Screening and evaluation of antioxidants for retinal pigment epithelial cell protection: L-ergothioneine as a novel therapeutic candidate through NRF2 activation.

The continual exposure of retinal tissues to oxidative stress leads to discernible anatomical and physiological alterations. Specifically, the onslaught of oxidative damage escalates the irreversible death of retinal pigmented epithelium (RPE) cells, pinpointed as the fundamental pathological event in dry age-related macular degeneration (AMD). There is a conspicuous lack of effective therapeutic strategies to counteract this degenerative process. This study screened a library of antioxidants for their ability to protect RPE cells against oxidative stress and identified L-ergothioneine (EGT) as a potent cytoprotective agent. L-ergothioneine provided efficient protection against oxidative stress-damaged RPE and maintained cell redox homeostasis and normal physiological functions. It maintained the normal structure of the retina in mice under oxidative stress conditions. Transcriptomic analysis revealed that EGT counteracted major gene expression changes induced by oxidative stress. It upregulated antioxidant gene expression and inhibited NRF2 translocation. The inhibition of NRF2 abolished EGT's protective effects, suggesting that NRF2 activation contributes to its mechanism of action. In conclusion, we identified EGT as a safe and effective small-molecule compound that is expected to be a novel antioxidative agent for treating AMD.

The single-cell transcriptomic atlas and RORA-mediated 3D epigenomic remodeling in driving corneal epithelial differentiation.

Proper differentiation of corneal epithelial cells (CECs) from limbal stem/progenitor cells (LSCs) is required for maintenance of ocular homeostasis and clear vision. Here, using a single-cell transcriptomic atlas, we delineate the comprehensive and refined molecular regulatory dynamics during human CEC development and differentiation. We find that RORA is a CEC-specific molecular switch that initiates and drives LSCs to differentiate into mature CECs by activating PITX1. RORA dictates CEC differentiation by establishing CEC-specific enhancers and chromatin interactions between CEC gene promoters and distal regulatory elements. Conversely, RORA silences LSC-specific promoters and disrupts promoter-anchored chromatin loops to turn off LSC genes. Collectively, our work provides detailed and comprehensive insights into the transcriptional dynamics and RORA-mediated epigenetic remodeling underlying human corneal epithelial differentiation.

Sex-Related Gene Network Revealed by Transcriptome Differentiation of Bisexual and Unisexual Flowers of Orchid Cymbidium tortisepalum.

Despite extensive research on orchid reproductive strategies, the genetic studies of sex differentiation in the orchid family are still lacking. In this study, we compared three sexual phenotypes of Cymbidium tortisepalum bisexual flowers as well as female and male unisexual mutants. Through comparative transcriptomes, we analyzed the sex-biased differentially expressed genes (DEGs) and gene co-expression networks of sex organs (gynostemium and ovary) among them, identified the candidate genes of sex differentiation, and validated their expression by qRT-PCR. The C. tortisepalum unisexual mutants with degenerated phenotypes were compared to the bisexual plants with respect to both the flower organs and plant morphologies. Totally, 12,145, 10,789, and 14,447 genes were uniquely expressed in the female, male, and hermaphrodite sex organs, respectively. A total of 4291 sex-biased DEGs were detected among them, with 871, 2867, and 1937 DEGs in the comparisons of bisexual vs. female, bisexual vs. male, and male vs. female flowers, respectively. Two co-expressed network modules, with 81 and 419 genes were tightly correlated with female sexual traits, while two others with 265 and 135 genes were highly correlated with male sexual traits. Two female-biased hub genes (CtSDR3b and CtSDR3b-like) nested in the female modules, the homologs of maize sex determinant tasselseed2, may control the feminization of C. tortisepalum. At the same time, two male-biased hub genes (CtYAB2 and CtYAB5) nested in the male modules, the homologs of grape sex determinant VviYABBY3, may control the androphany of C. tortisepalum. This study discovered the molecular regulation networks and proposed a model for orchid sex differentiation, therefore providing for the first time the genetic basis for the sex separation in the orchid family.

Predictive value of serum ß2-microglobulin in cardiac valve calcification in maintenance hemodialysis patients.

Background: Cardiac valve calcification (CVC) is associated with adverse cardiovascular events. We studied the risk factors of CVC in maintenance hemodialysis (MHD) patients and the value of serum ß2-microglobulin (ß2-MG) levels in predicting the incidence of CVC. ß2-MG is a middle molecular weight toxin. In recent years, researchers found that elevated blood ß2-MG was associated with coronary, thoracic, and abdominal aortic calcifications with significant correlations. ß2-MG has been emerging as a strong biomarker for cardiovascular mortality in uremic patients but its role in CVC is not well studied. This study looked specifically at CVC occurrence in relation to ß2-MG for MHD patients. Methods: Patients who underwent MHD for more than 3 months in the First People's Hospital of Nantong City from November 2012 to November 2019 with complete available data were included in the study. The patients were divided into the CVC group and the non-CVC group. The general information and clinical laboratory indicators of the patients were collected in a retrospective manner. We analyzed the risk factors for developing CVC in MHD patients using binary logistic regression method. Receiver operating characteristic (ROC) curves were used to calculate the cut-off value of ß2-MG for predicting CVC. The decision tree (DT) method was used to classify and explore the probability of CVC in patients with MHD. Results: The ß2-MG in the CVC group was significantly higher than that in the non-CVC group (t=6.750, P<0.001). Multivariate binary logistic regression analysis showed that gender, age, serum ß2-MG, and hemodialysis (HD) adequacy (Kt/V urea) were independent risk factors for CVC in MHD patients. ROC analysis showed that a ß2-MG value of 25 µg/L was the best cut-off point for predicting CVC in MHD patients. According to binary logistic regression analysis, the ß2-MG ≥25 µg/L group was 3.39 times more likely to develop CVC than the ß2-MG <25 µg/L group [odds ratio (OR), 3.39; 95% confidence interval (CI), 1.63-7.06; P=0.001]. The DT model determined that serum ß2-MG ≥25 µg/L and age >69 years were important determinants for predicting CVC in MHD patients. Conclusions: Serum ß2-MG in MHD patients has a positive correlation with the severity and occurrence of CVC.

Integrated metabolome and transcriptome analyses provide insight into the effect of red and blue LEDs on the quality of sweet potato leaves.

Red and blue light-emitting diodes (LEDs) affect the quality of sweet potato leaves and their nutritional profile. Vines cultivated under blue LEDs had higher soluble protein contents, total phenolic compounds, flavonoids, and total antioxidant activity. Conversely, chlorophyll, soluble sugar, protein, and vitamin C contents were higher in leaves grown under red LEDs. Red and blue light increased the accumulation of 77 and 18 metabolites, respectively. Alpha-linoleic and linolenic acid metabolism were the most significantly enriched pathways based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. A total of 615 genes were differentially expressed between sweet potato leaves exposed to red and blue LEDs. Among these, 510 differentially expressed genes were upregulated in leaves grown under blue light compared with those grown under red light, while the remaining 105 genes were expressed at higher levels in the latter than in the former. Among the KEGG enrichment pathways, blue light significantly induced anthocyanin and carotenoid biosynthesis structural genes. This study provides a scientific reference basis for using light to alter metabolites to improve the quality of edible sweet potato leaves.

Oxidative Stress Initiates Receptor-Interacting Protein Kinase-3/Mixed Lineage Kinase Domain-Like-Mediated Corneal Epithelial Necroptosis and Nucleotide-Binding Oligomerization Domain-Like Receptor Protein 3 Inflammasome Signaling during Fungal Keratitis.

Fungal keratitis remains a major cause of severe visual loss in developing countries because of limited choices of therapy. The progression of fungal keratitis is a race between the innate immune system and the outgrowth of fungal conidia. Programmed necrosis (necroptosis), a type of proinflammatory cell death, has been recognized as a critical pathologic change in several diseases. However, the role and potential regulatory mechanisms of necroptosis have not been investigated in corneal diseases. The current study showed, for the first time, that fungal infection triggered significant corneal epithelial necroptosis in human/mouse/in vitro models. Moreover, a reduction in excessive reactive oxygen species release effectively prevented necroptosis. NLRP3 knockout did not affect necroptosis in vivo. In contrast, ablation of necroptosis via RIPK3 knockout significantly delayed migration and inhibited the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome in macrophages, which enhanced the progression of fungal keratitis. Taking these findings together, the study indicated that overproduction of reactive oxygen species in fungal keratitis leads to significant necroptosis in the corneal epithelium. Furthermore, the necroptotic stimuli-mediated NLRP3 inflammasome serves as a driving force in host defense against fungal infection.

Weighted Gene Co-Expression Network Analysis to Explore Hub Genes of Resveratrol Biosynthesis in Exocarp and Mesocarp of 'Summer Black' Grape.

Resveratrol is a polyphenol compound beneficial to human health, and its main source is grapes. In the present study, the molecular regulation of resveratrol biosynthesis in developing grape berries was investigated using weighted gene co-expression network analysis (WGCNA). At the same time, the reason for the resveratrol content difference between grape exocarp (skin) and mesocarp (flesh) was explored. Hub genes (CHS, STS, F3'5'H, PAL, HCT) related to resveratrol biosynthesis were screened with Cytoscape software. The expression level of hub genes in the exocarp was significantly higher than that in the mesocarp, and the expressions of the hub genes and the content of resveratrol in exocarp peaked at the maturity stage. While the expression levels of PAL, CHS and STS in the mesocarp, reached the maximum at the maturity stage, and F3'5'H and HCT decreased. These hub genes likely play a key role in resveratrol biosynthesis. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis further indicated that resveratrol biosynthesis was related to flavonoid biosynthesis, phenylalanine metabolism, phenylpropanoid biosynthesis, and stilbene biosynthesis pathways. This study has theoretical significance for exploring genes related to resveratrol biosynthesis in the exocarp and mesocarp of grapes, and provides a theoretical basis for the subsequent function and regulatory mechanism of hub genes.

Single-cell transcriptomics implicates the FEZ1-DKK1 axis in the regulation of corneal epithelial cell proliferation and senescence.

Limbal stem/progenitor cells (LSC) represent the source of corneal epithelium renewal. LSC proliferation and differentiation are essential for corneal homeostasis, however, the regulatory mechanism remains largely unexplored. Here, we performed single-cell RNA sequencing and discovered proliferation heterogeneity as well as spontaneously differentiated and senescent cell subgroups in multiply passaged primary LSC. Fasciculation and elongation protein zeta 1 (FEZ1) and Dickkopf-1 (DKK1) were identified as two significant regulators of LSC proliferation and senescence. These two factors were mainly expressed in undifferentiated corneal epithelial cells (CECs). Knocking down the expression of either FEZ1 or DKK1 reduced cell division and caused cell cycle arrest. We observed that DKK1 acted as a downstream target of FEZ1 in LSC and that exogenous DKK1 protein partially prevented growth arrest and senescence upon FEZ1 suppression in vitro. In a mouse model of corneal injury, DKK1 also rescued the corneal epithelium after recovery was inhibited by FEZ1 suppression. Hence, the FEZ1-DKK1 axis was required for CEC proliferation and the juvenile state and can potentially be targeted as a therapeutic strategy for promoting recovery after corneal injury.

ETS1-HMGA2 Axis Promotes Human Limbal Epithelial Stem Cell Proliferation.

Purpose: This study aimed to investigate the role and molecular mechanism of ETS1 in the proliferation and differentiation of human limbal epithelial stem cells (LESCs). Methods: RNA-seq and quantitative real-time PCR were used to determine gene expression changes when ETS1 and HMGA2 was knocked down using short-hairpin RNAs or overexpressed by lentivirus. Immunofluorescence and flow cytometry experiments were performed to assess the roles of ETS1 and HMGA2 in LESC proliferation. ETS1-bound cis-regulatory elements and target genes in LESCs were identified using chromatin immunoprecipitation sequencing. The epigenetic features of ETS1-binding sites were assessed by the published histone modification and chromatin accessibility profiles. Results: ETS1 was robustly expressed in LESCs but dramatically reduced on differentiation into corneal epithelial cells (CECs). ETS1 knockdown in LESCs inhibited cellular proliferation and activated CEC markers (KRT3, KRT12, CLU, and ALDH3A1). When ETS1 was overexpressed during CEC differentiation, LESC-associated genes were upregulated while CEC-associated genes were downregulated. The genome-wide binding profile of ETS1 was identified in LESCs. ETS1 occupied H3K4me3-marked promoters and H3K27ac/H3K4me1-marked enhancers. ETS1-binding sites were also enriched for chromatin accessibility signal. HMGA2 showed a consistent expression pattern with ETS1. ETS1 activates HMAG2 by binding to its promoter. Knockdown and overexpression experiments suggested that HMGA2 can promote LESC proliferation and inhibits its differentiation. Conclusions: ETS1 promotes LESC proliferation and inhibits its differentiation via activating HMGA2.

Pistachio genomes provide insights into nut tree domestication and ZW sex chromosome evolution.

Pistachio is a nut crop domesticated in the Fertile Crescent and a dioecious species with ZW sex chromosomes. We sequenced the genomes of Pistacia vera cultivar (cv.) Siirt, the female parent, and P. vera cv. Bagyolu, the male parent. Two chromosome-level reference genomes of pistachio were generated, and Z and W chromosomes were assembled. The ZW chromosomes originated from an autosome following the first inversion, which occurred approximately 8.18 Mya. Three inversion events in the W chromosome led to the formation of a 12.7-Mb (22.8% of the W chromosome) non-recombining region. These W-specific sequences contain several genes of interest that may have played a pivotal role in sex determination and contributed to the initiation and evolution of a ZW sex chromosome system in pistachio. The W-specific genes, including defA, defA-like, DYT1, two PTEN1, and two tandem duplications of six VPS13A paralogs, are strong candidates for sex determination or differentiation. Demographic history analysis of resequenced genomes suggest that cultivated pistachio underwent severe domestication bottlenecks approximately 7640 years ago, dating the domestication event close to the archeological record of pistachio domestication in Iran. We identified 390, 211, and 290 potential selective sweeps in 3 cultivar subgroups that underlie agronomic traits such as nut development and quality, grafting success, flowering time shift, and drought tolerance. These findings have improved our understanding of the genomic basis of sex determination/differentiation and horticulturally important traits and will accelerate the improvement of pistachio cultivars and rootstocks.

Correlation between lactate dehydrogenase and QTc interval prolongation in maintenance hemodialysis patients: a cross-sectional study.

BACKGROUND: Corrected QT (QTc) interval prolongation is one of the common causes of sudden cardiac death in patients with maintenance hemodialysis (MHD) patients. However, there are few studies on QTc prolongation in MHD patients. The concentration of lactate dehydrogenase (LDH) in hemodialysis population increased, and LDH was associated with the mortality of MHD patients. This study aimed to investigate the relationship between QTc interval prolongation and LDH in MHD patients. METHODS: This is a cross-sectional observational study. Patients who underwent MHD for more than 3 months in the Second Affiliated Hospital of Nantong University from November 2012 to November 2019 with complete data were selected as the research subjects. The patients were divided into the normal QTc interval group and the QTc interval prolongation group. The general data of patients and clinical laboratory indicators were collected retrospectively from the electronic medical record system. Pearson correlation analysis and binary logistic regression were used to analyze the correlation between LDH and QTc interval prolongation; the cut-off value of LDH predicting QTc interval prolongation was calculated by receiver operating characteristic (ROC) curve. RESULTS: The LDH level in the prolonged QTc interval group was significantly higher than that in the normal group (301.96±110.91 vs. 215.39±67.65, t=-8.03, P<0.001). QTc interval and LDH (r=0.386) were positively correlated. Binary logistic regression analysis showed that LDH, serum potassium <4 mmol/L, serum phosphorus, and left ventricular end-diastolic diameter (LVDd) were independent related factors for QTc interval prolongation. The ROC curve results showed that LDH =220 U/L was the best cutoff point for predicting QTc interval prolongation in MHD patients, with a sensitivity of 81.45% and a specificity of 59.35%. Binary logistic regression analysis showed that the LDH >220 U/L group was 6.34 times more likely to have QTc interval prolongation than the LDH ≤220 U/L group (OR 6.34, 95% CI: 3.47-11.58, P<0.001). CONCLUSIONS: LDH in MHD patients is closely related to QTc interval prolongation. Serum LDH, ionic calcium, serum phosphorus and potassium may predict QTc interval prolongation. Monitoring related indicators can remind clinicians to intervene as soon as possible to reduce the potential risk of arrhythmia and sudden cardiac death (SCD).

Pantranscriptome combined with phenotypic quantification reveals germplasm kinship and regulation network of bract color variation in Bougainvillea.

Bracts are the metamorphic non-flower organ in angiosperm plants. The variation of the color and shape of bracts was found to be neo-functionalized (i.e., similar to petals), garnering research interest as a pollinator attractor. Bougainvillea is known for its specialized, large, and colorful bracts, which contrast with its tiny colorless flowers. As a plant whose bracts vary greatly in terms of coloration, the molecular mechanisms for Bougainvillea bract coloration and polychroism are largely unknown. The lack of genomic information for Bougainvillea largely hinders studies into the evolution and genetic basis of bract color variation. In this study, a pan-transcriptome of bracts obtained from 18 Bougainvillea glabra accessions was employed to investigate the global population-level germplasm kinship and the gene regulation network for bract color variation. Our results showed that the bracts of B. glabra accessions have largely differentiated International Commission on Illumination (CIE) L-a-b values. Moreover, germplasm kinship detected using principal component analysis, phylogeny, and admixture analysis showed three optimal subgroups, two of them distinctly clustered, which were not directly correlated with bract color variation at the population level. Differentially expressed genes (DEGs) between accessions of high vs. low L-a-b values revealed several considerable upregulated genes related to bract color L-a-b variation. A weighted gene co-expression network was constructed, and eight co-expressed regulation modules were identified that were highly correlated with variation in bract CIE L-a-b color values. Several candidate DEGs and co-expressed hub genes (e.g., GERD, SGR, ABCA3, GST, CYP76AD1, CYP76C, and JAZ) that were tightly associated with bract color variation were eventually determined responsible for L-a-b colorations, which might be the core regulation factors contributing to the B. glabra bract color variation. This study provides valuable insights into the research on germplasm kinship, population-level pan-transcriptome expression profiles, and the molecular basis of color variation of key innovative bracts in horticultural Bougainvillea.

Cotransplantation of Limbal Epithelial and Stromal Cells for Ocular Surface Reconstruction.

Purpose: To propose an improved stem cell-based strategy for limbal stem cell deficiency (LSCD) treatment. Design: Experimental randomized or parallel-group animal study. Subjects: Fifty adult male New Zealand white rabbits. Methods: Human limbal stem/progenitor cells (LSCs) and limbal stromal stem/progenitor cells (LSSCs) were cultured in serum-free conditions and further differentiated into corneal epithelial cells and keratocytes, respectively. All cell types were characterized with lineage-specific markers. Gene expression analysis was performed to identify the potential function of LSSCs in corneal regeneration. Two LSCD models of rabbits for transplantations were used: transplantation performed at the time of limbal and corneal epithelial excision (LSCD model) and transplantation performed after clinical signs were induced in an LSCD model (pLSCD model). The pLSCD model better mimics the pathologic changes and symptoms of human LSCD. Rabbit models received LSC or LSC plus LSSC treatment. Corneal epithelial defects, neovascularization, and opacity were assessed every 3 weeks for 24 weeks. ZsGreen-labeled LSSCs were used for short-term tracking in vivo. Main Outcome Measures: Rates of corneal epithelial defect area, corneal neovascularization and opacity scores, graft survival rate, and immunofluorescence staining of specific markers. Results: Both LSC transplantation and LSC plus LSSC cotransplantation effectively repaired the corneal surface in the LSCD model. These 2 strategies showed no significant differences in terms of graft survival rate or epithelial repair. However, corneal opacity was observed in the LSC group (in 3 of 8 rabbits), but not in the LSC plus LSSC group. Notably, when treating LSCD rabbits with distinguishable stromal opacification and neovascularization, cotransplantation of LSCs and LSSCs exhibited significantly better therapeutic effects than transplantation of LSCs alone, with graft survival rates of 87.5% and 37.5%, respectively. The implanted LSSCs could differentiate into keratocytes during the wound-healing process. RNA sequencing analysis showed that the stromal cells produced not only a collagen-rich extracellular matrix to facilitate reconstruction of the lamellar structure, but also niche factors that accelerated epithelial cell growth and inhibited angiogenesis and inflammation. Conclusions: These findings highlight the support of stromal cells in niche homeostasis and tissue regeneration, providing LSC plus LSSC cotransplantation as a new treatment strategy for corneal blindness.

Prognostic value and potential biological functions of ferroptosis-related gene signature in bladder cancer.

Bladder cancer (BC), as a genitourinary system tumor, is a highly prevalent tumor type. Ferroptosis is an iron-dependent oxidative cell death mechanism that is becoming increasingly recognized as a promising avenue for cancer therapy. However, further determination of the prospective prognostic value of ferroptosis for BC and investigation of the underlying mechanisms is required. The mRNA expression profiles and associated clinical data were downloaded from public databases such as The Cancer Genome Atlas, Gene Expression Omnibus and the IMvigor210 database. To construct a predictive formula, the least absolute shrinkage and selection operator Cox regression algorithm was used. In addition, a prognostic multigene signature was constructed using previously selected ferroptosis-related genes (FRGs). A total of 28 FRGs were differentially expressed between tumor and normal samples with |log2 fold change| >1 and adjusted P<0.05. A prognostic model was then established and it was validated in the GEO cohort using six genes: Glutamate-cysteine ligase modifier subunit, crystallin α-B, transferrin receptor, zinc finger E-box binding homeobox 1, squalene epoxidase and glucose-6-phosphate dehydrogenase (G6PD). Numerous important pathways involved in the development of the immune system and cancer were indicated to be significantly different between the two risk groups. In addition, it was discovered that G6PD expression subgroups that were associated with immunotherapy response in patients with BC had similar prognostic features to risk score subgroups. In the present study, a gene signature with a prognostic value for ferroptosis in BC was successfully developed and the potential value of G6PD was identified for future research.

A Retrospective Study from a Single Center in China to Develop a Nomogram to Predict One-Year Mortality in Patients with End-Stage Renal Disease Who Are Receiving Hemodialysis.

BACKGROUND The prognosis of end-stage renal disease (ESRD) patients receiving hemodialysis (HD) remains Poor. This retrospective study from a single center in China aimed to develop a nomogram to predict one-year mortality in patients with ESRD on HD. MATERIAL AND METHODS We enrolled 299 ethnic Han Chinese ESRD patients undergoing HD at the Second Affiliated Hospital of Nantong University from April 29, 2011 to January 30, 2021. Univariate and multivariate Cox regression analyses were used to select the predictors incorporated in the prediction model to assess the one-year mortality for ESRD patients receiving HD. We used receiver operating characteristic curves, C-index, and calibration curves to evaluate the performance of the nomogram. The predictive performance of the nomogram was also verified in different subgroup populations. RESULTS The median follow-up time was 23.30 months. The 299 ESRD patients receiving HD were divided into a death group (n=96) and a survival group (n=203), and the incidence of death was 32.11%. The main causes of death were cardiovascular disease, inflammation and cancer. A nomogram containing age, alkaline phosphatase, albumin, cystatin C, total bilirubin, and hypersensitive c-reactive protein was established. The performance of this nomogram was reflected by its moderate predictive ability, especially for patients who were male, had a primary disease of chronic glomerulonephritis, and had no history of comorbidities. CONCLUSIONS We developed and validated an easy-to-use nomogram for predicting the one-year mortality of ESRD patients undergoing HD.

Cis-regulatory chromatin loops analysis identifies GRHL3 as a master regulator of surface epithelium commitment.

Understanding the regulatory network of cell fate acquisition remains a major challenge. Using the induction of surface epithelium (SE) from human embryonic stem cells as a paradigm, we show that the dynamic changes in morphology-related genes (MRGs) closely correspond to SE fate transitions. The marked remodeling of cytoskeleton indicates the initiation of SE differentiation. By integrating promoter interactions, epigenomic features, and transcriptome, we delineate an SE-specific cis-regulatory network and identify grainyhead-like 3 (GRHL3) as an initiation factor sufficient to drive SE commitment. Mechanically, GRHL3 primes the SE chromatin accessibility landscape and activates SE-initiating gene expression. In addition, the evaluation of GRHL3-mediated promoter interactions unveils a positive feedback loop of GRHL3 and bone morphogenetic protein 4 on SE fate decisions. Our work proposes a concept that MRGs could be used to identify cell fate transitions and provides insights into regulatory principles of SE lineage development and stem cell-based regenerative medicine.

Healing Ability of Central Corneal Epithelium in Rabbit Ocular Surface Injury Models.

Purpose: Wound healing of the corneal epithelium mainly involves two types of cells: limbal stem/progenitor cells (LSCs) and differentiated central corneal epithelial cells (CECs). The healing ability of CECs is still debatable, and its correlated transcriptomic alterations during wound healing are yet to be elucidated. This study aimed to determine the healing ability and mechanisms underlying the actions of CECs using rabbit ocular surface injury models. Methods: A central corneal ring-like residual epithelium model was used to investigate the healing ability of CECs. Uninjured and injury-stimulated LSCs and CECs were collected for transcriptomic analysis. The analysis results were verified by quantitative reverse transcriptase polymerase chain reaction, immunofluorescence staining, and two types of rabbit corneal injury models. Results: During wound healing, the upregulated genes in LSCs were mostly enriched in the mitotic cell cycle-related processes, but those in CECs were mostly enriched in cell adhesion and migration. CECs could repair the epithelial defects successfully at one-time injuries. However, after repetitive injuries, the CECs repaired notably slower and failed to completely heal the defect, but the LSCs repaired even faster than the one-time injury. Conclusions: Our results indicated rabbit CECs repair the epithelial defect mainly depending on migration and its proliferative ability is limited, and LSCs are the main source of regenerative epithelial cells. Translational Relevance: This study provides information on gene expression in the corneal epithelium during wound healing, indicating that regulation of the cell cycle, cell adhesion, and migration may be the basis for future treatment strategies for corneal wound healing.

Comprehensive 3D epigenomic maps define limbal stem/progenitor cell function and identity.

The insights into how genome topology couples with epigenetic states to govern the function and identity of the corneal epithelium are poorly understood. Here, we generate a high-resolution Hi-C interaction map of human limbal stem/progenitor cells (LSCs) and show that chromatin multi-hierarchical organisation is coupled to gene expression. By integrating Hi-C, epigenome and transcriptome data, we characterize the comprehensive 3D epigenomic landscapes of LSCs. We find that super-silencers mediate gene repression associated with corneal development, differentiation and disease via chromatin looping and/or proximity. Super-enhancer (SE) interaction analysis identified a set of SE interactive hubs that contribute to LSC-specific gene activation. These active and inactive element-anchored loop networks occur within the cohesin-occupied CTCF-CTCF loops. We further reveal a coordinated regulatory network of core transcription factors based on SE-promoter interactions. Our results provide detailed insights into the genome organization principle for epigenetic regulation of gene expression in stratified epithelia.

The spinach YY genome reveals sex chromosome evolution, domestication, and introgression history of the species.

BACKGROUND: Spinach (Spinacia oleracea L.) is a dioecious species with an XY sex chromosome system, but its Y chromosome has not been fully characterized. Our knowledge about the history of its domestication and improvement remains limited. RESULTS: A high-quality YY genome of spinach is assembled into 952 Mb in six pseudo-chromosomes. By a combination of genetic mapping, Genome-Wide Association Studies, and genomic analysis, we characterize a 17.42-Mb sex determination region (SDR) on chromosome 1. The sex chromosomes of spinach evolved when an insertion containing sex determination genes occurred, followed by a large genomic inversion about 1.98 Mya. A subsequent burst of SDR-specific repeats (0.1-0.15 Mya) explains the large size of this SDR. We identify a Y-specific gene, NRT1/PTR 6.4 which resides in this insertion, as a strong candidate for the sex determination or differentiation factor. Resequencing of 112 spinach genomes reveals a severe domestication bottleneck approximately 10.87 Kya, which dates the domestication of spinach 7000 years earlier than the archeological record. We demonstrate that a strong selection signal associated with internode elongation and leaf area expansion is associated with domestication of edibility traits in spinach. We find that several strong genomic introgressions from the wild species Spinacia turkestanica and Spinacia tetrandra harbor desirable alleles of genes related to downy mildew resistance, frost resistance, leaf morphology, and flowering-time shift, which likely contribute to spinach improvement. CONCLUSIONS: Analysis of the YY genome uncovers evolutionary forces shaping nascent sex chromosome evolution in spinach. Our findings provide novel insights about the domestication and improvement of spinach.

dsRNA Induced IFNß-MMP13 Axis Drives Corneal Wound Healing.

Purpose: Cornea, the outermost transparent layer of the eye, is the first line of defense against external threats. Following injury, the wound healing response is crucial to corneal repair and regeneration, yet its underlying mechanism is poorly understood. Our study was designed to investigate the role of dsRNA and its regulatory network in corneal wound healing. Methods: A corneal wound healing model was established via the surgical removal of half of the corneal surface and adjoining limbus. RNase III was then used to clarify the role of dsRNA in corneal wound closure and RNA-seq was performed to investigate the mechanism of dsRNA in the healing process. Related gene expression was assessed using immunofluorescence staining, qPCR, and Western blot. Flow cytometry and scratch assay were used to analyze the proliferation and migration of limbal stem/progenitor cells (LSCs) in vitro and functional analysis of the target genes was completed using the corneal wound healing model. Results: Corneal wound healing was delayed and impaired when the dsRNAs were removed or damaged following RNase III digestion. The dsRNAs released following corneal damage activate type I interferon (IFN-I) signaling, primarily IFNß, via the corneal epithelium and neutralizing IFNß or blocking IFN-I signaling delays corneal wound closure. Moreover, our data identified MMP13 as a downstream effector of IFNß where its expression promotes LSC proliferation and enhances corneal epithelial reconstruction in vivo. Conclusions: The dsRNA induced IFNß-MMP13 axis plays a key role in corneal wound healing.