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1.
Int J Mol Sci ; 22(16)2021 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-34445067

RESUMO

Zebrafish is a vertebrate teleost widely used in many areas of research. As embryos, they develop quickly and provide unique opportunities for research studies owing to their transparency for at least 48 h post fertilization. Zebrafish have many ciliated organs that include primary cilia as well as motile cilia. Using zebrafish as an animal model helps to better understand human diseases such as Primary Ciliary Dyskinesia (PCD), an autosomal recessive disorder that affects cilia motility, currently associated with more than 50 genes. The aim of this study was to validate zebrafish motile cilia, both in mono and multiciliated cells, as organelles for PCD research. For this purpose, we obtained systematic high-resolution data in both the olfactory pit (OP) and the left-right organizer (LRO), a superficial organ and a deep organ embedded in the tail of the embryo, respectively. For the analysis of their axonemal ciliary structure, we used conventional transmission electron microscopy (TEM) and electron tomography (ET). We characterised the wild-type OP cilia and showed, for the first time in zebrafish, the presence of motile cilia (9 + 2) in the periphery of the pit and the presence of immotile cilia (still 9 + 2), with absent outer dynein arms, in the centre of the pit. In addition, we reported that a central pair of microtubules in the LRO motile cilia is common in zebrafish, contrary to mouse embryos, but it is not observed in all LRO cilia from the same embryo. We further showed that the outer dynein arms of the microtubular doublet of both the OP and LRO cilia are structurally similar in dimensions to the human respiratory cilia at the resolution of TEM and ET. We conclude that zebrafish is a good model organism for PCD research but investigators need to be aware of the specific physical differences to correctly interpret their results.


Assuntos
Cílios/patologia , Transtornos da Motilidade Ciliar/patologia , Peixe-Zebra , Animais , Transtornos da Motilidade Ciliar/fisiopatologia , Modelos Animais de Doenças , Humanos , Microscopia Eletrônica de Transmissão , Peixe-Zebra/fisiologia
2.
Cells ; 10(8)2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34440816

RESUMO

The mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) egress, similar to those of other coronaviruses, remain poorly understood. The virus buds in intracellular compartments and is therefore thought to be released by the biosynthetic secretory pathway. However, several studies have recently challenged this hypothesis. It has been suggested that coronaviruses, including SARS-CoV-2, use lysosomes for egress. In addition, a focused ion-beam scanning electron microscope (FIB/SEM) study suggested the existence of exit tunnels linking cellular compartments rich in viral particles to the extracellular space resembling those observed for the human immunodeficiency (HIV) in macrophages. Here, we analysed serial sections of Vero cells infected with SARS-CoV-2 by transmission electron microscopy (TEM). We found that SARS-CoV-2 was more likely to exit the cell in small secretory vesicles. Virus trafficking within the cells involves small vesicles, with each generally containing a single virus particle. These vesicles then fuse with the plasma membrane to release the virus into the extracellular space. This work sheds new light on the late stages of the SARS-CoV-2 infectious cycle of potential value for guiding the development of new antiviral strategies.


Assuntos
COVID-19/fisiopatologia , SARS-CoV-2/fisiologia , Vesículas Secretórias/ultraestrutura , Replicação Viral , Animais , Chlorocebus aethiops , Microscopia Eletrônica de Transmissão , Células Vero , Vírion/fisiologia
3.
Anal Chem ; 93(33): 11347-11356, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34370455

RESUMO

For over 25 years, transmission electron microscopy (TEM) has provided a method for the study of aerosol particles with sizes from below the optical diffraction limit to several microns, resolving the particles as well as smaller features. The wide use of this technique to study aerosol particles has contributed important insights about environmental aerosol particle samples and model atmospheric systems. TEM produces an image that is a 2D projection of aerosol particles that have been impacted onto grids and, through associated techniques and spectroscopies, can contribute additional information such as the determination of elemental composition, crystal structure, and 3D particle structures. Soot, mineral dust, and organic/inorganic particles have all been analyzed using TEM and spectroscopic techniques. TEM, however, has limitations that are important to understand when interpreting data including the ability of the electron beam to damage and thereby change the structure and shape of particles, especially in the case of particles composed of organic compounds and salts. In this paper, we concentrate on the breadth of studies that have used TEM as the primary analysis technique. Another focus is on common issues with TEM and cryogenic-TEM. Insights for new users on best practices for fragile particles, that is, particles that are easily susceptible to damage from the electron beam, with this technique are discussed. Tips for readers on interpreting and evaluating the quality and accuracy of TEM data in the literature are also provided and explained.


Assuntos
Poluentes Atmosféricos , Material Particulado , Aerossóis/análise , Poluentes Atmosféricos/análise , Monitoramento Ambiental , Microscopia Eletrônica de Transmissão , Tamanho da Partícula , Material Particulado/análise
4.
Int J Mol Sci ; 22(16)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34445789

RESUMO

The SARS-CoV-2 pseudovirus is a commonly used strategy that mimics certain biological functions of the authentic virus by relying on biological legitimacy at the molecular level. Despite the fact that spike (S), envelope (E), and membrane (M) proteins together wrap up the SARS-CoV-2 virion, most of the reported pseudotype viruses consist of only the S protein. Here, we report that the presence of E and M increased the virion infectivity by promoting the S protein priming. The S, E, and M (SEM)-coated pseudovirion is spherical, containing crown-like spikes on the surface. Both S and SEM pseudoviruses packaged the same amounts of viral RNA, but the SEM virus bound more efficiently to cells stably expressing the viral receptor human angiotensin-converting enzyme II (hACE2) and became more infectious. Using this SEM pseudovirus, we examined the infectivity and antigenic properties of the natural SARS-CoV-2 variants. We showed that some variants have higher infectivity than the original virus and that some render the neutralizing plasma with lower potency. These studies thus revealed possible mechanisms of the dissemination advantage of these variants. Hence, the SEM pseudovirion provides a useful tool to evaluate the viral infectivity and capability of convalescent sera in neutralizing specific SARS-CoV-2 S dominant variants.


Assuntos
Anticorpos Antivirais/metabolismo , COVID-19/imunologia , Proteínas do Envelope de Coronavírus/metabolismo , SARS-CoV-2/patogenicidade , Proteínas da Matriz Viral/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Antivirais/imunologia , COVID-19/sangue , COVID-19/virologia , Linhagem Celular , Proteínas do Envelope de Coronavírus/genética , Proteínas do Envelope de Coronavírus/imunologia , Proteínas do Envelope de Coronavírus/ultraestrutura , Cricetinae , Humanos , Microscopia Eletrônica de Transmissão , Mutação , Testes de Neutralização , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/imunologia , Proteínas da Matriz Viral/ultraestrutura , Vírion/genética , Vírion/imunologia , Vírion/metabolismo , Vírion/ultraestrutura
5.
BMC Gastroenterol ; 21(1): 334, 2021 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-34445965

RESUMO

BACKGROUND: SARS-CoV-2 may produce intestinal symptoms that are generally mild, with a small percentage of patients developing more severe symptoms. The involvement of SARS-CoV-2 in the physiopathology of bowel damage is poorly known. Transmission electron microscopy (TEM) is a useful tool that provides an understanding of SARS-CoV-2 invasiveness, replication and dissemination in body cells but information outside the respiratory tract is very limited. We report two cases of severe intestinal complications (intestinal lymphoma and ischaemic colitis) in which the presence of SARS-CoV-2 in intestinal tissue was confirmed by TEM. These are the first two cases reported in the literature of persistence of SARS-CoV-2 demonstrated by TEM in intestinal tissue after COVID 19 recovery and SARS-CoV-2 nasopharyngeal clearance. CASE PRESENTATION: During the first pandemic peak (1st March-30th April 2020) 932 patients were admitted in Hospital Universitari Mútua Terrassa due to COVID-19, 41 (4.4%) required cross-sectional imaging techniques to assess severe abdominal pain and six of them (0.64%) required surgical resection. SARS-CoV-2 in bowel tissue was demonstrated by TEM in two of these patients. The first case presented as an ileocaecal inflammatory mass which turned to be a B-cell lymphoma. Viral particles were found in the cytoplasm of endothelial cells of damaged mucosa. In situ hybridization was negative in tumour cells, thus ruling out an oncogenic role for the virus. SARS-CoV-2 remained in intestinal tissue 6 months after nasopharyngeal clearance, suggesting latent infection. The second patient had a severe ischaemic colitis with perforation and SARS-CoV-2 was also identified in endothelial cells. CONCLUSIONS: Severe intestinal complications associated with COVID-19 are uncommon. SARS-CoV-2 was identified by TEM in two cases, suggesting a causal role in bowel damage.


Assuntos
COVID-19 , SARS-CoV-2 , Dor Abdominal , Células Endoteliais , Humanos , Microscopia Eletrônica de Transmissão
6.
Sensors (Basel) ; 21(16)2021 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-34450748

RESUMO

TiO2 nanoparticles doped with different amounts of Nd3+ (0.5, 1, and 3 wt.%) were synthetized by the sol-gel method, and evaluated as potential temperature nanoprobes using the fluorescence intensity ratio between thermal-sensitive radiative transitions of the Nd3+. XRD characterization identified the anatase phase in all the doped samples. The morphology of the nanoparticles was observed with SEM, TEM and HRTEM microscopies. The relative amount of Nd3+ in TiO2 was obtained by EDXS, and the oxidation state of titanium and neodymium was investigated via XPS and NEXAFS, respectively. Nd3+ was present in all the samples, unlike titanium, where besides Ti4+, a significantly amount of Ti3+ was observed; the relative concentration of Ti3+ increased as the amount of Nd3+ in the TiO2 nanoparticles increased. The photoluminescence of the synthetized nanoparticles was investigated, with excitation wavelengths of 350, 514 and 600 nm. The emission intensity of the broad band that was associated with the presence of defects in the TiO2, increased when the concentration of Nd3+ was increased. Using 600 nm for excitation, the 4F7/2→4I9/2, 4F5/2→4I9/2 and 4F3/2→4I9/2 transitions of Nd3+ ions, centered at 760 nm, 821 nm, and 880 nm, respectively, were observed. Finally, the effect of temperature in the photoluminescence intensity of the synthetized nanoparticles was investigated, with an excitation wavelength of 600 nm. The spectra were collected in the 288-348 K range. For increasing temperatures, the emission intensity of the 4F7/2→4I9/2 and 4F5/2→4I9/2 transitions increased significantly, in contrast to the 4F3/2→4I9/2 transition, in which the intensity emission decreased. The fluorescence intensity ratio between the transitions I821I880=F5/24I49/2F43/2I49/2 and I760I880=F47/2I49/2F43/2I49/2 were used to calculate the relative sensitivity of the sensors. The relative sensitivity was near 3% K-1 for I760I880 and near 1% K-1 for I821I880.


Assuntos
Nanopartículas , Titânio , Microscopia Eletrônica de Transmissão , Temperatura
7.
Int J Mol Sci ; 22(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34360842

RESUMO

IBMPFD/ALS is a genetic disorder caused by a single amino acid mutation on the p97 ATPase, promoting ATPase activity and cofactor dysregulation. The disease mechanism underlying p97 ATPase malfunction remains unclear. To understand how the mutation alters the ATPase regulation, we assembled a full-length p97R155H with its p47 cofactor and first visualized their structures using single-particle cryo-EM. More than one-third of the population was the dodecameric form. Nucleotide presence dissociates the dodecamer into two hexamers for its highly elevated function. The N-domains of the p97R155H mutant all show up configurations in ADP- or ATPγS-bound states. Our functional and structural analyses showed that the p47 binding is likely to impact the p97R155H ATPase activities via changing the conformations of arginine fingers. These functional and structural analyses underline the ATPase dysregulation with the miscommunication between the functional modules of the p97R155H.


Assuntos
Demência Frontotemporal/metabolismo , Modelos Moleculares , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Mutação , Miosite de Corpos de Inclusão/metabolismo , Osteíte Deformante/metabolismo , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Proteína com Valosina/genética , Demência Frontotemporal/genética , Humanos , Microscopia Eletrônica de Transmissão , Distrofia Muscular do Cíngulo dos Membros/genética , Miosite de Corpos de Inclusão/genética , Osteíte Deformante/genética , Conformação Proteica , Proteína com Valosina/metabolismo
8.
Comput Methods Programs Biomed ; 209: 106318, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34375851

RESUMO

BACKGROUND AND OBJECTIVE: To achieve the full potential of deep learning (DL) models, such as understanding the interplay between model (size), training strategy, and amount of training data, researchers and developers need access to new dedicated image datasets; i.e., annotated collections of images representing real-world problems with all their variations, complexity, limitations, and noise. Here, we present, describe and make freely available an annotated transmission electron microscopy (TEM) image dataset. It constitutes an interesting challenge for many practical applications in virology and epidemiology; e.g., virus detection, segmentation, classification, and novelty detection. We also present benchmarking results for virus detection and recognition using some of the top-performing (large and small) networks as well as a handcrafted very small network. We compare and evaluate transfer learning and training from scratch hypothesizing that with a limited dataset, transfer learning is crucial for good performance of a large network whereas our handcrafted small network performs relatively well when training from scratch. This is one step towards understanding how much training data is needed for a given task. METHODS: The benchmark dataset contains 1245 images of 22 virus classes. We propose a representative data split into training, validation, and test sets for this dataset. Moreover, we compare different established DL networks and present a baseline DL solution for classifying a subset of the 14 most-represented virus classes in the dataset. RESULTS: Our best model, DenseNet201 pre-trained on ImageNet and fine-tuned on the training set, achieved a 0.921 F1-score and 93.1% accuracy on the proposed representative test set. CONCLUSIONS: Public and real biomedical datasets are an important contribution and a necessity to increase the understanding of shortcomings, requirements, and potential improvements for deep learning solutions on biomedical problems or deploying solutions in clinical settings. We compared transfer learning to learning from scratch on this dataset and hypothesize that for limited-sized datasets transfer learning is crucial for achieving good performance for large models. Last but not least, we demonstrate the importance of application knowledge in creating datasets for training DL models and analyzing their results.


Assuntos
Aprendizado Profundo , Redes Neurais de Computação , Benchmarking , Microscopia Eletrônica de Transmissão
9.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445504

RESUMO

Although previous studies continuously report an increased risk of hearing loss in diabetes patients, the impact of the disease on the inner ear remains unexplored. Herein, we examine the pathophysiology of diabetes-associated hearing impairment and cochlear synaptopathy in a mouse model of diabetes. Male B6.BKS(D)-Leprdb/J (db/db, diabetes) and heterozygote (db/+, control) mice were assigned into each experimental group (control vs. diabetes) based on the genotype and tested for hearing sensitivity every week from 6 weeks of age. Each cochlea was collected for histological and biological assays at 14 weeks of age. The diabetic mice exerted impaired hearing and a reduction in cochlear blood flow and C-terminal-binding protein 2 (CtBP2, a presynaptic ribbon marker) expression. Ultrastructural images revealed severely damaged mitochondria from diabetic cochlea accompanied by a reduction in Cytochrome c oxidase subunit 4 (COX4) and CR6-interacting factor 1 (CRIF1). The diabetic mice presented significantly decreased levels of platelet endothelial cell adhesion molecule (PECAM-1), B-cell lymphoma 2 (BCL-2), and procaspase-9, but not procaspase-8. Importantly, significant changes were not found in necroptotic programmed cell death markers (receptor-interacting serine/threonine-protein kinase 1, RIPK1; RIPK3; and mixed lineage kinase domain-like pseudokinase, MLKL) between the groups. Taken together, diabetic hearing loss is accompanied by synaptopathy, microangiopathy, damage to the mitochondrial structure/function, and activation of the intrinsic apoptosis pathway. Our results imply that mitochondrial dysfunction is deeply involved in diabetic hearing loss, and further suggests the potential benefits of therapeutic strategies targeting mitochondria.


Assuntos
Diabetes Mellitus Experimental/complicações , Perda Auditiva/fisiopatologia , Mitocôndrias/ultraestrutura , Receptores para Leptina/genética , Animais , Apoptose , Biomarcadores/metabolismo , Cóclea/irrigação sanguínea , Cóclea/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Modelos Animais de Doenças , Regulação para Baixo , Perda Auditiva/etiologia , Perda Auditiva/genética , Perda Auditiva/metabolismo , Humanos , Masculino , Camundongos , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo
10.
Int J Mol Sci ; 22(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34445338

RESUMO

Recently, another new cell type was found in the perivascular space called a novel desmin-immunopositive perivascular (DIP) cell. However, the differences between this novel cell type and other nonhormone-producing cells have not been clarified. Therefore, we introduced several microscopic techniques to gain insight into the morphological characteristics of this novel DIP cell. We succeeded in identifying novel DIP cells under light microscopy using desmin immunocryosection, combining resin embedding blocks and immunoelectron microscopy. In conventional transmission electron microscopy, folliculostellate cells, capsular fibroblasts, macrophages, and pericytes presented a flat cisternae of rough endoplasmic reticulum, whereas those of novel DIP cells had a dilated pattern. The number of novel DIP cells was greatest in the intact rats, though nearly disappeared under prolactinoma conditions. Additionally, focused ion beam scanning electron microscopy showed that these novel DIP cells had multidirectional processes and some processes reached the capillary, but these processes did not tightly wrap the vessel, as is the case with pericytes. Interestingly, we found that the rough endoplasmic reticulum was globular and dispersed throughout the cytoplasmic processes after three-dimensional reconstruction. This study clearly confirms that novel DIP cells are a new cell type in the rat anterior pituitary gland, with unique characteristics.


Assuntos
Desmina/metabolismo , Pericitos , Adeno-Hipófise/diagnóstico por imagem , Animais , Desmina/análise , Imuno-Histoquímica , Masculino , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Pericitos/citologia , Pericitos/metabolismo , Adeno-Hipófise/citologia , Adeno-Hipófise/metabolismo , Ratos , Ratos Wistar
11.
Int J Mol Sci ; 22(16)2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34445372

RESUMO

The synthesis of ester compounds is one of the most important chemical processes. In this work, Zn-Mg-Al mixed oxides with different Zn2+/Mg2+ molar ratios were prepared via co-precipitation method and supported gold nanoclusters to study the direct oxidative esterification of aldehyde and alcohol in the presence of molecular oxygen. Various characterization techniques such as N2-physical adsorption, X-ray diffraction (XRD), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and CO2 temperature programmed desorption (TPD) were utilized to analyze the structural and electronic properties. Based on the results, the presence of small amounts of Zn2+ ions (~5 wt.%) provoked a remarkable modification of the binary Mg-Al system, which enhanced the interaction between gold with the support and reduced the particle size of gold. For oxidative esterification reaction, the Au25/Zn0.05MgAl-400 catalyst showed the best performance, with the highest turnover frequency (TOF) of 1933 h-1. The active center was believed to be located at the interface between metallic gold with the support, where basic sites contribute a lot to transformation of the substrate.


Assuntos
Aldeídos/química , Hidróxido de Alumínio/química , Ouro/química , Hidróxido de Magnésio/química , Óxidos/química , Esterificação , Nanopartículas Metálicas , Microscopia Eletrônica de Transmissão , Oxirredução , Estresse Oxidativo , Tamanho da Partícula , Difração de Raios X , Zinco
12.
Int J Mol Sci ; 22(16)2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34445378

RESUMO

(1) Background: Several properties of silver nanoparticles (AgNPs), such as cytotoxic, anticancer, and antimicrobial activities, have been subjects of intense research; however, important aspects such as nanoparticle aggregation are generally neglected, although a decline in colloidal stability leads to a loss of the desired biological activities. Colloidal stability is affected by pH, ionic strength, or a plethora of biomolecules that interact with AgNPs under biorelevant conditions. (2) Methods: As only a few studies have focused on the relationship between aggregation behavior and the biological properties of AgNPs, here, we have systematically evaluated this issue by completing a thorough analysis of sterically (via polyvinyl-pyrrolidone (PVP)) stabilized AgNPs that were subjected to different circumstances. We assessed ultraviolet-visible light absorption, dynamic light scattering, zeta potential measurements, in vitro cell viability, and microdilution assays to screen both colloidal stability as well as bioactivity. (3) Results: The results revealed that although PVP provided outstanding biorelevant colloidal stability, the chemical stability of AgNPs could not be maintained completely with this capping material. (4) Conclusion: These unexpected findings led to the realization that stabilizing materials have more profound importance in association with biorelevant applications of nanomaterials than just being simple colloidal stabilizers.


Assuntos
Anti-Infecciosos/farmacologia , Antineoplásicos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Povidona/química , Prata/farmacologia , Anti-Infecciosos/química , Antineoplásicos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Difusão Dinâmica da Luz , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Nanopartículas Metálicas , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Transmissão , Prata/química
13.
Front Public Health ; 9: 699822, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34395371

RESUMO

The number of proton therapy facilities and the clinical usage of high energy proton beams for cancer treatment has substantially increased over the last decade. This is mainly due to the superior dose distribution of proton beams resulting in a reduction of side effects and a lower integral dose compared to conventional X-ray radiotherapy. More recently, the usage of metallic nanoparticles as radiosensitizers to enhance radiotherapy is receiving growing attention. While this strategy was originally intended for X-ray radiotherapy, there is currently a small number of experimental studies indicating promising results for proton therapy. However, most of these studies used low proton energies, which are less applicable to clinical practice; and very small gold nanoparticles (AuNPs). Therefore, this proof of principle study evaluates the radiosensitization effect of larger AuNPs in combination with a 200 MeV proton beam. CHO-K1 cells were exposed to a concentration of 10 µg/ml of 50 nm AuNPs for 4 hours before irradiation with a clinical proton beam at NRF iThemba LABS. AuNP internalization was confirmed by inductively coupled mass spectrometry and transmission electron microscopy, showing a random distribution of AuNPs throughout the cytoplasm of the cells and even some close localization to the nuclear membrane. The combined exposure to AuNPs and protons resulted in an increase in cell killing, which was 27.1% at 2 Gy and 43.8% at 6 Gy, compared to proton irradiation alone, illustrating the radiosensitizing potential of AuNPs. Additionally, cells were irradiated at different positions along the proton depth-dose curve to investigate the LET-dependence of AuNP radiosensitization. An increase in cytogenetic damage was observed at all depths for the combined treatment compared to protons alone, but no incremental increase with LET could be determined. In conclusion, this study confirms the potential of 50 nm AuNPs to increase the therapeutic efficacy of proton therapy.


Assuntos
Nanopartículas Metálicas , Terapia com Prótons , Radiossensibilizantes , Ouro , Humanos , Microscopia Eletrônica de Transmissão , Radiossensibilizantes/farmacologia
14.
Nat Commun ; 12(1): 4898, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385431

RESUMO

Hedgehog (Hh) signaling is essential during development and in organ physiology. In the canonical pathway, Hh binding to Patched (PTCH) relieves the inhibition of Smoothened (SMO). Yet, PTCH may also perform SMO-independent functions. While the PTCH homolog PTC-3 is essential in C. elegans, worms lack SMO, providing an excellent model to probe non-canonical PTCH function. Here, we show that PTC-3 is a cholesterol transporter. ptc-3(RNAi) leads to accumulation of intracellular cholesterol and defects in ER structure and lipid droplet formation. These phenotypes were accompanied by a reduction in acyl chain (FA) length and desaturation. ptc-3(RNAi)-induced lethality, fat content and ER morphology defects were rescued by reducing dietary cholesterol. We provide evidence that cholesterol accumulation modulates the function of nuclear hormone receptors such as of the PPARα homolog NHR-49 and NHR-181, and affects FA composition. Our data uncover a role for PTCH in organelle structure maintenance and fat metabolism.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Colesterol/metabolismo , Homeostase/genética , Metabolismo dos Lipídeos/genética , Receptor Patched-1/genética , Animais , Western Blotting , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/ultraestrutura , Proteínas de Caenorhabditis elegans/metabolismo , Regulação da Expressão Gênica , Microscopia Eletrônica de Transmissão , Receptor Patched-1/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
Nat Commun ; 12(1): 4883, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385449

RESUMO

Pure organic room-temperature phosphorescent (RTP) materials have been suggested to be promising bioimaging materials due to their good biocompatibility and long emission lifetime. Herein, we report a class of RTP materials. These materials are developed through the simple introduction of an aromatic carbonyl to a tetraphenylpyrrole molecule and also exhibit aggregation-induced emission (AIE) properties. These molecules show non-emission in solution and purely phosphorescent emission in the aggregated state, which are desirable properties for biological imaging. Highly crystalline nanoparticles can be easily fabricated with a long emission lifetime (20 µs), which eliminate background fluorescence interference from cells and tissues. The prepared nanoparticles demonstrate two-photon absorption characteristics and can be excited by near infrared (NIR) light, making them promising materials for deep-tissue optical imaging. This integrated aggregation-induced phosphorescence (AIP) strategy diversifies the existing pool of bioimaging agents to inspire the development of bioprobes in the future.


Assuntos
Corantes Fluorescentes/química , Luminescência , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Nanopartículas/química , Pirróis/química , Imagem com Lapso de Tempo/métodos , Animais , Células HeLa , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia Confocal/métodos , Microscopia Eletrônica de Transmissão/métodos , Nanopartículas/ultraestrutura , Tamanho da Partícula
16.
Photodiagnosis Photodyn Ther ; 35: 102463, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34325078

RESUMO

The novel approach for imaging of antimicrobial photodynamic therapy processes presented in this work is based on transmission electron microscopy methods. With the use of liquid cell, illumination system, and lowered electron dose the successful light-induced in-situ observations on Staphylococcus aureus encapsulated with methylene blue were performed. Results showed that with specified imaging parameters it is possible to conduct reliable research on bacteria in electron microscope despite the unfavorable damaging effect of the highly energetic electron beam used for imaging. This approach differs from the common methods, as it provides direct observations of the processes occurring upon light illumination. The effects obtained with the proposed method are very promising and may serve to answer why different microorganisms respond to the therapy differently.


Assuntos
Anti-Infecciosos , Fotoquimioterapia , Azul de Metileno/farmacologia , Azul de Metileno/uso terapêutico , Microscopia Eletrônica de Transmissão , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico
17.
Int J Mol Sci ; 22(14)2021 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-34298998

RESUMO

The purpose of the present study was to investigate molecular compositions of lipid droplets changing in live hepatic cells stimulated with major fatty acids in the human body, i.e., palmitic, stearic, oleic, and linoleic acids. HepG2 cells were used as the model hepatic cells. Morphological changes of lipid droplets were observed by optical microscopy and transmission electron microscopy (TEM) during co-cultivation with fatty acids up to 5 days. The compositional changes in the fatty chains included in the lipid droplets were analyzed via Raman spectroscopy and chemometrics. The growth curves of the cells indicated that palmitic, stearic, and linoleic acids induced cell death in HepG2 cells, but oleic acid did not. Microscopic observations suggested that the rates of fat accumulation were high for oleic and linoleic acids, but low for palmitic and stearic acids. Raman analysis indicated that linoleic fatty chains taken into the cells are modified into oleic fatty chains. These results suggest that the signaling pathway of cell death is independent of fat stimulations. Moreover, these results suggest that hepatic cells have a high affinity for linoleic acid, but linoleic acid induces cell death in these cells. This may be one of the causes of inflammation in nonalcoholic fatty liver disease (NAFLD).


Assuntos
Morte Celular/efeitos dos fármacos , Meios de Cultura/química , Ácidos Graxos/efeitos adversos , Hepatócitos/metabolismo , Gotículas Lipídicas/química , Análise Espectral Raman , Ácidos Graxos/metabolismo , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Humanos , Ácido Linoleico/farmacologia , Ácido Linoleico/toxicidade , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Microscopia Eletrônica de Transmissão , Ácido Oleico/farmacologia , Ácido Palmítico/farmacologia , Ácido Palmítico/toxicidade , Transdução de Sinais/efeitos dos fármacos , Ácidos Esteáricos/farmacologia , Ácidos Esteáricos/toxicidade
18.
Int J Mol Sci ; 22(14)2021 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-34299003

RESUMO

Here, we designed paper sheets coated with chitosan, bacterial cellulose (nanofibers), and ZnO with boosted antibacterial and mechanical activity. We investigated the compositions, with ZnO exhibiting two different sizes/shapes: (1) rods and (2) irregular sphere-like particles. The proposed processing of bacterial cellulose resulted in the formation of nanofibers. Antimicrobial behavior was tested using E. coli ATCC® 25922™ following the ASTM E2149-13a standard. The mechanical properties of the paper sheets were measured by comparing tearing resistance, tensile strength, and bursting strength according to the ISO 5270 standard. The results showed an increased antibacterial response (assigned to the combination of chitosan and ZnO, independent of its shape and size) and boosted mechanical properties. Therefore, the proposed composition is an interesting multifunctional mixture for coatings in food packaging applications.


Assuntos
Biopolímeros/química , Biopolímeros/farmacologia , Celulose/química , Quitosana/química , Nanocompostos/química , Embalagem de Produtos/métodos , Óxido de Zinco/química , Anti-Infecciosos , Celulose/ultraestrutura , Escherichia coli , Testes Mecânicos , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Nanocompostos/ultraestrutura , Nanofibras/química , Nanofibras/ultraestrutura , Propriedades de Superfície , Resistência à Tração , Difração de Raios X
19.
J Immunol ; 207(3): 888-901, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34290105

RESUMO

Because most of animal viruses are enveloped, cytoplasmic entry of these viruses via fusion with cellular membrane initiates their invasion. However, the strategies in which host cells counteract cytoplasmic entry of such viruses are incompletely understood. Pore-forming toxin aerolysin-like proteins (ALPs) exist throughout the animal kingdom, but their functions are mostly unknown. In this study, we report that ßγ-crystallin fused aerolysin-like protein and trefoil factor complex (ßγ-CAT), an ALP and trefoil factor complex from the frog Bombina maxima, directly blocks enveloped virus invasion by interfering with cytoplasmic entry. ßγ-CAT targeted acidic glycosphingolipids on the HSV type 1 (HSV-1) envelope to induce pore formation, as indicated by the oligomer formation of protein and potassium and calcium ion efflux. Meanwhile, ßγ-CAT formed ring-like oligomers of ∼10 nm in diameter on the liposomes and induced dye release from liposomes that mimic viral envelope. Unexpectedly, transmission electron microscopy analysis showed that the ßγ-CAT-treated HSV-1 was visibly as intact as the vehicle-treated HSV-1, indicating that ßγ-CAT did not lyse the viral envelope. However, the cytoplasmic entry of the ßγ-CAT-treated HSV-1 into HeLa cells was totally hindered. In vivo, topical application of ßγ-CAT attenuated the HSV-1 corneal infection in mice. Collectively, these results uncovered that ßγ-CAT possesses the capacity to counteract enveloped virus invasion with its featured antiviral-acting manner. Our findings will also largely help to illustrate the putative antiviral activity of animal ALPs.


Assuntos
Proteínas de Anfíbios/metabolismo , Antivirais/metabolismo , Córnea/patologia , Herpes Simples/imunologia , Herpesvirus Humano 1/fisiologia , Complexos Multiproteicos/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Fatores Trefoil/metabolismo , Proteínas de Anfíbios/genética , Animais , Anuros , Toxinas Bacterianas/genética , Córnea/virologia , Feminino , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Microscopia Eletrônica de Transmissão , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Envelope Viral/metabolismo , Envelope Viral/ultraestrutura , Internalização do Vírus , gama-Cristalinas/química
20.
Anal Chem ; 93(34): 11859-11867, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34319694

RESUMO

Counterfeits in the supply chain of high-value advanced materials such as graphene and their derivatives have become a concerning problem with a potential negative impact on this growing and emerging industry. Recent studies have revealed alarming facts that a large percentage of manufactured graphene materials on market are not graphene, raising considerable concerns for the end users. The common and recommended methods for the characterization of graphene materials, such as transmission electron microscopy (TEM), atomic force microscopy (AFM), and Raman spectroscopy based on spot analysis and probing properties of individual graphene particles, are limited to provide the determination of the properties of "bulk" graphene powders at a large scale and the identification of non-graphene components or purposely included additives. These limitations are creating counterfeit opportunities by adding low-cost black carbonaceous materials into manufactured graphene powders. To address this problem, it is critical to have reliable characterization methods, which can probe the specific properties of graphene powders at bulk scale, confirm their typical graphene signature, and detect the presence of unwanted additional compounds, where the thermogravimetric analysis (TGA) method is one of the most promising methods to perform this challenging task. This paper presents the evaluation of the TGA method and its ability to detect low-cost carbon additives such as graphite, carbon black, biochar, and activated carbon as potential counterfeiting materials to graphene materials and their derivatives such as graphene oxide (GO) and reduced GO. The superior performance of the TGA method is demonstrated here, showing its excellent capability to successfully detect these additives when mixed with graphene materials, which is not possible by two other comparative methods (Raman spectroscopy and powder X-ray diffraction (XRD)), which are used as the common characterization methods for graphene materials.


Assuntos
Grafite , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Análise Espectral Raman , Difração de Raios X
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