Role of N6-methyladenosine-related lncRnas in pseudoexfoliation glaucoma.

To explore the role of lncRNA m6A methylation modification in aqueous humour (AH) of patients with pseudoexfoliation glaucoma (PXG). Patients with open-angle PXG under surgery from June 2021 to December 2021 were selected. Age- and gender-matched patients with age-related cataract (ARC) were chosen as control. Patients underwent detailed ophthalmic examinations. 0.05-0.1 ml AH were extracted during surgery for MeRIP-Seq and RNA-Seq. Joint analysis was used to screen lncRNAs with differential m6A methylation modification and expression. Online software tools were used to draw lncRNA-miRNA-mRNA network (ceRNA). Expression of lncRNAs and mRNAs was confirmed using quantitative real-time PCR. A total of 4151 lncRNAs and 4386 associated m6A methylation modified peaks were identified in the PXG group. Similarly, 2490 lncRNAs and 2595 associated m6A methylation modified peaks were detected in the control. Compared to the ARC group, the PXG group had 234 hypermethylated and 402 hypomethylated m6A peaks, with statistically significant differences (| Fold Change (FC) |≥2, p < 0.05). Bioinformatic analysis revealed that these differentially methylated lncRNA enriched in extracellular matrix formation, tight adhesion, TGF- ß signalling pathway, AMPK signalling pathway, and MAPK signalling pathway. Joint analysis identified 10 lncRNAs with differential m6A methylation and expression simultaneously. Among them, the expression of ENST000000485383 and ROCK1 were confirmed downregulated in the PXG group by RT-qPCR. m6A methylation modification may affect the expression of lncRNA and participate in the pathogenesis of PXG through the ceRNA network. ENST000000485383-hsa miR592-ROCK1 May be a potential target pathway for further investigation in PXG m6A methylation.

Critical role of transcriptome-wide m6A methylation in the aqueous humor of patients with pseudoexfoliation glaucoma.

N6-methyladenosine (m6A) modification is one of the most common types of methylation modifications in eukaryotic mRNA. However, its role in the pathogenesis of pseudoexfoliation glaucoma (PXG) has not yet been reported. To enhance understanding in this regard, we assessed the m6A methylome in the aqueous humor of patients with PXG. MeRIP-Seq and RNA-Seq analyses were performed to compare the m6A methylomes and gene expression profiles of the aqueous humor of patients with PXG with those of patients with age-related cataract (ARC). Colorimetric m6A quantification was performed to detect global m6A levels. Quantitative reverse transcription PCR confirmed the expression of m6A-related enzymes and mRNAs in both groups. Results showed significantly higher aqueous humor m6A levels in the PXG group than in the ARC group. Five m6A-related enzymes, including METTL3, YTHDC2, HNRNPA2B1, HNRNPC, and LRPPRC, were significantly up-regulated in PXG specimens. We also observed 9728 m6A-modified peaks related to 6126 gene transcripts in the PXG group, with more than 250 genes containing one m6A peak (hypomethylated or hypermethylated). The distribution of the m6A peaks was enriched in coding sequences and 3'-untranslated regions for both groups. GGAC motif structures were also significantly enriched. Bioinformatics analysis further revealed that m6A plays a critical role in extracellular matrix formation and histone deacetylation. Additionally, MMP14, ADAMTSL1, FN1, and HDAC1 showed significant changes in m6A methylation and mRNA expression in the PXG group. Therefore, m6A methylation may regulate extracellular matrix composition in PXG and METTL3 may be a pivotal regulator of this process. In the future, it would be necessary to investigate MMP14, ADAMTSL1, FN1, and HDAC1, which are potential target genes.

Prevalence Patterns and Onset Prediction of High Myopia for Children and Adolescents in Southern China via Real-World Screening Data: Retrospective School-Based Study.

BACKGROUND: Patients with high myopia have an increased lifetime risk of complications. The prevalence patterns of high myopia in children and adolescents in southern China are unclear. Early identification of high-risk individuals is critical for reducing the occurrence and development of high myopia and avoiding the resulting complications. OBJECTIVE: This study aimed to determine the prevalence of high myopia in children and adolescents in southern China via real-world screening data and to predict its onset by studying the risk factors for high myopia based on machine learning. METHODS: This retrospective school-based study was conducted in 13 cities with different gross domestic products in southern China. Through data acquisition and filtering, we analyzed the prevalence of high myopia and its association with age, school stage, gross domestic product, and risk factors. A random forest algorithm was used to predict high myopia among schoolchildren and then assessed in an independent hold-out group. RESULTS: There were 1,285,609 participants (mean age 11.80, SD 3.07, range 6-20 years), of whom 658,516 (51.2%) were male. The overall prevalence of high myopia was 4.48% (2019), 4.88% (2020), and 3.17% (2021), with an increasing trend from the age of 11 to 17 years. The rates of high myopia increased from elementary schools to high schools but decreased at all school stages from 2019 to 2021. The coastal and southern cities had a higher proportion of high myopia, with an overall prevalence between 2.60% and 5.83%. Age, uncorrected distance visual acuity, and spherical equivalents were predictive factors for high myopia onset in schoolchildren. The random forest algorithm achieved a high accuracy of 0.948. The area under the receiver operator characteristic curve (AUC) was 0.975. Both indicated sufficient model efficacy. The performance of the model was validated in an external test with high accuracy (0.971) and a high AUC (0.957). CONCLUSIONS: High myopia had a high incidence in Guangdong Province. Its onset in children and adolescents was well predicted with the random forest algorithm. Efficient use of real-world data can contribute to the prevention and early diagnosis of high myopia.

Necroptosis signaling and NLRP3 inflammasome cross-talking in epithelium facilitate Pseudomonas aeruginosa mediated lung injury.

Pseudomonas aeruginosa induced acute lung injury is such a serious risk to public health, but the pathological regulation remains unclear. Here, we reported that PA mediated epithelial necroptosis plays an important role in pathological process. Pharmacological and genomic ablation of necroptosis signaling ameliorate PA mediated ALI and pulmonary inflammation. Our results further proved NLRP3 inflammasome to involve in the process. Mechanism investigation revealed the cross-talking between inflammasome activation and necroptosis that MLKL-dependent necroptosis signaling promotes the change of mitochondrial membrane potential for the release of reactive oxygen species (ROS), which is the important trigger for functional inflammasome activation. Furthermore, antioxidants such as Mito-TEMPO was confirmed to significantly restrain inflammasome activation in epithelium, resulting in a reduction in PA induced pulmonary inflammation. Taken together, our findings revealed that necroptosis-triggered NLRP3 inflammasome in epithelium plays a crucial role in PA mediated injury, which could be a potential therapeutic target for pulmonary inflammation.

DDR1 activation in macrophage promotes IPF by regulating NLRP3 inflammasome and macrophage reaction.

BACKGROUND: Discoidin Domain Receptor1 (DDR1) is a member of receptor tyrosine kinases (RTKs) which have been reported to be associated with idiopathic pulmonary fibrosis (IPF), but the mechanism remains unclear. METHODS: Bleomycin-induced IPF mice model was performed in this study, and two DDR1 inhibitors were administered in vivo, to investigate the role of DDR1 in IPF. Lentivirus mediated DDR1-/- stable Raw264.7 macrophage cell line or DDR1 inhibitors treatment in vitro, to study the effect of DDR1 on inflammasome activation and macrophage responses. All of the mechanisms were further tested in the lung sections of IPF patients. RESULT: Here, we reported that: (i) Both specific inhibitors of DDR1 dramatically alleviated the symptoms of bleomycin-induced IPF models. (ii) Immunofluorescence staining showed that DDR1 signaling is activated in macrophages. In vivo molecular biological analysis proved that DDR1 activation exacerbates IPF inflammation through inflammasome signaling, macrophage activation, and M1/M2 polarization. (iii) Extracellular matrix (ECM) such as Collagen 1 activates DDR1 in macrophage cell line Raw264.7 in vitro, to mediate inflammasome activation and macrophage responses. (iv) DDR1 activation in macrophage was confirmed in IPF patients' samples, which could be one of the mechanisms for the pathogenesis of IPF. DISCUSSION: In this study, we firstly reported DDR1 activation in macrophages to play a role in IPF via inflammasome activation and macrophage responses. In addition, DDR1 inhibitors DDR1-IN-1 and DDR1-IN-2 exerted significant anti-inflammatory and anti-fibrotic effects in IPF, all of which provide a potentially effective therapeutic medication for clinical IPF treatment.

Lens nucleus dislocation in hypermature cataract: Case report and literature review.

INTRODUCTION: Hypermature cataract is a form of late-stage cataract progression that can lead to a variety of complications. Spontaneous capsular rupture with lens nucleus displacement in hypermature cataracts has rarely been reported. We describe 2 cases of spontaneous dislocation of the lens nucleus in a hypermature cataract and perform a review of the literature on this complication. PATIENT CONCERNS: We report 2 rural men aged 50 and 76 years with deteriorating vision. DIAGNOSIS: The final diagnosis was senile hypermature cataract with dislocation of the lens nucleus in both patients and secondary glaucoma for the second patient. INTERVENTIONS AND OUTCOMES: During admission, both patients complained of deteriorating vision. Slit-lamp examination showed lens nucleus dislocation into the anterior chamber. The 50-year-old patient exhibited a residual lens capsule and a turbid cortex, with a normal anterior chamber and intraocular pressure. The 76-year-old patient presented a shrunken and ruptured capsule and no cortex in the pupillary area, mild inflammation in the anterior chamber, and high intraocular pressure. Both patients underwent intracapsular cataract extraction combined with anterior vitrectomy and achieved good postoperative recovery. CONCLUSION: Lens nucleus dislocation in hypermature cataracts can be seen in clinical practice, particularly in underdeveloped areas. Early recognition and surgery can improve vision.

Germinal peptide eye drop promotes corneal epithelial and stromal defect healing in rabbit model.

OBJECTIVE: Corneal defect is a common disease in ophthalmology caused by trauma, inflammation, drug toxicity, or surgery. To investigate the effect of germinal peptide eye drop on corneal epithelial and stromal defects after lamellar keratectomy in rabbit model. METHODS: Eighty-five male New Zealand white rabbits were divided into five groups: Germinal Peptide eye drop at three different concentration groups, normal saline (negative control group), recombinant human epidermal growth factor (rh-EGF) eye drop (positive control group). Corneal epithelial and stromal defects of around 150-200 µm in depth were created with an 8 mm diameter trephine in the center of the right eyes of all animals. RESULTS: Germinal peptide eye drop with the concentration of 0.001%, 0.002%, and 0.004% and rh-EGF eye drop were more effective in promoting healing, reducing opacity, and edema during the process of corneal epithelial and stromal defect regeneration compared with 0.9% normal saline. No significant difference was observed among the three different doses of germinal peptide eye drop. Compared with the saline control group, the structures of the regenerated corneas were more orderly and less inflammatory cell infiltration was observed in the germinal peptide eye drop groups and the rh-EGF eye drop group. CONCLUSION: Germinal peptide eye drop (0.001%, 0.002%, and 0.004%) can significantly stimulate the regeneration of corneal epithelia and stroma and reduce corneal opacity and edema. Dose dependency was not observed in the current study.

TNF-α stimulation enhances the neuroprotective effects of gingival MSCs derived exosomes in retinal ischemia-reperfusion injury via the MEG3/miR-21a-5p axis.

Retinal ischemia-reperfusion injury (IRI) is one of the main pathogenic mechanisms of glaucoma, which are largely unknown, including neuroinflammation and neuronal death in the pathological process. In our previous studies, mesenchymal stem cells (MSCs) have been reported to play anti-inflammatory and neuroprotective roles. Additionally, conditioned culture medium (CM) of MSCs stimulated by TNF-α have achieved better antiallergic effects in an experimental allergic conjunctivitis mouse model. However, there is an urgent need for cell-free therapy approaches, like exosomes, to reduce the side effects of autoimmunity. The present study aimed to elucidate the pathways involving TNF-α-stimulated gingival MSC (GMSC)-exosomes (TG-exos), in modulating inflammatory microglia and alleviating apoptosis. In this study, exosomes from the CM of GMSCs were isolated by ultracentrifugation and were injected into the vitreous of mice. The results showed that intraocular injection of TG-exos into mice with IRI notably reduced inflammation and cell loss than that with G-exos (GMSC-exosomes). Similar results were observed in vitro. Additionally, with the microRNA (miR) arrays, it was found that miR-21-5p acted as a crucial factor in TG-exos for neuroprotection and anti-inflammation. Following target prediction and dual-luciferase assay suggested that miR-21-5p played a role by combining with programmed cell death 4 (PDCD4), which was regulated by the long non-coding RNA (lncRNA) maternally expressed gene 3 (MEG3) as a competing endogenous RNA (ceRNA). This study demonstrates a new therapeutic pathway for neuroprotection against IRI by delivering miR-21-5p-enriched exosomes through MEG3/miR-21-5p/PDCD4 axis and paves the way for the establishment of a cell-free therapeutic approach for glaucoma.

TSSK4 upregulation in alveolar epithelial type-II cells facilitates pulmonary fibrosis through HSP90-AKT signaling restriction and AT-II apoptosis.

Alveolar epithelial injury is one of the important pathological changes in idiopathic pulmonary interstitial fibrosis (IPF), but the regulatory mechanism remains unclear. Here, we reported that alveolar epithelial type-II cells (AT II) play important roles in pathological process of pulmonary fibrosis. Through iTRAQ (isobaric tagging for relative and absolute quantification) quantitative proteomics, TSSK4 was identified to be upregulated in bleomycin-induced fibrotic mice model, which was further confirmed in clinical IPF patients' tissue specimens. TSSK4 is a germ-related protein, but its expression in other tissues and the association with other diseases are not reported. Immunofluorescence staining showed that TSSK4 selectively expressed in AT-II cells, which are essential for inflammation-induced AT-II loss during fibrosis. Luciferase assay and other molecular biological experiments proved that TSSK4 expression is regulated by TNF-α-mediated NF-κB signaling. The TSSK4 kinase activity is found to be closely related to the function of HSP90-AKT pathway that TSSK4 can phosphorylate its substrate HSP90ß on serine 255, to inhibit the ATPase activity of HSP90ß and reduce its molecular chaperone function on AKT. Under this condition, kinase activity of AKT is diminished to interfere its survival function, subsequently facilitating AT-II cellular apoptosis through the mitochondrial death machinery. Our findings highlight the importance of TSSK4 in regulating pulmonary fibrosis by facilitating AT-II loss through HSP90-AKT signaling, all of which suggest TSSK4 and the regulating mechanism as attractive targets for the clinical intervention of pulmonary injury and fibrosis.

Germinal peptide eye drops promote corneal wound healing and decrease inflammation after alkali injury.

Germinal peptide is being developed to treat corneal injuries. The purpose of this study was to investigate its effect on corneal epithelial cells in vitro and its ability to promote healing in an alkali injury model in vivo. Cultured rabbit corneal epithelial cells were treated with germinal peptide at three concentrations. Cell proliferation and migration were assessed and compared with the effect of recombinant human epidermal growth factor (rh-EGF). In vivo, the corneas of New Zealand albino rabbits were chemically burned with 1 mol/l NaOH for 30 s. The injured eyes were topically treated with germinal peptide (10, 20, and 40 µg/ml), rh-EGF, or phosphate-buffered saline thrice daily. At fixed time points post injury, the healing of the cornea and its histopathology were evaluated. There was no difference in the effect of germinal peptide on cultured cell proliferation. However, cell migration was significantly higher than that in the control groups, with germinal peptide at concentrations of 20 and 40 µg/ml being the most efficacious. In vivo, 20 and 40 µg/ml germinal peptide significantly alleviated corneal opacity and edema. By day 21, the areas of corneal neovascularization in the germinal peptide-treated groups were smaller than those in the rh-EGF and control groups. The repaired corneas in the germinal peptide- and rh-EGF-treated groups also had more corneal epithelial layers and fewer inflammatory cells than the controls. Germinal peptide may be developed as a novel topical treatment agent for corneal wound healing in clinical settings.

High infiltration of CD20+ B lymphocytes in extranodal natural killer/T-cell lymphoma is associated with better prognosis.

Immune cells have an uncertain function during the progression of extranodal natural killer/T-cell lymphoma (ENKTL). The present study determined the distribution, phenotype, and clinical significance of B lymphocytes in ENKTL. Immunohistochemistry indicated high infiltration of CD20+ B lymphocytes in the tumour tissues of 40% of the patients, and that a high infiltration correlated with better overall survival. Moreover, B lymphocytes had an active mature phenotype in situ and suppressed the proliferation of ENKTL cells in vitro. These results suggest that tumour infiltration of CD20+ B lymphocytes may be a new prognostic indicator for patients with ENKTL.

Combination of cetuximab with met inhibitor in control of cetuximab-resistant oral squamous cell carcinoma.

Objective: To investigate the underlying molecular mechanisms contributing to oral squamous cell carcinoma (OSCC) cell resistance to the epidermal growth factor receptor (EGFR) inhibitor. Materials and methods: OSCC cell lines HSC-2 and HSC-3 were assessed in vitro for drug treatment, cell viability, and gene expression and the online gene expression in OSCC tissues was analyzed for association with OSCC prognosis. Results: HSC-2 and HSC-3 cells expressed high EGFR levels, but hepatocyte growth factor (HGF) treatment induced cetuximab resistance, whereas the Met inhibitor PHA-665752 as well as Met siRNA was able to restore OSCC cell sensitivity to cetuximab. HGF treatment induced tumor cells to express p-Akt and p-ERK1/2. In contrast, the activity of Akt and ERK1/2 was suppressed by treatment with PHA-665752, Met siRNA, or their combination. Furthermore, Met was highly expressed in OSCC tissues and associated with a poor patient survival, while Met/HGF-activated Akt also was associated with a poor patient survival. Conclusions: This study demonstrates that Met/HGF expression results in OSCC resistance to cetuximab and tumor recurrence after cetuximab therapy; thus, inhibition of Met/HGF activity could restore OSCC sensitivity to cetuximab.

Effects of Adoptive Transferring Different Sources of Myeloid-Derived Suppressor Cells in Mice Corneal Transplant Survival.

BACKGROUND: Adoptively transferring different sources of myeloid-derived suppressor cells (MDSCs) may assist in mice corneal transplant survival. METHODS: Allogeneic full thickness corneal transplantation (donor C57BL/6 to recipient Balb/c mice) was performed. Naive myeloid cells, inflammation-induced MDSCs (iMDSCs), and tumor-induced MDSCs (tMDSCs) were purified from bone marrow of naive, cecal ligation and puncture, or tumor-bearing Balb/c mice, respectively. The inhibitory abilities of myeloid cells toward CD4(+) T cell proliferation were accessed by in vitro carboxyfluorescein diacetate, succinimidyl ester (CFSE) assays. Myeloid cells were adoptively transferred to corneal recipients by retroorbital injection after corneal transplantation. Corneal grafts were examined and photographed for a period of 45 days. The growth of corneal graft neovascularization was quantitatively measured by image editing software. Histopathology was performed to evaluate corneal graft inflammation. RESULTS: The iMDSCs and tMDSCs significantly inhibited T cell proliferation in vitro and significantly prolonged corneal allograft survival in vivo. Strikingly, iMDSC transferring significantly reduced neovascularization that was comparable to transferring of tMDSCs, without additional immunosuppression. However, additional adoptive transfer of MDSCs did not further ameliorate corneal survival in these allogeneic corneal transplantation mice. CONCLUSIONS: Inflammation-induced MDSC transfer could reduce corneal neovascularization and prolong corneal allograft survival.