RESUMEN

Disrupted lipid metabolism is a characteristic of gliomas. This study utilizes an ultrastructural approach to characterize the prevalence and distribution of lipids within gliomas. This study made use of tissue from IDH1 wild type (IDH1-wt) glioblastoma (n = 18) and IDH1 mutant (IDH1-mt) astrocytoma (n = 12) tumors. We uncover a prevalent and intriguing surplus of lipids. The bulk of the lipids manifested as sizable cytoplasmic inclusions and extracellular deposits in the tumor microenvironment (TME); in some tumors the lipids were stored in the classical membraneless spheroidal lipid droplets (LDs). Frequently, lipids accumulated inside mitochondria, suggesting possible dysfunction of the beta-oxidation pathway. Additionally, the tumor vasculature have lipid deposits in their lumen and vessel walls; this lipid could have shifted in from the tumor microenvironment or have been produced by the vessel-invading tumor cells. Lipid excess in gliomas stems from disrupted beta-oxidation and dysfunctional oxidative phosphorylation pathways. The implications of this lipid-driven environment include structural support for the tumor cells and protection against immune responses, non-lipophilic drugs, and free radicals.

Asunto(s)

RESUMEN

In the context of global climate change, recurrent freeze-thaw cycles (FTC) and concurrent exposure to polystyrene nanoplastics (PSNPs) directly impact crop growth and indirectly affect resilience to abiotic stress. In January 2023, experiments at the Environmental Biology Laboratory, Jilin University, Changchun, China, exposed rye seedlings to 100 nm PSNPs at concentrations of 0, 10, 50, and 100 mg/L for seven days, followed by three FTC. Scanning electron microscopy (SEM) demonstrated that PSNPs migrated from the roots to the leaves, with FTC significantly exacerbating their accumulation within plant tissues. Transmission electron microscopy (TEM) observations showed that FTC disrupted normal cell division, and combined stress from NPs damaged plant organs, particularly chloroplasts, thereby substantially inhibiting photosynthesis. FTC delayed plant phenological stages. Under combined stress, malondialdehyde (MDA) accumulation in plant tissues increased by 15.6%, while hydrogen peroxide (H2O2) content decreased. Simultaneously, the activities of peroxidase (POD) and catalase (CAT) increased by 34.2% and 38.6%, respectively. Molecular docking unveiled that PSNPs could bind to the active center of POD/CAT through hydrogen bonding or hydrophobic interactions. The Integrated Biomarker Response (IBR) index highlighted FTC as a crucial determinant for pronounced effects. Moreover, an apparent dose-dependent effect was observed, with antioxidant enzyme activities in rye seedlings induced by low pollutant concentrations and inhibited by high concentrations. These results indicate that FTC and PSNPs can disrupt plant membrane systems and cause severe oxidative damage. Overall, this study provides compelling scientific evidence of the risks associated with NPs exposure in plants subjected to abiotic stress.

Asunto(s)

RESUMEN

The mechanisms of the pathogenesis of neck paraganglioma (PGL) and the possible role of mast cells (MCs) in its development and metastasis are still poorly understood. We analyzed MCs' morphologic characterization, activation, and the properties of their cytoplasmic/released granules in PGLs, using light and transmission electron microscopy. Paragangliomas showed a large tumor-associated MC population both in the connective tissue layers of the tumor and between the tumor cells. Notably, MCs were presented by a high expression of specific proteases, size variation, polymorphism, and variable ultrastructural phenotype of granules. A massive number of granules were released surrounding the degranulated MCs while the integrity of MC membrane was maintained. Granules were electron-dense with or without a membrane, ranging from 0.2 to 0.8 µm in diameter. MC plasmalemma was not found at the site of MC-collagen fibrils contact, whereas the secretome and fibrils were directly contacted. We observed direct and mediator-based interactions between MCs and paraganglioma cells. The latter preserved their membrane integrity when MC granules were not in proximity. The effects of the MC secretome on the paraganglioma microenvironment demonstrated its pathogenetic role in tumor progression and allow its application to new diagnostic criteria and the development of protocols for personalized therapy. RESEARCH HIGHLIGHTS: Ultrastructural analysis reveals novel regulatory effects of mast cells via diverse secretory pathways on the pathogenesis of parasympathetic paraganglioma, including fibrous extracellular matrix remodeling and mediator-based interactions between MCs and cells of the tumor microenvironment.

Asunto(s)

RESUMEN

Large areas of crop yields in northern China have faced with cadmium (Cd) contamination problems. Mercapto-modified palygorskite (MP), as a highly efficient immobilization material, could reduce Cd absorption in wheat and alleviate its biotoxicity. However, the molecular mechanism underlying MP-mediated Cd reduction and detoxification processes in wheat is not well understood. This aim of this study was to investigate the biochemical and molecular mechanisms underlying the reduction in Cd accumulation in wheat (Triticum aestivum L.). The results showed that MP application decreased the Cd concentration by 68.91-74.32% (root) and 70.68-77.2% (shoot), and significantly increased the glutathione (GSH) and phytochelatins (PCs) contents in root and shoot. In addition, with the application of MP, the percentage of Cd in the cell walls and organelles of wheat decreased, while that of Cd in soluble components was increased. The content of Cd in all components was significantly reduced. Ultrastructural analysis revealed that MP thickened the cell wall, promoted vesicle formation in the membrane and protected the integrity of intracellular organelles in wheat. Transcriptome analysis further confirmed the above results. MP upregulated the expression of several genes (CCR, CAD COMT and SUS) involved in cell wall component biosynthesis and promoted vesicle formation on cell membranes by upregulating the expression of PLC and IPMK genes. In addition, genes related to antioxidant synthesis (PGD, glnA and GSS) and photosynthesis (Lhca, Lhcb) were altered by MP to alleviate Cd toxicity in wheat. This present work will help to more thoroughly elucidate the molecular mechanism by which wheat defends against Cd contamination under MP application and provide and important research basis for the application of this material in the future.

Asunto(s)

RESUMEN

The design of the implant prosthesis-abutment complex appears crucial for shaping healthy and stable peri-implant soft tissues. The aim of the present animal study was to compare two implants with different healing abutment geometries: a concave design (TEST) and a straight one (CTRL). Transmission electron microscopy (TEM) was used to quantify the three-dimensional topography and morphological properties of collagen at nanoscale resolution. 2 swine were included in the experiment and 6 implants per animal were randomly placed in the left or right hemimandible in either the physiologically mature bone present between the lower canine and first premolar or in the mandibular premolar area, within tooth extraction sites. Each CTRL implant was positioned across from its respective TEST implant on the other side of the jaw. After 12 weeks of healing, 8 specimens (4 CTRL and 4 TEST) were retrieved and prepared for histological and TEM analysis. The results showed a significantly higher percentage of area covered by collagen bundles and average bundle size in TEST implants, as well as a significant decrease in the number of longitudinally oriented bundles with respect to CTRL implants, which is potentially due to the larger size of TEST bundles. These data suggest that a concave transmucosal abutment design serves as a scaffold, favoring the deposition and growth of a well-organized peri-implant collagen structure over the implant platform in the early healing phase, also promoting the convergence of collagen fibers toward the abutment collar.

RESUMEN

Chikungunya virus (CHIKV) is an arthropod-borne virus recently associated with large outbreaks in many parts of the world. Infection is typically manifested as a febrile and self-limited illness, characterized by joint pain and myalgia, albeit severe neurological manifestations are also reported. Although CHIKV is not recognized as a truly neurotropic virus, neurons, astrocytes, and oligodendrocytes are susceptible to infection in vitro. Here we employed a model of 3D cell culture to obtain neurospheres from ATRA/BNDF differentiated human neuroblastoma cells. We demonstrate that CHIKV is able to establish a productive infection, resulting in ultrastructural changes in cell morphology and impaired neuronal differentiation. Ultrastructural analysis of neurospheres infected with CHIKV during neuronal differentiation revealed diminished neuron dendrite formation, accumulation of viral particles associated with the plasma membrane, numerous cell vacuoles, and swollen mitochondria. Apoptotic cells were significantly increased at 72 h post-infection. Compared to Zika virus, a well-characterized neurotropic arbovirus, CHIKV infection resulted in a more discrete, albeit detectable upregulation of IL-6 levels. Finally, we found that CHIKV infection resulted in an altered profile expression, mainly downregulation, of a group of transcription factors named Hox genes. Altogether our findings highlight important features of CHIKV in the CNS, as well as the feasibility of neurospheres as robust experimental models that can support further studies for novel pharmacological interventions.

RESUMEN

In this protocol, we describe the specific steps required to prepare human postmortem brain samples for ultrastructural microglial analysis. A detailed procedure is provided to improve the ultrastructural quality of the samples, using aldehyde fixatives followed by immunoperoxidase staining of allograft inflammatory factor 1 (AIF1, also known as IBA1), a marker of myeloid cells, and cluster of differentiation 68 (CD68), a marker of phagolysosomal activity. Additionally, we describe an osmium-thiocarbohydrazide-osmium (OTO) post-fixation method that preserves and increases the contrast of cellular membranes in human postmortem brain samples, as well as the steps necessary to acquire scanning electron microscopy (SEM) images of microglial cell bodies. In the last section, we cover the quantitative analysis of various microglial cytoplasmic organelles and their interactions with other parenchymal elements.

Asunto(s)

RESUMEN

Disrupted lipid metabolism is a characteristic of gliomas. This study utilizes an ultrastructural approach to characterize the prevalence and distribution of lipids within gliomas. This study made use of tissue from IDH1 wild type (IDH1-wt) glioblastoma (n = 18) and IDH1 mutant (IDH1-mt) astrocytoma (n = 12) tumors. We uncover a prevalent and intriguing surplus of lipids. The bulk of the lipids manifested as sizable cytoplasmic inclusions and extracellular deposits in the tumor microenvironment (TME); in some tumors the lipids were stored in the classical membraneless spheroidal lipid droplets (LDs). Frequently, lipids accumulated inside mitochondria, suggesting possible dysfunction of the beta-oxidation pathway. Additionally, the tumor vasculature have lipid deposits in their lumen and vessel walls; this lipid could have shifted in from the tumor microenvironment or have been produced by the vessel-invading tumor cells. Lipid excess in gliomas stems from disrupted beta-oxidation and dysfunctional oxidative phosphorylation pathways. The implications of this lipid-driven environment include structural support for the tumor cells and protection against immune responses, non-lipophilic drugs, and free radicals.

RESUMEN

The invasive nature of Toxoplasma gondii is closely related to the properties of its cytoskeleton, which is constituted by a group of diverse structural and dynamic components that play key roles during the infection. Even if there have been numerous reports about the composition and function of the Toxoplasma cytoskeleton, the ultrastructural organization of some of these components has not yet been fully characterized. This study used a detergent extraction process and several electron microscopy contrast methods that allowed the successful isolation of the cytoskeleton of Toxoplasma tachyzoites. This process allowed for the conservation of the structures known to date and several new structures that had not been characterized at the ultrastructural level. For the first time, characterization was achieved for a group of nanofibers that allow the association between the polar apical ring and the conoid as well as the ultrastructural characterization of the apical cap of the parasite. The ultrastructure and precise location of the peripheral rings were also found, and the annular components of the basal complex were characterized. Finally, through immunoelectron microscopy, the exact spatial location of the subpellicular network inside the internal membrane system that forms the pellicle was found. The findings regarding these new structures contribute to the knowledge concerning the biology of the Toxoplasma gondii cytoskeleton. They also provide new opportunities in the search for therapeutic strategies aimed at these components with the purpose of inhibiting invasion and thus parasitism.

Asunto(s)

RESUMEN

Cryo-electron tomography provides detailed views of macromolecules in situ. However, imaging a large field of view to provide more cellular context requires reducing magnification during data collection, which in turn restricts the resolution. To circumvent this trade-off between field of view and resolution, we have developed a montage data collection scheme that uniformly distributes the dose throughout the specimen. In this approach, sets of slightly overlapping circular tiles are collected at high magnification and stitched to form a composite projection image at each tilt angle. These montage tilt-series are then reconstructed into massive tomograms with a small pixel size but a large field of view. For proof-of-principle, we applied this method to the thin edge of HeLa cells. Thon rings to better than 10 Å were detected in the montaged tilt-series, and diverse cellular features were observed in the resulting tomograms. These results indicate that the additional dose required by this technique is not prohibitive to performing structural analysis to intermediate resolution across a large field of view. We anticipate that montage tomography will prove particularly useful for lamellae, increase the likelihood of imaging rare cellular events, and facilitate visual proteomics.

Asunto(s)

RESUMEN

The endoplasmic reticulum (ER) presents unique properties to establishing bacterium symbiosis in eukaryotic cells since it synthesizes and glycosylates essential molecules like proteins and lipids. Tunicamycin (TM) is an antibiotic that inhibits the first step in the N-linked glycosylation in eukaryotes and has been used as an ER stress inducer to activate the Unfolded Protein Response (UPR). Mutualistic symbiosis in trypanosomatids is characterized by structural adaptations and intense metabolic exchanges, thus we investigated the effects of TM in the association between Angomonas deanei and its symbiotic bacterium, through ultrastructural and proteomic approaches. Cells treated with the inhibitor showed a decrease in proliferation, enlargement of the ER and Golgi cisternae and an increased distance between the symbiont and the ER. TM proved to be an important tool to better understand ER stress in trypanosomatids, since changes in protein composition were observed in the host protozoan, especially the expression of the Hsp90 chaperone. Furthermore, data obtained indicates the importance of the ER for the adaptation and maintenance of symbiotic associations between prokaryotes and eukaryotes, considering that this organelle has recognized importance in the biogenesis and division of cell structures.

Asunto(s)

RESUMEN

Despite the advancement of molecular biology techniques, morphological studies using transmission electron microscopy (TEM) are still being of great importance to elucidate some aspects of viral structures, morphogenesis, and pathogenesis. In relation to dengue viruses (DENV), several studies report the use of TEM to obtain a clearer definition of viral morphology, the events involved in its morphogenesis, aspects of pathogenesis, and cell tropism. In this chapter, the main technical protocols and their respective reagents for studies of DENV infection by TEM are described, both in cell culture and in biological tissue samples.

Asunto(s)

RESUMEN

A precise quantitative description of the ultrastructural characteristics underlying biological mechanisms is often key to their understanding. This is particularly true for dynamic extra- and intracellular filamentous assemblies, playing a role in cell motility, cell integrity, cytokinesis, tissue formation and maintenance. For example, genetic manipulation or modulation of actin regulatory proteins frequently manifests in changes of the morphology, dynamics, and ultrastructural architecture of actin filament-rich cell peripheral structures, such as lamellipodia or filopodia. However, the observed ultrastructural effects often remain subtle and require sufficiently large datasets for appropriate quantitative analysis. The acquisition of such large datasets has been enabled by recent advances in high-throughput cryo-electron tomography (cryo-ET) methods. This also necessitates the development of complementary approaches to maximize the extraction of relevant biological information. We have developed a computational toolbox for the semi-automatic quantification of segmented and vectorized filamentous networks from pre-processed cryo-electron tomograms, facilitating the analysis and cross-comparison of multiple experimental conditions. GUI-based components simplify the processing of data and allow users to obtain a large number of ultrastructural parameters describing filamentous assemblies. We demonstrate the feasibility of this workflow by analyzing cryo-ET data of untreated and chemically perturbed branched actin filament networks and that of parallel actin filament arrays. In principle, the computational toolbox presented here is applicable for data analysis comprising any type of filaments in regular (i.e. parallel) or random arrangement. We show that it can ease the identification of key differences between experimental groups and facilitate the in-depth analysis of ultrastructural data in a time-efficient manner.

Asunto(s)

RESUMEN

Certain active drugs and excipients of nasal formulations may impair ciliary function and mucociliary clearance. The ciliary beat frequency (CBF) is a key parameter for determining mucociliary clearance rate, and in vitro assessments of CBF have proven to be accurate and reproducible. Since topical nasal formulations are applied with repeated doses, it is essential to elucidate their chronic, as opposed to acute, effect on mucociliary clearance and nasal mucosa. The aim of this study was to assess for the first time the ciliotoxicity and cytotoxicity of nasal sprays intended for chronic treatment (with repeated doses) using a previously designed set-up for CBF measurements. For 2 weeks, the 3D nasal MucilAir™ in vitro models were treated daily with undiluted or clinically relevant doses of mometasone nasal spray, placebo nasal spray, culture medium, or they were untreated. We demonstrated a dose-dependent and time-dependent (cumulative) effect of the nasal sprays on ciliary activity and cytotoxicity using CBF measurements and ultrastructural analysis, respectively. Our results indicate that repeated administration of clinically relevant doses of mometasone nasal spray is safe for in vivo use, which is in good agreement with a previous clinical study. Overall, our study suggests that such in vitro assays have great potential for topical nasal drug screening.

RESUMEN

INTRODUCTION: COVID-19, the disease caused by the novel coronavirus SARS-CoV-2, is a severe systemic thrombotic syndrome that emerged in 2019, with an ensuing pandemic. To evaluate the impact of this disease on placental tissue and perinatal outcome, histological, immunohistochemical and ultrastructural analyses of placental tissue were performed for five cases of pregnant women with COVID-19. CASE REPORTS: All five pregnant women in this series developed COVID-19 in late pregnancy. Two patients experienced respiratory distress, and computed tomography revealed signs of pneumonia, with bilateral involvement, multiple lobular and subsegmental areas of consolidation and ground-glass opacities. Histological studies of placental tissue revealed the presence of slight signs of maternal vascular underperfusion (MVUs) or foetal vascular underperfusion (FVUs) lesions and mild inflammatory lesions. CD15 immunoreactivity in the placental tissue was low in all cases, demonstrating that in these cases there was not severe foetal hypoxia/asphyxia risk for newborns or distal vascular immaturity. In all cases examined, ultrastructural analyses showed spherical-like coronavirus particles with an electron intermediate-density core as well as projections from the surface as spike-like structures in the syncytiotrophoblasts. At term, all of the women delivered newborns who were negative for SARS-CoV-2 by nasopharyngeal testing in their first day of life. All newborns were exclusively breastfed and were discharged on the 3rd day of life. CONCLUSIONS: In conclusion, placental patterns in pregnancy due to COVID-19 in the late stage of gestation indicate no evidence of vertical trans-placental SARS-CoV-2 transmission or a significant impact on the perinatal outcome of newborns, in both mild and more severe cases.

Asunto(s)

RESUMEN

BACKGROUND: The diseases for which Aedes aegypti is a vector are worrisome. The high vector competence of this mosquito, as well as its anthropophilia and ability to adapt to the urban environment, allows it to exploit many habitats, making its prevention an arduous task. Despite current disease control measures focused on the mosquito, the effectiveness in containing its dispersion still requires improvement; thus greater knowledge about this insect is fundamental. METHODS: Aedes aegypti egg morphology and embryonic development were analyzed from eggs of the insectary of the Institute of Biomedical Sciences of the University of São Paulo. Optical (light and confocal) and electronic (transmission and scanning) microscopy were used to analyze the morphological and ultrastructural features of the eggs. Embryos were observed in the initial (0-20.5 h after egg-laying), intermediate (20.6-40.1 h after egg-laying), and final (40.2-61.6 h) stages of development, and kept at a temperature of 28 °C ± 1 °C until collection for processing. RESULTS: Eggs of Ae. aegypti were whitish at the time of oviposition, and then quickly became black. The egg length was 581.45 ± 39.73 µm and the width was 175.36 ± 11.59. Access to the embryo was difficult due to the egg morphology, point of embryonic development, and difficult permeability of the exochorion (mainly in fixation). Only about 5% of the collected eggs were successfully processed. In the initial stage of embryonic development, characteristics suggestive of intense cellular activity were found. In the intermediate stage, the beginning of the segmentation process was evident. In the final phase, it was possible to differentiate the cephalic region and the thoracic and abdominal segments. CONCLUSION: The chorion was found to be an important protective barrier and a limiting factor for the evaluation of the embryos and mosquito embryonic cells, indicating that further studies need to be carried out to identify the reason that this occurs.

Asunto(s)

RESUMEN

The current study has been designed to appraise the efficacy of developed combinatorial lipid-nanosystem-based gel (linogel) of 5-fluorouracil and resveratrol for skin cancer treatment. Initially, linogel was prepared and characterized for different parameters, namely pH, texture, drug content uniformity, occlusiveness, etc. Then in vivo efficacy studies (tumor number, area, and volume, histopathology, ultrastructural and immunohistochemical analysis) of linogel were determined over-developed skin tumors. Developed linogel possessed significantly (p < 0.05) better texture and occlusiveness than conventional gel formulation. Decreased tumor number, area, and volume showed significant results (p < 0.05) in favor of linogel. Histopathological and ultrastructural analysis confirmed superior efficacy of linogel in terms of marked improvement in the nucleus and subcellular structures in photomicrographs. The antioxidants and anti-inflammatory analysis findings showed a significantly (p < 0.05) potent effectiveness of linogel. The apoptotic and anti-proliferation activity of linogel was confirmed by analysis of caspase-3 and ki-67, which showed significant (p < 0.05) elevation in the level of cleaved caspase-3 and reduction in the level of ki-67 than untreated and conventional gel formulation treated tumors, indicating antitumor effect due to cancerous cell death. Thus, developed linogel fulfilled all the criteria of dermal application and exhibited efficacious therapeutic results, which could be a beneficial therapeutic approach against skin cancer.

Asunto(s)

RESUMEN

The pathophysiological role of intracellular bacteria in osteomyelitis is still a matter of debate. Here, we demonstrate for the first time the presence of Staphylococcus aureus internalized into osteoblasts in human tissue samples of a case with a chronic osteomyelitis using ultrastructural transmission electron microscope analysis.

RESUMEN

In this study we evaluated the in vitro effect of divaricatic acid against coupled worms of Schistosoma mansoni. The schistosomicidal effect was evaluated through the bioassay of motility and mortality, cellular viability of the worms and ultrastructural analysis through Scanning Electron Microscopy. To evaluate the cytotoxicity of divaricatic acid, a cell viability assay was performed with human peripheral blood mononuclear cells. Divaricatic acid proved effect against S. mansoni after 3 hours of exposure. At the end of 24 h the concentrations of 100 - 200 µM presented lethality to the worms. Motility changes were observed at sublethal concentrations. The IC50 obtained by the cell viability assay for S. mansoni was 100.6 µM (96.24 - 105.2 µM). Extensive damage to the worm's tegument was observed such as peeling, erosion, bubbles, edema, damage and loss of tubercles and spines, fissures and tissue ruptures. No cytotoxicity was observed in human peripheral blood mononuclear cells. This report provides data showing the schistosomicidal effect of divaricatic acid on S. mansoni, causing death, motile changes and ultrastructural damage to worms. In addition, divaricatic acid was shown to be non-toxic to human peripheral blood mononuclear cells at concentrations effective on S. mansoni.

Asunto(s)