ABSTRACT
Benthic eukaryotic microalgae were analyzed by metabarcoding the partial 18S rRNA gene in Daya Bay bi-monthly in 2021. Altogether 941 eukaryotic microalgal OTUs were detected, belonging to 27 classes of 8 phyla. Dinophyta and Chlorophyta were the dominant phyla. Microalgal community in the mariculture zone differed significantly from those in non-mariculture zone, reflected by low alpha diversity indexes and increasing abundance and richness of chlorophytes and correspondingly decreasing of dinoflagellates. The abundant occurrences of the pico- and nano-sized taxa such as the chlorophyte Picochlorum in the mariculture zone suggested that nutrient enrichment might result in the miniaturization of the benthic eukaryotic microalgae. The co-occurrence network suggested more negative interactions between taxa in the mariculture zone. A total of 41 algal bloom and/or harmful algal bloom (HAB) species were detected in this study, suggesting a high potential risk of HABs in Daya Bay, especially for the recurrent bloom species Scrippsiella acuminata.
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The corneal epithelium is the outermost transparent barrier of the eyeball and undergoes continuous self-renewal by limbal stem cells (LSCs) during its lifetime; however, the impact of aging on LSCs remains largely unknown. Here, we showed that the healing ability of the cornea in elderly macaques (Macaca fascicularis) was significantly decreased compared to that of younger macaques. This delayed wound closure accompanied a disordered cell arrangement and corneal opacity. A novel cytokine, Secreted and Transmembrane 1 (SECTM1), was found to facilitate corneal healing and was upregulated in young macaques upon wounding. Mechanistically, SECTM1 is essential for LSC migration and proliferation, and may partially function through Cell Division Cycle Associated 7 (CDCA7). Notably, the topical application of SECTM1 to aged wounded corneas dramatically promoted re-epithelialization and improved corneal transparency in both mice and macaques. Our work suggests that aging may impair the expression of healing response factors and injury repair in non-human primate corneas, and that SECTM1 application could potentially benefit corneal wound healing in clinical treatment.
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Purpose: N6-methyladenosine (m6A) methylation is a chemical modification that occurs on RNA molecules, where the hydrogen atom of adenine (A) nucleotides is replaced by a methyl group, forming N6-methyladenosine. This modification is a dynamic and reversible process that plays a crucial role in regulating various biological processes, including RNA stability, transport, translation, and degradation. Currently, there is a lack of research on the role of m6A modifications in maintaining the characteristics of RPE cells. m6A readers play a crucial role in executing the functions of m6A modifications, which prompted our investigation into their regulatory roles in the RPE. Methods: Phagocytosis assays, immunofluorescence staining, flow cytometry experiments, ß-galactosidase staining, and RNA sequencing (RNA-seq) were conducted to assess the functional and cellular characteristics changes in retinal pigment epithelium (RPE) cells following short-hairpin RNA-mediated knockdown of insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2). RNA-seq and ultraviolet crosslinking immunoprecipitation with high-throughput sequencing (HITS-CLIP) were employed to identify the target genes regulated by IGF2BP2. adeno-associated virus (AAV) subretinal injection was performed in 6- to 8-week-old C57 mice to reduce IGF2BP2 expression in the RPE, and the impact of IGF2BP2 knockdown on mouse visual function was assessed using immunofluorescence, quantitative real-time PCR, optical coherence tomography, and electroretinography. Results: IGF2BP2 was found to have a pronounced effect on RPE phagocytosis. Subsequent in-depth exploration revealed that IGF2BP2 modulates the mRNA stability of PAX6 and OTX2, and the loss of IGF2BP2 induces inflammatory and aging phenotypes in RPE cells. IGF2BP2 knockdown impaired RPE function, leading to retinal dysfunction in vivo. Conclusions: Our data suggest a crucial role of IGF2BP2 as an m6A reader in maintaining RPE homeostasis by regulating the stability of PAX6 and OTX2, making it a potential target for preventing the occurrence of retinal diseases related to RPE malfunction.
Subject(s)
Otx Transcription Factors , PAX6 Transcription Factor , RNA-Binding Proteins , Retinal Pigment Epithelium , Animals , Mice , Cells, Cultured , Electroretinography , Flow Cytometry , Gene Expression Regulation/physiology , Homeostasis , Mice, Inbred C57BL , Otx Transcription Factors/metabolism , Otx Transcription Factors/genetics , PAX6 Transcription Factor/genetics , PAX6 Transcription Factor/metabolism , Phagocytosis/physiology , Retinal Pigment Epithelium/metabolism , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Tomography, Optical CoherenceABSTRACT
Photoperiod is an important environmental factor that influences seasonal reproduction behavior in birds. Birds translate photoperiodic information into neuroendocrine signals through deep brain photoreceptors (DBPs). OPN5 has been considered candidate DBPs involved in regulating seasonal reproduction in birds. We found that OPN5 could mediate light to regulate the follicle development in ducks. In this study, we further verified the effect of OPN5 on follicular development in Shan Partridge ducks by immunizing against the extracellular domain (ECD) of OPN5. We investigated the specific regulatory mechanism of photoperiod mediated by OPN5 on the reproductive activity of ducks. The trial randomly divided 120 Shan Partridge ducks into 3 groups with different treatments: the immunization of OPN5 group was done at d0, d15, d30, and d40 with 1 mL of vaccine containing OPN5 protein (thus containing 1, 1, 0.5, and 0.5 mg of OPN5-KLH protein), and the control group (CS and CL groups) was injected at the same time with the same dose of OPN5-uncontained blank vaccine. The group of CS (900 lux), OPN5 (600 lux), and CL (600 lux) lasted for 40 d in 12 L:12 D photoperiods, respectively. Then, the groups of CS, OPN5, and CL subsequently received 12 L:12 D, 12 L:12 D, and 17 L:7 D light treatments for 33 d, respectively. The ducks were caged in 3 constant rooms with the same feeding conditions for each group, free water, and limited feeding (150 g per duck each day). Duck serum and tissue samples were collected at d 40, d 62, and d 73 (n = 12). It was found that before prolonged light, the group of immunization (group OPN5) and the group of strong light intensity (group CS) were higher than the group of CL in egg production. Subsequent to prolonged light, the group CL in egg production rose about the same as the group immunization, while the strong light group (group CS) was lower. Group OPN5 increased the ovarian index of ducks, and both the immunization of group OPN5 and group CL (extended light) increased the thickness of the granular layer and promoted the secretion of E2, P4, LH, and PRL hormones. Compared with group CS, group CL and OPN5 increased the mRNA level and protein expression of OPN5 in the hypothalamus on d 62 and d 73 (P < 0.05). The gene or protein expression patterns of GnRH, TRH, TSHß, DIO2, THRß, VIP, and PRL were positively correlated with OPN5, whereas the gene expression patterns of GnIH and DIO3 were negatively correlated with OPN5. The results showed that immunization against OPN5 could activate the corresponding transmembrane receptors to promote the expression of OPN5, up-regulate the expression of TSHß and DIO2, and then regulate the HPG axis-related genes to facilitate the follicular development of Shan Partridge ducks. In addition, in this experiment, prolonging the photoperiod or enhancing the light intensity could also enhance follicle development, but the effect was not as significant as immunizing against OPN5. Our results will offer beneficial data and more supportive shreds of evidence in favor of elucidating the role of OPN5 in relation to photoperiods and reproduction.
Subject(s)
Photoperiod , Vaccines , Animals , Ducks/physiology , Chickens , Reproduction , Immunization/veterinaryABSTRACT
Gastrointestinal (GI) tumors are a significant global health threat, with high rates of morbidity and mortality. Exosomes contain various biologically active molecules like nucleic acids, proteins, and lipids and can serve as messengers for intercellular communication. They play critical roles in the exchange of information between tumor cells and the tumor microenvironment (TME). The TME consists of mesenchymal cells and components of the extracellular matrix (ECM), with fibroblasts being the most abundant cell type in the tumor mesenchyme. Cancer-associated fibroblasts (CAFs) are derived from normal fibroblasts and mesenchymal stem cells that are activated in the TME. CAFs can secrete exosomes to modulate cell proliferation, invasion, migration, drug resistance, and other biological processes in tumors. Additionally, tumor cells can manipulate the function and behavior of fibroblasts through direct cell-cell interactions. This review provides a summary of the intercellular crosstalk between GI tumor cells and CAFs through exosomes, along with potential underlying mechanisms.
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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.
Subject(s)
Antioxidants , Ergothioneine , NF-E2-Related Factor 2 , Oxidative Stress , Retinal Pigment Epithelium , Retinal Pigment Epithelium/drug effects , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/pathology , NF-E2-Related Factor 2/metabolism , NF-E2-Related Factor 2/genetics , Animals , Ergothioneine/pharmacology , Antioxidants/pharmacology , Oxidative Stress/drug effects , Mice , Mice, Inbred C57BL , Macular Degeneration/drug therapy , Macular Degeneration/metabolism , Macular Degeneration/pathology , Cells, Cultured , Humans , Blotting, Western , Disease Models, Animal , Gene Expression Regulation/drug effects , Reactive Oxygen Species/metabolismABSTRACT
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.
Subject(s)
Cornea , Epigenomics , Humans , Cell Differentiation/genetics , Gene Expression Profiling , Chromatin/genetics , Nuclear Receptor Subfamily 1, Group F, Member 1ABSTRACT
Groundwater is a vital resource for local human life and production in the oasis zone of the Cherchen River Basin of Xinjiang. Understanding the controlling factors of groundwater salinization and pollution is of great significance for the sustainable utilization of groundwater resources and protection of the ecological environment in desert oasis areas. In light of this, a total of 36 single structure unconfined groundwater samples and 54 multi-layered structure unconfined and confined groundwater samples were collected from the oasis zone of the Cherchen River Basin and evaluated for the distribution characteristics and pollution status of major ions. Hydrogeochemical methods (e.g., Piper diagram, multivariate statistics, Gibbs model, and relationships among ions) were used to determine the main controlling factors of groundwater salinization and pollution. Differences in hydrogeochemical zonation were found from the single structure unconfined aquifers in sloping plains of piedmont areas to the multi-layered structure unconfined and confined aquifers in alluvial-proluvial plain areas, and Cl-Na (87.8%) was the main hydrochemical type in the groundwater of the study area. The quality of single structure unconfined groundwater was starkly better than that of the multi-layered unconfined and confined groundwater, which was mainly caused by Na+ (mean value of 9 969 mg·L-1), Cl- (13 687 mg·L-1), and SO42- (5 840 mg·L-1). Moreover, the natural hydrogeochemical process was the main reason for the deterioration of groundwater quality. The hydrochemistry was mainly controlled by the water-rock interaction and evaporation processes. The mineral dissolution of silicates and evaporites was an important source of chemical ions in the groundwater. Furthermore, the chemical weathering of evaporites combined with the processes of evaporation and cation exchange had a significant influence on the salinization of multi-layered unconfined and confined groundwater in alluvial-proluvial plain areas. In addition, synthetic fertilizers were the main pollution sources of NO3- in groundwater in the intensive agricultural zones.
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A small molecule screen identified several cardiotonic steroids (digitoxin and ouabain) and the ionophore monensin as potent inhibitors of HCoV-229E, HCoV-OC43, and SARS-CoV-2 replication with EC50s in the low nM range. Subsequent tests confirmed antiviral activity in primary cell models including human nasal epithelial cells and lung organoids. Addition of digitoxin, ouabain, or monensin strongly reduced viral gene expression as measured by both viral protein and RNA accumulation. Furthermore, the compounds acted post virus entry. While the antiviral activity of digitoxin was dependent upon activation of the MEK and JNK signaling pathways but not signaling through GPCRs, the antiviral effect of monensin was reversed upon inhibition of several signaling pathways. Together, the data demonstrates the potent anti-coronavirus properties of two classes of FDA approved drugs that function by altering the properties of the infected cell, rendering it unable to support virus replication.
Subject(s)
Cardiac Glycosides , Coronavirus 229E, Human , Humans , Cardiac Glycosides/pharmacology , Monensin/pharmacology , Ouabain/pharmacology , Digitoxin/pharmacology , Antiviral Agents/pharmacologyABSTRACT
Acute necrotizing encephalopathy 1 (ANE1) is a very rare disorder associated with a dominant heterozygous mutation in the RANBP2 (RAN binding protein 2) gene. ANE1 is frequently triggered by a febrile infection and characterized by serious and irreversible neurological damage. Although only a few hundred cases have been reported, mutations in RANBP2 are only partially penetrant and can occur de novo, suggesting that their frequency may be higher in some populations. Genetic diagnosis is a lengthy process, potentially delaying definitive diagnosis. We therefore developed a rapid bedside qPCR-based tool for early diagnosis and screening of ANE1 mutations. Primers were designed to specifically assess RANBP2 and not RGPD (RANBP2 and GCC2 protein domains) and discriminate between wild-type or mutant RANBP2. Nasal epithelial cells were obtained from two individuals with known RANBP2 mutations and two healthy control individuals. RANBP2-specific reverse transcription followed by allele-specific primer qPCR amplification confirmed the specific detection of heterozygously expressed mutant RANBP2 in the ANE1 samples. This study demonstrates that allele-specific qPCR can be used as a rapid and inexpensive diagnostic tool for ANE1 using preexisting equipment at local hospitals. It can also be used to screen non-hospitalized family members and at risk-population to better establish the frequency of non-ANE-associated RANBP2 mutations, as well as possible tissue-dependent expression patterns. Systematic review registration: The protocol was registered in the international prospective register of systematic reviews (PROSPERO- CRD42023443257).
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Age is closely related to human health and disease risks. However, chronologically defined age often disagrees with biological age, primarily due to genetic and environmental variables. Identifying effective indicators for biological age in clinical practice and self-monitoring is important but currently lacking. The human lens accumulates age-related changes that are amenable to rapid and objective assessment. Here, using lens photographs from 20 to 96-year-olds, we develop LensAge to reflect lens aging via deep learning. LensAge is closely correlated with chronological age of relatively healthy individuals (R2 > 0.80, mean absolute errors of 4.25 to 4.82 years). Among the general population, we calculate the LensAge index by contrasting LensAge and chronological age to reflect the aging rate relative to peers. The LensAge index effectively reveals the risks of age-related eye and systemic disease occurrence, as well as all-cause mortality. It outperforms chronological age in reflecting age-related disease risks (p < 0.001). More importantly, our models can conveniently work based on smartphone photographs, suggesting suitability for routine self-examination of aging status. Overall, our study demonstrates that the LensAge index may serve as an ideal quantitative indicator for clinically assessing and self-monitoring biological age in humans.
Subject(s)
Deep Learning , Lens, Crystalline , Humans , Child, Preschool , Aging/geneticsABSTRACT
Background and study aims Undifferentiated early gastric cancer (UD-EGC) represents an extended indication for endoscopic submucosal dissection (ESD) based on the existing guidelines. This study evaluated the prevalence of UD-EGC recurrence after ESD, and potentially implicated risk factors. Patients and methods Data from 17 centers were collected retrospectively including demographics, endoscopic and pathological findings, and follow-up data from UD-EGC cases treated by ESD. Patients with incomplete resection or advanced disease were excluded. Descriptive statistics quantified variables and calculated the incidence of recurrence. Chi-square test was applied to assess any link between independent variables and relapse; significantly associated variables were inserted to a multivariable regression model. Results Seventy-one patients were eligible, with 2:1 female to male ratio and age of 65.8 ± 11.8 years. Mean lesion size was 33.5 ± 18.8 mm and the most frequent histological subtype was signet ring-cells UGC (2:1). Patients were followed-up every 5.6 ± 3.7 months with a mean surveillance period of 29.3 ± 15.3 months until data collection. Four patients (5.6%) developed local recurrence 8.8 ± 6.5 months post-ESD, with no lymph node or distal metastases been reported. Lesion size was not associated with recurrence ( P = 0.32), in contrast to lymphovascular and perineural invasion which were independently associated with local recurrence ( P = 0.006 and P < 0.001, respectively). Conclusions ESD could be considered as the initial step to manage UD-EGC, providing at least an "entire-lesion" biopsy to guide therapeutic strategy. When histology confirms absence of lymphovascular and perineural invasion, this modality could be therapeutic, providing low recurrence rates.
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The epithelial-mesenchymal transition (EMT) is closely associated with Crohn's disease (CD) related intestinal fibrosis, a condition whose prevalence is increasing annually among children. Recently, the CD marker gene microarray screening revealed an upregulation of circ_0001666 in the colon tissues of CD patients, but its underlying mechanisms remain unclear. In this study, we explored the molecular mechanism of circ_0001666 in regulating EMT-mediated fibrosis in CD in vitro. The levels of circ_0001666 and EMT-associated proteins were assessed in CD clinical samples, and a CD cell model was established using TGF-ß1 to induce human intestinal epithelial cells (HIECs). Additionally, the expression levels of genes and proteins related to EMT and fibrosis were analyzed by quantitative real-time PCR and western blot, cell migration, and invasion were assessed via wound healing assay and transwell, respectively, and RNA pull-down and RNA immunoprecipitation assays were performed to verify the relationship between SRSF1 and BMP7 or circ_0001666. Circ_0001666 was overexpressed in the intestinal mucosal tissues of CD patients and was positively correlated with EMT. Silencing circ_0001666 inhibited the migration, invasion, EMT, and fibrosis of HIECs induced by TGF-ß1. Mechanistically, circ_0001666 regulated BMP7 expression by interacting with SRSF1. Furthermore, the effects of inhibiting circ_0001666 on HIECs could be partially reversed by overexpressing SRSF1 or silencing BMP7. Collectively, circ_0001666 regulates TGF-ß1-induced HIEC migration, invasion, EMT, and fibrosis. Circ_0001666 also promoted EMT-mediated fibrosis by interacting with SRSF1 to accelerate BMP7 mRNA decay. These findings provide new insights into the pathogenesis of CD and suggest that circ_0001666 might be a potential therapeutic target for CD.
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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.
Subject(s)
Inflammasomes , Keratitis , Humans , Animals , Mice , Inflammasomes/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Reactive Oxygen Species/metabolism , Necroptosis , Apoptosis/physiology , Protein Kinases/metabolism , Oxidative Stress , Receptor-Interacting Protein Serine-Threonine Kinases/metabolismABSTRACT
It has been suggested that in vitro studies of the rescue effect of CFTR modulator drugs in nasal epithelial cultures derived from people with cystic fibrosis have the potential to predict clinical responses to the same drugs. Hence, there is an interest in evaluating different methods for measuring in vitro modulator responses in patient-derived nasal cultures. Commonly, the functional response to CFTR modulator combinations in these cultures is assessed by bioelectric measurements, using the Ussing chamber. While this method is highly informative, it is time-consuming. A fluorescence-based, multi-transwell method for assaying regulated apical chloride conductance (Fl-ACC) promises to provide a complementary approach to theratyping in patient-derived nasal cultures. In the present work, we compared Ussing chamber measurements and fluorescence-based measurements of CFTR-mediated apical conductance in matching, fully differentiated nasal cultures derived from CF patients, homozygous for F508del (n = 31) or W1282X (n = 3), or heterozygous for Class III mutations G551D or G178R (n = 5). These cultures were obtained through a bioresource called the Cystic Fibrosis Canada-Sick Kids Program in Individual CF Therapy (CFIT). We found that the Fl-ACC method was effective in detecting positive responses to interventions for all genotypes. There was a correlation between patient-specific drug responses measured in cultures harbouring F508del, as measured using the Ussing chamber technique and the fluorescence-based assay (Fl-ACC). Finally, the fluorescence-based assay has the potential for greater sensitivity for detecting responses to pharmacological rescue strategies targeting W1282X.
Subject(s)
Cystic Fibrosis , Humans , Cystic Fibrosis/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Fluorescence , Mutation , GenotypeABSTRACT
Purpose: Persistent fetal vasculature (PFV) is a pathological condition accounting for 4.8% of children's blindness in the United States. However, the PFV cell composition and pathogenetic mechanisms are poorly understood. This study aims to characterize PFV cell composition and associated molecular features and attempts to lay a foundation for further understanding the disease. Methods: Immunohistochemistry was conducted to characterize cell types at the tissue level. Single-cell RNA sequencing (sc-RNAseq) was performed on the vitreous cells derived from normal and Fz5 mutant mice at two early postnatal ages and human PFV samples. Bioinformatic tools were used to cluster cells and analyze their molecular features and functions. Results: The findings of this study are as follows: (1) a total of 10 defined and one undefined cell types were characterized in both the hyaloid vessel system and PFV by sc-RNAseq and immunohistochemistry; (2) neural crest-derived melanocytes, astrocytes, and fibroblasts were specifically retained in the mutant PFV; (3) Fz5 mutants were found to possess more vitreous cells at early postnatal age 3 but returned to similar levels as the wild type at postnatal age 6; (4) altered phagocytic and proliferation environments and cell-cell interactions were detected in the mutant vitreous; (5) the human PFV samples shared fibroblast, endothelial and macrophage cell types with the mouse, but having distinct immune cells including T cells, NK cells and Neutrophils; and last, (6) some neural crest features were also shared between certain mouse and human vitreous cell types. Conclusions: We characterized PFV cell composition and associated molecular features in the Fz5 mutant mice and two human PFV samples. The excessively migrated vitreous cells, intrinsic molecular properties of these cells, phagocytic environment, and cell-cell interactions may together contribute to PFV pathogenesis. Human PFV shares certain cell types and molecular features with the mouse.
Subject(s)
Astrocytes , Blindness , Frizzled Receptors , Animals , Child , Child, Preschool , Humans , Mice , Cell Communication , Computational Biology , FibroblastsABSTRACT
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.
Subject(s)
Adaptor Proteins, Signal Transducing , Corneal Injuries , Intercellular Signaling Peptides and Proteins , Limbal Stem Cells , Nerve Tissue Proteins , Transcriptome , Animals , Mice , Cell Proliferation , Corneal Injuries/metabolism , Limbal Stem Cells/metabolism , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolismABSTRACT
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.
Subject(s)
Epithelium, Corneal , Humans , Epithelium, Corneal/metabolism , Stem Cells , Cell Differentiation/physiology , Cell Proliferation , Chromatin/metabolism , Proto-Oncogene Protein c-ets-1/genetics , Proto-Oncogene Protein c-ets-1/metabolismABSTRACT
Corneal transplantation is impeded by donor shortages, immune rejection, and ethical reservations. Pre-made cornea prostheses (keratoprostheses) offer a proven option to alleviate these issues. Ideal keratoprostheses must possess optical clarity and mechanical robustness, but also high permeability, processability, and recyclability. Here, it is shown that rationally controlling the extent of arrested phase separation can lead to optimized multiscale structure that reconciles permeability and transparency, a previously conflicting goal by common pore-forming strategies. The process is simply accomplished by hydrothermally treating a dense and transparent hydrophobic association hydrogel. The examination of multiscale structure evolution during hydrothermal treatment reveals that the phase separation with upper miscibility gap evolves to confer time-dependent pore growth due to slow dynamics of polymer-rich phase which is close to vitrification. Such a process can render a combination of multiple desired properties that equal or surpass those of the state-of-the-art keratoprostheses. In vivo tests confirm that the keratoprosthesis can effectively repair corneal perforation and restore a transparent cornea with treatment outcomes akin to that of allo-keratoplasty. The keratoprosthesis is easy to access and convenient to carry, and thus would be an effective temporary substitute for a corneal allograft in emergency conditions.
Subject(s)
Cornea , Corneal Diseases , Humans , Cornea/surgery , Prostheses and Implants , Corneal Diseases/surgery , Hydrogels/chemistry , Treatment OutcomeABSTRACT
Background: With the increasing incidence of diabetes, diabetic foot ulcer(DFU) has become one of the most common and serious complications in people with diabetes. DFU is associated with significant morbidity and mortality, and can also result in significant economic, social and public health burdens. Due to peripheral neuropathy, peripheral vascular disease, hyperglycemic environment, inflammatory disorders and other factors, the healing of DFU is impaired or delayed, resulting in the formation of diabetic chronic refractory ulcer. Because of these pathological abnormalities in DFU, it may be difficult to promote wound healing with conventional therapies or antibiotics, whereas platelet-rich plasma(PRP) can promote wound healing by releasing various bioactive molecules stored in platelets, making it more promising than traditional antibiotics. Therefore, the purpose of this systematic review is to summarize and analyze the efficacy of PRP in the treatment of DFU. Methods: A literature search was undertaken in PubMed, CNKI, EMB-ASE, the Cochrane Library, the WanFang Database and the WeiPu Database by computer. Included controlled studies evaluating the efficacy of PRP in the treatment of diabetic foot ulcers. The data extraction and assessment are on the basis of PRISMA. Results: Twenty studies were evaluated, and nineteen measures for the evaluation of the efficacy of PRP in DFU treatment were introduced by eliminating relevant duplicate measures. The efficacy measures that were repeated in various studies mainly included the rate of complete ulcer healing, the percentage of ulcer area reduction, the time required for ulcer healing, wound complications (including infection rate, amputation rate, and degree of amputation), the rate of ulcer recurrence, and the cost and duration of hospitalization for DFU, as well as subsequent survival and quality of life scores. One of the most important indicators were healing rate, ulcer area reduction and healing time. The meta-analysis found that PRP was significantly improve the healing rate(OR = 4.37, 95% CI 3.02-6.33, P < 0.001) and shorten the healing time(MD = -3.21, 95% CI -3.83 to -2.59,P < 0.001)of patients with DFU when compared to the conventional treatment, but there was no significant difference in reducing the of ulcer area(MD = 5.67, 95% CI -0.77 to 12.11,P =0.08>0.05 ). Conclusion: The application of PRP to DFU can improve ulcer healing rate and shorten ulcer healing time, but more clinical data are needed to clarify some efficacy measures. At the same time, a standardized preparation process for PRP is essential.