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The mitotic spindle contains many bundles of microtubules (MTs) including midzones and kinetochore fibers, but little is known about how bundled structures are formed. Here, we show that the chromosomal passenger complex (CPC) purified from Escherichia coli undergoes liquid-liquid demixing in vitro. An emergent property of the resultant condensates is to generate parallel MT bundles when incubated with free tubulin and GTP in vitro. We demonstrate that MT bundles emerge from CPC droplets with protruding minus ends that then grow into long and tapered MT structures. During this growth, we found that the CPC in these condensates apparently reorganize to coat and bundle the resulting MT structures. CPC mutants attenuated for liquid-liquid demixing or MT binding prevented the generation of parallel MT bundles in vitro and reduced the number of MTs present at spindle midzones in HeLa cells. Our data demonstrate that an in vitro biochemical activity to produce MT bundles emerges after the concentration of the CPC and provides models for how cells generate parallel-bundled MT structures that are important for the assembly of the mitotic spindle. Moreover, these data suggest that cells contain MT-organizing centers that generate MT bundles that emerge with the opposite polarity from centrosomes.
Assuntos
Cromossomos , Microtúbulos , Fuso Acromático , Humanos , Células HeLa , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Mitose , Fuso Acromático/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Animais , Xenopus laevisRESUMO
Two styryl-lactone derivatives (1 and 2) were isolated from the aerial parts of Goniothalamus elegans. Compound 1 is a newly discovered natural product, and compound 2 is reported in this plant for the first time. The absolute configuration of 1 was determined based on the ECD spectrum. The two styryl-lactone derivatives were tested for cytotoxicity activity against five cancer cell lines and human embryonic kidney cells. The newly discovered compound demonstrated potent cytotoxicity, with IC50 values ranging from 2.05 to 3.96 µM. Computational methods were also applied to investigate the mechanism of the cytotoxic activity of the two compounds. Density functional theory and molecular mechanisms were used to assess the interaction between protein targets to compound 1 and 2, respectively, through the EGF/EGFR signaling pathway. The results indicated that 1 showed a strong binding affinity for two proteins EGFR and HER-2. Finally, ADMET predictions were used to validate the pharmacokinetics and toxicity of these compounds. The results showed that both compounds are likely to be absorbed in the gastrointestinal tract and penetrate the blood-brain barrier. Based on our findings, these compounds may have potential for further studies to be developed into active ingredients for cancer treatment.
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Sirtuin 1 (SIRT1) belongs to the histone deacetylase enzyme family and its activity regulates various signaling networks associated with aging. SIRT1 is widely involved in a large number of biological processes, including senescence, autophagy, inflammation, and oxidative stress. In addition, SIRT1 activation may improve lifespan and health in numerous experimental models. Therefore, SIRT1 targeting is a potential strategy to delay or reverse aging and age-related diseases. Although SIRT1 is activated by a wide array of small molecules, only a limited number of phytochemicals that directly interact with SIRT1 have been identified. Using the Geroprotectors.org database and a literature search, the aim of this study was to identify geroprotective phytochemicals that might interact with SIRT1. We performed molecular docking, density functional theory studies, molecular dynamic simulations (MDS), and absorption, distribution, metabolism, excretion, and toxicity (ADMET) prediction to screen potential candidates against SIRT1. After the initial screening of 70 phytochemicals, crocin, celastrol, hesperidin, taxifolin, vitexin, and quercetin had significant binding affinity scores. These six compounds established multiple hydrogen-bonding and hydrophobic interactions with SIRT1 and showed good drug-likeness and ADMET properties. In particular, crocin was further analyzed using MDS to study its complex with SIRT1 during simulation. Crocin has a high reactivity to SIRT1 and can form a stable complex with it, showing a good ability to fit into the binding pocket. Although further investigations are required, our results suggest that these geroprotective phytochemicals, especially crocin, are novel interacting partners of SIRT1.
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Simulação de Dinâmica Molecular , Sirtuína 1 , Simulação de Acoplamento Molecular , Sirtuína 1/metabolismo , Compostos Fitoquímicos/farmacologia , Compostos Fitoquímicos/químicaRESUMO
Hypervalent chloranes are a class of rare and poorly explored reagents. Their unique electronic properties confer reactivity that is complementary to that of the common iodanes and emerging bromanes. Highly chemo- and regioselective, metal-free, and mild C-C and C-O couplings are reported here. Experimental and computational mechanistic studies elucidate the unprecedented reactivities and selectivities of these systems and the intermediacy of aryne intermediates. The synthetic potential of these transformations is further demonstrated via the post-functionalization of C-C and C-O coupling products obtained from reactions of chloranes with phenols under different conditions.
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Fenóis , Indicadores e ReagentesRESUMO
SIGNIFICANCE: This study evaluated the effects scleral lens wear has on corneal health using fluorometry and in vivo confocal microscopy. No subclinical changes on healthy corneas of young subjects were observed during 3 months of scleral lens wear. PURPOSE: This study aimed to evaluate the effects 3 months of scleral lens wear has on the corneal epithelial barrier function, dendritic cell density, and nerve fiber morphology. METHODS: Twenty-seven neophytes (mean [standard deviation] age, 21.4 [3.9] years) wore scleral lenses of a fluorosilicone acrylate material bilaterally (97 Dk, 15.6 to 16.0-mm diameter) for 3 months without overnight wear. Subjects were randomized to use either Addipak (n = 12) or PuriLens Plus (n = 15) during lens insertion. Measurements of corneal epithelial permeability to fluorescein were performed with automated scanning fluorophotometer (Fluorotron Master; Ocumetrics, Mountain View, CA) on the central cornea of the right eye and the temporal corneal periphery of the left eye. Images of the distributions of corneal nerve fibers and dendritic cells and nerve fibers were captured in vivo with a confocal laser scanning microscope (Heidelberg Retina Tomograph, Rostock Cornea Module; Heidelberg Engineering, Heidelberg, Germany) on the central and inferior peripheral cornea of the left eye. Corneal measurements and imaging were performed at baseline and after 1 and 3 months of lens wear. RESULTS: The corneal permeability values in natural log, dendritic cell densities, and nerve fiber morphology did not significantly change from baseline to 1 and 3 months of lens wear, for both central and peripheral corneal regions (P > .05). Dendritic cell density at the inferior cornea was higher than the central cornea throughout the study (P < .001). No relationships were observed between each outcome measurements and the saline solution groups (P > .05). CONCLUSIONS: Scleral lens wear for 3 months on healthy cornea of young subjects did not affect corneal epithelial barrier function, nerve fiber, and dendritic cell densities. Buffered and nonbuffered saline solutions impacted the corneal health in similar ways.
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Lentes de Contato , Córnea/fisiologia , Esclera , Contagem de Células , Córnea/inervação , Células Dendríticas/citologia , Método Duplo-Cego , Epitélio Corneano/fisiologia , Feminino , Fluorofotometria , Humanos , Masculino , Microscopia Confocal , Nervo Oftálmico/anatomia & histologia , Estudos Prospectivos , Fatores de Tempo , Adulto JovemRESUMO
IMPORTANCE: Atropine eyedrops are a promising treatment for slowing myopia progression in East Asian children. However, its effects on children in Australia, including those of non-Asian background, have not been well-studied. BACKGROUND: The Western Australia Atropine for the Treatment of Myopia (WA-ATOM) study aims to determine the efficacy and long-term effects of low-dose atropine eyedrops in myopia control. This paper describes the study rationale, methodology and participant baseline characteristics. DESIGN: Single-centre, double-masked, randomized controlled trial. PARTICIPANTS: Children (6-16 years) with spherical equivalent ≤-1.50 D in each eye, astigmatism ≤1.50 D and myopia progression by ≥0.50 D/year. METHODS: Enrolled children were randomly assigned 2:1 to receive 0.01% atropine or placebo eyedrops. Participants are examined every 6 months during first 3 years of the study (2-year treatment phase followed by a 1-year washout phase), and then at a 5-year follow-up (2 years after the end of the washout phase). MAIN OUTCOME MEASURES: Annual progression rate of myopia and axial length, tolerability to eyedrops and incidence and severity of unwanted effects. RESULTS: Out of 311 children who were referred, 242 were suitable for study participation, and 153 were subsequently enrolled. The baseline characteristics of enrolled participants are presented. CONCLUSIONS AND RELEVANCE: Outcomes of the WA-ATOM study will inform on the efficacy, tolerability, safety and long-term effects of low-dose atropine eyedrops in myopia control in Australian children. The impact of ocular sun exposure, iris colour and parental myopia on the efficacy of low-dose atropine will also be assessed.
Assuntos
Atropina , Miopia , Austrália/epidemiologia , Criança , Progressão da Doença , Humanos , Miopia/tratamento farmacológico , Soluções Oftálmicas , Refração Ocular , Austrália Ocidental/epidemiologiaRESUMO
Research with animal models of Pseudomonas aeruginosa keratitis has shown that use of a topical corticosteroid alone against an established infection can significantly increase the number of colonizing bacteria or worsen clinical disease. Moreover, retrospective analysis has suggested that corticosteroid use in humans is associated with an increased risk of keratitis in eyes with pre-existing disease. Thus, while corticosteroids are often used to reduce ocular inflammation in the absence of infection, the risk of opportunistic infection remains a concern. However, the effect of corticosteroids on the intrinsic barrier function of uninfected corneas is unknown. Here, we tested if short-term topical corticosteroid treatment of an uninfected murine cornea would increase susceptibility to P. aeruginosa colonization or infection after epithelial injury. Topical prednisolone acetate (1%) was administered to one eye of C57BL/6 mice three times a day for 3 days; control eyes were treated with sterile PBS. Prior to inoculation with a cytotoxic P. aeruginosa corneal isolate strain 6206, corneas were subject to superficial-injury by tissue paper blotting, or scratch-injured followed by 12â¯h of healing. Previously we have shown that blotting renders mouse corneas susceptible to P. aeruginosa adhesion, but not infection, while 12â¯h healing reduces susceptibility to infection after scratching. Corneas were evaluated at 48â¯h for bacterial colonization and microbial keratitis (MK). To monitor impact on wound healing, corneal integrity was examined by fluorescein staining immediately after scarification and after 12â¯h healing. For both the tissue paper blotting and scratch-injury models, there was no significant difference in P. aeruginosa colonization at 48â¯h between corticosteroid-pretreated eyes and controls. With the blotting model, one case of MK was observed in a control (PBS-pretreated) cornea; none in corticosteroid-pretreated corneas. With the 12â¯h healing model, MK occurred in 6 of 17 corticosteroid-pretreated eyes versus 2 of 17 controls, a difference not statistically significant. Corticosteroid-pretreated eyes showed greater fluorescein staining 12â¯h after scarification injury, but this did not coincide with increased colonization or MK. Together, these data show that short-term topical corticosteroid therapy on an uninfected murine cornea does not necessarily enhance its susceptibility to P. aeruginosa colonization or infection after injury, even when it induces fluorescein staining.
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Lesões da Córnea/microbiologia , Infecções Oculares Bacterianas/microbiologia , Glucocorticoides/uso terapêutico , Prednisolona/análogos & derivados , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/isolamento & purificação , Administração Oftálmica , Animais , Córnea/efeitos dos fármacos , Lesões da Córnea/diagnóstico , Úlcera da Córnea/diagnóstico , Úlcera da Córnea/microbiologia , Modelos Animais de Doenças , Suscetibilidade a Doenças , Epitélio Corneano/lesões , Infecções Oculares Bacterianas/diagnóstico , Feminino , Fluoresceína/metabolismo , Corantes Fluorescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Prednisolona/uso terapêutico , Pré-Medicação , Infecções por Pseudomonas/diagnóstico , Estudos Retrospectivos , CicatrizaçãoRESUMO
PURPOSE: Contact lens wear carries a risk of complications, including corneal infection. Solving these complications has been hindered by limitations of existing animal models. Here, we report development of a new murine model of contact lens wear. METHODS: C57BL/6 mice were fitted with custom-made silicone-hydrogel contact lenses with or without prior inoculation with Pseudomonas aeruginosa (PAO1-GFP). Contralateral eyes served as controls. Corneas were monitored for pathology, and examined ex vivo using high-magnification, time-lapse imaging. Fluorescent reporter mice allowed visualization of host cell membranes and immune cells. Lens-colonizing bacteria were detected by viable counts and FISH. Direct-colony PCR was used for bacterial identification. RESULTS: Without deliberate inoculation, lens-wearing corneas remained free of visible pathology, and retained a clarity similar to non-lens wearing controls. CD11c-YFP reporter mice revealed altered numbers, and distribution, of CD11c-positive cells in lens-wearing corneas after 24â¯h. Worn lenses showed bacterial colonization, primarily by known conjunctival or skin commensals. Corneal epithelial cells showed vacuolization during lens wear, and after 5 days, cells with phagocyte morphology appeared in the stroma that actively migrated over resident keratocytes that showed altered morphology. Immunofluorescence confirmed stromal Ly6G-positive cells after 5 days of lens wear, but not in MyD88 or IL-1R gene-knockout mice. P. aeruginosa-contaminated lenses caused infectious pathology in most mice from 1 to 13 days. CONCLUSIONS: This murine model of contact lens wear appears to faithfully mimic events occurring during human lens wear, and could be valuable for experiments, not possible in humans, that help solve the pathogenesis of lens-related complications.
Assuntos
Lentes de Contato , Córnea/microbiologia , Infecções Oculares Bacterianas/microbiologia , Ceratite/microbiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/isolamento & purificação , Receptores Tipo I de Interleucina-1/genética , Animais , Contagem de Colônia Microbiana , Lentes de Contato/efeitos adversos , Córnea/patologia , Modelos Animais de Doenças , Infecções Oculares Bacterianas/metabolismo , Infecções Oculares Bacterianas/patologia , Ceratite/metabolismo , Ceratite/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/patologia , Receptores Tipo I de Interleucina-1/metabolismo , Tomografia de Coerência ÓpticaRESUMO
Silver nanoparticles (Ag NPs) were synthesized by an improved green synthesis method via a photo-reduction process using low-power UV light in the presence of poly (vinyl pyrrolidone) (PVP) as the surface stabilizer. The effective synthesis process was achieved by optimized synthesis parameters such as C2H5OH: H2O ratio, AgNO3: PVP ratio, pH value, and reducing time. The formation of Ag NPs was identified by Ultraviolet-visible (UV-vis) absorption spectra, X-ray diffraction pattern (XRD) and Fourier transform infrared spectroscopy (FTIR) spectra. Ag NPs were crystallized according to (111), (200), and (220) planes of the face-centered cubic. The transmission electron microscopy (TEM) image showed that the morphology of Ag NPs was uniform spherical with the average particle size of 16⯱â¯2â¯nm. The results of XRD pattern, TEM image, and dynamic light scattering (DLS) analysis proved that Ag crystals with uniform size were formed after the reduction process. The mechanism of the formation of Ag NPs was proposed and confirmed by FTIR spectra. The antibacterial activity of Ag NPs against Escherichia coli (E. coli) was tested and approximately 100% of E. coli was eliminated by Ag NPs 35â¯ppm. In the future, this study can become a new process for the application of Ag NPs as an antibiotic in the industrial scale.
Assuntos
Antibacterianos , Escherichia coli/efeitos dos fármacos , Química Verde , Microbiologia Industrial/métodos , Nanopartículas Metálicas/química , Prata , Antibacterianos/síntese química , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Transmissão , Estrutura Molecular , Tamanho da Partícula , Prata/química , Prata/farmacologia , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Fluorescent protein-based biosensors are indispensable molecular tools for life science research. The invention and development of high-fidelity biosensors for a particular molecule or molecular event often catalyze important scientific breakthroughs. Understanding the structural and functional organization of brain activities remain a subject for which optical sensors are in desperate need and of growing interest. Here, we review genetically encoded fluorescent sensors for imaging neuronal activities with a focus on the design principles and optimizations of various sensors. New bioluminescent sensors useful for deep-tissue imaging are also discussed. By highlighting the protein engineering efforts and experimental applications of these sensors, we can consequently analyze factors influencing their performance. Finally, we remark on how future developments can fill technological gaps and lead to new discoveries.
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OBJECTIVE: Previous genetic lineage tracing studies showed that Sox10+ cells differentiate into vascular mural cells, limited to neural crest-derived blood vessels in craniofacial tissues, aortic arch, pulmonary arch arteries, brachiocephalic, carotid arteries, and thymus. The purpose of this study was to investigate the contribution of Sox10+ cells to the vascular development in other tissues and organs and their relationship with neural crest. APPROACH AND RESULTS: Using genetic lineage tracing technique based on Cre/LoxP system, we examined blood vessels in the adult organs of the mice expressing Sox10-Cre/Rosa-LoxP-red fluorescent protein or Wnt1-Cre/Rosa-LoxP-red fluorescent protein by immunohistological analysis. In addition to previously reported tissues and organs derived from neural crest, we showed that Sox10+ cells also contributed to vascular mural cells in the lung, spleen, and kidney, which are derived from non-neural crest origin as evidenced by red fluorescent protein-negative blood vessels in these 3 organs of Wnt1-Cre/Rosa-LoxP-red fluorescent protein mice. CONCLUSIONS: This study demonstrates that Sox10+ cells contribute to pericytes and smooth muscle cells in most parts of the body, including those from neural crest and non-neural crest, which has significant implications in vascular remodeling under physiological and pathological conditions.
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Linhagem da Célula , Rim/irrigação sanguínea , Pulmão/irrigação sanguínea , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Crista Neural/metabolismo , Pericitos/metabolismo , Fatores de Transcrição SOXE/metabolismo , Baço/irrigação sanguínea , Animais , Imunofluorescência , Genótipo , Integrases/genética , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Camundongos Transgênicos , Morfogênese , Músculo Liso Vascular/citologia , Neovascularização Fisiológica , Crista Neural/citologia , Fenótipo , Fatores de Transcrição SOXE/genética , Remodelação Vascular , Proteína Wnt1/genética , Proteína Vermelha FluorescenteRESUMO
PURPOSE: The lymphatic pathway mediates transplant rejection. We recently reported that lymphatic vessels develop luminal valves in the cornea during lymphangiogenesis, and these valves express integrin alpha 9 (Itga-9) and play a critical role in directing lymph flow. In this study, we used an allogeneic corneal transplantation model to investigate whether Itga-9 blockade could suppress valvulogenesis after transplantation, and how this effect would influence the outcomes of the transplants. METHODS: Orthotopic corneal transplantation was performed between fully mismatched C57BL/6 (donor) and BALB/c (recipient) mice. The recipients were randomized to receive subconjunctival injections of either Itga-9 blocking antibody or isotype control twice a week for 8 weeks. Corneal grafts were assessed in vivo by ophthalmic slit-lamp biomicroscopy and analyzed using Kaplan-Meier survival curves. Additionally, whole-mount full-thickness corneas were evaluated ex vivo by immunofluorescent microscopy on both lymphatic vessels and valves. RESULTS: Anti-Itga-9 treatment suppressed lymphatic valvulogenesis after transplantation. Our treatment did not affect lymphatic vessel formation or their nasal polarized distribution in the cornea. More importantly, Itga-9 blockade led to a significant promotion of graft survival. CONCLUSIONS: Lymphatic valvulogenesis is critically involved in transplant rejection. Itga-9 targeting may offer a new and effective strategy to interfere with the immune responses and promote graft survival.
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Anticorpos Neutralizantes/uso terapêutico , Neovascularização da Córnea/tratamento farmacológico , Transplante de Córnea , Cadeias alfa de Integrinas/antagonistas & inibidores , Linfangiogênese/imunologia , Vasos Linfáticos/patologia , Animais , Neovascularização da Córnea/imunologia , Modelos Animais de Doenças , Cadeias alfa de Integrinas/imunologia , Estimativa de Kaplan-Meier , Vasos Linfáticos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Distribuição AleatóriaRESUMO
Lymphatic research signifies a field of rapid progression in recent years. Though lymphatic dysfunction has been found in a myriad of disorders, to date, few effective treatments are available for lymphatic diseases. It is therefore urgent to develop new experimental approaches and therapeutic protocols. The cornea offers an ideal site for lymphatic research due to its transparent nature, accessible location, and lymphatic-free but -inducible features. Moreover, we have recently discovered that corneal lymphatic vessels develop luminal valves as lymphangiogenesis proceeds. This tissue thus provides an optimal tool to study both lymphangiogenesis and valvulogenesis upon a pathological insult. In this paper, we show that the modified Prox-1-GFP mice carrying wildtype C57BL/6 background provide a valuable tool for intravital imaging of corneal lymphatic vessels and valves and can be used to study pathological lymphangiogenesis induced by various insults. Further, we demonstrate the multifaceted dynamics of lymphangiogenesis and valvulogenesis associated with transplantation, from the initiation to regression phases, and report several novel and critical phenomena and mechanisms that cannot be detected by conventional ex vivo approaches. Further investigation holds the great potential for divulging new mechanisms and therapeutic strategies for lymphangiogenesis and lymphangiogenesis-related diseases at various stages and inside or outside the eye.
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Microscopia Intravital , Linfangiogênese , Vasos Linfáticos , Animais , Córnea/patologia , Células Endoteliais/metabolismo , Genes Reporter , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Linfangiogênese/efeitos dos fármacos , Linfangiogênese/genética , Vasos Linfáticos/efeitos dos fármacos , Vasos Linfáticos/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Animais , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
We utilized several computational approaches to evaluate the binding energies of tyrosine (Tyr) and several unnatural Tyr analogs, to several orthogonal aaRSes derived from Methanocaldococcus jannaschii and Escherichia coli tyrosyl-tRNA synthetases. The present study reveals the following: (1) AutoDock Vina and ROSETTA were able to distinguish binding energy differences for individual pairs of favorable and unfavorable aaRS-amino acid complexes, but were unable to cluster together all experimentally verified favorable complexes from unfavorable aaRS-Tyr complexes; (2) MD-MM/PBSA provided the best prediction accuracy in terms of clustering favorable and unfavorable enzyme-substrate complexes, but also required the highest computational cost; and (3) MM/PBSA based on single energy-minimized structures has a significantly lower computational cost compared to MD-MM/PBSA, but still produced sufficiently accurate predictions to cluster aaRS-amino acid interactions. Although amino acid-aaRS binding is just the first step in a complex series of processes to acylate a tRNA with its corresponding amino acid, the difference in binding energy, as shown by MD-MM/PBSA, is important for a mutant orthogonal aaRS to distinguish between a favorable unnatural amino acid (unAA) substrate from unfavorable natural amino acid substrates. Our computational study should assist further designing and engineering of orthogonal aaRSes for the genetic encoding of novel unAAs.
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Aminoácidos/metabolismo , Proteínas Arqueais/metabolismo , Proteínas de Escherichia coli/metabolismo , Tirosina-tRNA Ligase/metabolismo , Tirosina/metabolismo , Aminoácidos/química , Proteínas Arqueais/química , Proteínas Arqueais/genética , Biologia Computacional/métodos , Transferência de Energia , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Mathanococcus/enzimologia , Mathanococcus/genética , Mathanococcus/metabolismo , Simulação de Dinâmica Molecular , Estrutura Molecular , Ligação Proteica , Engenharia de Proteínas , Estrutura Terciária de Proteína , Reprodutibilidade dos Testes , Tirosina/química , Tirosina-tRNA Ligase/química , Tirosina-tRNA Ligase/genéticaRESUMO
PURPOSE: Lymphatic research has progressed rapidly in recent years. Lymphatic dysfunction has been found in myriad disorders from cancer metastasis to transplant rejection; however, effective treatment for lymphatic disorders is still limited. This study investigates the role of angiopoietin-2 (Ang-2) in corneal inflammatory lymphangiogenesis (LG) in vivo and in lymphatic endothelial cell (LEC) functions in vitro. METHODS: Standard suture placement model was used to study Ang-2 expression in inflamed cornea, and corneal LG and hemangiogenesis (HG) responses in Ang-2 knockout mice. Moreover, human LEC culture system was used to examine the effect of Ang-2 gene knockdown on LEC functions using small interfering RNAs (siRNAs). The effect of siRNA treatment on corneal LG was also assessed in vivo. RESULTS: Angiopoietin-2 was expressed on lymphatic vessels and macrophages in inflamed cornea. While corneal LG response was abolished in Ang-2 knockout mice, the HG response was also significantly suppressed with disorganized patterning. Moreover, anti-Ang-2 treatment inhibited LEC proliferation and capillary tube formation in vitro and corneal LG in vivo. CONCLUSIONS: Angiopoietin-2 is critically involved in lymphatic processes in vivo and in vitro. Further investigation of the Ang-2 pathway may provide novel insights and therapeutic strategies for lymphatic-related disorders, which occur both inside and outside the eye.
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Angiopoietina-2/genética , Córnea/irrigação sanguínea , Neovascularização da Córnea/genética , Regulação da Expressão Gênica , Linfangiogênese/genética , Vasos Linfáticos/patologia , RNA/genética , Angiopoietina-2/biossíntese , Animais , Córnea/metabolismo , Córnea/patologia , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/patologia , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , Vasos Linfáticos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Schlemm's canal is an important structure of the conventional aqueous humor outflow pathway and is critically involved in regulating the intraocular pressure. In this study, we report a novel finding that prospero homeobox protein 1 (Prox-1), the master control gene for lymphatic development, is expressed in Schlemm's canal. Moreover, we provide a novel in vivo method of visualizing Schlemm's canal using a transgenic mouse model of Prox-1-green fluorescent protein (GFP). The anatomical location of Prox-1⺠Schlemm's canal was further confirmed by in vivo gonioscopic examination and ex vivo immunohistochemical analysis. Additionally, we show that the Schlemm's canal is distinguishable from typical lymphatic vessels by lack of lymphatic vessel endothelial hyaluronan receptor (LYVE-1) expression and absence of apparent sprouting reaction when inflammatory lymphangiogenesis occurred in the cornea. Taken together, our findings offer new insights into Schlemm's canal and provide a new experimental model for live imaging of this critical structure to help further our understanding of the aqueous humor outflow. This may lead to new avenues toward the development of novel therapeutic intervention for relevant diseases, most notably glaucoma.
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Humor Aquoso/fisiologia , Proteínas de Homeodomínio/genética , Pressão Intraocular/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Supressoras de Tumor/genética , Animais , Córnea/fisiologia , Córnea/ultraestrutura , Expressão Gênica , Glicoproteínas/deficiência , Glicoproteínas/genética , Gonioscopia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica , Linfangiogênese/fisiologia , Vasos Linfáticos/fisiologia , Vasos Linfáticos/ultraestrutura , Proteínas de Membrana Transportadoras , Camundongos , Camundongos Transgênicos , Imagem Molecular , Proteínas Recombinantes de Fusão/metabolismo , Esclera/fisiologia , Esclera/ultraestrutura , Malha Trabecular/fisiologia , Malha Trabecular/ultraestrutura , Proteínas Supressoras de Tumor/metabolismoRESUMO
Esthetic demand from patients continues to increase. Consequently, the treatments we offer are moving towards more discreet or invisible techniques using lingual brackets in order to achieve harmonious, balanced results in line with our treatment goals. As orthodontists, we act upon relationships between teeth and bone. And the equilibrium they create impacts the entire face via the smile. A balanced smile is essential to an esthetic outcome and is governed by rules, which guide both the practitioner and patient. A smile can be described in terms of mathematical ratios and proportions but beauty cannot be calculated. For the smile to sit harmoniously within the face, we need to take into account facial proportions and the possibility of their being modified by our orthopedic appliances or by surgery.
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Estética Dentária , Face/anatomia & histologia , Sorriso , Adolescente , Adulto , Fatores Etários , Envelhecimento , Arte/história , Beleza , Cefalometria/métodos , Criança , Técnicas Cosméticas , Expressão Facial , Feminino , História do Século XV , História Antiga , História Medieval , Humanos , Incisivo/patologia , Lábio/patologia , Masculino , Má Oclusão/terapia , Ortodontia Corretiva , Satisfação do Paciente , Autoimagem , Clareamento Dental/métodos , Traumatismos Dentários/terapiaRESUMO
PURPOSE: We have recently provided evidence showing that luminal lymphatic valves are formed right after the onset of corneal inflammatory lymphangiogenesis (LG). The purpose of this study was to further characterize the long-term time course, spatial distribution, directional orientation, and functional implications of the valve formation in relation to corneal LG. METHODS: Corneal LG was induced in normal adult BALB/c mice by a modified suture placement model with equal distribution in the nasal and temporal side. Whole-mount corneas were harvested every 2 weeks for up to 8 weeks post suturing for immunofluorescent microscopic assays. Quantitative analysis on both lymphatic vessels and valves was performed by using National Institutes of Health ImageJ software. Corneal lymphatic live imaging was performed to show functional drainage of the valves. RESULTS: Lymphatic vessel invasion areas at 4, 6, and 8 weeks were significantly less than the peak at 2 weeks post corneal suturing. In contrast, the ratio of lymphatic valves to vessel invasion area was at its lowest at 2 weeks with a peak approximately at 6 weeks post suturing. Lymphatic valves were more localized in the nasal quadrant at all time points studied, and most of the well-formed valves were directionally oriented toward the limbus. The lymphatic valves function to guide lymphatic drainage outside the cornea. CONCLUSIONS: This study presents new insights into corneal lymphatic valve formation and function in inflammatory LG. Further investigation on lymphatic valves may provide novel strategies to interfere with lymphatic maturation and function and to treat lymphatic-related disorders.
Assuntos
Córnea/irrigação sanguínea , Neovascularização da Córnea/patologia , Linfangiogênese , Vasos Linfáticos/patologia , Animais , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
PURPOSE: This study aimed to examine the effects of demographic, lens performance, and ocular surface response measures on contact lens-related discomfort and dryness, using a large contact lens study database. METHODS: A total of 4164 records were extracted from a database of 220 subjects participating in 46 silicone hydrogel contact lens studies. Subjects discontinued lens wear for 24 hours and were then fit with either comfilcon A or enfilcon A lenses. The fit and performance of the lenses, along with subjective comfort and dryness, were assessed on insertion and after 3 and 6 hours of wear. After 6 hours of wear, ocular surface health was also assessed by fluorescein slitlamp examination. RESULTS: Decreased comfort at 3 hours after insertion was associated with excessive lens movement (p < 0.001), front surface deposits (p = 0.004), poor wettability (p = 0.014), and Asian ethnicity (p < 0.001). After 6 hours of wear, decreased comfort remained associated with greater lens movement (p = 0.032) and Asian ethnicity (p < 0.001), along with inferior corneal staining (p < 0.001). Dryness after 3 hours of wear was associated with greater lens movement (p < 0.001), Asian ethnicity (p < 0.001), increased deposits (p < 0.001), and poor wettability (p < 0.001). Dryness after 6 hours of wear remained associated with greater lens movement (p < 0.001) and Asian ethnicity (p < 0.001), along with inferior corneal staining (p < 0.001) and inferior lens decentration (p = 0.001). CONCLUSIONS: Excessive lens movement, inferior lens decentration, poor surface wettability and deposits, inferior corneal staining, and Asian ethnicity are associated with discomfort and dryness. Clinicians should consider all these factors to achieve the most comfortable and successful contact lens fit.
Assuntos
Lentes de Contato Hidrofílicas/efeitos adversos , Síndromes do Olho Seco/etiologia , Transtornos da Visão/etiologia , Adolescente , Adulto , Bases de Dados Factuais , Síndromes do Olho Seco/fisiopatologia , Feminino , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato , Masculino , Satisfação do Paciente , Ajuste de Prótese , Silicones , Fatores de Tempo , Transtornos da Visão/fisiopatologia , Visão Ocular/fisiologia , Adulto JovemRESUMO
With an increase in sexual activity among young adults in Vietnam and associated risks, there is a need for evidence-based sexual health interventions. This evaluation of three sexual health programs based on the Protection Motivation Theory (PMT) was conducted in 12 communes in Ha Noi, Nha Trang City, and Ninh Hoa District. Inclusion criteria included unmarried youth 15-20 years residing in selected communes. Communes were randomly allocated to an intervention, and participants were randomly selected within each commune. The intervention programs included Vietnamese Focus on Kids (VFOK), the gender-based program Exploring the World of Adolescents (EWA), and EWA plus parental and health provider education (EWA+). Programs were delivered over a ten-week period in the communities by locally trained facilitators. The gender-based EWA program with parental involvement (EWA+) compared to VFOK showed significantly greater increase in knowledge. EWA+ in comparison to VFOK also showed significant decrease at immediate postintervention for intention to have sex. Sustained changes are observed in all three interventions for self-efficacy condom use, self-efficacy abstinence, response efficacy for condoms, extrinsic rewards, and perceived vulnerability for HIV. These findings suggest that theory-based community programs contribute to sustained changes in knowledge and attitudes regarding sexual risk among Vietnamese adolescents.