RESUMO
Regulated cell death (such as apoptosis, necroptosis, pyroptosis, autophagy, cuproptosis, ferroptosis, disulfidptosis) involves complex signaling pathways and molecular effectors, and has been proven to be an important regulatory mechanism for regulating neuronal aging and death. However, excessive activation of regulated cell death may lead to the progression of aging-related diseases. This review summarizes recent advances in the understanding of seven forms of regulated cell death (RCD) in age-related diseases. Notably, the newly identified ferroptosis and cuproptosis have been implicated in the risk of cognitive impairment and neurodegenerative diseases. These forms of cell death exacerbate disease progression by promoting inflammation, oxidative stress, and pathological protein aggregation. The review also provides an overview of key signaling pathways and crosstalk mechanisms among these RCD forms, with a focus on ferroptosis, cuproptosis, and disulfidptosis. For instance, FDX1 directly induces cuproptosis by regulating copper ion valency and dihydrolipoamide S-acetyltransferase (DLAT) aggregation, while copper mediates glutathione peroxidase 4 (GPX4) degradation, enhancing ferroptosis sensitivity. Additionally, inhibiting the Xc- transport system to prevent ferroptosis can increase disulfide formation and shift the NADP+/NADPH ratio, transitioning ferroptosis to disulfidptosis. These insights help to uncover the potential connections among these novel RCD forms and differentiate them from traditional RCD mechanisms. In conclusion, identifying key targets and their crosstalk points among various RCD pathways may aid in developing specific biomarkers to reverse the aging clock and treat age-related neurodegenerative conditions.
RESUMO
Tissue regeneration technology has been rapidly developed and widely applied in tissue engineering and repair. Compared with traditional approaches like surgical treatment, the rising gene therapy is able to have a durable effect on tissue regeneration, such as impaired bone regeneration, articular cartilage repair and cancer-resected tissue repair. Gene therapy can also facilitate the production of in situ therapeutic factors, thus minimizing the diffusion or loss of gene complexes and enabling spatiotemporally controlled release of gene products for tissue regeneration. Among different gene delivery vectors and supportive gene-activated matrices, advanced gene/drug nanocarriers attract exceptional attraction due to their tunable physiochemical properties, as well as excellent adaptive performance in gene therapy for tissue regeneration, such as bone, cartilage, blood vessel, nerve and cancer-resected tissue repair. This paper reviews the recent advances on nonviral-mediated gene delivery systems with an emphasis on the important role of advanced nanocarriers in gene therapy and tissue regeneration.
Assuntos
Técnicas de Transferência de Genes , Terapia Genética , Regeneração , Engenharia Tecidual , Alicerces Teciduais , Humanos , Animais , Terapia Genética/métodos , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Nanopartículas/química , Portadores de Fármacos/química , Vetores GenéticosRESUMO
Bietti crystalline corneoretinal dystrophy is an inherited retinal disease caused by mutations in CYP4V2, which results in blindness in the working-age population, and there is currently no available treatment. Here, we report the results of the first-in-human clinical trial (NCT04722107) of gene therapy for Bietti crystalline corneoretinal dystrophy, including 12 participants who were followed up for 180-365 days. This open-label, single-arm exploratory trial aimed to assess the safety and efficacy of a recombinant adeno-associated-virus-serotype-2/8 vector encoding the human CYP4V2 protein (rAAV2/8-hCYP4V2). Participants received a single unilateral subretinal injection of 7.5 × 1010 vector genomes of rAAV2/8-hCYP4V2. Overall, 73 treatment-emergent adverse events were reported, with the majority (98.6%) being of mild or moderate intensity and considered to be procedure- or corticosteroid-related; no treatment-related serious adverse events or local/systemic immune toxicities were observed. Compared with that measured at baseline, 77.8% of the treated eyes showed improvement in best-corrected visual acuity (BCVA) on day 180, with a mean ± standard deviation increase of 9.0 ± 10.8 letters in the 9 eyes analyzed (p = 0.021). By day 365, 80% of the treated eyes showed an increase in BCVA, with a mean increase of 11.0 ± 10.6 letters in the 5 eyes assessed (p = 0.125). Importantly, the patients' improvement observed using multifocal electroretinogram, microperimetry, and Visual Function Questionnaire-25 further supported the beneficial effects of the treatment. We conclude that the favorable safety profile and visual improvements identified in this trial encourage the continued development of rAAV2/8-hCYP4V2 (named ZVS101e).
Assuntos
Distrofias Hereditárias da Córnea , Família 4 do Citocromo P450 , Dependovirus , Terapia Genética , Doenças Retinianas , Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Adulto , Distrofias Hereditárias da Córnea/genética , Distrofias Hereditárias da Córnea/terapia , Distrofias Hereditárias da Córnea/patologia , Dependovirus/genética , Família 4 do Citocromo P450/genética , Vetores Genéticos/genética , Acuidade VisualRESUMO
Parkinson's disease (PD) is characterized by the progressive death of dopamine (DA) neurons and the pathological accumulation of α-synuclein (α-syn) fibrils. In our previous study, simulated PHB2 phosphorylation was utilized to clarify the regulatory role of c-Abl in PHB2-mediated mitophagy in PD models. In this investigation, we employed an independently patented PHB2Y121 phosphorylated antibody in the PD model to further verify that the c-Abl inhibitor STI571 can impede PHB2Y121 phosphorylation, decrease the formation of α-Syn polymers, and improve autophagic levels. The specific involvement of Nur77 in PD pathology has remained elusive. We also investigate the contribution of Nur77, a nuclear transcription factor, to α-syn and mitophagy in PD. Our findings demonstrate that under α-syn, Nur77 translocates from the cytoplasm to the mitochondria, improving PHB-mediated mitophagy by regulating c-Abl phosphorylation. Moreover, Nur77 overexpression alleviates the expression level of pS129-α-syn and the loss of DA neurons in α-syn PFF mice, potentially associated with the p-c-Abl/p-PHB2 Y121 axis. This study provides initial in vivo and in vitro evidence that Nur77 protects PD DA neurons by modulating the p-c-Abl/p-PHB2 Y121 axis, and STI571 holds promise as a treatment for PD.
Assuntos
Neuroblastoma , Doença de Parkinson , Camundongos , Humanos , Animais , alfa-Sinucleína/metabolismo , Mitofagia , Mesilato de Imatinib , Neuroblastoma/patologia , Doença de Parkinson/metabolismo , Neurônios Dopaminérgicos/metabolismoRESUMO
JOURNAL/nrgr/04.03/01300535-202408000-00037/figure1/v/2023-12-16T180322Z/r/image-tiff Endoplasmic reticulum stress and mitochondrial dysfunction play important roles in Parkinson's disease, but the regulatory mechanism remains elusive. Prohibitin-2 (PHB2) is a newly discovered autophagy receptor in the mitochondrial inner membrane, and its role in Parkinson's disease remains unclear. Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is a factor that regulates cell fate during endoplasmic reticulum stress. Parkin is regulated by PERK and is a target of the unfolded protein response. It is unclear whether PERK regulates PHB2-mediated mitophagy through Parkin. In this study, we established a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of Parkinson's disease. We used adeno-associated virus to knockdown PHB2 expression. Our results showed that loss of dopaminergic neurons and motor deficits were aggravated in the MPTP-induced mouse model of Parkinson's disease. Overexpression of PHB2 inhibited these abnormalities. We also established a 1-methyl-4-phenylpyridine (MPP+)-induced SH-SY5Y cell model of Parkinson's disease. We found that overexpression of Parkin increased co-localization of PHB2 and microtubule-associated protein 1 light chain 3, and promoted mitophagy. In addition, MPP+ regulated Parkin involvement in PHB2-mediated mitophagy through phosphorylation of PERK. These findings suggest that PHB2 participates in the development of Parkinson's disease by interacting with endoplasmic reticulum stress and Parkin.
RESUMO
Recent human decedent model studies1,2 and compassionate xenograft use3 have explored the promise of porcine organs for human transplantation. To proceed to human studies, a clinically ready porcine donor must be engineered and its xenograft successfully tested in nonhuman primates. Here we describe the design, creation and long-term life-supporting function of kidney grafts from a genetically engineered porcine donor transplanted into a cynomolgus monkey model. The porcine donor was engineered to carry 69 genomic edits, eliminating glycan antigens, overexpressing human transgenes and inactivating porcine endogenous retroviruses. In vitro functional analyses showed that the edited kidney endothelial cells modulated inflammation to an extent that was indistinguishable from that of human endothelial cells, suggesting that these edited cells acquired a high level of human immune compatibility. When transplanted into cynomolgus monkeys, the kidneys with three glycan antigen knockouts alone experienced poor graft survival, whereas those with glycan antigen knockouts and human transgene expression demonstrated significantly longer survival time, suggesting the benefit of human transgene expression in vivo. These results show that preclinical studies of renal xenotransplantation could be successfully conducted in nonhuman primates and bring us closer to clinical trials of genetically engineered porcine renal grafts.
Assuntos
Rejeição de Enxerto , Transplante de Rim , Macaca fascicularis , Suínos , Transplante Heterólogo , Animais , Humanos , Animais Geneticamente Modificados , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Transplante de Rim/métodos , Polissacarídeos/deficiência , Suínos/genética , Transplante Heterólogo/métodos , Transgenes/genéticaRESUMO
Oxidative stress plays a crucial role in the occurrence and development of Parkinson's disease (PD). Rutin, a natural botanical ingredient, has been shown to have antioxidant properties. Therefore, the aim of this study was to investigate the neuroprotective effects of rutin on PD and the underlying mechanisms. MPP+(1-methyl-4-phenylpyridinium ions)-treated SH-SY5Y cells were used as an in vitro model of PD. Human PHB2-shRNA lentiviral particles were transfected into SH-SY5Y cells to interfere with the expression of Prohibitin2 (PHB2). The oxidative damage of cells was analyzed by detecting intracellular reactive oxygen species (ROS), malondialdehyde (MDA), and mitochondrial membrane potential (MMP). Western blotting was used to detect the protein expression of antioxidant factors such as nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), NADPH quinone oxidoreductase-1 (NQO-1), and mitophagy factors PHB2, translocase of outer mitochondrial membrane 20 (TOM20), and LC3II/LC3I (microtubule-associated protein II light chain 3 (LC3II) to microtubule-associated protein I light chain 3 (LC3I)). In addition, we also examined the expression of PHB2 and LC3II/LC3I by immunofluorescence staining. MPP+ treatment significantly increased the generation of ROS and MDA and the level of MMP depolarization and decreased the protein expression of Nrf2, HO-1, NQO1, TOM20, PHB2, and LC3II/LC3I. In MPP+-treated SH-SY5Y cells, rutin significantly decreased the generation of ROS and MDA and the level of MMP depolarization and increased the protein expression of Nrf2, HO-1, NQO-1, TOM20, PHB2, and LC3II/LC3I. However, the protective role of rutin was inhibited in PHB2-silenced cells. Rutin attenuates oxidative damage which may be associated with PHB2-mediated mitophagy in MPP+-induced SH-SY5Y cells. Rutin might be used as a potential drug for the prevention and treatment of PD.
Assuntos
Neuroblastoma , Doença de Parkinson , Humanos , Espécies Reativas de Oxigênio/metabolismo , Antioxidantes/farmacologia , Mitofagia , 1-Metil-4-fenilpiridínio/toxicidade , Rutina/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Linhagem Celular Tumoral , Doença de Parkinson/tratamento farmacológico , Proteínas Associadas aos Microtúbulos/metabolismo , ApoptoseRESUMO
A new virtual synchronous generator (VSG) control strategy was researched and proposed for a VSC-HVDC (High Voltage Direct Current Based on Voltage Source Converter) transmission system. It can be applied to half-bridge or full-half-bridge hybrid topology modular multi-level converter (MMC) to improve the stability and reliability of the system. First, it is proposed that the energy stored in the equivalent capacitor of MMC power module was used to imitate the rotor inertial of synchronous generator. It can buffer transient power fluctuations and synchronize autonomously with the grid. Then the impedance characteristics of the proposed control method have been deduced and analyzed. The results show that the VSG control loop mainly improves the low frequency characteristics of the converter. Secondly, the ability to suppress transient fault current is weak. So, a method, that the given values of inner current loop are calculated by grid impedance matrix, was used. A double closed loop control structure composed by a power outer loop based on VSG control and a current inner loop is obtained. The simulation results show that it can effectively improve the current control capability during the transient process for systems with a 1:2 ratio of converter capacity to grid capacity (The grid short-circuit capacity is 60MW and the MMC is 30 MW). Finally, a hybrid MMC simulation model was built based on PSCAD and the steady-state and transient fault ride-through simulations were performed. The power adjustment time of MMC under the proposed VSG control is about 1s, while the adjustment time under the conventional control strategy is greater than 4s.
Assuntos
Fontes de Energia Elétrica , Modelos Teóricos , Reprodutibilidade dos Testes , Eletricidade , Simulação por ComputadorRESUMO
Mitophagy and oxidative stress play important roles in Parkinson's disease (PD). Dysregulated mitophagy exacerbates mitochondrial oxidative damage; however, the regulatory mechanism of mitophagy is unclear. Here, we provide a potential mechanistic link between c-Abl, a nonreceptor tyrosine kinase, and mitophagy in PD progression. We found that c-Abl activation reduces the interaction of prohibitin 2 (PHB2) and microtubule-associated protein 1 light chain 3 (LC3) and decreases the expressive level of antioxidative stress proteins, including nuclear factor erythroid 2-related factor 2 (Nrf2), NADPH quinone oxidoreductase-1 (NQO-1), and the antioxidant enzyme heme oxygenase-1 (HO-1) in 1-methyl-4-phenylpyridinium- (MPP+-) lesioned SH-SY5Y cells. Importantly, we found that MPP+ can increase the expression of phosphorylated proteins at the tyrosine site of PHB2 and the interaction of c-Abl with PHB2. We showed for the first time that PHB2 by changing tyrosine (Y) to aspartate (D) at site 121 resulted in impaired binding of PHB2 and LC3 in vitro. Moreover, silencing of PHB2 can decrease the interaction of PHB2 and LC3 and exacerbate the loss of dopaminergic neurons. We also found that STI 571, a c-Abl family kinase inhibitor, can decrease dopaminergic neuron damage and ameliorate MPTP-induced behavioral deficits in PD mice. Taken together, our findings highlight a novel molecular mechanism for aberrant PHB2 phosphorylation as an inhibitor of c-Abl activity and suggest that c-Abl and PHB2 are potential therapeutic targets for the treatment of individuals with PD. However, these results need to be further validated in PHB2 Y121D mice.
Assuntos
Neuroblastoma , Doença de Parkinson , Animais , Humanos , Camundongos , Mitofagia , Fosforilação , Doença de Parkinson/tratamento farmacológico , TYK2 Quinase/metabolismo , TYK2 Quinase/uso terapêutico , Proibitinas , 1-Metil-4-fenilpiridínio , Tirosina/metabolismoRESUMO
PURPOSE: To assess the performance of a deep learning (DL) algorithm for evaluating and supervising cataract extraction using phacoemulsification with intraocular lens (IOL) implantation based on cataract surgery (CS) videos. MATERIALS AND METHODS: DeepSurgery was trained using 186 standard CS videos to recognize 12 CS steps and was validated in two datasets that contained 50 and 21 CS videos, respectively. A supervision test including 50 CS videos was used to assess the DeepSurgery guidance and alert function. In addition, a real-time test containing 54 CSs was used to compare the DeepSurgery grading performance to an expert panel and residents. RESULTS: DeepSurgery achieved stable performance for all 12 recognition steps, including the duration between two pairs of adjacent steps in internal validation with an ACC of 95.06% and external validations with ACCs of 88.77% and 88.34%. DeepSurgery also recognized the chronology of surgical steps and alerted surgeons to order of incorrect steps. Six main steps are automatically and simultaneously quantified during the evaluation process (centesimal system). In a real-time comparative test, the DeepSurgery step recognition performance was robust (ACC of 90.30%). In addition, DeepSurgery and an expert panel achieved comparable performance when assessing the surgical steps (kappa ranged from 0.58 to 0.77). CONCLUSIONS: DeepSurgery represents a potential approach to provide a real-time supervision and an objective surgical evaluation system for routine CS and to improve surgical outcomes.
Assuntos
Extração de Catarata , Catarata , Aprendizado Profundo , Facoemulsificação , Algoritmos , HumanosRESUMO
Robust protocols to examine 3D chromatin structure have greatly advanced knowledge of gene regulatory mechanisms. Here we focus on the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which provides a paradigm for validating models of gene regulation built upon genome-wide analysis. We examine the mechanisms by which multiple cis-regulatory elements (CREs) at the CFTR gene coordinate its expression in intestinal epithelial cells. Using CRISPR/Cas9 to remove CREs, individually and in tandem, followed by assays of gene expression and higher-order chromatin structure (4C-seq), we reveal the cross-talk and dependency of two cell-specific intronic enhancers. The results suggest a mechanism whereby the locus responds when CREs are lost, which may involve activating transcription factors such as FOXA2. Also, by removing the 5' topologically-associating domain (TAD) boundary, we illustrate its impact on CFTR gene expression and architecture. These data suggest a multi-layered regulatory hierarchy that is highly sensitive to perturbations.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Elementos Facilitadores Genéticos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Cromatina , Regulação da Expressão Gênica , Fatores Ativadores da Transcrição/genética , Fatores Ativadores da Transcrição/metabolismoRESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) gene lies within a topologically associated domain (TAD) in which multiple cis-regulatory elements (CREs) and transcription factors (TFs) regulate its cell-specific expression. The CREs are recruited to the gene promoter by a looping mechanism that depends upon both architectural proteins and specific TFs. An siRNA screen to identify TFs coordinating CFTR expression in airway epithelial cells suggested an activating role for BTB domain and CNC homolog 1 (BACH1). BACH1 is a ubiquitous master regulator of the cellular response to oxidative stress. Here, we show that BACH1 may have a dual effect on CFTR expression by direct occupancy of CREs at physiological oxygen (â¼8%), while indirectly modulating expression under conditions of oxidative stress. Hence BACH1, can activate or repress the same gene, to fine tune expression in response to environmental cues such as cell stress. Furthermore, our 4C-seq data suggest that BACH1 can also directly regulate CFTR gene expression by modulating locus architecture through occupancy at known enhancers and structural elements, and depletion of BACH1 alters the higher order chromatin structure.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Estresse Oxidativo/genética , Fatores de Transcrição de Zíper de Leucina Básica/antagonistas & inibidores , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Perfilação da Expressão Gênica , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Glutationa/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Oxigênio/metabolismo , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Cell-specific and developmental mechanisms contribute to expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene; however, its developmental regulation is poorly understood. Here we use human induced pluripotent stem cells differentiated into pseudostratified airway epithelial cells to study these mechanisms. RESULTS: Changes in gene expression and open chromatin profiles were investigated by RNA-seq and ATAC-seq, and revealed that alterations in CFTR expression are associated with differences in stage-specific open chromatin. Additionally, two novel open chromatin regions, at +19.6 kb and +22.6 kb 3' to the CFTR translational stop signal, were observed in definitive endoderm (DE) cells, prior to an increase in CFTR expression in anterior foregut endoderm (AFE) cells. Chromatin studies in DE and AFE cells revealed enrichment of active enhancer marks and occupancy of OTX2 at these sites in DE cells. Loss of OTX2 in DE cells alters histone modifications across the CFTR locus and results in a 2.5-fold to 5-fold increase in CFTR expression. However, deletion of the +22.6 kb site alone does not affect CFTR expression in DE or AFE cells. CONCLUSIONS: These results suggest that a network of interacting cis-regulatory elements recruit OTX2 to the locus to impact CFTR expression during early endoderm differentiation.
Assuntos
Diferenciação Celular , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fatores de Transcrição Otx/metabolismo , Elementos Reguladores de Transcrição , Mucosa Respiratória/citologia , Células Cultivadas , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Endoderma/citologia , Humanos , Células-Tronco Pluripotentes InduzidasRESUMO
Spermatozoa released from the testis are unable to fertilize an egg without a coordinated process of maturation in the lumen of the epididymis. Relatively little is known about the molecular events that integrate this critical progression along the male genital ducts in man. Here, we use single cell RNA-sequencing to construct an atlas of the human proximal epididymis. We find that the CFTR, which is pivotal in normal epididymis fluid transport, is most abundant in surface epithelial cells in the efferent ducts and in rare clear cells in the caput epididymis, suggesting region-specific functional properties. We reveal transcriptional signatures for multiple cell clusters, which identify the individual roles of principal, apical, narrow, basal, clear, halo, and stromal cells in the epididymis. A marked cell type-specific distribution of function is seen along the duct with local specialization of individual cell types integrating processes of sperm maturation.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Epididimo/citologia , Adulto , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Epididimo/metabolismo , Epididimo/fisiologia , Células Epiteliais/metabolismo , Epitélio/metabolismo , Genitália Masculina , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Maturação do Esperma , Espermatozoides/citologia , Espermatozoides/fisiologia , TestículoRESUMO
PURPOSE: The present study highlighted the value of anterior segment optical coherence tomography (AS-OCT) for different types of corneal foreign bodies in humans. METHODS: This study was a prospective observational study. The patients included were divided into two groups. If the patients were directly diagnosed based on eye injury history and slit-lamp examination, then they were assigned to Group A. Otherwise, the patients were assigned to Group B. We compared and described the characteristics of the corneal foreign body in both groups using AS-OCT. RESULTS: From October 2017 to January 2020, 36 eyes of 36 patients (9 females and 27 males) with a mean age of 37.8 ± 11.7 years were included in the study. Patients in Group A were the majority and accounted for 72.2% (26/36). High signals on AS-OCT images were the main constituent and accounted for 92.3% (24/26) in Group A and 70.0% (7/10) in Group B. Most of the patients in Group A, 96.2% (25/26), had clear boundaries. A blurred boundary was observed in 70.0% (7/10) of the patients in Group B. The foreign bodies on AS-OCT images had key characteristics of a high signal followed by a central zone shadowing effect and a low signal followed by a marginal zone shadowing effect. Further, all of the lesions could be directly located in Group B, and 92.3% (24/26) of the patients in Group A did not have directly located lesions. Six representative cases are described in detail. CONCLUSIONS: AS-OCT is a valuable tool in the diagnosis and management of corneal foreign bodies, especially for unusual corneal foreign body.
RESUMO
BACKGROUND: Deep learning has had a large effect on medical fields, including ophthalmology. The goal of this study was to quantitatively analyze the functional filtering bleb size with Mask R-CNN. METHODS: This observational study employed eighty-three images of post-trabeculectomy functional filtering blebs. The images were divided into training and test groups and scored according to the Indiana Bleb Appearance Grading Scale (IBAGS) system. Then, 70 images from the training group were used to train an automatic detection system based on Mask R-CNN and perform a quantitative analysis of the function bleb size. Thirteen images from the test group were used to evaluate the model. During the training process, left and right image-flipping algorithms were used for data augmentation. Finally, the correlation between the functional filtering bleb area and the intraocular pressure (IOP) was analyzed. RESULTS: The 83 functional filtering blebs have similar morphological features. According to IBAGS, the functional filtering blebs have a high incidence of E1/E2, H1/H2, and V0/V1. Our Mask R-CNN-based model using the selected parameters achieves good results on the training group after a 200-epoch training process. All the Intersection over Union (IoU) scores exceeded 93% on the test group. The Spearman correlation coefficient between the area of functional filtering blebs and the IOP value was -0.757 (P<0.05). CONCLUSIONS: Deep learning is a powerful tool for quantitatively analyzing the functional filtering bleb size. This technique is suitable for use in monitoring post-trabeculectomy filtering blebs in the future.
RESUMO
The availability of robust protocols to differentiate induced pluripotent stem cells (iPSCs) into many human cell lineages has transformed research into the origins of human disease. The efficacy of differentiating iPSCs into specific cellular models is influenced by many factors including both intrinsic and extrinsic features. Among the most challenging models is the generation of human bronchial epithelium at air-liquid interface (HBE-ALI), which is the gold standard for many studies of respiratory diseases including cystic fibrosis. Here, we perform open chromatin mapping by ATAC-seq and transcriptomics by RNA-seq in parallel, to define the functional genomics of key stages of the iPSC to HBE-ALI differentiation. Within open chromatin peaks, the overrepresented motifs include the architectural protein CTCF at all stages, while motifs for the FOXA pioneer and GATA factor families are seen more often at early stages, and those regulating key airway epithelial functions, such as EHF, are limited to later stages. The RNA-seq data illustrate dynamic pathways during the iPSC to HBE-ALI differentiation, and also the marked functional divergence of different iPSC lines at the ALI stages of differentiation. Moreover, a comparison of iPSC-derived and lung donor-derived HBE-ALI cultures reveals substantial differences between these models.
Assuntos
Fator de Ligação a CCCTC/genética , Diferenciação Celular/genética , Fator 3-alfa Nuclear de Hepatócito/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Pulmão/metabolismo , Linhagem Celular , Células Cultivadas , Cromatina/genética , Fibrose Cística/genética , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Epitélio/metabolismo , Fatores de Transcrição GATA/genética , Genômica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Pulmão/citologia , Pulmão/patologia , RNA-Seq , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologiaRESUMO
There is a strong rationale to consider future cell therapeutic approaches for cystic fibrosis (CF) in which autologous proximal airway basal stem cells, corrected for CFTR mutations, are transplanted into the patient's lungs. We assessed the possibility of editing the CFTR locus in these cells using zinc-finger nucleases and have pursued two approaches. The first, mutation-specific correction, is a footprint-free method replacing the CFTR mutation with corrected sequences. We have applied this approach for correction of ΔF508, demonstrating restoration of mature CFTR protein and function in air-liquid interface cultures established from bulk edited basal cells. The second is targeting integration of a partial CFTR cDNA within an intron of the endogenous CFTR gene, providing correction for all CFTR mutations downstream of the integration and exploiting the native CFTR promoter and chromatin architecture for physiologically relevant expression. Without selection, we observed highly efficient, site-specific targeted integration in basal cells carrying various CFTR mutations and demonstrated restored CFTR function at therapeutically relevant levels. Significantly, Omni-ATAC-seq analysis revealed minimal impact on the positions of open chromatin within the native CFTR locus. These results demonstrate efficient functional correction of CFTR and provide a platform for further ex vivo and in vivo editing.
Assuntos
Brônquios/citologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/terapia , Células Epiteliais/transplante , Edição de Genes/métodos , Brônquios/metabolismo , Brônquios/transplante , Diferenciação Celular , Células Cultivadas , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , DNA Complementar/genética , DNA Complementar/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Mutação , Regiões Promotoras Genéticas , Análise de Sequência de DNARESUMO
Organoids are a valuable three-dimensional (3D) model to study the differentiated functions of the human intestinal epithelium. They are a particularly powerful tool to measure epithelial transport processes in health and disease. Though biological assays such as organoid swelling and intraluminal pH measurements are well established, their underlying functional genomics are not well characterized. Here we combine genome-wide analysis of open chromatin by ATAC-Seq with transcriptome mapping by RNA-Seq to define the genomic signature of human intestinal organoids (HIOs). These data provide an important tool for investigating key physiological and biochemical processes in the intestinal epithelium. We next compared the transcriptome and open chromatin profiles of HIOs with equivalent data sets from the Caco2 colorectal carcinoma line, which is an important two-dimensional (2D) model of the intestinal epithelium. Our results define common features of the intestinal epithelium in HIO and Caco2 and further illustrate the cancer-associated program of the cell line. Generation of Caco2 cysts enabled interrogation of the molecular divergence of the 2D and 3D cultures. Overrepresented motif analysis of open chromatin peaks identified caudal type homeobox 2 (CDX2) as a key activating transcription factor in HIO, but not in monolayer cultures of Caco2. However, the CDX2 motif becomes overrepresented in open chromatin from Caco2 cysts, reinforcing the importance of this factor in intestinal epithelial differentiation and function. Intersection of the HIO and Caco2 transcriptomes further showed functional overlap in pathways of ion transport and tight junction integrity, among others. These data contribute to understanding human intestinal organoid biology.
Assuntos
Cromatina/genética , Colo/fisiologia , Mucosa Intestinal/fisiologia , Organoides/metabolismo , Fatores de Transcrição/genética , Sequência de Bases , Fator de Transcrição CDX2/genética , Fator de Transcrição CDX2/metabolismo , Células CACO-2 , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Cromatina/metabolismo , Colo/anatomia & histologia , Colo/metabolismo , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Humanos , Mucosa Intestinal/metabolismo , Organoides/citologia , Fatores de Transcrição/metabolismo , TranscriptomaRESUMO
Organoid cultures derived from primary human tissues facilitate the study of disease processes and the development of new therapeutics. Most men with cystic fibrosis (CF) are infertile due to defects in the epididymis and vas deferens; however, the causative mechanisms are still unclear. We used human epididymis epithelial cell (HEE) organoids and polarized HEE cell cultures to assay the CF transmembrane conductance regulator (CFTR) in the human epididymis. 3D HEE organoids and polarized 2D HEE cell cultures on membrane inserts were established from human caput epididymis. Single-cell RNA sequencing (scRNA-seq) was performed to map cell type-specific gene expression in the organoids. Using forskolin (FSK) to activate CFTR and inhibitor CFTRinh172 to block its activity, we assessed how CFTR contributes to organoid swelling and epithelial barrier function. The scRNA-seq data showed key caput epididymis cell types present in HEE organoid cultures. FSK at 10 µM induced HEE organoid swelling by 20% at 16 h, while 5 and 10 µM CFTRinh172 treatment significantly reduced HEE organoid size. In transepithelial resistance (TER) measurements, FSK reduced TER, while inhibition of CFTR increased TER; also, depletion of CFTR with specific siRNAs significantly increased TER. FSK treatment significantly increased the flux of 4-kDa but not 70-kDa dextran, suggesting activation of CFTR mainly enhances transcellular diffusion. We have demonstrated that CFTR contributes to the maintenance of HEE cell TER and that cultured HEE organoids are a useful model to investigate human epididymis function. These results facilitate progress in elucidating how CFTR-dependent cellular processes impair fertility in CF.