RESUMO
Mitochondrial DNA damage in retinal ganglion cells (RGCs) may be closely related to lesions of glaucoma. RGCs were cultured with different concentrations of glucose and grouped into 3 groups, namely normal control (NC) group, Low-Glu group, and High-Glu group. Cell viability was measured with cell counting kit-8, and cell apoptosis was measured using flow cytometry. The DNA damage was measured with comet assay, and the morphological changes of damaged mitochondria in RGCs were observed using TEM. Western blot analyzed the expression of MRE11, RAD50, and NBS1 protein. Cell viability of RGCs in Low-Glu and High-Glu groups were lower than that of NC group in 48 and 96 h. The cell apoptosis in NC group was 4.9%, the Low-Glu group was 12.2% and High-Glu group was 24.4%. The comet imaging showed that NC cells did not have tailings, but the low-Glu and high-Glu group cells had tailings, indicating that the DNA of RGCs had been damaged. TEM, mitochondrial membrane potential, ROS, mitochondrial oxygen consumption, and ATP content detection results showed that RGCs cultured with high glucose occurred mitochondrial morphology changes and dysfunction. MRE11, RAD50, and NBS1 protein expression associated with DNA damage repair pathway in High-Glu group declined compared with Low-Glu group. Mitochondrial DNA damage caused by high glucose will result in apoptosis of retinal ganglion cells in glaucoma.
Assuntos
Apoptose , Sobrevivência Celular , Dano ao DNA , DNA Mitocondrial , Glucose , Potencial da Membrana Mitocondrial , Espécies Reativas de Oxigênio , Células Ganglionares da Retina , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/patologia , Glucose/toxicidade , Glucose/farmacologia , DNA Mitocondrial/metabolismo , DNA Mitocondrial/genética , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Trifosfato de Adenosina/metabolismo , Proteína Homóloga a MRE11/metabolismo , Proteína Homóloga a MRE11/genética , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Hidrolases Anidrido Ácido/genética , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Humanos , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Ensaio Cometa , AnimaisRESUMO
OBJECTIVES: Age-related cataract is the most common type of adult cataract and a leading cause of blindness. Currently, there are few reports on the establishment of animal models for age-related cataract. During the experimental breeding of Microtus fortis (M. fortis), we first observed that M. fortis aged 12 to 15 months could naturally develop cataracts. This study aims to explore the possibility of developing them as an animal model for age-related cataract via identifing and analyzing spontaneous cataract in M. fortis. METHODS: The 12-month-old healthy M. fortis were served as a control group and 12-month-old cataractous M. fortis were served as an experimental group. The lens transparency was observed using the slit-lamp biomicroscope. Hematoxylin and eosin staining was used to detect pathological changes in the lens. Biochemical detection methods were applied to detect blood routine, blood glucose levels, the serum activities of superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) in both groups. Finally, real-time RT-PCR was used to detect the transcription levels of cataract-related genes in the lens of 2 groups. RESULTS: Compared with the control group, the lens of cataract M. fortis showed severely visible opacity, the structure of lens was destroyed seriously, and some pathological damage, such as swelling, degeneration/necrosis, calcification, hyperplasia, and fiber liquefaction were found in lens epithelial cells (LECs). The fibrous structure was disorganized and irregularly distributed with morgagnian globules (MGs) aggregated in the degenerated lens fibers. There was no statistically significant difference in blood glucose levels between the experimental and control groups (P>0.05). However, white blood cell (WBC) count (P<0.05), lymphocyte count (P<0.01), and lymphocyte ratio (P<0.05) were significantly decreased, while neutrophil percentage (P<0.05) and monocyte ratio (P<0.01) were significantly increased. The serum activities of SOD and GSH-Px (both P<0.05) were both reduced. The mRNAs of cataract-related genes, including CRYAA, CRYBA1, CRYBB3, Bsfp1, GJA3, CRYBA2, MIP, HspB1, DNase2B, and GJA8, were significantly downregultaed in the lenses of the experimental group (all P<0.05). CONCLUSIONS: There are significant differences in lens pathological changes, peroxidase levels, and cataract-related gene expression between cataract and healthy M. fortis. The developed cataract spontaneously in M. fortis is closely related to age, the cataract M. fortis might be an ideal animal model for the research of age-related cataract.
Assuntos
Arvicolinae , Catarata , Glutationa Peroxidase , Cristalino , Superóxido Dismutase , Animais , Catarata/genética , Catarata/patologia , Catarata/etiologia , Cristalino/patologia , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Glutationa Peroxidase/sangue , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Envelhecimento , Modelos Animais de DoençasRESUMO
Flavan-3-ols are abundant in the tea plant (Camellia sinensis) and confer tea with flavor and health benefits. We recently found that alternative splicing of genes is likely involved in the regulation of flavan-3-ol biosynthesis; however, the underlying regulatory mechanisms remain unknown. Here, we integrated metabolomics and transcriptomics to construct metabolite-gene networks in tea leaves, collected over five different months and from five spatial positions, and found positive correlations between endogenous jasmonic acid (JA), flavan-3-ols, and numerous transcripts. Transcriptome mining further identified CsJAZ1, which is negatively associated with flavan-3-ols formation and has three CsJAZ1 transcripts, one full-length (CsJAZ1-1), and two splice variants (CsJAZ1-2 and -3) that lacked 3' coding sequences, with CsJAZ1-3 also lacking the coding region for the Jas domain. Confocal microscopy showed that CsJAZ1-1 was localized to the nucleus, while CsJAZ1-2 and CsJAZ1-3 were present in both the nucleus and the cytosol. In the absence of JA, CsJAZ1-1 was bound to CsMYC2, a positive regulator of flavan-3-ol biosynthesis; CsJAZ1-2 functioned as an alternative enhancer of CsJAZ1-1 and an antagonist of CsJAZ1-1 in binding to CsMYC2; and CsJAZ1-3 did not interact with CsMYC2. In the presence of JA, CsJAZ1-3 interacted with CsJAZ1-1 and CsJAZ1-2 to form heterodimers that stabilized the CsJAZ1-1-CsMYC2 and CsJAZ1-2-CsMYC2 complexes, thereby repressing the transcription of four genes that act late in the flavan-3-ol biosynthetic pathway. These data indicate that the alternative splicing variants of CsJAZ1 coordinately regulate flavan-3-ol biosynthesis in the tea plant and improve our understanding of JA-mediated flavan-3-ol biosynthesis.
Assuntos
Camellia sinensis , Processamento Alternativo/genética , Camellia sinensis/genética , Camellia sinensis/metabolismo , Flavonoides/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Chá/metabolismoRESUMO
BACKGROUND: PYL (Pyrabactin resistance 1-like) protein is a receptor of abscisic acid (ABA), which plays an important role in ABA signaling and influences plant growth and development and stress response. However, studies on PYL gene family in tea plants have not been reported. RESULTS: In this study, we identified 20 PYL genes from the reference genome of tea plant ('Shuchazao'). Phylogeny analysis indicated that PYLs from tea and other plant species were clustered into seven groups. The promoter region of PYL genes contains a large number of cis-elements related to hormones and stresses. A large number of PYL genes responding to stress were found by analyzing the expression levels of abiotic stress and biotic stress transcriptome data. For example, CSS0047272.1 were up-regulated by drought stress, and CSS0027597.1 could respond to both anthracnose disease and geometrid feeding treatments. In addition, 10 PYL genes related to growth and development were verified by RT-qPCR and their tissue expression characteristics were revealed. CONCLUSIONS: Our results provided a comprehensive characteristic of the PYL gene family in tea plants and provided an important clue for further exploring its functions in the growth and development, and resistance to stress of tea plants.
Assuntos
Camellia sinensis , Camellia sinensis/genética , Ácido Abscísico , Secas , Transcriptoma , CháRESUMO
Phosphoinositide 3-kinases (PI3Ks) generate lipids that control multitudinous intracellular cell signaling events which participate in cell survival and proliferation. In addition, PI3K signaling also contributes to metabolism, immunity, angiogenesis and cardiovascular homeostasis, and many diseases. The diverse actions of PI3K stem from the existence of their various isoforms and a variety of protein effectors. Hence, PI3K isoform-specific inhibitors have already achieved a wonderful effect on treating cancer. Herein, we summarize the molecular mechanism of PI3K inhibitors in preventing the permeability of vessels and neovascularization. Additionally, we briefly illustrate how PI3K signaling modulates blood vessel growth and discuss the different roles that PI3K isoforms play in angiogenesis.
Assuntos
Fosfatidilinositol 3-Quinase , Fosfatidilinositol 3-Quinases , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Isoformas de Proteínas/metabolismoRESUMO
Knowledge of the epidemiology and clinical characteristics of fungemia in southern China is limited. We conducted a six-year retrospective descriptive study to analyze the epidemiological and clinical characteristics of fungemia at the largest tertiary hospital in Guangxi, southern China. Data were obtained from the laboratory registry of patients with fungemia between January 2014 and December 2019. Demographic characteristics, underlying medical conditions, and outcomes for each case were analyzed. A total of 455 patients with fungemia were identified. Unexpectedly, Talaromyces marneffei (T. marneffei) was the most frequently isolated agent causing fungemia in the region (149/475, 31.4%), and Candida albicans (C. albicans) was the most commonly isolated Candida spp. (100/475, 21.1%). We identified that more than 70% of talaromycosis fungemia developed in AIDS patients, whereas candidemia was most commonly associated with a history of recent surgery. Notably, the total mortality rate of fungemia and the mortality rate in patients with T. marneffei and Cryptococcus neoformans (C. neoformans) fungemia were significantly higher in HIV-uninfected patients than in HIV-infected patients. In conclusion, the clinical pattern of fungemia in Guangxi is different from that in previous studies. Our study may provide new guidance for the early diagnosis and prompt treatment of fungemia in similar geographic regions.
Assuntos
Candidemia , Cryptococcus neoformans , Fungemia , Infecções por HIV , Humanos , Estudos Retrospectivos , China/epidemiologia , Fungemia/diagnóstico , Centros de Atenção Terciária , Candidemia/epidemiologia , Infecções por HIV/complicaçõesRESUMO
Fusarium head blight (FHB) is primarily caused by Fusarium graminearum and severely reduces wheat yield, causing mycotoxin contamination in grains and derived products. F. graminearum-secreted chemical toxins stably accumulate in plant cells, disturbing host metabolic homeostasis. We determined the potential mechanisms underlying FHB resistance and susceptibility in wheat. Three representative wheat varieties (Sumai 3, Yangmai 158, and Annong 8455) were inoculated with F. graminearum and their metabolite changes were assessed and compared. In total, 365 differentiated metabolites were successfully identified. Amino acids and derivatives, carbohydrates, flavonoids, hydroxycinnamate derivatives, lipids, and nucleotides constituted the major changes in response to fungal infection. Changes in defense-associated metabolites, such as flavonoids and hydroxycinnamate derivatives, were dynamic and differed among the varieties. Nucleotide and amino acid metabolism and the tricarboxylic acid cycle were more active in the highly and moderately resistant varieties than in the highly susceptible variety. We demonstrated that two plant-derived metabolites, phenylalanine and malate, significantly suppressed F. graminearum growth. The genes encoding the biosynthetic enzymes for these two metabolites were upregulated in wheat spike during F. graminearum infection. Thus, our findings uncovered the metabolic basis of resistance and susceptibility of wheat to F. graminearum and provided insights into engineering metabolic pathways to enhance FHB resistance in wheat.
Assuntos
Fusarium , Micotoxinas , Triticum/genética , Fusarium/fisiologia , Micotoxinas/metabolismo , Metabolômica , Doenças das Plantas/microbiologiaRESUMO
Gray blight (GB) is one of the most destructive diseases of tea plants, causing considerable damage and productivity losses; however, the dynamic roles of defense genes during pathogen infection remain largely unclear. To explore the numerous molecular interactions associated with GB stress in tea plants, we employed transcriptome, sRNAome and degradome sequencing from 1 to 13 days post-inoculation (dpi) at 3-day intervals. The transcriptomics results showed that differentially expressed genes (DEGs) related to flavonoid synthesis, such as chalcone synthase (CHS) and phenylalanine ammonia-lyase (PAL), were particularly induced at 4 dpi. Consistent with this, the contents of catechins (especially gallocatechin), which are the dominant flavonoids in tea plants, also increased in the leaves of tea plants infected with GB. Combined analysis of the sRNAome and degradome revealed that microRNAs could mediate tea plant immunity by regulating DEG expression at the post-transcriptional level. Co-expression network analysis demonstrated that miR530b-ethylene responsive factor 96 (ERF96) and miRn211-thaumatin-like protein (TLP) play crucial roles in the response to GB. Accordingly, gene-specific antisense oligonucleotide assays suggested that suppressing ERF96 decreased the levels of reactive oxygen species (ROS), whereas suppressing TLP increased the levels of ROS. Furthermore, ERF96 was induced, but TLP was suppressed, in susceptible tea cultivars. Our results collectively demonstrate that ERF96 is a negative regulator and TLP is a positive regulator in the response of tea plants to GB. Taken together, our comprehensive integrated analysis reveals a dynamic regulatory network linked to GB stress in tea plants and provides candidate genes for improvement of tea plants.
Assuntos
Camellia sinensis/genética , Genes de Plantas/genética , Doenças das Plantas/microbiologia , Transcriptoma/genética , Camellia sinensis/imunologia , Camellia sinensis/microbiologia , Regulação da Expressão Gênica de Plantas , Genes de Plantas/fisiologia , Sequenciamento de Nucleotídeos em Larga Escala , MicroRNAs/genética , MicroRNAs/fisiologia , Pestalotiopsis , Doenças das Plantas/imunologia , RNA de Plantas/genética , RNA de Plantas/fisiologiaRESUMO
N6 -methyladenosine (m6 A), the sixth N methylation of adenylate (A) in RNA, is the most abundant transcriptome modification in eukaryotic messenger RNA (mRNAs). m6 A modification exists in both coding mRNA and noncoding RNAs, and its functions are controlled by methyltransferase, demethylase, and m6 A reading proteins. Methylation modification of m6 A can regulate RNA cleavage, transport, stability, and expression. This review summarizes the enzymes involved in RNA m6 A methylation and the commonly used detection methods. The role of m6 A modification in physiological processes is described, and its impact on tumorigenesis, viral infection, and diabetes is further highlighted. Moreover, up-to-date knowledge of the implications of RNA m6 A modification in ocular diseases such as uveal melanoma and diabetic retinopathy is introduced. Clarifying the mechanism of RNA m6 A methylation will help elucidate the pathogenesis of various diseases, providing options for subsequent treatment.
Assuntos
Adenosina , Oftalmopatias , Metiltransferases , RNA , Adenosina/análogos & derivados , Adenosina/metabolismo , Oftalmopatias/metabolismo , Humanos , Metilação , Metiltransferases/metabolismo , RNA/metabolismo , RNA MensageiroRESUMO
Polypyrimidine tract-binding protein (PTB), as a member of the heterogeneous nuclear ribonucleoprotein family, functions by rapidly shuttling between the nucleus and the cytoplasm. PTB is involved in the alternative splicing of pre-messenger RNA (mRNA) and almost all steps of mRNA metabolism. PTB regulation is organ-specific; brain- or muscle-specific microRNAs and long noncoding RNAs partially contribute to regulating PTB, thereby modulating many physiological and pathological processes, such as embryonic development, cell development, spermatogenesis, and neuron growth and differentiation. Previous studies have shown that PTB knockout can inhibit tumorigenesis and development. The knockout of PTB in glial cells can be reprogrammed into functional neurons, which shows great promise in the field of nerve regeneration but is controversial.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas , Proteína de Ligação a Regiões Ricas em Polipirimidinas , Processamento Alternativo/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Neurônios/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , RNA Mensageiro/genéticaRESUMO
Pyroptosis is a programmed cell death characterized by swift plasma membrane disruption and subsequent release of cellular contents and pro-inflammatory mediators (cytokines), including IL-1ß and IL-18. It differs from other types of programmed cell death such as apoptosis, autophagy, necroptosis, ferroptosis, and NETosis in terms of its morphology and mechanism. As a recently discovered form of cell death, pyroptosis has been demonstrated to be involved in the progression of multiple diseases. Recent studies have also suggested that pyroptosis is linked to various ocular diseases. In this review, we systematically summarized and discussed recent scientific discoveries of the involvement of pyroptosis in common ocular diseases, including diabetic retinopathy, age-related macular degeneration, AIDS-related human cytomegalovirus retinitis, glaucoma, dry eye disease, keratitis, uveitis, and cataract. We also organized new and emerging evidence suggesting that pyroptosis signaling pathways may be potential therapeutic targets in ocular diseases, hoping to provide a summary of overall intervention strategies and relevant multi-dimensional evaluations for various ocular diseases, as well as offer valuable ideas for further research and development from the perspective of pyroptosis.
Assuntos
Inflamassomos , Piroptose , Apoptose , Humanos , Inflamassomos/metabolismo , Mediadores da Inflamação , Necroptose , Piroptose/fisiologiaRESUMO
BACKGROUND: Glaucoma, the major cause of irreversible blindness worldwide, is characterized by progressive degeneration of retinal ganglion cells (RGCs). Current treatments for glaucoma only slow or partially prevent the disease progression, failing to prevent RGCs death and visual field defects completely. Glutamate excitotoxicity via N-methyl-D-aspartic acid (NMDA) receptors plays a vital role in RGCs death in glaucoma, which is often accompanied by oxidative stress and NLRP3 inflammasome activation. However, the exact mechanisms remain unclear. METHODS: The glutamate-induced R28 cell excitotoxicity model and NMDA-induced mouse glaucoma model were established in this study. Cell counting kit-8, Hoechst 33342/PI dual staining and lactate dehydrogenase release assay were performed to evaluate cell viability. Annexin V-FITC/PI double staining was used to detect apoptosis and necrosis rate. Reactive oxygen species (ROS) and glutathione (GSH) were used to detect oxidative stress in R28 cells. Levels of proinflammatory cytokines were measured by qRT-PCR. Transmission electron microscopy (TEM) was used to detect necroptotic morphological changes in RGCs. Retinal RGCs numbers were detected by immunofluorescence. Hematoxylin and eosin staining was used to detect retinal morphological changes. The expression levels of RIP1, RIP3, MLKL and NLRP3 inflammasome-related proteins were measured by immunofluorescence and western blotting. RESULTS: We found that glutamate excitotoxicity induced necroptosis in RGCs through activation of the RIP1/RIP3/MLKL pathway in vivo and in vitro. Administration of the RIP3 inhibitor GSK872 and RIP1 inhibitor necrostatin-1 (Nec-1) prevented glutamate-induced RGCs loss, retinal damage, neuroinflammation, overproduction of ROS and a decrease in GSH. Furthermore, after suppression of the RIP1/RIP3/MLKL pathway by GSK872 and Nec-1, glutamate-induced upregulation of key proteins involved in NLRP3 inflammasome activation, including NLRP3, pro-caspase-1, cleaved-caspase-1, and interleukin-1ß (IL-1ß), was markedly inhibited. CONCLUSIONS: Our findings suggest that the RIP1/RIP3/MLKL pathway mediates necroptosis of RGCs and regulates NLRP3 inflammasome activation induced by glutamate excitotoxicity. Moreover, GSK872 and Nec-1 can protect RGCs from necroptosis and suppress NLRP3 inflammasome activation through inhibition of RIP1/RIP3/MLKL pathway, conferring a novel neuroprotective treatment for glaucoma.
Assuntos
Glaucoma , Necroptose , Camundongos , Animais , Espécies Reativas de Oxigênio/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Interleucina-1beta/metabolismo , N-Metilaspartato , Inflamassomos/metabolismo , Caspase 1/metabolismo , Células Ganglionares da Retina/metabolismo , Ácido Glutâmico/toxicidade , Hematoxilina , Amarelo de Eosina-(YS) , Apoptose , Glaucoma/induzido quimicamente , Glaucoma/tratamento farmacológico , Glutationa/metabolismo , Lactato Desidrogenases/metabolismoRESUMO
There are many theories surrounding the pathogenesis of glaucoma, and glutamate excitatory toxicity has been suggested to play an important role. Some studies have shown that glutamate excitatory toxicity is associated with mitochondrial dynamics; however, the relationship between glutamate excitatory toxicity and mitochondrial dynamics in the pathogenesis of glaucoma remains unclear. In this study, the glutamate transporter inhibitor, threohydroxyaspartate, was used to simulate the glutamate excitatory toxicity cell model of rat retinal neurons in vitro, and the changes in the level of proteins related to mitochondrial dynamics, mitochondrial morphology, and length of neuronal axons were measured. We found that in the glutamate excitotoxicity model, retinal neurons can promote mitochondrial fusion by reducing the phosphorylation of ERK1/2 and its downstream protein DRP1 S585, and enhance its ability to resist the excitotoxicity of glutamate. At the same time, the DRP1-specific inhibitor, Mdivi-1, could promote the mitochondrial fusion of retinal neurons.
Assuntos
Glaucoma , Neurônios Retinianos , Animais , Ratos , Dinâmica Mitocondrial , Ácido Glutâmico/toxicidade , Ácido Glutâmico/metabolismo , Dinaminas/metabolismo , Sistema de Sinalização das MAP Quinases , Glaucoma/patologia , Neurônios Retinianos/metabolismoRESUMO
Vitreous has been reported to prevent tumor angiogenesis, but our previous findings indicate that vitreous activate the signaling pathway of phosphoinositide 3-kinase (PI3K)/Akt, which plays a critical role in angiogenesis. The goal of this research is to determine which role of vitreous plays in angiogenesis-related cellular responses in vitro. We found that in human retinal microvascular endothelial cells (HRECs) vitreous activates a number of receptor tyrosine kinases including Anexelekto (Axl), which plays an important role in angiogenesis. Subsequently, we discovered that depletion of Axl using CRISPR/Cas9 and an Axl-specific inhibitor R428 suppress vitreous-induced Akt activation and cell proliferation, migration, and tuber formation of HRECs. Therefore, this line of research not only demonstrate that vitreous promotes angiogenesis in vitro, but also reveal that Axl is one of receptor tyrosine kinases to mediate vitreous-induced angiogenesis in vitro, thereby providing a molecular basis for removal of vitreous as cleanly as possible when vitrectomy is performed in treating patients with proliferative diabetic retinopathy.
Assuntos
Neovascularização Patológica/enzimologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Vasos Retinianos/enzimologia , Corpo Vítreo/enzimologia , Animais , Benzocicloeptenos/farmacologia , Sistemas CRISPR-Cas , Retinopatia Diabética/enzimologia , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Células HEK293 , Humanos , Camundongos , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Vasos Retinianos/patologia , Triazóis/farmacologia , Vitreorretinopatia Proliferativa/enzimologia , Vitreorretinopatia Proliferativa/genética , Vitreorretinopatia Proliferativa/patologia , Corpo Vítreo/patologia , Receptor Tirosina Quinase AxlRESUMO
In vivo genome editing meets numerous challenges including efficiency and safety. Here we describe an efficient in vivo genome editing method of delivering CRISPR-Cas9 into vascular endothelial cells with adeno-associated viruses (AAVs). In this system, expression of SpCas9 is driven by a specific endothelial promoter of intercellular adhesion molecule 2 (pICAM2) to restrict this foreign enzyme in vascular endothelial cells, which can be efficiently infected by AAV1. We exemplify this approach by editing VEGFR2 in retinal vascular endothelial cells in a mouse model of oxygen-induced retinopathy, and expect that this simplified protocol can be expanded to other researches on editing endothelial genome in vivo.
Assuntos
Células Endoteliais , Edição de Genes , Animais , Sistemas CRISPR-Cas/genética , Dependovirus/genética , Camundongos , Regiões Promotoras Genéticas , RetinaRESUMO
The purpose of this study was to investigate the effects of valdecoxib on the retina in retinal ischemia-reperfusion injury (IRI) and R28 cells following oxygen-glucose deprivation/recovery (OGD/R) injury, as well as the underlying mechanisms. Immunofluorescence and Cell Counting Kit-8 (CCK-8) analyses were used to identify the proper timepoint and concentration of valdecoxib's protective effect on the R28 cells in the OGD/R model. Hematoxylin-eosin (HE) staining and immunofluorescence were used to explore valdecoxib's effect on the retina and retina ganglion cell (RGC) in IRI. Cell apoptosis was determined by a TUNEL Apoptosis Detection Kit and Annexin V-FITC/PI flow cytometry. The expression levels of p-PERK, transcription factor 4 (ATF4), GRP78, CHOP, cleaved caspase 3, bax and bcl-2 were measured by Western blot analyses. The valdecoxib protected the R28 cells from OGD/R injury by decreasing the cell apoptosis rate, and it exerted a protective effect on retinas in I/R injury by inhibiting RGC apoptosis. The valdecoxib pretreatment reversed the expression of p-PERK, ATF4, CHOP, GRP78, cleaved caspase 3 and bax induced by the glaucomatous model. Meanwhile, the CCT020312 reversed the valdecoxib's anti-apoptosis effect by activating PERK-ATF4-CHOP pathway-mediated endoplasmic reticulum (ER) stress. These findings suggest that valdecoxib protects against glaucomatous injury by inhibiting ER stress-induced apoptosis via the inhibition of the PERK-ATF4-CHOP pathway.
Assuntos
Estresse do Retículo Endoplasmático , Glaucoma , Animais , Ratos , Caspase 3/metabolismo , Proteína X Associada a bcl-2 , Transdução de Sinais , Ratos Sprague-Dawley , Glucose/metabolismo , Oxigênio/metabolismo , Glaucoma/tratamento farmacológico , Fator de Transcrição CHOP/metabolismo , eIF-2 Quinase/metabolismo , Fator 4 Ativador da Transcrição/metabolismoRESUMO
Wall-associated kinases (WAKs) are important receptor-like proteins that play major roles in plant defense against pathogens. Fusarium head blight (FHB), one of the most widespread and devastating crop diseases, reduces wheat yield and leads to quality deterioration. Although WAK gene families have been studied in many plants, systematic research on bread wheat (Triticum aestivum) and its role in FHB resistance, in particular, is lacking. In this study, we identified and characterized 320 genes of the TaWAK family in wheat distributed across all chromosomes except 4B and divided them into three phylogenetic groups. Duplication and synteny analyses provided valuable information on the evolutionary characteristics of the TaWAK genes. The gene expression pattern analysis suggested that TaWAK genes play diverse roles in plant biological processes and that at least 30 genes may be involved in the response to Fusarium infection in wheat spikes, with most of the genes contributing to pectin- and chitin-induced defense pathways. Furthermore, 45 TaWAK genes were identified within 17 hcmQTLs that are related to wheat FHB resistance. Our findings provide potential candidate genes for improving FHB resistance and insights into the future functional analysis of TaWAK genes in wheat.
Assuntos
Fusarium , Pão , Resistência à Doença/genética , Fusarium/fisiologia , Filogenia , Doenças das Plantas/genética , Triticum/genética , Triticum/metabolismoRESUMO
OBJECTIVES: The weakness and dialysis of lens zonule after cataract surgery may lead to dislocation of intraocular lens (IOL). It has been shown that cataract surgery could induce or aggravate posterior vitreous detachment (PVD) due to postoperative inflammation and increased volume of vitreous cavity. PVD is associated with the occurrence of several vitreoretinal diseases, such as rhegmatogenous retinal detachment and macular hole. This study aims to explore risk factors for dislocation of IOL concurring with vitreoretinal disease, such as retinal detachment and macular hole, and to evaluate the efficacy and complications of surgical intervention for these abnormalities concurrently. METHODS: Ten patients (10 eyes) who diagnosed as rhegmatogenous retinal detachment, traumatic macular hole, high myopic macular hole, and combined with IOL dislocation at the Department of Ophthalmology of Xiangya Hospital from January 2004 to December 2020 were enrolled. The patients received vitreoretinal surgery and reposition of IOL by scleral suturing. Medical records were reviewed to figure out the time and type of IOL dislocation. Preoperative and 1 year of postoperative best corrected visual acuity, intraocular pressure, corneal endothelial density, and complications of surgical management were analyzed. RESULTS: Ten patients including 4 high myopia, 4 ocular contusion, and 2 who experienced IOL dislocation during the posterior capsulotomy were included in this study. Coexistence of IOL dislocation and vitreoretinal abnormalities occurred in patients with high myopia, ocular contusion, and capsulotomy. IOL dislocation happened in the vitreoretinal surgery in patients with high myopia or intraoperative capsulotomy. IOL dislocation occurred preoperatively in patients with ocular contusion. IOL capsular bag complex dislocation and out-of-the-bag IOL dislocation were found in 4 and 6 patients, respectively. Surgical relocation of dropped IOL and repair of vitreoretinal disease improved the best corrected visual acuity from preoperative 1.79±0.39 to postoperative 1.13±0.45 (P<0.001). The density of corneal endothelial cells in patients was lower than that before surgery [(1 806.40±181.20) cells/mm2 vs (1 914.00±182.22) cells/mm2, P<0.001]. There was no significant difference in intraocular pressure before and after surgery (P=0.099). Postoperative complications included high intraocular pressure and recurrent retinal detachment. CONCLUSIONS: Dislocation of IOL may be concurrent with vitreoretinal disease. High myopia, blunt contusion, and capsulectomy might be the risk factors for intraocular lens dislocation. The surgical technique used in the present study is successful in manipulating these disorders with optimal functional results and less severe complications.
Assuntos
Catarata , Contusões , Subluxação do Cristalino , Miopia , Descolamento Retiniano , Perfurações Retinianas , Catarata/etiologia , Contusões/complicações , Células Endoteliais , Humanos , Implante de Lente Intraocular , Subluxação do Cristalino/complicações , Subluxação do Cristalino/cirurgia , Miopia/complicações , Miopia/cirurgia , Complicações Pós-Operatórias/epidemiologia , Descolamento Retiniano/complicações , Perfurações Retinianas/complicações , Estudos Retrospectivos , Fatores de Risco , Resultado do Tratamento , Acuidade Visual , Vitrectomia/efeitos adversos , Vitrectomia/métodosRESUMO
Presbyopia, a progressive visual difficulty caused by weakened physiological regulation, is one of the main causes of visual impairment in people over 40 years old. Currently, the main methods of correction of presbyopia include optical correction, surgical correction, and drug treatment, which can improve the visual nearness disorder to some extent. Optical correction is the most common way with advantages of safety, which can adjust the lens parameters at any time, while cause kinds of inconvenience in life by wearing and taking off glasses frequently. Surgical intervention, including corneal surgery, lens surgery and scleral surgery, with certain advantages and disadvantages in each operation style. New pharmaceutical agents are expected to be a new and effective method for the treatment of presbyopia, but it lacks multicenter randomized controlled trials and evidence-based medicine evidence to evaluate the safety and effectiveness.
RESUMO
Noncoding ribonucleic acids (ncRNAs) are an increasingly studied class of RNA molecules with extensive biological activities, including important roles in human development, health, and disease. Glaucoma is a neurodegenerative disease of the retina, and one of the leading causes of blindness worldwide. However, the specific roles of ncRNAs in the development and progression of glaucoma are unclear, and related reports are fragmented. An in-depth understanding of ncRNAs participating in the pathogenesis and progression of glaucoma would be helpful for opening up new avenues to facilitate the early diagnosis and clinical treatment. Therefore, in this review, we aimed to discuss the current research progress, the potentialfuture clinical applications and the research limitations of three critical classes of ncRNAs in glaucoma, namely microRNAs, long noncoding RNAs, and circular RNAs.