Fluorescence Imaging of Diabetic Cataract-Associated Lipid Droplets in Living Cells and Patient-Derived Tissues.

Diabetic cataract (DC) surgery carries risks such as slow wound healing, macular edema, and progression of retinopathy and is faced with a deficiency of effective drugs. In this context, we proposed a protocol to evaluate the drug's efficacy using lipid droplets (LDs) as the marker. For this purpose, a fluorescent probe PTZ-LD for LDs detection is developed based on the phenothiazine unit. The probe displays polarity-dependent emission variations, i.e., lower polarity leading to stronger intensity. Especially, the probe exhibits photostability superior to that of Nile Red, a commercial LDs staining dye. Using the probe, the formation of LDs in DC-modeled human lens epithelial (HLE) cells is validated, and the interplay of LDs-LDs and LDs-others are investigated. Unexpectedly, lipid transfer between LDs is visualized. Moreover, the therapeutic efficacy of various drugs in DC-modeled HLE cells is assessed. Ultimately, more LDs were found in lens epithelial tissues from DC patients than in cataract tissues for the first time. We anticipate that this work can attract more attention to the important roles of LDs during DC progression.

Observation of HOCl generation associated with diabetic cataract using a highly sensitive fluorescent probe.

Diagnosis of diabetic cataract (DC) in the early stage is of great significance for drug intervention and surgery circumvention for DC patients. However, the lack of reliable imaging tools greatly limits the diagnosis of early DC. In this context, a fluorescent probe BBPy for hypochlorous acid (HOCl) is presented based on the oxidation of phenothiazine. The probe displays apparent emission enhancement at 562 nm toward HOCl with high selectivity, superb sensitivity (detection limit: 12.6 nM), and rapid response (within seconds). Using the probe, the HOCl generation in diabetic human lens epithelial cells was monitored, as well as the HOCl down-regulation during antioxidant treatment. Therefore, it is proposed that HOCl can be a promising biomarker for DC and fluorescence imaging technique can be regarded as a candidate tool for DC diagnosis.

iUMRG: multi-layered network-guided propagation modeling for the inference of susceptibility genes and potential drugs against uveal melanoma.

Uveal melanoma (UM) is the most common primary malignant intraocular tumor. The use of precision medicine for UM to enable personalized diagnosis, prognosis, and treatment require the development of computer-aided strategies and predictive tools that can identify novel high-confidence susceptibility genes (HSGs) and potential therapeutic drugs. In the present study, a computational framework via propagation modeling on integrated multi-layered molecular networks (abbreviated as iUMRG) was proposed for the systematic inference of HSGs in UM. Under the leave-one-out cross-validation experiments, the iUMRG achieved superior predictive performance and yielded a higher area under the receiver operating characteristic curve value (0.8825) for experimentally verified SGs. In addition, using the experimentally verified SGs as seeds, genome-wide screening was performed to detect candidate HSGs using the iUMRG. Multi-perspective validation analysis indicated that most of the top 50 candidate HSGs were indeed markedly associated with UM carcinogenesis, progression, and outcome. Finally, drug repositioning experiments performed on the HSGs revealed 17 potential targets and 10 potential drugs, of which six have been approved for UM treatment. In conclusion, the proposed iUMRG is an effective supplementary tool in UM precision medicine, which may assist the development of new medical therapies and discover new SGs.

MiR-223 inhibits hyperosmolarity-induced inflammation through downregulating NLRP3 activation in human corneal epithelial cells and dry eye patients.

We previously showed that increases in reactive oxygen species (ROS) generation upregulate NLRP3 inflammasome and inflammation through increases in both caspase-1 activity and rises in IL-1ß expression levels in animal models of dry eye (DE). As changes in microRNA (miRNAs) expression levels can modulate inflammasome function, we determine here if there is a relationship in DE between changes in miR-223 expression levels and NLRP3 activation induced in an intelligent controlled environmental system (ICES) in mice. In parallel, ROS, miR-223 and NLRP3 expression levels were assessed in conjunctival impression cytology and tear fluid samples obtained from DE patients and normal subjects. MiR-223 expression levels were modulated by transfection of either a mimic or its negative control (NC) in a human corneal epithelial cell line (HCECs) exposed to a 500 mOsm hyperosmotic medium for 4 h. The dual-luciferase reporter assay confirmed that miR-223 controls NLRP3 gene expression readout through directly interacting with the 3' UTR of its mRNA. Hyperosmolarity-induced NLRP3 activation was confirmed based on recruitment and colocalization of NLRP3 with ASC as well as increases in IL-1ß expression. The miR-223 expression level decreased by 55% in the conjunctiva and cornea of the murine DE model from the level in the control group (P ≤ 0.047), while NLRP3 protein expression rose by 30% (P ≤ 0.017). In DE patients, miR-223 expression decreased in conjunctival impression cytology samples (P = 0.002), whereas IL-1ß tear content rose significantly (P < 0.001).The relevance of this decline was confirmed by showing that exposure to a 500 mOsm stress decreased the miR-223 expression level whereas ROS generation as well as the NLRP3, and IL-1ß expression levels rose in HCECs (P ≤ 0.037). In contrast, miR-223 mimic transfection reduced the NLRP3 protein expression level by 30% (P = 0.037), whereas both ROS generation and IL-1ß secretion rose compared to their corresponding levels in the control group (P ≤ 0.043). Thus, miR-223 negatively regulates NLRP3 inflammasome activity via suppressing NLRP3 translation in DE. This inverse regulation between miR-223 and NLRP3 expression levels suggests that selective upregulation of miR-223 expression may be a novel option to suppress chronic inflammation in DE.

Synthesis of Fe0/Fe3O4@porous carbon through a facile heat treatment of iron-containing candle soots for peroxymonosulfate activation and efficient degradation of sulfamethoxazole.

Developing highly efficient, reusable, non-toxic and low-cost catalysts is of great importance for persulfate-based advanced oxidation processes (AOPs). In this work, ferrocene was mixed into paraffin to prepare a candle, and the iron-containing candle soots were collected and heated at 500 °C~900 °C under N2 atmosphere for 1 h to prepare magnetically recyclable Fe0/Fe3O4@porous carbon (Fe0/Fe3O4@PC) catalysts. The Fe0/Fe3O4@PC-700 obtained after pyrolysis at 700 °C exhibited the best catalytic activity for sulfamethoxazole (SMX) degradation. 10 mg/L SMX could be completely degraded within 10 min by 0.2 g/L of Fe0/Fe3O4@PC-700 and 0.5 mM PMS at pH 5.0. The carbon shell effectively inhibited the Fe leaching of Fe0/Fe3O4@PC-700, and 99.73% of Fe was retained after five consecutive cycles. In the Fe0/Fe3O4@PC-700/PMS system, SMX was degraded through the sulfate radical (SO4·¯), hydroxyl radical (·OH), superoxide radical (O2·¯) dominated radical pathway, and the singlet oxygen (1O2) dominated non-radical pathway. The coexisting inorganic ions and natural organic matters (NOM) in actual water inhibited the degradation of SMX. Finally, four possible degradation pathways were proposed based on the degradation intermediates of SMX. This work provides a facile heat treatment of iron-containing candle soots strategy to prepare the metal@carbon catalysts for PMS-based AOP.

Thermally treated candle soot as a novel catalyst for hydrogen peroxide in-situ production enhancement in the bio-electro-Fenton system.

Thermally treated candle soot (TCS) was used as a two-electron (2e¯) oxygen reduction reaction (ORR) catalyst to in situ produce H2O2 in a bio-electro-Fenton (BEF) system. Compared with the pristine candle soot (CS), TCS showed larger Brunauer-Emmett-Teller (BET) surface area (102.54 m2 g-1 vs. 61.79 m2 g-1), higher mesoporous ratio (50.39% vs. 34.98%), and improved hydrophilicity. X-ray photoelectron spectra (XPS) results revealed that the C-O-C was the dominant oxygen-containing group of the CS, and its percentage reached at 80.55%. However, the C-O-C ratio of the TCS decreased to 48.93%, whilst it's CO and OC-O ratios significantly increased to 27.92% and 23.15%. The TCS showed a high H2O2 selectivity (87.5%∼97.0%) at the neutral pH condition, which was much higher than that of the commonly used carbon black (CB) catalyst. Finally, the H2O2 concentration maxima (Cmax-H2O2) of the bio-electro-Fenton system running with the TCS air-cathode (BEF-TCS) achieved at 32.02 mg/L, which was 6.29 times higher than that of the BEF-CB (5.09 mg/L). The removal and mineralization ratios of the SMX in the BEF-TCS reached at 83.0% and 79.0%, respectively. This paper reported a novel 2e¯ ORR electro-catalyst which was low-cost, easily available and highly efficiency.

A triple-chamber microbial fuel cell enabled to synchronously recover iron and sulfur elements from sulfide tailings.

Bioleaching by coupling iron oxidization with microbial growth is a process frequently used to extract target metals from sulfide tailing piles. However, the slower leaching, longer operational times, and lower efficiency compared to those of other extracting processes are the most important reasons that make this approach unattractive for the recovery of target elements. A triple-chamber microbial fuel cell (MFC) was explored to elevate the dissolution of sulfide tailings via in-situ removal of bioleached Fe3+/Fe2+ and SO42-, during which iron and SO42- ions were synchronously recovered as Fe(OH)3 and S° in the first and second cathode chambers, respectively. 107.9 % of iron and 99.8 % of sulfur contained in the sulfide tailings was bioleached over 50 h, with 80.0 % iron and 22.1 % sulfur elements synchronously recovered. The purities of the Fe(OH)3 and S° precipitates with high metallurgical values were up to 93.1 % and 90.2 %, respectively. The excellent leaching performance of the explored triple-chamber MFC was attributed to the synergistic effect of Acidithiobacillia catalysis and electrochemical oxidation. The explored approach, by virtue of having the higher bioleaching efficiency, less aggressive conditions and shorter operating times than the conventional bioleaching, is of potential commercial value.

Substance P induces sympathetic immune response in the contralateral eye after the first eye cataract surgery in type 2 diabetic patients.

BACKGROUND: Substance P (SP) is a nociceptive tachykinin which regulates the immune inflammatory reactions including monocyte chemoattractant protein 1 (MCP-1) production. Sequential second-eye cataract surgery patients often suffer more pain than the first one partly because of the MCP-1 increase in aqueous humor (AH). This study aims to illustrate whether SP is involved in sympathetic inflammatory responses in the contralateral eye in patients with or without type 2 diabetes. METHODS: This prospective randomized clinical study included 51 cataract patients (22 with diabetes and 29 without). Bilateral sequential cataract surgeries were conducted with 1-day or 1-week interval randomly. More than 100 µl of AH were obtained before surgery and stored for later analysis using magnetic Luminex assays to detect interleukin (IL)-1ß, IL-1ra,IL-6, IL-8, IL-10, MCP-1, vascular endothelial growth factor, spinal macrophage inflammatory protein (MIP-1a), interferon-inducible protein 10 (IP-10), regulated on activation, normal T expressed and secreted (RANTES), as well as the enzyme-linked immunosorbent assay for SP. RESULTS: Among the 4 groups, no significant differences were found in age, sex distribution, the R/L ration of the first surgery eye, or the lens nucleus hardness (P ≥ 0.802). Over 100 µl of AH samples were collected safely in all cases without intraoperative complications. SP and MCP-1 levels were both increased significantly in the second eye of the diabetic patients with 1-day and 1-week interval (P ≤ 0.040). The SP increase in the second eye of the diabetic patients were significantly higher than that of the patients without diabetes (P ≤ 0.030) both in the groups with 1-day and 1-week interval. Similarly, the MCP-1 increase was significantly higher in the diabetic patients in the group with 1-week interval (P = 0.042). CONCLUSIONS: Substance P and MCP-1 productions elevated in the AH of the contralateral eye after the first-eye cataract surgery in diabetic patients, indicating that SP and MCP-1 were involved in the sympathetic inflammatory responses. Diabetic patients are susceptible to noninfectious inflammation after cataract surgery, and perceive more pain in the second-eye phacoemulsification. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR1900028374 , retrospectively registered on 20th December, 2019.

Phacoemulsification With 3.0 and 2.0 mm Opposite Clear Corneal Incisions for Correction of Corneal Astigmatism.

PURPOSE: To compare the effect of 3.0 and 2.0 mm opposite clear corneal incisions (OCCIs) in phacoemulsification on reduction of preexisting corneal astigmatism, and their impact on corneal aberrations. METHODS: This study is a prospective randomized controlled study that included 140 patients with age-related cataract and regular corneal astigmatism ≥0.75 diopter (D). Phacoemulsification was performed using on-meridian 3.0 or 2.0 mm corneal incision with or without an OCCI. Cases were divided into 4 groups: 3.0 mm OCCIs, 3.0 mm single clear corneal incision (3.0 mm SCCI), 2.0 mm OCCIs, and 2.0 mm SCCI. Keratometry and topography were performed at 3 months postoperatively. The variations in corneal astigmatism and aberrations were recorded. Surgically induced astigmatism was calculated using vector analysis. RESULTS: The corneal astigmatism reduction was 0.61 ± 0.38 D in the 3.0 mm OCCIs group, significantly higher than the other groups (P ≤ 0.004); and 0.29 ± 0.29 D in the 2.0 mm OCCIs group. The mean surgically induced astigmatism was 1.07 ± 0.51 D in the 3.0 mm OCCIs group, higher than 0.61 ± 0.35 D in the 2.0 mm OCCIs group (P = 0.001). The root mean square values of corneal trefoil, spherical aberration, and total higher order aberrations increased at 3 months postoperatively, but there were no significant differences between OCCI and SCCI groups. CONCLUSIONS: On-meridian 3.0 mm OCCIs are effective for correcting mild-to-moderate corneal astigmatism during cataract surgery, exerting no additional impact on corneal aberration compared with SCCI.

Reactive oxygen species activated NLRP3 inflammasomes initiate inflammation in hyperosmolarity stressed human corneal epithelial cells and environment-induced dry eye patients.

In studies on dry eye (DE) disease, an association has been identified between tear film hyperosmolarity and inflammation severity elicited through receptor-induced increases in proinflammatory cytokine and chemokine release. These immune reactions might be mediated by inflammasomes, macromolecular complexes mounted around the NLRP3 protein and can be activated by reactive oxygen species (ROS) over-generation. Hence in this study we determine whether: a) ROS activated NLRP3 inflammasomes mediate hyperosmotic stress-induced inflammation in human corneal epithelial cells (HCECs); b) the ROS-NLRP3-IL-1ß axis activation is associated with environment-induced DE. Immortalized HCECs were exposed to 500 mOsm medium in the presence and absence of a ROS inhibitor, N-acetyl-l-cysteine (NAC). HCECs transfected with NLRP3 siRNA or a negative control (NC) siRNA. Intracellular ROS was measured by fluorometric analysis using the probe 2',7'-dichlorofluorescin diacetate (DCFH-DA). Real-time PCR evaluated NLRP3, ASC, pro-caspase-1 and pro-IL-1ß mRNA levels. Western blot analysis assessed NLRP3 protein expression whereas caspase-1 activity was determined with a fluorometric assay. Bioactive IL-1ß release was assessed by ELISA. ROS production, NLRP3 inflammasome and pro-IL-1ß gene expression as well as IL-1ß secretion were also evaluated in the conjunctival epithelial cells and tear fluid samples of environment-induced DE patients and normal subjects. NAC suppressed hyperosmolarity-induced rises in ROS levels, NLRP3 inflammasome formation and activation, caspase-1 activity and IL-1ß release. On the other hand, NLRP3 siRNA knockdown inhibited hyperosmotic stress-induced NLRP3 activation, which led to ASC, pro-caspase-1 and pro-IL-1ß mRNA down-regulation followed by suppression of associated caspase-1 activity and IL-1ß secretion. In addition, in ocular surface samples of environment-induced DE patients, ROS generation, NLRP3, ASC, pro-caspase-1 and pro-IL-1ß gene expression as well as IL-1ß secretion were upregulated. Taken together, NLRP3 mediated innate immune responses triggered by rises in ROS generation induce inflammation in hyperosmotic stressed HCECs. ROS-NLRP3-IL-1ß signaling pathway might play a priming role in environment-induced DE development.

Stabilized triple procedure for management of coexisting corneal opacity and cataract.

We describe a surgical technique combining penetrating keratoplasty and cataract surgery with a secure anterior chamber to manage coexisting corneal opacity and cataract. Lamellar corneal dissection is performed to 80% of the corneal depth to provide sufficient visibility for cataract surgery. Manual small-incision cataract extraction and intraocular lens implantation is performed through a frown-shaped scleral tunnel. Subsequently, 4 small penetrating incisions are made along the edge of the trephination mark at the 3, 6, 9, and 12 o'clock positions. A graft is placed on the ophthalmic viscosurgical device-coated stromal bed, and the 4 penetration sites are sutured. The stromal bed is cut along the trephination groove, the graft is sutured quadrant by quadrant, and the stromal bed is drawn out of the anterior chamber. The surgery is completed with 16 sutures.

Reactive oxygen species activated NLRP3 inflammasomes prime environment-induced murine dry eye.

Tear film hyperosmolarity along with exposure to oxidant stress are factors that can induce chronic ocular surface inflammation and pain. However, there is limited information on how increases in reactive oxygen species (ROS) generated by oxidant exposure can induce inflammation. There is emerging evidence in other tissues that innate immune responses to a variety of environmental stresses stem from ROS-induced cytosolic NLRP3 inflammasome activation. Once this occurs, pro-caspase-1 is converted into its catalytic active form, which in turn cleaves pro-IL-1ß thereby generating its bioactive form. We determined the role of ROS generation in mediating increases in IL-1ß secretion through caspase-1 activation caused by NLRP3 inflammasome activation in an environment-induced murine dry eye (DE) model. An intelligently controlled environmental system (ICES) induced evaporative DE in female 4-6 week old C57BL/6J mice. Increases in ROS production preceded rises in corneal and conjunctival gene expression of NLRP3 inflammasome components and IL-1ß that were identified using real-time PCR. Confocal microscopy evaluated concomitant increases in NLPR3, caspase-1 and IL-1ß immunostaining. Increases in caspase-1 activity were used as an indicator of inflammasome activation. Rises in ROS generation occurred after 1 week of ICES exposure, which preceded increases in gene expression of three NLRP3 inflammasome components (i.e. NLRP3, ASC and caspase-1) leading to rises in bioactive IL-1ß release. Increases in caspase-1 activity occurred after 2 weeks of ICES exposure. Eyedrops containing 0.3% N-acetyl-l-cysteine (NAC) were applied to quench ROS generation by mice kept in the ICES for 2 weeks. This scavenger reduced corneal fluorescein staining and decreased ROS production. NAC also down-regulated both increases in NLRP3, ASC, caspase-1 and IL-1ß mRNA levels, along with their immunostaining. It robustly attenuated rises in inflammasome mediated increases in caspase-1 catalytic activity. We show in a dessicating DE disease murine model that rises in ROS generation trigger NLRP3 inflammasome complexation and activation leading to increases in bioactive IL-1ß secretion. These results prompt us to suggest that the ROS-NLRP3-IL-1ß signaling pathway might play a priming role in environment-induced DE progression. Finally, our findings provide a basis for developing novel strategies that may improve the management of patients requiring treatment for environment-induced dry eye disease.

Antiproliferative and apoptotic effects of indomethacin on human retinoblastoma cell line Y79 and the involvement of ß-catenin, nuclear factor-κB and Akt signaling pathways.

BACKGROUND: To determine in vitro if indomethacin inhibits proliferation and induces apoptosis in human retinoblastoma cell line Y79, and to explore possibly involved signaling pathways. METHODS: The human retinoblastoma cell line Y79 was cultured with indomethacin at various concentrations (0, 25, 50, 100, 200 and 400 µmol/l). The effect of indomethacin on cell proliferation and apoptosis was examined by the Cell Counting Kit-8 and TUNEL test, respectively. The mRNA level of survivin, ß-catenin and Bcl-2 was detected by RT-PCR. The protein level of survivin was measured by ELISA. Western blot was used to analyze ß-catenin, nuclear factor (NF)-κB/p65, phosphorylated Akt (pAkt) and total Akt (tAkt) expression in cultured cells. RESULTS: Indomethacin treatment inhibits proliferation (at concentrations from 25 to 400 µmol/l) and induces apoptosis (at concentrations from 100 to 400 µmol/l) of human retinoblastoma cell line Y79 in a dose-dependent manner. RT-PCR showed that the mRNA expression of Bcl-2 (F = 20.497; p < 0.001) and of ß-catenin (F = 14.835; p < 0.001) was significantly different among the treated groups. Survivin mRNA levels remained steady, but its protein levels decreased significantly as measured by ELISA (F = 67.633; p < 0.001). Western blot analysis showed a dose-dependent downregulation of ß-catenin (F = 37.411; p < 0.001), NF-κB/p65 (F = 16.302; p < 0.001) and of pAkt (F = 27.700; p < 0.001) after indomethacin treatment, while tAkt protein expression was steady among the groups. CONCLUSIONS: Treatment with indomethacin can potently suppress proliferation and induce apoptosis in the retinoblastoma Y79 cell line. Wnt/ß-catenin, NF-κB and Akt/PKB pathways might be implicated in the process.

Differential proteomics and functional research following gene therapy in a mouse model of Leber congenital amaurosis.

Leber congenital amaurosis (LCA) is one of the most severe forms of inherited retinal degeneration and can be caused by mutations in at least 15 different genes. To clarify the proteomic differences in LCA eyes, a cohort of retinal degeneration 12 (rd12) mice, an LCA2 model caused by a mutation in the RPE65 gene, were injected subretinally with an AAV vector (scAAV5-smCBA-hRPE65) in one eye, while the contralateral eye served as a control. Proteomics were compared between untreated rd12 and normal control retinas on P14 and P21, and among treated and untreated rd12 retinas and control retinas on P42. Gene therapy in rd12 mice restored retinal function in treated eyes, which was demonstrated by electroretinography (ERG). Proteomic analysis successfully identified 39 proteins expressed differently among the 3 groups. The expression of 3 proteins involved in regulation of apoptosis and neuroptotection (alpha A crystallin, heat shock protein 70 and peroxiredoxin 6) were investigated further. Immunofluorescence, Western blot and real-time PCR confirmed the quantitative changes in their expression. Furthermore, cell culture studies suggested that peroxiredoxin 6 could act in an antioxidant role in rd12 mice. Our findings support the feasibility of gene therapy in LCA2 patients and support a role for alpha A crystallin, heat shock protein 70 and peroxiredoxin 6 in the pathogenetic mechanisms involved in LCA2 disease process.

[Expression of dopamine receptor D2 and adenosine receptor A2A in human retinal pigment epithelium].

OBJECTIVE: The aim of this study was to identify the presence of dopamine receptor D2 and adenosine receptor A2A in human retinal pigment epithelium (RPE) in order to determine whether the RPE is a potential site of action for these two receptors. METHODS: RPE Cells were isolated and cultured. Reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry were performed to detect the expression of D2 and A2A receptors in the RPE. RESULTS: RT-PCR analysis showed that mRNA of D2 and A2A receptors were expressed in the RPE. D2 and A2A receptors were distributed in the RPE by immunocytochemistry. CONCLUSIONS: This study demonstrated the presence of D2 and A2A receptors in human RPE at both mRNA and protein levels. D2 and A2A receptors may play an important role in the maintenance of the function of human retinal pigment epithelium. Furthermore, their cooperation appeared to be important in the development of axial myopia and retinal and choroidal neovascularization.