The unusual microtubule polarity in teleost retinal pigment epithelial cells.

In cells of the teleost retinal pigment epithelium (RPE), melanin granules disperse into the RPE cells long apical projections in response to light onset, and aggregate toward the base of the RPE cell in response to dark onset. The RPE cells possess numerous microtubules, which in the apical projections are aligned longitudinally. Nocodazole studies have shown that pigment granule aggregation is microtubule-dependent (Troutt, L. L., and B. Burnside, 1988b Exp. Eye Res. In press.). To investigate further the mechanism of microtubule participation in RPE pigment granule aggregation, we have used the tubulin hook method to assess the polarity of microtubules in the apical projections of teleost RPE cells. We report here that virtually all microtubules in the RPE apical projections are uniformly oriented with plus ends toward the cell body and minus ends toward the projection tips. This orientation is opposite that found for microtubules of dermal melanophores, neurons, and most other cell types

Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking alphavbeta5 integrin.

Daily phagocytosis by the retinal pigment epithelium (RPE) of spent photoreceptor outer segment fragments is critical for vision. In the retina, early morning circadian photoreceptor rod shedding precedes synchronized uptake of shed photoreceptor particles by RPE cells. In vitro, RPE cells use the integrin receptor alphavbeta5 for particle binding. Here, we tested RPE phagocytosis and retinal function in beta5 integrin--deficient mice, which specifically lack alphavbeta5 receptors. Retinal photoresponses severely declined with age in beta5-/- mice, whose RPE accumulated autofluorescent storage bodies that are hallmarks of human retinal aging and disease. beta5-/- RPE in culture failed to take up isolated photoreceptor particles. beta5-/- RPE in vivo retained basal uptake levels but lacked the burst of phagocytic activity that followed circadian photoreceptor shedding in wild-type RPE. Rhythmic activation of focal adhesion and Mer tyrosine kinases that mediate wild-type retinal phagocytosis was also completely absent in beta5-/- retina. These results demonstrate an essential role for alphavbeta5 integrin receptors and their downstream signaling pathways in synchronizing retinal phagocytosis. Furthermore, they identify the beta5-/- integrin mouse strain as a new animal model of age-related retinal dysfunction

Profound Re-Organization of Cell Surface Proteome in Equine Retinal Pigment Epithelial Cells in Response to In Vitro Culturing

The purpose of this study was to characterize the cell surface proteome of native compared to cultured equine retinal pigment epithelium (RPE) cells. The RPE plays an essential role in visual function and represents the outer blood-retinal barrier. We are investigating immunopathomechanisms of equine recurrent uveitis, an autoimmune inflammatory disease in horses leading to breakdown of the outer blood-retinal barrier and influx of autoreactive T-cells into affected horses' vitrei. Cell surface proteins of native and cultured RPE cells from eye-healthy horses were captured by biotinylation, analyzed by high resolution mass spectrometry coupled to liquid chromatography (LC MS/MS), and the most interesting candidates were validated by PCR, immunoblotting and immunocytochemistry. A total of 112 proteins were identified, of which 84% were cell surface membrane proteins. Twenty-three of these proteins were concurrently expressed by both cell states, 28 proteins exclusively by native RPE cells. Among the latter were two RPE markers with highly specialized RPE functions: cellular retinaldehyde-binding protein (CRALBP) and retinal pigment epithelium-specific protein 65kDa (RPE65). Furthermore, 61 proteins were only expressed by cultured RPE cells and absent in native cells. As we believe that initiating events, leading to the breakdown of the outer blood-retinal barrier, take place at the cell surface of RPE cells as a particularly exposed barrier structure, this differential characterization of cell surface proteomes of native and cultured equine RPE cells is a prerequisite for future studies

The sustainable delivery of sexual violence prevention education in schools

Sexual violence is a crime that cannot be ignored: it causes our communities significant consequences including heavy economic costs, and evidence of its effects can be seen in our criminal justice system, public health system, Accident Compensation Corporation (ACC), and education system, particularly in our schools. Many agencies throughout New Zealand work to end sexual violence. Auckland-based Rape Prevention Education: Whakatu Mauri (RPE) is one such agency, and is committed to preventing sexual violence by providing a range of programmes and initiatives, information, education, and advocacy to a broad range of audiences. Up until early 2014 RPE employed one or two full-time positions dedicated to co-ordinating and training a large pool (up to 15) of educators on casual contracts to deliver their main school-based programmes, BodySafe – approximately 450 modules per year, delivered to some 20 high schools. Each year several of the contract educators, many of whom were tertiary students, found secure full time employment elsewhere. To retain sufficient contract educators to deliver its BodySafe contract meant that RPE had to recruit, induct and train new educators two to three times every year. This model was expensive, resource intense, and ultimately untenable. The Executive Director and core staff at RPE wanted to develop a more efficient and stable model of delivery that fitted its scarce resources. To enable RPE to know what the most efficient model was nationally and internationally, with Ministry of Justice funding, RPE commissioned Massey University to undertake this report reviewing national and international research on sexual violence prevention education (SVPE). [Background from Executive Summary.]Rape Prevention Education: Whakatu Maur

Decellularised extracellular matrix-derived peptides from neural retina and retinal pigment epithelium enhance the expression of synaptic markers and light responsiveness of human pluripotent stem cell derived retinal organoids

Tissue specific extracellular matrices (ECM) provide structural support and enable access to molecular signals and metabolites, which are essential for directing stem cell renewal and differentiation. To mimic this phenomenon in vitro, tissue decellularisation approaches have been developed, resulting in the generation of natural ECM scaffolds that have comparable physical and biochemical properties of the natural tissues and are currently gaining traction in tissue engineering and regenerative therapies due to the ease of standardised production, and constant availability. In this manuscript we report the successful generation of decellularised ECM-derived peptides from neural retina (decel NR) and retinal pigment epithelium (decel RPE), and their impact on differentiation of human pluripotent stem cells (hPSCs) to retinal organoids. We show that culture media supplementation with decel RPE and RPE-conditioned media (CM RPE) significantly increases the generation of rod photoreceptors, whilst addition of decel NR and decel RPE significantly enhances ribbon synapse marker expression and the light responsiveness of retinal organoids. Photoreceptor maturation, formation of correct synapses between retinal cells and recording of robust light responses from hPSC-derived retinal organoids remain unresolved challenges for the field of regenerative medicine. Enhanced rod photoreceptor differentiation, synaptogenesis and light response in response to addition of decellularised matrices from RPE and neural retina as shown herein provide a novel and substantial advance in generation of retinal organoids for drug screening, tissue engineering and regenerative medicine

The sustainable delivery of sexual violence prevention education in schools

Sexual violence is a crime that cannot be ignored: it causes our communities significant consequences including heavy economic costs, and evidence of its effects can be seen in our criminal justice system, public health system, Accident Compensation Corporation (ACC), and education system, particularly in our schools. Many agencies throughout New Zealand work to end sexual violence. Auckland-based Rape Prevention Education: Whakatu Mauri (RPE) is one such agency, and is committed to preventing sexual violence by providing a range of programmes and initiatives, information, education, and advocacy to a broad range of audiences. Up until early 2014 RPE employed one or two full-time positions dedicated to co-ordinating and training a large pool (up to 15) of educators on casual contracts to deliver their main school-based programmes, BodySafe – approximately 450 modules per year, delivered to some 20 high schools. Each year several of the contract educators, many of whom were tertiary students, found secure full time employment elsewhere. To retain sufficient contract educators to deliver its BodySafe contract meant that RPE had to recruit, induct and train new educators two to three times every year. This model was expensive, resource intense, and ultimately untenable. The Executive Director and core staff at RPE wanted to develop a more efficient and stable model of delivery that fitted its scarce resources. To enable RPE to know what the most efficient model was nationally and internationally, with Ministry of Justice funding, RPE commissioned Massey University to undertake this report reviewing national and international research on sexual violence prevention education (SVPE)

The reproducibility of perceptually regulated exercise responses during short-term cycle ergometry

This is the author's PDF version of an article published in International journal of sports medicine in 2004. The definitive version is available at www.thieme-connect.com.The purpose of this study was to assess the reproducibility over four trials of perceptually regulated exercise intensity during short-term cycle ergometry. Recent research has suggested that an improvement in the reproducibility (better agreement) of the exercise output would be observed with a repeated practice of using regulatory tools such as Borg’s 6-20 rating of perceived exertion (RPE) scale. Eighteen healthy active volunteers (nine males mean age (± SD) 24.7 ± 3.4 yr, and nine females 27.6 ± 5.4 yr) completed four identical intermittent effort production trials on a cycle ergometer, over a period of two-three weeks, with all trials being between three and five days apart. After warm-up, the volunteers were asked to produce four x three-minute bouts of exercise at RPE levels: 13, 15, 9, and 17 (in this order). Power output (W), percentage maximum heart rate reserve (%MHRR), and oxygen consumption (VO2; ml•kg-1•min-1) were recorded in the final minute of each bout. Analysis revealed that the 95% limits of agreement (LoA) between repeated trials did not decrease for the objective markers of exercise intensity, remaining wide throughout. In the worst case comparisons the LoA represented changes (expressed as a proportion of the mean of two trials) of up to 58.3% in power output (T2 vs. T3 at RPE 9), 65.5% in %MHRR (T1 vs. T2 at RPE 13) and 36.5% in VO2 (T3 vs. T4 at RPE 17). These findings question the use of ratings of perceived exertion to regulate exercise effort. That the reproducibility of effort is also not seen to improve with practice raises doubts over the validity of using the RPE scale for providing training intensities for this type of exercise.This article was submitted to the RAE2008 for the University of Chester - Allied Health Professions and Studies

A constitutively active Gαi3 protein corrects the abnormal retinal pigment epithelium phenotype of Oa1-/- mice.

PurposeOcular Albinism type 1 (OA1) is a disease caused by mutations in the OA1 gene and characterized by the presence of macromelanosomes in the retinal pigment epithelium (RPE) as well as abnormal crossing of the optic axons at the optic chiasm. We showed in our previous studies in mice that Oa1 activates specifically Gαi3 in its signaling pathway and thus, hypothesized that a constitutively active Gαi3 in the RPE of Oa1-/- mice might keep on the Oa1 signaling cascade and prevent the formation of macromelanosomes. To test this hypothesis, we have generated transgenic mice that carry the constitutively active Gαi3 (Q204L) protein in the RPE of Oa1-/- mice and are now reporting the effects that the transgene produced on the Oa1-/- RPE phenotype.MethodsTransgenic mice carrying RPE-specific expression of the constitutively active Gαi3 (Q204L) were generated by injecting fertilized eggs of Oa1-/- females with a lentivirus containing the Gαi3 (Q204L) cDNA. PCR, Southern blots, Western blots and confocal microscopy were used to confirm the presence of the transgene in the RPE of positive transgenic mice. Morphometrical analyses were performed using electron microscopy to compare the size and number of melanosomes per RPE area in putative Oa1-/-, Gαi3 (Q204L) transgenic mice with those of wild-type NCrl and Oa1-/- mice.ResultsWe found a correlation between the presence of the constitutively active Gαi3 (Q204L) transgene and the rescue of the normal phenotype of RPE melanosomes in Oa1-/-, Gαi3 (Q204L) mice. These mice have higher density of melanosomes per RPE area and a larger number of small melanosomes than Oa1-/- mice, and their RPE phenotype is similar to that of wild-type mice.ConclusionsOur results show that a constitutively active Gαi3 protein can by-pass the lack of Oa1 protein in Oa1-/- mice and consequently rescue the RPE melanosomal phenotype

Appropriately differentiated ARPE-19 cells regain phenotype and gene expression profiles similar to those of native RPE cells.

PurposeThe RPE cell line ARPE-19 provides a dependable and widely used alternative to native RPE. However, replication of the native RPE phenotype becomes more difficult because these cells lose their specialized phenotype after multiple passages. Compounding this problem is the widespread use of ARPE-19 cells in an undifferentiated state to attempt to model RPE functions. We wished to determine whether suitable culture conditions and differentiation could restore the RPE-appropriate expression of genes and proteins to ARPE-19, along with a functional and morphological phenotype resembling native RPE. We compared the transcriptome of ARPE-19 cells kept in long-term culture with those of primary and other human RPE cells to assess the former's inherent plasticity relative to the latter.MethodsARPE-19 cells at passages 9 to 12 grown in DMEM containing high glucose and pyruvate with 1% fetal bovine serum were differentiated for up to 4 months. Immunocytochemistry was performed on ARPE-19 cells grown on filters. Total RNA extracted from ARPE-19 cells cultured for either 4 days or 4 months was used for RNA sequencing (RNA-Seq) analysis using a 2 × 50 bp paired end protocol. The RNA-Seq data were analyzed to identify the affected pathways and recognize shared ontological classification among differentially expressed genes. RPE-specific mRNAs and miRNAs were assessed with quantitative real-time (RT)-PCR, and proteins with western blotting.ResultsARPE-19 cells grown for 4 months developed the classic native RPE phenotype with heavy pigmentation. RPE-expressed genes, including RPE65, RDH5, and RDH10, as well as miR-204/211, were greatly increased in the ARPE-19 cells maintained at confluence for 4 months. The RNA-Seq analysis provided a comprehensive view of the relative abundance and differential expression of the genes in the differentiated ARPE-19 cells. Of the 16,757 genes with detectable signals, nearly 1,681 genes were upregulated, and 1,629 genes were downregulated with a fold change of 2.5 or more differences between 4 months and 4 days of culture. Gene Ontology analysis showed that the upregulated genes were associated with visual cycle, phagocytosis, pigment synthesis, cell differentiation, and RPE-related transcription factors. The majority of the downregulated genes play a role in cell cycle and proliferation.ConclusionsThe ARPE-19 cells cultured for 4 months developed a phenotype characteristic of native RPE and expressed proteins, mRNAs, and miRNAs characteristic of the RPE. Comparison of the ARPE-19 RNA-Seq data set with that of primary human fetal RPE, embryonic stem cell-derived RPE, and native RPE revealed an important overall similar expression ratio among all the models and native tissue. However, none of the cultured models reached the absolute values in the native tissue. The results of this study demonstrate that low-passage ARPE-19 cells can express genes specific to native human RPE cells when appropriately cultured and differentiated