Exosomes from adipose-derived stem cells attenuate UVB-induced apoptosis, ROS, and the Ca2+ level in HLEC cells.

Cartilage acid protein 1 (CRTAC1) encodes a protein containing the Ca2+binding domain, which can promote apoptosis of human lens epithelial cells (HLECs) induced by ultraviolet B radiation. Exosomes secreted from adipose-derived stem cells (ASC-exo) have been used to treat many diseases, but the effect of ASC-exo on cataracts has not been established. We hypothesized that ASC-exo has a therapeutic effect on cataracts by regulating CRTAC1. We established the UVB-induced injured HLECs model to test the interactions between CRTAC1 and miR-10a-5p, and the effect on the Ca2+ level and reactive oxygen species (ROS) generation in apoptotic HLECs. We found that UVB significantly increased the level of CRTAC1 expression and induced HLEC apoptosis, while ASC-exo inhibited the induction of UVB and exosome inhibitor reduced the inhibition of ASC-exo. The qRT-PCR results showed that miR-10a-5p had a low level of expression in cataract lesions, whereas CRTAC1 was highly expressed. There was a negative correlation between the expression of CRTAC1 and miR-10a-5p. ASC-exo reversed UVB-inhibited miR-10a-5p expression and miR-10a-5p negatively regulated CRTAC1. In vitro data showed that miR-10a-5p reversed UVB-induced ROS, apoptosis, and the Ca2+ level in HLECs. Overexpression of CRTAC1 reversed the induction of ASC-exo in UVB-injured HLECs, and low expression of CRTAC1 reversed the induction of miR-10a-5p inhibitor. By upregulating the level of miR-10a-5p expression and downregulating the level of CRTAC1 expression, exosomes from ASCs attenuated UVB-induced apoptosis, ROS generation, and the Ca2+ level in HLECs. Our research provides novel insight into the treatment methods and associated mechanisms underlying cataracts.

Down-regulation of CRTAC1 attenuates UVB-induced pyroptosis in HLECs through inhibiting ROS production.

Pyroptosis has been found to be related to diverse ocular diseases, including cataract. Abnormal CRTAC1 expression has been reported to involve in cataract formation by affecting cell apoptosis. Whether CRTAC1 regulates pyroptosis in the formation progress of cataract is completely unknown. Here, we aimed to investigate the regulatory effects of CRTAC1 on pyroptosis and the potential mechanism in the UVB-induced cell damage model. The results showed that the levels of the established pyroptosis markers (NLRP3, active Caspase-1, pro Caspase-1, GSDMD-N, IL-1ß and IL-18) were significantly increased in cataract patients. The above pyroptosis markers could be obviously induced by UVB-irradiation in human lens epithelial cells (HLECs), while down-regulation of CRTAC1 significantly reversed the UVB-induced pyroptosis. Up-regulation of CRTAC1 promoted HLECs pyroptosis, while the ROS inhibitor N-acetyl-l-cysteine blocked the effects of CRTAC1 overexpression. In conclusion, our findings further suggested that the prominent role of CRTAC1 in cataract formation.

Evaluation of the antioxidant effects of different histone deacetylase inhibitors (HDACis) on human lens epithelial cells (HLECs) after UVB exposure.

BACKGROUND: To compare the protective effects of the histone deacetylase inhibitors (HDACis) ß-hydroxybutyrate (ßOHB), trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA) on human lens epithelial cells(HLECs) following ultraviolet-B (UVB) exposure. METHODS: HLECs were divided into subgroups: four HDACi groups, a control group, a UVB-treated group and a DMSO group (cells treated with DMSO and UVB irradiation). In the HDACi groups, HLECs were cultured with different concentrations of HDACis 12 h prior to UVB irradiation. The protective effects of the HDACis were evaluated by assessing apoptosis rates, cell activity and expression levels of genes associated with apotosis (caspase-3, Bcl-2, BAX, SOD1, FOXO3A and MT2). The levels of superoxide dismutase (SOD), reactive oxygen species (ROS), malondialdehyde (MDA) and total antioxidant capacity (T-AOC) were detected in order to evaluate oxidative stress. RESULTS: The results showed that SAHA (1 µmol/L, 2 µmol/L) and TSA (0.2 µmol/L) had mild protective effects on cell viability. ßOHB (4 mmol/L) and TSA (0.2 mol/L) demonstrated protective effects on BCL-2 expression. TSA (0.2 mol/L) showed protective effects on SOD1 expression. TSA (0.2 mol/L) and SAHA (1 µmol/L) suppressed BAX and caspase-3 expression. TSA (0.2 mol/L, 0.8 mol/L) and SAHA (1 µmol/L, 2 µmol/L) suppressed the expression of FOXO3A and MT2. SOD levels were increased after treatment with ßOHB (4 mmol/L), SAHA (8 µmol/L) and TSA (0.1 mol/L, 0.2 mol/L). T-AOC levels were increased in UVB-treated HLECs after treatment with SAHA (2 µmol/L). MDA levels decreased in UVB-treated HLECs following treatment with TSA (0.2 mol/L, 0.8 mol/L). ROS levels decreased in UVB-treated HLECs following treatment with ßOHB (4 mmol/L), SAHA (1 µmol/L, 2 µmol/L) and TSA (0.2 mol/L). Western blotting results demonstrated that SOD1 levels significantly increased in the ßOHB (4 mmol/L), SAHA (1 µmol/L, 2 µmol/L), TSA (0.1 mol/L, 0.2 mol/L) and VPA (5 mmol/L) groups. Only SAHA (1 µmol/L) had an anti-apoptotic effect on UVB-treated HLECs. CONCLUSIONS: Our findings indicate that low concentrations of HDACis (1 µmol/L of SAHA) mildly inhibit oxidative stress, thus protecting HLECs from oxidation. These results may suggest that there is a possibility to explore the clinical applications of HDACis for treatment and prevention of cataracts.

Case Series: Slight Intraocular Lens Malpositioning as a Potential Cause of Vision Loss.

SIGNIFICANCE: The cases illustrate an insidious cause of decreased visual acuity after cataract surgery. PURPOSE: The purpose of this study was to identify cases of postoperative vision loss caused by slight intraocular lens (IOL) malpositioning after cataract surgery. CASE REPORTS: Three patients presented with visual acuity decreased after cataract surgery. Silt-lamp examination before mydriasis revealed no abnormalities in two of the patients; mild IOL inferonasal decentration was found by the trifocal IOL diffraction ring in the third patient. Manifest refraction of these patients showed remarkable astigmatism with low corneal astigmatism. After pupil dilation, slight IOL decentration and tilt were observed in all patients, which were further confirmed using the Scheimpflug imaging system. Wavefront aberrometry showed a high level of ocular higher-order aberrations, most of which were derived from intraocular aberrations. CONCLUSIONS: Inconspicuous IOL malpositioning is one of the reasons responsible for decreased vision acuity after cataract surgery, which may not be easily identified by slit-lamp examination. High astigmatism and ocular higher-order aberrations derived from malpositioned IOL can be important clues.

Metabolic characterization of human aqueous humor in the cataract progression after pars plana vitrectomy.

BACKGROUND: While pars plana vitrectomy (PPV) has become the third most commonly performed surgery in the world, it can also induce multiple post complications easily. Among them, cataract progression is the most frequent one that can lead to blindness eventually. METHODS: To understand the underlying mechanisms of post PPV cataract progression, we performed comprehensive metabolic characterization of aqueous humor (AH) samples from 20 cataract patients (10 post PPV complication and 10 none PPV cataract) by a non-targeted metabolomic analysis using gas chromatography combined with time-of-flight mass spectrometer (GC/TOF MS). RESULTS: A total of 263 metabolites were identified and eight of them are determined to be significantly different (VIP ≥ 1 and p ≤ 0.05) between post PPV group and none PPV control group. The significantly changed metabolites included glutaric acid and pelargonic acid that play key roles in the regulation of oxidative stress and inflammatory responses. Furthermore, we constructed a metabolic regulatory network in each group based on metabolite-metabolite correlations, which reveals key metabolic pathways and regulatory elements including amino acids and lipids metabolisms that are related to cataract progression. CONCLUSIONS: Altogether, this work discovered some potential metabolite biomarkers for post PPV cataract diagnostics, as well as casted some novel insights into the underlying mechanisms of cataract progression after PPV.

Metabolic characterization of human aqueous humor in relation to high myopia.

High myopia is the common eye disorder worldwide, which may contribute to increase the risk of serious disorders including glaucoma and cataract. Although various studies including genomics, transcriptomics, and proteomics have been implicated to identify potential biomarkers (genes or proteins) for predicting high myopia and to reveal the underlying mechanism, the comprehensive metabolomics in relation to high myopia is very limited. In this study, we identified 242 metabolites in aqueous humor (AH) from a set of 40 cataract patients (including 20 with high myopia and 20 for controls), using a non-targeted metabolomic technology, gas chromatography coupled to time-of-flight mass spectrometer (GC/TOF MS). Further statistical analysis showed that 29 metabolites were significantly changed (Variable important for the projection, VIP ≥ 1 and p ≤ 0.05), between those two groups, while only 2 decreased metabolites were included. Moreover, for the first time, metabolite-metabolite correlations for AH were analyzed, which may dissect key regulatory elements or pathways involved in metabolism of amino acids, carbohydrates, and lipids. Accordingly, metabolic network was constructed based on those 29 changed metabolites in patients with high myopia. More than half of the changed metabolites were highly and positively associated, suggesting important roles of pathways involved in the metabolism of these metabolites in relation to high myopia. Altogether, this work not only provided potential biomarkers for the diagnosis and monitoring of high myopia formation, but also provided new insights into the underlying mechanisms.

Inhibition of Cartilage Acidic Protein 1 Reduces Ultraviolet B Irradiation Induced-Apoptosis through P38 Mitogen-Activated Protein Kinase and Jun Amino-Terminal Kinase Pathways.

BACKGROUND/AIMS: Ultraviolet B (UVB) irradiation can easily induce apoptosis in human lens epithelial cells (HLECs) and further lead to various eye diseases including cataract. Here for the first time, we investigated the role of cartilage acidic protein 1 (CRTAC1) gene in UVB irradiation induced-apoptosis in HLECs. METHODS: Three groups of HLECs were employed including model group, empty vector group, and CRTAC1 interference group. RESULTS: After UVB irradiation, the percentage of primary apoptotic cells was obviously fewer in CRTAC1 interference group. Meanwhile, inhibition of CRTAC1 also reduced both reactive oxygen species (ROS) production and intracellular Ca2+ concentration, but the level of mitochondrial membrane potential (Δψm) was increased in HLECs. Further studies indicated that superoxide dismutase (SOD) activity and total antioxidative (T-AOC) level were significantly increased in CRTAC1-inhibited cells, while the levels of malondialdehyde (MDA) and lactate dehydrogenase (LDH) were significantly decreased. ELISA analysis of CRTAC1-inhibited cells showed that the concentrations of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were significantly decreased, but the concentration of interleukin-10 (IL-10) was significantly increased. Western blot analyses of eight apoptosis-associated proteins including Bax, Bcl-2, p38, phospho-p38 (p-p38), Jun amino-terminal kinases (JNK1/2), phospho-JNK1/2 (p-JNK1/2), calcium-sensing receptor (CasR), and Ca2+/calmodulin-dependent protein kinase II (CaMKII) indicated that the inhibition of CRTAC1 alleviated oxidative stress and inflammation response, inactivated calcium-signaling pathway, p38 and JNK1/2 signal pathways, and eventually reduced UVB irradiation induced-apoptosis in HLECs. CONCLUSION: These results provided new insights into the mechanism of cataract development, and demonstrated that CRTAC1 could be a potentially novel target for cataract treatment.

Distribution of gene mutations in sporadic congenital cataract in a Han Chinese population.

PURPOSE: This study aimed to investigate the genetic effects underlying non-familial sporadic congenital cataract (SCC). METHODS: We collected DNA samples from 74 patients with SCC and 20 patients with traumatic cataract (TC) in an age-matched group and performed genomic sequencing of 61 lens-related genes with target region capture and next-generation sequencing (NGS). The suspected SCC variants were validated with MassARRAY and Sanger sequencing. DNA samples from 103 healthy subjects were used as additional controls in the confirmation examination. RESULTS: By filtering against common variants in public databases and those associated with TC cases, we identified 23 SCC-specific variants in 17 genes from 19 patients, which were predicted to be functional. These mutations were further confirmed by examination of the 103 healthy controls. Among the mutated genes, CRYBB3 had the highest mutation frequency with mutations detected four times in four patients, followed by EPHA2, NHS, and WDR36, the mutation of which were detected two times in two patients. We observed that the four patients with CRYBB3 mutations had three different cataract phenotypes. CONCLUSIONS: From this study, we concluded the clinical and genetic heterogeneity of SCC. This is the first study to report broad spectrum genotyping for patients with SCC.

Ambient mass spectrometry imaging metabolomics method provides novel insights into the action mechanism of drug candidates.

Elucidation of the mechanism of action for drug candidates is fundamental to drug development, and it is strongly facilitated by metabolomics. Herein, we developed an imaging metabolomics method based on air-flow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) under ambient conditions. This method was subsequently applied to simultaneously profile a novel anti-insomnia drug candidate, N(6)-(4-hydroxybenzyl)-adenosine (NHBA), and various endogenous metabolites in rat whole-body tissue sections after the administration of NHBA. The principal component analysis (PCA) represented by an intuitive color-coding scheme based on hyperspectral imaging revealed in situ molecular profiling alterations in response to stimulation of NHBA, which are in a very low intensity and hidden in massive interferential peaks. We found that the abundance of six endogenous metabolites changed after drug administration. The spatiotemporal distribution indicated that five altered molecules­including neurotransmitter γ-aminobutyric acid, neurotransmitter precursors choline and glycerophosphocholine, energy metabolism-related molecules adenosine (an endogenous sleep factor), and creatine­are closely associated with insomnia or other neurological disorders. These findings not only provide insights into a deep understanding on the mechanism of action of NHBA, but also demonstrate that the AFADESI-MSI-based imaging metabolomics is a powerful technique to investigate the molecular mechanism of drug action, especially for drug candidates with multitarget or undefined target in the preclinical study stage.

The mechanism of UVB irradiation induced-apoptosis in cataract.

Cataract is the most common eye disease that causes blindness in patients. Ultraviolet B (UVB) irradiation is considered an important factor leading to cataract by inducing apoptosis in human lens epithelial cells (HLECs), but the mechanism is currently unclear. In this study, we investigated HLECs under different intensities of UVB irradiation and different exposure time. The annexin V-FITC/propidium iodide staining results showed that UVB irradiation could efficiently lead to HLECs apoptosis in time- and dose-dependent manner. The expression of pro-apoptotic Bax gene was promoted by UVB irradiation, while anti-apoptotic Bcl-2 gene expression was inhibited at both transcript and protein levels. Notably, the ratio of Bax/Bcl-2 displayed a high and positive correlation to the proportion of apoptotic HLECs. Mitochondrial dysfunction was also observed with rapid loss of potential (∆Ψ m), as well as changes of the levels of reactive oxygen species, malondialdehyde, total antioxidative capabilities, and superoxide dismutase. In caspase pathway, the level of caspase-3 protein increased after UVB irradiation. All these discovered changes may play important roles in UVB-induced HLECs apoptosis, and would be helpful in understanding the mechanism of UVB-induced cataract and providing potential prevention and treatment strategies.

Potassium 2-(1-hydroxypentyl)-benzoate improves memory deficits and attenuates amyloid and τ pathologies in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by amyloid-ß (Aß) deposition and neurofibrillary tangles. Dl-PHPB [potassium 2-(1-hydroxypentyl)-benzoate], has been shown to have neuroprotective effects on cerebral ischemic, vascular dementia, and Aß-induced animal models by inhibiting oxidative injury, neuronal apoptosis, and glial activation. The aim of the present study was to examine the effect of dl-PHPB on learning and memory in amyloid precursor protein (APP) and presenilin 1 (PS1) double-transgenic AD mouse models (APP/PS1) and the mechanisms of dl-PHPB in reducing Aß accumulation and τ phosphorylation. Twelve-month-old APP/PS1 mice were given 30 mg/kg dl-PHPB by oral gavage for 3 months. Dl-PHPB treatment significantly improved the spatial learning and memory deficits compared with the vehicle-treated APP/PS1 mice. In the meantime, dl-PHPB obviously reduced τ hyperphosphorylation at Ser199, Thr205, and Ser396 sites in APP/PS1 mice. This reduction was accompanied by APP phosphorylation reduction and protein kinase C activation. In addition, expression of cyclin-dependent kinase and glycogen synthase kinase 3ß, the most important kinases involved in τ phosphorylation, was markedly decreased by dl-PHPB treatment. Phosphorylated protein kinase B and phosphoinositide 3-kinase levels of APP/PS1 mice were significantly reduced compared with levels in wild-type mice, and dl-PHPB reversed the reduction. The effects of dl-PHPB effecting a decrease in τ phosphorylation and kinase activation were further confirmed in neuroblastoma SK-N-SH cells overexpressing wild-type human APP695. These data raised the possibility that dl-PHPB might be a promising multitarget neuronal protective agent for the treatment of AD.

The impact of sucralose and neotame on the safety of metal precipitation in electronic cigarettes.

This study investigated the impact of sweeteners on the release of heavy metals during the heating and atomization processes in electronic cigarettes. Based on a PG/VG base e-liquid with the addition of 2% and 5% neotame or sucralose, we quantitatively analyzed the impact of sweetener content on the levels of heavy metals such as Ni, Cr, and Fe in the e-liquid and aerosol after heating and atomization. Additionally, the heated e-liquid samples were used to culture SH-SY-5Y and Beas-2B cells, and their cytotoxic effects were assessed using the CCK-8 assay. The results indicated that the e-liquid with 5% sucralose had the highest average levels of heavy metals after heating and atomization, particularly nickel (13.36 ± 2.50 mg/kg in the e-liquid and 12,109 ± 3,229 ng/200 puffs in the aerosol), whereas the e-liquid with neotame had significantly lower average heavy metal content in comparison. Additionally, it was measured that the chloride ion concentration in the e-liquid with 5% sucralose reached 191 mg/kg after heating at 200°C for 1 h, indicating that heating sucralose generated chloride ions, Which might corrode metal parts components leading to heavy metal release. Cytotoxicity tests revealed that the base e-liquid without sweeteners exhibited the highest average cell viability after heating, at 64.80% ± 2.84% in SH-SY-5Y cells and 63.24% ± 0.86% in Beas-2B cells. Conversely, the e-liquid variant with 5% sucralose showed a significant reduction in average cell viability, reducing it to 50.74% ± 0.88% in SH-SY-5Y cells and 53.03% ± 0.76% in Beas-2B cells, highlighting its more pronounced cytotoxic effects compared to other tested e-liquids. In conclusion, sucralose in e-liquids should be limited preferably less than 2%, or replaced with neotame, a safer alternative, to minimize health risks.

[Ginkgolide B promotes axonal growth of retina ganglion cells by anti-apoptosis in vitro].

One common feature of glaucoma, optic neuritis and some other optic nerve diseases is sustained and irreversible apoptosis of retinal ganglion cells (RGCs). Ginkgolide B is believed to protect neurons in brain and contribute to neurite outgrowth and synapse formation. The aim of the present study was to explore the effects of Ginkgo biloba extract (EGB761) and ginkgolide B on axonal growth of RCGs. Retina explants were cultured in three-dimensional tissue culture system, and the number and length of neurites were analyzed. Immunohistochemistry staining was performed to confirm that the neurite observed was axon of RGCs. TUNEL and activated caspase-3 staining were also applied to observe RGCs apoptosis. The result shows that neurites of RGCs treated with EGB761 or ginkgolide B were more and longer than those in control. The neurite is proved to be the axon of RGCs by immunostaining. Furthermore, compared with control group, RGCs treated with ginkgolide B showed decreased cellular apoptosis and inhibited caspase-3 activation. These results suggest ginkgolide B can promote RGCs axon growth by protecting RGCs against apoptosis.

[An overview of biomarkers in Alzheimer's disease].

Alzheimer's disease (AD), also called presenile dementia, is one of the most common neurodegenerative diseases in elderly people. The main pathological features of AD include senile plaques (SPs), neurofibrillary tangles (NFTs) and neuron loss. A biomarker is a characteristic that can be objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. Class biomarkers of AD such as Abeta and phosphorylated tau have been widely used in clinical diagnosis of AD patients. Recently, novel technologies like proteomics, genomics, and imaging techniques have expanded the role of a biomarker from early diagnosis to monitoring the progression of diseases and evaluating the response to various treatments. In this article, we will review the progress of various biomarkers of AD.

Presbyopia-Correcting Intraocular Lenses Implantation in Eyes After Corneal Refractive Laser Surgery: A Meta-Analysis and Systematic Review.

Purpose: To assess the efficacy, safety, and predictability of presbyopia-correcting intraocular lenses (IOLs) in cataract patients with previous corneal refractive surgery. Methods: A systematic literature search was performed to identify studies evaluating the clinical outcomes of presbyopia-correcting IOLs implantation in cataract surgery after laser refractive surgery. Outcomes were efficacy, safety and predictability parameters. Results: The authors identified 13 studies, involving a total of 128 patients and 445 eyes. Presbyopia-correcting IOLs were effective at improving distance, intermediate and near visual acuity aftercataract surgery. The proportion of post-laser surgery eyes with uncorrected distance visual acuity (UDVA) ≥ 20/25 was 0.82 [95% confidence interval (CI), 0.74-0.90] and the pooled rates of spectacle independence at near, intermediate, and far distances were 0.98 (95% CI, 0.94-1.00), 0.99 (95% CI, 0.95-1.00) and 0.78 (95% CI, 0.65-0.94) respectively. The percentage of participants who suffered from halos and glare was 0.40 (95% CI, 0.25-0.64) and 0.31 (95% CI, 0.16-0.60), respectively. The predictability had a percentage of 0.66 (95% CI, 0.57-0.75) and 0.90 (95% CI, 0.85-0.96) of eyes within ±0.5 diopters (D) and ±1.0 D from the targeted spherical equivalent. Conclusions: Presbyopia-correcting IOLs provide satisfactory results in terms of efficacy, safety and predictability in patients with previous corneal refractive surgery, but have a higher risk of photopic side effects such as halos and glare.

Lens Opacities Classification System III-based artificial intelligence program for automatic cataract grading.

PURPOSE: To establish and validate an artificial intelligence (AI)-assisted automatic cataract grading program based on the Lens Opacities Classification System III (LOCS III). SETTING: Eye and Ear, Nose, and Throat Hospital, Fudan University, Shanghai, China. DESIGN: AI training. METHODS: Advanced deep-learning algorithms, including Faster R-CNN and ResNet, were applied to the localization and analysis of the region of interest. An internal dataset from the EENT Hospital of Fudan University and an external dataset from the Pujiang Eye Study were used for AI training, validation, and testing. The datasets were automatically labeled on the AI platform regarding the capture mode and cataract grading based on the LOCS III. RESULTS: The AI program showed reliable capture mode recognition, grading, and referral capability for nuclear and cortical cataract grading. In the internal and external datasets, 99.4% and 100% of automatic nuclear grading, respectively, had an absolute prediction error of ≤1.0, with a satisfactory referral capability (area under the curve [AUC]: 0.983 for the internal dataset; 0.977 for the external dataset); 75.0% (internal dataset) and 93.5% (external dataset) of the automatic cortical grades had an absolute prediction error of ≤1.0, with AUCs of 0.855 and 0.795 for referral, respectively. Good consistency was observed between automatic and manual grading when both nuclear and cortical cataracts were evaluated. However, automatic grading of posterior subcapsular cataracts was impractical. CONCLUSIONS: The AI program proposed in this study showed robust grading and diagnostic performance for both nuclear and cortical cataracts, based on LOCS III.

Neuroprotective effect of transcorneal electrical stimulation on ischemic damage in the rat retina.

Some previous studies have showed that transcorneal electrical stimulation (TES) could protect retinal neurons in certain rodent models. However, it is not yet clear whether TES could also definitely protect retinal neurons against ischemic insults. In the present study, we hypothesized that TES had such a neuroprotective effect and further investigated its underlying mechanism. Adult female Sprague-Dawley (SD) rats received TES treatment every other day after ocular ischemia was induced by elevating the intraocular pressure to 120 mm Hg for 60 min. Retinal ganglion cells (RGCs) were labeled retrogradely 7 days before ischemia and were counted 7 and 14 days later. At the same time points, retinal function was assessed by scotopic electroretinography (ERG), combined with retinal histological analysis. The glutamine synthetase (GS) immunoreactivity was compared between ischemic retinas with TES and those with sham stimulation under identical confocal laser microscope conditions. The immunohistochemical indications were confirmed by Western blot analysis. Higher mean density of RGCs was quantified in TES treated retinas compared to retinas with sham stimulation on days 7 and 14 after ischemia. Similarly, histological analysis showed that TES better preserved the mean thickness of separate retinal layers. ERG studies indicated that by undergoing TES treatment, the b-wave amplitude was also significantly preserved on day 7 after ischemia and recovered robustly on day 14. Immunohistochemical and Western blot analysis both revealed that GS levels remarkably increased after TES and lasted for at least 7 days. Our results indicate that TES can protect retinal neurons against ischemic insults, probably related to increasing levels of GS localized in Müller cells. These findings suggest a new approach for potential clinical application to ocular ischemic diseases.

Conjunctival Microbiota in Patients With Type 2 Diabetes Mellitus and Influences of Perioperative Use of Topical Levofloxacin in Ocular Surgery.

Background: Patients with type 2 diabetes mellitus (T2DM) are prone to ocular surface infections. We therefore characterized the conjunctival microbiome of T2DM patients and the influence of topical levofloxacin to investigate whether a dysbiosis is associated with this phenomenon. Methods: Conjunctival microbiome of 79 T2DM patients and 113 non-diabetic controls was profiled using the 16S rDNA sequencing approach. Furthermore, 21 T2DM and 14 non-diabetic patients who underwent cataract surgeries were followed up perioperatively and the influence of pre- and post-operative levofloxacin on the conjunctival microbiome was further investigated prospectively and compared longitudinally. Results: The α-diversity of the conjunctival microbiota was significantly higher in T2DM patients than in controls (P < 0.05). Significant differences in both composition and function of the conjunctival microbiome were identified on the ocular surface of T2DM patients as compared to non-diabetic controls. Particularly, phylum Bacteroidetes and Fusobacteria, genus Pseudomonas, Haemophilus, and Empedobacter were enriched, while genus Streptococcus was reduced on the T2DM ocular surface. Microbial genes functioning of bacterial chemotaxis was elevated in the conjunctival microbiome of T2DM patients. Furthermore, compared to the initial status, several genera including Staphylococcus were more abundant in the conjunctival microbiome of T2DM patients after 3-days use of preoperative levofloxacin topically, while no genus was more abundant in the non-diabetic follow-up group. No difference was observed between initial status and 7 days after ceasing all postoperative medications in both diabetic and non-diabetic follow-up groups. Conclusions: The conjunctival microbiome of T2DM patients was more complex and may respond differently to topical antibiotics.

Cofilin 2 Acts as an Inflammatory Linker Between Chronic Periodontitis and Alzheimer's Disease in Amyloid Precursor Protein/Presenilin 1 Mice.

Increasing evidence has shown a correlation between chronic periodontitis (CP) and Alzheimer's disease (AD). Nevertheless, there is still a lack of direct evidence, and especially key molecules to connect the two diseases. This study aims to investigate potential protein links between CP and AD within the inflammatory aspect. The hippocampus of CP model mice and controls were collected, and changes in protein expression were evaluated using two-dimensional differential in-gel electrophoresis (2D-DIGE) analysis combined with liquid chromatography tandem mass spectrometry. A total of 15 differentially expressed proteins were identified in CP model mice, as compared with the controls. Among them, S100-A9, transthyretin, Cofilin 2, peroxiredoxin 2, and lipocalin-2 were validated by Western blot according to their dual function both in inflammation and AD. Based on 2D-DIGE analysis, CP animal model had higher levels of S100-A9, Cofilin 2, peroxiredoxin 2, and lipocalin-2 compared to controls. The level of Cofilin 2, one of the well-established proteins in the pathology of AD, was strongly correlated with the time course of CP pathology, indicating a specific molecular correlation between CP and AD. Moreover, the in vivo results showed the level of Cofilin 2 increased significantly along with a prominent increase of the phosphorylation of protein phosphatase 2 (PP2A) and tau protein in the cell lysates of Porphyromonas gingivalis (P.g-LPS)-treated SK-N-SH APPwt cells. Cofilin 2 inhibition resulted in a sharp decrease in PP2A dependent of tau phosphorylation. Furthermore, tumor growth factor (TGF)-ß1 was one of the most important inflammatory cytokines for the Pg-LPS-induced Cofilin 2 upregulation in SK-N-SH APPwt cells. These results showed inflammation served as the bond between CP and AD, whereas inflammatory related proteins could be the key linkers between the two diseases. Determining the association between CP and AD at the molecular mechanism will not only hold the direct evidence of the association between the two diseases but also provide a new way of preventing and treating AD: the effective prevention and treatment of CP could serve as a useful method to alleviate the development of AD.

Variants of WNT7A and GPR124 are associated with hemorrhagic transformation following intravenous thrombolysis in ischemic stroke.

AIMS: The canonical Wnt signaling pathway plays an essential role in blood-brain barrier integrity and intracerebral hemorrhage in preclinical stroke models. Here, we sought to explore the association between canonical Wnt signaling and hemorrhagic transformation (HT) following intravenous thrombolysis (IVT) in acute ischemic stroke (AIS) patients as well as to determine the underlying cellular mechanisms. METHODS: 355 consecutive AIS patients receiving IVT were included. Blood samples were collected on admission, and HT was detected at 24 hours after IVT. 117 single-nucleotide polymorphisms (SNPs) of 28 Wnt signaling genes and exon sequences of 4 core cerebrovascular Wnt signaling components (GPR124, RECK, FZD4, and CTNNB1) were determined using a customized sequencing chip. The impact of identified genetic variants was further studied in HEK 293T cells using cellular and biochemical assays. RESULTS: During the study period, 80 patients experienced HT with 27 parenchymal hematoma (PH). Compared to the non-PH patients, WNT7A SNPs (rs2163910, P = .001, OR 2.727; rs1124480, P = .002, OR 2.404) and GPR124 SNPs (rs61738775, P = .012, OR 4.883; rs146016051, P < .001, OR 7.607; rs75336000, P = .044, OR 2.503) were selectively enriched in the PH patients. Interestingly, a missense variant of GPR124 (rs75336000, c.3587G>A) identified in the PH patients resulted in a single amino acid alteration (p.Cys1196Tyr) in the intracellular domain of GPR124. This variant substantially reduced the activity of WNT7B-induced canonical Wnt signaling by decreasing the ability of GPR124 to recruit cytoplasmic DVL1 to the cellular membrane. CONCLUSION: Variants of WNT7A and GPR124 are associated with increased risk of PH in patients with AIS after intravenous thrombolysis, likely through regulating the activity of canonical Wnt signaling.