Integrative Analysis of miRNA and circRNA Expression Profiles and Interaction Network in HSV-1-Infected Primary Corneal Epithelial Cells.

PURPOSE: Circular RNAs (circRNAs) are products of alternative splicing with roles as competitive endogenous RNAs or microRNA sponges, regulating gene expression and biological processes. However, the involvement of circRNAs in herpes simplex keratitis remains largely unexplored. METHODS: This study examines circRNA and miRNA expression profiles in primary human corneal epithelial cells infected with HSV-1, compared to uninfected controls, using microarray analysis. Bioinformatic analysis predicted the potential function of the dysregulated circRNAs and microRNA response elements (MREs) in these circRNAs, forming an interaction network between dysregulated circRNAs and miRNAs. RESULTS: A total of 332 circRNAs and 16 miRNAs were upregulated, while 80 circRNAs and six miRNAs were downregulated (fold change ≥2.0 and p < 0.05). Gene ontology (GO) and KEGG pathway analyses were performed on parental genes of dysregulated circRNAs to uncover potential functions in HSV-1 infection. Notably, miR-181b-5p, miR-338-3p, miR-635, and miR-222-3p emerged as pivotal miRNAs interacting with multiple dysregulated circRNAs. CONCLUSIONS: This comprehensive study offers insights into differentially expressed circRNAs and miRNAs during HSV-1 infection in corneal epithelial cells, shedding light on circRNA-miRNA interactions' potential role in herpes simplex keratitis pathogenesis.

Accelerating the speed of innovative anti-tumor drugs to first-in-human trials incorporating key de-risk strategies.

Pharmaceutical companies have recently focused on accelerating the timeline for initiating first-in-human (FIH) trials to allow quick assessment of biologic drugs. For example, a stable cell pool can be used to produce materials for the toxicology (Tox) study, reducing time to the clinic by 4-5 months. During the coronavirus disease 2019 (COVID-19) pandemic, the anti-COVID drugs timeline from DNA transfection to the clinical stage was decreased to 6 months using a stable pool to generate a clinical drug substrate (DS) with limited stability, virus clearance, and Tox study package. However, a lean chemistry, manufacturing, and controls (CMC) package raises safety and comparability risks and may leave extra work in the late-stage development and commercialization phase. In addition, whether these accelerated COVID-19 drug development strategies can be applied to non-COVID projects and established as a standard practice in biologics development is uncertain. Here, we present a case study of a novel anti-tumor drug in which application of "fast-to-FIH" approaches in combination with BeiGene's de-risk strategy achieved successful delivery of a complete CMC package within 10 months. A comprehensive comparability study demonstrated that the DS generated from a stable pool and a single-cell-derived master cell bank were highly comparable with regards to process performance, product quality, and potency. This accomplishment can be a blueprint for non-COVID drug programs that approach the pace of drug development during the pandemic, with no adverse impact on the safety, quality, and late-stage development of biologics.

Near infrared II laser controlled free radical releasing nanogenerator for synergistic nitric oxide and alkyl radical therapy of breast cancer.

Recently, alkyl radicals have attracted much attention in cancer therapy due to their oxygen-independent generation property. For the first time, alkyl radical and nitric oxide (NO) combined therapy is demonstrated as an effective strategy for tumor inhibition. As a proof of concept, a biocompatible free radical nanogenerator with near-infrared (NIR) II laser-induced simultaneous NO and alkyl radical release property was elaborately fabricated. In particular, an NIR II molecule (IR 1061), NO donor (BNN6) and alkyl radical initiator (AIPH) were firstly encapsulated in a natural lecithin stabilized phase change material, and then further functionalized by an amphiphile of DSPE-PEG-RGD with specific tumor targeting ability, finally obtaining biocompatible P(IR/BNN6/AIPH)@Lip-RGD. Upon NIR II laser irradiation, the photothermal effect generated from IR 1061 could trigger the phase change of the nanogenerator by releasing the encapsulated BNN6 and AIPH, and subsequently decompose them to generate highly active NO and alkyl radicals. Remarkably, NO and alkyl radical release profiles of P(IR/BNN6/AIPH)@Lip-RGD could be precisely controlled using intermittent NIR II laser irradiation. Moreover, P(IR/BNN6/AIPH)@Lip-RGD displayed a synergistic NO and alkyl radicals' anticancer effect by significantly inhibiting the growth of breast tumors, upon NIR II laser exposure. Furthermore, an in depth mechanistic study revealed that synergistic NO and alkyl radical effect induced cancer cell apoptosis through a mitochondria-mediated apoptotic pathway. The synergistic effect jointly caused a burst generation of mitochondrial ROS, which significantly down-regulated Bcl-2 protein expression, accelerated cytochrome c release and triggered a cascade of apoptosis-related proteins of Caspase-3 and Caspase-9.

Cyanidin-3-O-glucoside Protects Lens Epithelial Cells against High Glucose-Induced Apoptosis and Prevents Cataract Formation via Suppressing NF-κB Activation and Cox-2 Expression.

Diabetic cataract is one of the most important causes of blindness worldwide. Cyanidin-3-O-glucoside (C3G) is found to exert beneficial effects on many diabetic complications. However, its effect on diabetic cataract is not well known. Herein, we investigated the effect of C3G on high glucose-induced lens epithelial cell (SRA01/04) apoptosis and cataract formation as well as the involved mechanisms. We found C3G (20 µM) could preserve cell viability in SRA01/04 cells exposed to high glucose (100 µM). Meanwhile, C3G inhibited SRA01/04 cell apoptosis and regulated the Bcl-2/Bax ratio. Additionally, C3G suppressed NF-κB activation and subsequent cyclooxygenases-2 (Cox-2) expression, which are associated with the protection against apoptosis. Moreover, C3G attenuated lens opacity and protein aggregation in lens culture exposed to high glucose. In conclusion, C3G protected against high glucose-induced SRA01/04 cell apoptosis and cataract formation, which indicated the potential protection of anthocyanins on diabetic cataract.

Posttranscriptional regulation of MMP-9 by HuR contributes to IL-1ß-induced pterygium fibroblast migration and invasion.

Inflammation is considered to be critical in the pterygium progression and recurrence. However, the underlying molecular mechanism is not well understood. Herein, we investigated the potential role of RNA binding protein human antigen R (HuR) responsible for the impact of inflammation on pterygium development. The expression of HuR and matrix metallopeptidase-9 (MMP-9) in pterygium and normal conjunctiva was detected with immunohistochemistry and quantitative reverse transcription polymerase chain reaction (qRT-PCR). The influence of interleukin-1ß (IL-1ß) on HuR expression and cellular distribution was determined with western blot and immunofluorescence. The pterygium fibroblast (PTF) migration was determined with scratch wound healing assay and Transwell migration assay. MMP-9 production was determined with qRT-PCR and gelatin zymography. The interaction between HuR and MMP-9 was investigated with RNP immunoprecipitation (IP) followed by RT-PCR and messenger RNA (mRNA) stability analysis. HuR and MMP-9 expression are elevated in pterygium, especially progressive pterygium compared with normal conjunctiva. IL-1ß could increase the expression and nucleus-cytoplasm shuttle of HuR in cultured PTFs. HuR mediated the stimulatory effect of IL-1ß on PTF migration and MMP-9 production. HuR bound to MMP-9 mRNA and in turn increased it stability. Our results suggest that posttranscriptional regulation of MMP-9 via stabilizing mRNA by HuR might contribute to the stimulatory effect of inflammatory factor IL-1ß on pterygium progression. These findings shed light on the pathogenesis of pterygium and provide a promising target for adjuvant treatment of pterygium.

Airborne particulate matter impairs corneal epithelial cells migration via disturbing FAK/RhoA signaling pathway and cytoskeleton organization.

BACKGROUND: Cornea is the outmost structure of the eye and exposed directly to the air pollution. However, little is known about the effect of PM2.5 on corneal epithelium, which is critical for maintenance of cornea homeostasis and visual function. OBJECTIVE: We investigated the influence of PM2.5 exposure on corneal epithelial migration and the possible mechanisms involved in the process. METHODS: We observed wound healing in mouse model of cornea abrasion, evaluated the migration and mobility of cultured corneal epithelial cells with wound scratch assay and Transwell migration assay, detected the phosphorylation and interaction of FAK/paxillin with immunofluorescence and immunoprecipitation, and determined the RhoA activity and actin reorganization, in response to PM2.5 exposure. RESULTS: Exposure to PM2.5 remarkably inhibited corneal epithelial cell migration both in mouse model of corneal abrasion and in cell culture model. We found the phosphorylation and interaction of FAK/paxillin, RhoA activity as well as actin reorganization were suppressed by PM2.5 exposure. Moreover, formation of ROS might play a role in the action of PM2.5. CONCLUSIONS: PM2.5 exposure could result in delay of corneal epithelium wound healing by inhibiting cell migration, thus more attention should be paid to the potential risk of corneal infection and effort should be made to protect eyes against impairment induced by PM2.5.

Aberrant expression of genes and proteins in pterygium and their implications in the pathogenesis.

Pterygium is a common ocular surface disease induced by a variety of factors. The exact pathogenesis of pterygium remains unclear. Numbers of genes and proteins are discovered in pterygium and they function differently in the occurrence and development of this disease. We searched the Web of Science and PubMed throughout history for literatures about the subject. The keywords we used contain pterygium, gene, protein, angiogenesis, fibrosis, proliferation, inflammation, pathogenesis and therapy. In this review, we summarize the aberrant expression of a range of genes and proteins in pterygium compared with normal conjunctiva or cornea, including growth factors, matrix metalloproteinases and tissue inhibitors of metalloproteinases, interleukins, tumor suppressor genes, proliferation related proteins, apoptosis related proteins, cell adhesion molecules, extracellular matrix proteins, heat shock proteins and tight junction proteins. We illustrate their possible mechanisms in the pathogenesis of pterygium as well as the related intervention based on them for pterygium therapy.

Assessment of DNA Damage and Cell Senescence in Corneal Epithelial Cells Exposed to Airborne Particulate Matter (PM2.5) Collected in Guangzhou, China.

PURPOSE: To assess the genotoxic effect of airborne particulate matter on corneal epithelial cells and investigate the role of reactive oxygen species (ROS) formation in this process. METHODS: Immortalized human corneal epithelial cells (HCECs) and primary bovine corneal epithelial cells were exposed to airborne particulate matter collected from Guangzhou for 24 hours. The cell viability and toxicity were measured by the CCK-8 test and lactate dehydrogenase (LDH) release, respectively. The DNA breaks and DNA repair were examined by alkaline comet assay and by immunofluorescence staining of the phosphorylated histone variant H2AX (γH2AX), respectively. Reactive oxygen species production was assessed by the fluorescent probe, CM-H2DCFDA. Cell senescence was evaluated with senescence-associated ß-Galactosidase staining, and cell ultrastructure was observed with transmission electron microscopy. RESULTS: Exposure to PM2.5 at the concentration of 20 µg/mL to 200 µg/mL decreased cell viability and increased LDH release. Remarkably increased DNA double-stand breaks, increased expression of DNA repair-related protein γH2AX, elevated ROS formation, and altered cell ultrastructure were observed in HCECs after treatment with PM2.5. The genotoxic effect of PM2.5 was attenuated by the ROS inhibitor N-acetyl-l-cysteine (NAC). CONCLUSIONS: Particulate matter 2.5 could induce DNA damage and cell senescence in corneal epithelial cells, probably by promoting ROS formation. Thus, whether long-term exposure of PM2.5 might be related to potential risk of abnormality in corneal epithelium renewal and regeneration should be further investigated.

Fast gas chromatography analysis of N-carbamates with cold on-column injection.

Direct gas chromatographic (GC) analysis of thermally labile N-carbamates is studied by using fast GC with cold on-column injection. With the greatly reduced injection temperature, short column lengths, high flow rate, and fast temperature-programming rate, the exposure of carbamates to high temperature is reduced and degradation can be avoided. Nine N-carbamates in EPA Method 531 are eluted without thermal decomposition. The relative standard deviation percentage for peak areas average 1.9% for all of the carbamates analyzed. A conventional GC instrument is employed to simplify the experiments. GC-mass spectrometry is used to monitor the decomposition peaks.

Fast gas chromatography analysis of N-carbamates with cold on-column injection.

Direct gas chromatographic (GC) analysis of thermally labile N-carbamates is studied by using fast GC with cold on-column injection. With the greatly reduced injection temperature, short column lengths, high flow rate, and fast temperature-programming rate, the exposure of carbamates to high temperature is reduced and degradation can be avoided. Nine N-carbamates in EPA Method 531 are eluted without thermal decomposition. The relative standard deviation percentage for peak areas average 1.9% for all of the carbamates analyzed. A conventional GC instrument is employed to simplify the experiments. GC-mass spectrometry is used to monitor the decomposition peaks.