The use of a SOX10 reporter toward ameliorating oligodendrocyte lineage differentiation from human induced pluripotent stem cells.

Oligodendrocytes (OLs) are key players in the central nervous system, critical for the formation and maintenance of the myelin sheaths insulating axons, ensuring efficient neuronal communication. In the last decade, the use of human induced pluripotent stem cells (iPSCs) has become essential for recapitulating and understanding the differentiation and role of OLs in vitro. Current methods include overexpression of transcription factors for rapid OL generation, neglecting the complexity of OL lineage development. Alternatively, growth factor-based protocols offer physiological relevance but struggle with efficiency and cell heterogeneity. To address these issues, we created a novel SOX10-P2A-mOrange iPSC reporter line to track and purify oligodendrocyte precursor cells. Using this reporter cell line, we analyzed an existing differentiation protocol and shed light on the origin of glial cell heterogeneity. Additionally, we have modified the differentiation protocol, toward enhancing reproducibility, efficiency, and terminal maturity. Our approach not only advances OL biology but also holds promise to accelerate research and translational work with iPSC-derived OLs.

CHARACTERISTICS OF 'NOTCH' IN RETINOPATHY OF PREMATURITY FOLLOWING INTRAVITREAL RANIBIZUMAB MONOTHERAPY.

PURPOSE: To explore the clinical features and significance of "notch" in reactivation of retinopathy of prematurity (ROP) post-intravitreal ranibizumab (IVR) monotherapy. METHODS: Ninety-six infants (173 eyes) with type 1 or aggressive ROP (A-ROP) post-IVR monotherapy were retrospectively analyzed; 51 eyes were notch (+) and 122 eyes were notch (-). General demographics and clinical outcomes were compared by notch status for type 1 and A-ROP. RESULTS: The notch primarily appeared in stage 2 ROP (84.4 and 78.9%) at the junction of zones I and II (68.8 and 63.2%) on the temporal side in type 1 ROP and A-ROP. Notch was present in the type 1 ROP group before first IVR but post-treatment in the A-ROP group. A significantly higher reactivation rate, longer follow-up duration, and postmenstrual age at last follow-up were seen in the notch (+) versus the notch (-) group. In the notch (+) ROP group, the mean gestational age (28.34±0.93 vs. 29.94±1.48 weeks) was significantly lower in reactivated versus regressed eyes. CONCLUSION: Notches appeared at different times but similar locations in type 1 ROP and A-ROP. The reactivation rate after IVR was increased in ROP with notches. Notch may be a useful biomarker for reactivation after IVR in ROP.

Internal Limiting Peeling in Conjunction with Subretinal Injection of a Balanced Salt Solution in the Macular Region to Treat Refractory Diabetic Macular Edema.

Background: Diabetic macular edema (DME) is one of the primary causes of decreased visual acuity in patients with diabetic retinopathy (DR). Rapid, effective, and safe treatment of DME is important to ensure patients' vision. Objective: In this study, we observed the efficacy and safety of internal limiting membrane (ILM) peeling in conjunction with subretinal injection of balanced salt solution (BSS) in treating refractory DME. Methods: A prospective, non-case-control study. Patients diagnosed with refractory DME in our hospital between October 2021 and June 2022 were included. All patients received 23G PPV in conjunction with internal limiting membrane removal and subretinal injection of BSS. During and after surgery, intravitreal injections of an anti-VEGF drug were administered. We compared and analyzed the best-corrected visual acuities (BCVA), central macular thickness (CMT), recurrence rate, complications, and other observation indicators. Results: The investigation included 32 patients (32 eyes). The BCVA at each time point after surgery was significantly higher than it was before the surgery (P < .001). One month after the surgery, the BCVA was significantly higher than it was before surgery and one week after the surgery. Postoperative CMT was statistically significantly lower than before surgery, and the difference was statistically significant (P < .001). One month after surgery, CMT was significantly lower than before and one week after surgery, and the difference was statistically significant (P < .001). Still, there was no significant difference between three and six months after surgery. Three times of intravitreal injections of anti-VEGF drugs were administered. At the most recent follow-up, DME recurred in three eyes (9.4%). During the follow-up period, no complications such as vitreous hemorrhage, retinal detachment, macular epiretinal membrane, or macular hole were observed. Conclusion: Subretinal injection of BSS can be an effective treatment for refractory DME and is recommended for clinical use.

An Inducible Luminescent System to Explore Parkinson's Disease-Associated Genes.

With emerging genetic association studies, new genes and pathways are revealed as causative factors in the development of Parkinson's disease (PD). However, many of these PD genes are poorly characterized in terms of their function, subcellular localization, and interaction with other components in cellular pathways. This represents a major obstacle towards a better understanding of the molecular causes of PD, with deeper molecular studies often hindered by a lack of high-quality, validated antibodies for detecting the corresponding proteins of interest. In this study, we leveraged the nanoluciferase-derived LgBiT-HiBiT system by generating a cohort of tagged PD genes in both induced pluripotent stem cells (iPSCs) and iPSC-derived neuronal cells. To promote luminescence signals within cells, a master iPSC line was generated, in which LgBiT expression is under the control of a doxycycline-inducible promoter. LgBiT could bind to HiBiT when present either alone or when tagged onto different PD-associated proteins encoded by the genes GBA1, GPNMB, LRRK2, PINK1, PRKN, SNCA, VPS13C, and VPS35. Several HiBiT-tagged proteins could already generate luminescence in iPSCs in response to the doxycycline induction of LgBiT, with the enzyme glucosylceramidase beta 1 (GCase), encoded by GBA1, being one such example. Moreover, the GCase chaperone ambroxol elicited an increase in the luminescence signal in HiBiT-tagged GBA1 cells, correlating with an increase in the levels of GCase in dopaminergic cells. Taken together, we have developed and validated a Doxycycline-inducible luminescence system to serve as a sensitive assay for the quantification, localization, and activity of HiBiT-tagged PD-associated proteins with reliable sensitivity and efficiency.

Mendelian randomization analysis reveals causal relationships between gut microbiome and optic neuritis.

BACKGROUND: It is unclear whether gut microbiota (GM) affects the risk of optic neuritis (ON) through the "gut-brain" axis and the "gut-retina" axis. To examine the causal relationship between GM and ON, we conducted Mendelian randomization (MR) study. METHODS: Up to 18,340 samples of 24 population-based cohorts were included in genome-wide association study (GWAS) of 196 GM taxa. ON outcomes were selected from the FinnGen GWAS (951 ON cases and 307,092 controls). In addition, the GWAS based on UK Biobank (UKB) (105 ON cases and 456,243 controls) was used for further exploration. Inverse variance weighted (IVW) was carried out to estimate their effects on ON risk and the MR assumptions were evaluated in sensitivity analyses. RESULTS: Among the 196 GM taxa, the IVW results confirmed that Family -Peptococcaceae (P = 2.17 × 10-3), Genus- Hungatella (P = 4.57 × 10-3) and genus-Eubacterium_rectale_group (P = 0.02) were correlated with the risk of ON based on Finngen GWAS. Based on data from UKB, Genus- Eubacterium_hallii_group (P = 1.50 × 10-3) and Genus- Ruminococcaceae_UCG_002 (P = 0.02) were correlated with the risk of ON. At the phylum, class and order levels, no GM taxa were causally related to ON (P > 0.05). Heterogeneity (P > 0.05) and pleiotropy (P > 0.05) analysis confirmed the robustness of the MR results. CONCLUSION: Our MR findings support the causal effect of specific GM taxa on ON. GM may affect the risk of ON through the "gut-brain" axis and the "gut-retina" axis. However, further research is needed to confirm the relevant mechanism of the relationship between GM and ON.

TFEB attenuates hyperglycemia-induced retinal capillary endothelial cells injury via autophagy regulation.

Diabetic retinopathy is a common microvascular complication of diabetes mellitus. The maintenance of retinal capillary endothelial cell homeostasis requires a complete and unobtrusive flow of autophagy because it may help combat the inflammatory response, apoptosis, and oxidative stress damage of cells in diabetes mellitus. The transcription factor EB is a master regulator of autophagy and lysosomal biogenesis, but its role in diabetic retinopathy remains unknown. This study aimed to confirm the involvement of transcription factor EB in diabetic retinopathy and explore the role of transcription factor EB in hyperglycemia-linked endothelial injury in vitro. First, the expression levels, including the nuclear location of transcription factor EB and autophagy, were reduced in diabetic retinal tissues and high glucose-treated human retinal capillary endothelial cells. Subsequently, autophagy was mediated by transcription factor EB in vitro. Moreover, transcription factor EB overexpression reversed high glucose-induced autophagy inhibition and lysosomal dysfunction and protected human retinal capillary endothelial cells from inflammation, apoptosis, and oxidative stress damage caused by high glucose treatment. Additionally, under high-glucose stimulation, the autophagy inhibitor chloroquine attenuated transcription factor EB overexpression-mediated protection, and the autophagy agonist Torin1 rescued transcription factor EB knockdown-induced damage effects. Taken together, these results suggest that transcription factor EB is involved in the development of diabetic retinopathy. In addition, transcription factor EB protects human retinal capillary endothelial cells from high glucose-induced endothelial damage via autophagy.

An approach to measuring protein turnover in human induced pluripotent stem cell organoids by mass spectrometry.

Patient-derived organoids from induced pluripotent stem cells have emerged as a model for studying human diseases beyond conventional two-dimensional (2D) cell culture. Briefly, these three-dimensional organoids are highly complex, capable of self-organizing, recapitulate cellular architecture, and have the potential to model diseases in complex organs, such as the brain. For example, the hallmark of Parkinson's disease (PD) - proteostatic dysfunction leading to the selective death of neurons in the substantia nigra - present a subtle distinction in cell type specificity that is lost in 2D cell culture models. As such, the development of robust methods to study global proteostasis and protein turnover in organoids will remain essential as organoid models evolve. To solve this problem, we have designed a workflow to reproducibly extract proteins from brain organoids, measure global turnover using mass spectrometry, and statistically investigate turnover differences between genotypes. We also provide robust methodology for data filtering and statistical treatment of turnover data. Using human midbrain organoids (hMO) as a model system, our method accurately characterized the half-lives of 773 midbrain proteins. We compared these half-lives both to Parkin knockout hMOs and to previously reported data from primary cell cultures and in vivo models. Overall, this method will facilitate the study of proteostasis in organoid models of human disease and will provide an analytical and statistical framework to measure protein turnover in organoids of all cell types.

Internal limiting membrane peeling combined with mouse nerve growth factor injection for idiopathic macular hole.

BACKGROUND: The study was intended to confirm whether Pars Plana Vitrectomy (PPV) with Internal Limiting Membrane (ILM) peeling and intravitreal injection mouse Nerve Growth Factor(mNGF) was effective for the treatment of Idiopathic Macular Hole(IMH) by Optical Coherence Tomography Angiography(OCTA) and microperimetry. METHODS: A retrospective study was performed in adults' patients. A total of 44 eyes (March 2021-October 2021) with IMH who received surgical treatment in the Affiliated Eye Hospital of Nanchang University in Nanchang City, Jiangxi Province were selected. The subjects were treated using PPV combined with ILM peeling and intravitreal mNGF (combined group) or PPV combined with ILM peeling (placebo group). The Best Corrected Visual Acuity (BCVA), Optical Coherence Tomography Angiography (OCTA) and MP-3 microperimetry were carried out and observed at baseline, 1 week(1W), 1,3 and 6 months (1 M,3 M,6 M) postoperatively. RESULTS: The minimum diameter of MH were (568.650 ± 215.862)µm and (533.348 ± 228.836)µm in the Placebo and Combine group pre-operative. During the observation, the macular hole closure rate in the placebo group and combined group were 90% and 95.8% respectively and the difference was not statistically significant(p = 0.583). Compared to pre-surgery, the perimeter and circularity of Foveal Avascular Zone (FAZ) in the placebo group decreased at 1,3,6 M (p = 0.001, < 0.001, < 0.001) and 1W,1,6 M (p = 0.045,0.010, < 0.001) post-surgery respectively. And the perimeter and circularity of FAZ showed significant reduction in the combined group at 1,3,6 M (p = 0.005,0.004, < 0.001) and at each follow-up time point (all values of p < 0.001). The vascular density of SCP increased at 1W(p = 0.031) and 6 M(p = 0.007), the perfusion density of SCP was significantly improved at each follow-up time point (p = 0.028, 0.011, 0.046, 0.004) in the combined group. The BCVA in the combined group was more obvious than that in the placebo group at 1 M, 3 M and 6 M after operation (t1 = 2.248, p1 = 0.030; t3 = 3.546, p3 = 0.001; t6 = 3.054, p6 = 0.004). The changes of BCVA in the combined group was more conspicuous than that in the placebo group at each follow-up time point, and the difference was statistically significant (t1 = 2.206,p1 = 0.033;t2 = 2.54,p2 = 0.015;t3 = 3.546,p3 = 0.001;t6 = 3.124,p6 = 0.003).At 1 M, 3 M and 6 M, the MRS of 2° and 4° in the combined group was better than that in the placebo group(t = -2.429,-2.650,-3.510,-2.134,-2.820,-3.099 p = 0.020,0.011,0.001,0.039,0.007,0.004). During various time points, the MRS of 12°in the combined group was better than that in the placebo group, the difference was statistically significant (t = -3.151, -3.912, -4.521, -4.948, p1 = 0.003, < 0.001, < 0.001 < 0.001). The integrity of External Limiting Membrane (ELM) in combination group was better than that in placebo group at 6 M postoperative(p = 0.022) and that of Ellipsoid Zone(EZ) was preferable in the combined group at 3 M and 6 M after surgery(p = 0.012,0.004). Correlation analysis showed that the integrity of EZ was correlated with 12°MRS at 1 M, 3 M and 6 M after surgery(r = -0.318, -0.343,-0.322;p = 0.023,0.033, < 0.001). There was no correlation between postoperative ELM integrity and postoperative BCVA and 12°MRS(p > 0.05). CONCLUSIONS: Our results manifested that PPV combined with ILM peeling and intravitreal injection mNGF might be more effective for initial IMH. This method increased the blood flow, MRS and promoted the recovery of ELM and EZ in the macular and might improve the visual function of patients postoperatively.

Comparison of anterior sclera thickness in emmetropes and myopes.

BACKGROUND: This study aimed to compare anterior scleral thicknesses (ASTs) in people with emmetropia and myopia to explore the effect of myopia on AST. METHODS: In this cross-sectional study, 93 participants (i.e., 93 eyes) with emmetropia and myopia underwent ocular imaging via anterior segment optical coherence tomography. We acquired raw B-scan OCT images along each of the four meridians (superior, inferior, nasal, and temporal), The AST was estimated from the limbus to a distance of 6 mm. The participants were aged between 20 and 50 years (mean age: 30.2 ± 8.8 years). The axial length (AL) was 22.50 ~ 33.04 mm (mean AL: 26.51 ± 2.65 mm), and the spherical equivalent (SE) was + 0.50 ~ 27.5 D (mean SE: -7.20 ± 6.5 D). The selected sample comprised 37 males and 56 females who were categorized as emmetropes, mild-moderate myopes, or high myopes. The four meridians of AST, AL, and refractive error were observed. RESULTS: The AL was significantly negatively correlated with the four meridians of AST (the r value ranged between - 0.511 and - 0.228, P < 0.05). There was no significant correlation between age and inferior diameter (r = 0.113, P = 0.314), but age was positively correlated with the average AST of the superior, temporal, and nasal diameters (the r value ranged between 0.452 and 0.552, P < 0.05). There was no significant correlation between sex and AST (the T value ranged between - 1.816 and - 0.130, P > 0.05). Except for the inferior diameters of 1 mm, 5 mm, and 6 mm and the temporal diameter of 1 mm, the four diameters in the emmetropia group and the high myopia group were statistically significant at a distance of 0 ~ 6 mm from the limbus (P < 0.05). CONCLUSION: The AST is negatively correlated with AL and positively correlated with age. Compared with emmetropic eyes, the AST is thinner in highly myopic eyes. Myopia affects AST, which may be useful for monitoring progression in cases of myopia.

Transcriptional Dysregulation and Impaired Neuronal Activity in FMR1 Knock-Out and Fragile X Patients' iPSC-Derived Models.

Fragile X syndrome (FXS) is caused by a repression of the FMR1 gene that codes the Fragile X mental retardation protein (FMRP), an RNA binding protein involved in processes that are crucial for proper brain development. To better understand the consequences of the absence of FMRP, we analyzed gene expression profiles and activities of cortical neural progenitor cells (NPCs) and neurons obtained from FXS patients' induced pluripotent stem cells (IPSCs) and IPSC-derived cells from FMR1 knock-out engineered using CRISPR-CAS9 technology. Multielectrode array recordings revealed in FMR1 KO and FXS patient cells, decreased mean firing rates; activities blocked by tetrodotoxin application. Increased expression of presynaptic mRNA and transcription factors involved in the forebrain specification and decreased levels of mRNA coding AMPA and NMDA subunits were observed using RNA sequencing on FMR1 KO neurons and validated using quantitative PCR in both models. Intriguingly, 40% of the differentially expressed genes were commonly deregulated between NPCs and differentiating neurons with significant enrichments in FMRP targets and autism-related genes found amongst downregulated genes. Our findings suggest that the absence of FMRP affects transcriptional profiles since the NPC stage, and leads to impaired activity and neuronal differentiation over time, which illustrates the critical role of FMRP protein in neuronal development.

Evaluation of retinal microcirculation alterations using OCTA in hyperopic ametropic amblyopia patients before and after treatment.

PURPOSE: We aimed to compare retinal microcirculation in hyperopic ametropic amblyopia patients before and after treatment and in healthy children using optical coherence tomography angiography (OCTA), and to explore the pathogenesis of hyperopic ametropic amblyopia. METHODS: Eighteen patients with hyperopic ametropic amblyopia aged 4-8 years were selected as the patient group, and 18 age-matched healthy children were randomly selected as controls. The foveal avascular zone (FAZ) area, perimeter and circularity, vessel density (VD) and perfusion density (PD) of macular superficial retinal capillary plexus, macular thickness, peripapillary retinal nerve fiber layer thickness, and ganglion cell-inner plexiform layer thickness were compared between both groups. After 6 months of amblyopia treatment, the same parameters were measured again. RESULTS: The VD and PD in the central, inner, inner nasal, and inner inferior regions in hyperopic ametropic amblyopia were lower than in the control group after adjustment for axial length. After 6 months of treatment, the VD increased significantly, except in the outer nasal and outer inferior regions. The PD in the central (p < 0.001), inner superior (p = 0.001), inner inferior (p = 0.011) and inner temporal (p = 0.026) regions increased. The FAZ perimeter and circularity significantly differed between the groups. After 6 months of treatment, the FAZ area and perimeter decreased, but circularity increased. CONCLUSION: Hyperopic ametropic amblyopia eyes showed a significant decrease in vessel and perfusion densities. After amblyopia treatment, the vessel and perfusion densities of patients with hyperopic ametropic amblyopia increased, suggesting that abnormalities in the microvascular system are a pathogenic factor of amblyopia.

Sirtuin (Sirt) 3 Overexpression Prevents Retinopathy in Streptozotocin-Induced Diabetic Rats.

BACKGROUND Sirtuin (Sirt) 3 could promote autophagy by downregulating the expression of genes related to neovascularization in retinal endothelial cells. In this study, we aimed to investigate the effect of Sirt3 overexpression on retinopathy in streptozotocin (STZ)-induced diabetic rats, and to assess its mechanisms. MATERIAL AND METHODS Ntraperitoneal injection of STZ in rats was used to produce a diabetic model. The study rats were divided into 4 groups (n=6 for each group): a control group; a model group; a model+scrambled adenovirus group; and a model+Sirt3 overexpression group. Hematoxylin and eosin (H&E) staining determined the pathological changes of retina tissues. Immunohistochemistry, fluorescence quantitative polymerase chain reaction, and western blotting were used to detect the expression of Sirt3, vascular endothelial growth factor (VEGF), and microtubule-associated protein 1A/1B-light chain 3 (LC3). RESULTS In the model group, the inner limiting membrane was swollen, uneven and thickened, and the capillary endothelial cells occasionally protruded into the inner limiting membrane. These abnormalities were prevented by Sirt3 overexpression. Compared with the control group, the expression of Sirt3 at both mRNA and protein levels in the model group was significantly reduced, while the expression of VEGF was increased versus the control group (P.

A study of dry eye after cataract surgery in MGD patients.

OBJECTIVE: To investigate the dry eye symptoms after cataract surgery in MGD patients and their relationships METHODS: The study included 115 patients (115 eyes) with age-related cataract that underwent uncomplicated cataract surgery, and the patients were divided into two groups according to the MGD diagnostic criteria: group A (MGD group) and group B (control group). Schirmer I test (ST-I), tear breakup time (TBUT), and corneal fluorescein staining (CFS) were performed preoperatively and at 3 days, 7 days, 14 days, and 30 days postoperatively. We also measured eyelid meibomian gland morphology, meibomian gland expression, and meibum character scores before and after the cataract surgery. RESULTS: Postoperatively, in group A, TBUT decreased and CFS scores increased significantly. ST-I increased in the early postoperative course but decreased later. The eyelid margin morphology scores and meibomian gland expression scores of group A significantly increased after the cataract operation. Thus, patients with MGD may have a greater chance of developing the dry eye disease after cataract surgery. Cataract surgery may aggravate the signs of MGD, and the severity of MGD may positively correlate with TBUT, CFS, and corneal lesions after surgery. CONCLUSIONS: The characteristics of dry eye after cataract surgery in patients with MGD are different from common cataract patients, changes in the early postoperative phase to the ocular surface were caused by surgical factors, and the damages to epithelial function in the later postoperative phase were mainly associated with the inflammation of the meibomian gland and eyelid.

GEP100/ARF6 regulates VEGFR2 signaling to facilitate high-glucose-induced epithelial-mesenchymal transition and cell permeability in retinal pigment epithelial cells.

Cell permeability and epithelial-mesenchymal transition (EMT) were found to be enhanced in diabetic retinopathy, and the aim of this study was to investigate the underlying mechanism. ARPE-19 cell line or primary retinal pigment epithelial (RPE) cells were cultured under high or normal glucose conditions. Specific shRNAs were employed to knock down ADP-ribosylation factor 6 (ARF6), GEP100, or VEGF receptor 2 (VEGFR2) in ARPE-19 or primary RPE cells. Cell migration ability was measured using Transwell assay. Western blotting was used to measure indicated protein levels. RPE cells treated with high glucose showed increased cell migration, paracellular permeability, EMT, and expression of VEGF. Knockdown of VEGFR2 inhibited the high-glucose-induced effects on RPE cells via inactivation of ARF6 and MAPK pathways. Knockdown ARF6 or GEP100 led to inhibition of high-glucose-induced effects via inactivation of VEGFR2 pathway. Knockdown of ARF6, but not GEP100, decreased high-glucose-induced internalization of VEGFR2. High-glucose enhances EMT and cell permeability of RPE cells through activation of VEGFR2 and ARF6/GEP100 pathways, which form a positive feedback loop to maximize the activation of VEGF/VEGFR2 signaling.

Otx2 enhances transdifferentiation of Müller cells-derived retinal stem cells into photoreceptor-like cells.

Retinal Müller glial cells have the potential of neurogenic retinal progenitor cells, and could reprogram into retinal-specific cell types such as photoreceptor cells. How to promote the differentiation of Müller cells into photoreceptor cells represents a promising therapy strategy for retinal degeneration diseases. This study aimed to enhance the transdifferentiation of rat Müller cells-derived retinal stem cells (MC-RSCs) into photoreceptor-like cells and explore the signalling mechanism. We dedifferentiated rat Müller cells into MC-RSCs which were infected with Otx2 overexpression lentivirus or control. The positive rate of photoreceptor-like cells among MC-RSCs treated with Otx2 overexpression lentivirus was significantly higher compared to control. Furthermore, pre-treatment with Crx siRNA, Nrl siRNA, or GSK-3 inhibitor SB-216763 reduced the positive rate of photoreceptor-like cells among MC-RSCs treated with Otx2 overexpression lentivirus. Finally, Otx2 induced photoreceptor precursor cells were injected into subretinal space of N-methyl-N-nitrosourea induced rat model of retinal degeneration and partially recovered retinal degeneration in the rats. In conclusion, Otx2 enhances transdifferentiation of MC-RSCs into photoreceptor-like cells and this is associated with the inhibition of Wnt signalling. Otx2 is a potential target for gene therapy of retinal degenerative diseases.

Transplantation of rat embryonic stem cell-derived retinal cells restores visual function in the Royal College of Surgeons rats.

AIM OF STUDY: To evaluate the feasibility of transplantation of embryonic stem cell (ESC)-derived retinal cells in the treatment of retinal degeneration. MATERIALS AND METHODS: Rat ESCs were isolated and induced into retinal progenitor cells (RPCs) in vitro, which were subsequently induced into retinal pigment epithelium cells (RPEs) and photoreceptors (PRCs). All cells were identified by Western blot detection of their specific markers. RPEs and PRCs were, respectively, injected into the retina of Royal College of Surgeons (RCSs) rats. Control group was injected with PBS. Post-transplantation visual function was determined by electroretinography (ERG). The histology of the whole eye was compared by H&E staining. RESULTS: RPEs and PRCs were successfully derived from rat ESCs through the two-step differentiation as indicated by the presence of ESC- (Oct-3/4, Nanog, TRA-1-60 and TRA-1-81), RPC- (Rx, Mitf, Pax6 and Chx10), RPE- (RPE65 and keratin) and PRC-specific markers (blue opsin, red/green opsin, recoverin and rhodopsin) in Western blot. The amplitude of ERG a- and b-wave in RPE- and PRC-transplanted groups at week 2 and 10 after transplantation was markedly higher compared with PBS controls. Retinal injury and vascular stress response was not detected in any of the RCS rats after transplantation. CONCLUSION: The developed stepwise protocol can derive retinal cells from ESCs. Transplantation of these retinal cells can restore visual function of RCS rats. Our study provides evidence for potential clinical application of ESC-based cell therapy for retinal degeneration.

Potential suppression of the high glucose and insulin-induced retinal neovascularization by Sirtuin 3 in the human retinal endothelial cells.

Retinal neovascularization generally play roles in the formation of various severe eye diseases, such as age-related macular degeneration and diabetic retinopathy. The regulation of neovascularization-related pathways by Sirtuin 3 (Sirt3), a major mitochondrial NAD+-dependent deacetylase, give us a cue that Sirt3 may participate in the retinal neovascularization. However, the mechanism remains unclear. Here, we established a retinal neovascularization model by using human retinal endothelial cells (HRECs) under the induction of high glucose and insulin (HGI). With this model, Sirt3-expressing lentivirus was constructed and then used to investigate the effect of Sirt3 overexpression on the expression of migration-, neovascularization- and autophagy-related genes. After the treatment of HGI on HRECs, the mRNA and protein levels of migration-related genes, including matrix metalloproteinase-2 (MMP-2) and MMP-9, were significantly upregulated. Meanwhile, angiogenesis-related genes, including vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1α (HIF-1α), and insulin-like growth factor-1 (IGF-1) were promoted at both mRNA and protein levels. However, HGI had no clear effect on the mRNA and protein levels of microtubule associated protein 1 light chain 3 (LC3), an autophagy-related gene. When Sirt3 was overexpressed by lentivirus infection after HGI, the upregulation of MMP-2, MMP-9, VEGF, HIF-1α, and IGF-1 were suppressed at both transcription and translation levels. At the same time, LC3 mRNA and LC3-II protein increased. These results suggest that Sirt3 may inhibit retinal neovascularization by regulating the migration-, neovascularization- and autophagy-related factors expression. Thus we argue that Sirt3 may be a potential candidate drug for curing various eye diseases induced by retinal neovascularization.

MiR-21 inhibitor suppressed the progression of retinoblastoma via the modulation of PTEN/PI3K/AKT pathway.

MicroRNA-21 (miR-21) was reported to act as an oncogene during the development of many human tumors. However, little was revealed about the function of miR-21 in retinoblastoma (RB). In this study, we examined the expression of miR-21 in RB tissues and explored the relationship between miR-21 and phosphatase and tensin homolog (PTEN)/phosphatidylinositol-3-OH kinase (PI3K)/AKT signal. Quantitative real-time PCR (qRT-PCR) results showed that the level of miR-21 in RB tissues was higher than that in retinal normal tissues. In Weri-Rb-1 cells, miR-21 inhibitor suppressed the expression of miR-21 and cell viability, but improved cell apoptotic rates by modulating the levels of PDCD4, Bax, and Bcl-2. Meanwhile, miR-21 inhibitor suppressed cell migration and invasion via inhibiting the protein levels of MMP2 and MMP9 and significantly affected the expression of PTEN, PI3K, and p-AKT. Taken together, miR-21 inhibitor suppressed cell proliferation, migration, and invasion via the PTEN/PI3K/AKT signal. These findings revealed the molecular basis of miR-21 functioning in the progression of RB and provided a new means for cell therapy in RB.

Characterization of the minimal length of functional SecA in Escherichia coli.

Previous studies showed that certain regions of E. coli SecA can be deleted from its N- and/or C-termini to complement a SecA amber ts mutant. In this study, we determined and characterized the dispensability of both ends of SecA molecules. With N-terminal intact or 9-aa deleted, 826aa (SecA1-826 and SecA10-826, respectively) is the minimum for complementation activity, while with N-terminus deleted by 2-21aa, SecA22-829 is the minimum. Further deletion at the C-terminus of SecA1-826/SecA10-826/SecA22-829 abolished the complementation activity in the cells. A hydrophobic amino acid is required for the 826th residue in the minimal-length SecAs. Chemical crosslinking and gel filtration result showed that both purified SecA22-828 and SecA22-829 could form a dimer. Moreover, the in vitro ATPase and protein translocation activities of SecA22-828 and SecA22-829 were similar, though lower than wild-type SecA. The active mutants had more truncated SecA in soluble than membrane-bound form, but was more stably embedded in membranes. In contrast, the inactive mutants tended to have truncated SecA more membrane-bound than soluble form, and were more loosely bound and easily chased out. Thus, the loss of complementation appears to be related to their altered subcellular localization and stability in the membranes. This study defines the substantial regions of N- and C-termini of SecA that may be deleted without losing complementation activity.

Blocking the urotensin II receptor pathway ameliorates the metabolic syndrome and improves cardiac function in obese mice.

The metabolic syndrome is defined by the presence of hyperlipidemia, obesity, hypertension, and diabetes. The syndrome is associated with significant cardiovascular morbidity and mortality. The aim of the present study was to determine the role of the vasoactive peptide urotensin II (UII) in the pathogenesis of the metabolic syndrome. We used obese mice (ob/ob) to determine the effect of UII receptor (UT) blockage on the different facets of the metabolic syndrome with special emphasis on cardiac function. Our data demonstrate a significant increase in UII and UT expression in the myocardium of obese mice accompanied by a significant decrease in sarco/endoplasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) expression, as well as intracellular Na(+) and Ca(2+) compared with wild-type mice (P<0.05). Treatment of ob/ob mice with the UII receptor antagonist SB657510 significantly improved glucose levels, blood pressure, hyperlipidemia, expression of myocardial SERCA2a, intracellular Na(+) and Ca(2+) and cardiac function in association with a decrease in weight gain, and mammalian target of rapamycin (mTOR) and sodium/hydrogen exchanger 1 (NHE-1) protein expression compared with vehicle (P<0.05). These findings demonstrate an important role for UII in the pathogenesis of the metabolic syndrome and suggest that the use of UT receptor antagonists may provide a new therapeutic tool for the treatment of this syndrome.