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BACKGROUND: Retinal ischemia/reperfusion (RIR) is implicated in various forms of optic neuropathies, yet effective treatments are lacking. RIR leads to the death of retinal ganglion cells (RGCs) and subsequent vision loss, posing detrimental effects on both physical and mental health. Apigenin (API), derived from a wide range of sources, has been reported to exert protective effects against ischemia/reperfusion injuries in various organs, such as the brain, kidney, myocardium, and liver. In this study, we investigated the protective effect of API and its underlying mechanisms on RGC degeneration induced by retinal ischemia/reperfusion (RIR). METHODS: An in vivo model was induced by anterior chamber perfusion following intravitreal injection of API one day prior to the procedure. Meanwhile, an in vitro model was established through 1% oxygen and glucose deprivation. The neuroprotective effects of API were evaluated using H&E staining, spectral-domain optical coherence tomography (SD-OCT), Fluoro-Gold retrograde labeling, and Photopic negative response (PhNR). Furthermore, transmission electron microscopy (TEM) was employed to observe mitochondrial crista morphology and integrity. To elucidate the underlying mechanisms of API, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, flow cytometry assay, western blot, cell counting kit-8 (CCK-8) assay, lactate dehydrogenase (LDH) assay, JC-1 kit assay, dichlorofluorescein-diacetate (DCFH-DA) assay, as well as TMRE and Mito-tracker staining were conducted. RESULTS: API treatment protected retinal inner plexiform layer (IPL) and ganglion cell complex (GCC), and improved the function of retinal ganglion cells (RGCs). Additionally, API reduced RGC apoptosis and decreased lactate dehydrogenase (LDH) release by upregulating Bcl-2 and Bcl-xL expression, while downregulating Bax and cleaved caspase-3 expression. Furthermore, API increased mitochondrial membrane potential (MMP) and decreased extracellular reactive oxygen species (ROS) production. These effects were achieved by enhancing mitochondrial function, restoring mitochondrial cristae morphology and integrity, and regulating the expression of OPA1, MFN2, and DRP1, thereby regulating mitochondrial dynamics involving fusion and fission. CONCLUSION: API protects RGCs against RIR injury by modulating mitochondrial dynamics, promoting mitochondrial fusion and fission.
Assuntos
Apigenina , Dinâmica Mitocondrial , Fármacos Neuroprotetores , Traumatismo por Reperfusão , Células Ganglionares da Retina , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/patologia , Células Ganglionares da Retina/metabolismo , Apigenina/farmacologia , Apigenina/uso terapêutico , Animais , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/patologia , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Dinâmica Mitocondrial/efeitos dos fármacos , Masculino , Apoptose/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Modelos Biológicos , Camundongos Endogâmicos C57BLRESUMO
Pure-state single photons and large-bandwidth-correlation biphotons are fundamental resources for quantum information processing. The dispersion properties of micro/nanofiber (MNF) can be tailored by carefully choosing its diameter, resulting in a flexibly tailored biphoton spectrum. We theoretically investigate pure-state single photons and large-bandwidth-correlation biphotons produced by degenerate spontaneous four-wave mixing in MNF. In our simulation, a single-photon state with a purity of 99% will be theoretically attained by choosing the appropriate pump bandwidth and center wavelength with respect to the diameter and length of the MNF. Further, when an appropriate diameter with a negligible curvature at the zero dispersion wavelength is chosen, even a narrow pump bandwidth is capable of motivating remarkably broadband correlation biphotons, e.g., for a MNF diameter of 0.7â µm, the theoretical full width at half maximum is 473â nm. In practice, the application of a MNF-based quantum light source is dependent on the technologies that precisely control and measure the diameter. Our theoretical investigation will guide the experimental realization of high-quality quantum light sources based on MNF.
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On the basis of the "seeing is believing" concept and the existing theory of Hg2+ coordination chemistry, for the first time, we innovatively designed and synthesized a visual-volumetric sensor platform with fluorescein and uracil functionalized polyacrylamide hydrogel. Without the aid of any complicated instruments and power sources, the sensor-enabled quantitative µM-level Hg2+ detection Hg2+ by reading graduation on a pipette with the naked eye. The sensor undergoes volumetric response and shows a wide linear response range to Hg2+ (1.0 × 10-6-5.0 × 10-5 mol L-1) with 2.8 × 10-7 mol L-1 as the detection limit. The highly selective (easily distinguished Hg2+ from other common metal ions), rapid response (â¼30 min), and acceptable repeatability (RSD < 5% in all cases) demonstrated that the developed sensor is suitable for onsite practical use for the determination of Hg2+ while being low-cost, simple, and portable. The design principles of the obtained materials and the construction techniques and methods of the sensors described in our study provide a new idea for the research and development of smart materials and a series of visual-volumetric sensors for other analytes.
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Determination of 1p/19q co-deletion status is important for the classification, prognostication, and personalized therapy in diffuse lower-grade gliomas (LGG). We developed and validated a deep learning imaging signature (DLIS) from preoperative magnetic resonance imaging (MRI) for predicting the 1p/19q status in patients with LGG. The DLIS was constructed on a training dataset (n = 330) and validated on both an internal validation dataset (n = 123) and a public TCIA dataset (n = 102). The receiver operating characteristic (ROC) analysis and precision recall curves (PRC) were used to measure the classification performance. The area under ROC curves (AUC) of the DLIS was 0.999 for training dataset, 0.986 for validation dataset, and 0.983 for testing dataset. The F1-score of the prediction model was 0.992 for training dataset, 0.940 for validation dataset, and 0.925 for testing dataset. Our data suggests that DLIS could be used to predict the 1p/19q status from preoperative imaging in patients with LGG. The imaging-based deep learning has the potential to be a noninvasive tool predictive of molecular markers in adult diffuse gliomas.
Assuntos
Neoplasias Encefálicas/genética , Deleção Cromossômica , Cromossomos Humanos Par 19/genética , Cromossomos Humanos Par 1/genética , Aprendizado Profundo , Glioma/genética , Imageamento por Ressonância Magnética/métodos , Adulto , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/diagnóstico por imagem , Feminino , Glioma/diagnóstico , Glioma/diagnóstico por imagem , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Prognóstico , Curva ROC , Reprodutibilidade dos TestesRESUMO
OBJECTIVES: To develop and validate a deep learning model for predicting overall survival from whole-brain MRI without tumor segmentation in patients with diffuse gliomas. METHODS: In this multicenter retrospective study, two deep learning models were built for survival prediction from MRI, including a DeepRisk model built from whole-brain MRI, and an original ResNet model built from expert-segmented tumor images. Both models were developed using a training dataset (n = 935) and an internal tuning dataset (n = 156) and tested on two external test datasets (n = 194 and 150) and a TCIA dataset (n = 121). C-index, integrated Brier score (IBS), prediction error curves, and calibration curves were used to assess the model performance. RESULTS: In total, 1556 patients were enrolled (age, 49.0 ± 13.1 years; 830 male). The DeepRisk score was an independent predictor and can stratify patients in each test dataset into three risk subgroups. The IBS and C-index for DeepRisk were 0.14 and 0.83 in external test dataset 1, 0.15 and 0.80 in external dataset 2, and 0.16 and 0.77 in TCIA dataset, respectively, which were comparable with those for original ResNet. The AUCs at 6, 12, 24, 26, and 48 months for DeepRisk ranged between 0.77 and 0.94. Combining DeepRisk score with clinicomolecular factors resulted in a nomogram with a better calibration and classification accuracy (net reclassification improvement 0.69, p < 0.001) than the clinical nomogram. CONCLUSIONS: DeepRisk that obviated the need of tumor segmentation can predict glioma survival from whole-brain MRI and offers incremental prognostic value. KEY POINTS: ⢠DeepRisk can predict overall survival directly from whole-brain MRI without tumor segmentation. ⢠DeepRisk achieves comparable accuracy in survival prediction with deep learning model built using expert-segmented tumor images. ⢠DeepRisk has independent and incremental prognostic value over existing clinical parameters and IDH mutation status.
Assuntos
Glioma , Adulto , Humanos , Masculino , Pessoa de Meia-Idade , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Glioma/diagnóstico por imagem , Glioma/patologia , Imageamento por Ressonância Magnética/métodos , Estudos Retrospectivos , FemininoRESUMO
BACKGROUND: Glaucoma, the leading cause of irreversible blindness worldwide, is a type of retinal disease characterized by the selective death of retinal ganglion cells (RGCs). However, the pathogenesis of glaucoma has not been fully elucidated. Transient receptor potential vanilloid 4 (TRPV4) is a pressure-sensitive and calcium-permeable cation channel. TRPV4 is widely distributed in the retina and its sustained activation leads to RGC death; indicating that TRPV4 may be a possible target for glaucoma treatment. Here, we investigated the effects of TRPV4 on RGC apoptosis in a rat model of chronic ocular hypertension (COH), then examined the mechanism underlying these effects. METHODS: The COH model was established by injection of micro-magnetic beads into the anterior chamber of adult male rats. The expression levels of TRPV4, glial fibrillary acidic protein, and inflammatory factors were assessed by immunohistochemistry and immunoblotting. RGC apoptosis and visual dysfunction were evaluated by TUNEL assay and photopic negative response. Functional expression of TRPV4 was examined by electrophysiology and calcium imaging. Real-time polymerase chain reaction and immunoblotting were employed to investigate the molecular mechanism underlying the effects of TRPV4 on tumor necrosis factor-α (TNF-α) release. RESULTS: We found that TRPV4 played an essential role in glaucoma, such that high levels of TRPV4 expression were associated with elevated intraocular pressure. Furthermore, TRPV4 activation was involved in glaucoma-induced RGC apoptosis and RGC-related reductions in visual function. Mechanistic investigation demonstrated that TRPV4 activation led to enhanced Müller cell gliosis and TNF-α release via the JAK2/STAT3/NF-kB pathway, while TRPV4 inhibition could reverse these effects. Finally, TRPV4 activation could lead to elevated expression of TNF receptor 1 in RGCs, while inhibition of TNF-α could reduce TRPV4-mediated RGC apoptosis. CONCLUSIONS: TRPV4 activation induces Müller cell gliosis and TNF-α elevation via the JAK2/STAT3/NF-κB pathway, which may exacerbate RGC apoptosis in glaucoma; these results suggest that TRPV4 can serve as a therapeutic target in glaucoma treatment.
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Apoptose/genética , Células Ependimogliais/patologia , Glaucoma/genética , Janus Quinase 2/genética , NF-kappa B/genética , Células Ganglionares da Retina/patologia , Fator de Transcrição STAT3/genética , Transdução de Sinais/genética , Canais de Cátion TRPV/genética , Fator de Necrose Tumoral alfa/metabolismo , Animais , Glaucoma/patologia , Gliose/patologia , Pressão Intraocular , Masculino , Hipertensão Ocular/genética , Hipertensão Ocular/patologia , Ratos , Ratos Wistar , Transtornos da Visão/genética , Transtornos da Visão/patologiaRESUMO
The transient receptor potential vanilloid 4 (TRPV4) channel is widely distributed in the retina. Activation of the TRPV4 channel enhances excitatory signaling from bipolar cells to retinal ganglion cells (RGCs), thereby increasing RGC firing rate and membrane excitability. In this study, we investigated the effect of TRPV4 channel activation on the miniature inhibitory postsynaptic current (mIPSC) in rat RGCs. Our results showed that perfusion with HC-067047, a TRPV4-channel antagonist, significantly reduced the amplitude of RGC mIPSCs. Extracellular application of the TRPV4 channel agonist GSK1016790A (GSK101) enhanced the frequency and amplitude of mIPSCs in ON- and OFF-type RGCs; pre-application of HC-067047 blocked the effect of GSK101 on mIPSCs. Furthermore, TRPV4 channels were able to enhance the frequency and amplitude of glycine receptor (GlyR)-mediated mIPSCs and inhibit the frequency of type A γ-aminobutyric acid receptor (GABAA R)-mediated mIPSCs. Upon intracellular administration or intravitreal injection of GSK101, TRPV4 channel activation reduced the release of presynaptic glycine and enhanced the function and expression of postsynaptic GlyRs; however, it inhibited presynaptic release of GABA, but did not affect postsynaptic GABAA Rs. Our study results provide insight regarding the effect of TRPV4 channel activation on RGCs and offer a potential interventional target for retinal diseases involving TRPV4 channels.
Assuntos
Potenciais Pós-Sinápticos Inibidores , Células Ganglionares da Retina/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Leucina/análogos & derivados , Leucina/farmacologia , Masculino , Potenciais Pós-Sinápticos em Miniatura , Ratos , Ratos Sprague-Dawley , Receptores de GABA/metabolismo , Receptores de Glicina/metabolismo , Células Ganglionares da Retina/fisiologia , Sulfonamidas/farmacologia , Canais de Cátion TRPV/agonistasRESUMO
We experimentally reveal the potential security risks for chaotic semiconductor lasers caused by time-delay signature (TDS). In order to eliminate TDS and enhance effective bandwidth, we propose a new scheme by introducing phase-modulation injection using Gaussian noise or pseudo-random binary sequence (PRBS) as phase-modulation signals. Our numerical results indicate that TDS elimination and bandwidth enhancement are obtained over a wide-parameter region of injection strength and frequency detuning. We further improve the scheme by using two channels of chaotic lasers to generate phase-modulation signals. Numerical simulation results show that the improved scheme is feasible, and TDS cancellation quality is always excellent in wide phase-modulation depth ranges.
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Serotonin (5-hydroxytryptamine, 5-HT) receptor agonists are neuroprotective in CNS injury models. However, the neuroprotective functional implications and synaptic mechanism of 8-hydroxy-2- (di-n-propylamino) tetralin (8-OH-DPAT), a serotonin receptor (5-HT1A) agonist, in an adult male Wistar rat model of chronic glaucoma model remain unknown. We found that ocular hypertension decreased 5-HT1A receptor expression in rat retinas because the number of retinal ganglion cells (RGCs) was significantly reduced in rats with induced ocular hypertension relative to that in control retinas and 8-OH-DPAT enhanced the RGC viability. The protective effects of 8-OH-DPAT were blocked by intravitreal administration of the selective 5-HT1A antagonist WAY-100635 or the selective GABAA receptor antagonist SR95531. Using patch-clamp techniques, spontaneous and miniature GABAergic IPSCs (sIPSCs and mIPSCs, respectively) of RGCs in rat retinal slices were recorded. 8-OH-DPAT significantly increased the frequency and amplitude of GABAergic sIPSCs and mIPSCs in ON- and OFF-type RGCs. Among the signaling cascades mediated by the 5-HT1A receptor, the role of cAMP-protein kinase A (PKA) signaling was investigated. The 8-OH-DPAT-induced changes at the synaptic level were enhanced by PKA inhibition by H-89 and blocked by PKA activation with bucladesine. Furthermore, the density of phosphorylated PKA (p-PKA)/PKA was significantly increased in glaucomatous retinas and 8-OH-DPAT significantly decreased p-PKA/PKA expression, which led to the inhibition of PKA phosphorylation upon relieving neurotransmitter GABA release. These results showed that the activation of 5-HT1A receptors in retinas facilitated presynaptic GABA release functions by suppressing cAMP-PKA signaling and decreasing PKA phosphorylation, which could lead to the de-excitation of RGC circuits and suppress excitotoxic processes in glaucoma.SIGNIFICANCE STATEMENT We found that serotonin (5-HT) receptors in the retina (5-HT1A receptors) were downregulated after intraocular pressure elevation. Patch-clamp recordings demonstrated differences in the frequencies of miniature GABAergic IPSCs (mIPSCs) in ON- and OFF-type retinal ganglion cells (RGCs) and RGCs in normal and glaucomatous retinal slices. Therefore, phosphorylated protein kinase A (PKA) inhibition upon release of the neurotransmitter GABA was eliminated by 8-hydroxy-2- (di-n-propylamino) tetralin (8-OH-DPAT), which led to increased levels of GABAergic mIPSCs in ON- and OFF-type RGCs, thus enhancing RGC viability and function. These protective effects were blocked by the GABAA receptor antagonist SR95531 or the 5-HT1A antagonist WAY-100635. This study identified a novel mechanism by which activation of 5-HT1A receptors protects damaged RGCs via the cAMP-PKA signaling pathway that modulates GABAergic presynaptic activity.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , AMP Cíclico/fisiologia , Proteínas do Olho/metabolismo , Glaucoma/metabolismo , Receptor 5-HT1A de Serotonina/fisiologia , Células Ganglionares da Retina/metabolismo , Ácido gama-Aminobutírico/metabolismo , 8-Hidroxi-2-(di-n-propilamino)tetralina/farmacologia , Animais , Bucladesina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Isoquinolinas/farmacologia , Masculino , Hipertensão Ocular/metabolismo , Fosforilação/efeitos dos fármacos , Piperazinas/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Piridinas/farmacologia , Ratos , Ratos Wistar , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Sistemas do Segundo Mensageiro/fisiologia , Agonistas do Receptor 5-HT1 de Serotonina/farmacologia , Antagonistas do Receptor 5-HT1 de Serotonina/farmacologia , Sulfonamidas/farmacologiaRESUMO
BACKGROUND: Stargardt disease (STGD1) is a common recessive hereditary macular dystrophy in early adulthood or childhood, with an estimated prevalence of 1:8000 to 1:10,000. ABCA4 is the causative gene for STGD1. The current study aims at identifying the novel disease-related ABCA4 variants in Han Chinese families with STGD1 using next-generation sequencing (NGS). METHODS: In the present study, 12 unrelated Han Chinese families (19 males and 17 females) with STGD1 were tested by panel-based NGS. In order to capture the coding exons and the untranslated regions (UTRs) plus 30 bp of intronic flanking sequences of 792 genes, which were closely associated with usual ophthalmic genetic disease, we designed a customized panel, namely, Target_Eye_792_V2 chip. STGD1 patients were clinically diagnosed by experienced ophthalmologists. All the detected variants were filtered and analyzed through the public databases and in silico programs to assess potential pathogenicity. RESULTS: Twenty-one ABCA4 mutant variants were detected in 12 unrelated Han Chinese families with STGD1, containing 14 missense, three splicing, two frameshift, one small deletion, and one nonsense variants. Base on the American College of Medical Genetics (ACMG) guidelines, 8 likely pathogenic and 13 pathogenic variants were determined. The functional consequences of these mutant variants were predicted through in silico programs. Of the 21 mutant variants in ABCA4, two novel coding variants c.3017G > A and c.5167 T > C and one novel null variant c.3051-1G > A were detected in three unrelated probands. CONCLUSIONS: By panel-based NGS, 21 ABCA4 variants were confirmed in 12 unrelated Han Chinese families. Among them, 3 novel mutant variants were found, which further expanded the ABCA4 mutation spectrum in STGD1 patients.
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Transportadores de Cassetes de Ligação de ATP/genética , Mutação , Doença de Stargardt/genética , Transportadores de Cassetes de Ligação de ATP/deficiência , Adolescente , Adulto , Sequência de Aminoácidos , Animais , Povo Asiático , Criança , Éxons , Família , Feminino , Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Íntrons , Masculino , Linhagem , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Doença de Stargardt/diagnóstico , Doença de Stargardt/etnologia , Doença de Stargardt/patologiaRESUMO
BACKGROUND: Stickler syndrome is the most common genetic cause of rhegmatogenous retinal detachment (RRD) in children, and has a high risk of blindness. Type I (STL1) is the most common subtype, caused by COL2A1 mutations. This study aims to analyze the mutation spectrum of COL2A1 and further elucidate the genotype-phenotype relationships in the East Asian populations with STL1, which is poorly studied at present. METHODS: By searching MEDLINE, Web of Science, CNKI, Wanfang Data, HGMD and Clinvar, all publications associated with STL1 were collected. Then, they were carefully screened to obtain all reported STL1-related variants in COL2A1 and clinical features in East Asian patients with STL1. RESULTS: There were 274 COL2A1 variants identified in 999 patients with STL1 from 466 unrelated families, and more than half of them were truncation mutations. Of the 107 STL1 patients reported in the East Asian population, it was found that patients with truncation mutations had milder systemic phenotypes, whereas patients with splicing mutations had severer phenotypes. In addition, several recurrent variants (c.3106C > T, c.1833 + 1G > A, c.2710C > T and c.1693C > T) were found. CONCLUSIONS: Genotype-phenotype correlations should certainly be studied carefully, contributed to making personalized follow-up plans and predicting prognosis of this disorder. Genome editing holds great potential for treating inherited diseases caused by pathogenic mutations. In this study, several recurrent variants were found, providing potential candidate targets for genetic manipulation in the future.
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Artrite/genética , Doenças do Tecido Conjuntivo/genética , Análise Mutacional de DNA , Perda Auditiva Neurossensorial/genética , Mutação/genética , Descolamento Retiniano/genética , Artrite/epidemiologia , Artrite/patologia , Doenças do Tecido Conjuntivo/epidemiologia , Doenças do Tecido Conjuntivo/patologia , Oftalmopatias Hereditárias , Estudos de Associação Genética , Perda Auditiva Neurossensorial/epidemiologia , Perda Auditiva Neurossensorial/patologia , Humanos , Fenótipo , Descolamento Retiniano/epidemiologia , Descolamento Retiniano/patologiaRESUMO
Sensing nonradiation-induced singlet oxygen (1 O2 ) in whole-animal is deemed as one of the most challenging tasks in noninvasive techniques due to the µs level lifetime of 1 O2 and quenching by numerous reductants in tissues. Here a distinct chemiluminescent (CL) nanosensor (NTPE-PH) that boasts ultrahigh concentrated CL units in one nanoparticle is reported. Taking advantage of the intramolecular energy transfer mechanism that promises high energy transfer efficiency and the aggregation-induced emission behavior that guarantees high CL amplification, the NTPE-PH sensor is sensitive to a nm level 1 O2 . Experiments demonstrate that the NTPE-PH yields a highly selective CL response toward 1 O2 among common reactive oxygen species. With proved low cytotoxicity and good animal compatibility, real-time mapping of ultratrace 1 O2 in whole-animal during acute and chronic inflammations is first achieved. It is anticipated that the NTPE-PH sensor can be a useful tool for monitoring 1 O2 variation during immune response and pathological processes corresponding to different stimuli, even with drug treatment included.
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Técnicas Biossensoriais , Inflamação/metabolismo , Oxigênio Singlete/análise , Animais , Feminino , Sistema Imunitário , Luminescência , Medições Luminescentes , Espectroscopia de Ressonância Magnética , Nanopartículas , Oxigênio/química , Ratos , Espécies Reativas de Oxigênio/metabolismoRESUMO
PURPOSE: To characterize the genetic landscape of patients with suspected retinitis pigmentosa (RP) in the Chinese population. DESIGN: Cohort study. PARTICIPANTS: A total of 1243 patients of Chinese origin with clinically suspected RP and their available family members (n = 2701) were recruited. METHODS: All patients and available family members were screened using multigene panel testing (including 586 eye disease-associated genes), followed by clinical variant interpretation. MAIN OUTCOME MEASURES: Diagnostic yield, the 17 most commonly implicated genes, age at onset, de novo mutations, and clinical usefulness of genetic testing. RESULTS: Overall, 72.08% of patients received a molecular diagnosis, and the 17 top genes covered 75.63% of diagnostic cases. Diagnostic yield was higher among patients in the early-onset subgroup (≤5 years old, 79.58%) than in the childhood or adolescence-onset subgroup (6-16 years old, 73.74%) and late-onset subgroup (≥17 years old, 65.99%). Moreover, different genes associated with different onset ages and subgroups with different onset ages showed a diverse mutation spectrum. Only 11 de novo mutations (3.18%) were identified. Furthermore, 16.84% of the patients who received a molecular diagnosis had refinement of the initial clinical diagnoses, and the remaining 83.16% received definite genetic subtypes of RP. CONCLUSIONS: This large cohort study provides population-based data of the genome landscape of patients with suspected RP in China. The diagnostic yield was significantly higher than that in previous studies, and the mutation spectrum is completely different with other populations. Genetic testing improves the chance to establish a precise diagnosis, identifies features not previously determined, and allows a more accurate refinement of risk to family members. Our results not only expand the existing genotypic spectrum but also serve as an efficient reference for the design of panel-based genetic diagnostic testing and genetic counseling for patients with suspected RP in China.
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Povo Asiático/genética , Proteínas do Olho/genética , Retinose Pigmentar/genética , Adolescente , Adulto , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , China/epidemiologia , Estudos de Coortes , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética , Testes Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Mutação , Retinose Pigmentar/diagnósticoRESUMO
BACKGROUND: The vascular complications of diabetes mellitus are the major causes of morbidity and mortality among people with diabetes. Circular RNAs are a class of endogenous noncoding RNAs that regulate gene expression in eukaryotes. In this study, we investigated the role of circular RNA in retinal vascular dysfunction induced by diabetes mellitus. METHODS: Quantitative polymerase chain reactions, Sanger sequencing, and Northern blots were conducted to detect circular HIPK3 (circHIPK3) expression pattern on diabetes mellitus-related stresses. MTT (3-[4,5-dimethythiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assays, EdU (5-ethynyl-2'-deoxyuridine) incorporation assays, Transwell migration assays, and Matrigel assays were conducted to detect the role of circHIPK3 in retinal endothelial cell function in vitro. Retinal trypsin digestion, vascular permeability assays, and ELISA assays were conducted to detect the role of circHIPK3 in retinal vascular dysfunction in vivo. Bioinformatics analysis, luciferase activity assays, RNA pull-down assays, and in vitro studies were conducted to reveal the mechanism of circHIPK3-mediated retinal vascular dysfunction. RESULTS: circHIPK3 expression was significantly upregulated in diabetic retinas and retinal endothelial cells following stressors related to diabetes mellitus. circHIPK3 silencing or overexpressing circHIPK3 changed retinal endothelial cell viability, proliferation, migration, and tube formation in vitro. circHIPK3 silencing in vivo alleviated retinal vascular dysfunction, as shown by decreased retinal acellular capillaries, vascular leakage, and inflammation. circHIPK3 acted as an endogenous miR-30a-3p sponge to sequester and inhibit miR-30a-3p activity, which led to increased vascular endothelial growth factor-C, FZD4, and WNT2 expression. Ectopic expression of miR-30a-3p mimicked the effect of circHIPK3 silencing on vascular endothelial phenotypes in vivo and in vitro. CONCLUSIONS: The circular RNA circHIPK3 plays a role in diabetic retinopathy by blocking miR-30a function, leading to increased endothelial proliferation and vascular dysfunction. These data suggest that circular RNA is a potential target to control diabetic proliferative retinopathy.
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Diabetes Mellitus Experimental/metabolismo , Retinopatia Diabética/metabolismo , Células Endoteliais/metabolismo , RNA não Traduzido/metabolismo , Vasos Retinianos/metabolismo , Animais , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Células Endoteliais/fisiologia , Receptores Frizzled/biossíntese , Receptores Frizzled/genética , Regulação da Expressão Gênica , Masculino , Camundongos , MicroRNAs/biossíntese , MicroRNAs/genética , RNA não Traduzido/genética , Vasos Retinianos/patologia , Fator C de Crescimento do Endotélio Vascular/biossíntese , Fator C de Crescimento do Endotélio Vascular/genética , Proteínas Wnt/biossíntese , Proteínas Wnt/genéticaRESUMO
We report the unique self-catalyzing chemiluminescence (CL) of luminol-diazonium ion (N2+-luminol) and its analytical potential. Visual CL emission was initially observed when N2+-luminol was subjected to alkaline aqueous H2O2 without the aid of any catalysts. Further experimental investigations found peroxidase-like activity of N2+-luminol on the cleavage of H2O2 into OH⢠radical. Together with other experimental evidence, the CL mechanism is suggested as the activation of N2+-luminol and its dediazotization product 3-hydroxyl luminol by OH⢠radical into corresponding intermediate radicals, and then further oxidation to excited-state 3-N2+-phthalic acid and 3-hydroxyphthalic acid, which finally produce 415 nm CL. The self-catalyzing CL of N2+-luminol provides us an opportunity to achieve the attractive catalyst-free CL detection of H2O2. Experiments demonstrated the 10-8 M level detection sensitivity to H2O2 as well as to glucose or uric acid if presubjected to glucose oxidase or uricase. With the exampled determination of serum glucose and uric acid, N2+-luminol shows its analytical potential for other analytes linking the production or consumption of H2O2. Under physiological condition, N2+-luminol exhibits highly selective and sensitive CL toward 1O2 among the common reactive oxygen species. This capacity supports the significant application of N2+-luminol for detecting 1O2 in live animals. By imaging the arthritis in LEW rats, N2+-luminol CL is demonstrated as a potential tool for mapping the inflammation-relevant biological events in a live body.
Assuntos
Artrite/diagnóstico , Compostos de Diazônio/química , Peróxido de Hidrogênio/análise , Substâncias Luminescentes/química , Luminol/análogos & derivados , Luminol/química , Animais , Catálise , Feminino , Glucose/análise , Limite de Detecção , Imagem Óptica/métodos , Ratos Endogâmicos Lew , Ácido Úrico/análiseRESUMO
Progressive retinal ganglion cell (RGC) death is the major cause of retinal nerve fiber layer thinning and visual field defects in glaucoma. The purpose of this study was to compare RGC damage in three commonly used glaucomatous rat models. These models were generated by (i) injection of paramagnetic microbeads into the anterior chamber; (ii) cauterization of three episcleral veins of the eye (EVC); and (iii) intravitreal injection of N-Methyl-D-Aspartate (NMDA). Intraocular pressure (IOP) was measured with a rebound tonometer at 6, 12, and 18â¯h; 1, 3, and 5 days; and 1, 2, 3, 4, 6, and 8 weeks. We measured the RGC density of the three glaucomatous models in the flat-mounted retina by immunofluorescence. Subsequently, the thicknesses of both retinal ganglion cell layer (GCL) and inner retinal layer (IRL) were analyzed by hematoxylin and eosin staining of retinal sections. The visual functional deterioration was evaluated by measurement of the photopic negative response (PhNR) of different models. The IOP averages during three weeks were 22.35⯱â¯1.23â¯mmHg (mean⯱â¯SD), 20.91⯱â¯1.97â¯mmHg, and 9.67⯱â¯0.42â¯mmHg, with 50.2%, 44.00% and 66.76% RGC loss by 8 weeks, respectively, in the microbead group, EVC group and NMDA group. Decreased thickness in the GCL was observed in all three groups, while the thickness of IRL and ONL was decreased in the EVC and NMDA groups. Significant positive correlation of RGC loss rate with ΔIOP integral were demonstrated in both microbead and EVC models. Moreover, we found that the PhNR amplitudes declined early by the first day in the NMDA group, 5 days later in the EVC group and by 7 days in the microbead group. Each glaucomatous rat model has its strength and weakness. Our study provides detailed data for choosing suitable animal models to advance glaucoma research.
Assuntos
Modelos Animais de Doenças , Glaucoma/fisiopatologia , Doenças Retinianas/fisiopatologia , Células Ganglionares da Retina/patologia , Animais , Eletrorretinografia , Técnica Indireta de Fluorescência para Anticorpo , Glaucoma/etiologia , Pressão Intraocular , Ratos , Ratos Wistar , Tonometria OcularRESUMO
PURPOSE: Familial exudative vitreoretinopathy (FEVR) is a genetically and clinically heterogeneous disease, characterized by failure of vascular development of the peripheral retina. The symptoms of FEVR vary widely among patients in the same family, and even between the two eyes of a given patient. This study was designed to identify the genetic defect in a patient cohort of ten Chinese families with a definitive diagnosis of FEVR. METHODS: To identify the causative gene, next-generation sequencing (NGS)-based target capture sequencing was performed. Segregation analysis of the candidate variant was performed in additional family members by using Sanger sequencing and quantitative real-time PCR (QPCR). RESULTS: Of the cohort of ten FEVR families, six pathogenic variants were identified, including four novel and two known heterozygous mutations. Of the variants identified, four were missense variants, and two were novel heterozygous deletion mutations [LRP5, c.4053 DelC (p.Ile1351IlefsX88); TSPAN12, EX8Del]. The two novel heterozygous deletion mutations were not observed in the control subjects and could give rise to a relatively severe FEVR phenotype, which could be explained by the protein function prediction. CONCLUSIONS: We identified two novel heterozygous deletion mutations [LRP5, c.4053 DelC (p.Ile1351IlefsX88); TSPAN12, EX8Del] using targeted NGS as a causative mutation for FEVR. These genetic deletion variations exhibit a severe form of FEVR, with tractional retinal detachments compared with other known point mutations. The data further enrich the mutation spectrum of FEVR and enhance our understanding of genotype-phenotype correlations to provide useful information for disease diagnosis, prognosis, and effective genetic counseling.
Assuntos
Povo Asiático/genética , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Mutação de Sentido Incorreto , Doenças Retinianas/genética , Deleção de Sequência , Tetraspaninas/genética , Adolescente , Adulto , China/epidemiologia , Estudos de Coortes , Análise Mutacional de DNA , Oftalmopatias Hereditárias , Vitreorretinopatias Exsudativas Familiares , Feminino , Estudos de Associação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Adulto JovemRESUMO
Mutations in the optineurin gene (OPTN) have been found to be associated with glaucoma and amyotrophic lateral sclerosis (ALS). However, the mechanism by which this single gene mutation leads to neurodegeneration in those two diseases remains unrevealed. To study the roles of wild-type (WT) OPTN and its pathogenic mutants in neuronal survival, here we overexpressed SH-SY5Y cells with WT OPTN or its four mutants (E50K, M98K, Q398X and E478G), and detected their effects on neuronal viability under normal or oxidative stress conditions. We found that overexpression of WT OPTN or its glaucoma-linked mutants (E50K and M98K) causes little harm in SH-SY5Y cells, while ALS-associated OPTN mutants (Q398X and E478G) leads to remarkably increased oxidative status and decreased antioxidase activity, which might result in severe mitochondrial dysfunction and neuronal injury. Further investigation suggests that overexpression of WT OPTN promotes endogenous antioxidase activation in the SH-SY5Y cells under oxidative stress and increases neuronal survival. Nevertheless, this neuroprotective effect of WT OPTN is abolished by its four mutants. Our results indicate that oxidative stress may play a central role in the pathogenesis of glaucoma and ALS caused by OPTN mutation.
Assuntos
Apoptose , Mutação de Sentido Incorreto , Neurônios/metabolismo , Fator de Transcrição TFIIIA/genética , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Humanos , Proteínas de Membrana Transportadoras , Fator de Transcrição TFIIIA/metabolismoRESUMO
Glaucoma is a chronic neurodegenerative disease characterized by the progressive loss of retinal ganglion cells (RGCs). Mitochondrial DNA (mtDNA) alterations have been documented as a key component of many neurodegenerative disorders. However, whether mtDNA alterations contribute to the progressive loss of RGCs and the mechanism whereby this phenomenon could occur are poorly understood. We investigated mtDNA alterations in RGCs using a rat model of chronic intraocular hypertension and explored the mechanisms underlying progressive RGC loss. We demonstrate that the mtDNA damage and mutations triggered by intraocular pressure (IOP) elevation are initiating, crucial events in a cascade leading to progressive RGC loss. Damage to and mutation of mtDNA, mitochondrial dysfunction, reduced levels of mtDNA repair/replication enzymes, and elevated reactive oxygen species form a positive feedback loop that produces irreversible mtDNA damage and mutation and contributes to progressive RGC loss, which occurs even after a return to normal IOP. Furthermore, we demonstrate that mtDNA damage and mutations increase the vulnerability of RGCs to elevated IOP and glutamate levels, which are among the most common glaucoma insults. This study suggests that therapeutic approaches that target mtDNA maintenance and repair and that promote energy production may prevent the progressive death of RGCs.