Crosstalk of protein clearance, inflammasome, and Ca2+ channels in retinal pigment epithelium derived from age-related macular degeneration patients.

Degeneration and/or dysfunction of retinal pigment epithelium (RPE) is generally detected as the formation of intracellular and extracellular protein aggregates, called lipofuscin and drusen, respectively, in patients with age-related macular degeneration (AMD), the leading cause of blindness in the elderly population. These clinical hallmarks are linked to dysfunctional protein homeostasis and inflammation and furthermore, are both regulated by changes in intracellular Ca2+ concentration. While many other cellular mechanisms have been considered in the investigations of AMD-RPE, there has been relatively little work on understanding the interactions of protein clearance, inflammation, and Ca2+ dynamics in disease pathogenesis. Here we established induced pluripotent stem cell-derived RPE from two patients with advanced AMD and from an age- and gender-matched control subject. We studied autophagy and inflammasome activation under disturbed proteostasis in these cell lines and investigated changes in their intracellular Ca2+ concentration and L-type voltage-gated Ca2+ channels. Our work demonstrated dysregulated autophagy and inflammasome activation in AMD-RPE accompanied by reduced intracellular free Ca2+ levels. Interestingly, we found currents through L-type voltage-gated Ca2+ channels to be diminished and showed these channels to be significantly localized to intracellular compartments in AMD-RPE. Taken together, the alterations in Ca2+ dynamics in AMD-RPE together with dysregulated autophagy and inflammasome activation indicate an important role for Ca2+ signaling in AMD pathogenesis, providing new avenues for the development of therapeutic approaches.

VR-haptic and phantom head dental training: Does the order matter? A comparative study from a preclinical fixed prostho-dontics course.

Learning tooth preparation techniques and the finesse required is an important part of preclinical dental education. Being able to practice surgical skills without loss of Frasaco® teeth while being provided with performance analysis data is a boon to students and educators. We investigated the combination of haptics-enhanced virtual reality (Simodont®) and conventional phantom head practice in a preclinical dental course, evaluating the students' performances and perceptions. Forty students were randomized into two groups: Group One began within a VR-haptic setting while Group Two worked with Frasaco® teeth. Halfway through the course the scenarios were switched. A crown preparation test on Frasaco® teeth was conducted at the end of the course. Students' performances and satisfaction were assessed anonymously. Analysis of the students' performances included clinical metrics (occlusal and axial reduction, convergence angle, damage to adjacent teeth). The perceived usefulness of VR-haptic and phantom head simulations was assessed. In Group One, the tooth preparation metrics were more in line with the requested parameters compared to Group Two. All students ranked VR-haptics highly regarding manual dexterity improvement. In conclusion, this study shows that by combining VR-haptics with conventional dental procedures, it is possible to improve important preparation metrics in fixed prosthodontics tooth preparation.

Epithelial-Mesenchymal Transition and Senescence in the Retinal Pigment Epithelium of NFE2L2/PGC-1α Double Knock-Out Mice.

Age-related macular degeneration (AMD) is the most prevalent form of irreversible blindness worldwide in the elderly population. In our previous studies, we found that deficiencies in the nuclear factor, erythroid 2 like 2 (NFE2L2) and peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) genes caused AMD-like pathological phenotypes in mice. In the present work, we show hijacked epithelial-mesenchymal transition (EMT) due to the common loss of PGC-1α and NFE2L2 (double knock-out, dKO) genes in aged animals. The implanted area was assessed by histology, immunohistochemistry and transmission electron microscopy. Confocal microscopy revealed altered regions in the filamentous actin ring. This contrasted with hexagonal RPE morphology in wild-type mice. The ultrastructural RPE features here illustrated loss of apical microvilli, alteration of cell-cell contact, loss of basal in-folding with deposits on Bruch's membrane, and excessive lipofuscin deposition in dKO samples. We also found the expression of epithelial-mesenchymal transition transcription factors, such as Snail, Slug, collagen 1, vimentin and OB-cadherin, to be significantly different in dKO RPEs. An increased immunoreactivity of senescence markers p16, DEC1 and HMGB1 was also noted. These findings suggest that EMT and senescence pathways may intersect in the retinas of dKO mice. Both processes can be activated by damage to the RPE, which may be caused by increased oxidative stress resulting from the absence of NFE2L2 and PGC-1α genes, important for antioxidant defense. This dKO model may provide useful tools for studying AMD pathogenesis and evaluating novel therapies for this disease.

The combination of listening to background music and deep breathing as a stress reduction strategy during dental preclinical study.

OBJECTIVES: According to our earlier study, background music can help foster dental preclinical manual dexterity training and learning and has the potential to increase student satisfaction and productivity. Deep diaphragmatic breathing helps produce a relaxed mental state and is considered a behavioral coping strategy to reduce anxiety. Experimental data on whether background music combined with deep belly breathing reduces the stress and anxiety of preclinical dental students during practice is lacking. Thus, we focused on evaluating the effects of the combined use of these two techniques on dental students during preclinical manual skills training. METHODS: Thirty-one voluntary 3rd-year dental students' anxiety levels and their behavioral changes were monitored via anonymous, voluntary surveys including the Patient Health Questionnaire-4, General Health Questionnaire-12 (GHQ-12), and Visual Analogue Scale for Anxiety (VAS-A). RESULTS: Overall, stress measured by GHQ-12 decreased significantly in female students (p = 0.025), but not in males. Both background music and deep belly breathing significantly reduced all students' fear and stress based on their VAS-A scores in practice. However, a clear difference from the nonuse of coping strategies could not be conclusively shown. The students regarded the course positively based on survey replies. CONCLUSIONS: The combined use of the two coping strategies delivered beneficial effects to the preclinical dental students: they reduced the amount of stress in the supervised preclinical students and considerably cut down the students' anxiety. Further investigation of this new dental training scenario may help professionals provide better student education and care management during the dental preclinical phase.

Influence of background music on stress reduction and impact on performances during students' simulation exercises.

BACKGROUND: The acquisition of manual skills is essential in preclinical dental training. Background music improves the learning of many manual skills, though we found no data on the consequences of background music on preclinical manual skills training of dental students. OBJECTIVE: The first aim of this project was to explore whether listening to slow background music could reduce the stress of students when learning how to perform cavity preparations and restorations in a simulation laboratory. The second aim of this study was to determine the impact of slow background music on the quality and time used during cavity preparation. METHOD: We invited all of the 40 third-year dental students to participate in the study, of whom 88% chose to anonymously fill in questionnaires on their subjective evaluations of the effects of slow background music on the stress or anxiety levels experienced during the course. Twenty-four students further volunteered to participate in a cross-over study on the impact of slow background music on the quality of and time used during cavity preparation. RESULTS: The overall satisfaction with the slow background music was high. In particular, the music reduced stress but also increased motivation to learn and practice. Communication in the classroom went well despite the music. Time use and quality of cavity preparation were enhanced. CONCLUSION: This study lends support to the use of slow background music in preclinical cariology training, as it appeared to have helpful effects on dental skills education and practice.

Epithelial-mesenchymal transition-related serum markers ET-1, IL-8 and TGF-ß2 are elevated in a Finnish wet age-related macular degeneration cohort.

PURPOSE: It has been hypothesized that epithelial-mesenchymal transition (EMT) may occur in the retinal pigment epithelium of advanced stage age-related macular degeneration (AMD). Various serum and plasma growth factors and inflammatory mediators have been linked to AMD. We were interested in finding out whether systemic levels of EMT-associated markers were altered in the serum of wet AMD patients. Serum biomarkers associated with the various pathological processes of AMD may present an avenue towards identifying and characterizing the birth mechanisms of wet AMD, its progression and severity, paving the way towards the application of precision medicine. METHODS: We chose to measure the serum levels of known biomarkers of EMT - EGF (epidermal growth factor), ET-1 (endothelin 1), IL-8 (interleukin 8), TGF-ß1 and TGF-ß2 (transforming growth factor-beta 1 and 2) and VEGF-A (vascular endothelial growth factor A) - using enzyme-linked immunosorbent assays. We measured them from 71 Finnish wet AMD patients who were receiving intravitreal anti-VEGF-A injection treatments, as well as 64 age-adjusted controls. RESULTS: We found significantly elevated levels of ET-1, IL-8 and TGF-ß2 in the serums of wet AMD patients. CONCLUSIONS: ET-1, IL-8 and TGF-ß2 appear to be useful serum biomarkers in understanding active wet AMD. However, we cannot conclude that local retinal EMT-processes could be observed from the corresponding systemic serum biomarkers in patients undergoing anti-VEGF-A treatments.

Nature-Inspired Hybrids (NIH) Improve Proteostasis by Activating Nrf2-Mediated Protective Pathways in Retinal Pigment Epithelial Cells.

Antioxidant systems play key roles in many elderly diseases, including age-related macular degeneration (AMD). Oxidative stress, autophagy impairment and inflammation are well-described in AMD, especially in retinal pigment epithelial (RPE) cells. The master regulator of antioxidant defense Nrf2 has been linked to AMD, autophagy and inflammation. In this study, in human ARPE-19 cells, some nature-inspired hybrids (NIH1-3) previously shown to induce Nrf2-mediated protection against oxidative stress were further investigated for their potential against cellular stress caused by dysfunction of protein homeostasis. NIH1-3 compounds increased the expression of two Nrf2-target genes coding defense proteins, HO-1 and SQSTM1/p62, in turn exerting beneficial effects on intracellular redox balance without modification of the autophagy flux. NIH1-3 treatments predisposed ARPE-19 cells to a better response to following exposure to proteasome and autophagy inhibitors, as revealed by the increase in cell survival and decreased secretion of the pro-inflammatory IL-8 compared to NIH-untreated cells. Interestingly, NIH4 compound, through an Nrf2-independent pathway, also increased cell viability and decreased IL-8 secretion, although to a lesser extent than NIH1-3, suggesting that all NIHs are worthy of further investigation into their cytoprotective properties. This study confirms Nrf2 as a valuable pharmacological target in contexts characterized by oxidative stress, such as AMD.

Inhibition of prolyl oligopeptidase: A promising pathway to prevent the progression of age-related macular degeneration.

Dry age-related macular degeneration (AMD) is a currently untreatable vision threatening disease. Impaired proteasomal clearance and autophagy in the retinal pigment epithelium (RPE) and subsequent photoreceptor damage are connected with dry AMD, but detailed pathophysiology is still unclear. In this paper, we discover inhibition of cytosolic protease, prolyl oligopeptidase (PREP), as a potential pathway to treat dry AMD. We showed that PREP inhibitor exposure induced autophagy in the RPE cells, shown by increased LC3-II levels and decreased p62 levels. PREP inhibitor treatment increased total levels of autophagic vacuoles in the RPE cells. Global proteomics was used to examine the phenotype of a commonly used cell model displaying AMD characteristics, oxidative stress and altered protein metabolism, in vitro. These RPE cells displayed induced protein aggregation and clear alterations in macromolecule metabolism, confirming the relevance of the cell model. Differences in intracellular target engagement of PREP inhibitors were observed with cellular thermal shift assay (CETSA). These differences were explained by intracellular drug exposure (the unbound cellular partition coefficient, Kpuu). Importantly, our data is in line with previous observations regarding the discrepancy between PREP's cleaving activity and outcomes in autophagy. This highlights the need to further explore PREP's role in autophagy so that more effective compounds can be designed to battle diseases in which autophagy induction is needed. The present work is the first report investigating the PREP pathway in the RPE and we predict that the PREP inhibitors can be further optimized for treatment of dry AMD.

Pinosylvin Extract Retinari™ Sustains Electrophysiological Function, Prevents Thinning of Retina, and Enhances Cellular Response to Oxidative Stress in NFE2L2 Knockout Mice.

Chronic oxidative stress eventually leads to protein aggregation in combination with impaired autophagy, which has been observed in age-related macular degeneration. We have previously shown an effective age-related macular degeneration disease model in mice with nuclear factor-erythroid 2-related factor-2 (NFE2L2) knockout. We have also shown pinosylvin, a polyphenol abundant in bark waste, to increase human retinal pigment epithelium cell viability in vitro. In this work, the effects of commercial natural pinosylvin extract, Retinari™, were studied on the electroretinogram, optical coherence tomogram, autophagic activity, antioxidant capacity, and inflammation markers. Wild-type and NFE2L2 knockout mice were raised until the age of 14.8 ± 3.8 months. They were fed with either regular or Retinari™ chow (141 ± 17.0 mg/kg/day of pinosylvin) for 10 weeks before the assays. Retinari™ treatment preserved significant retinal function with significantly preserved a- and b-wave amplitudes in the electroretinogram responses. Additionally, the treatment prevented thinning of the retina in the NFE2L2 knockout mice. The NFE2L2 knockout mice showed reduced ubiquitin-tagged protein accumulation in addition to local upregulation of complement factor H and antioxidant enzymes superoxide dismutase 1 and catalase. Therefore, the treatment in the NFE2L2 KO disease model led to reduced chronic oxidative stress and sustained retinal function and morphology. Our results demonstrate that pinosylvin supplementation could potentially lower the risk of age-related macular degeneration onset and slow down its progression.