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In situ cryo-electron tomography enables investigation of macromolecules in their native cellular environment. Samples have become more readily available owing to recent software and hardware advancements. Data collection, however, still requires an experienced operator and appreciable microscope time to carefully select targets for high-throughput tilt series acquisition. Here, we developed smart parallel automated cryo-electron tomography (SPACEtomo), a workflow using machine learning approaches to fully automate the entire cryo-electron tomography process, including lamella detection, biological feature segmentation, target selection and parallel tilt series acquisition, all without the need for human intervention. This degree of automation will be essential for obtaining statistically relevant datasets and high-resolution structures of macromolecules in their native context.
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A 56-year-old woman. She was underwent a lower gastrointestinal endoscopy for bloody stool, and type 2 advanced rectal cancer was found. In CT scan, although distant metastasis is not found, the tumor has been expanded to the dorsal side. So, infiltration into the sacrum was suspected. For the risk of bleeding and residual tumor in circumferential resection surface, it was decided to perform pre-operative adjuvant chemotherapy. Because RAS gene has no mutation, the regimen chose CAPOX plus cetuximab. Although skin damage and cytopenia were observed, there was no appearance of adverse events that were intolerant, and 4 courses were performed. Although scar stenosis was observed in the endoscope after 4 courses, tumor size decreased. Even in CT, the wall thickening was significantly reduced, and progress to the tumor dorsal side was also reduced, so laparoscopic lower anterior resection was performed. During surgery, the tumor dorsal side sacral infiltration was suspected, although observed a sclerotic change, it is relatively easily peelable, it was possible to safely complete the laparoscopic operation. Even after the operation, the course was good, and it was discharged from the hospital lightly on the 12th day after the operation. In pathological diagnosis, medium-differentiated adenocarcinoma, T3, N0, histological therapeutic effect of chemotherapy was grade 2. Cetuximab combination regimen was considered to be an effective option.
Assuntos
Laparoscopia , Neoplasias Retais , Feminino , Humanos , Pessoa de Meia-Idade , Capecitabina/uso terapêutico , Cetuximab/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Resultado do Tratamento , Neoplasias Retais/tratamento farmacológico , Neoplasias Retais/cirurgia , Neoplasias Retais/patologia , Oxaliplatina/uso terapêuticoRESUMO
A 50-year-old male was referred to our hospital for the further evaluation and treatment of abdominal pain. He was diagnosed with complicated appendicitis using computed tomography. After conservative treatment, he underwent an interval appendectomy. A histopathological examination revealed a goblet cell carcinoid(GCC)of the appendix with subserosal invasion. He underwent laparoscopic ileocecal resection with D3 lymph node dissection. Histopathological findings showed neither residual tumor nor lymph node metastasis. The patients is currently followed as an outpatient without recurrence. Here we report our experience with GCC, a rare disease.
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Neoplasias do Apêndice , Apendicite , Apêndice , Tumor Carcinoide , Masculino , Humanos , Pessoa de Meia-Idade , Apendicite/complicações , Apendicite/cirurgia , Neoplasias do Apêndice/complicações , Neoplasias do Apêndice/cirurgia , Neoplasias do Apêndice/patologia , Tumor Carcinoide/complicações , Tumor Carcinoide/cirurgia , Apendicectomia/métodosRESUMO
Tripeptidyl peptidase II (TPPII) is a eukaryotic protease acting downstream of the 26S proteasome; it removes tripeptides from the degradation products released by the proteasome. Structural studies in vitro have revealed the basic architecture of TPPII, a two-stranded linear polymer that assembles to form a spindle-shaped complex of â¼6 MDa. Dependent on protein concentration, TPPII has a distinct tendency for polymorphism. Therefore, its structure in vivo has remained unclear. To resolve this issue, we have scrutinized cryo-electron tomograms of rat hippocampal neurons for the occurrence and spatial distribution of TPPII by template matching. The quality of the tomograms recorded with the Volta phase plate enabled a detailed structural analysis of TPPII despite its low abundance. Two different assembly states (36-mers and 32-mers) coexist as well as occasional extended forms with longer strands. A distance analysis of the relative locations of TPPII and 26S proteasomes confirmed the visual impression that these two complexes spatially associate in agreement with TPPII's role in postproteasomal degradation.
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Aminopeptidases/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Serina Endopeptidases/metabolismo , Aminopeptidases/genética , Animais , Animais Recém-Nascidos , Células Cultivadas , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Modelos Moleculares , Neurônios/ultraestrutura , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Conformação Proteica , Ratos , Serina Endopeptidases/genéticaRESUMO
Neurotransmitter release is orchestrated by synaptic proteins, such as SNAREs, synaptotagmin, and complexin, but the molecular mechanisms remain unclear. We visualized functionally active synaptic proteins reconstituted into proteoliposomes and their interactions in a native membrane environment by electron cryotomography with a Volta phase plate for improved resolvability. The images revealed individual synaptic proteins and synaptic protein complex densities at prefusion contact sites between membranes. We observed distinct morphologies of individual synaptic proteins and their complexes. The minimal system, consisting of neuronal SNAREs and synaptotagmin-1, produced point and long-contact prefusion states. Morphologies and populations of these states changed as the regulatory factors complexin and Munc13 were added. Complexin increased the membrane separation, along with a higher propensity of point contacts. Further inclusion of the priming factor Munc13 exclusively restricted prefusion states to point contacts, all of which efficiently fused upon Ca2+ triggering. We conclude that synaptic proteins have evolved to limit possible contact site assemblies and morphologies to those that promote fast Ca2+-triggered release.
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Proteínas de Fusão de Membrana/metabolismo , Fusão de Membrana , Neurônios/metabolismo , Membranas Sinápticas/metabolismo , Animais , Cálcio/metabolismo , Microscopia Crioeletrônica/métodos , Proteínas de Fusão de Membrana/química , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Domínios Proteicos , Proteolipídeos/metabolismo , Proteolipídeos/ultraestrutura , Proteínas SNARE/química , Proteínas SNARE/metabolismo , Membranas Sinápticas/ultraestrutura , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestrutura , Sinaptotagmina I/química , Sinaptotagmina I/metabolismoRESUMO
Electron cryotomography provides a means of studying the three dimensional structure of pleomorphic objects, such as organelles or cells, with a resolution of 1-3nm. A limitation in the study of radiation sensitive biological samples is the low signal-to-noise ratio of the tomograms which may obscure fine details. To overcome this limitation, the recently developed Volta phase plate (VPP) was applied in electron cryotomographic studies of a wide range of cellular structures, from magnetotactic bacteria to primary cultured neurons. The results show that the VPP improves contrast significantly and consequently the signal-to-noise ratio of the tomograms, moreover it avoids disturbing fringing artifacts typical for Zernike phase plates. The contrast improvement provided by the VPP was also confirmed in projection images of relatively thick (â¼400nm) samples. In order to investigate the respective contributions of the VPP and the energy filter, images acquired with different combinations of the two were compared. Zero-loss energy filtering reduced the background noise in thicker areas of the sample and improved the contrast of features such as poly-ß-hydroxybutyrate granules in magnetotactic bacteria, whereas the VPP provided an overall contrast improvement for all sample areas. After 3D reconstruction, tomograms acquired with the combination of a VPP and an energy filter showed structural features in neuronal processes with outstanding clarity. We also show that the VPP can be combined with focused ion beam milling to examine structures embedded deeply inside cells. Thus, we expect that VPP will become a standard element of the electron cryotomography workflow.
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Células/citologia , Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Imageamento Tridimensional/métodos , Vitrificação , Meios de Contraste , Razão Sinal-RuídoRESUMO
Integration of fluorescence light and transmission electron microscopy into the same device would represent an important advance in correlative microscopy, which traditionally involves two separate microscopes for imaging. To achieve such integration, the primary technical challenge that must be solved regards how to arrange two objective lenses used for light and electron microscopy in such a manner that they can properly focus on a single specimen. To address this issue, both lateral displacement of the specimen between two lenses and specimen rotation have been proposed. Such movement of the specimen allows sequential collection of two kinds of microscopic images of a single target, but prevents simultaneous imaging. This shortcoming has been made up by using a simple optical device, a reflection mirror. Here, we present an approach toward the versatile integration of fluorescence and electron microscopy for simultaneous imaging. The potential of simultaneous hybrid microscopy was demonstrated by fluorescence and electron sequential imaging of a fluorescent protein expressed in cells and cathodoluminescence imaging of fluorescent beads.
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Microscopia Crioeletrônica/instrumentação , Microscopia Crioeletrônica/métodos , Microscopia Eletrônica de Transmissão/métodos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Animais , Células Cultivadas , Elétrons , Fluorescência , Fótons , PotoroidaeRESUMO
Since the Human Genome Project, drug discovery via structure-based drug design and development has significantly accelerated. Therefore, generating high-resolution structural information from biological macromolecules and macromolecular complexes, such as proteins and nucleic acids, is paramount in structural biology, medicine and the pharmaceutical industry. Recently, electron cryomicroscopy (cryo-EM) has undergone a technological revolution and attracted much attention in the structure-based drug discovery pipeline. This recognition is primarily due to its ability to analyze and reconstruct high-resolution structures of previously unattainable large target macromolecular complexes captured in various functional and dynamic states. Previously, cryo-EM was a niche method in the structure determination field, and research was limited to a small number of laboratories and produced low-resolution structures incomplete for detailed and unambiguous structural interpretation. However, with the development of new camera technology, software and computational algorithms that now seamlessly integrate these new developments, the achievable resolutions produced from cryo-EM-determined structures have dramatically improved. This has solidified cryo-EM as one of the main structural determination methods widely used in the field. In this review, we introduce the evolution of two essential techniques incorporated into the cryo-EM workflow-single particle analysis and tomography-focusing on achievable resolution and the technological innovations that have become indispensable tools for high-resolution reconstruction and structural analysis of biological macromolecules. Here, we also describe challenges and discuss future prospects that have fixed cryo-EM as a dominant feature in the landscape of high-resolution structure determination methods and the structure-based drug discovery pipeline.
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Algoritmos , Software , Humanos , Microscopia Crioeletrônica/métodos , Descoberta de Drogas , Substâncias Macromoleculares/químicaRESUMO
Cryo-electron tomography of frozen hydrated cells has provided cell biologists with an indispensable tool for delineating three-dimensional arrangements of cellular ultrastructure. To avoid the damage induced by electron irradiation, images of frozen hydrated biological specimens are generally acquired under low-dose conditions, resulting in weakly contrasted images that are difficult to interpret, and in which ultrastructural details remain ambiguous. Zernike phase contrast transmission electron microscopy can improve contrast, and can also fix a fatal problem related to the inherent low contrast of conventional electron microscopy, namely, image modulation due to the unavoidable setting of deep defocus. In this study, we applied cryo-electron tomography enhanced with a Zernike phase plate, which avoids image modulation by allowing in-focus setting. The Zernike phase contrast cryo-electron tomography has a potential to suppress grainy background generation. Due to the smoother background in comparison with defocus phase contrast cryo-electron tomography, Zernike phase contrast cryo-electron tomography could yield higher visibility for particulate or filamentous ultrastructure inside the cells, and allowed us to clearly recognize membrane protein structures.
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Membrana Celular/ultraestrutura , Animais , Linhagem Celular , Microscopia Crioeletrônica/métodos , Dipodomys , Tomografia com Microscopia Eletrônica/métodos , Processamento de Imagem Assistida por Computador , Proteínas de Membrana/ultraestrutura , Microscopia de Contraste de Fase/métodos , Análise de Célula Única/métodosRESUMO
We report a case of multiple liver metastases from colon cancer initially thought to be incurable and review several literature cases. The patient has survived for 3 years without tumor relapse after multimodality therapy and surgical intervention was performed.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Neoplasias Hepáticas/terapia , Idoso , Neoplasias do Colo/patologia , Neoplasias do Colo/cirurgia , Terapia Combinada , Intervalo Livre de Doença , Feminino , Humanos , Neoplasias Hepáticas/secundárioRESUMO
Intestinal malrotation is a rare congenital disease caused by abnormal intestinal rotation and fixation of the intestinal tract in the early embryonic state. Adult cases are rare. A laparoscopic Ladd procedure for adult intestinal malrotation is increasingly reported, but owing to the rarity, some important aspects of the disease and its treatment may be overlooked. Three adult cases of intestinal malrotation that underwent surgery at our hospital between January 2019 and October 2020 were retrospectively examined about patient backgrounds, short-term results, and complications. All patients were male, median age was 54.6 years, and the complaints were abdominal pain and/or distention. No midgut volvulus was observed. The laparoscopic Ladd procedure was performed for all cases. One patient underwent reoperation (duodenoduodenostomy) because of impaired passage of the duodenal descending section due to postoperative pancreatic fistula. The postoperative courses of the other two patients were good. No recurrence of symptoms was observed in any of the cases. The reason for reoperation in one of the cases is considered to be pancreatic injury when the severe curve from the duodenum to the upper jejunum near the pancreatic head was straightened. Correction of the curve is important to improve passage disorder of the duodenum, but special care is required to avoid organ damage, especially during a laparoscopic procedure with forceps. The laparoscopic Ladd procedure for adult intestinal malrotation is recommended if there is no midgut volvulus; it is minimally invasive and a comparatively simple technique, but surgeons should take special care to avoid organ damage.
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The prognosis for intervertebral disc disease (IVDD), a common neurologic disease in dogs, varies, with some cases requiring long-term rehabilitation. Corsets are used as part of the physical rehabilitation of dogs, and one of these, the Anifull Dog's Corset Pro, is manufactured and sold by Daiya Industry Co., Ltd. This corset is used to relieve pain caused by spinal cord and vertebral diseases, and to prevent neurological conditions from worsening, by limiting spinal movement. However, the effect of the Anifull Dog's Corset Pro on gait has not yet been clarified. Therefore, we aimed to evaluate the effects of this corset on the gait of dogs using kinematic and kinetic analyses. Five healthy beagle dogs wearing corsets were trotted, kinematic and kinetic parameters were measured using motion capture and force plates, and the results were compared to those obtained when the dogs were not wearing a corset. The range of motion of the angle formed by the 13th thoracic vertebra and the 7th lumbar vertebra at the apex of the 7th cervical vertebra was significantly reduced in the corset-wearing dogs. Thus, the Anifull Dog's Corset Pro may improve trunk stability without affecting gait.
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In order to acquire phase-contrast images with adequate contrast, conventional TEM requires large amount of defocus. Increasing the defocus improves the low-frequency components but attenuates the high-frequency ones. On the other hand, Zernike phase-contrast TEM (ZPC-TEM) can recover low-frequency components without losing the high-frequency ones under in-focus conditions. ZPC-TEM however, has another problem, especially in imaging of complex biological specimens such as cells and tissues; strong halos appear around specimen structures, and these halos hinder the interpretation of images. Due to this problem, the application of ZPC-TEM has been restricted to imaging of smaller particles. In order to improve the halo appearance, we fabricated a new quarter-wave thin film phase-plate with a smaller central hole and tested it on vitreous biological specimens. ZPC-TEM with the new plate could successfully visualize, in in-focus images, the intracellular fine features of cultured cells and brain tissues. This result indicates that reduction of the central hole diameter makes ZPC-TEM applicable on size scales ranging from protein particles to tissue sections. The application of ZPC-TEM to vitreous biological specimens will be a powerful method to advance the new field of imaging science for ultrastructures in close-to-physiological state.
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Microscopia Crioeletrônica/métodos , Microscopia Eletrônica de Transmissão/métodos , Microscopia de Contraste de Fase/métodos , Animais , Encéfalo/ultraestrutura , Células Cultivadas , Técnicas In Vitro , Masculino , CamundongosRESUMO
Actin waves are dynamic supramolecular structures involved in cell migration, cytokinesis, adhesion, and neurogenesis. Although wave-like propagation of actin networks is a widespread phenomenon, the actin architecture underlying wave propagation remained unknown. In situ cryo-electron tomography of Dictyostelium cells unveils the wave architecture and provides evidence for wave progression by de novo actin nucleation. Subtomogram averaging reveals the structure of Arp2/3 complex-mediated branch junctions in their native state, and enables quantitative analysis of the 3D organization of branching within the waves. We find an excess of branches directed toward the substrate-attached membrane, and tent-like structures at sites of branch clustering. Fluorescence imaging shows that Arp2/3 clusters follow accumulation of the elongation factor VASP. We propose that filament growth toward the membrane lifts up the actin network as the wave propagates, until depolymerization of oblique filaments at the back causes the collapse of horizontal filaments into a compact layer.
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Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Moléculas de Adesão Celular/metabolismo , Dictyostelium/ultraestrutura , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Protozoários/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/química , Dictyostelium/metabolismo , Tomografia com Microscopia Eletrônica , Modelos Moleculares , Proteínas de Protozoários/químicaRESUMO
We developed an assist suit with lightweight, flexible artificial muscles of pneumatic rubber for reducing muscle load in the lumbar region. We designed two assist forces to control the artificial muscles with pulse width modulation based on the measured EMG of the spinal column muscle and estimated the torque of the hip joint. The experimental results confirmed the developed work assist suit could unload muscle activity during bending and stretching exercises. We also proposed to use an EMG measurement device at the wearer's temple to control the assist timing and confirmed the feasibility of detecting the intention of the wearer.
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Borracha , Eletromiografia , Articulação do Quadril , Região Lombossacral , Músculo Esquelético , TorqueRESUMO
Many cellular processes depend on a precise structural organization of molecular components. Here, we established that neurons grown in culture provide a suitable system for in situ structural investigations of cellular structures by cryo-electron tomography, a method that allows high resolution, three-dimensional imaging of fully hydrated, vitrified cellular samples. A higher level of detail of cellular components present in our images allowed us to quantitatively characterize presynaptic and cytoskeletal organization, as well as structures involved in axonal transport and endocytosis. In this way we provide a structural framework into which information from other methods need to fit. Importantly, we show that short pleomorphic linkers (tethers and connectors) extensively interconnect different types of spherical vesicles and other lipid membranes in neurons imaged in a close-to-native state. These linkers likely serve to organize and precisely position vesicles involved in endocytosis, axonal transport and synaptic release. Hence, structural interactions via short linkers may serve as ubiquitous vesicle organizers in neuronal cells.
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Axônios/metabolismo , Rede Nervosa/citologia , Vesículas Sinápticas/metabolismo , Animais , Axônios/ultraestrutura , Transporte Biológico , Microscopia Crioeletrônica , Citoesqueleto/metabolismo , Hipocampo/citologia , Rede Nervosa/ultraestrutura , RatosRESUMO
In the field of neuroscience, low-invasive in vivo imaging would be a very useful method of monitoring the morphological dynamics of intact neurons in living animals. At present, there are two widely used in vivo imaging methods; one is the two-photon microscope method, and the other is the fiber optics method. However, these methods are not suitable for the in vivo imaging of deeper subcortical structures. In our study, we have developed a novel method for the in vivo imaging of pyramidal neurons in layer V of the cerebral cortex, utilizing a MicroLSM system and a stick-type objective lens that can be directly inserted into the target tissue. By using this method, we succeeded in obtaining clear images of pyramidal neurons in layer V of the cerebral cortex under a low-invasive condition. The MicroLSM system is a useful and versatile in vivo imaging system that will be applicable not only to the brain but also to other organs.
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Córtex Cerebral/citologia , Dendritos/ultraestrutura , Lentes , Microscopia Confocal/instrumentação , Células Piramidais/citologia , Animais , Animais Recém-Nascidos , Dendritos/metabolismo , Diagnóstico por Imagem/métodos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos TransgênicosRESUMO
The 26S proteasome is a key player in eukaryotic protein quality control and in the regulation of numerous cellular processes. Here, we describe quantitative in situ structural studies of this highly dynamic molecular machine in intact hippocampal neurons. We used electron cryotomography with the Volta phase plate, which allowed high fidelity and nanometer precision localization of 26S proteasomes. We undertook a molecular census of single- and double-capped proteasomes and assessed the conformational states of individual complexes. Under the conditions of the experimentthat is, in the absence of proteotoxic stressonly 20% of the 26S proteasomes were engaged in substrate processing. The remainder was in the substrate-accepting ground state. These findings suggest that in the absence of stress, the capacity of the proteasome system is not fully used.
Assuntos
Hipocampo/citologia , Neurônios/enzimologia , Neurônios/ultraestrutura , Complexo de Endopeptidases do Proteassoma/química , Animais , Células Cultivadas , Hipocampo/enzimologia , Conformação Proteica , Ratos , Estresse FisiológicoRESUMO
Correlative microscopy allows imaging of the same feature over multiple length scales, combining light microscopy with high resolution information provided by electron microscopy. We demonstrate two procedures for coordinate transformation based correlative microscopy of vitrified biological samples applicable to different imaging modes. The first procedure aims at navigating cryo-electron tomography to cellular regions identified by fluorescent labels. The second procedure, allowing navigation of focused ion beam milling to fluorescently labeled molecules, is based on the introduction of an intermediate scanning electron microscopy imaging step to overcome the large difference between cryo-light microscopy and focused ion beam imaging modes. These methods make it possible to image fluorescently labeled macromolecular complexes in their natural environments by cryo-electron tomography, while minimizing exposure to the electron beam during the search for features of interest.