vitro against the asexual erythrocytic stages of Plasmodium falciparum and the pre-erythrocytic (i.e., liver) stages of various Plasmodium species. Low concentrations of either infusion accounted for significant inhibitory activities across every parasite species and stage studied. We show that these antiplasmodial effects were essentially artemisinin-independent and were additionally monitored by observations of the parasite apicoplast and mitochondrion. In particular, the infusions significantly incapacitated sporozoites, and for Plasmodium vivax and P. cynomolgi, disrupted the hypnozoites. This provides the first indication that compounds other than 8-aminoquinolines could be effective antimalarials against relapsing parasites. These observations advocate for further screening to uncover urgently needed novel antimalarial lead compounds.

Transcriptional differentiation of Trypanosoma brucei during in vitro acquisition of resistance to acoziborole.

Subspecies of the protozoan parasite Trypanosoma brucei are the causative agents of Human African Trypanosomiasis (HAT), a debilitating neglected tropical disease prevalent across sub-Saharan Africa. HAT case numbers have steadily decreased since the start of the century, and sustainable elimination of one form of the disease is in sight. However, key to this is the development of novel drugs to combat the disease. Acoziborole is a recently developed benzoxaborole, currently in advanced clinical trials, for treatment of stage 1 and stage 2 HAT. Importantly, acoziborole is orally bioavailable, and curative with one dose. Recent studies have made significant progress in determining the molecular mode of action of acoziborole. However, less is known about the potential mechanisms leading to acoziborole resistance in trypanosomes. In this study, an in vitro-derived acoziborole-resistant cell line was generated and characterised. The AcoR line exhibited significant cross-resistance with the methyltransferase inhibitor sinefungin as well as hypersensitisation to known trypanocides. Interestingly, transcriptomics analysis of AcoR cells indicated the parasites had obtained a procyclic- or stumpy-like transcriptome profile, with upregulation of procyclin surface proteins as well as differential regulation of key metabolic genes known to be expressed in a life cycle-specific manner, even in the absence of major morphological changes. However, no changes were observed in transcripts encoding CPSF3, the recently identified protein target of acoziborole. The results suggest that generation of resistance to this novel compound in vitro can be accompanied by transcriptomic switches resembling a procyclic- or stumpy-type phenotype.

Drug repurposing for Chagas disease: In vitro assessment of nimesulide against Trypanosoma cruzi and insights on its mechanisms of action.

Chagas disease is a neglected illness caused by Trypanosoma cruzi and its treatment is done only with two drugs, nifurtimox and benznidazole. However, both drugs are ineffective in the chronic phase, in addition to causing serious side effects. This context of therapeutic limitation justifies the continuous research for alternative drugs. Here, we study the in vitro trypanocidal effects of the non-steroidal anti-inflammatory drug nimesulide, a molecule that has in its chemical structure a toxicophoric nitroaromatic group (NO2). The set of results obtained in this work highlights the potential for repurposing nimesulide in the treatment of this disease that affects millions of people around the world.

Metabolomics reveal alterations in arachidonic acid metabolism in Schistosoma mekongi after exposure to praziquantel.

BACKGROUND: Mekong schistosomiasis is a parasitic disease caused by the blood-dwelling fluke Schistosoma mekongi. This disease contributes to human morbidity and mortality in the Mekong region, posing a public health threat to people in the area. Currently, praziquantel (PZQ) is the drug of choice for the treatment of Mekong schistosomiasis. However, the molecular mechanisms of PZQ action remain unclear, and Schistosoma PZQ resistance has been reported occasionally. Through this research, we aimed to use a metabolomic approach to identify the potentially altered metabolic pathways in S. mekongi associated with PZQ treatment. METHODOLOGY/PRINCIPAL FINDINGS: Adult stage S. mekongi were treated with 0, 20, 40, or 100 µg/mL PZQ in vitro. After an hour of exposure to PZQ, schistosome metabolites were extracted and studied with mass spectrometry. The metabolomic data for the treatment groups were analyzed with the XCMS online platform and compared with data for the no treatment group. After low, medium (IC50), and high doses of PZQ, we found changes in 1,007 metabolites, of which phosphatidylserine and anandamide were the major differential metabolites by multivariate and pairwise analysis. In the pathway analysis, arachidonic acid metabolism was found to be altered following PZQ treatment, indicating that this pathway may be affected by the drug and potentially considered as a novel target for anti-schistosomiasis drug development. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that arachidonic acid metabolism is a possible target in the parasiticidal effects of PZQ against S. mekongi. Identifying potential targets of the effective drug PZQ provides an interesting viewpoint for the discovery and development of new agents that could enhance the prevention and treatment of schistosomiasis.

Differential expression of calcium-dependent protein kinase 4, tubulin tyrosine ligase, and methyltransferase by xanthurenic acid-induced Babesia bovis sexual stages.

BACKGROUND: Babesia bovis is one of the most significant tick-transmitted pathogens of cattle worldwide. Babesia bovis parasites have a complex lifecycle, including development within the mammalian host and tick vector. Each life stage has developmental forms that differ in morphology and metabolism. Differentiation between these forms is highly regulated in response to changes in the parasite's environment. Understanding the mechanisms by which Babesia parasites respond to environmental changes and the transmission cycle through the biological vector is critically important for developing bovine babesiosis control strategies. RESULTS: In this study, we induced B. bovis sexual stages in vitro using xanthurenic acid and documented changes in morphology and gene expression. In vitro induced B. bovis sexual stages displayed distinctive protrusive structures and surface ruffles. We also demonstrated the upregulation of B. bovis calcium-dependent protein kinase 4 (cdpk4), tubulin-tyrosine ligase (ttl), and methyltransferase (mt) genes by in vitro induced sexual stages and during parasite development within tick midguts. CONCLUSIONS: Similar to other apicomplexan parasites, it is likely that B. bovis upregulated genes play a vital role in sexual reproduction and parasite transmission. Herein, we document the upregulation of cdpk4, ttl, and mt genes by both B. bovis in vitro induced sexual stages and parasites developing in the tick vector. Understanding the parasite's biology and identifying target genes essential for sexual reproduction will enable the production of non-transmissible live vaccines to control bovine babesiosis.

An Overview on Target-Based Drug Design against Kinetoplastid Protozoan Infections: Human African Trypanosomiasis, Chagas Disease and Leishmaniases.

The protozoan diseases Human African Trypanosomiasis (HAT), Chagas disease (CD), and leishmaniases span worldwide and therefore their impact is a universal concern. The present regimen against kinetoplastid protozoan infections is poor and insufficient. Target-based design expands the horizon of drug design and development and offers novel chemical entities and potential drug candidates to the therapeutic arsenal against the aforementioned neglected diseases. In this review, we report the most promising targets of the main kinetoplastid parasites, as well as their corresponding inhibitors. This overview is part of the Special Issue, entitled "Advances of Medicinal Chemistry against Kinetoplastid Protozoa (Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp.) Infections: Drug Design, Synthesis and Pharmacology".

Further investigation of harmicines as novel antiplasmodial agents: Synthesis, structure-activity relationship and insight into the mechanism of action.

The rise of the resistance of the malaria parasite to the currently approved therapy urges the discovery and development of new efficient agents. Previously we have demonstrated that harmicines, hybrid compounds composed from ß-carboline alkaloid harmine and cinnamic acid derivatives, linked via either triazole or amide bond, exert significant antiplasmodial activity. In this paper, we report synthesis, antiplasmodial activity and cytotoxicity of expanded series of novel triazole- and amide-type harmicines. Structure-activity relationship analysis revealed that amide-type harmicines 27, prepared at N-9 of the ß-carboline core, exhibit superior potency against both erythrocytic stage of P. falciparum and hepatic stages of P. berghei. Notably, harmicine 27a, m-(trifluoromethyl)cinnamic acid derivative, exhibited the most favourable selectivity index (SI = 1105). Molecular dynamics simulations revealed the ATP binding site of P. falciparum heat shock protein 90 as a druggable binding location, confirmed the usefulness of the harmine's N-9 substitution and identified favourable N-H … π interactions involving Lys45 and the aromatic phenyl unit in the attached cinnamic acid fragment as crucial for the enhanced biological activity. Thus, those compounds were identified as promising and valuable leads for further derivatization in the search of novel, more efficient antiplasmodial agents.

Novel halogenated arylvinyl-1,2,4 trioxanes as potent antiplasmodial as well as anticancer agents: Synthesis, bioevaluation, structure-activity relationship and in-silico studies.

Herein, we have synthesized a series of lipophilic, halogenated-arylvinyl-1,2,4-trioxanes 8a-g (28 compounds) and assessed for their in vitro anti-plasmodial activity in Plasmodium falciparum culture using SYBRgreen-I fluorescence assay against chloroquine-resistant Pf INDO and artemisinin-resistant Pf Cam 3.1R539T (MRA-1240) strains. Alongside, the cell cytotoxic potential of 8a-g has also been determined against the HEK293 cell line in vitro. Out of twenty-eight halogenated-arylvinyl-1,2,4-trioxanes; ten analogues (8a2, 8a4, 8b2, 8b4, 8d4, 8e1, 8e2, 8e4,8f2, and 8g4) have shown potent in vitro antiplasmodial activity with IC50 < 27 nM (IC50 range = 4.48-26.58 nM). Also, the selectivity index (SI) for these ten analogues were found in the range of 72.00-3972.50 which indicates their selective potential towards Plasmodium cells. Results of the cell cycle stage specificity with two of the most potent compounds 8a4 {(IC50 = 4.48 nM; SI = 3972.50) more potent than chloroquine (IC50 = 546 nM; SI = 36.64) and artesunate (IC50 = 6.6 nM; SI = 4333.33)} and 8e2 (IC50 = 9.69 nM; SI = 1348) against Pf INDO indicated all three stages to be the target of the action of 8e2 while only rings and trophozoites appeared to be targeted by 8a4. Ring stage survival assay against artemisinin-resistant Pf Cam 3.1R539T indicated that 8a4 may be well suited to replace artemisinin from current ACTs which are experiencing in vivo delayed parasite clearance. With intraperitoneal (i.p.) and oral (p.o.) route at the dose of 50 mg/kg/day × 4 days; 8a4 has also shown 100% suppression of parasitemia in P. berghei ANKA infected Balb C mice. Further, the in vitro anticancer activity of 8a-g performed against human lung (A549) and liver (HepG2) cancer cell lines as also against immortalized normal lung (BEAS-2B) and liver (LO2) cell lines has revealed that most of the derivatives are endowed also with promising anticancer activity (IC50 = 0.69-15 µM; SI = 1.02-20.61) in comparison with standard drugs such as chloroquine (IC50 = 100 µM; SI = 0.03), artemisinin (IC50 = 100 µM), and artesunic acid (IC50 = 9.85 µM; SI = 0.76), respectively. All the derivatives have shown moderate anticancer activity against liver (HepG2) cancer cell lines. Arylvinyl-1,2,4-trioxanes 8f2 (IC50 = 0.69 µM; SI = 16.66), the most active compound of the series, has shown ∼145 fold more cytotoxic potential with higher selectivity in comparison to reference drugs chloroquine (IC50 = 100 µM; SI = 0.03) and artemisinin (IC50 = 100 µM), respectively against the lung (A549) cancer cell line. Finally, the in-silico docking studies of the potent halogenated 1,2,4-trioxanes along with reference drug molecules against epidermal growth factor receptor (EGFR; PDB ID: 1M17) have demonstrated the strong virtual interaction.

Exposure to dexamethasone modifies transcriptomic responses of free-living stages of Strongyloides stercoralis.

Hyperinfection and disseminated infection by the parasitic nematode Strongyloides stercoralis can be induced by iatrogenic administration of steroids and immunosuppression and lead to an elevated risk of mortality. Responses of free-living stages of S. stercoralis to the therapeutic corticosteroid dexamethasone (DXM) were investigated using RNA-seq transcriptomes of DXM-treated female and male worms. A total of 17,950 genes representing the transcriptome of these free-living adult stages were obtained, among which 199 and 263 were differentially expressed between DXM-treated females and DXM-treated males, respectively, compared with controls. According to Gene Ontology analysis, differentially expressed genes from DXM-treated females participate in developmental process, multicellular organismal process, cell differentiation, carbohydrate metabolic process and embryonic morphogenesis. Others are involved in signaling and signal transduction, including cAMP, cGMP-dependent protein kinase pathway, endocrine system, and thyroid hormone pathway, as based on Kyoto Encyclopedia of Genes and Genomes analysis. The novel findings warrant deeper investigation of the influence of DXM on growth and other pathways in this neglected tropical disease pathogen, particularly in a setting of autoimmune and/or allergic disease, which may require the clinical use of steroid-like hormones during latent or covert strongyloidiasis.

Oral combination of eugenol oleate and miltefosine induce immune response during experimental visceral leishmaniasis through nitric oxide generation with advanced cytokine demand.

Conventional therapy of visceral leishmaniasis (VL) remains challenging with the pitfall of toxicity, drug resistance, and expensive. Hence, urgent need for an alternative approach is essential. In this study, we evaluated the potential of combination therapy with eugenol oleate and miltefosine in Leishmania donovani infected macrophages and in the BALB/c mouse model. The interactions between eugenol oleate and miltefosine were found to be additive against promastigotes and amastigotes with xΣFIC 1.13 and 0.68, respectively. Significantly (p < 0.001) decreased arginase activity, increased nitrite generation, improved pro-inflammatory cytokines, and phosphorylated p38MAPK were observed after combination therapy with eugenol oleate and miltefosine. >80% parasite clearance in splenic and hepatic tissue with concomitant nitrite generation, and anti-VL cytokines productions were observed after orally administered miltefosine (5 mg/kg body weight) and eugenol oleate (15 mg/kg body weight) in L. donovani-infected BALB/c mice. Altogether, this study suggested the possibility of an oral combination of miltefosine with eugenol oleate against visceral leishmaniasis.

Acute and chronic toxicity of microcystin-LR and phenanthrene alone or in combination to the cladoceran (Daphnia magna).

Hazardous substances, such as microcystin-LR (MC-LR) and phenanthrene (Phe) are ubiquitous co-contaminants in eutrophic freshwaters, which cause harms to aquatic organisms. However, the risks associated with the co-exposure of aquatic biota to these two chemicals in the environment have received little attention. In this study, the single and mixture toxic effects of MC-LR and Phe mixtures were investigated in Daphnia magna after acute and chronic exposure. Acute tests showed that the median effective concentrations (48 h) for MC-LR, Phe and their mixtures were 13.46, 0.57 and 8.84 mg/L, respectively. Mixture toxicity prediction results indicated that the independent action model was more applicable than the concentration addition model. Moreover, combination index method suggested that the mixture toxicity was concentration dependent. Synergism was elicited at low concentrations of MC-LR and Phe exposure (≤4.04 + 0.17 mg/L), whereas antagonistic or additive effects were induced at higher concentrations. The involved mechanism of antagonism was presumably attributable to the protective effects of detoxification genes activated by high concentrations of MC-LR in mixtures. Additionally, chronic results also showed that exposure to a MC-LR and Phe mixture at low concentrations (≤50 +2 µg/L) resulted in greater toxic effects on D. magna life history than either chemical acting alone. The significant inhibition on detoxification genes and increased accumulation of MC-LR could be accounted for their synergistic toxic effects on D. magna. Our findings revealed the exacerbated ecological hazard of MC-LR and Phe at environmental concentrations (≤50 +2 µg/L), and provided new insights to the potential toxic mechanisms of MC-LR and Phe in aquatic animals.

Design and synthesis of Mannich base-type derivatives containing imidazole and benzimidazole as lead compounds for drug discovery in Chagas Disease.

The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, the most important parasitic infection in Latin America. The only treatments currently available are nitro-derivative drugs that are characterised by high toxicity and limited efficacy. Therefore, there is an urgent need for more effective, less toxic therapeutic agents. We have previously identified the potential for Mannich base derivatives as novel inhibitors of this parasite. To further explore this family of compounds, we synthesised a panel of 69 new analogues, based on multi-parametric structure-activity relationships, which allowed optimization of both anti-parasitic activity, physicochemical parameters and ADME properties. Additionally, we optimized our in vitro screening approaches against all three developmental forms of the parasite, allowing us to discard the least effective and trypanostatic derivatives at an early stage. We ultimately identified derivative 3c, which demonstrated excellent trypanocidal properties, and a synergistic mode of action against trypomastigotes in combination with the reference drug benznidazole. Both its druggability and low-cost production make this derivative a promising candidate for the preclinical, in vivo assays of the Chagas disease drug-discovery pipeline.

Tadalafil impacts the mechanical properties of Plasmodium falciparum gametocyte-infected erythrocytes.

Plasmodium falciparum gametocytes modify the mechanical properties of their erythrocyte host to persist for several weeks in the blood circulation and to be available for mosquitoes. These changes are tightly regulated by the plasmodial phosphodiesterase delta that decreases both the stiffness and the permeability of the infected host cell. Here, we address the effect of the phosphodiesterase inhibitor tadalafil on deformability and permeability of gametocyte-infected erythrocytes. We show that this inhibitor drastically increases isosmotic lysis of gametocyte-infected erythrocytes and impairs their ability to circulate in an in vitro model for splenic retention. These findings indicate that tadalafil represents a novel drug lead potentially capable of blocking malaria parasite transmission by impacting gametocyte circulation.