Transient expression of anti-HrpE scFv antibody reduces the hypersensitive response in non-host plant against bacterial phytopathogen Xanthomonas citri subsp. citri.

Citrus canker is a bacterial disease caused by Xanthomonas citri subsp. citri (Xcc) that affects the citrus industry worldwide. Hrp pili subunits (HrpE), an essential component of Type III secretion system (T3SS) bacteria, play a crucial role in the pathogenesis of Xcc by transporting effector proteins into the host cell and causing canker symptoms. Therefore, development of antibodies that block HrpE can suppress disease progression. In this study, a specific scFv detecting HrpE was developed using phage display technique and characterized using sequencing, ELISA, Western blotting, and molecular docking. In addition, a plant expression vector of pCAMBIA-scFvH6 was constructed and agroinfiltrated into Nicotiana tabacum cv. Samson leaves. The hypersensitive response (HR) in the leaves of transformed and non-transformed plants was evaluated by inoculating leaves with Xcc. After three rounds of biopanning of the phage library, a specific human scFv antibody, named scFvH6, was identified that showed high binding activity against HrpE in ELISA and Western blotting. Molecular docking results showed that five intermolecular hydrogen bonds are involved in HrpE-scFvH6 interaction, confirming the specificity and high binding activity of scFvH6. Successful transient expression of pCAMBIA-scFvH6 in tobacco leaves was verified using immunoassay tests. The binding activity of plant-produced scFvH6 to detect HrpE in Western blotting and ELISA was similar to that of bacterial-produced scFvH6 antibody. Interestingly, tobacco plants expressing scFvH6 showed a remarkable reduction in HR induced by Xcc compared with control plants, so that incidence of necrotic lesions was significantly higher in non-transformed controls (≥ 1.5 lesions/cm2) than in the plants producing scFvH6 (≤ 0.5 lesions/cm2) after infiltration with Xcc inoculum. Our results revealed that the expression of scFvH6 in tobacco leaves can confer resistance to Xcc, indicating that this approach could be considered to provide resistance to citrus bacterial canker disease.

Whole-Genome-Sequence-Based Classification of Xanthomonas euvesicatoria pv. eucalypti and Computational Analysis of the Type III Secretion System.

Xanthomonas spp. infect a wide range of annual and perennial plants. Bacterial blight in young seedlings of Eucalyptus spp. in Indonesia was originally identified as X. perforans. However, these strains failed to elicit a hypersensitive response (HR) on either tomatoes or peppers. Two of the strains, EPK43 and BCC 972, when infiltrated into tomato and pepper leaves, failed to grow to significant levels in comparison with well-characterized X. euvesicatoria pv. perforans (Xp) strains. Furthermore, spray inoculation of 'Bonny Best' tomato plants with a bacterial suspension of the Eucalyptus strains resulted in no obvious symptoms. We sequenced the whole genomes of eight strains isolated from two Eucalyptus species between 2007 and 2015. The strains had average nucleotide identities (ANIs) of at least 97.8 with Xp and X. euvesicatoria pv. euvesicatoria (Xeu) strains, both of which are causal agents of bacterial spot of tomatoes and peppers. A comparison of the Eucalyptus strains revealed that the ANI values were >99.99% with each other. Core genome phylogeny clustered all Eucalyptus strains with X. euvesicatoria pv. rosa. They formed separate clades, which included X. euvesicatoria pv. alangii, X. euvesicatoria pv. citrumelonis, and X. euvesicatoria pv. alfalfae. Based on ANI, phylogenetic relationships, and pathogenicity, we designated these Eucalyptus strains as X. euvesicatoria pv. eucalypti (Xee). Comparative analysis of sequenced strains provided unique profiles of type III secretion effectors. Core effector XopD, present in all pathogenic Xp and Xeu strains, was absent in the Xee strains. Comparison of the hrp clusters of Xee, Xp, and Xeu genomes revealed that HrpE in Xee strains was very different from that in Xp and Xeu. To determine if it was functional, we deleted the gene and complemented with the Xee hrpE, confirming it was essential for secretion of type III effectors. HrpE has a hypervariable N-terminus in Xanthomonas spp., in which the N-terminus of Xee strains differs significantly from those of Xeu and Xp strains.

Potential involvement of miR-183/96/182 cluster-gene target interactions in transdifferentiation of human retinal pigment epithelial cells into retinal neurons.

miR-183/96/182 cluster plays a critical role in the developing retina by regulating many target genes involved in signaling pathways. This study aimed to survey the miR-183/96/182 cluster-target interactions that, potentially contribute to human retinal pigmented epithelial (hRPE) cell differentiation into photoreceptors. Target genes of the miR-183/96/182 cluster were obtained from miRNA-target databases and applied to construct miRNA-target networks. Gene ontology and KEGG pathway analysis was performed. miR-183/96/182 cluster sequence was cloned into an eGFP-intron splicing cassette in an AAV2 vector and overexpressed in hRPE cells. The expression level of target genes including HES1, PAX6, SOX2, CCNJ, and RORΒ was evaluated using qPCR. Our results showed that miR-183, miR-96, and miR-182 share 136 target genes that are involved in cell proliferation pathways such as PI3K/AKT and MAPK pathway. qPCR data indicated a 22-, 7-, and 4-fold overexpression of miR-183, miR-96, and miR-182, respectively, in infected hRPE cells. Consequently, the downregulation of several key targets such as PAX6, CCND2, CDK5R1, and CCNJ and upregulation of a few retina-specific neural markers such as Rhodopsin, red opsin, and CRX was detected. Our findings suggest that the miR-183/96/182 cluster may induce hRPE transdifferentiation by targeting key genes that involve in the cell cycle and proliferation pathways.

Essential Acidovorax citrulli Virulence Gene hrpE Activates Host Immune Response against Pathogen.

Bacterial fruit blotch (BFB) caused by Acidovorax citrulli (Ac) is a devastating watermelon disease that severely impacts the global watermelon industry. Like other Gram-negative bacteria, the type three secretion system (T3SS) is the main pathogenicity factor of A. citrulli. The T3SS apparatus gene hrpE codes for the Hrp pilus and serves as a conduit to secret effector proteins into host cells. In this study, we found that the deletion of hrpE in A. citrulli results in the loss of pathogenicity on hosts and the hypersensitive response on non-hosts. In addition, the A. citrulli hrpE mutant showed a reduction in in vitro growth, in planta colonization, swimming and twitching motility, and displayed increases in biofilm formation ability compared to the wild type. However, when HrpE was transiently expressed in hosts, the defense responses, including reactive oxygen species bursts, callose deposition, and expression of defense-related genes, were activated. Thus, the A. Citrulli growth in HrpE-pretreated hosts was suppressed. These results indicated that HrpE is essential for A. citrulli virulence but can also be used by hosts to help resist A. citrulli. Our findings provide a better understanding of the T3SS pathogenesis in A. citrulli, thus providing a molecular basis for biopesticide development, and facilitating the effective control of BFB.

Correlation between ELF-PEMF exposure and Human RPE Cell Proliferation, Apoptosis and Gene Expression.

PURPOSE: Emerging evidence implies that electromagnetic fields (EMFs) can negatively affect angiogenesis. In this regard, the effects of extremely low frequency pulsed electromagnetic field (ELF-PEMF) exposure on the relative expression level of angiogenic factors involved in the pathogenesis of ocular disorders were evaluated in human retinal pigment epithelial (hRPE) cells in order to investigate a noninvasive therapeutic method for patients with several ocular diseases associated with neovascularization. METHODS: After separating hRPE cells from globes, hRPE cells were exposed to 15 mT of ELF-PEMF (120 Hz) at 5, 10, and 15 min for seven days. Cell proliferation and apoptosis of treated cells were evaluated via ELISA assay. Moreover, relative expression changes of HIF-1α, CTGF, VEGFA, MMP-2, cathepsin D, and E2F3 were performed using real-time RT-PCR. RESULTS: ELF-PEMF exposure had no significant effects on the apoptosis and proliferation rate of hRPE cells. Expression level of HIF-1α, CTGF, VEGFA, MMP-2, cathepsin D, and E2F3 was downregulated following 5 min of ELF-PEMF exposure. CONCLUSION: As ELF-PEMF showed inhibitory effects on the expression of angiogenic genes in hRPE cells with no cytotoxic or proliferative side effects, it can be introduced as a useful procedure for managing angiogenesis induced by retinal pathogenesis, although more studies with adequate follow-up in animal models are needed.

Protective Effect of a Bispecific Fc-Fusion Protein on the Barrier of Human Retinal Pigment Epithelial Cells.

INTRODUCTION: The aim of the study was to evaluate the protective effects of IBI302, a bispecific Fc-fusion protein that theoretically can bind vascular endothelial growth factor (VEGF), complement C3b, and C4b in the barrier of the cultured human retinal pigment epithelial (hRPE) cells. METHODS: Primary hRPE cells were isolated and cultured to monolayer barrier. hRPE monolayers were divided into the PBS control group, VEGF-Trap group, complement receptor 1 (CR1) group, and IBI302 group. Identification of hRPE cells, barrier function, inflammation factors, and immune response products was tested by immunofluorescent staining, transepithelial resistance (TER), and ELISA. RESULTS: IBI302 treatment significantly improved the TER of the barrier of hRPE cells after complement-activated oxidative stress compared with the PBS control group, VEGF-Trap group, and CR1 group. The maximum effect of IBI302 on protecting hRPE cell viability was observed at the concentration of 1 µg/mL. The elevated expression of VEGF, chemokine (C-C Motif) ligand 2, C3a, C5a, and membrane attack complex was reduced by IBI302. CONCLUSION: IBI302 could protect the barrier function of hRPE cells. IBI302 might be a potentially effective drug for the RPE barrier-associated ocular diseases.

WRKY33 interacts with WRKY12 protein to up-regulate RAP2.2 during submergence induced hypoxia response in Arabidopsis thaliana.

Tolerance of hypoxia is essential for most plants, but the underlying mechanisms are largely unknown. Here we show that adaptation to submergence induced hypoxia in Arabidopsis involves up-regulation of RAP2.2 through interactive action of WRKY33 and WRKY12. WRKY33- or WRKY12-overexpressing plants showed enhanced resistance to hypoxia. Y2H, BiFC, Co-IP and pull-down experiments confirmed the interaction of WRKY33 with WRKY12. Genetic experiments showed that RAP2.2 acts downstream of WRKY33/WRKY12. WRKY33 and WRKY12 can bind to and activate RAP2.2 individually. Genetic and molecular experiments demonstrate that the two WRKYs can synergistically enhance activation towards RAP2.2 to increase hypoxia tolerance. WRKY33 expression is increased in RAP2.2-overexpressing plants, indicating a feedback regulation by RAP2.2 during submergence process, which was corroborated by EMSA, ChIP, dual-LUC and genetic experiments. Our results show that a regulatory cascade module involving WRKY33, WRKY12 and RAP2.2 plays a key role in submergence induced hypoxia response of Arabidopsis and illuminate functions of WRKYs in hypoxia tolerance.

A Single-Cell Transcriptome Atlas of the Human Retinal Pigment Epithelium.

Human retinal pigment epithelium cells are arranged in a monolayer that plays an important supporting role in the retina. Although the heterogeneity of specific retinal cells has been well studied, the diversity of hRPE cells has not been reported. Here, we performed a single-cell RNA sequencing on 9,302 hRPE cells from three donors and profiled a transcriptome atlas. Our results identified two subpopulations that exhibit substantial differences in gene expression patterns and functions. One of the clusters specifically expressed ID3, a macular retinal pigment epithelium marker. The other cluster highly expressed CRYAB, a peripheral RPE marker. Our results also showed that the genes associated with oxidative stress and endoplasmic reticulum stress were more enriched in the macular RPE. The genes related to light perception, oxidative stress and lipid metabolism were more enriched in the peripheral RPE. Additionally, we provided a map of disease-related genes in the hRPE and highlighted the importance of the macular RPE and peripheral RPE clusters P4 and P6 as potential therapeutic targets for retinal diseases. Our study provides a transcriptional landscape for the human retinal pigment epithelium that is critical to understanding retinal biology and disease.

An Improved HRPE-Based Transcriptional Output Reporter to Detect Hypoxia and Anoxia in Plant Tissue.

Oxygen levels in plant tissues may vary, depending on metabolism, diffusion barriers, and environmental availability. Current techniques to assess the oxic status of plant cells rely primarily on invasive microoptodes or Clark-type electrodes, which are not optimally suited for experiments that require high spatial and temporal resolution. In this case, a genetically encoded oxygen biosensor is required instead. This article reports the design, test, and optimization of a hypoxia-signaling reporter, based on five-time repeated hypoxia-responsive promoter elements (HRPE) driving the expression of different reporter proteins. Specifically, this study aimed to improve its performance as a reporter of hypoxic conditions by testing the effect of different untranslated regions (UTRs) at the 5' end of the reporter coding sequence. Next, we characterized an optimized version of the HRPE promoter (HRPE-Ω) in terms of hypoxia sensitivity and time responsiveness. We also observed that severe oxygen deficiency counteracted the reporter activity due to inhibition of GFP maturation, which requires molecular oxygen. To overcome this limitation, we therefore employed an oxygen-independent UnaG fluorescent protein-coupled to an O2-dependent mCherry fluorophore under the control of the optimized HRPE-Ω promoter. Remarkably, this sensor, provided a different mCherry/UnaG ratiometric output depending on the externally imposed oxygen concentration, providing a solution to distinguish between different degrees of tissue hypoxia. Moreover, a ubiquitously expressed UnaG-mCherry fusion could be used to image oxygen concentrations directly, albeit at a narrow range. The luminescent and fluorescent hypoxia-reporters described here can readily be used to conduct studies that involve anaerobiosis in plants.

Nanoscopic Approach to Study the Early Stages of Epithelial to Mesenchymal Transition (EMT) of Human Retinal Pigment Epithelial (RPE) Cells In Vitro.

The maintenance of visual function is supported by the proper functioning of the retinal pigment epithelium (RPE), representing a mosaic of polarized cuboidal postmitotic cells. Damage factors such as inflammation, aging, or injury can initiate the migration and proliferation of RPE cells, whereas they undergo a pseudo-metastatic transformation or an epithelial to mesenchymal transition (EMT) from cuboidal epithelioid into fibroblast-like or macrophage-like cells. This process is recognized as a key feature in several severe ocular pathologies, and is mimicked by placing RPE cells in culture, which provides a reasonable and well-characterized in vitro model for a type 2 EMT. The most obvious characteristic of EMT is the cell phenotype switching, accompanied by the cytoskeletal reorganization with changes in size, shape, and geometry. Atomic force microscopy (AFM) has the salient ability to label-free explore these characteristics. Based on our AFM results supported by the genetic analysis of specific RPE differentiation markers, we elucidate a scheme for gradual transformation from the cobblestone to fibroblast-like phenotype. Structural changes in the actin cytoskeletal reorganization at the early stages of EMT lead to the development of characteristic geodomes, a finding that may reflect an increased propensity of RPE cells to undergo further EMT and thus become of diagnostic significance.

The Type III Accessory Protein HrpE of Xanthomonas oryzae pv. oryzae Surpasses the Secretion Role, and Enhances Plant Resistance and Photosynthesis.

Many species of plant-pathogenic gram-negative bacteria deploy the type III (T3) secretion system to secrete virulence components, which are mostly characteristic of protein effectors targeting the cytosol of the plant cell following secretion. Xanthomonas oryzae pv. oryzae (Xoo), a rice pathogen causing bacterial blight disease, uses the T3 accessory protein HrpE to assemble the pilus pathway, which in turn secretes transcription activator-like (TAL) effectors. The hrpE gene can execute extensive physiological and pathological functions beyond effector secretion. As evidenced in this study, when the hrpE gene was deleted from the Xoo genome, the bacteria incur seriouimpairments in multiplication, motility, and virulence. The virulence nullification is attributed to reduced secretion and translocation of PthXo1, which is a TAL effector that determines the bacterial virulence in the susceptible rice varieties. When the HrpE protein produced by prokaryotic expression is applied to plants, the recombinant protein is highly effective at inducing the defense response. Moreover, leaf photosynthesis efficiency is enhanced in HrpE-treated plants. These results provide experimental avenues to modulate the plant defense and growth tradeoff by manipulating a bacterial T3 accessory protein.

Optimization of an in vitro bilayer model for studying the functional interplay between human primary retinal pigment epithelial and choroidal endothelial cells isolated from donor eyes.

OBJECTIVE: The microenvironment of outer retina is largely regulated by retinal pigment epithelium (RPE) and choroid. Damage to either of these layers lead to the development of age related macular degeneration (AMD). A simplified cell culture model that mimics the RPE/Bruch's membrane (BM) and choroidal layers of the eye is a prerequisite for elucidating the molecular mechanism of disease progression. RESULTS: We have isolated primary retinal pigment epithelial cells (hRPE) and human primary choroidal endothelial cells (hCEC) from donor eyes to construct a bilayer of hCEC/hRPE on transwell inserts. Secretion of VEGF in the insert grown bilayer was significantly higher (22 pg/ml) than hCEC monolayer (3 pg/ml). To mimic the disease condition the model was treated with 100 ng/ml of VEGF, which increased the permeability of bilayer for 20 kDa FITC dextran while addition of bevacizumab, a humanized anti-VEGF drug, reversed the effect. To conclude the transwell insert based human primary hCEC/hRPE bilayer model would be an ideal system for studying the disease mechanisms and the crosstalk between RPE and choroid. This model will also be useful in screening small molecules and performing drug permeability kinetics.

The effects of electromagnetic fields on cultured human retinal pigment epithelial cells.

A great deal of evidence has confirmed that electromagnetic fields (EMFs) can affect the central nervous system. In this study, cultured neonatal human retinal pigment epithelial (hRPE) cells were exposed to pulsed EMF of 1 mT intensity and 50 Hz frequency 8 h daily for 3 days. In addition to cell proliferation and cell death assays, immunocytochemistry for RPE65, PAX6, nestin, and cytokeratin 8/18 proteins were performed. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed for NES, PAX6, RPE65, and ACTA2 gene expression. Exposed hRPE cells did not demonstrate significant change in terms of cytomorphology, cell proliferation, or cell death. Protein expression of PAX6 was decreased in treated cells compared to controls and remained unchanged for RPE65, cytokeratin 8/18, and nestin. Gene expressions of NES, RPE65, and PAX6 were decreased in treated cells as compared to controls. Gene expression of ACTA2 did not significantly change. In conclusion, viability of cultivated neonatal hRPE cells did not change after short exposure to a safe dose of pulsed EMF albeit that both gene and protein expressions of retinal progenitor cell markers were reduced. Whether longer exposure durations that are being constantly produced by widely-used electronic devices may induce significant changes in these cells, needs further investigation. Bioelectromagnetics. 39:585-594, 2018. © 2018 Wiley Periodicals, Inc.

Design, development and characterization of synthetic Bruch's membranes.

Age-related macular degeneration (AMD) is a leading cause of blindness, and dry AMD has no effective treatment. Retinal constructs comprising retinal pigment epithelium (RPE) cells supported by electrospun scaffolds have been investigated to treat dry AMD. However, electrospun scaffolds studied to-date do not mimic the structural microenvironment of human Bruch's membrane (BM), essential for native-like RPE monolayers. The aim of this study was to develop a structurally biomimetic scaffold designed to support a functional RPE monolayer, comprising porous, electrospun nanofibrous membranes (ENMs), coated with laminin, mimicking the inner collagenous layer (ICL) and basal RPE lamina respectively, the cell supporting layers of the BM. In vitro evaluation showed 70nm PLLA ENMs adsorbed high amounts of laminin and supported functional RPE monolayers, exhibiting 3D polygonal-cobblestone morphology, apical microvilli, basal infoldings, high transepithelial resistance (TER), phagocytic activity and expression of signature RPE markers. 70nm PLLA ENMs were successfully implanted into the subretinal space of RCS-rdy+p+/LAV rats, also commonly know as rdy rats. At week 4, in the absence of immunosuppressants, implanted PLLA ENMs were surrounded by a significantly low number of activated microglial cells, compared to week 1, indicating no adverse long-term immune response. In conclusion, we successfully designed and tested ENMs emulating the RPE cell supporting layers of the BM, and found 70nm PLLA ENMs to be best suited as scaffolds for fabricating retinal constructs. STATEMENT OF SIGNIFICANCE: Age related macular degeneration (AMD) is a leading cause of vision loss in the developed world, with an increasing number of people suffering from blindness or severe visual impairment. Transplantation of retinal pigment epithelium (RPE) cells supported on a synthetic, biomimetic-like Bruch's membrane (BM) is considered a promising treatment. However, the synthetic scaffolds used do not mimic the microenvironment of the RPE cell supporting layers, required for the development of a functional RPE monolayer. This study indicated that porous, laminin coated, 70nm PLLA ENMs supported functional RPE monolayers, exhibiting 3D polygonal-cobblestone morphology, apical microvilli, basal infoldings, high transepithelial resistance (TER), phagocytic activity and expression of signature RPE markers. These findings indicate the potential clinical use of porous, laminin coated, 70nm PLLA ENMs in fabricating retinal constructs aimed at treating dry AMD.

Comparative genomics of host adaptive traits in Xanthomonas translucens pv. graminis.

BACKGROUND: Xanthomonas translucens pathovars differ in their individual host ranges among Poaceae. As the causal agent of bacterial wilt in Italian ryegrass (Lolium multiflorum Lam.), X. translucens pv. graminis (Xtg) is one of the most important bacterial pathogens in temperate grassland regions. The genomes of six Xtg strains from Switzerland, Norway, and New Zealand were sequenced in order to gain insight into conserved genomic traits from organisms covering a wide geographical range. Subsequent comparative analysis with previously published genome data of seven non-graminis X. translucens strains including the pathovars arrhenatheri, poae, phlei, cerealis, undulosa, and translucens was conducted to identify candidate genes linked to the host adaptation of Xtg to Italian ryegrass. RESULTS: Phylogenetic analysis revealed a tight clustering of Xtg strains, which were found to share a large core genome. Conserved genomic traits included a non-canonical type III secretion system (T3SS) and a type IV pilus (T4P), which both revealed distinct primary structures of the pilins when compared to the non-graminis X. translucens strains. Xtg-specific traits that had no homologues in the other X. translucens strains were further found to comprise several hypothetical proteins, a TonB-dependent receptor, transporters, and effector proteins as well as toxin-antitoxin systems and DNA methyltransferases. While a nearly complete flagellar gene cluster was identified in one of the sequenced Xtg strains, phenotypic analysis pointed to swimming-deficiency as a common trait of the pathovar graminis. CONCLUSION: Our study suggests that host adaptation of X. translucens pv. graminis may be conferred by a combination of pathovar-specific effector proteins, regulatory mechanisms, and adapted nutrient acquisition. Sequence deviations of pathogen-associated molecular patterns (PAMPs), as observed for the pilins of the T4P and T3SS, are moreover likely to impede perception by the plant defense machinery and thus facilitate successful host colonization of Italian ryegrass.

Overexpression of miR-183/-96/-182 triggers neuronal cell fate in Human Retinal Pigment Epithelial (hRPE) cells in culture.

miR-183 cluster, composed of miR-183/-96/-182 genes, is highly expressed in the adult retina, particularly in photoreceptors. It involves in development, maturation and normal function of neuroretina. Ectopic overexpression of miR-183/-96/-182 genes was performed to assess reprogramming of hRPE cells. They were amplified from genomic DNA and cloned independently or in tandem configuration into pAAV.MCS vector. hRPE cells were then transfected with the recombinant constructs. Real-Time PCR was performed to measure the expression levels of miR-183/-96/-182 and that of several retina-specific neuronal genes such as OTX2, NRL, PDC and DCT. The transfected cells also were immunocytochemically examined for retina-specific neuronal markers, including Rhodopsin, red opsin, CRX, Thy1, CD73, recoverin and PKCα, to determine the cellular fate of the transfected hRPE cells. Data showed that upon miR-183/-96/-182 overexpression in hRPE cultures, the expression of neuronal genes including OTX2, NRL, PDC and DCT was also upregulated. Moreover, miR-183 cluster-treated hRPE cells were immunoreactive for neuronal markers such as Rhodopsin, red opsin, CRX and Thy1. Both transcriptional and translational upregulation of neuronal genes in miR-183 cluster-treated hRPE cells suggests that in vitro overexpression of miR-183 cluster could trigger reprogramming of hRPE cells to retinal neuron fate.

Bioactive compounds isolated from apple, tea, and ginger protect against dicarbonyl induced stress in cultured human retinal epithelial cells.

BACKGROUND: Methylglyoxal (MGO) is known to be a major precursor of advanced glycation end products (AGEs) which are linked to diabetes and its related complications. Naturally occurring bioactive compounds could play an important role in countering AGEs thereby minimizing the risk associated with their formation. METHODS: In this study, eight specific bioactive compounds isolated from apple, tea and ginger were evaluated for their AGEs scavenging activity using Human Retinal Pigment Epithelial (H-RPE) cells treated with MGO. RESULTS: Among the eight specific compounds evaluated, (-)-epigallocatechin 3-gallate (EGCG) from tea, phloretin in apple, and [6]-shogaol and [6]-gingerol from ginger were found to be most effective in preventing MGO-induced cytotoxicity in the epithelial cells. Investigation of possible underlying mechanisms suggests that that these compounds could act by modulating key regulative detoxifying enzymes via modifying nuclear factor-erythroid 2-related factor 2 (Nrf2) function. MGO-induced cytotoxicity led to increased levels of AGEs causing increase in Nε-(Carboxymethyl) lysine (CML) and glutathione (GSH) levels and over expression of receptor for advanced glycation end products (RAGE). Data also showed that translocation of Nrf2 from cytosol to nucleus was inhibited, which decreased the expression of detoxifying enzyme like heme oxygenase-1 (HO-1). The most potent bioactive compounds scavenged dicarbonyl compounds, inhibited AGEs formation and significantly reduced carbonyl stress by Nrf2 related pathway and restoration of HO-1 expression. CONCLUSIONS: These findings demonstrated the protective effect of bioactive compounds derived from food sources against MGO-induced carbonyl stress through activation of the Nrf2 related defense pathway, which is of significant importance for therapeutic interventions in complementary treatment/management of diabetes-related complications.

Bioactive substrates for human retinal pigment epithelial cell growth from elastin-like recombinamers.

The aim of this study was to investigate the use of bioactive RGD-containing elastin-like recombinamers (ELR-RGDs) as a substrate that can maintain human retinal pigment epithelial cell (hRPE) phenotype and growth pattern. Results obtained are compared with previously published behavior of ARPE19 cells. The extension of these results to hRPE is required because ARPE19 cells cannot be used clinically to treat age-related macular degeneration. hRPE cells were isolated, cultured, seeded, and grown on surface of glass, treated polystyrene (TCP), and solvent-cast ELR-RGD and ELR-IK film with no specific sequence. Cells were analyzed to study cell adhesion, proliferation, morphology, and RPE65 protein expression by staining with diamidino-2-phenylindole, Rhodamine-Phalloidin, and anti-RPE65 antibody at 12, 24, 72, 120, 168, and 360 h. hRPE cells always grew better on ELR-RGD than on glass and ELR-IK but not on TCP. The kinetic hRPE growth curves confirmed that growth differences started to appear at 24 h for these surfaces in ascending order of cell growths, namely glass, ELR-IK, ELR-RGD, and TCP. There was a clear difference at 360 h. ELR-RGD maintained hRPE cells stable morphology and RPE65 protein expression. ELR-RGD seems to be a good substrate for growing hRPE cells with stable morphology and RPE65 protein expression. As such, this work confirms our hypothesis regarding ELR-RGD substrates viability, which can be used as a Bruch's membrane prosthesis for further studies in animals. However, these results must subsequently be extrapolated to use of hRPE cells in animals to evaluate them as a transplantation vehicle in human.

High glucose-induced barrier impairment of human retinal pigment epithelium is ameliorated by treatment with Goji berry extracts through modulation of cAMP levels.

Human retinal pigment epithelium cells were used to investigate the mechanisms underlying blood-retinal barrier disruption under conditions of chronic hyperglycemia. The treatment with 25 mM glucose caused a rapid drop in the transepithelial electrical resistance (TEER), which was reversed by the addition of either a methanolic extract from Goji (Lycium barbarum L.) berries or its main component, taurine. Intracellular cAMP levels increased concurrently with the high glucose-induced TEER decrease, and were correlated to an increased activity of the cytosolic isoform of the enzyme adenylyl cyclase. The treatment with plant extract or taurine restored control levels. Data are discussed in view of a possible prevention approach for diabetic retinopathy.