Chronic undernutrition impairs renal mitochondrial respiration accompanied by intense ultrastructural damage in juvenile rats.

This study investigated whether chronic undernutrition alters the mitochondrial structure and function in renal proximal tubule cells, thus impairing fluid transport and homeostasis. We previously showed that chronic undernutrition downregulates the renal proximal tubules (Na++K+)ATPase, the main molecular machine responsible for fluid transport and ATP consumption. Male rats received a multifactorial deficient diet, the so-called Regional Basic Diet (RBD), mimicking those used in impoverished regions worldwide, from weaning to a juvenile age (3 months). The diet has a low content (8 %) of poor-quality proteins, low lipids, and no vitamins compared to control (CTR). We investigated citrate synthase activity, mitochondrial respiration (oxygraphy) in phosphorylating and non-phosphorylating conditions with different substrates/inhibitors, potential across the internal membrane (Δψ), and anion superoxide/H2O2 formation. The data were correlated with ultrastructural alterations evaluated using transmission electron microscopy (TEM) and focused ion beam scanning electron microscopy (FIB-SEM). Citrate synthase activity decreased (∼50 %) in RBD rats, accompanied by a similar reduction in respiration in non-phosphorylating conditions, maximum respiratory capacity, and ATP synthesis. The Δψ generation and its dissipation after carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone remained unmodified in the survival mitochondria. H2O2 production increased (∼100 %) after Complex II energization. TEM demonstrated intense matrix vacuolization and disruption of cristae junctions in a subpopulation of RBD mitochondria, which was also demonstrated in the 3D analysis of FIB-SEM tomography. In conclusion, chronic undernutrition impairs mitochondrial functions in renal proximal tubules, with profound alterations in the matrix and internal membrane ultrastructure that culminate with the compromise of ATP supply for transport processes.

Quantitative assessment of the nanoanatomy of the contractile vacuole complex in Trypanosoma cruzi.

Trypanosoma cruzi uses various mechanisms to cope with osmotic fluctuations during infection, including the remodeling of organelles such as the contractile vacuole complex (CVC). Little is known about the morphological changes of the CVC during pulsation cycles occurring upon osmotic stress. Here, we investigated the structure-function relationship between the CVC and the flagellar pocket domain where fluid discharge takes place-the adhesion plaque-during the CVC pulsation cycle. Using TcrPDEC2 and TcVps34 overexpressing mutants, known to have low and high efficiency for osmotic responses, we described a structural phenotype for the CVC that matches their corresponding physiological responses. Quantitative tomography provided data on the volume of the CVC and spongiome connections. Changes in the adhesion plaque during the pulsation cycle were also quantified and a dense filamentous network was observed. Together, the results suggest that the adhesion plaque mediates fluid discharge from the central vacuole, revealing new aspects of the osmoregulatory system in T. cruzi.

Knockdown of Sec16 causes early lethality and defective deposition of the protein Rp30 in the eggshell of the vector Rhodnius prolixus.

In nearly every species of insect, embryonic development takes place outside of the mother's body and is entirely dependent on the elements that the mother had previously stored within the eggs. It is well known that the follicle cells (FCs) synthesize the eggshell (chorion) components during the process of choriogenesis, the final step of oogenesis before fertilization. These cells have developed a specialization in the massive production of chorion proteins, which are essential for the protection and survival of the embryo. Here, we investigate the function of Sec16, a protein crucial for the endoplasmic reticulum (ER) to Golgi traffic, in the oocyte development in the insect Rhodnius prolixus. We discovered that Sec16 is strongly expressed in vitellogenic females' ovaries, particularly in the choriogenic oocyte and it is mainly associated with the FCs. Silencing of Sec16 by RNAi caused a sharp decline in oviposition rates, F1 viability, and longevity in adult females. In the FCs, genes involved in the unfolded protein response (UPR), the ubiquitin-proteasome system (UPS), and autophagy were massively upregulated, whereas the mRNAs of Rp30 and Rp45-which code for the two major chorion proteins - were downregulated as a result of Sec16 silencing, indicating general proteostasis disturbance. As a result, the outer surface ultrastructure of Sec16-silenced chorions was altered, with decreased thickness, dityrosine crosslinking, sulfur signals, and lower amounts of the chorion protein Rp30. These findings collectively demonstrate the critical role Sec16 plays in the proper functioning of the FCs, which impacts the synthesis and deposition of particular components of the chorion as well as the overall reproduction of this vector.

Combination therapy of itraconazole and an acylhydrazone derivative (D13) for the treatment of sporotrichosis in cats.

Acylhydrazone (AH) derivatives represent a novel category of anti-fungal medications that exhibit potent activity against Sporothrix sp., both in vitro and in a murine model of sporotrichosis. In this study, we demonstrated the anti-fungal efficacy of the AH derivative D13 [4-bromo-N'-(3,5-dibromo-2-hydroxybenzylidene)-benzohydrazide] against both planktonic cells and biofilms formed by Sporothrix brasiliensis. In a clinical study, the effect of D13 was then tested in combination with itraconazole (ITC), with or without potassium iodide, in 10 cats with sporotrichosis refractory to the treatment of standard of care with ITC. Improvement or total clinical cure was achieved in five cases after 12 weeks of treatment. Minimal abnormal laboratory findings, e.g., elevation of alanine aminotransferase, were observed in four cats during the combination treatment and returned to normal level within a week after the treatment was ended. Although highly encouraging, a larger and randomized controlled study is required to evaluate the effectiveness and the safety of this new and exciting drug combination using ITC and D13 for the treatment of feline sporotrichosis. IMPORTANCE: This paper reports the first veterinary clinical study of an acylhydrazone anti-fungal (D13) combined with itraconazole against a dimorphic fungal infection, sporotrichosis, which is highly endemic in South America in animals and humans. Overall, the results show that the combination treatment was efficacious in ~50% of the infected animals. In addition, D13 was well tolerated during the course of the study. Thus, these results warrant the continuation of the research and development of this new class of anti-fungals.

The parabasal filaments of Trichomonas vaginalis: A new filament and observations using 0.8 nm-resolution scanning electron microscopy.

Trichomonas vaginalis is the etiologic agent of trichomoniasis, the most common nonviral sexually transmitted infection worldwide, with an estimated 260 million new cases annually. T. vaginalis contains organelles common to all eukaryotic cells, uncommon cell structures such as hydrogenosomes, and a complex and elaborate cytoskeleton constituting the mastigont system. The mastigont system is mainly formed by several proteinaceous structures associated with basal bodies, the pelta-axostylar complex made of microtubules, and striated filaments named the costa and the parabasal filaments (PFs). Although the structural organization of trichomonad cytoskeletons has been analyzed using several techniques, observation using a new generation of scanning electron microscopes with a resolution exceeding 1 nm has allowed more detailed visualization of the three-dimensional organization of the mastigont system. In this study, we have investigated the cytoskeleton of T. vaginalis using a diverse range of scanning probe microscopy techniques, which were complemented by electron tomography and Fast-Fourier methods. This multi-modal approach has allowed us to characterize an unknown parabasal filament and reveal the ultrastructure of other striated fibers that have not been published before. Here, we show the differences in origin, striation pattern, size, localization, and additional details of the PFs, thus improving the knowledge of the cell biology of this parasite.

Characterization of a new extra-axonemal structure in the Giardia intestinalis flagella.

The inner structure of the flagella of Giardia intestinalis is similar to that of other organisms, consisting of nine pairs of outer microtubules and a central pair containing radial spokes. Although the 9+2 axonemal structure is conserved, it is not clear whether subregions, including the transition zone, are present in the flagella of this parasite. Giardia axonemes originate from basal bodies and have a lengthy cytosolic portion before becoming active flagella. The region of the emergence of the flagellum is not accompanied by any membrane specialization, as seen in other protozoa. Although Giardia is an intriguing model of study, few works focused on the ultrastructural analysis of the flagella of this parasite. Here, we analyzed the externalization region of the G. intestinalis flagella using ultra-high resolution scanning microscopy (with electrons and ions), atomic force microscopy in liquid medium, freeze fracture, and electron tomography. Our data show that this region possesses a distinctive morphological feature - it extends outward and takes on a ring-like shape. When the plasma membrane is removed, a structure surrounding the axoneme becomes visible in this region. This new extra-axonemal structure is observed in all pairs of flagella of trophozoites and remains attached to the axoneme even when the interconnections between the axonemal microtubules are disrupted. High-resolution scanning electron microscopy provided insights into the arrangement of this structure, contributing to the characterization of the externalization region of the flagella of this parasite.

SARS-CoV-2 egress from Vero cells: a morphological approach.

Despite being extensively studied because of the current coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) interactions with mammalian cells are still poorly understood. Furthermore, little is known about this coronavirus cycle within the host cells, particularly the steps that lead to viral egress. This study aimed to shed light on the morphological features of SARS-CoV-2 egress by utilizing transmission and high-resolution scanning electron microscopy, along with serial electron tomography, to describe the route of nascent virions towards the extracellular medium. Electron microscopy revealed that the clusters of viruses in the paracellular space did not seem to result from collective virus release. Instead, virus accumulation was observed on incurved areas of the cell surface, with egress primarily occurring through individual vesicles. Additionally, our findings showed that the emission of long membrane projections, which could facilitate virus surfing in Vero cells infected with SARS-CoV-2, was also observed in non-infected cultures, suggesting that these are constitutive events in this cell lineage.

A spatially resolved elemental nanodomain organization within acidocalcisomes in Trypanosoma cruzi.

How are ions distributed in the three-dimensional (3D) volume confined in a nanoscale compartment? Regulation of ionic flow in the intracellular milieu has been explained by different theoretical models and experimentally demonstrated for several compartments with microscale dimensions. Most of these models predict a homogeneous distribution of ions seconds or milliseconds after an initial diffusion step formed at the ion translocation site, leaving open questions when it comes to ion/element distribution in spaces/compartments with nanoscale dimensions. Due to the influence of compartment size on the regulation of ionic flow, theoretical variations of classical models have been proposed, suggesting heterogeneous distributions of ions/elements within nanoscale compartments. Nonetheless, such assumptions have not been fully proven for the 3D volume of an organelle. In this work, we used a combination of cutting-edge electron microscopy techniques to map the 3D distribution of diffusible elements within the whole volume of acidocalcisomes in trypanosomes. Cryofixed cells were analyzed by scanning transmission electron microscopy tomography combined with elemental mapping using a high-performance setup of X-ray detectors. Results showed the existence of elemental nanodomains within the acidocalcisomes, where cationic elements display a self-excluding pattern. These were validated by Pearson correlation analysis and in silico molecular dynamic simulations. Formation of element domains within the 3D space of an organelle is demonstrated. Distribution patterns that support the electrodiffusion theory proposed for nanophysiology models have been found. The experimental pipeline shown here can be applied to a variety of models where ion mobilization plays a crucial role in physiological processes.

Modelling premature cardiac aging with induced pluripotent stem cells from a hutchinson-gilford Progeria Syndrome patient.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic disorder that causes accelerated aging and a high risk of cardiovascular complications. However, the underlying mechanisms of cardiac complications of this syndrome are not fully understood. This study modeled HGPS using cardiomyocytes (CM) derived from induced pluripotent stem cells (iPSC) derived from a patient with HGPS and characterized the biophysical, morphological, and molecular changes found in these CM compared to CM derived from a healthy donor. Electrophysiological recordings suggest that the HGPS-CM was functional and had normal electrophysiological properties. Electron tomography showed nuclear morphology alteration, and the 3D reconstruction of electron tomography images suggests structural abnormalities in HGPS-CM mitochondria, however, there was no difference in mitochondrial content as measured by Mitotracker. Immunofluorescence indicates nuclear morphological alteration and confirms the presence of Troponin T. Telomere length was measured using qRT-PCR, and no difference was found in the CM from HGPS when compared to the control. Proteomic analysis was carried out in a high-resolution system using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). The proteomics data show distinct group separations and protein expression differences between HGPS and control-CM, highlighting changes in ribosomal, TCA cycle, and amino acid biosynthesis, among other modifications. Our findings show that iPSC-derived cardiomyocytes from a Progeria Syndrome patient have significant changes in mitochondrial morphology and protein expression, implying novel mechanisms underlying premature cardiac aging.

Anti-Leishmania amazonensis activity of the marine sponge Dercitus (Stoeba) latex (Porifera) from São Pedro and São Paulo Archipelago, Pernambuco, Brazil.

The search for new therapeutic strategies for leishmaniasis treatment is essential due to the side effects of available drugs and the increasing incidence of resistance to them. Marine sponges use chemical compounds as a defense mechanism, and several of them present interesting pharmacological properties. The aim of this study was to evaluate the in vitro activity of the aqueous extract of the marine sponge Dercitus (Stoeba) latex against Leishmania amazonensis. MIC and toxicity against mammal cells were evaluated through broth microdilution assays. Transmission electron microscopy analysis was performed to assess possible effects on L. amazonensis ultrastructure. Arginase and proteolytic activities were measured by spectrometric methodologies. The extract of Dercitus (Stoeba) latex displayed antileishmanial activity and moderate toxicity against peritonial macrophages. Ultrastructural changes were observed after the growth of L. amazonensis promastigotes in the presence of the extract at 150 µg.ml-1 (IC50), mainly on acidocalcysomes. The extract was able to inhibit the activity of arginase and serine proteases. This study shows that Dercitus (Stoeba) latex aqueous extract may be a novel potential source of protozoa protease inhibitors and drugs that are less toxic to be used in the treatment of L. amazonensis infections.

Overexpression of mir-135b and mir-210 in mesenchymal stromal cells for the enrichment of extracellular vesicles with angiogenic factors.

Extracellular vesicles (EVs) are known as molecular carriers involved in cell communication and the regulation of (patho)physiological processes. miRNAs and growth factors are the main contents of EVs which make them a good candidate for the treatment of diseases caused by ischemia, but the low production of EVs by a cell producer and a significant variation of the molecular contents in EVs according to the cell source are the main limitations of their widespread use. Here, we show how to improve the therapeutic properties of mesenchymal stromal cell (MSC)-derived EVs (MSC-EVs) by modifying MSCs to enrich these EVs with specific angiomiRs (miR-135b or miR-210) using lentiviral vectors carrying miR-135b or miR-210. MSCs were obtained from the mouse bone marrow and transduced with a corresponding lentivector to overexpress miR-135b or miR-210. The EVs were then isolated by ultracentrifugation and characterized using a flow cytometer and a nanoparticle tracking analyzer. The levels of 20 genes in the MSCs and 12 microRNAs in both MSCs and EVs were assessed by RT‒qPCR. The proangiogenic activity of EVs was subsequently assessed in human umbilical vein endothelial cells (HUVECs). The results confirmed the overexpression of the respective microRNA in modified MSCs. Moreover, miR-135b overexpression upregulated miR-210-5p and follistatin, whereas the overexpression of miR-210 downregulated miR-221 and upregulated miR-296. The tube formation assay showed that EVs from MSCs overexpressing miR-210-5p (EVmiR210) significantly promoted tubular structure formation in HUVECs. A significant increase in angiogenic proteins (PGF, endothelin 1, and artemin) and genes (VEGF, activin A, and IGFBP1) in HUVECs treated with VEmiR210 justifies the better tubular structure formation of these cells compared with that of EVmiR135b-treated HUVECs, which showed upregulated expression of only artemin. Collectively, our results show that the EV cargo can be modified by lentiviral vectors to enrich specific miRNAs to achieve a specific angiogenic potential.

The monoterpene 1,8-cineole prevents cerebral edema in a murine model of severe malaria.

1,8-Cineole is a naturally occurring compound found in essential oils of different plants and has well-known anti-inflammatory and antimicrobial activities. In the present work, we aimed to investigate its potential antimalarial effect, using the following experimental models: (1) the erythrocytic cycle of Plasmodium falciparum; (2) an adhesion assay using brain microvascular endothelial cells; and (3) an experimental cerebral malaria animal model induced by Plasmodium berghei ANKA infection in susceptible mice. Using the erythrocytic cycle of Plasmodium falciparum, we characterized the schizonticidal effect of 1,8-cineole. This compound decreased parasitemia in a dose-dependent manner with a half maximal inhibitory concentration of 1045.53 ± 63.30 µM. The inhibitory effect of 972 µM 1,8-cineole was irreversible and independent of parasitemia. Moreover, 1,8-cineole reduced the progression of intracellular development of the parasite over 2 cycles, inducing important morphological changes. Ultrastructure analysis revealed a massive loss of integrity of endomembranes and hemozoin crystals in infected erythrocytes treated with 1,8-cineole. The monoterpene reduced the adhesion index of infected erythrocytes to brain microvascular endothelial cells by 60%. Using the experimental cerebral malaria model, treatment of infected mice for 6 consecutive days with 100 mg/kg/day 1,8-cineole reduced cerebral edema with a 50% reduction in parasitemia. Our data suggest a potential antimalarial effect of 1,8-cineole with an impact on the parasite erythrocytic cycle and severe disease.

Early Effects of Extracellular Vesicles Secreted by Adipose Tissue Mesenchymal Cells in Renal Ischemia Followed by Reperfusion: Mechanisms Rely on a Decrease in Mitochondrial Anion Superoxide Production.

Acute kidney injury (AKI) caused by ischemia followed by reperfusion (I/R) is characterized by intense anion superoxide (O2•-) production and oxidative damage. We investigated whether extracellular vesicles secreted by adipose tissue mesenchymal cells (EVs) administered during reperfusion can suppress the exacerbated mitochondrial O2•- formation after I/R. We used Wistar rats subjected to bilateral renal arterial clamping (30 min) followed by 24 h of reperfusion. The animals received EVs (I/R + EVs group) or saline (I/R group) in the kidney subcapsular space. The third group consisted of false-operated rats (SHAM). Mitochondria were isolated from proximal tubule cells and used immediately. Amplex Red™ was used to measure mitochondrial O2•- formation and MitoTracker™ Orange to evaluate inner mitochondrial membrane potential (Δψ). In vitro studies were carried out on human renal proximal tubular cells (HK-2) co-cultured or not with EVs under hypoxic conditions. Administration of EVs restored O2•- formation to SHAM levels in all mitochondrial functional conditions. The gene expression of catalase and superoxide dismutase-1 remained unmodified; transcription of heme oxygenase-1 (HO-1) was upregulated. The co-cultures of HK-2 cells with EVs revealed an intense decrease in apoptosis. We conclude that the mechanisms by which EVs favor long-term recovery of renal structures and functions after I/R rely on a decrease of mitochondrial O2•- formation with the aid of the upregulated antioxidant HO-1/Nuclear factor erythroid 2-related factor 2 system, thus opening new vistas for the treatment of AKI.

Three-dimensional architecture of Cyrilia lignieresi gametocyte-stage development inside red blood cells.

The Haemogregarinidae family (Apicomplexa: Adeleina) comprises hemoprotozoa that infect mammals, birds, amphibians, fish, and reptiles. Some morphological characteristics of the Cyrilia lignieresi have been described previously, but the parasite-erythrocyte relationship is still poorly understood. In order to understand the structural architecture of C. lignieresi-infected red blood cells, electron microscopy-based three-dimensional reconstruction was carried out using TEM as well as FIB-SEM tomography. Results showed that development of the macrogametocyte-stage inside the red blood cell is related to an increase in cleft-like structures in the host cell cytoplasm. Furthermore, other aspects related to parasite intraerythrocytic development were explored by 3D visualization techniques. We observed the invagination of a large extension of the Inner Membrane Complex (IMC) on the parasite body, which results from or induces a folding of the posterior end of the parasite. Small tubular structures were seen associated with areas related to IMC folding. Taken together, results provide new information on the remodeling of erythrocytes induced by the protozoan C. lignieresi.

Myelination of Callosal Axons Is Hampered by Early and Late Forelimb Amputation in Rats.

Deafferentation is an important determinant of plastic changes in the CNS, which consists of a loss of inputs from the body periphery or from the CNS itself. Although cortical reorganization has been well documented, white matter plasticity was less explored. Our goal was to investigate microstructural interhemispheric connectivity changes in early and late amputated rats. For that purpose, we employed diffusion-weighted magnetic resonance imaging, as well as Western blotting, immunohistochemistry, and electron microscopy of sections of the white matter tracts to analyze the microstructural changes in the corticospinal tract and in the corpus callosum (CC) sector that contains somatosensory fibers integrating cortical areas representing the forelimbs and compare differences in rats undergoing forelimb amputation as neonates, with those amputated as adults. Results showed that early amputation induced decreased fractional anisotropy values and reduction of total myelin amount in the cerebral peduncle contralateral to the amputation. Both early and late forelimb amputations induced decreased myelination of callosal fibers. While early amputation affected myelination of thinner axons, late amputation disrupted axons of all calibers. Since the CC provides a modulation of inhibition and excitation between the hemispheres, we suggest that the demyelination observed among callosal fibers may misbalance this modulation.

Silver chitosan nanocomposites as a potential treatment for superficial candidiasis.

Silver compounds are widely known for their antimicrobial activity, but can exert toxic effects to the host. Among the strategies to reduce its toxicity, incorporation into biopolymers has shown promising results. We investigated the green syntheses of silver nanoparticles (AgNPs) and their functionalization in a chitosan matrix (AgNPs@Chi) as a potential treatment against Candida spp. Inhibitory concentrations ranging between 0.06 and  1 µg/ml were observed against distinct Candida species. Nanocomposite-treated cells displayed cytoplasmic degeneration and a cell membrane and wall disruption. Silver nanocomposites in combination with fluconazole and amphotericin B showed an additive effect when analyzed by the Bliss method. The low cytotoxicity displayed in mammalian cells and in the Galleria mellonella larvae suggested their potential use in vivo. When tested as a topical treatment against murine cutaneous candidiasis, silver nanocomposites reduced the skin fungal burden in a dose-response behavior and favored tissue repair. In addition, the anti-biofilm effect of AgNPs@Chi in human nail model was demonstrated, suggesting that the polymeric formulation of AgNPs does not affect antifungal activity even against sessile cells. Our results suggest that AgNPs@Chi seems to be a less toxic and effective topical treatment for superficial candidiasis. LAY SUMMARY: This study demonstrated the efficacy of silver nanoparticles (AgNPs) in inhibiting the growth of Candida. AgNPs incorporated in chitosan displayed a reduced toxicity. Tests in infected mice showed the effectiveness of the treatment. AgNPs-chitosan could be an alternative to combat candidiasis.

Antifungal activity of Acylhydrazone derivatives against Sporothrix spp.

Sporotrichosis is an emerging mycosis caused by members of the genus Sporothrix The disease affects humans and animals, particularly cats, which plays an important role in the zoonotic transmission. Feline sporotrichosis treatment options include itraconazole (ITC), potassium iodide and amphotericin B, drugs usually associated with deleterious adverse reactions and refractoriness in cats, especially when using ITC. Thus, affordable, non-toxic and clinically effective anti-Sporothrix agents are needed. Recently, acylhydrazones (AH), molecules targeting vesicular transport and cell cycle progression, exhibited a potent antifungal activity against several fungal species and displayed low toxicity when compared to the current drugs. In this work, the AH derivatives D13 and SB-AF-1002 were tested against Sporothrix schenckii and Sporothrix brasiliensis Minimal inhibitory concentrations of 0.12 - 1 µg/mL were observed for both species in vitro D13 and SB-AF-1002 showed an additive effect with itraconazole. Treatment with D13 promoted yeast disruption with release of intracellular components, as confirmed by transmission electron microscopy of S. brasiliensis exposed to the AH derivatives. AH-treated cells displayed thickening of the cell wall, discontinuity of the cell membrane and an intense cytoplasmic degeneration. In a murine model of sporotrichosis, treatment with AH derivatives was more efficient than ITC, the drug of choice for sporotrichosis. The results of the preliminary clinical study in cats indicate that D13 is safe and has potential to become a therapeutic option for sporotrichosis when associated to ITC. Our results expand the antifungal broadness of AH derivatives and suggest that these drugs could be exploited to combat sporotrichosis.

Intracellular host cell membrane remodelling induced by SARS-CoV-2 infection in vitro.

BACKGROUND INFORMATION: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection induces an alteration in the endomembrane system of the mammalian cells. In this study, we used transmission electron microscopy and electron tomography to investigate the main structural alterations in the cytoplasm of Vero cells infected with a SARS-CoV-2 isolate from São Paulo state (Brazil). RESULTS: Different membranous structures derived from the zippered endoplasmic reticulum were observed along with virus assembly through membrane budding. Also, we demonstrated the occurrence of annulate lamellae in the cytoplasm of infected cells and the presence of virus particles in the perinuclear space. CONCLUSIONS AND SIGNIFICANCE: This study contributes to a better understanding of the cell biology of SARS-CoV-2 and the mechanisms of the interaction of the virus with the host cell that promote morphological changes, recruitment of organelles and cell components, in a context of a virus-induced membrane remodelling.

Current Microscopy Strategies to Image Fungal Vesicles: From the Intracellular Trafficking and Secretion to the Inner Structure of Isolated Vesicles.

Extracellular vesicles (EVs) are nano-sized structures that play important roles in a variety of biological processes among members of the Eukaryota domain. They have been studied since the 1940s and a broader use of different microscopy techniques to image either isolated vesicles or vesicles within the intracellular milieu (trafficking) has been limited by their nanometric size, usually below the resolution limit of most standard light microscopes. The development of genetically encoded fluorescent proteins and fluorescent probes able to switch between "on" and "off" states, as well the improvement in computer-assisted microscopy, photon detector devices, illumination designs, and imaging strategies in the late Twentieth century, boosted the use of light microscopes to provide structural and functional information at the sub-diffraction resolution, taking advantage of a nondestructive analytical probe such light, and opening new possibilities in the study of life at the nanoscale. As well, traditional and novel electron microscopy techniques have been widely used in the characterization of subcellular compartments, either isolated or in situ, providing a comprehensive understanding of their functional role in many cellular processes. Here, we present basic aspects of some of these techniques that have already been applied and their potential application to the study of fungal vesicles.

Eugenol disrupts Plasmodium falciparum intracellular development during the erythrocytic cycle and protects against cerebral malaria.

BACKGROUND: Malaria is a parasitic disease that compromises the human host. Currently, control of the Plasmodium falciparum burden is centered on artemisinin-based combination therapies. However, decreased sensitivity to artemisinin and derivatives has been reported, therefore it is important to identify new therapeutic strategies. METHOD: We used human erythrocytes infected with P. falciparum and experimental cerebral malaria (ECM) animal model to assess the potential antimalarial effect of eugenol, a component of clove bud essential oil. RESULTS: Plasmodium falciparum cultures treated with increasing concentrations of eugenol reduced parasitemia in a dose-dependent manner, with IC50 of 532.42 ± 29.55 µM. This effect seems to be irreversible and maintained even in the presence of high parasitemia. The prominent effect of eugenol was detected in the evolution from schizont to ring forms, inducing important morphological changes, indicating a disruption in the development of the erythrocytic cycle. Aberrant structural modification was observed by electron microscopy, showing the separation of the two nuclear membrane leaflets as well as other subcellular membranes, such as from the digestive vacuole. Importantly, in vivo studies using ECM revealed a reduction in blood parasitemia and cerebral edema when mice were treated for 6 consecutive days upon infection. CONCLUSIONS: These data suggest a potential effect of eugenol against Plasmodium sp. with an impact on cerebral malaria. GENERAL SIGNIFICANCE: Our results provide a rational basis for the use of eugenol in therapeutic strategies to the treatment of malaria.