Aß monomers protect lens epithelial cells against oxidative stress by upregulating CDC25B.

Our previous studies showed high ß-amyloid (Aß) expression levels in the nuclei of the lens epithelial cells (LECs) of healthy subjects and revealed that Aß monomers could protect LECs from oxidative damage. Here, we further explored the mechanism by which Aß monomers act as transcription factors to regulate the oxidative stress of LECs through high-throughput studies. First, we compared the Aß-binding sites in the lens epithelia (LE) of age-related cataract patients with those in the LE of healthy donors via chromatin immunoprecipitation-sequencing (ChIP-seq), and we identified comparable numbers (1648 and 1445, respectively) of Aß peaks. Then, the KEGG tool was used for gene function enrichment analysis of these genes, which were more highly enriched in healthy LE. Combining the literature review with these KEGG analysis results, in the current study, we chose four target genes related to oxidative stress, namely, CDC25B, SOS2, CTNNA1 and Cox6a1. Then, ChIP-PCR assays, dual-luciferase reporter assays, real-time PCR and Western blotting were performed to validate the regulatory effects of Aß on these targets. Our data suggested that Aß monomers could upregulate the mRNA and protein expression levels of CDC25B in LECs. We also confirmed that Aß monomers could activate the Akt/Nrf2 pathway in a CDC25B-dependent manner by knockdown experiments in cultured LECs. Furthermore, we performed functional verification of the CDC25B-mediated protective effects of Aß monomers against oxidative stress. We observed that Aß monomers significantly improved the antioxidant capacity (the GSH level, SOD activity and total antioxidant capacity) and decreased the oxidative stress (the ROS and MDA levels) of LECs, while CDC25B knockdown decreased the antioxidant effects of Aß, disrupting redox homeostasis. Therefore, we propose that Aß monomers activate the Akt/Nrf2 pathway by upregulating CDC25B expression, increase various downstream antioxidant enzyme levels, maintain peroxidation-antioxidant homeostasis in LECs, and prevent the cell damage caused by oxidative stress.

Synthesis and cytotoxicity evaluation of 3-amino-2-hydroxypropoxyisoflavone derivatives.

Soy isoflavones exert a wide variety of biological activities, such as antioxidant, anti-inflammatory and anti-cancer properties. Nuclear factor erythroid 2-related factor 2 (Nrf2), a bZip transcription factor, plays a key role in soy isoflavones induced protection against oxidative stress and cancer. To obtain more effective isofavones, a series of 7,4'-bis-(3-amino-2-hydroxypropoxy), 7 or 4'-(3-amino-2-hydroxypropoxy) isoflavone derivatives have been synthesized as potential antitumor agents and Nrf2/ARE (antioxidant response element) activators. The cytotoxicity of these compounds in human cancer cell lines MDA-MB-231, HT-29, HCT116, HepG2 and 7402 was tested by MTT assay. In this study, the cytotoxicity of compound 3b exhibited highest cytotoxic activity and at the safety dose range, it also strongly up-regulated antioxidant response element (ARE)-luciferase reporter activity. In addition, compound 3b induced Nrf2 nuclear translocation and upregulated its downstream target genes NQO-1 and HO-1 at protein level. Taken together, our results suggest that compound 3b could be a potential agent for cancer themotherapy or cancer chemoprevention.

Seismological constraints on the crustal structures generated by continental rejuvenation in northeastern China.

Crustal rejuvenation is a key process that has shaped the characteristics of current continental structures and components in tectonic active continental regions. Geological and geochemical observations have provided insights into crustal rejuvenation, although the crustal structural fabrics have not been well constrained. Here, we present a seismic image across the North China Craton (NCC) and Central Asian Orogenic Belt (CAOB) using a velocity structure imaging technique for receiver functions from a dense array. The crustal evolution of the eastern NCC was delineated during the Mesozoic by a dominant low seismic wave velocity with velocity inversion, a relatively shallow Moho discontinuity, and a Moho offset beneath the Tanlu Fault Zone. The imaged structures and geochemical evidence, including changes in the components and ages of continental crusts and significant continental crustal growth during the Mesozoic, provide insight into the rejuvenation processes of the evolving crust in the eastern NCC caused by structural, magmatic and metamorphic processes in an extensional setting. The fossil structural fabric of the convergent boundary in the eastern CAOB indicates that the back-arc action of the Paleo-Pacific Plate subduction did not reach the hinterland of Asia.

An in vivo confocal microscopy and impression cytology analysis of goblet cells in patients with chemical burns.

PURPOSE: To evaluate goblet cell density (GCD) on conjunctiva and cornea in patients with ocular chemical burns by in vivo laser scanning confocal microscopy (LSCM) and impression cytology (IC) and to explore the correlation between two methods. METHODS: Fifty-four patients (58 eyes) with chemical burn were enrolled in the study. LSCM was applied to identify the goblet cells on conjunctiva and cornea under in vivo conditions, and GCD was analyzed with the customized software. Impression cytology was then performed, and the biopsy specimens were stained to visualize goblet cells in vitro and to measure the density. Statistical software was used to analyze the correlation between GCD taken by two methods. RESULTS: Conjunctival goblet cells could be discriminated in 55 eyes and 57 eyes by in vivo LSCM and IC. They could be identified on the cornea in nine eyes and eight eyes by two methods. The positive rate of two methods had no significant difference. GCDs on conjunctiva measured by in vivo LSCM and IC were 136 +/- 79 cells/mm(2) and 121 +/- 66 cells/mm(2). Median GCDs on cornea detected by two methods were 30 cells/mm(2) and 23 cells/mm(2), respectively. A significant positive correlation was found between the GCDs on conjunctiva measured by these two methods as well as the GCDs on cornea. CONCLUSIONS: GCD decreased in patients with chemical burns. A positive correlation was found between GCD measured by in vivo LSCM and IC after chemical burns. In vivo LSCM was a promising device to study goblet cells in vivo under pathologic conditions.

[Normal human bulbar conjunctiva on confocal microscopy in vivo].

OBJECTIVE: To analyze the morphology of human bulbar conjunctiva by in vivo laser scanning confocal microscopy. METHODS: This research was a cross-sectional study. From February to July 2008, 50 eyes of 50 healthy subjects were enrolled in this study. They had no history of ocular trauma, infection or contact lens wear and had no found after routine slit-lamp examinations. In vivo laser scanning confocal microscopic examinations were performed on the superior, inferior, nasal and temporal bulbar conjunctiva and the images were recorded. The morphology of bulbar conjunctiva was analyzed and the density of epithelial cells, dendritic cells and goblet cells were calculated. One-way analysis of variance (ANOVA) was used to compare the means of epithelial cell densities in different layers and goblet cell densities in different positions. Subsequently the datum between two groups were analyzed by least significant difference (LSD). RESULTS: Superficial epithelial cells of bulbar conjunctiva were characterized as large loose-arranged cells with a hyporeflective nucleus. The mean density is (1643 +/- 206) cells/mm(2). Intermediate epithelial cells were captured with features of oval small tight-arranged cells with a punctiform hyperreflective nucleus. The mean density is (4693 +/- 228) cells/mm(2). Basal epithelial cells appeared to be polygonal and regular-arranged within hyperreflective cell borders. The mean density is (4420 +/- 230) cells/mm(2). There was a significant difference among three kinds of conjunctival epithelium (F = 1160.312, P = 0.000). The presumed goblet cell was defined as a large hyperreflective oval-shaped cell with relatively homogeneous brightness, crowded in groups or mainly dispersed. The mean density is (432 +/- 72) cells/mm(2). The dendritic cell appeared to be hyperreflective corpuscular particles with dendritic processes scattered among conjunctival epithelial cells. The mean density is (22 +/- 25) cells/mm(2). The basement membrane, a prominent hyperreflective band, separated epithelial cells from subepithelial structure. Bulbar conjunctival substantia propria, beneath the basement membrane, was mainly composed of highly vascularized, loose connective tissues which were irregularly arranged fibers or a network of fibers, punctiform hyperreflective immune cells and sharp flows of blood vessels. CONCLUSION: In vivo laser scanning confocal microscopy is a useful tool in the analysis of the bulbar conjunctival morphology, which provided a fast and noninvasive method for the diagnosis of ocular surface diseases.

[Proliferative ability and ultramicrostructure of basal limbal epithelial cells in different age group of normal subjects].

OBJECTIVE: To demonstrate the difference in proliferative ability and ultramicrostructure of basal limbal epithelial cells in different age group of normal subjects. METHODS: It is a case-control study.40 specimens of limbus from 40 eyes provided by the eye bank of Eye Ear Nose and Throat Hospital of Fudan University in 2007 - 2008 were enrolled in this study. Only one eye was enrolled from the same donner. Specimens were divided into 4 groups according to the donners' age: A (0 - 19 years), B (20 - 39 years), C (40 - 59 years) and D (60 - 79 years). Immunohistochemistry of proliferating cell nuclear antigen (PCNA) was performed on 10 specimens of each group. The staining status of limbal epithelium was observed and the staining positive rate of the limbal basal epithelial cells was graded. And transmission electric microscopy was performed on specimens of each group. The morphologic feature of the ultramicrostructure of limbal basal epithelial cells were observed. RESULTS: 50% of specimens in group A showed a strong PCNA staining of (++++), while a weak staining of (+) took a main part in all the other 3 groups (B 40%, C 60% and D 50%, respectively). Furthermore, positive nuclear staining of limbal superficial cells was observed in 3 specimens and positive cytoplasm staining was observed in 1 specimen, all in group A. Transmission electric microscopy showed that the stem cell-like cells in group A possessed 3 morphologic features: extremely small, stretching along the basal membrane, with densely packed heterochromatin. In contrast, the stem cell-like cells in the other 3 groups did not show great disparity in cell size with their neighbor epithelial cells, and they stretched vertically to the basal membrane, with dispersed euchromatin. CONCLUSION: Difference in proliferative ability and ultramicrostructure of basal limbal epithelial cells was found in different age group of normal subjects.

[The influence of lens and vitreous resection on anterior chamber associated immune deviation in mice model].

OBJECTIVE: To explore the impact of lens and vitreous resection on the induction of the anterior chamber associated immune deviation (ACAID) in the mice, and its relationship with the time duration postoperatively. METHODS: The current study was an experimental research. Fifty-one Wistar mice were chosen to involve this study. After the surgeries, 50 Wistar mice were randomly divided into 5 groups. Other 5 SD mice and 1 Wistar mouse were used as the providers of epithelium removed corneal grafts to induce ACAID. Group A: corneal grafts from wistar mouse were implanted into the anterior chamber 1 week after the lens and vitreous resection. Group B: the spleen cells were injected into the nape of neck 1 week after the lens and vitreous resection. The corneal grafts from SD mice were implanted into the anterior chamber 1 week, 4 week and 8 week after the lens and vitreous resection in group C, D and E respectively. To induce the delayed type hypersensitivity (DTH), spleen cells were injected into the right ear pinna 1 week after the neck injection in group B, 2 weeks after the corneal grafts anterior chamber implantation in other groups. At the same time point as the DTH inducement, aqueous humor was collected to measure the concentration of transforming growth factor (TGF) -beta2 and interleukin (IL)-10; heart blood was collected to detect the concentration of IL-4 and IL-10. And the spleens were removed to evaluate the expression of the GATA-3 mRNA by RT-PCR. The eyeballs were enucleated to evaluate the histopathologic changes. RESULTS: (1) The evaluation of DTH. The DTH were found in group B, C, D and E. (2) The analysis of the TGF-beta2 and IL-10 concentrations in aqueous humor and the IL-4, IL-10 concentrations in serum. The concentrations of IL-4 in serum were (4.073 +/- 0.198) ng/L in group A, (5.806 +/- 0.635) ng/L in group B, (5.535 +/- 0.278) ng/L in group C, (4.102 +/- 0.344) ng/L in group D and (5.313 +/- 0.317) ng/L in group E. The concentrations of IL-10 in serum in group A to group E were (7.854 +/- 2.349) ng/L, (25.633 +/- 6.307) ng/L, (40.103 +/- 16.010) ng/L, (14.321 +/- 2.983) ng/L and (28.620 +/- 5.251) ng/L. The IL-10 concentrations in the aqueous humor were (8.857 +/- 0.401) ng/L, (22.882 +/- 3.315) ng/L, (21.548 +/- 0.477) ng/L, (7.742 +/- 0.952) ng/L and (12.119 +/- 0.477) ng/L, respectively. The concentrations of TGF-beta in the aqueous humor were (5.800 +/- 2.899) ng/L in group A, (60.010 +/- 0.000) ng/L in group B, (57.055 +/- 4.179) ng/L in group C, (28.490 +/- 4.144) ng/L in group D and (36.370 +/- 3.169) ng/L in group E. Significant differences were found in concentrations of TGF-beta2 and IL-10 in aqueous humor and the concentrations of IL-4 and IL-10 in serum in group C, D and E. The concentrations of TGF-beta2, IL-4 and IL-10 were significantly lower in group D than those in group C and E. The trend that the concentrations of TGF-beta2, IL-4 and IL-10 increased after one week postoperatively, then decreased, and finally increased again was observed. (3) The expression of GATA-3 mRNA in spleen. The expression values of GATA-3 mRNA were 662.5 +/- 114.4 in group A, 730.7 +/- 53.8 in group B, 881.9 +/- 10.7 in group C, 1288.3 +/- 258.0 in group D and 1129.7 +/- 95.7 in group E. An obvious up-regulation of the GATA-3 mRNA was noted in group D and E. CONCLUSIONS: The eye after the lens and vitreous resection lost the ability to induce ACAID in the research duration of this study. But with the gradually decrease of the intraocular inflammation, the ability to induce ACAID might recover with the time.

[Evolving concepts on limbal stem cells and their niche].

As a concentrated location for adult somatic stem cells, the niche plays a crucial role on maintaining the high proliferative ability and the inducing the particular differentiation of the stem cells by providing the special microenvironment containing definite extracellular matrix and niche cells. Nowadays limbal stem cells (LSCs) are the acknowledged adult somatic stem cells of corneal epithelium. Rapid progress has been accomplished in the theory and clinical practice on LSCs. However, the understanding on its niche has obviously fallen behind. This review summarized and analyzed the status quo of the study on niche of LSCs, mainly through the following aspects: the precise location of the niche was described as limbal epithelial crypts; the blood-derived and stroma-derived cytokine could regulate the proliferation, differentiation and apoptosis of LSCs by triggering particular intracellular information pathway; the survival and function of LSCs could be influenced by damaged niche microenvironment, while the differentiation of LSCs could be veered by another type of niche; in ex vivo LSCs expansion, reconstruction of the niche is needed and could be achieved by 3T3 cells or using amniotic membrane as the carrier.

[Variations of the microstructure in human limbus by confocal microscopy in vivo].

OBJECTIVE: To demonstrate the variations in the niche of limbal stem cells and the limbal basal epithelial cells in vivo by confocal microscopy. METHODS: One hundred and twenty eyes of 120 healthy subjects were enrolled in this study after a routine slit-lamp examination. They were divided into four groups according to the age: group A (0-), group B (20-), group C (40-) and group D (60 - 79). Confocal microscopic examination in vivo was performed and the images of the inferior limbus were recorded. The morphologic features of each group were analyzed and the average size of limbal basal epithelial cells of each group were measured. RESULTS: The morphologic features of limbus varied among groups. 96.7% (29/30) of group A showed clear Vogt Palisades, while 3.3% (1/30) showed no Vogt Palisades. 96.7% (29/30) of group B also showed clear Vogt Palisades, while 3.3% (1/30) showed no Vogt Palisades.70.0% (21/30) of group C showed clear Vogt Palisades, while 13.3% (4/30) showed atrophic Vogt Palisades and 16.7% (5/30) showed no Vogt Palisades at all. 33.3% (10/30) of group C showed clear Vogt Palisades, while 10.0% (3/30) showed atrophic Vogt Palisades and 56.7% (17/30) showed no Vogt Palisades at all. The average size of limbal basal epithelial cells in each group was (9.89 +/- 1.12), (10.65 +/- 1.45), (10.70 +/- 1.39) and (12.22 +/- 1.42) microm in sequence. CONCLUSIONS: There is variations in the microenvironment of niche in human limbus. Meanwhile, limbal basal epithelial cells showed different quantity and cell size, demonstrating varied proliferative potential.

[Morphological changes of cornea in iridocorneal endothelial syndrome under the confocal microscopy].

OBJECTIVE: To evaluate the morphological changes of cornea in iridocorneal endothelial syndrome (ICE) under the examination of in vivo confocal microscopy. METHODS: The experimental design was retrospective observation case series. Twenty-three eyes of 23 consecutive patients, each diagnosed as ICE, had their both eyes examined with the in vivo confocal microscopy (NIDEK, confoscan 3.0). The images were recorded and analyzed by software NAVIS. Measurements were performed on endothelium density, average endothelial area, the percentage of hexagonal cells and the percentage of endothelium with nuclei, and the ANOVA was done to assess the differences. RESULTS: In vivo confocal microscopy highlighted two main patterns of endothelial changes: "kite-like" and "epithelial-like" abnormal endothelium, characterised by marked hyperreflective nuclei and loss of regularity in cellular size and shape. With the progression of disease, the endothelium density and the percentage of hexagonal endothelial cells decreased, which were (1687.1 +/- 122.6), (1210.6 +/- 168.7), (947.3 +/- 145.2), (856.8 +/- 73.4) cells/mm2 and (51.5 +/- 6.3)%, (39.8 +/- 9.2)%, (32.7 +/- 8.1)%, (24.1 +/- 5.6)% respectively in detail. In contrast, the average endothelial area the percentage of endothelium with nuclei increased, which were (678.3 +/- 56.3), (928.7 +/- 96.2), (1188.5 +/- 72.6), (1337.5 +/- 60.8) microm2 and (12.6 +/- 1.4)%, (56.8 +/- 3.7)%, (78.7 +/- 5.6)%, (84.3 +/- 2.8)%. The differences all had statistical significance (F = 7.158, 7.736, 6.876, 14.452 respectively, P = 0.000). The morphology of keratocyte and endothelium were normal, compared with the contralateral healthy eyes. However, the stromal nerves became thicker and more tortuous with the disease advancement. CONCLUSIONS: The application of confocal microscopy indicates that the ICE is characterised by epithelial-like endothelial cells with hyperreflective nuclei. The technique has great potential in diagnosing ICE, especially in evaluating the disease progression.