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1.
Urologiia ; (4): 68-72, 2021 Sep.
Artigo em Russo | MEDLINE | ID: mdl-34486277

RESUMO

INTRODUCTION: Chronic recurrent bacterial prostatitis (CRPD) is an urgent problem of modern urology and andrology. OBJECTIVE: To study the immunological features of the secretion of the prostate by electron microscopy in patients with chronic recurrent bacterial prostatitis. MATERIALS AND METHODS: The analysis of the morphometric study of neutrophils in the secretion of the prostate was carried out in 90 patients with chronic bacterial prostatitis, who were divided into two groups. Group I (study) (n=51) with chronic recurrent bacterial prostatitis (CRBP) and group II (control) (n=39) with chronic primary diagnosed bacterial prostatitis (CPDBP). RESULTS: At electron microscopy of ALE in most patients with CRBP of the group, the cytological picture of ALE was represented by inactive neutrophils with pathology of phagocytosis. The cells are of the correct rounded shape, without pseudopodia, with light cytoplasm. The cytological picture of ALE in CPDBP is characterized by the absence of impaired local immunity. The normal process of phagocytosis is recorded, where many pseudopodia of the segmented neutrophil completely complete the process of opsonization of microorganisms in the area of the inflammatory process. CONCLUSIONS: In patients with CRBP, in 100% of cases, dysfunction of immunocompetent prostate cells was noted, which is the basis for the appointment of immunoactive therapy for CRBP.


Assuntos
Prostatite , Doença Crônica , Eletrônica , Humanos , Masculino , Microscopia Eletrônica
2.
Nature ; 596(7871): 257-261, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34349261

RESUMO

An animal's nervous system changes as its body grows from birth to adulthood and its behaviours mature1-8. The form and extent of circuit remodelling across the connectome is unknown3,9-15. Here we used serial-section electron microscopy to reconstruct the full brain of eight isogenic Caenorhabditis elegans individuals across postnatal stages to investigate how it changes with age. The overall geometry of the brain is preserved from birth to adulthood, but substantial changes in chemical synaptic connectivity emerge on this consistent scaffold. Comparing connectomes between individuals reveals substantial differences in connectivity that make each brain partly unique. Comparing connectomes across maturation reveals consistent wiring changes between different neurons. These changes alter the strength of existing connections and create new connections. Collective changes in the network alter information processing. During development, the central decision-making circuitry is maintained, whereas sensory and motor pathways substantially remodel. With age, the brain becomes progressively more feedforward and discernibly modular. Thus developmental connectomics reveals principles that underlie brain maturation.


Assuntos
Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Caenorhabditis elegans/citologia , Conectoma , Modelos Neurológicos , Vias Neurais , Sinapses/fisiologia , Envelhecimento/metabolismo , Animais , Encéfalo/anatomia & histologia , Encéfalo/ultraestrutura , Caenorhabditis elegans/anatomia & histologia , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/ultraestrutura , Individualidade , Interneurônios/citologia , Microscopia Eletrônica , Neurônios/citologia , Comportamento Estereotipado
3.
Nano Lett ; 21(16): 6882-6890, 2021 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-34387492

RESUMO

Through its ability to image liquid-phase dynamics at nano/atomic-scale resolution, liquid-cell electron microscopy is essential for a wide range of applications, including wet-chemical synthesis, catalysis, and nanoparticle tracking, for which involved structural features are critical. However, statistical investigations by usual techniques remain challenging because of the difficulty in fabricating substantial liquid cells with appreciable efficiency. Here, we report a general approach for efficiently printing huge numbers of ready-to-use liquid cells (∼9000) within 30 s by electrospinning, with the unique feature of statistical liquid-phase studies requiring only one experimental time slot. Our solution efficiently resolves a complete transition picture of bubble evolution and also the induced nanoparticle motion. We statistically quantify the effect of the electron dose rate on the bubble variation and conclude that the bubble-driven nanoparticle motion is a ballistic-like behavior insignificant to morphological asymmetries. The versatile approach here is critical for statistical research, offering great opportunities in liquid-phase-associated dynamic studies.


Assuntos
Nanopartículas , Catálise , Microscopia Eletrônica , Movimento (Física) , Impressão Tridimensional
4.
Nano Lett ; 21(17): 7332-7338, 2021 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-34406014

RESUMO

Step edges are an important and prevalent topological feature that influence catalytic, electronic, vibrational, and structural properties arising from modulation of atomic-scale force fields due to edge-atom relaxation. Direct probing of ultrafast atomic-to-nanoscale lattice dynamics at individual steps poses a particularly significant challenge owing to demanding spatiotemporal resolution requirements. Here, we achieve such resolutions with femtosecond 4D ultrafast electron microscopy and directly image nanometer-variant softening of photoexcited phonons at individual surface steps. We find large degrees of softening precisely at the step position, with a thickness-dependent, strain-induced frequency modulation extending tens of nanometers laterally from the atomic-scale discontinuity. The effect originates from anisotropic bond dilation and photoinduced incoherent atomic displacements delineated by abrupt molecular-layer cessation. The magnitude and spatiotemporal extent of softening is quantitatively described with a finite-element transient-deformation model. The high spatiotemporal resolutions demonstrated here enable uncovering of new insights into atomic-scale structure-function relationships of highly defect-sensitive, functional materials.


Assuntos
Fônons , Microscopia Eletrônica
5.
Neuroimage Clin ; 31: 102735, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34247117

RESUMO

Diffuse low-grade gliomas (DLGG) display different preferential locations in eloquent and secondary associative brain areas. The reason for this tendency is still unknown. We hypothesized that the intrinsic architecture and water diffusion properties of the white matter bundles in these regions may facilitate gliomas infiltration. Magnetic resonance imaging of sixty-seven diffuse low-grade gliomas patients were normalized to/and segmented in MNI space to create three probabilistic infiltration weighted gradient maps according to the molecular status of each tumor group (IDH mutated, IDH wild-type and IDH mutated/1p19q co-deleted). Diffusion tensor imaging (DTI)- based parameters were derived for five major white matter bundles, displaying regional differences in the grade of infiltration, averaged over 20 healthy individuals acquired from the Human connectome project (HCP) database. Transmission electron microscopy (TEM) was used to analyze fiber density, fiber diameter and g-ratio in 100 human white matter regions, sampled from cadaver specimens, reflecting areas with different gliomas infiltration in each white matter bundle. Histological results and DTI-based parameters were compared in anatomical regions of high- and low grade of infiltration (HIF and LIF) respectively. We detected differences in the white matter infiltration of five major white matter bundles in three groups. Astrocytomas IDHm infiltrated left fronto-temporal subcortical areas. Astrocytomas IDHwt were detected in the posterior-temporal and temporo-parietal regions bilaterally. Oligodendrogliomas IDHm/1p19q infiltrated anterior subcortical regions of the frontal lobes bilaterally. Regional differences within the same white matter bundles were detected by both TEM- and DTI analysis linked to different topographical variables. Our multimodal analysis showed that HIF regions, common to all the groups, displayed a smaller fiber diameter, lower FA and higher RD compared with LIF regions. Our results suggest that the both morphological features and diffusion parameters of the white matter may be different in regions linked to the preferential location of DLGG.


Assuntos
Neoplasias Encefálicas , Glioma , Substância Branca , Neoplasias Encefálicas/diagnóstico por imagem , Imagem de Tensor de Difusão , Glioma/diagnóstico por imagem , Humanos , Microscopia Eletrônica , Substância Branca/diagnóstico por imagem
6.
Int J Mol Sci ; 22(14)2021 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-34299263

RESUMO

BACKGROUND: Charged-particle radiotherapy is an emerging treatment modality for radioresistant tumors. The enhanced effectiveness of high-energy particles (such as heavy ions) has been related to the spatial clustering of DNA lesions due to highly localized energy deposition. Here, DNA damage patterns induced by single and multiple carbon ions were analyzed in the nuclear chromatin environment by different high-resolution microscopy approaches. MATERIAL AND METHODS: Using the heavy-ion microbeam SNAKE, fibroblast monolayers were irradiated with defined numbers of carbon ions (1/10/100 ions per pulse, ipp) focused to micrometer-sized stripes or spots. Radiation-induced lesions were visualized as DNA damage foci (γH2AX, 53BP1) by conventional fluorescence and stimulated emission depletion (STED) microscopy. At micro- and nanoscale level, DNA double-strand breaks (DSBs) were visualized within their chromatin context by labeling the Ku heterodimer. Single and clustered pKu70-labeled DSBs were quantified in euchromatic and heterochromatic regions at 0.1 h, 5 h and 24 h post-IR by transmission electron microscopy (TEM). RESULTS: Increasing numbers of carbon ions per beam spot enhanced spatial clustering of DNA lesions and increased damage complexity with two or more DSBs in close proximity. This effect was detectable in euchromatin, but was much more pronounced in heterochromatin. Analyzing the dynamics of damage processing, our findings indicate that euchromatic DSBs were processed efficiently and repaired in a timely manner. In heterochromatin, by contrast, the number of clustered DSBs continuously increased further over the first hours following IR exposure, indicating the challenging task for the cell to process highly clustered DSBs appropriately. CONCLUSION: Increasing numbers of carbon ions applied to sub-nuclear chromatin regions enhanced the spatial clustering of DSBs and increased damage complexity, this being more pronounced in heterochromatic regions. Inefficient processing of clustered DSBs may explain the enhanced therapeutic efficacy of particle-based radiotherapy in cancer treatment.


Assuntos
Quebras de DNA de Cadeia Dupla/efeitos da radiação , DNA/efeitos da radiação , Radioterapia com Íons Pesados/efeitos adversos , Técnicas de Cultura de Células , Análise por Conglomerados , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Eucromatina/genética , Eucromatina/efeitos da radiação , Fibroblastos , Radioterapia com Íons Pesados/métodos , Íons Pesados/efeitos adversos , Heterocromatina/genética , Heterocromatina/efeitos da radiação , Humanos , Autoantígeno Ku/genética , Autoantígeno Ku/efeitos da radiação , Transferência Linear de Energia/efeitos da radiação , Microscopia Eletrônica/métodos , Radiação Ionizante
7.
eNeuro ; 8(4)2021.
Artigo em Inglês | MEDLINE | ID: mdl-34193510

RESUMO

In the central and peripheral nervous systems, the myelin sheath promotes neuronal signal transduction. The thickness of the myelin sheath changes during development and in disease conditions like multiple sclerosis. Such changes are routinely detected using electron microscopy through g-ratio quantification. While g-ratio is one of the most critical measurements in myelin studies, a major drawback is that g-ratio quantification is extremely laborious and time-consuming. Here, we report the development and validation of MyelTracer, an installable, stand-alone software for semi-automated g-ratio quantification based on the Open Computer Vision Library (OpenCV). Compared with manual g-ratio quantification, using MyelTracer produces consistent results across multiple tissues and animal ages, as well as in remyelination after optic nerve crush, and reduces total quantification time by 40-60%. With g-ratio measurements via MyelTracer, a known hypomyelination phenotype can be detected in a Williams syndrome mouse model. MyelTracer is easy to use and freely available for Windows and Mac OS X (https://github.com/HarrisonAllen/MyelTracer).


Assuntos
Bainha de Mielina , Software , Animais , Axônios , Modelos Animais de Doenças , Camundongos , Microscopia Eletrônica , Nervo Óptico
8.
Nano Lett ; 21(15): 6684-6689, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34283612

RESUMO

Phase-separation is commonly observed in multimetallic nanomaterials, yet it is not well understood how immiscible elements distribute in a thermodynamically stable nanoparticle. Herein, we studied the phase-separation of Au and Rh in nanoparticles using electron microscopy and tomography techniques. The nanoparticles were thermally annealed to form thermodynamically stable structures. HAADF-STEM and EDS characterizations reveal that Au and Rh segregate into two domains while their miscibility is increased. Using aberration-corrected HAADF-STEM and atomic electron tomography, we show that the increased solubility of Au in Rh is achieved by forming Au clusters and single atoms inside the Rh domains and on the Rh surface. Furthermore, based on the three-dimensional reconstruction of a AuRh nanoparticle, we can visualize the uneven interface that is embedded in the nanoparticle. The results advance our understanding on the nanoscale thermodynamic behavior of metal mixtures, which is crucial for the optimization of multimetallic nanostructures for many applications.


Assuntos
Nanopartículas , Nanoestruturas , Microscopia Eletrônica , Solubilidade , Termodinâmica
9.
Int J Mol Sci ; 22(14)2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34298979

RESUMO

Platelet extravasation during inflammation is under-appreciated. In wild-type (WT) mice, a central corneal epithelial abrasion initiates neutrophil (PMN) and platelet extravasation from peripheral limbal venules. The same injury in mice expressing low levels of the ß2-integrin, CD18 (CD18hypo mice) shows reduced platelet extravasation with PMN extravasation apparently unaffected. To better define the role of CD18 on platelet extravasation, we focused on two relevant cell types expressing CD18: PMNs and mast cells. Following corneal abrasion in WT mice, we observed not only extravasated PMNs and platelets but also extravasated erythrocytes (RBCs). Ultrastructural observations of engorged limbal venules showed platelets and RBCs passing through endothelial pores. In contrast, injured CD18hypo mice showed significantly less venule engorgement and markedly reduced platelet and RBC extravasation; mast cell degranulation was also reduced compared to WT mice. Corneal abrasion in mast cell-deficient (KitW-sh/W-sh) mice showed less venule engorgement, delayed PMN extravasation, reduced platelet and RBC extravasation and delayed wound healing compared to WT mice. Finally, antibody-induced depletion of circulating PMNs prior to corneal abrasion reduced mast cell degranulation, venule engorgement, and extravasation of PMNs, platelets, and RBCs. In summary, in the injured cornea, platelet and RBC extravasation depends on CD18, PMNs, and mast cell degranulation.


Assuntos
Plaquetas/fisiologia , Antígenos CD18/fisiologia , Degranulação Celular , Córnea/irrigação sanguínea , Eritrócitos/fisiologia , Hiperemia/fisiopatologia , Mastócitos/fisiologia , Neutrófilos/fisiologia , Migração Transendotelial e Transepitelial/fisiologia , Vasculite/imunologia , Vênulas/metabolismo , Animais , Antígenos CD18/deficiência , Movimento Celular , Quimiotaxia de Leucócito , Lesões da Córnea/metabolismo , Lesões da Córnea/patologia , Epitélio Corneano/fisiologia , Feminino , Hiperemia/sangue , Macrófagos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microcirculação , Microscopia Eletrônica , Modelos Animais , Fagocitose , Regeneração/fisiologia , Vasculite/sangue , Vênulas/patologia , Cicatrização/fisiologia
10.
Nat Commun ; 12(1): 4399, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285221

RESUMO

The decline of neuronal synapses is an established feature of ageing accompanied by the diminishment of neuronal function, and in the motor system at least, a reduction of behavioural capacity. Here, we have investigated Drosophila motor neuron synaptic terminals during ageing. We observed cumulative fragmentation of presynaptic structures accompanied by diminishment of both evoked and miniature neurotransmission occurring in tandem with reduced motor ability. Through discrete manipulation of each neurotransmission modality, we find that miniature but not evoked neurotransmission is required to maintain presynaptic architecture and that increasing miniature events can both preserve synaptic structures and prolong motor ability during ageing. Our results establish that miniature neurotransmission, formerly viewed as an epiphenomenon, is necessary for the long-term stability of synaptic connections.


Assuntos
Envelhecimento/fisiologia , Neurônios Motores/fisiologia , Terminações Pré-Sinápticas/fisiologia , Transmissão Sináptica/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Potencial Evocado Motor/fisiologia , Masculino , Microscopia Eletrônica , Modelos Animais , Neurônios Motores/ultraestrutura , Músculos/inervação , Músculos/fisiologia , Músculos/ultraestrutura , Terminações Pré-Sinápticas/ultraestrutura , Fatores de Tempo
11.
Methods Mol Biol ; 2314: 183-203, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235653

RESUMO

The Dictyostelium discoideum-Mycobacterium marinum host-pathogen system is a well-established and powerful alternative model system to study mycobacterial infections. In this chapter, we will describe three microscopy methods that allow the precise identification and quantification of very diverse phenotypes arising during infection of D. discoideum with M. marinum. First, at the lowest end of the scale, we use the InfectChip, a microfluidic device that enables the long-term monitoring of the integrated history of the infection course at the single-cell level. We use single-cell analysis to precisely map and quantitate the various fates of the host and the pathogen during infection. Second, a high-content microscopy setup was established to study the infection dynamics with high-throughput imaging of a large number of cells at the different critical stages of infection. The large datasets are then fed into a deep image analysis pipeline allowing the development of complex phenotypic analyses. Finally, as part of its life cycle, single D. discoideum amoebae aggregate by chemotaxis to form multicellular structures, which represent ordered assemblies of hundreds of thousands of cells. This transition represents a challenge for the monitoring of infection at multiple scales, from single cells to a true multicellular organism. In order to visualize and quantitate the fates of host cells and bacteria during the developmental cycle in a controlled manner, we can adjust the proportion of infected cells using live FAC-sorting. Then, cells are plated in defined humidity conditions on optical glass plates in order to image large fields, using tile scans, with the help of a spinning disc confocal microscope.


Assuntos
Dictyostelium/microbiologia , Interações Hospedeiro-Patógeno , Dispositivos Lab-On-A-Chip , Microscopia Eletrônica/métodos , Infecções por Mycobacterium não Tuberculosas/diagnóstico , Mycobacterium marinum/crescimento & desenvolvimento , Análise de Célula Única/métodos , Dictyostelium/ultraestrutura , Infecções por Mycobacterium não Tuberculosas/microbiologia
12.
BMC Plant Biol ; 21(1): 258, 2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34134628

RESUMO

BACKGROUND: Dionysia tapetodes, a small cushion-forming mountainous evergreen in the Primulaceae, possesses a vast surface-covering of long silky fibres forming the characteristic "woolly" farina. This contrasts with some related Primula which instead form a fine powder. Farina is formed by specialized cellular factories, a type of glandular trichome, but the precise composition of the fibres and how it exits the cell is poorly understood. Here, using a combination of cell biology (electron and light microscopy) and analytical chemical techniques, we present the principal chemical components of the wool and its mechanism of exit from the glandular trichome. RESULTS: We show the woolly farina consists of micron-diameter fibres formed from a mixture of flavone and substituted flavone derivatives. This contrasts with the powdery farina, consisting almost entirely of flavone. The woolly farina in D. tapetodes is extruded through specific sites at the surface of the trichome's glandular head cell, characterised by a small complete gap in the plasma membrane, cell wall and cuticle and forming a tight seal between the fibre and hole. The data is consistent with formation and thread elongation occurring from within the cell. CONCLUSIONS: Our results suggest the composition of the D. tapetodes farina dictates its formation as wool rather than powder, consistent with a model of thread integrity relying on intermolecular H-bonding. Glandular trichomes produce multiple wool fibres by concentrating and maintaining their extrusion at specific sites at the cell cortex of the head cell. As the wool is extensive across the plant, there may be associated selection pressures attributed to living at high altitudes.


Assuntos
Flavonas/análise , Primulaceae/ultraestrutura , Tricomas/ultraestrutura , Microscopia , Microscopia Eletrônica , Primulaceae/química
13.
Wiad Lek ; 74(6): 1297-1301, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34159907

RESUMO

OBJECTIVE: The aim: The paper was aimed at the study of the processes of mineralization of the enamel of the permanent tooth after its eruption. PATIENTS AND METHODS: Materials and methods: To study the structure of the enamel of permanent teeth has been carried out using light and electron microscopy. The study of the process of the development of the primordia of the permanent teeth involved 10 culled puppies of 30-40 days of age. Microscopic, electron microscopic, immunohistochemical methods of research have been used to study the processes of histogenesis. RESULTS: Results: The studies show that in the postnatal period, the formation of the crown, externally covered with cuticular epithelium, marks the formation of the primordium of the permanent tooth at the follicle stage. After eruption of a tooth, different parts of its crown have three individual structural and functional barriers to enamel biomineralization. The first one is provided by the cuticular epithelium of the pitted areas of the crown, which ensures filtering of the salivary fluid from the protein deposit in the form of a pellicle. The second barrier is defined on the lateral and cuspidate surfaces of the enamel, where the cuticle is erased or poorly expressed. The third structural and functional barrier of enamel biomineralization is located in the cervical portion of teeth of different classes. CONCLUSION: Conclusions: Different areas of the enamel in the tooth crown have specific filtration barriers, which can be distinguished as follows: pit-and-fissure-and-groove, cuspidateand-approximal, and cervical barriers. The cuticle is poorly expressed or totally absent on the cusps of the tooth crowns in contrast to pitted areas.


Assuntos
Erupção Dentária , Dente , Animais , Coroas , Esmalte Dentário , Cães , Microscopia Eletrônica
14.
Int J Mol Sci ; 22(11)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071337

RESUMO

Cellulose nanofibers (CNF) isolated from plant biomass have attracted considerable interests in polymer engineering. The limitations associated with CNF-based nanocomposites are often linked to the time-consuming preparation methods and lack of desired surface functionalities. Herein, we demonstrate the feasibility of preparing a multifunctional CNF-zinc oxide (CNF-ZnO) nanocomposite with dual antibacterial and reinforcing properties via a facile and efficient ultrasound route. We characterized and examined the antibacterial and mechanical reinforcement performances of our ultrasonically induced nanocomposite. Based on our electron microscopy analyses, the ZnO deposited onto the nanofibrous network had a flake-like morphology with particle sizes ranging between 21 to 34 nm. pH levels between 8-10 led to the formation of ultrafine ZnO particles with a uniform size distribution. The resultant CNF-ZnO composite showed improved thermal stability compared to pure CNF. The composite showed potent inhibitory activities against Gram-positive (methicillin-resistant Staphylococcus aureus (MRSA)) and Gram-negative Salmonella typhi (S. typhi) bacteria. A CNF-ZnO-reinforced natural rubber (NR/CNF-ZnO) composite film, which was produced via latex mixing and casting methods, exhibited up to 42% improvement in tensile strength compared with the neat NR. The findings of this study suggest that ultrasonically-synthesized palm CNF-ZnO nanocomposites could find potential applications in the biomedical field and in the development of high strength rubber composites.


Assuntos
Antibacterianos/química , Arecaceae/química , Celulose/química , Nanocompostos/química , Nanofibras/química , Óxido de Zinco/química , Antibacterianos/síntese química , Antibacterianos/farmacologia , Estabilidade de Medicamentos , Concentração de Íons de Hidrogênio , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Microscopia Eletrônica , Nanocompostos/ultraestrutura , Nanofibras/ultraestrutura , Tamanho da Partícula , Borracha/química , Salmonella/efeitos dos fármacos , Salmonella/crescimento & desenvolvimento , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura , Difração de Raios X
15.
Methods Mol Biol ; 2276: 31-39, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060030

RESUMO

As the powerhouse of the cell, mitochondria, plays a crucial role in many aspects of life, whereby mitochondrial dysfunctions are associated with pathogenesis of many diseases, like neurodegenerative diseases, obesity, cancer, and metabolic as well as cardiovascular disorders. Mitochondria analysis frequently starts with isolation and enrichment procedures, which have become increasingly important in biomedical research. Unfortunately, isolation procedures can easily cause changes in the structural integrity of mitochondria during in vitro handling having impact on their function. This carries the risk that conclusions about isolated mitochondria may be drawn on the basis of experimental artifacts. Here we critically review a commonly used isolation procedure for mitochondria utilizing differential (gradient) centrifugation and depict major challenges to achieve "functional" mitochondria as basis for comprehensive physiological studies.


Assuntos
Fracionamento Celular/métodos , Centrifugação/métodos , Microscopia Eletrônica/métodos , Mitocôndrias/metabolismo , Animais , Artefatos , Humanos
16.
Methods Mol Biol ; 2277: 277-287, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080157

RESUMO

Isolation of mitochondria is a crucial method for examining molecular details of this organelle's manifold functions. Historically, mitochondrial isolations required large amounts of sample material which impeded their isolation from cultured cells. We have therefore developed a method allowing for controlled and reproducible isolation of intact and functional mitochondria from diverse cell types in culture. Here we provide a methodological update of this approach together with a protocol for the subsequent analysis of such isolated mitochondria by electron microscopy. Combining the isolation procedure with this powerful imaging method can reveal ultrastructural mitochondrial peculiarities in disease settings that might not be evident in intact cells and allows for assessment of mitochondrial membrane integrity and sample purity.


Assuntos
Neoplasias Hepáticas/patologia , Microscopia Eletrônica/métodos , Mitocôndrias Hepáticas/patologia , Linhagem Celular Tumoral , Separação Celular/instrumentação , Separação Celular/métodos , Desenho de Equipamento , Células Hep G2 , Humanos , Processamento de Imagem Assistida por Computador/métodos
17.
Nano Lett ; 21(13): 5842-5849, 2021 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-34153185

RESUMO

Hybrids of graphene and metal plasmonic nanostructures are promising building blocks for applications in optoelectronics, surface-enhanced scattering, biosensing, and quantum information. An understanding of the coupling mechanism in these hybrid systems is of vital importance to its applications. Previous efforts in this field mainly focused on spectroscopic studies of strong coupling within the hybrids with no spatial resolution. Here we report direct imaging of the local plasmonic coupling between single Au nanocapsules and graphene step edges at the nanometer scale by photon-induced near-field electron microscopy in an ultrafast electron microscope for the first time. The proximity of a step in the graphene to the nanocapsule causes asymmetric surface charge density at the ends of the nanocapsules. Computational electromagnetic simulations confirm the experimental observations. The results reported here indicate that this hybrid system could be used to manipulate the localized electromagnetic field on the nanoscale, enabling promising future plasmonic devices.


Assuntos
Grafite , Nanoestruturas , Microscopia de Força Atômica , Microscopia Eletrônica , Nanotecnologia
18.
Nat Methods ; 18(7): 771-774, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34168373

RESUMO

We develop an automatic method for synaptic partner identification in insect brains and use it to predict synaptic partners in a whole-brain electron microscopy dataset of the fruit fly. The predictions can be used to infer a connectivity graph with high accuracy, thus allowing fast identification of neural pathways. To facilitate circuit reconstruction using our results, we develop CIRCUITMAP, a user interface add-on for the circuit annotation tool CATMAID.


Assuntos
Encéfalo/fisiologia , Processamento de Imagem Assistida por Computador/métodos , Sinapses/fisiologia , Animais , Encéfalo/citologia , Bases de Dados Factuais , Drosophila melanogaster , Microscopia Eletrônica , Vias Neurais
19.
Theranostics ; 11(14): 7029-7044, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34093869

RESUMO

Background: Increased IL-6 level, M2 macrophages and PD-1+CD8+ T cells in tumor microenvironments (TME) have been identified to correlate with resistance to checkpoint blockade immunotherapy, yet the mechanism remains poorly understood. Rab small GTPase-mediated trafficking of cytokines is critical in immuno-modulation. We have previously reported dysregulation of Rab37 in lung cancer cells, whereas the roles of Rab37 in tumor-infiltrating immune cells and cancer immunotherapy are unclear. Methods: The tumor growth of the syngeneic mouse allograft in wild type or Rab37 knockout mice was analyzed. Imaging analyses and vesicle isolation were conducted to determine Rab37-mediated IL-6 secretion. STAT3 binding sites at PD-1 promoter in T cells were identified by chromatin immunoprecipitation assay. Multiplex fluorescence immunohistochemistry was performed to detect the protein level of Rab37, IL-6 and PD-1 and localization of the tumor-infiltrating immune cells in allografts from mice or tumor specimens from lung cancer patients. Results: We revealed that Rab37 regulates the secretion of IL-6 in a GTPase-dependent manner in macrophages to trigger M2 polarization. Macrophage-derived IL-6 promotes STAT3-dependent PD-1 mRNA expression in CD8+ T cells. Clinically, tumors with high stromal Rab37 and IL-6 expression coincide with tumor infiltrating M2-macrophages and PD1+CD8+ T cells that predicts poor prognosis in lung cancer patients. In addition, lung cancer patients with an increase in plasma IL-6 level are found to be associated with immunotherapeutic resistance. Importantly, combined blockade of IL-6 and CTLA-4 improves survival of tumor-bearing mice by reducing infiltration of PD1+CD8+ T cells and M2 macrophages in TME. Conclusions: Rab37/IL-6 trafficking pathway links with IL-6/STAT3/PD-1 transcription regulation to foster an immunosuppressive TME and combined IL-6/CTLA-4 blockade therapy exerts potent anti-tumor efficacy.


Assuntos
Interleucina-6/metabolismo , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Fator de Transcrição STAT3/metabolismo , Microambiente Tumoral/imunologia , Proteínas rab de Ligação ao GTP/metabolismo , Aloenxertos , Animais , Linfócitos T CD8-Positivos/metabolismo , Antígeno CTLA-4/antagonistas & inibidores , Antígeno CTLA-4/metabolismo , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Interleucina-6/antagonistas & inibidores , Interleucina-6/sangue , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Prognóstico , Receptor de Morte Celular Programada 1/genética , Fator de Transcrição STAT3/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Microambiente Tumoral/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/ultraestrutura
20.
FEBS Lett ; 595(14): 1876-1885, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34060653

RESUMO

IM30, the inner membrane-associated protein of 30 kDa, is conserved in cyanobacteria and chloroplasts. Although its exact physiological function is still mysterious, IM30 is clearly essential for thylakoid membrane biogenesis and/or dynamics. Recently, a cryptic IM30 GTPase activity has been reported, albeit thus far no physiological function has been attributed to this. Yet, it is still possible that GTP binding/hydrolysis affects formation of the prototypical large homo-oligomeric IM30 ring and rod structures. Here, we show that the Synechocystis sp. PCC 6803 IM30 protein in fact is an NTPase that hydrolyzes GTP and ATP, but not CTP or UTP, with about identical rates. While IM30 forms large oligomeric ring complexes, nucleotide binding and/or hydrolysis are clearly not required for ring formation.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Membrana/metabolismo , Nucleosídeo-Trifosfatase/metabolismo , Synechocystis/enzimologia , Tilacoides/enzimologia , Trifosfato de Adenosina/química , Proteínas de Bactérias/genética , Clonagem Molecular , Ensaios Enzimáticos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Guanosina Trifosfato/química , Hidrólise , Cinética , Proteínas de Membrana/genética , Microscopia Eletrônica , Nucleosídeo-Trifosfatase/genética , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Synechocystis/genética , Synechocystis/ultraestrutura , Tilacoides/genética , Tilacoides/ultraestrutura
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