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The correct inheritance of chromatin structure is key for maintaining genome function and cell identity and preventing cellular transformation. DEK, a conserved non-histone chromatin protein, has recognized tumor-promoting properties, its overexpression being associated with poor prognosis in various cancer types. At the cellular level, DEK displays pleiotropic functions, influencing differentiation, apoptosis and stemness, but a characteristic oncogenic mechanism has remained elusive. Here, we report the identification of DEK bodies, focal assemblies of DEK that regularly occur at specific, yet unidentified, sites of heterochromatin replication exclusively in late S-phase. In these bodies, DEK localizes in direct proximity to active replisomes in agreement with a function in the early maturation of heterochromatin. A high-throughput siRNA screen, supported by mutational and biochemical analyses, identifies SUMO as one regulator of DEK body formation, linking DEK to the complex SUMO protein network that controls chromatin states and cell fate. This work combines and refines our previous data on DEK as a factor essential for heterochromatin integrity and facilitating replication under stress, and delineates an avenue of further study for unraveling the contribution of DEK to cancer development.
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Heterocromatina , Neoplasias , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , CromatinaRESUMO
Adult neural progenitor cells (aNPCs) ensure lifelong neurogenesis in the mammalian hippocampus. Proper regulation of aNPC fate has thus important implications for brain plasticity and healthy aging. Piwi proteins and the small noncoding RNAs interacting with them (piRNAs) have been proposed to control memory and anxiety, but the mechanism remains elusive. Here, we show that Piwil2 (Mili) is essential for proper neurogenesis in the postnatal mouse hippocampus. RNA sequencing of aNPCs and their differentiated progeny reveal that Mili and piRNAs are dynamically expressed in neurogenesis. Depletion of Mili and piRNAs in the adult hippocampus impairs aNPC differentiation toward a neural fate, induces senescence, and generates reactive glia. Transcripts modulated upon Mili depletion bear sequences complementary or homologous to piRNAs and include repetitive elements and mRNAs encoding essential proteins for proper neurogenesis. Our results provide evidence of a critical role for Mili in maintaining fitness and proper fate of aNPCs, underpinning a possible involvement of the piRNA pathway in brain plasticity and successful aging.
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Proteínas Argonautas , Hipocampo , Neurogênese , Animais , Camundongos , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Senescência Celular/genética , Hipocampo/metabolismo , Mamíferos/genética , Mamíferos/metabolismo , Neurogênese/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
The small GTPases, Rab5 and Rac, are essential for endocytosis and actin remodeling, respectively. Coordination of these processes is critical to achieve spatial restriction of intracellular signaling, which is essential for a variety of polarized functions. Here, we show that clathrin- and Rab5-mediated endocytosis are required for the activation of Rac induced by motogenic stimuli. Rac activation occurs on early endosomes, where the RacGEF Tiam1 is also recruited. Subsequent recycling of Rac to the plasma membrane ensures localized signaling, leading to the formation of actin-based migratory protrusions. Thus, membrane trafficking of Rac is required for the spatial resolution of Rac-dependent motogenic signals. We further demonstrate that a Rab5-to-Rac circuitry controls the morphology of motile mammalian tumor cells and primordial germinal cells during zebrafish development, suggesting that this circuitry is relevant for the regulation of migratory programs in various cells, in both in vitro settings and whole organisms.
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Movimento Celular , Endocitose , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Proteínas rab5 de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Linhagem Celular Tumoral , Clatrina/metabolismo , Embrião de Mamíferos/citologia , Embrião não Mamífero/citologia , Endossomos/metabolismo , Células Germinativas/citologia , Células Germinativas/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Camundongos , Peixe-ZebraRESUMO
The properties of signal strength and integrated intensity in a scanned imaging system are reviewed. These properties are especially applied to confocal imaging systems, including image scanning microscopy. The integrated intensity, equal to the image of a uniform planar (sheet) object, rather than the peak of the point spread function, is a measure of the flux in an image. Analytic expressions are presented for the intensity in the detector plane for a uniform volume object, and for the resulting background. The variation in the integrated intensity with defocus for an offset point detector is presented. This axial fingerprint is independent of any pixel reassignment. The intensity in the detector plane is shown to contain the defocus information, and simple processing of the recorded data can improve optical sectioning and background rejection.
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Microscopia Confocal , Microscopia Confocal/métodosRESUMO
We discuss the effects of image scanning microscopy using doughnut beam illumination on the properties of signal strength and integrated intensity. Doughnut beam illumination can give better optical sectioning and background rejection than Airy disk illumination. The outer pixels of a detector array give a signal from defocused regions, so digital processing of these (e.g., by simple subtraction) can further improve optical sectioning and background rejection from a single in-focus scan.
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BACKGROUND: The aim of this study was to evaluate the use of autologous fat graft injection to correct lower eyelid position METHODS: A retrospective, observational, single blind, case-control study was carried out on 94 patients, presenting with lower eyelid retraction in 159 eyes. In the sub-population with monolateral eyelid retraction, the not affected site has been considered as a control and compared with the outcomes recorded after treatment of the contralateral side Follow-up at 12 months was performed with a subjective assessment carried out by a questionnaire administered to patients while objective result assessment was performed 12 months after surgery by two independent blind examiners. RESULTS: The eyelid upward reposition has improved one year after fat grafting in both bilateral (1,52 mm) and unilateral (1,7 mm) population: the latter allowed to statistically validate (P<0.05) the result with respect to the not affected site. CONCLUSIONS: This is the first paper that highlights the outcomes of sole fat injection in the treatment of lower eyelid retraction: blind objective evaluation of surgical outcomes along with a patient assessment of both functional and aesthetic improvement one year after surgery confirm its efficacy and reliability along with the first case/control outcome evaluation of the technique carried on in the sub-population of 29 patients affected by unilateral lower eyelid retraction that validate the average improvement of the retracted eyelid one year after fat grafting as statistically significant. Nevertheless, longer follow-up periods and a larger sample size are needed to thoroughly confirm surgical outcomes and statistical results. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.
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Blefaroplastia , Doenças Palpebrais , Humanos , Estudos Retrospectivos , Blefaroplastia/métodos , Estudos de Casos e Controles , Reprodutibilidade dos Testes , Método Simples-Cego , Resultado do Tratamento , Doenças Palpebrais/cirurgia , Pálpebras/cirurgia , EstéticaRESUMO
DEAR EDITOR: We have read Dr Riccardo Fondrini and Colleagues commentary on our paper, and we are grateful for their remarks. We herewith would clarify the critical points mentioned about fat reabsorption and survival, along with the risk of periorbital chronic oedema as per the recorded outcomes in the presented case series. Lastly, we have unfortunately to disagree with the commentary's point related to recommended injection depth as it is mandatory to point out that in our experience the superficial injection of nano-fat is a crucial step for lower eyelid retraction management, otherwise a retraction may persist due to the untreated scar in the anterior lamella, thus missing the therapeutic goal that we aim to achieve. LEVEL OF EVIDENCE V: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .
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Blefaroplastia , Doenças Palpebrais , Humanos , Estudos Retrospectivos , Estudos de Casos e Controles , Método Simples-Cego , Pálpebras/cirurgiaRESUMO
Increasing evidence indicates that photobiomodulation, based on tissue irradiation with photons in the red to near-infrared spectrum, may be an effective therapeutic approach to central nervous system disorders. Although nervous system functionality has been shown to be affected by photons in animal models, as well as in preliminary evidence in healthy subjects or in patients with neuropsychiatric disorders, the mechanisms involved in the photobiomodulation effects have not yet been clarified. We previously observed that photobiomodulation could stimulate glutamate release. Here, we investigate mechanisms potentially involved in the glutamate-releasing effect of photons from adult mouse cerebrocortical nerve terminals. We report evidence of photon ability to induce an exocytotic vesicular release of glutamate from the terminals of glutamatergic neurons in a power-dependent way. It can be hypothesized that photobiomodulation, depending on the potency, can release glutamate in a potentially neurotoxic or physiological range.
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Ácido Glutâmico , Fótons , Animais , Camundongos , Córtex Cerebral , Ácido Glutâmico/farmacologia , Terminações Nervosas , Neurônios , SinaptossomosRESUMO
The molecular mechanisms that underlie oncogene-induced genomic damage are still poorly understood. To understand how oncogenes affect chromatin architecture, it is important to visualize fundamental processes such as DNA replication and transcription in intact nuclei and quantify the alterations of their spatiotemporal organization induced by oncogenes. Here, we apply superresolution microscopy in combination with image cross correlation spectroscopy to the U937-PR9 cell line, an in vitro model of acute promyelocytic leukemia that allows us to activate the expression of the PML-RARα oncogene and analyze its effects on the spatiotemporal organization of functional nuclear processes. More specifically, we perform Tau-stimulated emission depletion imaging, a superresolution technique based on the concept of separation of photons by lifetime tuning. Tau-stimulated emission depletion imaging is combined with a robust image analysis protocol that quickly produces a value of colocalization fraction on several hundreds of single cells and allows observation of cell-to-cell variability. Upon activation of the oncogene, we detect a significant increase in the fraction of transcription sites colocalized with PML/PML-RARα. This increase of colocalization can be ascribed to oncogene-induced disruption of physiological PML bodies and the abnormal occurrence of a relatively large number of PML-RARα microspeckles. We also detect a significant cell-to-cell variability of this increase of colocalization, which can be ascribed, at least in part, to a heterogeneous response of the cells to the activation of the oncogene. These results prove that our method efficiently reveals oncogene-induced alterations in the spatial organization of nuclear processes and suggest that the abnormal localization of PML-RARα could interfere with the transcription machinery, potentially leading to DNA damage and genomic instability.
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Leucemia Promielocítica Aguda , Proteínas de Fusão Oncogênica , Humanos , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/metabolismo , Oncogenes , Análise EspectralRESUMO
The antisecretory factor (AF) is an endogenous protein that counteracts intestinal hypersecretion and various inflammation conditions in vivo. It has been detected in many mammalian tissues and plasma, but its mechanisms of action are largely unknown. To study the pharmacological action of the AF on different GABAA receptor populations in cerebellar granule cells, we took advantage of the two-photon uncaging method as this technique allows to stimulate the cell locally in well-identified plasma membrane parts. We compared the electrophysiological response evoked by releasing a caged GABA compound on the soma, the axon initial segment and neurites before and after administering AF-16, a 16 amino acids long peptide obtained from the amino-terminal end of the AF protein. After the treatment with AF-16, we observed peak current increases of varying magnitude depending on the neuronal region. Thus, studying the effects of furosemide and AF-16 on the electrophysiological behaviour of cerebellar granules, we suggest that GABAA receptors, containing the α6 subunit, may be specifically involved in the increase of the peak current by AF, and different receptor subtype distribution may be responsible for differences in this increase on the cell.
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Neuropeptídeos , Receptores de GABA-A , Animais , Cerebelo/fisiologia , Mamíferos/metabolismo , Neurônios/fisiologia , Neuropeptídeos/metabolismo , Ratos , Receptores de GABA-A/metabolismo , Ácido gama-Aminobutírico/metabolismo , Ácido gama-Aminobutírico/farmacologiaRESUMO
Image scanning microscopy (ISM) can improve the effective spatial resolution of confocal microscopy to its theoretical limit. However, current implementations are not robust or versatile, and are incompatible with fluorescence lifetime imaging (FLIM). We describe an implementation of ISM based on a single-photon detector array that enables super-resolution FLIM and improves multicolor, live-cell and in-depth imaging, thereby paving the way for a massive transition from confocal microscopy to ISM.
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Processamento de Imagem Assistida por Computador/métodos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Algoritmos , Animais , Biologia Computacional , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Poro Nuclear/metabolismo , Imagem Óptica , Fótons , Software , Tubulina (Proteína)/químicaRESUMO
We report the development of a supramolecular structure endowed with photosensitizing properties and targeting capability for antimicrobial photodynamic inactivation. Our synthetic strategy uses the tetrameric bacterial protein streptavidin, labeled with the photosensitizer eosin, as the main building block. Biotinylated immunoglobulin G (IgG) from human serum, known to associate with Staphylococcus aureus protein A, was bound to the complex streptavidin-eosin. Fluorescence correlation spectroscopy and fluorescence microscopy demonstrate binding of the complex to S. aureus. Efficient photoinactivation is observed for S. aureus suspensions treated with IgG-streptavidin-eosin at concentrations higher than 0.5 µM and exposed to green light. The proposed strategy offers a flexible platform for targeting a variety of molecules and microbial species.
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Anti-Infecciosos , Fotoquimioterapia , Antibacterianos/farmacologia , Anti-Infecciosos/química , Amarelo de Eosina-(YS) , Humanos , Imunoglobulina G , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Staphylococcus aureus/fisiologia , EstreptavidinaRESUMO
Measures of purity for 3D partially polarized fields, and in particular, the separation into circularly and linearly polarized contributions, are reexamined, and a new degree of total linear polarization introduced. Explicit expressions for the characteristic decomposition in terms of coherency matrix elements are presented, including the special case of an intrinsic coherency matrix. Parameterization of the coherency matrix in terms of ellipticity, and the directions of the ellipse normal and major axis are investigated. Phase consistency is discussed. A comprehensive collection of results regarding intrinsic polarization properties is presented.
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BACKGROUND: Hyaluronic acid (HA) gel injections were first used to treat the tear trough in 2005 and since then it has been a mainstay of the approach to lower eyelid deformities. OBJECTIVE: The authors present this retrospective multicentric observational study based on single-blind objective and subjective evaluation and patient satisfaction in relation to the aesthetic improvement of a large group of patients treated. METHODS AND MATERIALS: Between January 2016 and December 2019, 600 patients (468 women and 132 men), were enrolled in this study, and 1200 tear trough deformities were treated with both needle and cannula techniques. RESULTS: Average follow-up time was 12 ± 1 months, and the outcomes were assessed both objectively and subjectively with respect to Hirmand's classification. Statistical analysis shows an inverse correlation between age and class amelioration. CONCLUSION: HA injection of the tear trough is most effective in patients between 30 and 40 years of age, while its benefits extend to up to 50 years old; afterward, it should no longer be the treatment of choice. This confirms that correction of tear trough with hyaluronic acid injections may provide an option to achieve immediate and durable results for up to one year after the injection. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .
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Blefaroplastia , Técnicas Cosméticas , Preenchedores Dérmicos , Blefaroplastia/métodos , Pálpebras/cirurgia , Feminino , Humanos , Ácido Hialurônico , Masculino , Estudos Retrospectivos , Método Simples-Cego , Resultado do TratamentoRESUMO
In spite of their value as genetically encodable reporters for imaging in living systems, fluorescent proteins have been used sporadically for stimulated emission depletion (STED) super-resolution imaging, owing to their moderate photophysical resistance, which does not enable reaching resolutions as high as for synthetic dyes. By a rational approach combining steady-state and ultrafast spectroscopy with gated STED imaging in living and fixed cells, we here demonstrate that F99S/M153T/V163A GFP (c3GFP) represents an efficient genetic reporter for STED, on account of no excited state absorption at depletion wavelengths <600 nm and a long emission lifetime. This makes c3GFP a valuable alternative to more common, but less photostable, EGFP and YFP/Citrine mutants for STED imaging studies targeting the green-yellow region of the optical spectrum.
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Corantes Fluorescentes , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/genética , Microscopia de Fluorescência/métodosRESUMO
Mueller matrix microscopy is an advanced imaging technique providing a full characterization of the optical polarization fingerprint of a sample. The Lu-Chipman (LC) decomposition, a method based on the modeling of elementary polarimetric arrangements and matrix inversions, is the gold standard to extract each polarimetric component separately. However, this models the optical system as a small number of discrete optical elements and requires a priori knowledge of the order in which these elements occur. In stratified media or when the ordering is not known, the interpretation of the LC decomposition becomes difficult. In this work, we propose a new, to our knowledge, representation dedicated to the study of biological tissues that combines Mueller matrix microscopy with a phasor approach. We demonstrate that this method provides an easier and direct interpretation of the retardance images in any birefringent material without the use of mathematical assumptions regarding the structure of the sample and yields comparable contrast to the LC decomposition. By validating this approach through numerical simulations, we demonstrate that it is able to give access to localized structural information, resulting in a simple determination of the birefringent parameters at the microscopic level. We apply our novel, to our knowledge, method to typical biological tissues that are of interest in the field of biomedical diagnosis.
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Dispositivos Ópticos , Humanos , Microscopia Confocal , Imagem Óptica , Análise EspectralRESUMO
Chromatin in the nucleus is organized in functional sites at variable level of compaction. Structured illumination microscopy (SIM) can be used to generate three-dimensional super-resolution (SR) imaging of chromatin by changing in phase and in orientation a periodic line illumination pattern. The spatial frequency domain is the natural choice to process SIM raw data and to reconstruct an SR image. Using an alternative approach, we demonstrate that the additional spatial information encoded in the knowledge of the position of the illumination pattern can be efficiently decoded using a generalized version of separation of photon by lifetime tuning (SPLIT) that does not require lifetime measurements. In the resulting SPLIT-SIM, the SR image is obtained by isolating a fraction of the intensity corresponding to the center of the diffraction-limited point spread function. This extends the use of the SPLIT approach from stimulated emission depletion microscopy to SIM. The SPLIT-SIM algorithm is based only on phasor analysis and does not require deconvolution. We show that SPLIT-SIM can be used to generate SR images of chromatin organizational motifs with tunable resolution and can be a valuable tool for the imaging of functional sites in the nucleus.
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Processamento de Imagem Assistida por Computador , Iluminação , Cromatina , Imageamento Tridimensional , Microscopia de FluorescênciaRESUMO
Stimulated emission depletion (STED) microscopy provides subdiffraction resolution while preserving useful aspects of fluorescence microscopy, such as optical sectioning, and molecular specificity and sensitivity. However, sophisticated microscopy architectures and high illumination intensities have limited STED microscopy's widespread use in the past. Here we summarize the progress that is mitigating these problems and giving substantial momentum to STED microscopy applications. We discuss the future of this method in regard to spatiotemporal limits, live-cell imaging and combination with spectroscopy. Advances in these areas may elevate STED microscopy to a standard method for imaging in the life sciences.
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Células/citologia , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , HumanosRESUMO
Volume restoration remains the principal target to be addressed when approaching midface rejuvenation, and absorbable sutures can create a suspension system that addresses ptotic skin located primarily in the midface. The aim of this pilot study was to investigate the effectiveness in lifting sagging tissue and to determine the outcome of thread lifting procedures of the midface. Six participants were enrolled and treated with suspension threads to correct mild to moderate ptosis of the flabby tissues of the midface. Volumetric changes were calculated at t0 (pretreatment), t1(120 days) and t2 (365 days), with a mean follow-up time of 349.64 days, (range from 304 to 380.5 days). Results showed an overall average improvement of 5.59 mL at 120 days (t0-t1) after thread implantation, up to a mean value of 4.16 mL at the end of the 350 days follow up (t0-t2). A comparison was made in between and statistical analysis was performed with level of significance set at P < .05. The records shown suggest that it is possible to achieve volume restoration which lasts up to 12 months as all parameters improved significantly (P < .05) at t1 and at t2 with respect to t0. Facial tissues suspension by means of threads is therefore safe and effective, as it is possible to achieve tissue repositioning which lasts up to 12 months for the correction of mild to moderate ptosis of the midface.
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Ritidoplastia , Face , Humanos , Projetos Piloto , Rejuvenescimento , SuturasRESUMO
In image scanning microscopy, the pinhole of a confocal microscope is replaced by a detector array. The point spread function for each detector element can be interpreted as the probability density function of the signal, the peak giving the most likely origin. This thus allows a form of maximum likelihood restoration, and compensation for aberrations, with similarities to adaptive optics. As an example of an aberration, we investigate theoretically and experimentally illumination with a vortex doughnut beam. After reassignment and summation over the detector array, the point spread function is compact, and the resolution and signal level higher than in a conventional microscope.