Ginsenoside compound K induces ferroptosis via the FOXO pathway in liver cancer cells.

Liver cancer is a common malignant tumor worldwide, traditional Chinese medicine is one of the treatment measures for liver cancer because of its good anti-tumor effects and fewer toxic side effects. Ginsenoside CK (CK) is an active component of ginseng. This study explored the mechanism by which CK induced ferroptosis in liver cancer cells. We found that CK inhibited the proliferation of HepG2 and SK-Hep-1 cells, induced ferroptosis of cells. Ferrostatin-1, an ferroptosis inhibitor, was used to verify the role of CK in inducing ferroptosis of liver cancer cells. Network pharmacological analysis identified the FOXO pathway as a potential mechanism of CK, and western blot showed that CK inhibited p-FOXO1. In cells treated with the FOXO1 inhibitor AS1842856, further verify the involvement of the FOXO pathway in regulating CK-induced ferroptosis in HepG2 and SK-Hep-1 cells. A HepG2 cell-transplanted tumor model was established in nude mice, and CK inhibited the growth of transplanted tumors in nude mice, p-FOXO1 was decreased in tumor tissues, and SLC7A11 and GPX4 expressions were also down-regulated after CK treatment. These findings suggested that CK induces ferroptosis in liver cancer cells by inhibiting FOXO1 phosphorylation and activating the FOXO signaling pathway, thus playing an antitumor role.

Intravitreal injection of new adeno-associated viral vector: Enhancing retinoschisin 1 gene transduction in a mouse model of X-linked retinoschisis.

Adeno-associated virus (AAV) vectors have been widely used in therapy to treat hereditary retinal diseases. But its transduction efficiency by intravitreal injection still needs to be improved. In this study, we investigated the transduction efficiency of AAV-DJ (K137R)-GFP in different retinal cells of normal mice, as well as the therapy effection of AAV-DJ (K137R)-Rs1 on retinal function and structure in Rs1-KO mice. The intravitreal injection of AAV-DJ (K137R)-GFP demonstrated that this vector transduced cells in all layers of the retina, including the inner nuclear layer and photoreceptor layer. The intravitreal injection of AAV-DJ (K137R)-Rs1 found that 3 months post-injection of this vector improved retinal function and structure in Rs1-KO mice. Our conclusion is that AAV-DJ (K137R) vector can efficiently and safely penetrate the inner limiting membrane and transduce different layers of retinal cells in the long term, as well as being able to continuously and efficiently express target therapeutic proteins, making it a candidate therapeutic vector for X-linked retinoschisis (XLRS).

Association of 10 Genetic Variations and 10 Environmental Factors with Myopia of Different Severities in Different Age Groups of People in Northeast China.

BACKGROUND: To investigate the association of 10 genetic variations and 10 environmental factors with myopia of different severities in different age groups of children and adolescents in northeast China. METHODS: Parental history and genetic testing for myopia-related susceptibility genes were carried out in a cohort of children and adolescents aged 2-17 years. In addition, 10 single nucleotide polymorphism (SNP) sites for genotyping and 10 environmental risk factors were selected, and the differences between site variation and environmental factors in different age groups with different degrees of myopia were explored. RESULTS: A total of 2497 volunteers were recruited, including 2023 myopes and 474 non-myopes in the control group. From the cohort, 1160 subjects were sequenced for myopia SNP sites. Compared with the non-myopic group, the myopia of parents, outdoor activity less than 60 min per day, and a high-sugar diet were risk factors for developing myopia. Two syntrophin beta 1 (SNTB1) sites, rs4455882 and rs6469937 were found to be significantly associated with moderate myopia; fibroblast growth factor 10 (FGF10) rs339501 was significantly correlated with high myopia; and insulin-like growth factor 1 (IGF1) rs5742714 was significantly correlated with different degrees of myopia in the age group of <6 years. Finally, the FGF10 gene rs339501 SNP was significantly associated with moderate myopia and mild myopia in the 6- to 12-year-old age group. CONCLUSIONS: Our results indicate that myopia is affected by both environmental and genetic factors. To prevent and control myopia, attention should be paid to the parental history of myopia, a high-sugar diet should be avoided, and outdoor time should be adjusted according to the average daily sunshine. In addition, it is necessary to pay attention to the increased risk of myopia in school-age children caused by SNTB1 rs4455882, FGF10 rs339501, and IGF1 rs5742714.

Towards genetic improvement of social behaviours in livestock using large-scale sensor data: data simulation and genetic analysis.

BACKGROUND: Harmful social behaviours, such as injurious feather pecking in poultry and tail biting in swine, reduce animal welfare and production efficiency. While these behaviours are heritable, selective breeding is still limited due to a lack of individual phenotyping methods for large groups and proper genetic models. In the near future, large-scale longitudinal data on social behaviours will become available, e.g. through computer vision techniques, and appropriate genetic models will be needed to analyse such data. In this paper, we investigated prospects for genetic improvement of social traits recorded in large groups by (1) developing models to simulate and analyse large-scale longitudinal data on social behaviours, and (2) investigating required sample sizes to obtain reasonable accuracies of estimated genetic parameters and breeding values (EBV). RESULTS: Latent traits were defined as representing tendencies of individuals to be engaged in social interactions by distinguishing between performer and recipient effects. Animal movement was assumed random and without genetic variation, and performer and recipient interaction effects were assumed constant over time. Based on the literature, observed-scale heritabilities ([Formula: see text]) of performer and recipient effects were both set to 0.05, 0.1, or 0.2, and the genetic correlation ([Formula: see text]) between those effects was set to - 0.5, 0, or 0.5. Using agent-based modelling, we simulated ~ 200,000 interactions for 2000 animals (~ 1000 interactions per animal) with a half-sib family structure. Variance components and breeding values were estimated with a general linear mixed model. The estimated genetic parameters did not differ significantly from the true values. When all individuals and interactions were included in the analysis, the accuracy of EBV was 0.61, 0.70, and 0.76 for [Formula: see text] = 0.05, 0.1, and 0.2, respectively (for [Formula: see text]= 0). Including 2000 individuals each with only ~ 100 interactions, already yielded promising accuracies of 0.47, 0.60, and 0.71 for [Formula: see text] = 0.05, 0.1, and 0.2, respectively (with [Formula: see text] = 0). Similar results were found with [Formula: see text] of - 0.5 or 0.5. CONCLUSIONS: We developed models to simulate and genetically analyse social behaviours for animals that are kept in large groups, anticipating the availability of large-scale longitudinal data in the near future. We obtained promising accuracies of EBV with ~ 100 interactions per individual, which would correspond to a few weeks of recording. Therefore, we conclude that animal breeding can be a promising strategy to improve social behaviours in livestock.

Case report: Familial foveal retinoschisis caused by CRB1 gene mutation in a family with recessive inheritance.

X-linked retinoschisis is more common in male children and rare in females. Clinically, male patients mainly present with early onset visual impairment or vision loss, and retinal retinoschisis due to division of the inner retina. We report a long-term observation of a female patient with familial foveal retinoschisis (FFR) caused by CRB1 gene with complex heterozygotic mutation. The initial symptoms of the female patient reported in this study were very similar to some early manifestations of X-linked retinoschisis (XLRS) caused by RS1 mutations involving macular fovea. However, as time going on, the splitting height at retinal fovea of FFR gradually decreased, and the splitting extent at retinal fovea of FFR gradually decreased.

De Novo Mutations Contributes Approximately 7% of Pathogenicity in Inherited Eye Diseases.

Purpose: The purpose of this study was to describe genotype-phenotype associations and novel insights into genetic characteristics in a trio-based cohort of inherited eye diseases (IEDs). Methods: To determine the etiological role of de novo mutations (DNMs) and genetic profile in IEDs, we retrospectively reviewed a large cohort of proband-parent trios of Chinese origin. The patients underwent a detailed examination and was clinically diagnosed by an ophthalmologist. Panel-based targeted exome sequencing was performed on DNA extracted from blood samples, containing coding regions of 792 IED-causative genes and their flanking exons. All participants underwent genetic testing. Results: All proband-parent trios were divided into 22 subgroups, the overall diagnostic yield was 48.67% (605/1243), ranging from 4% to 94.44% for each of the subgroups. A total of 108 IED-causative genes were identified, with the top 24 genes explaining 67% of the 605 genetically solved trios. The genetic etiology of 6.76% (84/1243) of the trio was attributed to disease-causative DNMs, and the top 3 subgroups with the highest incidence of DNM were aniridia (n = 40%), Marfan syndrome/ectopia lentis (n = 38.78%), and retinoblastoma (n = 37.04%). The top 10 genes have a diagnostic yield of DNM greater than 3.5% in their subgroups, including PAX6 (40.00%), FBN1 (38.78%), RB1 (37.04%), CRX (10.34%), CHM (9.09%), WFS1 (8.00%), RP1L1 (5.88%), RS1 (5.26%), PCDH15 (4.00%), and ABCA4 (3.51%). Additionally, the incidence of DNM in offspring showed a trend of correlation with paternal age at reproduction, but not statistically significant with paternal (P = 0.154) and maternal (P = 0.959) age at reproduction. Conclusions: Trios-based genetic analysis has high accuracy and validity. Our study helps to quantify the burden of the full spectrum IED caused by each gene, offers novel potential for elucidating etiology, and plays a crucial role in genetic counseling and patient management.

Study of the biological characteristics of human umbilical cord mesenchymal stem cells after long-time cryopreservation.

Human umbilical cord mesenchymal stem cells (hUC-MSCs) have considerable potential in cell therapy. Cryopreservation represents the gold standard in cell storage, but its effect on hUC-MSCs is still not well understood. The aim of this study was to investigate the effect of one year of cryopreservation and thawing on the biological characteristics of hUC-MSCs from the same donors. Fresh hUC-MSCs were cryopreserved in commercial freezing medium (serum-free CellBanker 2) at passage 2. After one year of cryopreservation, the hUC-MSCs were thawed and subcultured to passage 4. The comparison was performed in terms of followings: cell count, viability, morphology, proliferation capacity, differentiation potential and chromosomal stability. The total cell count and viability of hUC-MSCs before and after one year of cryopreservation were 1 × 107 and 96.34% and 0.943 × 107 and 93.81%, respectively. Cryopreserved and fresh hUC-MSCs displayed a similar cell doubling times, expressed the markers CD73, CD90, CD105 and were negative for the markers CD34, CD45, and HLA-DR. Karyotypes were found to be normal after one year of cryopreservation. The trilineage differentiation properties were maintained after cryopreservation. However, when compared to freshly isolated hUC-MSCs from the same donor, cryopreserved hUC-MSCs exhibited decreased expression of osteogenesis- and chondrogenesis-related genes including Runx2, Sox9, and Col1a1, and increased expression of adipogenesis-related genes. These results demonstrated that cryopreservation did not affect cell morphology, surface marker expression, cell viability, proliferative capacity, or chromosomal stability. However, the osteogenic and chondrogenic differentiation capacities of cryopreserved hUC-MSCs were slightly reduced compared with those of fresh cells from the same donor.

Evaluation of the Genetic Variation Spectrum Related to Corneal Dystrophy in a Large Cohort.

AIMS: To characterize the genetic landscape and mutation spectrum of patients with corneal dystrophies (CDs) in a large Han ethnic Chinese Cohort with inherited eye diseases (IEDs). METHODS: Retrospective study. A large IED cohort was recruited in this study, including 69 clinically diagnosed CD patients, as well as other types of eye diseases patients and healthy family members as controls. The 792 genes on the Target_Eye_792_V2 chip were used to screen all common IEDs in our studies, including 22 CD-related genes. RESULTS: We identified 2334 distinct high-quality variants on 22 CD-related genes in a large IEDs cohort. A total of 21 distinct pathogenic or likely pathogenic mutations were identified, and the remaining 2313 variants in our IED cohort had no evidence of CD-related pathogenicity. Overall, 81.16% (n = 56/69) of CD patients received definite molecular diagnoses, and transforming growth factor-beta-induced protein (TGFBI), CHTS6, and SLC4A11 genes covered 91.07, 7.14, and 1.79% of the diagnosed cases, respectively. Twelve distinct disease-associated mutations in the TGFBI gene were identified, 11 of which were previously reported and one is novel. Four of these TGFBI mutations (p.D123H, p.M502V, p.P501T, and p.P501A) were redefined as likely benign in our Han ethnic Chinese IED cohort after performing clinical variant interpretation. These four TGFBI mutations were detected in asymptomatic individuals but not in CD patients, especially the previously reported disease-causing mutation p.P501T. Among 56 CD patients with positive detected mutations, the recurrent TGFBI mutations were p.R124H, p.R555W, p.R124C, p.R555Q, and p.R124L, and the proportions were 32.14, 19.64, 14.29, 10.71, and 3.57%, respectively. Twelve distinct pathogenic or likely pathogenic mutations of CHTS6 were detected in 28 individuals. The recurrent mutations were p.Y358H, p.R140X, and p.R205W, and the proportions were 25.00, 21.43, and 14.29%, respectively. All individuals associated with TGFBI were missense mutations; 74.19% associated with CHTS6 mutations were missense mutations, and 25.81% were non-sense mutations. Hot regions were located in exons 4 and 12 of TGFBI individuals and located in exon 3 of CHTS6 individuals. No de novo mutations were identified. CONCLUSION: For the first time, our large cohort study systematically described the variation spectrum of 22 CD-related genes and evaluated the frequency and pathogenicity of all 2334 distinct high-quality variants in our IED cohort. Our research will provide East Asia and other populations with baseline data from a Han ethnic population-specific level.

Portable Handheld Slit-Lamp Based on a Smartphone Camera for Cataract Screening.

PURPOSE: As part of plans to provide help to people in remote and poor areas who have no medical resources, a portable slit-lamp based on a smartphone was proposed. This would help in early screening of cataract diseases. METHODS: This means a microlens is designed that would work with a phone's camera. The phone's photo taking function is used in capturing the image of the eyes lens to replace the observation system of the desktop slit-lamp. A simplified slit light band was designed. In order for the light source part to meet the portable requirements of the slit-lamp, the adjustable and diffused light functions of the ligaments were removed in this design. Furthermore, the images collected by the smartphone are uploaded to the deep learning cataract screening system, which can achieve real-time and effective screening of cataract. RESULTS: Unlike the desktop slit-lamp, which needs skilled personnel to operate, this device can be easily operated by less-skilled or inexperienced doctors. This eliminates the concerns of inaccurate diagnosis based on the use of unskilled professionals. Due to the portability, ease of use, and simplicity in obtaining crystal images of this device, it serves as a promising platform for nonhospital screening and telemedicine. CONCLUSIONS: In this paper, we invented a small portable device for screening cataract. This device is to make screening and diagnosis of cataract in remote areas very fast and effective. It will also solve the problem of inadequate specialized doctors and equipment in those areas as well. Translational Relevance. Smartphones can be used with portable slit-lamps to capture the images of the lens.

Intravitreal Injection of Human Retinal Progenitor Cells for Treatment of Retinal Degeneration.

BACKGROUND Retinal degeneration causes irreversible blindness. Human retinal progenitor cells (hRPCs) have the potential to treat retinal diseases. The vitreous cavity is a relatively immune-privileged site that is suitable for stem cell transplantation in the treatment of retinal diseases. This study aimed to evaluate the therapeutic efficacy and safety of intravitreal injection of hRPCs in retinal degeneration therapy. MATERIAL AND METHODS hRPCs were primary-cultured and injected into the vitreous cavity of RCS rats. To determine whether hRPCs formed teratomas in immune-deficient mice, hRPCs at different passages were transplanted into BALB/c-nu mice. The visual function was detected by electroretinography recording. Changes in the outer nuclear layer (ONL) were analyzed by histological testing and cell counting. The protective mechanism was further assessed by cytokine antibody array. RESULTS Intravitreal transplantation of hRPCs maintained retinal function and preserved retinal morphology. Importantly, grafted cells in the vitreous cavity were well tolerated, with no adverse effects. Teratoma was not formed in BALB/c-nu mice after hRPCs transplantation. The number of hRPCs-injected eyes and thickness of ONL in the hRPCs-treated group were higher than those in the untreated group and HBSS injection group. The cytokine antibody array revealed that hRPCs expressed GDF-15, PDGF-AA, EGF, and NT-4. CONCLUSIONS Our findings show that intravitreal injection of hRPCs is effective and safe in protecting photoreceptor cells in RCS rats, but were no longer effective at 12 weeks after transplantation. Moreover, hRPCs released multiple neurotrophic factors that may be involved in treating retinal disease.

Genetic and clinical findings of panel-based targeted exome sequencing in a northeast Chinese cohort with retinitis pigmentosa.

BACKGROUND: Panel-based targeted exome sequencing was used to analyze the genetic and clinical findings of targeted genes in a cohort of northeast Chinese with retinitis pigmentosa. METHODS: A total of 87 subjects, comprising 23 probands and their family members (total patients: 32) with confirmed retinitis pigmentosa were recruited in the study. Panel-based targeted exome sequencing was used to sequence the patients and family members, all subjects with retinitis pigmentosa underwent a complete ophthalmologic examination. RESULTS: Of the 23 probands, the clinical manifestations include night blindness, narrowing of vision, secondary cataracts, choroidal atrophy, color blindness, and high myopia, the average age of onset of night blindness is 12.9 ± 14 (range, 0-65; median, 8). Posterior subcapsular opacities is the most common forms of secondary cataracts (nine cases, 39.1%), and peripheral choroidal atrophy is the most common form of secondary choroidal atrophy (12 cases, 52.2%). Of these probands with complication peripheral choroidal atrophy, there were eight probands (66.7%, 8/12) caused by the pathogenic variation in USH2A gene. A total of 17 genes and 45 variants were detected in 23 probands. Among these genes, the commonest genes were USH2A (40%; 18/45), RP1 (15.6%; 7/45), and EYS (8.9%; 4/45), and the top three genes account for 56.5% (13/23) of diagnostic probands. Among these variants, comprising 22 (48.9%) pathogenic variants, 14 (31%) likely pathogenic variants, and nine (20%) uncertain clinical significance variants, and 22 variants was discovered first time. Most of the mutations associated with RP were missense (53.3%, 24/45), and the remaining mutation types include frameshift (35.6%, 16/45), nonsense (6.7%, 3/45), and spliceSite (4.4%, 2/45). Among the probands with mutations detected, compound heterozygous forms was detected in 13 (56.5%, 13/23) probands, and digenic inheritance (DI) forms was detected in five (21.7%, 5/23) probands. CONCLUSION: Panel-based targeted exome sequencing revealed 23 novel mutations, recognized different combinations forms of variants, and extended the mutational spectrum of retinitis pigmentosa and depicted common variants in northeast China.

Genetic and clinical analysis in Chinese patients with retinitis pigmentosa caused by EYS mutations.

BACKGROUND: Panel-based targeted exome sequencing was applied to identify the pathogenic variants and genetic characteristics of retinitis pigmentosa (RP) in two Chinese families, and to gain a deeper understanding of the relationship between clinical manifestations and genotypes. METHODS: A total of 17 subjects, comprising two probands (total patients: four subjects) and their family member, were recruited in this study. All subjects underwent comprehensive ophthalmic examinations and clinical evaluations, and the complete history and medical records were collected according to the standard procedures. All participants were screened using the multigene panel test (Target_Eye_792_V2 chip), and Sanger sequencing was used to confirm the candidate variants. RESULTS: Among these two families, a total of three novel mutations in the EYS gene were identified in patients, including a homozygous frameshift mutation c.9252_9253insT detected in two patients in one family, and the compound heterozygous splicesite mutation c.5644+2T>C and frameshift mutation c.1920_1923delTGAG detected in two patients in the another family. All patients in both families had early onset of night blindness and poor visual acuity, and with typical posterior capsule opacification. The mutation co-segregated within all recruited individuals. In addition, one patient with compound heterozygous mutations was found to have typical blue-blindness symptoms and detected a previously reported disease-causing mutation c.235G>A in OPN1SW gene, which caused blue blindness manifestations and was first discovered in patient combined with RP causative genes. CONCLUSIONS: Panel-based targeted exome sequencing was used to identify three novel variants of RP causative gene, and we also detected a known pathogenic variants of blue-blindness causative genes in two patients. Our finding will provide a powerful basis for genetic counseling and enhance our current understanding of the genetics factors for RP families.

A start codon mutation of the TSPAN12 gene in Chinese families causes clinical heterogeneous familial exudative vitreoretinopathy.

BACKGROUND: Familial exudative vitreoretinopathy (FEVR) is a severe clinically and genetically heterogeneous retinal disorder characterized with failure of vascular development of the peripheral retina. The symptoms of FEVR vary widely among patients in the same family, and even between the two eyes of a given patient. The purpose of this study was to investigate the molecular mechanisms by which the start codon mutation of the TSPAN12 causes difference in clinical manifestations between individuals in the same family. METHODS: Next-generation sequencing (NGS)-based target capture sequencing was performed in proband with a diagnosis of FEVR and their normal visual acuity family members. Cosegregation analysis of the candidate causative variant was performed in additional family members by using Sanger sequencing. Complete fundus examination, fundus fluorescein angiography (FFA), and family history collection were performed in all family members. Potential candidate causative variants were verified with reference to guidelines and standards from the American College of Medical Genetics and Genomics. RESULTS: We identified a novel heterozygous missense mutation (c.1A>G, p.M1V) localized in the start codon of the TSPAN12 and was detected as a potentially disease-causing variant for the proband. Retrospective analysis of clinical data, fundus examination, and FFA showed that the mutant carrier presented peripheral retinal vascular anomalies in early stages, and visual acuity did not show significant effects. However, the proband who carried this mutation and his cousin showed typical high-stage FEVR fundus changes coupled with a sharp decline in vision. CONCLUSIONS: We report a novel start codon mutation (c.1A>G, p.M1V) in the TSPAN12 that causes clinically heterogeneous manifestations. Our results expand the mutation spectrums of TSPAN12, and will be valuable for disease diagnosis, prognosis, genetic counseling, and enriching our understanding of the role of the tetraspanin-12 protein in the pathogenesis of FEVR.

The Effect of Age on the Regenerative Potential of Human Eyelid Adipose-Derived Stem Cells.

Human eyelid adipose-derived stem cells (HEASCs) are a new source of autologous mesenchymal stem cells, which are derived from neuroectoderm and potentially applied in the tissue regeneration and cell therapies. Based on the prevalence of blepharoplasty in Asia and the availability of HEASCs, we investigated the effect of donor age on their characteristics and regenerative potential of HEASCs in vitro. The HEASCs were isolated from patients of three groups: (1) <20 years (n = 4), (2) >20 years, <45 years (n = 5), and (3) >55 years (n = 4). For each group, the proliferative capacity, colony-forming ability, surface markers, differentiation ability, wound healing function, and secreted protein were contrastively evaluated and quantified for statistical analysis. It was found that HEASCs were successfully isolated and cultured by an explant culture method. The proliferative rates, osteogenic and chondrogenic differentiation potentials, wound healing ability, and the expression of TGF-ß1 and fibronectin protein of HEASCs significantly decreased as age increased. However, the expression of CD90 antigen and the adipogenic differentiation showed an age-related increase in HEASCs. As many degenerative diseases increase in prevalence with age, the age-related changes of the HEASCs proliferation potential, differentiation capacity, and wound healing ability should be taken into account whenever they are intended for use in research or cytotherapy.

The condition medium of mesenchymal stem cells promotes proliferation, adhesion and neuronal differentiation of retinal progenitor cells.

Retinal progenitor cell is a promising candidate in the treatment of retinal pigmentosa diseases. The limiting factors of stem cell transplantation are the proliferation and differentiation capacities of hRPCs, which may be governed by culture conditions. Previous studies have proved that the secretome of human Umbilical Cord Mesenchymal stem cells (hUCMSCs) and human Adipose derived stem cells (hADSCs), including more active cytokines and neurotrophic factors, have the paracrine potential of enhancing proliferation and differentiation in several cell types. The aim of this study was to investigate whether hRPCs could effectively proliferate, adhere and differentiate towards specific retinal cell types by treating with the condition medium (CM) of hUCMSCs (hUCMSCCM) or hADSCs (hADSCCM). Here, we show that hUCMSCCM or hADSCCM enhances the proliferation rate of the S and G2 phase cells, with an upregulation of Ki67 expression. Moreover, the upregulation expression of NF, Recoverin and Rhodopsin indicates that specialized retinal cells including ganglion cells and photoreceptors are favored over hRPCs differentiation due to hUCMSCCM or hADSCCM. Under FBS induced differentiation conditions, hRPCs treated with hUCMSCCM or hADSCCM increase the expression of retinal neuron and photoreceptor specific markers. These results suggest that hUCMSCCM and hADSCCM can stimulate the hRPC proliferation, promote its adherence and support hRPC neuronal and photoreceptor differentiation. These findings may provide a new strategy to improve the viability of hRPCs and photoreceptor differentiation capacities.

Inhibition of DNA Methylation and Methyl-CpG-Binding Protein 2 Suppresses RPE Transdifferentiation: Relevance to Proliferative Vitreoretinopathy.

PURPOSE: The purpose of this study was to evaluate expression of methyl-CpG-binding protein 2 (MeCP2) in epiretinal membranes from patients with proliferative vitreoretinopathy (PVR) and to investigate effects of inhibition of MeCP2 and DNA methylation on transforming growth factor (TGF)-ß-induced retinal pigment epithelial (RPE) cell transdifferentiation. METHODS: Expression of MeCP2 and its colocalization with cytokeratin and α-smooth muscle actin (α-SMA) in surgically excised PVR membranes was studied using immunohistochemistry. The effects of 5-AZA-2'-deoxycytidine (5-AZA-dC) on human RPE cell migration and viability were evaluated using a modified Boyden chamber assay and the colorimetric 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay. Expression of RASAL1 mRNA and its promoter region methylation were evaluated by real-time PCR and methylation-specific PCR. Effects of 5-AZA-dC on expression of α-SMA, fibronectin (FN), and TGF-ß receptor 2 (TGF-ß R2) and Smad2/3 phosphorylation were analyzed by Western blotting. Effect of short interfering RNA (siRNA) knock-down of MeCP2 on expression of α-SMA and FN induced by TGFß was determined. RESULTS: MeCP2 was abundantly expressed in cells within PVR membranes where it was double labeled with cells positive for cytokeratin and α-SMA. 5-AZA-dC inhibited expression of MeCP2 and suppressed RASAL1 gene methylation while increasing expression of the RASAL1 gene. Treatment with 5-AZA-dC significantly suppressed the expression of α-SMA, FN, TGF-ß R2 and phosphorylation of Smad2/3 and inhibited RPE cell migration. TGF-ß induced expression of α-SMA, and FN was suppressed by knock-down of MeCP2. CONCLUSIONS: MeCP2 and DNA methylation regulate RPE transdifferentiation and may be involved in the pathogenesis of PVR.

Liquid crystalline assembly of coil-rod-coil molecules with lateral methyl groups into 3-D hexagonal and tetragonal assemblies.

In this paper, we report the synthesis and self-assembly behavior of coil-rod-coil molecules, consisting of three biphenyls linked through a vinylene unit as a conjugated rod segment and poly(ethylene oxide) (PEO) with a degree of polymerization (DP) of 7, 12 and 17, incorporating lateral methyl groups between the rod and coil segments as the coil segment. Self-organized investigation of these molecules by means of differential scanning calorimetry (DSC), thermal polarized optical microscopy (POM) and X-ray diffraction (XRD) reveals that the lateral methyl groups attached to the surface of rod and coil segments, dramatically influence the self-assembling behavior in the liquid-crystalline mesophase. Molecule 1 with a relatively short PEO coil length (DP=7) self-assembles into rectangular and oblique 2-dimensional columnar assemblies, whereas molecules 2 and 3 with DP of 12 and 17 respectively, spontaneously self-organize into unusual 3-dimensional hexagonal close-packed or body-centered tetragonal assemblies.