Promastigote EPS secretion and haptomonad biofilm formation as evolutionary adaptations of trypanosomatid parasites for colonizing honeybee hosts.

Bees are major pollinators involved in the maintenance of all terrestrial ecosystems. Biotic and abiotic factors placing these insects at risk is a research priority for ecological and agricultural sustainability. Parasites are one of the key players of this global decline and the study of their mechanisms of action is essential to control honeybee colony losses. Trypanosomatid parasites and particularly the Lotmaria passim are widely spread in honeybees, however their lifestyle is poorly understood. In this work, we show how these parasites are able to differentiate into a new parasitic lifestyle: the trypanosomatid biofilms. Using different microscopic techniques, we demonstrated that the secretion of Extracellular Polymeric Substances by free-swimming unicellular promastigote forms is a prerequisite for the generation and adherence of multicellular biofilms to solid surfaces in vitro and in vivo. Moreover, compared to human-infective trypanosomatid parasites our study shows how trypanosomatid parasites of honeybees increases their resistance and thus resilience to drastic changes in environmental conditions such as ultralow temperatures and hypoosmotic shock, which would explain their success thriving within or outside their hosts. These results set up the basis for the understanding of the success of this group of parasites in nature and to unveil the impact of such pathogens in honeybees, a keystones species in most terrestrial ecosystems.

Antiprotozoal Activity of Benzoylthiourea Derivatives against Trypanosoma cruzi: Insights into Mechanism of Action.

For decades, only two nitroheterocyclic drugs have been used as therapeutic agents for Chagas disease. However, these drugs present limited effectiveness during the chronic phase, possess unfavorable pharmacokinetic properties, and induce severe adverse effects, resulting in low treatment adherence. A previous study reported that N-(cyclohexylcarbamothioyl) benzamide (BTU-1), N-(tert-butylcarbamothioyl) benzamide (BTU-2), and (4-bromo-N-(3-nitrophenyl) carbamothioyl benzamide (BTU-3) present selective antiprotozoal activity against all developmental forms of Trypanosoma cruzi Y strain. In this study, we investigated the mechanism of action of these compounds through microscopy and biochemical analyses. Transmission electron microscopy analysis showed nuclear disorganization, changes in the plasma membrane with the appearance of blebs and extracellular arrangements, intense vacuolization, mitochondrial swelling, and formation of myelin-like structures. Biochemical results showed changes in the mitochondrial membrane potential, reactive oxygen species content, lipid peroxidation, and plasma membrane fluidity. In addition, the formation of autophagic vacuoles was observed. These findings indicate that BTU-1, BTU-2, and BTU-3 induced profound morphological, ultrastructural, and biochemical alterations in epimastigote forms, triggering an autophagic-dependent cell death pathway.

Development of a TaqMan qPCR assay for trypanosomatid multi-species detection and quantification in insects.

BACKGROUND: Trypanosomatid parasites are widely distributed in nature and can have a monoxenous or dixenous life-cycle. These parasites thrive in a wide number of insect orders, some of which have an important economic and environmental value, such as bees. The objective of this study was to develop a robust and sensitive real-time quantitative PCR (qPCR) assay for detecting trypanosomatid parasites in any type of parasitized insect sample. METHODS: A TaqMan qPCR assay based on a trypanosomatid-conserved region of the α-tubulin gene was standardized and evaluated. The limits of detection, sensitivity and versatility of the α-tubulin TaqMan assay were tested and validated using field samples of honeybee workers, wild bees, bumblebees and grasshoppers, as well as in the human infective trypanosomatid Leishmania major. RESULTS: The assay showed a detection limit of 1 parasite equivalent/µl and successfully detected trypanosomatids in 10 different hosts belonging to the insect orders Hymenoptera and Orthoptera. The methodology was also tested using honeybee samples from four apiaries (n = 224 worker honeybees) located in the Alpujarra region (Granada, Spain). Trypanosomatids were detected in 2.7% of the honeybees, with an intra-colony prevalence of 0% to 13%. Parasite loads in the four different classes of insects ranged from 40.6 up to 1.1 × 108 cell equivalents per host. CONCLUSIONS: These results show that the α-tubulin TaqMan qPCR assay described here is a versatile diagnostic tool for the accurate detection and quantification of trypanosomatids in a wide range of environmental settings.

Administration of enteral nutrition and gastrointestinal complications in Covid-19 critical patients in prone position.

Background: The prone position (PP) used in the treatment of critically ill patients infected with SARS-CoV-2, may be a barrier to enteral nutrition (EN). This study aimed to analyze the effectiveness and complications of EN in the PP, as well as clinical outcomes. Methods: Prospective cohort study with patients in EN and coronavirus disease 2019 (COVID-19), on mechanical ventilation (MV), which whom needed or not PP. Gastrointestinal intolerances (GII) related to PP were evaluated, and correlated with possible confounding factors. EN, days on MV, Intensive Care Unit (ICU) length of stay, hospital length of stay, ventilator-associated pneumonia (VAP) and mortality were analyzed. The data were evaluated daily and compared prone group (PG=57) and supine group (SG=69). Results: The PP was associated with GII (P=0.000) and presented in 32 patients (26,44%) with no difference among groups. Association between epinephrine (P=0.003), vasopressin (P=0.018), and GII was observed. There was no difference between the total volume of enteral nutrition (TVEN) infused in the groups. However, the mean EN infused for the days when the patient was on PP was (70.0% ± 31.5) and for the days in supine position was (74.8% ± 27.3), P= 0.006. The PG had a longer time on MV (P=0.005) and ICU (P=0.003) and PP was associated with VAP (P=<0.001). The infused TVEN showed no association with VAP (P=0.09). Conclusion: PP was a determining factor in GII and proved to be a risk factor for VAP, but the EN protocol seems to have ensured an adequate EN supply in PP and be a safe alternative.

Exploring Honeybee Abdominal Anatomy through Micro-CT and Novel Multi-Staining Approaches.

Continuous improvements in morphological and histochemical analyses of Apis mellifera could improve our understanding of the anatomy and physiology of these insects at both the cellular and tissue level. In this work, two different approaches have been performed to add new data on the abdomen of worker bees: (i) Micro-computed tomography (Micro-CT), which allows the identification of small-scale structures (micrometers) with adequate/optimal resolution and avoids sample damage and, (ii) histochemical multi-staining with Periodic Acid-Schiff-Alcian blue, Lactophenol-Saphranin O and pentachrome staining to precisely characterize the histological structures of the midgut and hindgut. Micro-CT allowed high-resolution imaging of anatomical structures of the honeybee abdomen with particular emphasis on the proventriculus and pyloric valves, as well as the connection of the sting apparatus with the terminal abdominal ganglia. Furthermore, the histochemical analyses have allowed for the first-time description of ventricular telocytes in honeybees, a cell type located underneath the midgut epithelium characterized by thin and long cytoplasmic projections called telopodes. Overall, the analysis of these images could help the detailed anatomical description of the cryptic structures of honeybees and also the characterization of changes due to abiotic or biotic stress conditions.

The Screen of a Phage Display Library Identifies a Peptide That Binds to the Surface of Trypanosoma cruzi Trypomastigotes and Impairs Their Infection of Mammalian Cells.

Background: Chagas is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. On the order of seven million people are infected worldwide and current therapies are limited, highlighting the urgent need for new interventions. T. cruzi trypomastigotes can infect a variety of mammalian cells, recognition and adhesion to the host cell being critical for parasite entry. This study focuses on trypomastigote surface ligands involved in cell invasion. Methods: Three selection rounds of a phage peptide display library for isolation of phages that bind to trypomastigotes, resulted in the identification of the N3 dodecapeptide. N3 peptide binding to T. cruzi developmental forms (trypomastigotes, amastigotes and epimastigotes) was evaluated by flow cytometry and immunofluorescence assays. Parasite invasion of Vero cells was assessed by flow cytometry and immunofluorescence assays. Results: Phage display screening identified the N3 peptide that binds preferentially to the surface of the trypomastigote and amastigote infective forms as opposed to non-infective epimastigotes. Importantly, the N3 peptide, but not a control scrambled peptide, inhibits trypomastigote invasion of Vero cells by 50%. Conclusion: The N3 peptide specifically binds to T. cruzi, and by doing so, inhibits Vero cell infection. Follow-up studies will identify the molecule on the parasite surface to which the N3 peptide binds. This putative T. cruzi ligand may advance chemotherapy design and vaccine development.

Antileishmanial activity of neo-clerodane diterpenes from Croton echioides.

Nowadays, new leishmanicidal drugs are needed and natural products arise as a promising alternative source. Therefore, bioguided fractionation of a hydroethanolic extract from the stem bark of Croton echioides Baill. were conducted based on its antileishmanial activity. Two novel neo-clerodane diterpenoids methyl-15,16-epoxy-3,13(16),14-neo-clerodatrien-17,18-dicarboxylate (1) and dimethyl-3-oxo-15,16-epoxy-13(16),14-neo-clerodadien-17,18-dicarboxylate (2) were isolated, as well as four known compounds (3-6) and lupeol, from the hexane fraction. Their structures were established by NMR analysis. The crude extract, fractions and the compounds (1 and 3-6) were evaluated for their in vitro antileishmanial activity and cytotoxicity against macrophages J774A.1. The selectivity index (SI) were calculated. The most active compound against promastigote forms of L. amazonensis was the clerodane diterpene 4, with IC50 values of 8.3 µM and SI value of 80.9. Our results highlighted stem bark of Croton echioides Baill. as a promising source for the development of a new chemotherapeutic agent to combat leishmaniasis.

Membrane dynamics in Leishmania amazonensis and antileishmanial activities of ß-carboline derivatives.

Two ß-carboline compounds, 8i and 6d, demonstrated in vitro antileishmanial activity against Leishmania (L.) amazonensis promastigotes similar to that of miltefosine (MIL). Estimates of the membrane-water partition coefficient (KM/W) and the compound concentrations in the membrane (cm50) and aqueous phase (cw50) for half maximal inhibitory concentration were made. Whereas these biophysical parameters for 6d were not significantly different from those reported for MIL, 8i showed lower affinity for the parasite membrane (lower KM/W) and a lower concentration of the compound in the membrane required to inhibit the growth of the parasite (lower cm50). A 2-hour treatment of Leishmania promastigotes with the compounds 8i and 6d caused membrane rigidity in a concentration-dependent manner, as demonstrated by the electron paramagnetic resonance (EPR) technique and spin label method. This increased rigidity of the membrane was interpreted to be associated with the occurrence of cross-linking of oxidized cytoplasmic proteins to the parasite membrane skeleton. Importantly, the two ß-carboline-oxazoline derivatives showed low hemolytic action, both in experiments with isolated red blood cells or with whole blood, denoting their great Leishmania/erythrocyte selectivity index. Using electron microscopy, changes in the membrane of both the amastigote and promastigote form of the parasite were confirmed, and it was demonstrated that compounds 8i and 6d decreased the number of amastigotes in infected murine macrophages. Furthermore, 8i and 6d were more toxic to the protozoa than to J774A.1 macrophages, with treated promastigotes exhibiting a decrease in cell volume, mitochondrial membrane potential depolarization, accumulation of lipid bodies, increased ROS production and changes in the cell cycle.

Antiproliferative activity of the dibenzylideneacetone derivate (E)-3-ethyl-4-(4-nitrophenyl)but­3-en-2-one in Trypanosoma cruzi.

Chagas disease is one of the most prevalent neglected diseases in the world. The illness is caused by Trypanosoma cruzi, a protozoan parasite with a complex life cycle and three morphologically distinct developmental stages. Nowadays, the only treatment is based on two nitro-derivative drugs, benznidazole and nifurtimox, which cause serious side effects. Since the treatment is limited, the search for new treatment options for patients with Chagas disease is highly necessary. In this study we analyzed the substance A11K3, a dibenzylideneacetone (DBA). DBAs have an acyclic dienone attached to aryl groups in both ß-positions and studies have shown that they have biological activity against tumors cells, bacteria, and protozoa such as T. cruzi and Leishmania spp. Here we show that A11K3 is active against all three T. cruzi evolutionary forms: the epimastigote (IC50 = 3.3 ± 0.8), the trypomastigote (EC50 = 24 ± 4.3) and the intracellular amastigote (IC50 = 9.3 ± 0.5 µM). A cytotoxicity assay in LLCMK2 cells showed a CC50 of 239.2 ± 15.7 µM giving a selectivity index (CC50/IC50) of 72.7 for epimastigotes, 9.9 for trypomastigotes and 25.9 for intracellular amastigotes. Morphological and ultrastructural analysis of the parasites treated with A11K3 by TEM and SEM revealed alterations in the Golgi complex, mitochondria, plasma membrane and cell body, with an increase of autophagic vacuoles and lipid bodies. Biochemical assays of A11K3-treated T. cruzi showed an increase of ROS, plasma membrane ruptures, lipid peroxidation, mitochondrial membrane depolarization with a decrease in ATP and accumulation of autophagic vacuoles. The results lead to the hypothesis that A11K3 causes death of the protozoan through events such as plasma membrane and mitochondrial alterations and autophagy, characteristic of cell collapse.

Antimicrobial Potential of Essential Oils from Cerrado Plants against Multidrug-Resistant Foodborne Microorganisms.

Foodborne pathogens are a real public health concern in an escalating antimicrobial resistance scenario. Natural products represent a promising source of bioactive molecules, and essential oils have attracted much attention due to their myriad of biological properties, including antibacterial activities. In this context, essential oils obtained from the leaves of Chromolaena squalida, Campomanesia sessiliflora, Myrsine guianensis, Matayba guianensis, Siparuna guianensis, Ocotea minarum and Endlicheria paniculata-species from the Cerrado biome of Midwest Brazil-were extracted and evaluated for their antibacterial activity against a panel of four standard and three clinical multidrug-resistant bacterial strains. All tested oils showed moderate to good activity against at least four bacterial strains, including Salmonella Typhi and oxacillin-resistant Staphylococcus. The essential oils from C. squalida, C. sessiliflora, My. guianensis and Ma. guianensis showed strong inhibition of clinical Staphylococcus strains, which cause bovine mastitis and are related to milk-borne diseases. Their chemical profiles were investigated by gas chromatography coupled to mass spectrometry (GC/MS), which revealed a predominance of mono- and sesquiterpene hydrocarbons, some of which with well-known antimicrobial properties. The essential oil from Cerrado plants proved active against resistant Gram-positive and Gram-negative bacteria, revealing their potentialities for the development of new alternative agents to prevent the spreading of resistant bacterial contamination.

Novel nitroimidazole derivatives evaluated for their trypanocidal, cytotoxic, and genotoxic activities.

The current treatment of Chagas disease is based on the use of two drugs, nifurtimox (Nfx) and benznidazole (Bnz), both of which present limited efficacy in the chronic stage of the disease and toxic side effects. Thus, the discovery of novel compounds is urgently required. Herein, we report the successful synthesis of 4-nitroimidazole analogs of Bnz via nucleophilic aromatic substitution or cycloaddition reactions. The analogs were biologically evaluated, and compound 4 (4-cyclopropyl-1-(1-methyl-4-nitro-1H-imidazole-5-yl)-1H-1,2,3-triazole) was identified as the most potent against both the trypomastigote (IC50 = 5.4 µM) and amastigote (IC50 = 12.0 µM) forms of T. cruzi, showing activity in the same range as Bnz (IC50 = 8.8 and 8.7 µM, respectively). The cytotoxic and genotoxic activities of compounds 5, 4 and 11 were assessed. These three compounds were cytotoxic and genotoxic to RAW and HepG2 cells and mutagenic to Salmonella enterica strains. However, 4 exhibited toxic effects only at concentrations higher than those needed for trypanocidal activity. Molecular docking of 4 showed the importance of the size and π-π interactions between the nitroimidazole and the cofactor (flavin mononucleotide) of T.cruzi-nitroreductase (TcNTR). Moreover, the residues His503 and Tyr545 are relevant for binding to TcNTR. Our design strategy was capable of generating novel and active Bnz analogs.

From nano- to micrometer scale: the role of microwave-assisted acid and alkali pretreatments in the sugarcane biomass structure.

BACKGROUND: To date, great strides have been made in elucidating the role of thermochemical pretreatments in the chemical and structural features of plant cell walls; however, there is no clear picture of the plant recalcitrance and its relationship to deconstruction. Previous studies precluded full answers due to the challenge of multiscale features of plant cell wall organization. Complementing the previous efforts, we undertook a systematic, multiscale, and integrated approach to track the effect of microwave-assisted H2SO4 and NaOH treatments on the hierarchical structure of plants, i.e., from a nano- to micrometer scale. We focused on the investigation of the highly recalcitrant sclerenchyma cell walls from sugarcane bagasse. RESULTS: Through atomic force microscopy and X-ray diffraction analyses, remarkable details of the assembly of cellulose microfibrils not previously seen were revealed. Following the H2SO4 treatment, we observed that cellulose microfibrils were almost double the width of the alkali pretreated sample at the temperature of 160 °C. Such enlargement led to a greater contact between cellulose chains, with a subsequent molecule alignment, as indicated by the X-ray diffraction (XRD) results with the conspicuous expansion of the average crystallite size. The delignification process had little effect on the local nanometer-sized arrangement of cellulose molecules. However, the rigidity and parallel alignment of cellulose microfibrils were partially degraded. The XRD analysis also agrees with these findings as evidenced by large momentum transfer vectors (q > 20 nm-1), interpreted as indicators of the long-range order of cell wall components, which were similar for all the studied samples except with application of the NaOH treatment at 160 °C. These changes were followed by the eventual swelling of the fiber cell walls. CONCLUSIONS: Based on an integrated approach, we presented multidimensional architectural models of cell wall deconstruction resulting from microwave-assisted pretreatments. We provided direct evidence supporting the idea that hemicellulose is the main barrier for the swelling of cellulose microfibrils, whereas lignin adds rigidity to cell walls. Our findings shed light on the design of more efficient strategies, not only for the conversion of biomass to fuels but also for the production of nanocellulose, which has great potential for several applications such as composites, rheology modifiers, and pharmaceuticals.

Synthesis, antileishmanial activity and mechanism of action studies of novel ß-carboline-1,3,5-triazine hybrids.

A series of novel hybrids ß-carboline-1,3,5-triazine were synthesized and evaluated for their in vitro antileishmanial activity against promastigote and amastigote forms of Leishmania amazonensis. Among the compounds tested, the hybrids 9d, 9e, 16a and 16b showed potent activity against the promastigote forms with IC50 values less than 8 µM. Compounds 9e and 16b were also active against amastigote forms, displaying IC50 values of 1.0 ±â€¯0.1 µM and 1.2 ±â€¯0.5 µM, respectively. Besides that, the hybrid 16b bearing the 4-methoxyphenyl group at C-1 of ß-carboline and isopropylamino group at 1,3,5-triazine, showed low toxicity, being 23.5 and 121.4 times more toxic for promastigotes and axenic amastigotes, respectively, than for macrophage J774-A1 cell lines. Investigation of action mechanism in promastigotes showed that compound 16b caused alterations in cell division cycle and an increase of lipid-storage bodies, leading the cells to death through various factors. The accumulation of lipid bodies may be associated with apoptotic cell death.