On the structural organization of the bacillary band of Trichuris muris under cryopreparation protocols and three-dimensional electron microscopy.

Whipworms of the genus Trichuris are nematode parasites that infect mammals and can lead to various intestinal diseases of human and veterinary interest. The most intimate interaction between the parasite and the host intestine occurs through the anterior region of the nematode body, inserted into the intestinal mucosa during infection. One of the most prominent structures of the nematode surface found at the infection site is the bacillary band, a surface domain formed by a number of cells, mostly stichocytes and bacillary glands, whose structure and function are still under debate. Here, we used confocal microscopy, field emission scanning electron microscopy, helium ion microscopy, transmission electron microscopy and FIB-SEM tomography to unveil the functional role of the bacillary gland cell. We analyzed the surface organization as well as the intracellular milieu of the bacillary glands of Trichuris muris in high pressure frozen/freeze-substituted samples. Results showed that the secretory content is preserved in all gland openings, presenting a projected pattern. FIB-SEM analysis showed that the lamellar zone within the bacillary gland chamber is formed by a set of lacunar structures that may exhibit secretory or absorptive functions. In addition, incubation of parasites with the fluid phase endocytosis marker sulforhodamine B showed a time-dependent uptake by the parasite mouth, followed by perfusion through different tissues with ultimate secretion through the bacillary gland. Taken together, the results show that the bacillary gland possess structural characteristics of secretory and absorptive cells and unequivocally demonstrate that the bacillary gland cell functions as a secretory structure.

Development of an in vitro system to study the developmental stages of Toxoplasma gondii using a genetically modified strain expressing markers for tachyzoites and bradyzoites.

Toxoplasma gondii, the agent of toxoplasmosis, is an intracellular parasite that can infect a wide range of vertebrate hosts. Toxoplasmosis causes severe damage to immunocompromised hosts and its treatment is mainly based on the combination of pyrimethamine and sulfadiazine, which causes relevant side effects primarily observed in AIDS patients, including bone marrow suppression and hematological toxicity (pyrimethamine) and/or hypersensitivity and allergic skin reactions (sulfadiazine). Thus, it is important to investigate new compounds against T. gondii, particularly those that may act on bradyzoites, which are present in cysts during the chronic disease phase. We propose an in vitro model to simultaneously study new candidate compounds against the two main causative stages of Toxoplasma infection in humans, using the EGS-DC strain that was modified from a type I/III strain (EGS), isolated from a case of human congenital toxoplasmosis in Brazil and engineered to express markers for both stages of development. One feature of this strain is that it presents tachyzoite and bradyzoite in the same culture system and in the same host cell under normal culture conditions. Additionally, this strain presents stage-specific fluorescent protein expression, allowing for easy identification of both stages, thus making this strain useful in different studies. HFF cells were infected and after 4 and 7 days post infection the cells were treated with 10 µM of pyrimethamine or atovaquone, for 48 or 72 h. We used high-throughput screening to quantify the extent of parasite infection. Despite a reduction in tachyzoite infection caused by both treatments, the atovaquone treatment reduced the bradyzoite infection while the pyrimethamine one increased it. Ultrastructural analysis showed that after treatment with both drugs, parasites displayed altered mitochondria. Fluorescence microscopy of cells labeled with MitoTracker CMXRos showed that the cysts present inside the cells lost their mitochondrial membrane potential. Our results indicate that this experimental model is adequate to simultaneously analyze new active compounds against tachyzoite and bradyzoite forms.

Toxoplasma gondii reorganizes the host cell architecture during spontaneous cyst formation in vitro.

Toxoplasma gondii is an intracellular protozoan parasite that causes toxoplasmosis, a prevalent infection related to abortion, ocular diseases and encephalitis in immuno-compromised individuals. In the untreatable (and life-long) chronic stage of toxoplasmosis, parasitophorous vacuoles (PVs, containing T. gondii tachyzoites) transform into tissue cysts, containing slow-dividing bradyzoite forms. While acute-stage infection with tachyzoites involves global rearrangement of the host cell cytoplasm, focused on favouring tachyzoite replication, the cytoplasmic architecture of cells infected with cysts had not been described. Here, we characterized (by fluorescence and electron microscopy) the redistribution of host cell structures around T. gondii cysts, using a T. gondii strain (EGS) with high rates of spontaneous cystogenesis in vitro. Microtubules and intermediate filaments (but not actin microfilaments) formed a 'cage' around the cyst, and treatment with taxol (to inhibit microtubule dynamics) favoured cystogenesis. Mitochondria, which appeared adhered to the PV membrane, were less closely associated with the cyst wall. Endoplasmic reticulum (ER) profiles were intimately associated with folds in the cyst wall membrane. However, the Golgi complex was not preferentially localized relative to the cyst, and treatment with tunicamycin or brefeldin A (to disrupt Golgi or ER function, respectively) had no significant effect on cystogenesis. Lysosomes accumulated around cysts, while early and late endosomes were more evenly distributed in the cytoplasm. The endocytosis tracer HRP (but not BSA or transferrin) reached bradyzoites after uptake by infected host cells. These results suggest that T. gondii cysts reorganize the host cell cytoplasm, which may fulfil specific requirements of the chronic stage of infection.

Candida albicans biofilms: comparative analysis of room-temperature and cryofixation for scanning electron microscopy.

Biofilms are frequently related to invasive fungal infections and are reported to be more resistant to antifungal drugs than planktonic cells. The structural complexity of the biofilm as well as the presence of a polymeric extracellular matrix (ECM) is thought to be associated with this resistant behavior. Scanning electron microscopy (SEM) after room temperature glutaraldehyde-based fixation, have been used to study fungal biofilm structure and drug susceptibility but they usually fail to preserve the ECM and, therefore, are not an optimised methodology to understand the complexity of the fungal biofilm. Thus, in this work, we propose a comparative analysis of room-temperature and cryofixation/freeze substitution of Candida albicans biofilms for SEM observation. Our experiments showed that room-temperature fixative protocols using glutaraldehyde and osmium tetroxide prior to alcohol dehydration led to a complete extraction of the polymeric ECM of biofilms. ECM from fixative and alcohol solutions were recovered after all processing steps and these structures were characterised by biochemistry assays, transmission electron microscopy and mass spectrometry. Cryofixation techniques followed by freeze-substitution lead to a great preservation of both ECM structure and C. albicans biofilm cells, allowing the visualisation of a more reliable biofilm structure. These findings reinforce that cryofixation should be the indicated method for SEM sample preparation to study fungal biofilms as it allows the visualisation of the EMC and the exploration of the biofilm structure to its fullest, as its structural/functional role in interaction with host cells, other pathogens and for drug resistance assays.

A comprehensive assessment of cognitive function in the common genetic generalized epilepsy syndromes.

BACKGROUND AND PURPOSE: Considered to be benign conditions, the common genetic generalized epilepsy (GGE) syndromes are now known to be frequently accompanied by cognitive dysfunction. However, unresolved issues impede clinical management of this common comorbidity, including which cognitive abilities are most affected, whether there are differences between syndromes and how seizure type and mood symptoms affect cognitive dysfunction. We provide a detailed description of cognitive ability and evaluate factors contributing to cognitive dysfunction. METHODS: A total of 76 adults with GGE were assessed with the Woodcock Johnson III Tests of Cognitive Abilities. RESULTS: Scores on tests of overall cognitive ability, acquired knowledge, long-term retrieval and speed of information processing were significantly below the normative mean. Long-term retrieval was a pronounced weakness with a large reduction in scores (d = 0.84). GGE syndrome, seizure type and the presence of recent psychopathology symptoms were not significantly associated with cognitive function. CONCLUSIONS: This study confirms previous meta-analytic findings with a prospective study, offers new insights into the cognitive comorbidity of these common epilepsy syndromes and reinforces the need for cognitive interventions in people with GGE.

Role of demographic and job-related variables in determining work-related quality of life of hospital employees.

BACKGROUND: Considering a huge working population in health sector faced with stressful work life, limited autonomy in work and declining work contentment calls for an overemphasis on evaluating and monitoring their satisfaction associated with work-related quality of life (WRQoL). This study evaluates WRQoL of hospital employees and validates the bilingual (English and Marathi) version of WRQoL scale. METHODS: The study was conducted during March-April'2014 on employees of a corporate hospital of Pune, India after ethical approval and informed consent from employees. The bilingual WRQoL scale has been tested for reliability and validity, and WRQoL scores have been reported. RESULTS: A total of 132 hospital employees (mean age 31 [±8] years, 55% males) who participated in the study reported overall moderate WRQoL scores. The scale showed high internal consistency (Cronbach's alpha = 0.82, P < 0.0001) and moderate to high validity. WRQoL did not significantly vary across marital status, family size, and gender. "Stress at work" score of WRQoL increased with age of employees. Higher work experience, employment at higher positions and those working in clinical and diagnostic departments reported a higher WRQoL. CONCLUSION: WRQoL scale is a reliable and valid instrument. Better WRQoL in employees placed in higher organizational positions indicates a need for focused measures to enhance WRQoL of employees in lower hierarchical levels, especially in control at work and home life interface domains. WRQoL needs regular monitoring for employees in lower positions and aging employees.

Full-length genomic and molecular characterization of Canine parvovirus in dogs from North of Brazil.

With the objective of characterizing Canine parvovirus (CPV) from some suspected fecal samples of dogs collected from the Veterinarian Hospital in Belém city, five positive samples were found by PCR assay and an update molecular characterization was provided of the CPV-2 circulation in Belém. Through sequencing of the complete DNA sequences (NS1, NS2, VP1, and VP2 genes), the CPV-2 strain was identified as CPV-2b (Asn426Asp) circulating in Belém. The CPV-2b strain with a different change at the position Tyr324Leu was detected in all samples assessed and thus reported for the first time for the scientific community. Phylogenetic analysis indicated that Belém CPV-2b and CPV-2a strains would be related to a cluster with samples after the 1990s, suggesting that CPV-2b in Belém originated from CPV-2a circulating in Brazil after the 1990s. Potential recombination events were analyzed using RDP4 and SplitsTree4; therefore, results suggest that CPV-2 sequences here described were not potentially recombination events. Continuous monitoring and molecular characterization of CPV-2 samples are needed not only to identify possible genetic and antigenic changes that may interfere with the effectiveness of vaccines but also to bring a better understanding of the mechanisms that drive the evolution of CPV-2 in Brazil.

A Systematic Review of Psychiatric and Psychosocial Comorbidities of Genetic Generalised Epilepsies (GGE).

Psychiatric disorders and associated poor psychosocial outcomes are recognised to be a common sequelae of epilepsy. The extent to which this is true of genetic generalised epilepsies (GGE), particularly syndromes other than juvenile myoclonic epilepsy (JME) is unclear. This systematic review synthesises findings regarding psychiatric and associated comorbidities in adults and children with GGE. Systematic review yielded 34 peer-reviewed studies of psychiatric and psychosocial outcomes in adults and children with GGE. Clinically significant psychiatric comorbidity was reported in over half of all children and up to a third of all adults with GGE. There was no evidence to support the presence of personality traits specific to JME or other syndromes; rather rates mirrored community samples. A small number of studies report poor psychosocial outcomes in GGE, however the interpretation of these findings is limited by paucity of healthy comparison groups. Some evidence suggests that anti-epileptic drug polytherapy in children and seizure burden at all ages may constitute risk factors for psychopathology. Findings highlight the importance of early screening so as not to overlook early or developing symptoms of psychopathology.

Potent In Vitro Antiproliferative Synergism of Combinations of Ergosterol Biosynthesis Inhibitors against Leishmania amazonensis.

Leishmaniases comprise a spectrum of diseases caused by protozoan parasites of the Leishmania genus. Treatments available have limited safety and efficacy, high costs, and difficult administration. Thus, there is an urgent need for safer and more-effective therapies. Most trypanosomatids have an essential requirement for ergosterol and other 24-alkyl sterols, which are absent in mammalian cells. In previous studies, we showed that Leishmania amazonensis is highly susceptible to aryl-quinuclidines, such as E5700, which inhibit squalene synthase, and to the azoles itraconazole (ITZ) and posaconazole (POSA), which inhibit C-14α-demethylase. Herein, we investigated the antiproliferative, ultrastructural, and biochemical effects of combinations of E5700 with ITZ and POSA against L. amazonensis. Potent synergistic antiproliferative effects were observed against promastigotes, with fractional inhibitory concentration (FIC) ratios of 0.0525 and 0.0162 for combinations of E5700 plus ITZ and of E5700 plus POSA, respectively. Against intracellular amastigotes, FIC values were 0.175 and 0.1125 for combinations of E5700 plus ITZ and E5700 plus POSA, respectively. Marked alterations of the ultrastructure of promastigotes treated with the combinations were observed, in particular mitochondrial swelling, which was consistent with a reduction of the mitochondrial transmembrane potential, and an increase in the production of reactive oxygen species. We also observed the presence of vacuoles similar to autophagosomes in close association with mitochondria and an increase in the number of lipid bodies. Both growth arrest and ultrastructural/biochemical alterations were strictly associated with the depletion of the 14-desmethyl endogenous sterol pool. These results suggest the possibility of a novel combination therapy for the treatment of leishmaniasis.

Reduction of Toxoplasma gondii Development Due to Inhibition of Parasite Antioxidant Enzymes by a Dinuclear Iron(III) Compound.

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan that can infect a wide range of vertebrate cells. Here, we describe the cytotoxic effects of the dinuclear iron compound [Fe(HPCINOL)(SO4)]2-µ-oxo, in which HPCINOL is the ligand 1-(bis-pyridin-2-ylmethyl-amino)-3-chloropropan-2-ol, on T. gondii infecting LLC-MK2 host cells. This compound was not toxic to LLC-MK2 cells at concentrations of up to 200 µM but was very active against the parasite, with a 50% inhibitory concentration (IC50) of 3.6 µM after 48 h of treatment. Cyst formation was observed after treatment, as indicated by the appearance of a cyst wall, Dolichos biflorus lectin staining, and scanning and transmission electron microscopy characteristics. Ultrastructural changes were also seen in T. gondii, including membrane blebs and clefts in the cytoplasm, with inclusions similar to amylopectin granules, which are typically found in bradyzoites. An analysis of the cell death pathways in the parasite revealed that the compound caused a combination of apoptosis and autophagy. Fluorescence assays demonstrated that the redox environment in the LLC-MK2 cells becomes oxidant in the presence of the iron compound. Furthermore, a reduction in superoxide dismutase and catalase activities in the treated parasites and the presence of reactive oxygen species within the parasitophorous vacuoles were observed, indicating an impaired protozoan response against these radicals. These findings suggest that this compound disturbs the redox equilibrium of T. gondii, inducing cystogenesis and parasite death.

Mitochondrial respiration and genomic analysis provide insight into the influence of the symbiotic bacterium on host trypanosomatid oxygen consumption.

Certain trypanosomatids co-evolve with an endosymbiotic bacterium in a mutualistic relationship that is characterized by intense metabolic exchanges. Symbionts were able to respire for up to 4 h after isolation from Angomonas deanei. FCCP (carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone) similarly increased respiration in wild-type and aposymbiotic protozoa, though a higher maximal O2 consumption capacity was observed in the symbiont-containing cells. Rotenone, a complex I inhibitor, did not affect A. deanei respiration, whereas TTFA (thenoyltrifluoroacetone), a complex II activity inhibitor, completely blocked respiration in both strains. Antimycin A and cyanide, inhibitors of complexes III and IV, respectively, abolished O2 consumption, but the aposymbiotic protozoa were more sensitive to both compounds. Oligomycin did not affect cell respiration, whereas carboxyatractyloside (CAT), an inhibitor of the ADP-ATP translocator, slightly reduced O2 consumption. In the A. deanei genome, sequences encoding most proteins of the respiratory chain are present. The symbiont genome lost part of the electron transport system (ETS), but complex I, a cytochrome d oxidase, and FoF1-ATP synthase remain. In conclusion, this work suggests that the symbiont influences the mitochondrial respiration of the host protozoan.

Force of infection of dengue serotypes in a population-based study in the northeast of Brazil.

This study investigated anti-dengue serotype-specific neutralizing antibodies in a random sample of dengue IgG-positive individuals identified in a survey performed in a hyperendemic setting in northeastern Brazil in 2005. Of 323 individuals, 174 (53.8%) had antibodies to dengue virus serotype 1 (DENV-1), 104 (32.2%) to DENV-2 and 301 (93.2%) to DENV-3. Monotypic infections by DENV-3 were the most frequent infection (35.6%). Of 109 individuals aged <15 years, 61.5% presented multitypic infections. The force of infection estimated by a catalytic model was 0.9%, 0.4% and 2.5% person-years for DENV-1, DENV-2 and DENV-3, respectively. By the age of 5 years, about 70%, 30% and 40% of participants were immune to DENV-3, DENV-2 and DENV-1, respectively. The data suggest that infection with DENV-1, -2 and -3 is intense at early ages, demonstrating the need for research efforts to investigate dengue infection in representative population samples of Brazilian children during early infancy.

Clinical experience with using lacosamide for the treatment of epilepsy in a tertiary centre.

OBJECTIVE: Lacosamide is approved for the adjunctive treatment of partial-onset seizures in adults. Phase II/III clinical trials suggest that it is a safe, effective and well-tolerated medication. However, there is little post-marketing information available about this medication. METHODS: We report our clinical experience from a tertiary referral epilepsy centre, which has been using lacosamide for the past 18 months, with 128 patients treated during this time. RESULTS: Fifty-three patients (41%) achieved at least a 50% reduction in seizure frequency, with 14 patients (11%) achieving seizure freedom for a mean time of 35 weeks. This 50% responder rate matches, and the seizure free rate outperforms that seen in previous pooled trials. The efficacy of lacosamide did not vary with concurrent sodium channel blocking agent (SCB) use, and a statistically significant dose-dependent response was not shown, which is in contrast to previous trials. Treatment emergent adverse effects (TEAEs) were noted in 52 patients (41%), with 24 patients (19%) discontinuing the medication. TEAEs were more frequent in patients on concurrent SCBs, affecting 51% vs. 28% of patients not on other SCBs. This increased risk of TEAEs from concurrent SCB use was of statistical significance (P = 0.01). The most frequently noted TEAEs from lacosamide were dizziness, sedation and diplopia, which all appeared to be dose-related. CONCLUSION: This post-marketing analysis suggests that lacosamide in clinical practice at least mirrors, and possibly outperforms the results seen in previous phase II/III trials.

Seizures during high-dose antidepressant therapy: a case series with clinical and video-electroencephalography characteristics.

This case series identified drug causes of seizures in patients with depressive disorders. Three patients with seizures were admitted for video-electroencephalography (EEG) monitoring in the context of high-dose antidepressants and sodium valproate. Very high-dose antidepressants caused complex partial seizures with secondary generalisation that gave characteristic EEG discharges. This is the first reported series to capture the ictal EEG features associated with antidepressants.

Irrigation with saline water in the cultivation of mini watermelon under phosphate fertilization.

The objective of this study was to evaluate the water status, photosynthetic pigments, and photochemical efficiency of mini watermelon plants under salt stress and phosphate fertilization. The experiment was conducted in pots under greenhouse conditions in Pombal, PB, Brazil. The experimental design used was randomized blocks in a 5 × 4 factorial scheme, with five levels of electrical conductivity of irrigation water - ECw (0.3, 1.3, 2.3, 3.3, and 4.3 dS m-1) and four doses of phosphorus (60, 80, 100, and 120% of the recommendation), with three replicates. The relative water content in the tissues decreased with the increase in ECw levels in all phosphorus doses, with decreases of 7.05, 7.81 and 8.83% per unit increase in ECw, in plants fertilized with 80, 100 and 120% P2O5. On the other hand, ECw levels increased electrolyte leakage, regardless of phosphorus doses of the recommendation. The synthesis of photosynthetic pigments and the quantum efficiency of photosystem II were inhibited by increasing water salinity from 0.3 dS m-1 in plants grown under phosphorus doses above 60% of the recommendation. Water salinity from 0.3 dS m-1 reduced chlorophyll b contents, initial, maximum, and variable fluorescence of mini watermelon plants, with a decrease of 11.86, 4.51, 4.53, and 4.54% per unit increment of ECw, respectively.

Morphology and morphometry of Paratanaisia bragai () Freitas, 1959 (Digenea, Eucotylidae) through light and scanning electron microscopy.

Paratanaisia bragai is a digenetic trematode that reaches sexual maturity in the kidney collecting ducts of domestic and wild birds, while the snails Subulina octona and Leptinaria unilamellata serve as its intermediate hosts in Brazil. The present study analyzed the morphology and morphometry of P. bragai. Adult specimens of the parasite were collected from naturally infected Columba livia kidneys, fixed and prepared for observation via bright field and differential interference contrast light microscopy and scanning electron microscopy. The parasite has an elongated and flattened body, with a subterminal oral sucker located at the anterior end of the body, as observed by all techniques used. Staining the parasite with hematoxylin-eosin enabled observation of the pharynx, located posteriorly to the oral sucker, the vitelline glands, which are extra-cecal and extend anteriorly to the pre-ovarian region and later to the median region of the body, and intestinal caeca parallel to the vitelline glands. The presence and functionality of the acetabulum are controversial points in the literature, but it was observed in all specimens analyzed by scanning electron microscopy, with a major diameter of 38.36 ± 6.96 (28.77 - 45.39) and minor diameter of 31.59 ± 7.04 (21.75 - 38.16). Close to the acetabulum, scales were observed in the integument of the parasite. Scales with (1 - 5) blade divisions were identified. In the genital pore, it was possible to see the everted cirrus with rosette shape. The excretory pore (first morphometric record) is dorsal and subterminal, with major diameter of 12.27 ± 9.16 (5.79 - 18.75) and minor diameter of 3.95 ± 1.49 (2.89 - 5.00).

Mineral composition and production of guava under salt stress and salicylic acid.

The limitation in the quality of water sources for irrigation in the semi-arid region of northeastern Brazil is increasingly present, so it is necessary to use water with high concentrations of salts for agricultural production, which makes the use of elicitors essential to mitigate the harmful effects of salinity on plants. Given the above, the objective of this study was to evaluate the effects of foliar application of salicylic acid on the mineral composition and production of guava plants under salt stress conditions in the post-grafting phase. The experiment was carried out under greenhouse conditions, in a randomized block design, in a 2 × 4 factorial scheme, with two levels of electrical conductivity of irrigation water (0.6 and 3.2 dS m-1) and four concentrations of salicylic acid (0, 1.2, 2.4, and 3.6 mM), with three replicates. During the flowering stage of guava, N, P, and K contents accumulated in the leaves according to the following order of concentration: N > K > P. Foliar application of 1.2 mM of salicylic acid increases the leaf contents of N, P, and K in guava plants grown under irrigation with water of 0.6 dS m-1. Water salinity of 3.2 dS m-1 reduces the growth and production components of guava plants.

The prevalence and demographic distribution of treated epilepsy: a community-based study in Tasmania, Australia.

OBJECTIVES: To estimate the prevalence and demographic distribution of treated epilepsy in a community-based population. MATERIALS & METHODS: We surveyed all residents in Tasmania, Australia, who were supplied at least one antiepileptic drug prescription between July 1, 2001 and June 30, 2002, recorded on the national prescription database. We adjusted for the effect of disease-related non-response bias by imputation methods. RESULTS: After three mail contacts, 54.0% (4072/7541) responded, with 1774 (43.6%) indicating treatment for epilepsy, representing 86.0% of the estimated total possible cases in Tasmania. The adjusted treated epilepsy prevalence was 4.36 per 1000 (95% CI 4.34, 4.39); lower in women (prevalence ratio 0.92 (95% CI 0.84, 1.00)); greater with increasing age (P < 0.001); similar in the three main geographic regions; and similar with socioeconomic status of postcode of residence. CONCLUSIONS: Although our estimates are likely to be affected by access to health services, overall treated epilepsy prevalence of 4.4 per 1000 is similar to previous studies. Our finding of high elderly prevalence has been reported in a few recent studies in developed countries and has important clinical and public health implications in populations with similar aging demographics.

Tomatidine promotes the inhibition of 24-alkylated sterol biosynthesis and mitochondrial dysfunction in Leishmania amazonensis promastigotes.

Leishmaniasis is a set of clinically distinct infectious diseases caused by Leishmania, a genus of flagellated protozoan parasites, that affects ~12 million people worldwide, with ~2 million new infections annually. Plants are known to produce substances to defend themselves against pathogens and predators. In the genus Lycopersicon, which includes the tomato, L. esculentum, the main antimicrobial compound is the steroidal glycoalkaloid α-tomatine. The loss of the saccharide side-chain of tomatine yields the aglycone tomatidine. In the present study, we investigated the effects of tomatidine on the growth, mitochondrial membrane potential, sterol metabolism, and ultrastructure of Leishmania amazonensis promastigotes. Tomatidine (0·1 to 5 µM) inhibited parasite growth in a dose-dependent manner (IC(50)=124±59 nM). Transmission electron microscopy revealed lesions in the mitochondrial ultrastructure and the presence of large vacuoles and lipid storage bodies in the cytoplasm. These structural changes in the mitochondria were accompanied by an effective loss of mitochondrial membrane potential and a decrease in ATP levels. An analysis of the neutral lipid content revealed a large depletion of endogenous 24-alkylated sterols such as 24-methylene-cholesta-5, 7-dien-3ß-ol (5-dehydroepisterol), with a concomitant accumulation of cholesta-8, 24-dien-3ß-ol (zymosterol), which implied a perturbation in the cellular lipid content. These results are consistent with an inhibition of 24-sterol methyltransferase, an important enzyme responsible for the methylation of sterols at the 24 position, which is an essential step in the production of ergosterol and other 24-methyl sterols.

Dynamics of the orphan myosin MyoF over Trypanosoma cruzi life cycle and along the endocytic pathway.

Trypanosoma cruzi proliferative forms perform endocytosis through a specialized structure named the cytostome-cytopharynx complex (SPC). The SPC is a specialized invagination of the cell membrane that extends through the cell body towards the posterior regions, with its aperture close to the flagellar pocket. Recently, diverse proteins were found along the cytopharynx, including two myosin motors. One of these is the orphan myosin MyoF, that was proved to be essential for endocytosis in epimastigotes. However, the dynamics of MyoF localization along the endocytic pathway and through the T. cruzi life cycle remain unclear. Using CRISPR-Cas9 genome editing, we generated epimastigotes expressing MyoF fused to mNeonGreen from its endogenous locus. Using these cells, we observed that during the epimastigote cell cycle MyoF signal disappeared during G2, reappearing at early cytokinesis. Additionally, we show that MyoF localization during metacyclogenesis is compatible with the progressive disappearance of the SPC, being absent in metacyclic trypomastigotes. Detergent fractionation showed that MyoF was predominantly present in the insoluble fraction and immunolocalized at the SPC microtubules in whole-mount cytoskeleton preparations. Moreover, during tracer uptake through the SPC, MyoF followed the tracer along the endocytic pathway and was found in posterior compartments after 30 min. Taken together, the data suggest that MyoF may play a role not only at the cargo entry site but also along the endocytic pathway.