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Giardia intestinalis is a parasitic protozoan that inhabits its vertebrate hosts' upper small intestine and is the most common cause of waterborne diarrhoea worldwide. Giardia trophozoites present few organelles, and among them, they possess peripheral vesicles (PVs), which are considered an endosomal–lysosomal system. All experimental procedures carried out until now indicate that Giardia ingests macromolecules by fluid-phase and receptor-mediated endocytic pathways. Still, there is no description concerning the interaction and ingestion of large materials. Here, we tested Giardia's capacity to interact with large particles; once, in vivo, it inhabits an environment with a microbiota. We tested protozoan interaction with yeasts, bacteria, latex beads, ferritin and albumin, in different times of interaction and used several microscopy techniques (light microscopy, scanning electron microscopy and transmission electron microscopy) to follow their fate. Giardia interacted with all of the materials we tested. Projections of the plasma membrane similar to pseudopods were seen. As albumin, small markers were found in the PVs while the larger materials were not seen there. Large vacuoles containing large latex beads were detected intracellularly. Thus, we observed that: (1) Giardia interacts with large materials; (2) Giardia can display an amoeboid shape and exhibit membrane projections when in contact with microorganisms and large inorganic materials; (3) the region of the exit of the ventral flagella is very active when in contact with large materials, although all cell surface also present activity in the interactions; (4) intracellular vacuoles, which are not the PVs, present ingested large beads.

Toxoplasma gondii is a protozoan parasite that is the causative agent of toxoplasmosis, an infection with high prevalence worldwide. Most of the infected individuals are either asymptomatic or have mild symptoms, but T. gondii can cause severe neurologic damage and even death of the fetus when acquired during pregnancy. It is also a serious condition in immunodeficient patients. The life-cycle of T. gondii is complex, with more than one infective form and several transmission pathways. In two animated videos, we describe the main aspects of this cycle, raising questions about poorly or unknown issues of T. gondii biology. Original plates, based on electron microscope observations, are also available for teachers, students and researchers. The main goal of this review is to provide a source of learning on the fundamental aspects of T. gondii biology to students and teachers contributing for better knowledge and control on this important parasite, and unique cell model. In addition, drawings and videos point to still unclear aspects of T. gondii lytic cycle that may stimulate further studies. Graphical Abstract

The parasitic protozoan Giardia intestinalis, the causative agent of giardiasis, presents a stable and elaborated cytoskeleton, which shapes and supports several intracellular structures, including the ventral disc, the median body, the funis, and four pairs of flagella. Giardia trophozoite is the motile form that inhabits the host small intestine and attaches to epithelial cells, leading to infection. The ventral disc is considered one important element of adhesion to the intestinal cells. It is adjacent to the plasma membrane in the ventral region of the cell and consists of a spiral layer of microtubules and microribbons. In this work, we studied the organization of the cytoskeleton in the ventral disc of G. intestinalis trophozoites using high-resolution scanning electron microscopy or helium ion microscopy in plasma membrane-extracted cells. Here, we show novel morphological details about the arrangement of cross-bridges in different regions of the ventral disc. Results showed that the disc is a non-uniformly organized structure that presents specific domains, such as the margin and the ventral groove region. High-resolution scanning electron microscopy allowed observation of the labeling pattern for several anti-tubulin antibodies using secondary gold particle-labeled antibodies. Labeling in the region of the emergence of the microtubules and supernumerary microtubules using an anti-acetylated tubulin antibody was observed. Ultrastructural analysis and immunogold labeling for gamma-tubulin suggest that disc microtubules originate from a region bounded by the bands of the banded collar and merge with microtubules formed at the perinuclear region. Actin-like filaments and microtubules of the disc are associated, showing an interconnection between elements of the cytoskeleton of the trophozoite.

This review presents the main cell organelles and structures of two important protist parasites, Giardia intestinalis, and Trichomonas vaginalis; many are unusual and are not found in other eukaryotic cells, thus could be good candidates for new drug targets aimed at improvement of the chemotherapy of diseases caused by these eukaryotic protists. For example, in Giardia, the ventral disc is a specific structure to this parasite and is fundamental for the adhesion and pathogenicity to the host. In Trichomonas, the hydrogenosome, a double membrane-bounded organelle that produces ATP, also can be a good target. Other structures include mitosomes, ribosomes, and proteasomes. Metronidazole is the most frequent compound used to kill many anaerobic organisms, including Giardia and Trichomonas. It enters the cell by passive diffusion and needs to find a highly reductive environment to be reduced to the nitro radicals to be active. However, it provokes several side effects, and some strains present metronidazole resistance. Therefore, to improve the quality of the chemotherapy against parasitic protozoa is important to invest in the development of highly specific compounds that interfere with key steps of essential metabolic pathways or in the functional macromolecular complexes which are most often associated with cell structures and organelles.

Cardiac cells are organized in vivo in a complex tridimensional structural organization that is crucial for heart function. While in vitro studies can reveal details about cardiac cell biology, usually cells are grown on simplified two-dimensional (2D) environments. To address these differences, we established a cardiac cell culture composed of both 2D and three-dimensional (3D)-organized cells. Our results shows significant differences between the two culture contexts in relation to the overall morphology of the cells, contraction ability, proliferation rate, presence of intercellular adhesion structures, organization of myofibrils, mitochondria morphology, endoplasmic reticulum contents, cytoskeletal filaments and extracellular matrix distribution, and expression of markers of cardiac differentiation. Cardiac cells grown in 2D-context displayed a flattened and well spread shape, were mostly isolated and their cytoplasm was filled with a large network of microfilaments and microtubules. In contrast, 3D-cells were smaller in size, were always in close contact with each other with several cellular junctions, and displayed a less conspicuous cytoskeletal network. 3D-cells had more mitochondria and myofibrils and these cells contract spontaneously more often than 2D-cells. On the other hand, endoplasmic reticulum membranes were present in higher amounts in 2D-cells when compared to 3D-cells. The expression of desmin, cadherin and alpha-actinin was higher in 3D-aggregates compared to 2D-spread cells. These findings indicate that the tridimensional environment in which the cardiac cells are grown influence several aspects of cardiac differentiation, including cell adhesion, cell shape, myofibril assembly, mitochondria contents and protein expression. We suggest that the use of this cardiac culture model, with 2D and 3D-context cells, could be useful for studies on the effects of different drugs, or growth factors, giving valuable information on the biological response of cells grown in different spatial organizations.

This review presents the main cell characteristics altered after in vitro incubation of the parasite with commercial drugs used to treat the disease caused by Giardia intestinalis. This important intestinal parasite primarily causes diarrhea in children. Metronidazole and albendazole are the primary compounds used in therapy against Giardia intestinalis. However, they provoke significant side effects, and some strains have developed resistance to metronidazole. Benzimidazole carbamates, such as albendazole and mebendazole, have shown the best activity against Giardia. Despite their in vitro efficacy, clinical treatment with benzimidazoles has yielded conflicting results, demonstrating lower cure rates. Recently, nitazoxanide has been suggested as an alternative to these drugs. Therefore, to enhance the quality of chemotherapy against this parasite, it is important to invest in developing other compounds that can interfere with key steps of metabolic pathways or cell structures and organelles. For example, Giardia exhibits a unique cell structure called the ventral disc, which is crucial for host adhesion and pathogenicity. Thus, drugs that can disrupt the adhesion process hold promise for future therapy against Giardia. Additionally, this review discusses new drugs and strategies that can be employed, as well as suggestions for developing novel drugs to control the infection caused by this parasite.

Hydrogenosomes are spherical or slightly elongated organelles found in non-mitochondrial organisms. In Trichomonas hydrogenosomes measure between 200 to 500 nm, but under drug treatment they can reach 2 μm. Like mitochondria hydrogenosomes: (1) are surrounded by two closely apposed membranes and present a granular matrix: (2) divide in three different ways: segmentation, partition and the heart form; (3) they may divide at any phase of the cell cycle; (4) produce ATP; (5) participate in the metabolism of pyruvate formed during glycolysis; (6) are the site of molecular hydrogen formation; (7) present a relationship with the endoplasmic reticulum; (8) incorporate calcium; (9) import proteins post-translationally; (10) present cardiolipin. However, there are differences, such as: (1) absence of genetic material, at least in trichomonas; (2) lack a respiratory chain and cytochromes; (3) absence of the F0- F1 ATPase; (4) absence of the tricarboxylic acid cycle; (5) lack of oxidative phosphorylation; (6) presence of peripheral vesicles. Hydrogenosomes are considered an excellent drug target since their metabolic pathway is distinct from those found in mitochondria and thus medicines directed to these organelles will probably not affect the host-cell. The main drug used against trichomonads is metronidazole, although other drugs such as β-Lapachone, colchicine, Taxol, nocodazole, griseofulvin, cytochalasins, hydroxyurea, among others, have been used in trichomonad studies, showing: (1) flagella internalization forming pseudocyst; (2) dysfunctional hydrogenosomes; (3) hydrogenosomes with abnormal sizes and shapes and with an electron dense deposit called nucleoid; (4) intense autophagy in which hydrogenosomes are removed and further digested in lysosomes.

Trichomonas vaginalis is an extracellular protozoan parasite that causes human trichomoniasis, a sexually transmitted infection (STI) that affects approximately 270 million people worldwide. The phenomenon of T. vaginalis adhesion to inert substrates has been described in several reports. Still, very few studies on cluster formation have been conducted, and more detailed analyses of the contact regions between the parasites’ membranes in these aggregate formations have not been carried out. The present study aims to show that T. vaginalis forms a tight monolayer, similar to an epithelium, with parasites firmly adhered to the culture flask bottom by interdigitations and in the absence of host cells. In addition, we analyzed and compared the formation of the clusters, focusing on parasite aggregates that float in the culture flasks. We employed various imaging techniques, including high-resolution scanning electron microscopy, transmission electron microscopy, cytochemistry, TEM tomography, and dye injection. We analyzed whether the monolayer behaves as an epithelium, analyzing cell junctions, cell communication, and ultrastructural aspects, and concluded that monolayer formation differs from cluster formation in many aspects. The monolayers form strong adhesion, whereas the clusters have fragile attachments. We did not find fusion or the passage of molecules between neighbor-attached cells; there is no need for different strains to form filopodia, cytonemes, and extracellular vesicles during cluster and monolayer formation.

  Protozoan parasites of the Leishmania genus are the causative agent of leishmaniasis, one of the most important neglected tropical diseases as designated by the World Health Organization (WHO). Didactic View Using 3D Animations of Leishmania Life Cycle Leishmania parasites have a complex life cycle that involves both vertebrate and invertebrate hosts and two developmental stages: promastigotes, the proliferative form found in the lumen of the female sandfly, and amastigotes, the proliferative form found inside several types of mammalian host cells. [...]during subsequent insect bites, infective metacyclic promastigotes are released and can thus infect a new mammalian host, thereby starting the cycle over.

Background Trichomonas vaginalis is a protist parasite that causes trichomoniasis, a sexually transmitted disease. Metronidazole is the current treatment for trichomoniasis. However, this drug can provoke severe side effects, and some strains present resistance, making the development of alternative treatments for trichomoniasis urgent. Objectives We investigate the use of essential oil obtained from Dracocephalum kotschyi on T. vaginalis. D. kotschyi has antispasmodic and analgesic properties and is well known in Iran. Methods The essential oil was obtained by hydrodistillation from 1000 g of the powdered plant. Gas chromatography‐mass spectrometry analysis was used for the chemical composition of the essential oil, and 11 substances were identified, corresponding to 91.5% of the oil. Copaene (22.15%), Methyl geranate (16.31%), Geranial (13.78%) and Carvone (11.34%) were the main substances. A cell viability test was used to determine the percentage of growth inhibition (GI%) and the half‐maximal inhibitory concentration (IC50) on T. vaginalis after incubation with the prepared essential oil. Results The oil induced an IC50 of 84.07 µg/ml after 24 h contact with trophozoites. Cytotoxicity was determined by MTT assay on the J774.A1 haematopoietic cell line. In addition, the initial stage of apoptosis was assayed using the fluorescein isothiocyanate Annexin V Apoptosis Detection Kit. Evaluation of the in vitro anti‐trichomonal properties of D. kotschyi essential oils showed that it effectively induces apoptosis on T. vaginalis between 100 and 700 µg/ml after 48 h without toxicity on haematopoietic cells, suggesting that D. kotschyi essential oil can induce programmed death in T. vaginalis. Conclusions The anti‐trichomonal properties of D. kotschyi essential oil indicate that they could be suitable for new pharmacologic studies after new tests with human vaginal epithelial cells. The anti‐trichomonal properties of D. kotschyi essential oil indicate that they could be suitable for new pharmacologic studies after new tests with human vaginal epithelial cells.