The RNA N6-methyladenosine demethylase FTO regulates ATG5 to inhibit malignant progression of uveal melanoma.

PURPOSE: This research aimed to identify the function of fat mass- and obesity-associated protein (FTO), an eraser of N6-methyladenosine (m6A), and explore its possible mechanisms in uveal melanoma (UVM). METHODS: We performed quantitative real-time PCR (qPCR), Western blotting and gene correlation analysis with GEPIA2 to assess FTO expression and identify its potential targets in UVM. CCK-8, colony formation, cell cycle, cell apoptosis, wound healing and Transwell invasion assays were utilized to assess cell viability, cell cycle distribution, apoptosis, migration and invasion. Western blotting, qPCR and methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) were carried out to explore the underlying mechanism of FTO in 2 UVM cell lines. RESULTS: FTO, a key m6A demethylase, was found to be upregulated in human UVM tissues compared with normal choroid tissues. Knockdown of FTO in Mel270 and OMM2.3 cells significantly promoted proliferation and migration and suppressed apoptosis. Mechanistically, knockdown of FTO decreased the expression of ATG5, an autophagy-related gene, leading to attenuation of autophagosome formation, thereby inhibiting autophagy. Upon FTO knockdown, increased levels of methylated ATG5 and decreased ATG5 stability were detected. Furthermore, ATG5 dramatically alleviated FTO downregulation-induced tumor growth and metastasis. CONCLUSIONS: Our research highlights the importance of the m6A demethylase FTO in UVM by demonstrating that it direct regulates ATG5-induced autophagy in an m6A-dependent manner. These findings suggest that FTO may serve as a potential therapeutic target for UVM.

Hydromassage of macular hole edges for large and persistent full-thickness macular holes.

AIM: To introduce the macular hole (MH) hydromassage technique as a potentially beneficial approach for the treatment of large or persistent MH. METHODS: This retrospective observational case series comprised 16 consecutive patients (17 eyes) diagnosed with MH. Inclusion criteria involved a hole aperture diameter larger than 600 µm or the presence of an unclosed MH larger than 600 µm following the previous vitrectomy. Standard MH repair procedures were administered in all cases, involving the manipulation and aspiration of the hole margin through the application of water flow with a soft-tip flute needle. A comprehensive assessment was conducted for each case before and after surgery, and optical coherence tomography (OCT) images were captured at every follow-up point. RESULTS: The mean preoperative aperture diameter was 747±156 µm (range 611-1180 µm), with a mean base diameter of 1390±435 µm (range 578-2220 µm). Following surgery, all cases achieved complete anatomical closure of MH, with 13 cases (76.5%) exhibiting type 1 closure and 4 cases (23.5%) demonstrating type 2 closure. No significant differences were observed in the preoperative OCT variables between the two closure types. Eyes with type 1 closure showed a significantly improved visual acuity (0.70±0.10, range 0.50-0.80) compared to those with type 2 closure (0.90±0.12, range 0.80-1.00, P=0.014). CONCLUSION: The MH hydromassage technique demonstrates promising results, achieving acceptable closure rates in cases of large or persistent MH. This technique may serve as an effective adjunctive maneuver during challenging MH surgery.

Effect of High Energy Low Protein Diet on Lipid Metabolism and Inflammation in the Liver and Abdominal Adipose Tissue of Laying Hens.

The aim of this study was to evaluate the effects of a high-energy low-protein (HELP) diet on lipid metabolism and inflammation in the liver and abdominal adipose tissue (AAT) of laying hens. A total of 200 Roman laying hens (120 days old) were randomly divided into two experimental groups: negative control group (NC group) and HELP group, with 100 hens per group. The chickens in the NC group were fed with a basic diet, whereas those in the HELP group were given a HELP diet. Blood, liver, and AAT samples were collected from 20 chickens per group at each experimental time point (30, 60, and 90 d). The morphological and histological changes in the liver and AAT were observed, and the level of serum biochemical indicators and the relative expression abundance of key related genes were determined. The results showed that on day 90, the chickens in the HELP group developed hepatic steatosis and inflammation. However, the diameter of the adipocytes of AAT in the HELP group was significantly larger than that of the NC group. Furthermore, the results showed that the extension of the feeding time significantly increased the lipid contents, lipid deposition, inflammatory parameters, and peroxide levels in the HELP group compared with the NC group, whereas the antioxidant parameters decreased significantly. The mRNA expression levels of genes related to lipid synthesis such as fatty acid synthase (FASN), stearoyl-coA desaturase (SCD), fatty acid binding protein 4 (FABP4), and peroxisome proliferator-activated receptor gamma (PPARγ) increased significantly in the liver and AAT of the HELP group, whereas genes related to lipid catabolism decreased significantly in the liver. In addition, the expression of genes related to lipid transport and adipokine synthesis decreased significantly in the AAT, whereas in the HELP group, the expression levels of pro-inflammatory parameters such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1ß) increased significantly in the liver and AAT. Conversely, the expression level of the anti-inflammatory parameter interleukin-10 (IL-10) decreased significantly in the liver. The results indicated that the HELP diet induced lipid peroxidation and inflammation in the liver and AAT of the laying hens. Hence, these results suggest that chicken AAT may be involved in the development of fatty liver.

High expression of miR-22-3p in chicken hierarchical follicles promotes granulosa cell proliferation, steroidogenesis, and lipid metabolism via PTEN/PI3K/Akt/mTOR signaling pathway.

MicroRNAs (miRNAs) are a class of RNA macromolecules that play regulatory roles in follicle development by inhibiting protein translation through binding to the 3'UTR of its target genes. Granulosa cell (GC) proliferation, steroidogenesis, and lipid metabolism have indispensable effect during folliculogenesis. In this study, we found that miR-22-3p was highly expressed in the hierarchical follicles of the chickens, which indicated that it may be involved in follicle development. The results obtained suggested that miR-22-3p promoted proliferation, hormone secretion (progesterone and estrogen), and the content of lipid droplets (LDs) in the chicken primary GC. The results from the bioinformatics analysis, luciferase reporter assay, qRT-PCR, and Western blotting, confirmed that PTEN was directly targeted to miR-22-3p. Subsequently, it was revealed that PTEN inhibited proliferation, hormone secretion, and the content of LDs in GC. Therefore, this study showed that miR-22-3p could activate PI3K/Akt/mTOR pathway via targeting PTEN. Taken together, the findings from this study indicated that miR-22-3p was highly expressed in the hierarchical follicles of chickens, which promotes GC proliferation, steroidogenesis, and lipid metabolism by repressing PTEN to activate PI3K/AKT/mTOR pathway.

miR-128-3p regulates chicken granulosa cell function via 14-3-3ß/FoxO and PPAR-γ/LPL signaling pathways.

MicroRNAs (miRNAs) are class of 22 nt short RNA sequences which inhibit protein translation through binding to the 3'UTR of its target genes. The continuous ovulatory property of chicken follicle makes it a perfect model for studying granulosa cell (GC) functions. In this study, we found that large number of miRNAs including miR-128-3p, were differentially expressed in the GCs of F1 and F5 follicles of chicken. Subsequently, the results revealed that miR-128-3p inhibited proliferation, the formation of lipid droplets, and hormone secretion in chicken primary GCs through directly targeting YWHAB and PPAR-γ genes. To determine the effects of 14-3-3ß (encoded by YWHAB) protein on GCs functions, we overexpressed or inhibited the expression of YWHAB, and the results showed that YWHAB inhibited the function of FoxO proteins. Collectively, we found that miR-128-3p was highly expressed in the chicken F1 follicles compared to the F5 follicles. In addition, the results indicated that miR-128-3p promoted GC apoptosis through 14-3-3ß/FoxO pathway via repressing YWHAB, and inhibited lipid synthesis by impeding the PPAR-γ/LPL pathway, as well as reduced the secretion of progesterone and estrogen. Taken together, the results showed that miR-128-3p plays a regulatory role in chicken granulosa cell function via 14-3-3ß/FoxO and PPAR-γ/LPL signaling pathways.

Dietary supplementation of salidroside alleviates liver lipid metabolism disorder and inflammatory response to promote hepatocyte regeneration via PI3K/AKT/Gsk3-ß pathway.

Fatty liver hemorrhagic syndrome (FLHS) is a chronic hepatic disease which occurs when there is a disorder in lipid metabolism. FLHS is often observed in caged laying hens and characterized by a decrease in egg production and dramatic increase of mortality. Salidroside (SDS) is an herbal drug which has shown numerous pharmacological activities, such as protecting mitochondrial function, attenuating cell apoptosis and inflammation, and promoting antioxidant defense system. We aimed to determine the therapeutic effects of SDS on FLHS in laying hens and investigate the underlying mechanisms through which SDS operates these functions. We constructed oleic acid (OA)-induced fatty liver model in vitro and high-fat diet-induced FLHS of laying hens in vivo. The results indicated that SDS inhibited OA-induced lipid accumulation in chicken primary hepatocytes, increased hepatocyte activity, elevated the mRNA expression of proliferation related genes PCNA, CDK2, and cyclinD1 and increased the protein levels of PCNA and CDK2 (P < 0.05), as well as decreased the cleavage levels of Caspase-9, Caspase-8, and Caspase-3 and apoptosis in hepatocytes (P < 0.05). Moreover, SDS promoted the phosphorylation levels of PDK1, AKT, and Gsk3-ß, while inhibited the PI3K inhibitor (P < 0.05). Additionally, we found that high-fat diet-induced FLHS hens had heavier body weight, liver weight, and abdominal fat weight, and severe steatosis in histology, compared with the control group (Con). However, hens fed with SDS maintained lighter body weight, liver weight, and abdominal fat weight, as well as normal liver without hepatic steatosis. In addition, high-fat diet-induced FLHS hens had high levels of serum total cholesterol (TC), triglyceride (TG), alanine transaminase (ALT), and aspartate aminotransferase (AST) compared to the Con group, however, in the Model+SDS group, the levels of TC, TG, ALT, and AST decreased significantly, whereas the level of superoxide dismutase (SOD) increased significantly (P < 0.05). We also found that SDS significantly decreased the mRNA expression abundance of PPARγ, SCD, and FAS in the liver, as well as increased levels of PPARα and MTTP, and decreased the mRNA expression of TNF-α, IL-1ß, IL-6, and IL-8 in the Model+SDS group (P < 0.05). In summary, this study showed that 0.3 mg/mL SDS attenuated ROS generation, inhibited lipid accumulation and hepatocyte apoptosis, and promoted hepatocyte proliferation by targeting the PI3K/AKT/Gsk3-ß pathway in OA-induced fatty liver model in vitro, and 20 mg/kg SDS alleviated high-fat-diet-induced hepatic steatosis, oxidative stress, and inflammatory response in laying hens in vivo.

Dietary quercetin and vitamin E supplementation modulates the reproductive performance and antioxidant capacity of aged male breeder chickens.

Aged male chickens experience rapid declines in spermatogenesis, antioxidant capacity, immunity, and hormone synthesis. Vitamin E plays a significant role in reproduction, nervous system function, and disease resistance in animals. Quercetin also exerts many biological effects, such as antioxidant ability, immunostimulation, and protection of spermatozoal plasma membranes. This study evaluated the effects of combining dietary quercetin (Q) and vitamin E (VE) on sperm quality, antioxidant capacity, immunity, and expression of genes related to spermatogenesis, immunity, apoptosis, and inflammation in aged male chickens. A total of 120 Tianfu breeder male chickens (65 wk old) were randomly allotted to 4 treatments with 3 replicates (10 birds each). The birds were fed diets containing Q (0.4g/kg), VE (0.2g/kg), Q+VE (0.4g/kg + 0.2g/kg), and a basal diet for 11 wk. At the end of the experimental period, blood, semen, liver, testes, and spleen samples were collected from 2 birds per replicate. Serum hormones, antioxidant parameters, cytokines, and immunoglobulins were evaluated; and the mRNA expression of genes related to spermatogenesis, apoptosis, and inflammation are determined in the testes and liver tissues. The results showed that the combination quercetin and vitamin E significantly promoted the sperm count and motility, as well as elevated the levels of testosterone, follicle-stimulating hormone, and luteinizing hormone, antioxidant enzymes (Superoxide dismutase, Glutathione, and Total antioxidant capacity), and serum immunoglobulins (IgA and IgM) in the aged male chickens; also Q+VE showed protective effects on the liver against injury. In addition, Q+VE significantly increased the expression of genes related to spermatogenesis (AR, pgk2, Cyclin A1, and Cyclin A2), immunity (IFN-γ and IL-2), and anti-inflammatory cytokines (IL-10) (P < 0.05), whereas the expression of proinflammatory cytokines (IL-1ß and IL-6) was decreased (P < 0.05). Taken together, these data indicate that the combination of quercetin and vitamin E improved reproductive characteristics such as spermatogenesis, sperm quality, and hormone regulation, as well as promoted antioxidant defense, hepatoprotective capacity, and immune response in aged male chickens without any detrimental effects.

Systemic conbercept pharmacokinetics and VEGF pharmacodynamics following intravitreal injections of conbercept in patients with retinopathy of prematurity.

BACKGROUND: Data on serum vascular endothelial growth factor (VEGF) and drug levels in patients with retinopathy of prematurity (ROP) following intravitreal injections of conbercept (IVC) are lacking. METHODS: Multicentre, prospective, non-randomised study of patients with aggressive posterior retinopathy of prematurity (APROP) or type 1 ROP who had not received other treatment. All infants received therapy in both eyes plus intravitreal IVC 0.25 mg/0.025 mL in one eye and had at least 6 months of follow-up. Blood samples were collected before and 1 week and 4 weeks after IVC. The main outcome measures were serum conbercept and VEGF concentrations. RESULTS: Forty infants with APROP or type 1 ROP were enrolled. The mean serum VEGF at baseline and 1 week and 4 weeks after a total of 0.25 mg of IVC was 953.35±311.90 pg/mL, 303.46±181.89 pg/mL and 883.12±303.89 pg/mL, respectively. Serum VEGF 1 week after IVC was significantly lower (p<0.05) than baseline, and at 4 weeks after IVC, it was significantly higher (p<0.05) than at 1 week. There was no significant difference (p>0.05) between baseline and 4 weeks. Serum conbercept was below the limit of quantitation (BLOQ) at baseline and 4 weeks and was 19.81±7.60 ng/mL at 1 week. CONCLUSION: Serum VEGF 1 week after IVC was significantly lower than baseline but returned to baseline at 4 weeks. Serum conbercept increased at 1 week and was BLOQ at 4 weeks.

Exosomal miR-487a derived from m2 macrophage promotes the progression of gastric cancer.

Tumor-associated macrophages contribute to cell growth, development, and metastasis in various cancers. However, the underlying mechanisms of M2 macrophage that modulate the progression of gastric cancer (GC) remain largely unknown. In this study, we detected the ratio of macrophages in GC tissues and found that the proportion of M2 macrophages was increased in GC tissues. We then co-cultured GC cells with M1 and M2 macrophages, respectively, and then assessed cell proliferation and tumorigenicity of GC cells by MTT and colony formation assay. The results indicated that M2 macrophages promoted the proliferation of GC cells, but M1 not. Besides, GW4869, an exosomes inhibitor, reduced the effects induced by M2 macrophage. Then, we isolated and identified exosomes derived from M1 and M2 macrophage, and confirmed that the exosomes could be taken up by GC cells. We demonstrated that M2 macrophage-exosomes could induce the proliferation and tumorigenesis in vitro and in vivo. Moreover, miR-487a was enriched in M2 macrophage-exosomes and further determined that miR-487a exert the functions by targeting TIA1. In conclusion, exosomal miR-487a derived from M2 macrophage promotes the proliferation and tumorigenesis in gastric cancer, and the novel findings might be helpful to the development of novel diagnostic and therapeutic methods in GC.

TOMM40 rs2075650 polymorphism shows no association with neovascular age-related macular degeneration or polypoidal choroidal vasculopathy in a Chinese population.

PURPOSE: Age-related macular degeneration (AMD) and Alzheimer disease (AD) are age-related neurodegenerative diseases that share similar environmental risk factors, cellular pathologies, and genetic backgrounds. Recently, the rs2075650 single nucleotide polymorphism in the translocase of outer mitochondrial membrane 40 homolog (TOMM40) gene was identified as a risk factor for AMD and Alzheimer disease. We aimed to examine the associations between the TOMM40 rs2075650 polymorphism and neovascular age-related macular degeneration (nAMD) and polypoidal choroidal vasculopathy (PCV) in a Chinese population. METHODS: The study consisted of 900 subjects, including 300 controls, 300 cases with nAMD, and 300 cases with PCV. Genomic DNA was extracted from venous blood leukocytes. The allelic variant of rs2075650 was determined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Differences in the observed genotypic distributions between the case and control groups were tested using chi-square tests, with age and gender adjusted using logistic regression analysis. RESULTS: The TOMM40 rs2075650 polymorphism was not statistically significantly associated with the nAMD or PCV phenotype (p>0.05). The difference remained insignificant after correction for age and gender differences based on the logistic regression models (p>0.05). CONCLUSIONS: Our data provide no evidence to support an association of rs2075650 in TOMM40 with nAMD or PCV, suggesting that this gene is unlikely to be a major AMD and PCV susceptibility gene locus in the Chinese population.