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.

Dengue virus infection affects millions of people all over the world. Although the clinical manifestations of dengue virus-induced diseases are known, the physiopathological mechanisms involved in deteriorating cellular function are not yet understood. In this study we evaluated for the first time the associations between dengue virus-induced cell death and mitochondrial function in HepG2, a human hepatoma cell line. Dengue virus infection promoted changes in mitochondrial bioenergetics, such as an increase in cellular respiration and a decrease in DeltaPsim. These alterations culminated in a 20% decrease in ATP content and a 15% decrease in the energy charge of virus-infected cells. Additionally, virus-infected cells showed several ultrastructural alterations, including mitochondria swelling and other morphological changes typical of the apoptotic process. The alterations in mitochondrial physiology and energy homeostasis preceded cell death. These results indicate that HepG2 cells infected with dengue virus are under metabolic stress and that mitochondrial dysfunction and alterations in cellular ATP balance may be related to the pathogenesis of dengue virus infection.

Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogenital tract, where it remains extracellular and adheres to epithelial cells. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Despite the serious consequences associated with trichomoniasis disease, little is known about parasite or host factors involved in attachment of the parasite-to-host epithelial cells. Here, we report the identification of microvesicle-like structures (MVs) released by T. vaginalis. MVs are considered universal transport vehicles for intercellular communication as they can incorporate peptides, proteins, lipids, miRNA, and mRNA, all of which can be transferred to target cells through receptor-ligand interactions, fusion with the cell membrane, and delivery of a functional cargo to the cytoplasm of the target cell. In the present study, we demonstrated that T. vaginalis release MVs from the plasma and the flagellar membranes of the parasite. We performed proteomic profiling of these structures demonstrating that they possess physical characteristics similar to mammalian extracellular vesicles and might be selectively charged with specific protein content. In addition, we demonstrated that viable T. vaginalis parasites release large vesicles (LVs), membrane structures larger than 1 µm that are able to interact with other parasites and with the host cell. Finally, we show that both populations of vesicles present on the surface of T vaginalis are induced in the presence of host cells, consistent with a role in modulating cell interactions.

SummaryThe parasite Trichomonas vaginalis is the causative agent of trichomoniasis, a prevalent sexually transmitted infection. Here, we report the cellular analysis of T. vaginalis tetraspanin family (TvTSPs). This family of membrane proteins has been implicated in cell adhesion, migration and proliferation in vertebrates. We found that the expression of several members of the family is up-regulated upon contact with vaginal ectocervical cells. We demonstrate that most TvTSPs are localized on the surface and intracellular vesicles and that the C-terminal intracellular tails of surface TvTSPs are necessary for proper localization. Analyses of full-length TvTSP8 and a mutant that lacks the C-terminal tail indicates that surface-localized TvTSP8 is involved in parasite aggregation, suggesting a role for this protein in parasite : parasite interaction.

This study reports that the cytopathic effect of Trichomonas vaginalis, an important human parasite of the urogenital tract, occurs due to mechanical stress and subsequent phagocytosis of the necrotic cells. The investigation was done using a primary culture of bovine oviduct epithelial cells (BOECs), grown either in monolayers or as floating cells. Trophozoites displaying different virulence levels were co-incubated with BOECs for times varying between 1 min and 48 h. Analyses were performed using videomicroscopy, scanning and transmission electron microscopy, colourimetric assays and cytochemistry. Injury was observed as early as 1 h after incubation, while after 12 h the host cells were severely damaged when a fresh trichomonad isolate was used. Trichomonads attack the host cells by clustering around them. Mechanical stress on the microvilli of the host cells was observed and appeared to induce plasma membrane damage and cell death. After membrane injury and lysis, fragments of the necrotic cells were ingested by trichomonads. Phagocytosis occurred by trichomonads avidly eating large portions of epithelial cells containing the nucleus and other organelles, but living or intact cells were not ingested. Necrotic fragments were rapidly digested in lysosomes, as shown by acid phosphatase and ruthenium red assays where only the BOECs were labelled. The lytic capacity of the trichomonads was more pronounced in host cell suspensions.