RESUMO
Extracellular vesicles (EVs) are critical elements of cell-cell communication. Here, we characterized the outer membrane vesicles (OMVs) released by specific clones of Escherichia coli isolated from the Long-Term Evolution Experiment after 50,000 generations (50K) of adaptation to glucose minimal medium. Compared with their ancestor, the evolved clones produce small OMVs but also larger ones which display variable amounts of both OmpA and LPS. Tracking ancestral, fluorescently labelled OMVs revealed that they fuse with both ancestral- and 50K-evolved cells, albeit in different proportions. We quantified that less than 2% of the cells from one 50K-evolved clone acquired the fluorescence delivered by OMVs from the ancestral strain but that one cell concomitantly fuses with several OMVs. Globally, our results showed that OMV production in E. coli is a phenotype that varies along bacterial evolution and question the contribution of OMVs-mediated interactions in bacterial adaptation.
Assuntos
Escherichia coli , Vesículas Extracelulares , Escherichia coli/genética , Proteínas da Membrana Bacteriana Externa/genéticaRESUMO
An artificial amyloid-based redox hydrogel was designed for mediating electron transfer between a [NiFeSe] hydrogenase and an electrode. Starting from a mutated prion-forming domain of fungal protein HET-s, a hybrid redox protein containing a single benzyl methyl viologen moiety was synthesized. This protein was able to self-assemble into structurally homogenous nanofibrils. Molecular modeling confirmed that the redox groups are aligned along the fibril axis and are tethered to its core by a long, flexible polypeptide chain that allows close encounters between the fibril-bound oxidized or reduced redox groups. Redox hydrogel films capable of immobilizing the hydrogenase under mild conditions at the surface of carbon electrodes were obtained by a simple pH jump. In this way, bioelectrodes for the electrocatalytic oxidation of H2 were fabricated that afforded catalytic current densities of up to 270â µA cm-2 , with an overpotential of 0.33â V, under quiescent conditions at 45 °C.
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Amiloide/metabolismo , Hidrogéis/metabolismo , Hidrogênio/metabolismo , Hidrogenase/metabolismo , Amiloide/química , Biocatálise , Eletrodos , Transporte de Elétrons , Hidrogéis/química , Hidrogênio/química , Hidrogenase/química , Modelos Moleculares , Oxirredução , Tamanho da PartículaRESUMO
Living cells embedded in a complex extra-cellular matrix migrate in a sophisticated way thanks to adhesions to matrix fibres and contractility. It is important to know what kind of forces are exerted by the cells. Here, we use reflectance confocal microscopy to locate fibres accurately and determine displacement fields. Correlation techniques are used to this aim, coupled with proper digital image processing. Benchmark tests validate the method in the case of shear and stretching motions. Finally, the method is tested successfully for studying cancer cells migrating in collagen gels of different concentration.
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Movimento Celular , Colágeno , Géis , Processamento de Imagem Assistida por Computador , Microscopia Confocal/métodos , Adesão Celular , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Humanos , Imagem Óptica/métodosRESUMO
BACKGROUND: The 3D architecture of the ventricular mass is poorly known, although in vivo imaging techniques show the physiological inhomogeneity of ventricular walls mechanics. Polarized light imaging makes it possible to quantitatively analyse the myosin filament orientation. AIMS: In this paper, we focus on the study the 3D architecture and regional isotropy of myocardial cells. METHODS: Twenty normal human hearts, 10 from the perinatal period and 10 from the post-neonatal period were studied by polarized light microscopy. In each voxel of the ventricular mass (90 × 90 × 500 µm) the principal orientation segment was automatically and unambiguously extracted as well as a regional isotropy index (regional orientation tensor of the voxel neighbourhood). RESULTS: During the first months of postnatal age, the median regional isotropy values decreased in the ventricular mesh. This global decrease was not homogeneous across the ventricular walls. From the perinatal to the neonatal period, this decrease was more marked in the inner two-third of the lateral left ventricular wall and in the right part of the interventricular septum. There was a progressive post-neonatal appearance of a particularly inhomogeneous secondary arrangement of myocardial cells with alternation of thick low-RI and thin high-RI areas. CONCLUSIONS: This study has shown a postnatal change in ventricular myocardial architecture, which became more inhomogeneous. The cell rearrangements responsible for the inhomogeneity in ventricular myocardial architecture are revealed by a variation of the regional isotropy index. These major changes are probably an adaptive consequence of the major haemodynamic changes occurring after birth during the neonatal period that generates major parietal stress variations and parietal remodelling.
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Ventrículos do Coração/citologia , Miocárdio/citologia , Humanos , Imageamento Tridimensional , Lactente , Recém-NascidoRESUMO
Cardiac ischemia-reperfusion (IR) injury compromises mitochondrial oxidative phosphorylation (OxPhos) and compartmentalized intracellular energy transfer via the phosphocreatine/creatine kinase (CK) network. The restriction of ATP/ADP diffusion at the level of the mitochondrial outer membrane (MOM) is an essential element of compartmentalized energy transfer. In adult cardiomyocytes, the MOM permeability to ADP is regulated by the interaction of voltage-dependent anion channel with cytoskeletal proteins, particularly with ß tubulin II. The IR-injury alters the expression and the intracellular arrangement of cytoskeletal proteins. The objective of the present study was to investigate the impact of IR on the intracellular arrangement of ß tubulin II and its effect on the regulation of mitochondrial respiration. Perfused rat hearts were subjected to total ischemia (for 20min (I20) and 45min (I45)) or to ischemia followed by 30min of reperfusion (I20R and I45R groups). High resolution respirometry and fluorescent confocal microscopy were used to study respiration, ß tubulin II and mitochondrial arrangements in cardiac fibers. The results of these experiments evidence a heterogeneous response of mitochondria to IR-induced damage. Moreover, the intracellular rearrangement of ß tubulin II, which in the control group colocalized with mitochondria, was associated with increased apparent affinity of OxPhos for ADP, decreased regulation of respiration by creatine without altering mitochondrial CK activity and the ratio between octameric to dimeric isoenzymes. The results of this study allow us to highlight changes of mitochondrial interactions with cytoskeleton as one of the possible mechanisms underlying cardiac IR injury.
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Citoesqueleto/patologia , Mitocôndrias Cardíacas/patologia , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/patologia , Tubulina (Proteína)/metabolismo , Animais , Respiração Celular , Citoesqueleto/metabolismo , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Masculino , Mitocôndrias Cardíacas/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/metabolismo , Ratos Wistar , Tubulina (Proteína)/ultraestruturaRESUMO
L-Maurocalcine (L-MCa) is the first reported animal cell-penetrating toxin. Characterizing its cell penetration properties is crucial considering its potential as a vector for the intracellular delivery of drugs. Radiolabeling is a sensitive and quantitative method to follow the cell accumulation of a molecule of interest. An L-MCa analog containing an additional N-terminal tyrosine residue (Tyr-L-MCa) was synthesized, shown to fold and oxidize properly, and successfully radioiodinated to (125)I-Tyr-L-MCa. Using various microscopy techniques, the average volume of the rat line F98 glioma cells was evaluated at 8.9 to 18.9×10(-7)µl. (125)I-Tyr-L-MCa accumulates within cells with a dose-dependency similar to the one previously published using 5,6-carboxyfluorescein-L-MCa. According to subcellular fractionation of F98 cells, plasma membranes keep less than 3% of the peptide, regardless of the extracellular concentration, while the nucleus accumulates over 75% and the cytosol around 20% of the radioactive material. Taking into account both nuclear and cytosolic fractions, cells accumulate intracellular concentrations of the peptide that are equal to the extracellular concentrations. Estimation of (125)I-Tyr-L-MCa cell entry kinetics indicate a first rapid phase with a 5min time constant for the plasma membrane followed by slower processes for the cytoplasm and the nucleus. Once inside cells, the labeled material no longer escapes from the intracellular environment since 90% of the radioactivity remains 24h after washout. Dead cells were found to have a lower uptake than live ones. The quantitative information gained herein will be useful for better framing the use of L-MCa in biotechnological applications. This article is part of a Special Issue entitled: Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.
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Membrana Celular/metabolismo , Peptídeos Penetradores de Células/metabolismo , Venenos de Escorpião/metabolismo , Tirosina/química , Sequência de Aminoácidos , Animais , Transporte Biológico , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Núcleo Celular/metabolismo , Tamanho Celular , Peptídeos Penetradores de Células/síntese química , Citosol/metabolismo , Portadores de Fármacos , Radioisótopos do Iodo , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Dobramento de Proteína , Ratos , Venenos de Escorpião/síntese química , Técnicas de Síntese em Fase SólidaRESUMO
This review describes the recent experimental data on the importance of the VDAC-cytoskeleton interactions in determining the mechanisms of energy and metabolite transfer between mitochondria and cytoplasm in cardiac cells. In the intermembrane space mitochondrial creatine kinase connects VDAC with adenine nucleotide translocase and ATP synthase complex, on the cytoplasmic side VDAC is linked to cytoskeletal proteins. Applying immunofluorescent imaging and Western blot analysis we have shown that ß2-tubulin coexpressed with mitochondria is highly important for cardiac muscle cells mitochondrial metabolism. Since it has been shown by Rostovtseva et al. that αß-heterodimer of tubulin binds to VDAC and decreases its permeability, we suppose that the ß-tubulin subunit is bound on the cytoplasmic side and α-tubulin C-terminal tail is inserted into VDAC. Other cytoskeletal proteins, such as plectin and desmin may be involved in this process. The result of VDAC-cytoskeletal interactions is selective restriction of the channel permeability for adenine nucleotides but not for creatine or phosphocreatine that favors energy transfer via the phosphocreatine pathway. In some types of cancer cells these interactions are altered favoring the hexokinase binding and thus explaining the Warburg effect of increased glycolytic lactate production in these cells. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.
Assuntos
Creatina Quinase Mitocondrial/metabolismo , Citoesqueleto/metabolismo , Mitocôndrias/metabolismo , Células Musculares/citologia , Células Musculares/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismo , Animais , Respiração Celular , Humanos , Mitocôndrias/enzimologia , Ligação ProteicaRESUMO
Mitochondria are dynamic structures for which fusion and fission are well characterized for rapidly dividing cells in culture. Based on these data, it has recently been proposed that high respiratory activity is the result of fusion and formation of mitochondrial reticulum, while fission results in fragmented mitochondria with low respiratory activity. In this work we test the validity of this new hypothesis by analyzing our own experimental data obtained in studies of isolated heart mitochondria, permeabilized cells of cardiac phenotype with different mitochondrial arrangement and dynamics. Additionally, we reviewed published data including electron tomographic investigation of mitochondrial membrane-associated structures in heart cells. Oxygraphic studies show that maximal ADP-dependent respiration rates are equally high both in isolated heart mitochondria and in permeabilized cardiomyocytes. On the contrary, these rates are three times lower in NB HL-1 cells with fused mitochondrial reticulum. Confocal and electron tomographic studies show that there is no mitochondrial reticulum in cardiac cells, known to contain 5,000-10,000 individual, single mitochondria, which are regularly arranged at the level of sarcomeres and are at Z-lines separated from each other by membrane structures, including the T-tubular system in close connection to the sarcoplasmic reticulum. The new structural data in the literature show a principal role for the elaborated T-tubular system in organization of cell metabolism by supplying calcium, oxygen and substrates from the extracellular medium into local domains of the cardiac cells for calcium cycling within Calcium Release Units, associated with respiration and its regulation in Intracellular Energetic Units.
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Respiração Celular/fisiologia , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Cálcio/metabolismo , Metabolismo Energético , Microscopia Confocal , Dinâmica Mitocondrial , Miócitos Cardíacos/citologiaRESUMO
There is a variety of fluorescent probes for pH measurements and which are mainly used for biological systems. In general, they can be classified into two groups. The first group includes fluorescent pH probes which exhibit a single fluorescence emission peak. For these probes, the fluorescence excitation profile is pH-dependent and the shape of the emission spectra remains almost constant. Hence, the ratiometric pH measurement-which makes pH determination independent of the probe concentration-is implemented when the excitation is performed at two excitation wavelengths and the fluorescence emission is measured at one wavelength. The second group exhibits a dual fluorescence emission peak. Here, each protonated or deprotonated form exhibits characteristics emission and/or absorption spectra. Shifts between spectra obtained for protonated and deprotonated species can be exploited in order to perform a ratiometric measurement. In this study we present a methodology that evaluates the precision of the ratiometric measurements based on multiple wavelengths excitation to determine the optimum wavelengths combination for pH determination in biological samples. This methodology using the BCECF probe is applied to measure the pH drift in cell culture medium. It exhibits a high precision and significantly extends the range of validity for pH measurements spanning from very acidic to basic.
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Corantes Fluorescentes , Concentração de Íons de Hidrogênio , Fluoresceínas , Espectrometria de Fluorescência/métodosRESUMO
This article is the third in our series dedicated to the analysis of cardiac myoarchitecture as a nematic chiral liquid crystal (NCLC). Previously, we introduced the concept of topological defects (disclinations) and focused on their visual identification inside the compact myocardium. Herein, we investigate these using a mathematical and automated algorithm for the reproducible identification of a larger panel of topological defects throughout the myocardium of 13 perinatal and 11 early infant hearts. This algorithm identified an average of 29 ± 11 topological defects per slice with a 2D topological charge of m = +1/2 and an average of 27 ± 10 topological defects per slice with a 2D topological charge of m = -1/2. The excess of defects per slice with a 2D topological charge of m = +1/2 was statistically significant (p < 0.001). There was no significant difference in the distribution of defects with a 2D topological charge of m = +1/2 and m = -1/2 between perinatal and early infant hearts. These defects were mostly arranged in pairs, as expected in nematics, and located inside the trabecular myocardium. When isolated, defects with a 2D topological charge of m = +1/2 were located near the luminal extremity of the trabeculae and those with a 2D topological charge of m = -1/2 were located at the anterior and posterior part of the interventricular septum. These findings constitute an advance in the characterization of the deep cardiac myoarchitecture for application in developmental and pathological studies.
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A well-known feature of tumor cells is high glycolytic activity, leading to acidification of the tumor microenvironment through extensive lactate production. This acidosis promotes processes such as metastasis, aggressiveness, and invasiveness, which have been associated with a worse clinical prognosis. Moreover, the function and expression of transporters involved in regulation of intracellular pH might be altered. In this study, the capacity of tumor cells to regulate their intracellular pH when exposed to a range of pH from very acidic to basic was characterized in two glioma cell lines (F98 and U87) using a new recently published method of fluorescence imaging. Our results show that the regulation of acidity in tumors is not the same for the two investigated cell lines; U87 cells are able to reduce their intracellular acidity, whereas F98 cells do not exhibit this property. On the other hand, F98 cells show a higher level of resistance to acidity than U87 cells. Intracellular regulation of acidity appears to be highly cell-dependent, with different mechanisms activated to preserve cell integrity and function. This characterization was performed on 2D monolayer cultures and 3D spheroids. Spatial heterogeneities were exhibited in 3D, suggesting a spatially modulated regulation in this context. Based on the corpus of knowledge available in the literature, we propose plausible mechanisms to interpret our results, together with some new lines of investigation to validate our hypotheses. Our results might have implications on therapy, since the activity of temozolomide is highly pH-dependent. We show that the drug efficiency can be enhanced, depending on the cell type, by manipulating the extracellular pH. Therefore, personalized treatment involving a combination of temozolomide and pH-regulating agents can be considered.
RESUMO
Heat shock triggers a transient and ubiquitous response, the function of which is to protect cells against stress-induced damage. The heat-shock response is controlled by a key transcription factor known as heat shock factor 1 (HSF1). We have developed a multiconfocal fluorescence correlation spectroscopy setup to measure the dynamics of HSF1 during the course of the heat-shock response. The system combines a spatial light modulator, to address several points of interest, and an electron-multiplying charge-coupled camera for fast multiconfocal recording of the photon streams. Autocorrelation curves with a temporal resolution of 14 µs were analyzed before and after heat shock on eGFP and HSF1-eGFP-expressing cells. Evaluation of the dynamic parameters of a diffusion-and-binding model showed a slower HSF1 diffusion after heat shock. It is also observed that the dissociation rate decreases after heat shock, whereas the association rate is not affected. In addition, thanks to the multiconfocal fluorescence correlation spectroscopy system, up to five spots could be simultaneously located in each cell nucleus. This made it possible to quantify the intracellular variability of the diffusion constant of HSF1, which is higher than that of inert eGFP molecules and increases after heat shock. This finding is consistent with the fact that heat-shock response is associated with an increase of HSF1 interactions with DNA and cannot be explained even partially by heat-induced modifications of nuclear organization.
Assuntos
Resposta ao Choque Térmico , Espectrometria de Fluorescência/métodos , Sítios de Ligação , Calibragem , Linhagem Celular Tumoral , Sobrevivência Celular , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Cinética , FotodegradaçãoRESUMO
The aim of this study was to investigate the possible role of tubulin ßII, a cytoskeletal protein, in regulation of mitochondrial oxidative phosphorylation and energy fluxes in heart cells. This isotype of tubulin is closely associated with mitochondria and co-expressed with mitochondrial creatine kinase (MtCK). It can be rapidly removed by mild proteolytic treatment of permeabilized cardiomyocytes in the absence of stimulatory effect of cytochrome c, that demonstrating the intactness of the outer mitochondrial membrane. Contrary to isolated mitochondria, in permeabilized cardiomyocytes (in situ mitochondria) the addition of pyruvate kinase (PK) and phosphoenolpyruvate (PEP) in the presence of creatine had no effect on the rate of respiration controlled by activated MtCK, showing limited permeability of voltage-dependent anion channel (VDAC) in mitochondrial outer membrane (MOM) for ADP regenerated by MtCK. Under normal conditions, this effect can be considered as one of the most sensitive tests of the intactness of cardiomyocytes and controlled permeability of MOM for adenine nucleotides. However, proteolytic treatment of permeabilized cardiomyocytes with trypsin, by removing mitochondrial ßII tubulin, induces high sensitivity of MtCK-regulated respiration to PK-PEP, significantly changes its kinetics and the affinity to exogenous ADP. MtCK coupled to ATP synthasome and to VDAC controlled by tubulin ßII provides functional compartmentation of ATP in mitochondria and energy channeling into cytoplasm via phosphotransfer network. Therefore, direct transfer of mitochondrially produced ATP to sites of its utilization is largely avoided under physiological conditions, but may occur in pathology when mitochondria are damaged. This article is part of a Special Issue entitled ''Local Signaling in Myocytes''.
Assuntos
Metabolismo Energético/fisiologia , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Tubulina (Proteína)/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Respiração Celular , Creatina Quinase Mitocondrial/metabolismo , Masculino , Microscopia Confocal , Microscopia de Fluorescência , Membranas Mitocondriais/metabolismo , Consumo de Oxigênio , Transporte Proteico , Ratos , Ratos WistarRESUMO
This is our second article devoted to the cardiac myoarchitecture considered as a nematic chiral liquid crystal (NCLC). While the first article focused on the myoarchitecture of the left ventricle (LV), this new article extends to the whole ventricular mass and introduces the concept of disclinations and topological singularities, which characterize the differences and relationships between the left and right ventricles (RV). At the level of the ventricular apices, we constantly observed a vortex shape at the LV apex, corresponding, in the terminology of liquid crystals, to a "+1 disclination"; we never observed this at the RV apex. At the level of the interventricular septum (IVS), we identified "-1/2 disclinations" at the anterior and posterior parts. During the perinatal period, there was a significant difference in their distribution, with more "-1/2 disclinations" in the posterior part of the IVS. After birth, concomitant to major physiological changes, the number of "-1/2 disclinations" significantly decreased, both in the anterior and posterior parts of the IVS. Finally, the description of the disclinations must be considered in any attempt to segment the whole ventricular mass, in biomechanical studies, and, more generally, for the characterization of myocardial remodeling.
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Cancer cell migration is a widely studied topic but has been very often limited to two dimensional motion on various substrates. Indeed, less is known about cancer cell migration in 3D fibrous-extracellular matrix (ECM) including variations of the microenvironment. Here we used 3D time lapse imaging on a confocal microscope and a phase correlation method to follow fiber deformations, as well as cell morphology and live actin distribution during the migration of cancer cells. Different collagen concentrations together with three bladder cancer cell lines were used to investigate the role of the metastatic potential on 3D cell migration characteristics. We found that grade-3 cells (T24 and J82) are characterized by a great diversity of shapes in comparison with grade-2 cells (RT112). Moreover, grade-3 cells with the highest metastatic potential (J82) showed the highest values of migration speeds and diffusivities at low collagen concentration and the greatest sensitivity to collagen concentration. Our results also suggested that the small shape fluctuations of J82 cells are the signature of larger migration velocities. Moreover, the displacement fields generated by J82 cells showed significantly higher fiber displacements as compared to T24 and RT112 cells, regardless of collagen concentration. The analysis of cell movements enhanced the fact that bladder cancer cells were able to exhibit different phenotypes (mesenchymal, amoeboid). Furthermore, the analysis of spatio-temporal migration mechanisms showed that cancer cells are able to push or pull on collagen fibers, therefore producing efficient local collagen deformations in the vicinity of cells. Our results also revealed that dense actin regions are correlated with the largest displacement fields, and this correlation is enhanced for the most invasive J82 cancer cells. Therefore this work opens up new routes to understand cancer cell migration in soft biological networks.
Assuntos
Actinas , Neoplasias da Bexiga Urinária , Actinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Humanos , Microambiente Tumoral , Neoplasias da Bexiga Urinária/patologiaRESUMO
Fluorescence Lifetime Imaging Microscopy (FLIM) is a powerful technique to investigate the local environment of fluorophores in living cells. To correctly estimate all lifetime parameters, time domain FLIM imaging requires a high number of photons and consequently long laser exposure times. This is an issue because long exposure times are incompatible with the observation of dynamic molecular events and induce cellular stress. To minimize exposure time, we have developed an original approach that statistically inflates the number of collected photons. Our approach, called Adaptive Monte Carlo Data Inflation (AMDI), combines the well-known bootstrap technique with an adaptive Parzen kernel. We here demonstrate using both Monte Carlo simulations and live cells that our robust method accurately estimate fluorescence lifetimes with exposure time reduced up to 50 times for monoexponential decays (corresponding to a minimum of 20 photons/pixel), and 10 times for biexponential decays (corresponding to a minimum of 5,000 photons/pixel), compared to standard fitting method. Thanks to AMDI, in Förster resonance energy transfer experiments, it is possible to estimate all fitting parameters accurately without constraining any parameters. By reducing the commonly used spatial binning factor, our technique also improves the spatial resolution of FLIM images.
Assuntos
Algoritmos , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Método de Monte Carlo , Transferência Ressonante de Energia de Fluorescência/métodos , Fótons , SoftwareRESUMO
There are still grey areas in the understanding of the myoarchitecture of the ventricular mass. This is despite the progress of investigation methods since the beginning of the 21st century (diffusion tensor magnetic resonance imaging, microcomputed tomography, and polarised light imaging). The objective of this article is to highlight the specificities and the limitations of polarised light imaging (PLI) of the unstained myocardium embedded in methyl methacrylate (MMA). Thus, to better differentiate our method from other PLI modes, we will refer to it by the acronym PLI-MMA. PLI-MMA shows that the myosin mesh of the compact left ventricular wall behaves like a biological analogous of a nematic chiral liquid crystal. Results obtained by PLI-MMA are: the main direction of the myosin molecules contained in an imaged voxel, the crystal liquid director n, and a regional isotropy index RI that is an orientation tensor, the equivalent of the crystal liquid order parameter. The vector n is collinear with the first eigenvector of diffusion tensor imaging (DTI-MRI). The RI has not been confounded with the diffusion tensor of DTI that gives information about the three eigenvectors of the ellipsoid of diffusion. PLI-MMA gives no information about the collagen network. The physics of soft matter has allowed the revisiting of Streeter's conjecture on the myoarchitecture of the compact left ventricular wall: "geodesics on a nested set of toroidal surfaces". Once the torus topology is understood, this characterisation of the myoarchitecture is more accurate and parsimonious than former descriptions. Finally, this article aims to be an enthusiastic invitation to a transdisciplinary approach between physicists of liquid crystals, anatomists, and specialists of imaging.
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Shortwave infrared window (SWIR: 1000-1700 nm) represents a major improvement compared to the NIR-I region (700-900 nm) in terms of temporal and spatial resolutions in depths down to 4 mm. SWIR is a fast and cheap alternative to more precise methods such as X-ray and opto-acoustic imaging. Main obstacles in SWIR imaging are the noise and scattering from tissues and skin that reduce the precision of the method. We demonstrate that the combination of SWIR in vivo imaging in the NIR-IIb region (1500-1700 nm) with advanced deep learning image analysis allows to overcome these obstacles and making a large step forward to high resolution imaging: it allows to precisely segment vessels from tissues and noise, provides morphological structure of the vessels network, with learned pseudo-3D shape, their relative position, dynamic information of blood vascularization in depth in small animals and distinguish the vessels types: artieries and veins. For demonstration we use neural network IterNet that exploits structural redundancy of the blood vessels, which provides a useful analysis tool for raw SWIR images.
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Aprendizado Profundo , Animais , Raios Infravermelhos , Redes Neurais de Computação , Ondas de RádioRESUMO
We evaluated the impact of light-scattering effects on spatial resolution in different shortwave infrared (SWIR) sub-regions by analyzing two SWIR emissive phantoms made of polydimethylsiloxane (PDMS)-gold nanoclusters (Au NCs) composite covered with mice skin, or capillary tubes filled with Au NCs or IRDye 800CW at different depth in intralipids and finally, after administration of the Au NCs intravenously in mice. Our findings highlighted the benefit of working at the highest tested spectral range of the SWIR region with a 50% enhancement of spatial resolution measured in artificial model when moving from NIR-II (1000-1300 nm) to NIR-IIa (1300-1450 nm) region, and a 25% reduction of the scattering from the skin determined by point spread function analysis from the NIR-II to NIR-IIb region (1500-1700 nm). We also confirmed that a series of Monte Carlo restoration of images significantly improved the spatial resolution in vivo in mice in deep tissues both in the NIR-II and NIR-IIa spectral windows.
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Ouro , Ondas de Rádio , Animais , Raios Infravermelhos , Camundongos , Imagem Óptica , Imagens de FantasmasRESUMO
Microfibrils are macromolecular complexes associated with elastin to form elastic fibers that endow extensible tissues, such as arteries, lungs, and skin, with elasticity property. Fibrillin-1, the main component of microfibrils, is a 350-kDa glycoprotein for which genetic haploinsufficiency in humans can lead to Marfan syndrome, a severe polyfeatured pathology including aortic aneurysms and dissections. Microfibrils and fibrillin-1 fragments mediate adhesion of several cell types, including endothelial cells, while fibrillin-1 additionally triggers lung and mesangial cell migration. However, fibrillin-1-induced intracellular signaling is unknown. We have studied the signaling events induced in human umbilical venous endothelial cells (HUVECs) by aortic microfibrils as well as recombinant fibrillin-1 Arg-Gly-Asp (RGD)-containing fragments PF9 and PF14. Aortic microfibrils and PF14, not PF9, substantially and dose dependently increased HUVEC cytoplasmic and nuclear calcium levels measured using the fluorescent dye Fluo-3. This effect of PF14 was confirmed in bovine aortic endothelial cells. PF14 action in HUVECs was mediated by αvß3 and α5ß1 integrins, phospholipase-C, inosital 1,4,5-trisphosphate, and mobilization of intracellular calcium stores, whereas membrane calcium channels were not or only slightly implicated, as shown in patch-clamp experiments. Finally, PF14 enhanced endothelial cell proliferation and migration. Hence, fibrillin-1 sequences may physiologically activate endothelial cells. Genetic fibrillin-1 deficiency could alter normal endothelial signaling and, since endothelium dysfunction is an important contributor to Marfan syndrome, participate in the arterial anomalies associated with this developmental disease.