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The heterogeneity of endothelial cells (ECs) across tissues remains incompletely inventoried. We constructed an atlas of >32,000 single-EC transcriptomes from 11 mouse tissues and identified 78 EC subclusters, including Aqp7+ intestinal capillaries and angiogenic ECs in healthy tissues. ECs from brain/testis, liver/spleen, small intestine/colon, and skeletal muscle/heart pairwise expressed partially overlapping marker genes. Arterial, venous, and lymphatic ECs shared more markers in more tissues than did heterogeneous capillary ECs. ECs from different vascular beds (arteries, capillaries, veins, lymphatics) exhibited transcriptome similarity across tissues, but the tissue (rather than the vessel) type contributed to the EC heterogeneity. Metabolic transcriptome analysis revealed a similar tissue-grouping phenomenon of ECs and heterogeneous metabolic gene signatures in ECs between tissues and between vascular beds within a single tissue in a tissue-type-dependent pattern. The EC atlas taxonomy enabled identification of EC subclusters in public scRNA-seq datasets and provides a powerful discovery tool and resource value.
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Células Endoteliales/metabolismo , Análisis de la Célula Individual , Transcriptoma , Animales , Encéfalo/citología , Sistema Cardiovascular/citología , Células Endoteliales/clasificación , Células Endoteliales/citología , Tracto Gastrointestinal/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Músculos/citología , Especificidad de Órganos , RNA-Seq , Testículo/citologíaRESUMEN
Glutamine synthetase, encoded by the gene GLUL, is an enzyme that converts glutamate and ammonia to glutamine. It is expressed by endothelial cells, but surprisingly shows negligible glutamine-synthesizing activity in these cells at physiological glutamine levels. Here we show in mice that genetic deletion of Glul in endothelial cells impairs vessel sprouting during vascular development, whereas pharmacological blockade of glutamine synthetase suppresses angiogenesis in ocular and inflammatory skin disease while only minimally affecting healthy adult quiescent endothelial cells. This relies on the inhibition of endothelial cell migration but not proliferation. Mechanistically we show that in human umbilical vein endothelial cells GLUL knockdown reduces membrane localization and activation of the GTPase RHOJ while activating other Rho GTPases and Rho kinase, thereby inducing actin stress fibres and impeding endothelial cell motility. Inhibition of Rho kinase rescues the defect in endothelial cell migration that is induced by GLUL knockdown. Notably, glutamine synthetase palmitoylates itself and interacts with RHOJ to sustain RHOJ palmitoylation, membrane localization and activation. These findings reveal that, in addition to the known formation of glutamine, the enzyme glutamine synthetase shows unknown activity in endothelial cell migration during pathological angiogenesis through RHOJ palmitoylation.
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Células Endoteliales/enzimología , Células Endoteliales/patología , Glutamato-Amoníaco Ligasa/metabolismo , Glutamina/biosíntesis , Neovascularización Patológica , Actinas/metabolismo , Animales , Movimiento Celular , Células Endoteliales/metabolismo , Femenino , Glutamato-Amoníaco Ligasa/deficiencia , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/fisiología , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/enzimología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Lipoilación , Ratones , Ácido Palmítico/metabolismo , Procesamiento Proteico-Postraduccional , Fibras de Estrés/metabolismo , Proteínas de Unión al GTP rho/química , Proteínas de Unión al GTP rho/metabolismo , Quinasas Asociadas a rho/metabolismoRESUMEN
An impedimetric sensing strategy was developed for sensitively determining diethylstilbestrol (DES) based on a platform of porphyrin-containing covalent-organic framework (p-COF). The p-COF was synthesized using 5,10,15,20-tetra (4-aminophenyl) porphyrin (TAPP) and 1,3,6,8-tetrakis(4-formylphenyl) pyrene (TFPy) as building blocks via condensation reaction, for which p-COF was named as TAPP-TFPy-COF. Considering the large specific surface area (302.9 m2 g-1), high porosity, rich nitrogen functionality, superior electrochemical activity, and strong bioaffinity toward DNA strands, the TAPP-TFPy-COF-based platform exhibited enhanced, non-label, and amplified electrochemical signal, large number of immobilized DES-targeted aptamer strands, and fast-response toward the analyte. Electrochemical results reveal that the TAPP-TFPy-COF-based aptasensor promoted the sensing performance for the detection of DES, resulting in an extremely low limit of detection of 0.42 fg mL-1 within a DES concentration ranging from 1 fg mL-1 to 0.1 pg mL-1, which was substantially lower than those of most reported DES sensors. Furthermore, the TAPP-TFPy-COF-based aptasensor possessed outperformed stability, high selectivity, ascendant reproducibility, and acceptable applicability in diverse environments. The recovery values for DES detection in milk, tap water, and frozen shrimp were in the range 91.80-118.50% with low relative standard deviation of 0.11-4.26%. This work provides a new sensing electrochemical approach based on COF network for DES detection and shows a deep insight into the construction of COF-based biosensors, which can be extended to be used for other target compounds.
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Aptámeros de Nucleótidos , Estructuras Metalorgánicas , Porfirinas , Aptámeros de Nucleótidos/química , Dietilestilbestrol , Límite de Detección , Estructuras Metalorgánicas/química , Porfirinas/química , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Renal endothelial cells from glomerular, cortical, and medullary kidney compartments are exposed to different microenvironmental conditions and support specific kidney processes. However, the heterogeneous phenotypes of these cells remain incompletely inventoried. Osmotic homeostasis is vitally important for regulating cell volume and function, and in mammals, osmotic equilibrium is regulated through the countercurrent system in the renal medulla, where water exchange through endothelium occurs against an osmotic pressure gradient. Dehydration exposes medullary renal endothelial cells to extreme hyperosmolarity, and how these cells adapt to and survive in this hypertonic milieu is unknown. METHODS: We inventoried renal endothelial cell heterogeneity by single-cell RNA sequencing >40,000 mouse renal endothelial cells, and studied transcriptome changes during osmotic adaptation upon water deprivation. We validated our findings by immunostaining and functionally by targeting oxidative phosphorylation in a hyperosmolarity model in vitro and in dehydrated mice in vivo. RESULTS: We identified 24 renal endothelial cell phenotypes (of which eight were novel), highlighting extensive heterogeneity of these cells between and within the cortex, glomeruli, and medulla. In response to dehydration and hypertonicity, medullary renal endothelial cells upregulated the expression of genes involved in the hypoxia response, glycolysis, and-surprisingly-oxidative phosphorylation. Endothelial cells increased oxygen consumption when exposed to hyperosmolarity, whereas blocking oxidative phosphorylation compromised endothelial cell viability during hyperosmotic stress and impaired urine concentration during dehydration. CONCLUSIONS: This study provides a high-resolution atlas of the renal endothelium and highlights extensive renal endothelial cell phenotypic heterogeneity, as well as a previously unrecognized role of oxidative phosphorylation in the metabolic adaptation of medullary renal endothelial cells to water deprivation.
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Adaptación Fisiológica/genética , Células Endoteliales/metabolismo , Riñón/citología , Análisis de Secuencia de ARN , Privación de Agua/fisiología , Animales , Células Endoteliales/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , FenotipoRESUMEN
A novel label-free surface plasmon resonance (SPR) aptasensor has been constructed for the detection of N-gene of SARS-CoV-2 by using thiol-modified niobium carbide MXene quantum dots (Nb2C-SH QDs) as the bioplatform for anchoring N-gene-targeted aptamer. In the presence of SARS-CoV-2 N-gene, the immobilized aptamer strands changed their conformation to specifically bind with N-gene. It thus increased the contact area or enlarged the distance between aptamer and the SPR chip, resulting in a change of the SPR signal irradiated by the laser (He-Ne) with the wavelength (λ) of 633 nm. Nb2C QDs were derived from Nb2C MXene nanosheets via a solvothermal method, followed by functionalization with octadecanethiol through a self-assembling method. Subsequently, the gold chip for SPR measurements was modified with Nb2C-SH QDs via covalent binding of the Au-S bond also by self-assembling interaction. Nb2C-SH QDs not only resulted in high bioaffinity toward aptamer but also enhanced the SPR response. Thus, the Nb2C-SH QD-based SPR aptasensor had low limit of detection (LOD) of 4.9 pg mL-1 toward N-gene within the concentration range 0.05 to 100 ng mL-1. The sensor also showed excellent selectivity in the presence of various respiratory viruses and proteins in human serum and high stability. Moreover, the Nb2C-SH QD-based SPR aptasensor displayed a vast practical application for the qualitative analysis of N-gene from different samples, including seawater, seafood, and human serum. Thus, this work can provide a deep insight into the construction of the aptasensor for detecting SARS-CoV-2 in complex environments. A novel label-free surface plasmon resonance aptasensor has been constructed to detect sensitively and selectively the N-gene of SARS-CoV-2 by using thiol-modified niobium carbide MXene quantum dots as the scaffold to anchor the N-gene-targeted aptamer.
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Aptámeros de Nucleótidos , COVID-19/diagnóstico , Niobio/química , Nucleocápside/metabolismo , Puntos Cuánticos/química , SARS-CoV-2/aislamiento & purificación , Resonancia por Plasmón de Superficie/métodos , COVID-19/virología , Humanos , Límite de DetecciónRESUMEN
Oxidative stress, due to insufficiency of antioxidants or over-production of oxidants, can lead to severe cell and tissue damage. Oxidative stress occurs constantly and has been shown to be involved in innumerable diseases, such as degenerative, cardiovascular, neurological, and metabolic disorders, cancer, and aging, thus highlighting the vital need of antioxidant defense mechanisms. Vascular endothelial growth factor B (VEGF-B) was discovered a long time ago, and is abundantly expressed in most types of cells and tissues. VEGF-B remained functionally mysterious for many years and later on has been shown to be minimally angiogenic. Recently, VEGF-B is reported to be a potent antioxidant by boosting the expression of key antioxidant enzymes. Thus, one major role of VEGF-B lies in safeguarding tissues and cells from oxidative stress-induced damage. VEGF-B may therefore have promising therapeutic utilities in treating oxidative stress-related diseases. In this review, we discuss the current knowledge on the newly discovered antioxidant function of VEGF-B and the related molecular mechanisms, particularly, in relationship to some oxidative stress-related diseases, such as retinitis pigmentosa, age-related macular degeneration, diabetic retinopathy, glaucoma, amyotrophic lateral sclerosis, Alzheimer's disease, and Parkinson's disease.
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Antioxidantes/uso terapéutico , Oftalmopatías/tratamiento farmacológico , Enfermedades Neurodegenerativas/tratamiento farmacológico , Factor B de Crecimiento Endotelial Vascular/uso terapéutico , Animales , Antioxidantes/farmacología , Oftalmopatías/metabolismo , Humanos , Enfermedades Neurodegenerativas/metabolismo , Factor B de Crecimiento Endotelial Vascular/metabolismo , Factor B de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
Thermoplastic polyurethane (TPU)/polypropylene (PP) blends of different weight ratios were prepared with a self-made vane extruder (VE), which generates global dynamic elongational flow, and a traditional twin-screw extruder (TSE), which generates shear flow. High-resolution scanning electron microscopy and polarizing microscopy showed a structure feature of fiber morphology and a clear interlocking structure of spherulites of PP/TPU blends prepared with a VE. The wide-angle X-ray diffraction results showed that the TPU/PP blend based on dynamic elongational flow had evident crystalline structure of the ß form as a function of PP (90 wt %), compared to that of the conventional shear flow processing techniques. A significant improvement of the mechanical properties was obtained; the samples prepared with a VE had superior mechanical properties compared to those of the samples prepared with a TSE. Interestingly, differential scanning calorimetry curves showed that dynamic elongational flow could successfully improve the crystallinity of the PP/TPU blends. Furthermore, dynamic thermomechanical and thermogravimetric analysis curves revealed the apparent partial miscibility and strong interaction of the PP/TPU blends influenced by dynamic elongational flow, compared to that of TSE-extruded. Further research will provide significant understanding of the spherulite interface and high-performance manipulation of PP/TPU blends under dynamic elongational flow, achieving superior PP/TPU blends.
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Purpose: The underlying mechanism of congenital cataracts caused by deficiency or mutation of junctional adhesion molecule C (JAM-C) gene remains unclear. Our study aims to elucidate the abnormal developmental process in Jamc-/- lenses and reveal the genes related to lens development that JAM-C may regulate. Methods: Jamc knockout (Jamc-/-) mouse embryos and pups were generated for in vivo studies. Four key developmental stages from embryonic day (E) 12.5 to postnatal day (P) 0.5 were selected for the following experiments. Hematoxylin and eosin staining was used for histological analysis. The 5-bromo-2'-deoxyuridine (BrdU) incorporation assay and TUNEL staining were performed to label lens epithelial cell (LEC) proliferation and apoptosis, respectively. Immunofluorescence and Western blot were used to analyze the markers of lens epithelium, cell cycle exit, and lens fiber differentiation. Results: JAM-C was expressed throughout the process of lens development. Deletion of Jamc resulted in decreased lens size and disorganized lens fibers, which arose from E16.5 and aggravated gradually. The LECs of Jamc-/- lenses showed decreased quantity and proliferation, accompanied with reduction of key transcription factor, FOXE3. The fibers in Jamc-/- lenses were disorganized. Moreover, Jamc-deficient lens fibers showed significantly altered distribution patterns of Cx46 and Cx50. The marker of fiber homeostasis, γ-crystallin, was also decreased in the inner cortex and core fibers of Jamc-/- lenses. Conclusions: Deletion of JAM-C exhibits malfunction of LEC proliferation and fiber maturation during murine lens development, which may be related to the downregulation of FOXE3 expression and abnormal localization patterns of Cx46 and Cx50.
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Molécula C de Adhesión de Unión , Cristalino , Animales , Ratones , Diferenciación Celular/fisiología , Proliferación Celular , Células Epiteliales/metabolismo , Epitelio , Molécula C de Adhesión de Unión/metabolismo , Cristalino/metabolismo , Ratones NoqueadosRESUMEN
Although mitogen-inducible gene 6 (MIG6) is highly expressed in vascular endothelial cells, it remains unknown whether MIG6 affects vascular permeability. Here, we show for the first time a critical role of MIG6 in limiting vascular permeability. We unveil that genetic deletion of Mig6 in mice markedly increased VEGFA-induced vascular permeability, and MIG6 knockdown impaired endothelial barrier function. Mechanistically, we reveal that MIG6 inhibits VEGFR2 phosphorylation by binding to the VEGFR2 kinase domain 2, and MIG6 knockdown increases the downstream signaling of VEGFR2 by enhancing phosphorylation of PLCγ1 and eNOS. Moreover, MIG6 knockdown disrupted the balance between RAC1 and RHOA GTPase activation, leading to endothelial cell barrier breakdown and the elevation of vascular permeability. Our findings demonstrate an essential role of MIG6 in maintaining endothelial cell barrier integrity and point to potential therapeutic implications of MIG6 in the treatment of diseases involving vascular permeability. Xing et al. (2022) investigated the critical role of MIG6 in vascular permeability. MIG6 deficiency promotes VEGFA-induced vascular permeability via activation of PLCγ1-Ca2+-eNOS signaling and perturbation of the balance in RAC1/RHOA activation, resulting in endothelial barrier disruption.
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Although VEGF-B was discovered as a VEGF-A homolog a long time ago, the angiogenic effect of VEGF-B remains poorly understood with limited and diverse findings from different groups. Notwithstanding, drugs that inhibit VEGF-B together with other VEGF family members are being used to treat patients with various neovascular diseases. It is therefore critical to have a better understanding of the angiogenic effect of VEGF-B and the underlying mechanisms. Using comprehensive in vitro and in vivo methods and models, we reveal here for the first time an unexpected and surprising function of VEGF-B as an endogenous inhibitor of angiogenesis by inhibiting the FGF2/FGFR1 pathway when the latter is abundantly expressed. Mechanistically, we unveil that VEGF-B binds to FGFR1, induces FGFR1/VEGFR1 complex formation, and suppresses FGF2-induced Erk activation, and inhibits FGF2-driven angiogenesis and tumor growth. Our work uncovers a previously unrecognized novel function of VEGF-B in tethering the FGF2/FGFR1 pathway. Given the anti-angiogenic nature of VEGF-B under conditions of high FGF2/FGFR1 levels, caution is warranted when modulating VEGF-B activity to treat neovascular diseases.
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Factor 2 de Crecimiento de Fibroblastos , Factor B de Crecimiento Endotelial Vascular , Humanos , Factor 2 de Crecimiento de Fibroblastos/genética , Inmunoterapia , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genéticaRESUMEN
In the previous study of the mud crab (Scylla paramamosain) hemocyte proteins, which interacted with a bacterium, Vibrio parahaemolyticus, a protein known as antilipopolysaccharide factor (Sp-ALF) was isolated in addition to a serine proteinase homolog (Sp-SPH) protein. In the present study, we further reported the characterization of two isoforms of the mud crab ALF - Sp-ALFs genes (designated as Sp-ALF1 and Sp-ALF2, respectively) based on our previous result. The Sp-ALF1 and Sp-ALF2 cDNA contained 1070 bp and 731 bp, respectively, with 123 deduced amino acid residues. Alignment of deduced amino acid sequences showed that Sp-ALFs possessed high identity with other known ALFs from crustaceans and exhibited an overall similarity of 57.7% to those of ALFs compared. Phylogenetic tree analysis revealed a clear group of each species and also suggested that ALFs from Scylla genus and those from Portunus genus were closely related. Tissue distribution analysis in adult crab implied that both Sp-ALF1 and Sp-ALF2 were mainly expressed in hemocytes. The mRNA transcripts were also found in embryo (I, II, III and V), zoea-I and juvenile crab, but were rarely observed in the megalopa stage. To further identify the biological activity of Sp-ALFs, recombinant proteins (rSp-ALFs: designated as rSp-ALF1 and rSp-ALF2, respectively) were obtained by expression in Pichia pastris, and the synthetic peptide fragments (sSp-ALFs: designated as sSp-ALF1 and sSp-ALF2, respectively) including the putative LPS binding loop were also prepared for antimicrobial test. The results indicated that both rSp-ALFs and sSp-ALFs were highly effective against most of the Gram-positive bacteria and Gram-negative bacteria tested. In contrast to cecropin P1, a membrane integrity assay revealed that Sp-ALFs did not affect the Escherichia coli by disruption of membrane integrity. Additionally, the recombinant Sp-ALFs proteins exhibited strong antiviral activity against an important aquaculture pathogen, white spot syndrome virus, in crustaceans. Taken together, these data suggested that Sp-ALFs might play a key role in immune defense against microbial infection in the mud crab S. paramamosain.
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Péptidos Catiónicos Antimicrobianos/genética , Braquiuros/genética , Secuencia de Aminoácidos , Animales , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Astacoidea/citología , Astacoidea/virología , Bacterias/efectos de los fármacos , Secuencia de Bases , Braquiuros/clasificación , Braquiuros/metabolismo , Braquiuros/microbiología , Membrana Celular/metabolismo , Células Cultivadas , Perfilación de la Expresión Génica , Datos de Secuencia Molecular , Filogenia , Pichia/genética , Isoformas de Proteínas , Proteínas Recombinantes/farmacología , Alineación de Secuencia , Virus del Síndrome de la Mancha Blanca 1/efectos de los fármacosRESUMEN
Breast cancer is one of the most widespread and fatal cancers in women. At present, anticancer drug-inhibiting estrogen receptor α subtype (ERα) can greatly improve the cure rate for breast cancer patients, so the research and development of this kind of drugs are very urgent. In this paper, the problem of how to screen excellent anticancer drugs is abstracted as an optimization problem. Firstly, the graph model is used to extract low-dimensional features with strong distinguishing and describing ability according to various attributes of candidate compounds, and then, kernel functions are used to map these features to high-dimensional space. Then, the quantitative analysis model of ERα biological activity and the classification model based on ADMET properties of the support vector machine are constructed. Finally, sequential least square programming (SLSQP) is utilized to solve the ERα biological activity model. The experimental results show that for anticancer data sets, compared with principal component analysis (PCA), the error rate of the graph model constructed in this paper is reduced by 6.4%, 15%, and 7.8% on mean absolute error (MAE), mean squared error (MSE), and root mean square error (RMSE), respectively. In terms of classification prediction, compared with principal component analysis (PCA), the recall and precision rates of this method are enhanced by 19.5% and 12.41%, respectively. Finally, the optimal biological activity value (IC50_nM) 34.6 and inhibitory biological activity value (pIC50) 7.46 were obtained.
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Receptor alfa de Estrógeno , Neoplasias , Algoritmos , Femenino , Humanos , Análisis de Componente Principal , Máquina de Vectores de SoporteRESUMEN
Purpose: The malfunction of junctional adhesion molecule C (JAM-C) has been reported to induce congenital cataract in humans and mice; however, specific characters and the mechanism of this cataract are still unclear. This study aimed to characterize abnormal lens development in Jamc knockout mice and clarify the underlying mechanism. Methods: Jamc knockout mice backcrossed onto the C57BL/6 genetic background were used for this research. Slit-lamp and darkfield images showed the cataract phenotype of Jamc-/- mice. Hematoxylin and eosin staining was performed to visualize the morphological and histological features. RNA sequencing was applied to detect differentially expressed genes. Quantitative RT-PCR, western blot, and immunofluorescence were used to determine the level of unfolded protein response (UPR)-related genes. TUNEL staining was utilized to label cell death. Results: Jamc knockout mice exhibited nuclear cataract with abnormal lens morphology and defective degradation of nuclei and organelles in lens fiber cells. Compared with wild-type control mice, the expression level of BiP, CHOP, TRIB3, and CHAC1, genes involved in endoplasmic reticulum stress and the UPR, were highly upregulated in Jamc-/- lenses, suggesting that abnormal lens development was accompanied by UPR activation. Moreover, increased cell death was also found in Jamc-/- lenses. Conclusions: Congenital nuclear cataract caused by Jamc deficiency is accompanied by defective degradation of nuclei and organelles in lens fiber cells, lens structure disorder, and UPR activation, suggesting that JAM-C is required for maintaining normal lens development and that UPR activation is involved in cataract formation in Jamc-deficient lenses.
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Catarata , Cristalino , Animales , Catarata/metabolismo , Humanos , Cristalino/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Respuesta de Proteína DesplegadaRESUMEN
Mean Shift algorithm is a robust approach toward feature space analysis and it has been used wildly for natural scene image and medical image segmentation. However, high computational complexity of the algorithm has constrained its application in remote sensing images with massive information. A fast image segmentation algorithm is presented by extending traditional mean shift method to wavelet domain. In order to evaluate the effectiveness of the proposed algorithm, multispectral remote sensing image and synthetic image are utilized. The results show that the proposed algorithm can improve the speed 5-7 times compared to the traditional MS method in the premise of segmentation quality assurance.
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Algoritmos , Interpretación de Imagen Asistida por Computador/métodos , Telemetría/métodos , Reconocimiento de Normas Patrones Automatizadas/métodosRESUMEN
The mitogen-inducible gene 6 (MIG6) is an adaptor protein widely expressed in vascular endothelial cells. However, it remains unknown thus far whether it plays a role in angiogenesis. Here, using comprehensive in vitro and in vivo model systems, we unveil a potent anti-angiogenic effect of MIG6 in retinal development and neovascularization and the underlying molecular and cellular mechanisms. Loss of function assays using genetic deletion of Mig6 or siRNA knockdown increased angiogenesis in vivo and in vitro, while MIG6 overexpression suppressed pathological angiogenesis. Moreover, we identified the cellular target of MIG6 by revealing its direct inhibitory effect on vascular endothelial cells (ECs). Mechanistically, we found that the anti-angiogenic effect of MIG6 is fulfilled by binding to SHC1 and inhibiting its phosphorylation. Indeed, SHC1 knockdown markedly diminished the effect of MIG6 on ECs. Thus, our findings show that MIG6 is a potent endogenous inhibitor of angiogenesis that may have therapeutic value in anti-angiogenic therapy.
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Endothelial cells (ECs) from the small intestine, colon, liver, and heart have distinct phenotypes and functional adaptations that are dependent on their physiological environment. Gut ECs adapt to low oxygen, heart ECs to contractile forces, and liver ECs to low flow rates. Isolating high-purity ECs in sufficient quantities is crucial to study their functions. Here, we describe protocols combining magnetic and fluorescent activated cell sorting for rapid and reproducible EC purification from four adult murine tissues. For complete details on the use and execution of these protocols, please refer to Kalucka et al. (2020).
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Células Endoteliales/citología , Citometría de Flujo/métodos , Intestinos/citología , Hígado/citología , Miocardio/citología , Animales , Células Cultivadas , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Endothelial cells (ECs) harbor distinct phenotypical and functional characteristics depending on their tissue localization and contribute to brain, eye, lung, and muscle diseases such as dementia, macular degeneration, pulmonary hypertension, and sarcopenia. To study their function, isolation of pure ECs in high quantities is crucial. Here, we describe protocols for rapid and reproducible blood vessel EC purification established for scRNA sequencing from murine tissues using mechanical and enzymatic digestion followed by magnetic and fluorescence-activated cell sorting. For complete details on the use and execution of these protocol, please refer to Kalucka et al. (2020), Rohlenova et al. (2020), and Goveia et al. (2020).
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Encéfalo/citología , Coroides/citología , Células Endoteliales/citología , Pulmón/citología , Músculos/citología , Animales , Citometría de Flujo/métodos , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Endothelial cells (ECs) exhibit phenotypic and functional tissue specificities, critical for studies in the vascular field and beyond. Thus, tissue-specific methods for isolation of highly purified ECs are necessary. Kidney, spleen, and testis ECs are relevant players in health and diseases such as chronic kidney disease, acute kidney injury, myelofibrosis, and cancer. Here, we provide tailored protocols for rapid and reproducible EC purification established for scRNA sequencing from these adult murine tissues using the combination of magnetic- and fluorescence-activated cell sorting. For complete details on the use and execution of these protocols, please refer to Kalucka et al. (2020) and Dumas et al. (2020).
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Células Endoteliales/citología , Riñón/citología , Bazo/citología , Testículo/citología , Animales , Citometría de Flujo , Masculino , RatonesRESUMEN
White spot syndrome virus (WSSV) is a lethal pathogen of shrimp and many other crustaceans, which has been causing huge economic losses in global aquaculture. Laminin receptor (LR) is a cell surface receptor which participates in the interactions between cells as well as cells and extracellular matrix. Previously, we found that a CqLR-like gene was responsive to WSSV infection in the hematopoietic tissue (Hpt) cells from red claw crayfish Cherax quadricarinatus. To further reveal the role of CqLR-like gene involved in WSSV infection, the full-length cDNA of CqLR-like gene was cloned with 1000 bp, and the open reading frame encoded 308 amino acids with a conserved laminin-binding domain. Importantly, both the WSSV entry and viral replication were strongly reduced in Hpt cells after loss-of-function of CqLR-like gene by gene silencing. Protein interaction assay demonstrated that the recombinant CqLR-like protein could bind to WSSV virion in vitro by enzyme-linked immunosorbent assay and the binding affinity was in a dose-dependent manner. Furthermore, recombinant CqLR-like protein was found to bind to WSSV envelop protein VP28, but not other envelop proteins tested including VP19, VP24, and VP26, by pull down assay in HEK293T cells. In regarding to that LR is mainly localized on many types of cells' membrane, these data together suggested that CqLR-like protein was likely to function as a putative recognition molecule towards WSSV and act in the viral entry into a crustacean host cell, which may benefit the elucidation of the WSSV pathogenesis and further the pharmaceutical target for the possibly effective control of WSSV disease.