Longitudinal Changes in Choroidal Thickness Varied With Refractive Progression in Myopic and Non-Myopic Children: A Two-Year Cohort Study.

Purpose: To evaluate the longitudinal changes in subfoveal choroidal thickness (SFCT) in children with different refractive status. Methods: A total of 2290 children 3 to 14 years old who attended the first year of kindergarten (G0), first year of primary school (G1), fourth year of primary school (G4), or first year of junior high school (G7) in Guangzhou, China, were recruited and followed up for 2 years. All participants received cycloplegic autorefraction, axial length measurement and SFCT measurement using a CIRRUS HD-OCT device. Children were divided into groups of persistent non-myopia (PNM), persistent myopia (PM), or newly developed myopia (NDM). Children in the PNM and PM groups were further divided into subgroups of stable refraction (absolute mean annual spherical equivalent refraction [SER] change < 0.5 D) and refractive progression (absolute mean annual SER change ≥ 0.5 D). Results: The mean ± SD ages for the G1 to G7 cohorts were 3.89 ± 0.30, 6.79 ± 0.47, 9.71 ± 0.34, and 12.54 ± 0.38, years, respectively. SFCT consistently decreased in the NDM group across the G1 to G7 cohorts (all P < 0.001) and exhibited variability across different age cohorts in the PNM and PM groups. Further subgroup analysis revealed significant thickening of SFCT in the PNM-stable group among the G0, G1, and G7 cohorts (all P < 0.05), whereas it remained stable among all cohorts in the PM-stable group (all P > 0.05). Conversely, SFCT exhibited thinning in the G4 and G7 cohorts in the PM-progressive group (both P < 0.01) and for the entire cohort of children in the PNM-progressive group (P = 0.012). Conclusions: SFCT increased in nonmyopic children with stable refraction, remained stable in myopic children maintained stable refraction, and decreased in those with refractive progression, whether they were myopic or not.

Ocular trauma from Lawn mower accidents: Clinical insights, visual outcomes and microbial profiles.

AIMS: To characterize the clinical features of ocular trauma resulting from lawn mower, identify determinants of unfavorable final visual acuity (FVA), and assess the spectrum of microbial in posttraumatic endophthalmitis. METHODS: This retrospective study enrolled patients who experienced ocular trauma due to lawn mower at Zhongshan Ophthalmic Center from January 2013 to August 2021. Demographics, clinical features, isolated microorganisms, risk factors influencing reduced visual acuity, treatment regimens, and utilization of eyewear were collected. RESULTS: The study included 140 participants (140 eyes) (49.47 ± 12.03 years, 95% male). The predominant injury manifestations were penetrating globe injuries (75.7%) and intraocular foreign bodies (51.4%). Endophthalmitis occurred in 35 cases (25%) and Bacillus cereus (23.5%) was the primary pathogen, followed by Staphylococcus epidermidis (11.8%) and Streptococcus species (11.8%). Following the initial assessment, where 77.9% of patients had initial visual acuity (IVA) at grade IV (ranging from light perception to 4/200) and only 0.7% attained grade I (better than 20/40), post-treatment results revealed that 5.7% achieved FVA at grade I, with a concurrent decrease in patients with grade IV vision to 64.3%. Multivariate logistic regression revealed that injury protection (p < 0.001, OR = 0.237, 95% CI = 0.126-0.446), IVA (p = 0.001, OR = 4.102, 95% CI = 1.730-9.729), and retinal detachment (p = 0.042, OR = 8.105, 95% CI = 1.075-61.111) were significant independent risk factors impacting FVA. CONCLUSION: Lawn mower often cause severe ocular injuries, with high-velocity metal foreign bodies that can lead to infections, most commonly caused by Bacillus cereus. Correct use of protective gear, initial vision assessment, and detecting retinal detachment are crucial for visual prognosis.

Effect of Kaempferol on the Biological Behavior of Human Colon Cancer via Regulating MMP1, MMP2, and MMP9.

Kaempferol is a kind of flavonoid, which has a significant anticancer effect. MMPs were discovered with the function of cleaving the extracellular matrix. We utilized bioinformatics to analyze the association and bonding mode between the traditional Chinese medicine (TCM) monomer composition (i.e., kaempferol) and the target proteins. The purpose of our research was to verify the effect of kaempferol on the biological behavior of human colon cancer cells HCT116 and HT29 and the expression of matrix metalloproteinase (MMP) 1, 2, and 9 genes. We detected the changes in the biological behavior of colon cancer cells treated with kaempferol by CCK-8, wound healing assay, transwell migration/invasion assay, and flow cytometry. Meanwhile， we detected the expression difference of the target gene by qRT-PCR and western blot. Compared with the two control groups, the cell viability of the kaempferol group decreased, the rate of cell migration and the number of transmembrane cells in the kaempferol group decreased significantly, and the early apoptosis rate increased, the number of cells in the G1 phase increased and in the S phase decreased. The results of qRT-PCR and western blot showed that the expression of target genes MMP1, 2, and 9 in the kaempferol group was lower than that in the two control groups. Kaempferol can significantly inhibit the proliferation, invasion, and migration ability of colon cancer cells; induce their apoptosis; and block the cell cycle. Meanwhile, the expression of MMP1, 2, and 9 genes was downregulated, which verified the results of bioinformatic analysis.

Simple and rapid determination of homozygous transgenic mice via in vivo fluorescence imaging.

Setting up breeding programs for transgenic mouse strains require to distinguish homozygous from the heterozygous transgenic animals. The combinational use of the fluorescence reporter transgene and small animal in-vivo imaging system might allow us to rapidly and visually determine the transgenic mice homozygous for transgene(s) by the in vivo fluorescence imaging. RLG, RCLG or Rm17LG transgenic mice ubiquitously express red fluorescent protein (RFP). To identify homozygous RLG transgenic mice, whole-body fluorescence imaging for all of newborn F2-generation littermates produced by mating of RFP-positive heterozygous transgenic mice (F1-generation) derived from the same transgenic founder was performed. Subsequently, the immediate data analysis of the in vivo fluorescence imaging was carried out, which greatly facilitated us to rapidly and readily distinguish RLG transgenic individual(s) with strong fluorescence from the rest of F2-generation littermates, followed by further determining this/these RLG individual(s) showing strong fluorescence to be homozygous, as strongly confirmed by mouse mating. Additionally, homozygous RCLG or Rm17LG transgenic mice were also rapidly and precisely distinguished by the above-mentioned optical approach. This approach allowed us within the shortest time period to obtain 10, 8 and 2 transgenic mice homozygous for RLG, RCLG and Rm17LG transgene, respectively, as verified by mouse mating, indicating the practicality and reliability of this optical method. Taken together, our findings fully demonstrate that the in vivo fluorescence imaging offers a visual, rapid and reliable alternative method to the traditional approaches (i.e., mouse mating and real-time quantitative PCR) in identifying homozygous transgenic mice harboring fluorescence reporter transgene under the control of a ubiquitous promoter in the situation mentioned in this study.

Plasma arginine metabolites reflect airway dysfunction in a murine model of allergic airway inflammation.

L-arginine metabolism is important in the maintenance of airway tone. Shift of metabolism from the nitric oxide synthase to arginase pathways contributes to the increased airway responsiveness in asthma. We tested the hypothesis that systemic levels of L-arginine metabolites are biomarkers reflective of airway dysfunction. We used a mouse model of acute allergic airway inflammation to OVA that manifests with significant airway hyperresponsiveness to methacholine. To determine tissue arginase activity in vivo, the isotopic enrichment of an infused L-arginine stable isotope and its product amino acid L-ornithine were measured in lung and airway homogenates using liquid chromatography-tandem mass spectrometry. Tissue and plasma concentrations of other L-arginine metabolites, including L-citrulline and symmetric and asymmetric dimethylarginine, were measured and correlated with lung arginase activity and methacholine responsiveness of the airways. The effectiveness of intratracheal instillation of an arginase inhibitor (boronoethylcysteine) on pulmonary arginase activity and circulating concentrations of L-arginine metabolites was also studied. We demonstrate that 1) plasma indexes of L-arginine bioavailability and impairment of nitric oxide synthase function correlate with airway responsiveness to methacholine; 2) plasma levels of L-ornithine predict in vivo pulmonary arginase activity and airway function; and 3) acute arginase inhibition reduces in vivo pulmonary arginase activity to control levels and normalizes plasma L-ornithine, but not L-arginine, bioavailability in this model. We conclude that plasma L-ornithine may be useful as a systemic biomarker to predict responses to therapeutic interventions targeting airway arginase in asthma.

Multitracer stable isotope quantification of arginase and nitric oxide synthase activity in a mouse model of pseudomonas lung infection.

Cystic fibrosis airways are deficient for L-arginine, a substrate for nitric oxide synthases (NOSs) and arginases. The rationale for this study was to quantify NOS and arginase activity in the mouse lung. Anesthetized unventilated mice received a primed constant stable isotope intravenous infusion containing labeled L-arginine, ornithine, and citrulline. The isotopic enrichment of each of the infused isotopomers and its product amino acids were measured in plasma and organ homogenates using liquid chromatography-tandem mass spectrometry. The effect of infection was studied three days after direct tracheal instillation of Pseudomonas-coated agar beads. In the infusion model, lung infection resulted in a significant (28-fold) increase in NOS activity in lung but not in trachea, kidney, liver, or plasma. Absolute rates of arginase activity in solid tissues could not be calculated in this model. In an isolated lung perfusion model used for comparison increased NOS activity in infected lungs was confirmed (28.5-fold) and lung arginase activity was increased 9.7-fold. The activity of L-arginine metabolizing enzymes can be measured using stable isotope conversion in the mouse. Accumulation of L-ornithine in the whole mouse model hindered the exact quantification of arginase activity in the lung, a problem that was overcome utilizing an isolated lung perfusion model.

Lung arginase expression and activity is increased in cystic fibrosis mouse models.

The activity of arginase is increased in airway secretions of patients with cystic fibrosis (CF). Downstream products of arginase activity may contribute to CF lung disease. We hypothesized that pulmonary arginase expression and activity would be increased in mouse models of CF and disproportionally increased in CF mice with Pseudomonas aeruginosa pneumonia. Expression of arginase isoforms in lung tissue was quantified with reverse transcriptase-PCR in naive cystic fibrosis transmembrane conductance regulator (Cftr)-deficient mice and ß-epithelial sodium channel-overexpressing [ß-ENaC-transgenic (Tg)] mice. An isolated lung stable isotope perfusion model was used to measure arginase activity in Cftr-deficient mice before and after intratracheal instillation of Pseudomonas aeruginosa. The expression of arginase-2 in lung was increased in adult Cftr-deficient animals and in newborn ß-ENaC-Tg. Arginase-1 lung expression was normal in Cftr-deficient and in newborn ß-ENaC-Tg mice, but was increased in ß-ENaC-Tg mice at age 1, 3, and 6 wk. Arginase activity was significantly higher in lung (5.0 ± 0.7 vs. 3.2 ± 0.3 nmol·(-1)·h(-1), P = 0.016) and airways (204.6 ± 49.8 vs. 79.3 ± 17.2 nmol·(-1)·h(-1), P = 0.045) of naive Cftr-deficient mice compared with sex-matched wild-type littermate controls. Infection with Pseudomonas aeruginosa resulted in a far greater increase in lung arginase activity in Cftr-deficient mice (10-fold) than in wild-type controls (6-fold) (P = 0.01). This is the first ex vivo characterization of arginase expression and activity in CF mouse lung and airways. Our data show that pulmonary arginase expression and activity is increased in CF mice, especially with Pseudomonas aeruginosa infections.

Effect of arginase inhibition on pulmonary L-arginine metabolism in murine Pseudomonas pneumonia.

RATIONALE: Infection of the lung with Pseudomonas aeruginosa results in upregulation of nitric oxide synthases (NOS) and arginase expression, and both enzymes compete for L-arginine as substrate. Nitric oxide (NO) production may be regulated by arginase as it controls L-arginine availability for NOS. We here studied the effect of systemic arginase inhibition on pulmonary L-arginine metabolism in Pseudomonas pneumonia in the mouse. METHODS: Mice (C57BL/6, 8-10 weeks old, female) underwent direct tracheal instillation of Pseudomonas (PAO-1)-coated agar beads and were treated by repeated intra-peritoneal injections of the arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) or PBS until lungs were harvested on day 3 of the infection. L-arginine metabolites were quantified using liquid chromatography-tandem mass spectrometry, NO metabolites nitrate and nitrite by Griess reagent and cytokines by ELISA. RESULTS: NO metabolite concentrations (48.5±2.9 vs. 10.9±2.3 µM, p<0.0001), as well as L-ornithine (29.6±1.7 vs 2.3±0.4 µM, p<0.0001), the product of arginase activity, were increased in Pseudomonas infected lungs compared to naïve controls. Concentrations of the NOS inhibitor asymmetric dimethylarginine (ADMA) were also increased (0.44±0.02 vs. 0.16±0.01 µM, p<0.0001). Arginase inhibition in the infected animals resulted in a significant decrease in L-ornithine (14.6±1.6 µM, p<0.0001) but increase in L-arginine concentration (p<0.001), L-arginine/ADMA ratio (p<0.001), L-arginine availability for NOS (p<0.001), and NO metabolite concentrations (67.3±5.7 µM, p<0.05). Arginase inhibitor treatment also resulted in an increase in NO metabolite levels in animals following intratracheal injection of LPS (p = 0.015). Arginase inhibition was not associated with an increase in inflammatory markers (IFN-γ, IL-1ß, IL-6, MIP-2, KC or TNF-α) in lung. Concentrations of the L-ornithine-dependent polyamines putrescine, spermidine and spermine were increased in Pseudomonas infected lungs (p<0.001, respectively) but were unaffected by ABH treatment. CONCLUSIONS: Systemic arginase inhibition with ABH during Pseudomonas pneumonia in mice results in an increase in pulmonary NO formation but no pro-inflammatory effect.

Asymmetric dimethylarginine is increased in asthma.

RATIONALE: Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthase (NOS) inhibitor that competes with L-arginine for binding to NOS. It has been suggested that ADMA contributes to inflammation, collagen deposition, nitrosative stress, and lung function in murine models. OBJECTIVES: To test the hypothesis that ADMA is increased in asthma and that NOS inhibition by ADMA contributes to airways obstruction. METHODS: We assessed alterations of L-arginine, ADMA, and symmetric dimethylarginine (SDMA) levels in a murine model of allergic airways inflammation using LC-tandem mass spectrometry. Based on the levels of ADMA observed in the murine model, we further tested the direct effects of nebulized inhaled ADMA on airways responsiveness in naive control mice. We also assessed alterations of L-arginine, ADMA, and SDMA in humans in adult lung specimens and sputum samples from pediatric patients with asthma. MEASUREMENTS AND MAIN RESULTS: ADMA was increased in lungs from the murine model of allergic airways inflammation. Exogenous administration of ADMA to naive mice, at doses consistent with the levels observed in the allergically inflamed lungs, resulted in augmentation of the airways responsiveness to methacholine. ADMA levels were also increased in human asthma lungs and sputum samples. CONCLUSIONS: ADMA levels are increased in asthma and contribute to NOS-related pathophysiology.

Fog2 is required for normal diaphragm and lung development in mice and humans.

Congenital diaphragmatic hernia and other congenital diaphragmatic defects are associated with significant mortality and morbidity in neonates; however, the molecular basis of these developmental anomalies is unknown. In an analysis of E18.5 embryos derived from mice treated with N-ethyl-N-nitrosourea, we identified a mutation that causes pulmonary hypoplasia and abnormal diaphragmatic development. Fog2 (Zfpm2) maps within the recombinant interval carrying the N-ethyl-N-nitrosourea-induced mutation, and DNA sequencing of Fog2 identified a mutation in a splice donor site that generates an abnormal transcript encoding a truncated protein. Human autopsy cases with diaphragmatic defect and pulmonary hypoplasia were evaluated for mutations in FOG2. Sequence analysis revealed a de novo mutation resulting in a premature stop codon in a child who died on the first day of life secondary to severe bilateral pulmonary hypoplasia and an abnormally muscularized diaphragm. Using a phenotype-driven approach, we have established that Fog2 is required for normal diaphragm and lung development, a role that has not been previously appreciated. FOG2 is the first gene implicated in the pathogenesis of nonsyndromic human congenital diaphragmatic defects, and its necessity for pulmonary development validates the hypothesis that neonates with congenital diaphragmatic hernia may also have primary pulmonary developmental abnormalities.

Interacting genetic loci cause airway hyperresponsiveness.

Airway hyperresponsiveness (AHR) is a key physiological component of asthma, and the genetic basis of this complex trait has remained elusive. We created recombinant congenic mice with increased naive AHR by serially backcrossing A/J mice (which have elevated naive AHR) with C57BL/6J mice and selecting for mice with an elevated naive AHR phenotype. The seventh backcross-generation hyperresponsive mice retained A/J loci in three regions. Quantitative trait linkage (QTL) analysis of 123 unselected N8 progeny demonstrated that the AHR phenotype was not associated with any single locus but was significantly associated with an interaction of loci on chromosomes 2 and 6. These findings were confirmed in an independent analysis of chromosome substitution strain mice. The identification of genomic regions containing loci causally associated with AHR and the demonstration that this trait requires their interaction have important implications for the dissection of the genetic etiology of asthma in humans.

Modulation of inducible nitric oxide synthase by hypoxia in pulmonary artery endothelial cells.

The effects of hypoxia on the regulation of inducible nitric oxide synthase (NOS) 2 expression were examined in cultured rat pulmonary microvascular endothelial cells (EC). EC did not express NOS 2 mRNA or protein when exposed to normoxia or hypoxia unless they were pretreated with interleukin (IL)-1beta and/or tumor necrosis factor (TNF)-alpha for 24 h. Induction of NOS 2 by IL-1beta+TNF-alpha was significantly attenuated by concomitant exposure of EC to hypoxia or treatment of EC with antioxidants such as tiron, diphenyliodonium, and catalase, suggesting that NOS 2 expression is dependent on the production of reactive oxygen species. Degradation of IkappaB and activation of NF-kappaB, which were both induced by treatment of EC with cytokines, were not altered when the cells were exposed to hypoxia, suggesting that the modulation of NOS 2 expression by hypoxia is unrelated to NF-kappaB activation. Following stimulation with IL-1beta+TNF-alpha for 24 h, incubation of EC in normoxia resulted in a progressive decline in NOS 2 expression and a calculated half-life of approximately 6 h for NOS 2 mRNA. Hypoxia significantly prolonged the half-life of NOS 2 mRNA (17 h, P < 0.05 versus normoxic EC). The half-life of NOS 2 mRNA was also prolonged by actinomycin D treatment (19.5 and 29.5 h for normoxic and hypoxic EC, respectively), suggesting that transcription of an RNA destabilizing factor or RNAse contributes to NOS 2 mRNA degradation. In EC transiently transfected with the rat NOS 2 promoter, hypoxia and the combination of IL-1beta+TNF-alpha independently increased promoter activity 2.2- and 3-fold, respectively. As opposed to the attenuating effect that hypoxia had on IL-1beta+TNF-alpha- dependent induction of NOS 2 gene expression, the concomitant treatment with IL-1beta+TNF-alpha and hypoxia synergistically increased NOS 2 promoter activity 17.6-fold. Taken together, these results suggest that hypoxia alone does not induce NOS 2 expression in cultured pulmonary microvascular EC, but may modulate cytokine induction of this enzyme at pretranscriptional, transcriptional, and posttranscriptional levels.