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1.
Science ; 383(6690): 1471-1478, 2024 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-38547288

RESUMO

Consciousness is lost within seconds upon cessation of cerebral blood flow. The brain cannot store oxygen, and interruption of oxidative phosphorylation is fatal within minutes. Yet only rudimentary knowledge exists regarding cortical partial oxygen tension (Po2) dynamics under physiological conditions. Here we introduce Green enhanced Nano-lantern (GeNL), a genetically encoded bioluminescent oxygen indicator for Po2 imaging. In awake behaving mice, we uncover the existence of spontaneous, spatially defined "hypoxic pockets" and demonstrate their linkage to the abrogation of local capillary flow. Exercise reduced the burden of hypoxic pockets by 52% compared with rest. The study provides insight into cortical oxygen dynamics in awake behaving animals and concurrently establishes a tool to delineate the importance of oxygen tension in physiological processes and neurological diseases.


Assuntos
Córtex Cerebral , Circulação Cerebrovascular , Hipóxia Encefálica , Medições Luminescentes , Saturação de Oxigênio , Oxigênio , Animais , Camundongos , Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/diagnóstico por imagem , Córtex Cerebral/metabolismo , Oxigênio/sangue , Oxigênio/metabolismo , Pressão Parcial , Hipóxia Encefálica/sangue , Hipóxia Encefálica/diagnóstico por imagem , Hipóxia Encefálica/metabolismo , Vasodilatação , Medições Luminescentes/métodos , Luciferases/genética , Luciferases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipercapnia/sangue , Hipercapnia/diagnóstico por imagem , Hipercapnia/metabolismo
2.
Nat Commun ; 14(1): 6598, 2023 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-37891202

RESUMO

L-Lactate is increasingly appreciated as a key metabolite and signaling molecule in mammals. However, investigations of the inter- and intra-cellular dynamics of L-lactate are currently hampered by the limited selection and performance of L-lactate-specific genetically encoded biosensors. Here we now report a spectrally and functionally orthogonal pair of high-performance genetically encoded biosensors: a green fluorescent extracellular L-lactate biosensor, designated eLACCO2.1, and a red fluorescent intracellular L-lactate biosensor, designated R-iLACCO1. eLACCO2.1 exhibits excellent membrane localization and robust fluorescence response. To the best of our knowledge, R-iLACCO1 and its affinity variants exhibit larger fluorescence responses than any previously reported intracellular L-lactate biosensor. We demonstrate spectrally and spatially multiplexed imaging of L-lactate dynamics by coexpression of eLACCO2.1 and R-iLACCO1 in cultured cells, and in vivo imaging of extracellular and intracellular L-lactate dynamics in mice.


Assuntos
Técnicas Biossensoriais , Ácido Láctico , Camundongos , Animais , Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência , Células Cultivadas , Imagem Óptica , Mamíferos
3.
STAR Protoc ; 4(4): 102652, 2023 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-37883226

RESUMO

In bioluminescence imaging (BLI), the biochemical reaction between a substrate and enzyme triggers light emission upon convergence. Unlike fluorescence imaging, BLI does not require excitation. In this protocol, we utilize the high signal-to-background ratio of the reaction between luciferase and its substrate to study the exchange of molecules between blood and cerebrospinal fluid. We outline steps for skull window thinning, cisterna magna infusion, intravascular retro-orbital injection, and imaging. For complete details on the use and execution of this protocol, please refer to Møllgård et al. (2023).1.


Assuntos
Cisterna Magna , Diagnóstico por Imagem , Camundongos , Animais , Luciferases/genética , Injeções
4.
Radiologie (Heidelb) ; 63(10): 749-757, 2023 Oct.
Artigo em Alemão | MEDLINE | ID: mdl-37698653

RESUMO

Femoroacetabular impingement syndrome (FAIS) is caused by a repetitive mechanical conflict between the acetabulum and the proximal femur, occurring in flexion and internal rotation. In cam impingement, bony prominences of the femoral head-neck junction induce chondrolabral damage. The acetabular type of FAIS, termed pincer FAIS, may be either due to focal or global retroversion and/or acetabular overcoverage. Combinations of cam and pincer morphology are common. Pathological femoral torsion may aggravate or decrease the mechanical conflict in FAI but can also occur in isolation. Of note, a high percentage of adolescents with FAI-like shape changes remain asymptomatic. The diagnosis of FAIS is therefore made clinically, whereas imaging reveals the underlying morphology. X­rays in two planes remain the primary imaging modality, the exact evaluation of the osseous deformities of the femur and chondrolabral damage is assessed by magnetic resonance imaging (MRI). Acetabular coverage and version are primarily assessed on radiographs. Evaluation of the entire circumference of the proximal femur warrants MRI which is further used in the assessment of chondrolabral lesions, and also bone marrow and adjacent soft tissue abnormalities. The MRI protocol should routinely include measurements of femoral torsion. Fluid-sensitive sequences should be acquired to rule out degenerative or inflammatory extra-articular changes.


Assuntos
Impacto Femoroacetabular , Adolescente , Humanos , Impacto Femoroacetabular/diagnóstico por imagem , Impacto Femoroacetabular/patologia , Articulação do Quadril/patologia , Acetábulo/diagnóstico por imagem , Acetábulo/patologia , Fêmur/patologia , Cabeça do Fêmur/diagnóstico por imagem , Cabeça do Fêmur/patologia
5.
Nat Commun ; 14(1): 1871, 2023 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-37015909

RESUMO

Information transfer within neuronal circuits depends on the balance and recurrent activity of excitatory and inhibitory neurotransmission. Chloride (Cl-) is the major central nervous system (CNS) anion mediating inhibitory neurotransmission. Astrocytes are key homoeostatic glial cells populating the CNS, although the role of these cells in regulating excitatory-inhibitory balance remains unexplored. Here we show that astrocytes act as a dynamic Cl- reservoir regulating Cl- homoeostasis in the CNS. We found that intracellular chloride concentration ([Cl-]i) in astrocytes is high and stable during sleep. In awake mice astrocytic [Cl-]i is lower and exhibits large fluctuation in response to both sensory input and motor activity. Optogenetic manipulation of astrocytic [Cl-]i directly modulates neuronal activity during locomotion or whisker stimulation. Astrocytes thus serve as a dynamic source of extracellular Cl- available for GABAergic transmission in awake mice, which represents a mechanism for modulation of the inhibitory tone during sustained neuronal activity.


Assuntos
Astrócitos , Cloretos , Camundongos , Animais , Astrócitos/fisiologia , Transmissão Sináptica , Neuroglia , Encéfalo
6.
Science ; 379(6627): 84-88, 2023 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-36603070

RESUMO

The central nervous system is lined by meninges, classically known as dura, arachnoid, and pia mater. We show the existence of a fourth meningeal layer that compartmentalizes the subarachnoid space in the mouse and human brain, designated the subarachnoid lymphatic-like membrane (SLYM). SLYM is morpho- and immunophenotypically similar to the mesothelial membrane lining of peripheral organs and body cavities, and it encases blood vessels and harbors immune cells. Functionally, the close apposition of SLYM with the endothelial lining of the meningeal venous sinus permits direct exchange of small solutes between cerebrospinal fluid and venous blood, thus representing the mouse equivalent of the arachnoid granulations. The functional characterization of SLYM provides fundamental insights into brain immune barriers and fluid transport.


Assuntos
Encéfalo , Espaço Subaracnóideo , Animais , Humanos , Camundongos , Dura-Máter/citologia , Dura-Máter/fisiologia , Endotélio/citologia , Endotélio/fisiologia , Espaço Subaracnóideo/citologia , Espaço Subaracnóideo/fisiologia , Epitélio/fisiologia , Encéfalo/anatomia & histologia , Encéfalo/imunologia , Líquido Cefalorraquidiano/fisiologia
7.
Methods Mol Biol ; 2564: 143-183, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36107341

RESUMO

Flavin-based fluorescent proteins (FbFPs), a class of small fluorescent proteins derived from light-oxygen-voltage (LOV) domains, bind ubiquitous endogenous flavins as chromophores. Due to their unique properties, they can be used as versatile in vivo reporter proteins under aerobic and anaerobic conditions. This chapter presents methodologies for in-depth characterization of the biochemical, spectroscopic, photophysical, and photochemical properties of FbFPs.


Assuntos
Dinitrocresóis , Flavinas , Flavinas/metabolismo , Oxigênio/metabolismo , Proteínas
8.
J Vis Exp ; (136)2018 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-30010642

RESUMO

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a super-resolution technique that allows for the determination of the microcompartments that are accessible for proteins by generating localization and tracking maps of these proteins. Moreover, by multi-color localization microscopy, the localization and tracking profiles of proteins in different subcompartments are obtained simultaneously. The technique is specific for live cells and is based on the repetitive imaging of single mobile membrane proteins. Proteins of interest are genetically fused with specific, so-called self-labeling tags. These tags are enzymes that react with a substrate in a covalent manner. Conjugated to these substrates are fluorescent dyes. Reaction of the enzyme-tagged proteins with the fluorescence labeled substrates results in labeled proteins. Here, Tetramethylrhodamine (TMR) and Silicon Rhodamine (SiR) are used as fluorescent dyes attached to the substrates of the enzymes. By using substrate concentrations in the pM to nM range, sub-stoichiometric labeling is achieved that results in distinct signals. These signals are localized with ~15-27 nm precision. The technique allows for multi-color imaging of single molecules, whereby the number of colors is limited by the available membrane-permeable dyes and the repertoire of self-labeling enzymes. We show the feasibility of the technique by determining the localization of the quality control enzyme (Pten)-induced kinase 1 (PINK1) in different mitochondrial compartments during its processing in relation to other membrane proteins. The test for true physical interactions between differently labeled single proteins by single molecule FRET or co-tracking is restricted, though, because the low labeling degrees decrease the probability for having two adjacent proteins labeled at the same time. While the technique is strong for imaging proteins in membrane compartments, in most cases it is not appropriate to determine the localization of highly mobile soluble proteins.


Assuntos
Corantes Fluorescentes/química , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência/métodos , Organelas/metabolismo , Animais , Transfecção
10.
Nat Commun ; 7: 12549, 2016 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-27633552

RESUMO

We show here that computer game players can build high-quality crystal structures. Introduction of a new feature into the computer game Foldit allows players to build and real-space refine structures into electron density maps. To assess the usefulness of this feature, we held a crystallographic model-building competition between trained crystallographers, undergraduate students, Foldit players and automatic model-building algorithms. After removal of disordered residues, a team of Foldit players achieved the most accurate structure. Analysing the target protein of the competition, YPL067C, uncovered a new family of histidine triad proteins apparently involved in the prevention of amyloid toxicity. From this study, we conclude that crystallographers can utilize crowdsourcing to interpret electron density information and to produce structure solutions of the highest quality.


Assuntos
Crowdsourcing/métodos , Cristalografia/métodos , Currículo , Modelos Químicos , Software , Hidrolases/química , Hidrolases/classificação , Conformação Proteica
11.
ACS Chem Biol ; 10(9): 1970-6, 2015 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-26046594

RESUMO

The cytosolic phosphatase and tensin homologue Pten-kinase PINK1 involved in mitochondrial quality control undergoes a proteolytic process inside mitochondria. It has been suggested that the protein is not fully imported into mitochondria during this maturation. Here, we have established live cell triple-color super-resolution microscopy by combining FPALM and tracking and localization microscopy (TALM) in order to unravel the spatiotemporal organization of the C-terminal kinase domain of PINK1 during this process. We find that the kinase domain is imported into active mitochondria and colocalizes with respiratory complex I at the inner mitochondrial membrane. When the processing step inside mitochondria is inhibited or mitochondria are de-energized, full length PINK1 distributes between the outer and the inner mitochondrial membranes, indicating a holdup of import. These findings give the molecular base for a dual role of PINK1-inside energized mitochondria and outside of de-energized mitochondria.


Assuntos
Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Quinases/análise , Proteínas Quinases/metabolismo , Células HeLa , Humanos , Potencial da Membrana Mitocondrial , Microscopia de Fluorescência , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/ultraestrutura , Estrutura Terciária de Proteína
12.
J Comp Pathol ; 147(2-3): 267-74, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22304974

RESUMO

The larval stage of Taenia crassiceps has been used to study human cysticercosis as these larvae have antigenic similarity to the cysticerci of Taenia solium. The aim of this study was to evaluate the histopathological and immunological changes that followed the inoculation of T. crassiceps cysticerci into the subcutaneous tissue of C57BL/6 mice. Microscopically, granulomas formed of neutrophils and macrophages developed at the sites of inoculation. The serum concentration of the cytokine interferon (IFN)-γ increased throughout the course of infection, while the serum concentration of interleukin-4 increased during the period of transition from the initial phase (7-30 days postinoculation [dpi]) to the late phase (60-90 dpi) of infection. Destruction of the parasite therefore appears to be associated with an increase in IFN-γ, suggesting that a type 1 immune response is important in the control of the parasite.


Assuntos
Cisticercose/patologia , Granuloma/patologia , Doenças Parasitárias em Animais/patologia , Tela Subcutânea/patologia , Animais , Cisticercose/sangue , Cisticercose/imunologia , Modelos Animais de Doenças , Granuloma/sangue , Granuloma/imunologia , Interações Hospedeiro-Parasita/fisiologia , Interferon gama/sangue , Interleucina-4/sangue , Macrófagos/imunologia , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Neutrófilos/patologia , Doenças Parasitárias em Animais/sangue , Doenças Parasitárias em Animais/imunologia , Tela Subcutânea/imunologia , Tela Subcutânea/parasitologia , Fatores de Tempo
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