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Retinal pigment epithelial (RPE) cells maintain the health and functional integrity of both photoreceptors and the choroidal vasculature. Loss of RPE differentiation has long been known to play a critical role in numerous retinal diseases, including inherited rod-cone degenerations, inherited macular degeneration, age-related macular degeneration, and proliferative vitreoretinopathy. Recent studies in post-mortem eyes have found upregulation of critical epithelial-mesenchymal transition (EMT) drivers such as TGF-β, Wnt, and Hippo. As RPE cells become less differentiated, they begin to exhibit the defining characteristics of mesenchymal cells, namely, the capacity to migrate and proliferate. A number of preclinical studies, including animal and cell culture experiments, also have shown that RPE cells undergo EMT. Taken together, these data suggest that RPE cells retain the reprogramming capacity to move along a continuum between polarized epithelial cells and mesenchymal cells. We propose that movement along this continuum toward a mesenchymal phenotype be defined as RPE Dysfunction. Potential mechanisms include impaired tight junctions, accumulation of misfolded proteins and dysregulation of several key pathways and molecules, such as TGF-β pathway, Wnt pathway, nicotinamide, microRNA 204/211 and extracellular vesicles. This review synthesizes the evidence implicating EMT of RPE cells in post-mortem eyes, animal studies, primary RPE, iPSC-RPE and ARPE-19 cell lines.

Neurodegenerative retinal diseases, such as glaucoma and diabetic retinopathy, involve a gradual loss of neurons in the retina as the disease progresses. Central nervous system neurons are not able to regenerate in mammals, therefore, an often sought after course of treatment for neuronal loss follows a neuroprotective or regenerative strategy. Neuroprotection is the process of preserving the structure and function of the neurons that have survived a harmful insult; while regenerative approaches aim to replace or rewire the neurons and synaptic connections that were lost, or induce regrowth of damaged axons or dendrites. In order to test the neuroprotective effectiveness or the regenerative capacity of a particular agent, a robust experimental model of retinal neuronal damage is essential. Zebrafish are being used more often in this type of study because their eye structure and development is well-conserved between zebrafish and mammals. Zebrafish are robust genetic tools and are relatively inexpensive to maintain. The large array of functional and behavioral tests available in zebrafish makes them an attractive model for neuroprotection studies. Some common insults used to model retinal disease and study neuroprotection in zebrafish include intense light, chemical toxicity and mechanical damage. This review covers the existing retinal neuroprotection and regeneration literature in the zebrafish and highlights their potential for future studies.

Calsenilin is a calcium ion (Ca 2+ )-binding protein involved in regulating the intracellular concentration of Ca 2+ , a second messenger that controls multiple cellular signaling pathways. The ryanodine receptor (RyR) amplifies Ca 2+ signals entering the cytoplasm by releasing Ca 2+ from endoplasmic reticulum (ER) stores, a process termed calcium-induced calcium release (CICR). Here, we describe a novel mechanism, in which calsenilin controls the activity of neuronal RyRs. We show calsenilin co-localized with RyR2 and 3 in the ER of mouse hippocampal and cortical neurons using immunocytochemistry. The underlying protein-protein interaction between calsenilin and the RyR was determined in mouse central nervous system (CNS) neurons using immunoprecipitation studies. The functional relevance of this interaction was assayed with single-channel electrophysiology. At low physiological Ca 2+ concentrations, calsenilin binding to the cytoplasmic face of neuronal RyRs decreased the RyR’s open probability, while calsenilin increased the open probability at high physiological Ca 2+ concentrations. This novel molecular mechanism was studied further at the cellular level, where faster release kinetics of caffeine-induced Ca 2+ release were measured in SH-SY5Y neuroblastoma cells overexpressing calsenilin. The interaction between calsenilin and neuronal RyRs reveals a new regulatory mechanism and possibly a novel pharmacological target for the control of Ca 2+ release from intracellular stores.

Abstract Rhynchophorus palmarum (Coleoptera: Curculionidae) is a significant agricultural pest in palm plantations across tropical America, playing a critical role as a vector of the fungus Thielaviopsis paradoxa, which is the causative agent of stem bleeding disease in coconut palms. This disease has raised concerns due to its rapid spread and subsequent reduction in coconut production in northeastern Brazil. Additionally, this insect can establish mutualistic interactions with various fungi, including saprophytic, phytopathogenic, and entomopathogenic fungi, underscoring the importance of identifying its external mycobiota. The aim of this study was to assess the presence of T. paradoxa in the digestive tract and identify the cultivable mycobiota associated with the carapace of R. palmarum. To achieve this, a mycological study was conducted by culturing the external surface and digestive tract of field-caught adult insects (10 males and 10 females) on potato dextrose agar (PDA) in Maceió, Alagoas, Brazil. Fungal identification was performed by correlating microscopic features with the macroscopic characteristics of the obtained colonies. The results showed that T. paradoxa was detected in 15.0% of carapace isolates but was not found in the insects' intestinal tract. Additionally, nine fungal genera frequently associated with saprophytic or phytopathogenic behaviors were identified on the carapace. Eight of these genera belong to the Ascomycota phylum, while one is classified in the Basidiomycota phylum. The ubiquitous presence of Paecilomyces spp. and the occurrence of Trichosporon spp. in 95% of the assessed insects stand out. Furthermore, other potentially phytopathogenic fungi such as Penicillium spp., Fusarium spp., and Aspergillus spp., as well as fungi with entomopathogenic potential like Paecilomyces spp., Trichoderma spp., Metarhizium spp., and Beauveria bassiana, were detected. These findings enhance the understanding of the complex interactions between R. palmarum and its fungal hosts, providing insights for integrated pest management strategies. Resumo Rhynchophorus palmarum (Coleoptera: Curculionidae) é uma praga agrícola de plantações de palmeiras na América tropical, apresentando grande importância por ser vetor do fungo Thielaviopsis paradoxa, agente causal da doença de sangramento do tronco (resinose) em coqueiros, que tem causado grande preocupação devido à sua rápida disseminação e consequente redução na produção de coco no nordeste do Brasil. Além disso, este inseto pode apresentar interações mutualísticas com diferentes fungos, incluindo fungos saprofíticos, fitopatógenos e entomopatógenos, destacando a importância de identificar sua micobiota externa. O objetivo deste estudo foi verificar a presença de T. paradoxa no trato digestivo e identificar a micobiota cultivável associada à carapaça de R. palmarum. Para isso, um estudo micológico foi realizado através de cultivo em ágar batata dextrose (APD) da parte externa e do trato digestivo de insetos adultos capturados em campo (10 machos e 10 fêmeas) na cidade de Maceió, Alagoas, Brasil. A identificação dos fungos foi realizada associando os aspectos microscópicos às características macroscópicas das colônias obtidas. Os resultados revelaram que T. paradoxa foi detectado em 15,0% dos isolados da carapaça, mas não foi encontrado no trato intestinal dos insetos. Além disso, foram identificados na carapaça nove gêneros fúngicos frequentemente associados a comportamentos saprófitos ou fitopatogênicos. Oito desses gêneros pertencem ao filo Ascomycota, enquanto um é classificado no filo Basidiomycota. Destaca-se a presença ubíqua de Paecilomyces spp. e a ocorrência de Trichosporon spp. em 95% dos insetos avaliados. Adicionalmente, outros fungos potencialmente fitopatogênicos, como Penicillium spp., Fusarium spp. e Aspergillus spp., e fungos com potencial entomopatogênico, como Paecilomyces spp., Trichoderma spp., Metarhizium spp. e Beauveria bassiana, foram detectados. Essas descobertas ampliam a compreensão das complexas interações entre R. palmarum e seus fungos hospedeiros, fornecendo informações para estratégias de manejo integrado de pragas.

Background:Age-related macular degeneration (AMD) is the most common cause of vision loss in people above the age of 50, affecting approximately 10% of the population worldwide and the incidence is rising. Hyperreflective foci (HRF) are a major predictor of AMD progression. The purpose of this study was to use the sodium iodate mouse model to study HRF formation in retinal degeneration.Methods:Sodium iodate (NaIO3) treated rodents were studied to characterize HRF. 3-month-old male wild-type (WT) C57Bl/6J mice were injected with phosphate-buffered saline (PBS) or varying doses of NaIO3 (15–60 mg/kg). Optical Coherence Tomography (OCT) images were collected at baseline and several days post-NaIO3 injection. Retinal thicknesses were measured using Bioptigen software. Seven days post-injection, eyes were prepared for either transmission electron microscopy (TEM), Hematoxylin & Eosin (H&E), or immunofluorescence.Results:OCT imaging of the mice given higher doses of NaIO3 revealed HRF formation in the neural retina (n = 4). The amount of HRF correlated with the degree of retinal tissue loss. H&E and TEM imaging of the retinas seven days post-NaIO3 injection revealed several pigmented bodies in multiple layers of the retina (n = 3–5). Immunofluorescence revealed that some pigmented bodies were positive for macrophage markers and an epithelial-to-mesenchymal transition marker, while all were retinal pigment epithelium (RPE) 65-negative (n = 4).Conclusions:The data suggest that NaIO3 induces the formation of HRF in the outer retina and their abundance correlates with retinal tissue loss. The experiments in this study highlight NaIO3 as a clinically relevant model of intermediate AMD that can be used to study HRF formation and to discover new treatment targets.

Background/Objectives: The aim of this study was to investigate the association between stunting in the second year of life and metabolic syndrome components in early adulthood among subjects who have been prospectively followed-up since birth, in a city in Southern Brazil. Subjects/Methods: In 1984, we attempted to follow-up the entire cohort; the subjects were examined and their mothers interviewed. Stunting was defined by a length-for-age Z -score 2 s.d. or more below the mean, in accordance with the World Health Organization reference. Between 2004 and 2005, we again tried to follow the entire cohort; during this period the subjects were evaluated for the following metabolic syndrome components: high-density lipoprotein (HDL) cholesterol, triglycerides, random blood glucose, waist circumference and systolic and diastolic blood pressure. Family income at the time of the baby’s birth, asset index, mother’s education, mother’s smoking during pregnancy and duration of breastfeeding were considered possible confounders. Linear regression was used in the unadjusted and adjusted analyses. Results: Among men, stunting was inversely associated with triglycerides ( β =−11.90, confidence interval (CI)=−22.33 to −1.48) and waist circumference ( β =−4.29, CI=−5.62 to −2.97), whereas for women stunting was negatively related to HDL-cholesterol ( β =−4.50, CI=−6.47 to −2.52), triglycerides ( β =−9.61, CI=−17.66 to −1.56) and waist circumference ( β =−1.14, CI=−4.22 to −1.02). However, after controlling for confounding variables, these associations vanished. Conclusions: The findings suggest that stunting in childhood is not associated with metabolic syndrome components in young adults.

In patients with unstable angina, persistent or worsening symptoms and signs of ischemia despite full medical therapy indicate a poor prognosis 1 – 6 . However, at the time of hospital admission, it is not possible to predict whether unstable angina will remit or progress to myocardial infarction, because the causes of instability and the mechanisms underlying its evolution are not known. A role for inflammation in unstable angina is suggested by histologic studies of unstable coronary plaques, 7 – 10 evidence of the systemic release of thromboxanes and leukotrienes, 11 – 13 and the presence of activated circulating leukocytes 14 , 15 . Furthermore, increased concentrations of . . .

N-Palmitoylethanolamine (NAE 16:0) is an endogenous lipid signaling molecule that has limited water solubility, and its action is short-lived due to its rapid metabolism. This poses a problem for use in vivo as oral administration requires a high concentration for significant levels to reach target tissues, and injection of the compound in a dimethyl sulfoxide- or ethanol-based vehicle is usually not desirable during long-term treatment. A depot injection of NAE 16:0 was successfully emulsified in sterile corn oil (10 mg/kg) and administered in young DBA/2 mice in order to elevate baseline levels of NAE 16:0 in target tissues. NAE 16:0 levels were increased in various tissues, particularly in the retina, 24 and 48 hours following injections. Increases ranged between 22% and 215% (above basal levels) in blood serum, heart, brain, and retina and induced an entourage effect by increasing levels of other 18 carbon N-Acylethanolamines (NAEs), which ranged between 31% and 117% above baseline. These results indicate that NAE 16:0 can be used as a depot preparation, avoiding the use of inadequate vehicles, and can provide the basis for designing tissue-specific dosing regimens for therapies involving NAEs and related compounds.

We have shown previously that expression of R345W‐Fibulin‐3 induces epithelial‐mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells. The purpose of the current study was to determine if extracellular vesicles (EVs) derived from RPE cells expressing R345W‐Fibulin‐3 mutation are sufficient to induce EMT in recipient cells. ARPE‐19 cells were infected with luciferase‐tagged wild‐type (WT)‐ Fibulin‐3 or luciferase‐tagged R345W‐Fibulin‐3 (R345W) using lentiviruses. EVs were isolated from the media by ultracentrifugation or density gradient ultracentrifugation. Transmission electron microscopy and cryogenic electron microscopy were performed to study the morphology of the EVs. The size distribution of EVs were determined by nanoparticle tracking analysis (NTA). EV cargo was analysed using LC‐MS/MS based proteomics. EV‐associated transforming growth factor beta 1 (TGFβ1) protein was measured by enzyme‐linked immunosorbent assay. The capacity of EVs to stimulate RPE migration was evaluated by treating recipient cells with WT‐ or R345W‐EVs. The role of EV‐bound TGFβ was determined by pre‐incubation of EVs with a pan‐TGFβ blocking antibody or IgG control. EM imaging revealed spherical vesicles with two subpopulations of EVs: a group with diameters around 30 nm and a group with diameters over 100 nm, confirmed by NTA analysis. Pathway analysis revealed that members of the sonic hedgehog pathway were less abundant in R345W‐ EVs, while EMT drivers were enriched. Additionally, R345W‐EVs had higher concentrations of TGFβ1 compared to control. Critically, treatment with R345W‐EVs was sufficient to increase EMT marker expression, as well as cell migration in recipient cells. This EV‐increased cell migration was significantly inhibited by pre‐incubation of EVs with pan‐TGFβ‐neutralising antibody. In conclusion, the expression of R345W‐Fibulin‐3 alters the size and cargo of EVs, which are sufficient to enhance the rate of cell migration in a TGFβ dependent manner. These results suggest that EV‐bound TGFβ plays a critical role in the induction of EMT in RPE cells.

To investigate the role of protein misfolding in retinal pigment epithelial (RPE) cell dysfunction, the effects of R345W-Fibulin-3 expression on RPE cell phenotype were studied. Primary RPE cells were cultured to confluence on Transwells and infected with lentivirus constructs to express wild-type (WT)- or R345W-Fibulin-3. Barrier function was assessed by evaluating zonula occludens-1 (ZO-1) distribution and trans-epithelial electrical resistance (TER). Polarized secretion of vascular endothelial growth factor (VEGF), was measured by Enzyme-linked immunosorbent assay (ELISA). Differentiation status was assessed by qPCR of genes known to be preferentially expressed in terminally differentiated RPE cells, and conversion to an epithelial-mesenchymal transition (EMT) phenotype was assessed by a migration assay. Compared to RPE cells expressing WT-Fibulin-3, ZO-1 distribution was disrupted and TER values were significantly lower in RPE cells expressing R345W-Fibulin-3. In cells expressing mutant Fibulin-3, VEGF secretion was attenuated basally but not in the apical direction, whereas Fibulin-3 secretion was reduced in both the apical and basal directions. Retinal pigment epithelial signature genes were downregulated and multiple genes associated with EMT were upregulated in the mutant group. Migration assays revealed a faster recovery rate in ARPE-19 cells overexpressing R345W-Fibulin-3 compared to WT. The results suggest that expression of R345W-Fibulin-3 promotes EMT in RPE cells.