A feedback loop driven by H3K9 lactylation and HDAC2 in endothelial cells regulates VEGF-induced angiogenesis.

BACKGROUND: Vascular endothelial growth factor (VEGF) is one of the most powerful proangiogenic factors and plays an important role in multiple diseases. Increased glycolytic rates and lactate accumulation are associated with pathological angiogenesis. RESULTS: Here, we show that a feedback loop between H3K9 lactylation (H3K9la) and histone deacetylase 2 (HDAC2) in endothelial cells drives VEGF-induced angiogenesis. We find that the H3K9la levels are upregulated in endothelial cells in response to VEGF stimulation. Pharmacological inhibition of glycolysis decreases H3K9 lactylation and attenuates neovascularization. CUT& Tag analysis reveals that H3K9la is enriched at the promoters of a set of angiogenic genes and promotes their transcription. Interestingly, we find that hyperlactylation of H3K9 inhibits expression of the lactylation eraser HDAC2, whereas overexpression of HDAC2 decreases H3K9 lactylation and suppresses angiogenesis. CONCLUSIONS: Collectively, our study illustrates that H3K9la is important for VEGF-induced angiogenesis, and interruption of the H3K9la/HDAC2 feedback loop may represent a novel therapeutic method for treating pathological neovascularization.

Comparison of inebilizumab or rituximab in addition to glucocorticoid therapy for neuromyelitis optica spectrum disorders.

AIM: To investigate the short-term efficacy and safety of inebilizumab for neuromyelitis optica spectrum disorders (NMOSD). METHODS: A total of 33 patients with NMOSD treated with inebilizumab (Group INB, n=15) or rituximab (Group RTX, n=18) in addition to high-dose glucocorticoids were included. Both groups underwent hormone shock therapy during the acute phase. Subsequently, Group INB received inebilizumab injections during the remission phase, while Group RTX received rituximab injections. A comparison of aquaporins 4 (AQP4) titer values, peripheral blood B lymphocyte counts, and visual function recovery was conducted before and 8wk after treatment. Additionally, adverse reactions and patient tolerability were analyzed after using inebilizumab treatment regimes. RESULTS: Following inebilizumab treatment, there was a significantly improvement in the visual acuity of NMOSD patients (P<0.05), accompanied by a notable decrease in AQP4 titer values and B lymphocyte ratio (P<0.05). Moreover, inebilizumab treatment showed a partial effect in preventing optic nerve atrophy (P<0.05). However, there were no significant differences in other therapeutic effects compared to rituximab, which has previously demonstrated substantial therapeutic efficacy (P>0.05). Furthermore, inebilizumab exhibited higher safety levels than that of rituximab injections. CONCLUSION: The combination of inebilizumab and high-dose glucocorticoids proves to be effective. In comparison to rituximab injections, inebilizumab displays better tolerance and safety. Moreover, it demonstrates a partial effect in preventing optic nerve atrophy. Thus, it stands as an effective method to reduce the disability rates and improve the daily living ability of patients with NMOSD.

miR-504 knockout regulates tumor cell proliferation and immune cell infiltration to accelerate oral cancer development.

miR-504 plays a pivotal role in the progression of oral cancer. However, the underlying mechanism remains elusive in vivo. Here, we find that miR-504 is significantly down-regulated in oral cancer patients. We generate miR-504 knockout mice (miR-504-/-) using CRISPR/Cas9 technology to investigate its impact on the malignant progression of oral cancer under exposure to 4-Nitroquinoline N-oxide (4NQO). We show that the deletion of miR-504 does not affect phenotypic characteristics, body weight, reproductive performance, or survival in mice, but results in changes in the blood physiological and biochemical indexes of the mice. Moreover, with 4NQO treatment, miR-504-/- mice exhibit more pronounced pathological changes characteristic of oral cancer. RNA-seq shows that the differentially expressed genes observed in samples from miR-504-/- mice with oral cancer are involved in regulating cell metabolism, cytokine activation, and lipid metabolism-related pathways. Additionally, these differentially expressed genes are significantly enriched in lipid metabolism pathways that influence immune cell infiltration within the tumor microenvironment, thereby accelerating tumor development progression. Collectively, our results suggest that knockout of miR-504 accelerates malignant progression in 4NQO-induced oral cancer by regulating tumor cell proliferation and lipid metabolism affecting immune cell infiltration.

The miR-23b-3p from adipose-derived stem cell exosomes alleviate inflammation in mice experiencing kainic acid-induced epileptic seizures.

Epilepsy is a common neurologic disorder. While a good clinical solution is still missing, studies have confirmed that exosomes (Exos) derived from adipose-derived stem cells (ADSCs) had a therapeutic effect on various diseases, including neurological diseases. Therefore, this study aimed to reveal whether ADSC-Exo treatment could improve kainic acid (KA)-induced seizures in epileptic mice. ADSCs and Exos were isolated. Mice were generated with KA-induced epileptic seizures. ELISA was used to detect inflammatory factor expression. Luciferase reporter analysis detection showed a relationship among miR-23b-3p, STAT1, and glyoxylate reductase 1 (GlyR1). ADSC-Exos had a protective effect on KA-induced seizures by inhibiting inflammatory factor expression and the M1 microglia phenotype. The result showed that miR-23b-3p played an important role in the Exo-mediated protective effect in KA-induced seizures in epileptic mice by regulating STAT1 and GlyR1. Luciferase reporter analysis confirmed that miR-23b-3p interacted with the 3'-UTR of STAT1 and GlyR1. The miR-23b-3p inhibited M1 microglia-mediated inflammatory factor expression in microglial cells by regulating STAT1 and GlyR1. The downregulation of miR-23b-3p decreased the protective effect of ADSC-Exos on KA-induced seizures in epileptic mice. The miR-23b-3p from ADSC-Exos alleviated inflammation in mice with KA-induced epileptic seizures.

Icariin alleviates oxygen-induced retinopathy by targeting microglia hexokinase 2.

Retinopathy of prematurity (ROP) is a retinal disease-causing retinal neovascularization that can lead to blindness. Oxygen-induced retinopathy (OIR) is a widely used ROP animal model. Icariin (ICA) has anti-oxidative and anti-inflammation properties; however, whether ICA has a regulatory effect on OIR remains unclear. In this study, ICA alleviated pathological neovascularization, microglial activation and blood-retina barrier (BRB) damage in vivo. Further results indicated that endothelial cell tube formation, migration and proliferation were restored by ICA treatment in vitro. Proteomic microarrays and molecular mimicry revealed that ICA can directly bind to hexokinase 2 (HK2) and decrease HK2 protein expression in vivo and in vitro. In addition, ICA inhibited the AKT/mTOR/HIF1α pathway activation. The effects of ICA on pathological neovascularization, microglial activation and BRB damage disappeared after HK2 overexpression in vivo. Similarly, the endothelial cell function was revised after HK2 overexpression. HK2 overexpression reversed ICA-induced AKT/mTOR/HIF1α pathway inhibition in vivo and in vitro. Therefore, ICA prevented pathological angiogenesis in OIR in an HK2-dependent manner, implicating ICA as a potential therapeutic agent for ROP.

YY1 Lactylation Aggravates Autoimmune Uveitis by Enhancing Microglial Functions via Inflammatory Genes.

Activated microglia in the retina are essential for the development of autoimmune uveitis. Yin-Yang 1 (YY1) is an important transcription factor that participates in multiple inflammatory and immune-mediated diseases. Here, an increased YY1 lactylation in retinal microglia within in the experimental autoimmune uveitis (EAU) group is observed. YY1 lactylation contributed to boosting microglial activation and promoting their proliferation and migration abilities. Inhibition of lactylation suppressed microglial activation and attenuated inflammation in EAU. Mechanistically, cleavage under targets & tagmentation ï¼ˆCUT&Tag） analysis revealed that YY1 lactylation promoted microglial activation by regulating the transcription of a set of inflammatory genes, including STAT3, CCL5, IRF1, IDO1, and SEMA4D. In addition, p300 is identified as the writer of YY1 lactylation. Inhibition of p300 decreased YY1 lactylation and suppressed microglial inflammation in vivo and in vitro. Collectively, the results showed that YY1 lactylation promoted microglial dysfunction in autoimmune uveitis by upregulating inflammatory cytokine secretion and boosting cell migration and proliferation. Therapeutic effects can be achieved by targeting the lactate/p300/YY1 lactylation/inflammatory genes axis.

A single cell atlas of circulating immune cells involved in diabetic retinopathy.

This study focused on examining the exact role of circulating immune cells in the development of diabetic retinopathy (DR). In vitro co-culture experiments showed that peripheral blood mononuclear cells (PBMCs) from patients with type 1 DR crucially modulated the biological functions of human retinal microvascular endothelial cells (HRMECs), consequently disrupting their normal functionality. Single-cell RNA sequencing (scRNA-seq) study revealed unique differentially expressed genes and pathways in circulating immune cells among healthy controls, non-diabetic retinopathy (NDR) patients, and DR patients. Of significance was the observed upregulation of JUND in each subset of PBMCs in patients with type 1 DR. Further studies showed that downregulating JUND in DR patient-derived PBMCs led to the amelioration of HRMEC dysfunction. These findings highlighted the notable alterations in the transcriptomic patterns of circulating immune cells in type 1 DR patients and underscored the significance of JUND as a key factor for PBMCs in participating in the pathogenesis of DR.

Hepatic Stellate Cell-Targeting Micelle Nanomedicine for Early Diagnosis and Treatment of Liver Fibrosis.

Diagnosing and treating liver fibrosis is a challenging yet crucial endeavor due to its complex pathogenesis and risk of deteriorating into cirrhosis, liver failure, and even hepatic cancer. Herein, a silica cross-linked micelles (SCLMs) based nano-system is developed for both diagnosing and treating liver fibrosis. The SCLMs are first modified with peptide CTCE9908 (CT-SCLMs) and can actively target CXCR4, which is overexpressed in activated hepatic stellate cells (HSCs). To enable diagnosis, an ONOO--responded near-infrared fluorescent probe NOF2 is loaded into the CT-SCLMs. This nano-system can target the aHSCs and diagnose the liver fibrosis particularly in CCl4-induced liver damage, by monitoring the reactive nitrogen species. Furthermore, a step is taken toward treatment by co-encapsulating two anti-fibrosis drugs, silibinin and sorafenib, within the CT-SCLMs. This combined approach results in a significant alleviation of liver injury. Symptoms associated with liver fibrosis, such as deposition of collagen, expression of hydroxyproline, and raised serological indicators show notable improvement. In summary, the CXCR4-targeted nano-system can serve as a promising theragnostic system of early warning and diagnosis for liver fibrosis, offering hope against progression of this serious liver condition.

Current Status of Hedgehog Signaling Inhibitors.

The Hedgehog (Hh) signaling pathway plays a crucial role in diverse biological processes such as cell differentiation, proliferation, senescence, tumorigenesis, malignant transformation, and drug resistance. Aberrant Hh signaling, resulting from mutations and excessive activation, can contribute to the development of various diseases during different stages of biogenesis and development. Moreover, it has been linked to unfavorable outcomes in several human cancers, including basal cell carcinoma (BCC), multiple myeloma (MM), melanoma, and breast cancer. Hence, the presence of mutations and excessive activation of the Hh pathway presents obstacles and constraints in the realm of cancer treatment. Extant research has demonstrated that small molecule inhibitors are regarded as the most effective therapeutic approaches for targeting the Hh pathway in contrast to traditional chemotherapy and radiotherapy. Consequently, this review focuses on the present repertoire of small molecule inhibitors that target various components of the Hh pathway, including Hh ligands, Ptch receptors, Smo transmembrane proteins, and Gli nuclear transcription factors. This study provides a comprehensive analysis of small molecules' structural and functional aspects in the preclinical and clinical management of cancer. Additionally, it elucidates the obstacles encountered in targeting the Hh pathway for human cancer therapy and proposes potential therapeutic approaches.

OR11H1 Missense Variant Confers the Susceptibility to Vogt-Koyanagi-Harada Disease by Mediating Gadd45g Expression.

Vogt-Koyanagi-Harada (VKH) disease is a severe autoimmune disease. Herein, whole-exome sequencing (WES) study are performed on 2,573 controls and 229 VKH patients with follow-up next-generation sequencing (NGS) in a collection of 2,380 controls and 2,278 VKH patients. A rare c.188T>C (p Val63Ala) variant in the olfactory receptor 11H1 (OR11H1) gene is found to be significantly associated with VKH disease (rs71235604, Pcombined = 7.83 × 10-30 , odds ratio = 3.12). Functional study showes that OR11H1-A63 significantly increased inflammatory factors production and exacerbated barrier function damage. Further studies using RNA-sequencing find that OR11H1-A63 markedly increased growth arrest and DNA-damage-inducible gamma (GADD45G) expression. Moreover, OR11H1-A63 activates the MAPK and NF-κB pathways, and accelerates inflammatory cascades. In addition, inhibiting GADD45G alleviates inflammatory factor secretion, likely due to the regulatory effect of GADD45G on the MAPK and NF-κB pathways. Collectively, this study suggests that the OR11H1-A63 missense mutation may increase susceptibility to VKH disease in a GADD45G-dependent manner.

Association of Long Non-coding RNA C1RL-AS1 and PTPN6 Gene Polymorphisms with Ocular Behcet's Disease in Han Chinese.

PURPOSE: To explore the association of the polymorphisms in PTPN6 and LncRNA C1RL-AS1 genes with ocular BD in Han Chinese patients. METHODS: Correlation study was performed using the iPLEX system on a cohort of ocular BD patients andcontrols. The genotyping of 7 SNPs for LncRNA C1RL-AS1 and PTPN6 genes in ocular BD patients was performed using the iPLEX Gold genotype. RESULTS: The frequencies of rs4013722 AG genotype/A allele in LncRNA C1RL-AS1 were significantly decreased in BD patients, and the frequency of GG genotype was significantly increased in BD patients. The rs4013722 was associated with ocular BD in male patients, but not in female patients. The AG and GG genotype of rs4013722 were associated with skin lesions in male patients. The gene polymorphisms of PTPN6 were not associated with BD patients. CONCLUSIONS: The LncRNA C1RL-AS1/rs4013722 polymorphism conferred susceptibility to ocular BD in Han Chinese patients, which was influenced by sex.Abbreviations: LncRNA: Long Non-coding RNA; BD: Behcet's disease; SNP: single nucleotide polymorphism; PBMCs: peripheral blood mononuclear cells; PTPs: Protein tyrosine phosphatases; PTPN6: protein tyrosine phosphatase non-receptor 6; GWAS: genome-wide association study; HWE: Hardy-Weinberg equilibrium; LD: linkage disequilibrium; OR: odds ratio; CI: confidence interval; eQTL: expression quantitative trait loci; IBD: inflammatory bowel disease; RA: rheumatoid arthritis; Padj: Bonferroni corrected P value; NS: non-significant.

A de novo missense mutation in MPP2 confers an increased risk of Vogt-Koyanagi-Harada disease as shown by trio-based whole-exome sequencing.

Vogt-Koyanagi-Harada (VKH) disease is a leading cause of blindness in young and middle-aged people. However, the etiology of VKH disease remains unclear. Here, we performed the first trio-based whole-exome sequencing study, which enrolled 25 VKH patients and 50 controls, followed by a study of 2081 VKH patients from a Han Chinese population to uncover detrimental mutations. A total of 15 de novo mutations in VKH patients were identified, with one of the most important being the membrane palmitoylated protein 2 (MPP2) p.K315N (MPP2-N315) mutation. The MPP2-N315 mutation was highly deleterious according to bioinformatic predictions. Additionally, this mutation appears rare, being absent from the 1000 Genome Project and Genome Aggregation Database, and it is highly conserved in 10 species, including humans and mice. Subsequent studies showed that pathological phenotypes and retinal vascular leakage were aggravated in MPP2-N315 mutation knock-in or MPP2-N315 adeno-associated virus-treated mice with experimental autoimmune uveitis (EAU). In vitro, we used clustered regularly interspaced short palindromic repeats (CRISPR‒Cas9) gene editing technology to delete intrinsic MPP2 before overexpressing wild-type MPP2 or MPP2-N315. Levels of cytokines, such as IL-1ß, IL-17E, and vascular endothelial growth factor A, were increased, and barrier function was destroyed in the MPP2-N315 mutant ARPE19 cells. Mechanistically, the MPP2-N315 mutation had a stronger ability to directly bind to ANXA2 than MPP2-K315, as shown by LC‒MS/MS and Co-IP, and resulted in activation of the ERK3/IL-17E pathway. Overall, our results demonstrated that the MPP2-K315N mutation may increase susceptibility to VKH disease.

Global lactylome reveals lactylation-dependent mechanisms underlying TH17 differentiation in experimental autoimmune uveitis.

Dysregulation of CD4+ T cell differentiation is linked to autoimmune diseases. Metabolic reprogramming from oxidative phosphorylation to glycolysis and accumulation of lactate are involved in this process. However, the underlying mechanisms remain unclear. Our study showed that lactate-derived lactylation regulated CD4+ T cell differentiation. Lactylation levels in CD4+ T cells increased with the progression of experimental autoimmune uveitis (EAU). Inhibition of lactylation suppressed TH17 differentiation and attenuated EAU inflammation. The global lactylome revealed the landscape of lactylated sites and proteins in the CD4+ T cells of normal and EAU mice. Specifically, hyperlactylation of Ikzf1 at Lys164 promoted TH17 differentiation by directly modulating the expression of TH17-related genes, including Runx1, Tlr4, interleukin-2 (IL-2), and IL-4. Delactylation of Ikzf1 at Lys164 impaired TH17 differentiation. These findings exemplify how glycolysis regulates the site specificity of protein lactylation to promote TH17 differentiation and implicate Ikzf1 lactylation as a potential therapeutic target for autoimmune diseases.

Risk of stroke in patients with diabetic retinopathy: A systematic review and meta-analysis.

OBJECTIVE: Diabetic retinopathy (DR) is a microvascular complication of diabetes that occurs at high frequencies (more than 20%) during the course of the disease. Therefore, we conducted a meta-analysis of the incidence of stroke in DR to determine whether DR is associated with stroke. METHODS: The PubMed, Embase and Cochrane databases were systematically searched from their inception to December 1, 2022. Randomized controlled trials (RCTs) that reported DR and stroke events were included. The pooled risk ratio and 95% confidence interval (CI) were calculated. For the incidences of DR and stroke, risk difference and standard error were measured. Sensitivity analysis was performed to assess whether any single study could affect the overall outcome. RESULTS: Nine RCTs involving 46,599 patients with diabetes were included in this meta-analysis. The incidence of DR in all patients was 0.29 (95% CI 0.20-0.38). The incidence of any stroke in all patients was 0.03 (95% CI 0.03-0.04). The incidence of any stroke in patients with DR was 0.05 (95% CI 0.04-0.07), significant higher than that in all diabetes patients. The pooled risk ratio of stroke in patients with DR was 2.04 (95% CI 1.25-3.32). The estimated risk ratio of stroke in patients with DR without additional conditions was 1.70 (95% CI 1.43-2.03), which was lower than that in patients with DR with additional conditions (2.29, 95% CI 0.93-5.65). CONCLUSION: The presence of DR is associated with an increased risk of stroke. Our findings indicate that DR is an important biomarker for the prediction of stroke, and periodic eye examinations should be conducted for stroke prevention.

Frizzled 6 mutation regulates reserpine-induced depression-like behavior and Wnt signaling pathway in mice.

BACKGROUND: Frizzled 6 (Fzd6) is involved in the development of various disorders; however, its role in the etiology of depression remains unclear. We aimed to determine the potential regulatory mechanisms of Fzd6 as a Wnt receptor in depression. METHODS: Mice were divided into four groups: wild-type control (Fzd6WT-control), Fzd6 mutant control (Fzd6Q152E-control), wild-type reserpine (Fzd6WT-reserpine), and Fzd6 mutant reserpine (Fzd6Q152E-reserpine). Reserpine (0.5 mg/kg) was injected intraperitoneally for 10 days. Four behavioral experiments were performed to assess the effects of Fzd6Q152E on depression-like behaviors in the reserpine-treated mice. Blood samples were collected for an enzyme-linked immunosorbent assay (ELISA). Gene expression in the hippocampus was quantified using quantitative real-time polymerase chain reaction (qRT-PCR), and protein expression levels in the hippocampus were identified using western blotting. RESULTS: The Fzd6 mutation affected reserpine-induced depression-like behavioral changes in mice. ELISA revealed significantly reduced serum levels of 5-hydroxytryptamine (5-HT), brain-derived neurotrophic factor (BDNF), and norepinephrine in both Fzd6Q152E-reserpine and Fzd6WT-reserpine mice, with a more pronounced decrease in Fzd6Q152E-reserpine mice, especially in norepinephrine expression. The qRT-PCR results showed significantly decreased Fzd6 expression in Fzd6Q152E-reserpine mice and altered expression of Dkk2, Gsk-3ß, Lrp6, Wnt2, Wnt3, and Wnt3a in the Wnt pathway. Western blotting revealed decreased Fzd6 protein expression in Fzd6Q152E-control mice compared to Fzd6WT-control mice, whereas Fzd6 protein expression was restored in Fzd6Q152E-reserpine mice, and Gsk-3ß expression was significantly changed. CONCLUSION: Fzd6 potentially influences reserpine-induced depressive behavioral changes and serum depressive factor alterations and modulates the expression of the Wnt signaling pathway in the hippocampus of depressed mice.

FTO-mediated m6A modification alleviates autoimmune uveitis by regulating microglia phenotypes via the GPC4/TLR4/NF-κB signaling axis.

Uveitis, a vision-threatening inflammatory disease worldwide, is closely related to resident microglia. Retinal microglia are the main immune effector cells with strong plasticity, but their role in uveitis remains unclear. N6-methyladenosine (m6A) modification has been proven to be involved in the immune response. Therefore, we in this work aimed to identify the potentially crucial m6A regulators of microglia in uveitis. Through the single-cell sequencing (scRNA-seq) analysis and experimental verification, we found a significant decrease in the expression of fat mass and obesity-associated protein (FTO) in retinal microglia of uveitis mice and human microglia clone 3 (HMC3) cells with inflammation. Additionally, FTO knockdown was found to aggravate the secretion of inflammatory factors and the mobility/chemotaxis of microglia. Mechanistically, the RNA-seq data and rescue experiments showed that glypican 4 (GPC4) was the target of FTO, which regulated microglial inflammation mediated by the TLR4/NF-κB pathway. Moreover, RNA stability assays indicated that GPC4 upregulation was mainly regulated by the downregulation of the m6A "reader" YTH domain family protein 3 (YTHDF3). Finally, the FTO inhibitor FB23-2 further exacerbated experimental autoimmune uveitis (EAU) inflammation by promoting the GPC4/TLR4/NF-κB signaling axis, and this could be attenuated by the TLR4 inhibitor TAK-242. Collectively, a decreased FTO could facilitate microglial inflammation in EAU, suggesting that the restoration or activation of FTO function may be a potential therapeutic strategy for uveitis.

Increased METTL3 expression and m6A RNA methylation may contribute to the development of dry eye in primary Sjögren's syndrome.

BACKGROUND: Primary Sjögren's syndrome (pSS) is a chronic autoimmune disorder defined by xerostomia and keratoconjunctivitis sicca, and its etiology remains unknown. N6-methyladenosine (m6A) is the predominant posttranscriptional modification in eukaryotic mRNAs and is dynamically regulated by m6A regulators. Dysregulation of m6A modification is closely associated with several autoimmune disorders, but the role of m6A modification in pSS remains unknown. This study investigated the potential role of m6A and m6A-related regulators in pSS patients with dry eye. METHODS: This cross-sectional study included forty-eight pSS patients with dry eye and forty healthy controls (HCs). Peripheral blood mononuclear cells (PBMCs) were isolated, and the level of m6A in total RNA was measured. The expression of m6A regulators was determined utilizing real-time PCR and western blotting. The serological indicators detected included autoantibodies, immunoglobulins (Igs), complement factors (Cs), and inflammatory indicators. Dry eye symptoms and signs were measured, including the ocular surface disease index, Schirmer's test (ST), corneal fluorescein staining score (CFS), and tear break-up time. Spearman's correlation coefficient was employed to assess the associations of m6A and m6A-related regulator expression with clinical characteristics. RESULTS: The expression level of m6A was markedly increased in the PBMCs of pSS patients with dry eye compared to HCs (P value<0.001). The relative mRNA and protein expression levels of the m6A regulators methyltransferase-like 3 (METTL3) and YT521-B homology domains 1 were markedly elevated in pSS patients with dry eye (both P value<0.01). The m6A RNA level was found to be positively related to METTL3 expression in pSS patients (r = 0.793, P value<0.001). Both the m6A RNA level and METTL3 mRNA expression correlated with the anti-SSB antibody, IgG, ST, and CFS (all P values < 0.05). The m6A RNA level was associated with C4 (r = -0.432, P value = 0.002), while METTL3 mRNA expression was associated with C3 (r = -0.313, P value = 0.030). CONCLUSIONS: Our work revealed that the upregulation of m6A and METTL3 was associated with the performance of serological indicators and dry eye signs in pSS patients with dry eye. METTL3 may contribute to the pathogenesis of dry eye related to pSS.

Apigenin Alleviates Autoimmune Uveitis by Inhibiting Microglia M1 Pro-Inflammatory Polarization.

Purpose: Apigenin is a natural small molecule compound widely present in various vegetables and fruits. Recently, Apigenin was reported to inhibit lipopolysaccharide (LPS)-simulated microglial proinflammatory activation. Considering the important role of microglia in retinal disorders, we wonder whether Apigenin could exert a therapeutic effect on experimental autoimmune uveitis (EAU) through reprogramming retinal microglia to a beneficial subtype. Methods: EAU was induced in C57BL/6J mice by immunization with interphotoreceptor retinoid-binding protein (IRBP)651-670, followed by intraperitoneal administration of Apigenin. Disease severity was assessed based on clinical and pathological scores. In vivo, Western blotting was used to quantify protein levels of classical inflammatory factors, microglial M1/M2 markers and the tight junction protein of the blood-retinal-barrier (BRB). Immunofluorescence was used to determine the Apigenin's efficacy on microglial phenotype. In vitro, Apigenin was added in LPS and IFN-γ stimulated human microglial cell line. Western blotting and Transwell assays were used to analyze the phenotype of microglia. Results: In vivo, we found that Apigenin significantly reduced the clinical and pathological scores of EAU. The protein levels of inflammatory cytokines were significantly decreased in retina, and BRB disruption was ameliorated after Apigenin treatment. Meanwhile, Apigenin inhibited microglia M1 transition in EAU mice retina. In vitro functional studies showed that Apigenin decreased LPS and IFN-γ-induced microglial inflammatory factor production and M1-activation via the TLR4/MyD88 pathway. Conclusions: Apigenin can ameliorate retinal inflammation in IRBP induced autoimmune uveitis through inhibiting microglia M1 pro-inflammatory polarization via TLR4/MyD88 pathway.

Structure of the green heme isolated from allylbenzene-modified chloroperoxidase: A novel heme architecture implicating the mechanisms of CPO inactivation and epoxidation.

The chemical identification of the modified heme (the green heme) during chloroperoxidase catalyzed epoxidation of allylbenzene remains unestablished due to its high instability within the protein matrix, the absence of paramagnetically shifted signals, and the difficulty in obtaining crystals of the modified enzyme. We have established the unambiguous structure of the modified prosthetic heme group, which was extracted from the protein matrix using 2D NMR spectroscopy and LC-MS spectrometry. The modified heme was isolated as a µ-oxo dimer that can be quantitatively converted to the corresponding monomer. The depolymerized green heme displayed characteristic NMR signatures of iron porphyrin complexes, but no Nuclear Overhauser Effect was observable to assist in signal assignment. An alternative strategy was applied by removing the iron center of the green heme, resulting in a stable demetallated green porphyrin species. Complete assignment of all the NMR resonances in the demetallated green heme allowed us to establish the molecular architecture of the modified species as a novel N-alkylated heme. Decisive space correlations between the propyl protons of allylbenzene and the γ meso proton coupled with clear dipolar connectivities between the propyl-2H of the substrate and the ß proton in the side chain of the propionic acid at carbon-6 of the porphyrin ring, clearly indicate that allylbenzene was covalently attached to the nitrogen atom of the pyrrole ring III of the prosthetic heme. In this study, the mechanism of green CPO formation and its relation to CPO catalyzed chiral transformations are also discussed. It is concluded that the double-phenyl clamp formed by two phenylalanine residues at the distal heme pocket plays a critical role in fine-tuning substrate orientation that determines the outcome of CPO catalyzed epoxidation of substituted styrenes.

Integrative Single-Cell Transcriptomics and Epigenomics Mapping of the Fetal Retina Developmental Dynamics.

The underlying mechanisms that determine gene expression and chromatin accessibility in retinogenesis are poorly understood. Herein, single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin sequencing are performed on human embryonic eye samples obtained 9-26 weeks after conception to explore the heterogeneity of retinal progenitor cells (RPCs) and neurogenic RPCs. The differentiation trajectory from RPCs to 7 major types of retinal cells are verified. Subsequently, diverse lineage-determining transcription factors are identified and their gene regulatory networks are refined at the transcriptomic and epigenomic levels. Treatment of retinospheres, with the inhibitor of RE1 silencing transcription factor, X5050, induces more neurogenesis with the regular arrangement, and a decrease in Müller glial cells. The signatures of major retinal cells and their correlation with pathogenic genes associated with multiple ocular diseases, including uveitis and age-related macular degeneration are also described. A framework for the integrated exploration of single-cell developmental dynamics of the human primary retina is provided.