VEGF and ELAVL1/HuR protein levels are increased in dry and wet AMD patients. A new tile in the pathophysiologic mechanisms underlying RPE degeneration?

Age-related macular degeneration (AMD) is a common retinal pathology characterized by degeneration of macula's retinal pigment epithelium (RPE) and photoreceptors, visual impairment, or loss. Compared to wet AMD, dry AMD is more common, but lacks cures; therefore, identification of new potential therapeutic targets and treatments is urgent. Increased oxidative stress and declining antioxidant, detoxifying systems contribute to the pathophysiologic mechanisms underlying AMD. The present work shows that the Embryonic Lethal Abnormal Vision-Like 1/Human antigen R (ELAVL1/HuR) and the Vascular Endothelial Growth Factor (VEGF) protein levels are higher in the RPE of both dry and wet AMD patients compared to healthy subjects. Moreover, increased HuR protein levels are detected in the retina, and especially in the RPE layer, of a dry AMD model, the nuclear factor erythroid 2-related factor 2 (Nrf2) / peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) double knock-out mouse. The crosstalk among Nrf2, HuR and VEGF has been also studied in ARPE-19 cells in basal and stressful conditions related to the AMD context (i.e., oxidative stress, autophagy impairment, Nrf2 deficit), offering new evidence of the mutual influence between Nrf2 and HuR, of the dependence of VEGF expression and secretion by these two factors, and of the increased susceptibility of cells to stressful conditions in Nrf2- or HuR-impaired contexts. Overall, this study shows evidence of the interplay among Nrf2, HuR and VEGF, essential factors for RPE homeostasis, and represents an additional piece in the understanding of the complex pathophysiologic mechanisms underlying AMD.

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.

Autoimmune amelogenesis imperfecta in patients with APS-1 and coeliac disease.

Ameloblasts are specialized epithelial cells in the jaw that have an indispensable role in tooth enamel formation-amelogenesis1. Amelogenesis depends on multiple ameloblast-derived proteins that function as a scaffold for hydroxyapatite crystals. The loss of function of ameloblast-derived proteins results in a group of rare congenital disorders called amelogenesis imperfecta2. Defects in enamel formation are also found in patients with autoimmune polyglandular syndrome type-1 (APS-1), caused by AIRE deficiency3,4, and in patients diagnosed with coeliac disease5-7. However, the underlying mechanisms remain unclear. Here we show that the vast majority of patients with APS-1 and coeliac disease develop autoantibodies (mostly of the IgA isotype) against ameloblast-specific proteins, the expression of which is induced by AIRE in the thymus. This in turn results in a breakdown of central tolerance, and subsequent generation of corresponding autoantibodies that interfere with enamel formation. However, in coeliac disease, the generation of such autoantibodies seems to be driven by a breakdown of peripheral tolerance to intestinal antigens that are also expressed in enamel tissue. Both conditions are examples of a previously unidentified type of IgA-dependent autoimmune disorder that we collectively name autoimmune amelogenesis imperfecta.

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.

Oxidative Stress and Cellular Protein Accumulation Are Present in Keratoconus, Macular Corneal Dystrophy, and Fuchs Endothelial Corneal Dystrophy.

The aim of the study was to investigate oxidative stress as well as cellular protein accumulation in corneal diseases including keratoconus (KC), macular corneal dystrophy (MCD), and Fuchs endothelial corneal dystrophy (FECD) at their primary affecting sites. Corneal buttons from KC, MCD, and FECD patients, as well as healthy controls, were analyzed immunohistochemically to evaluate the presence of oxidative stress and the function of the proteostasis network. 4-Fydroxynonenal (4-HNE) was used as a marker of oxidative stress, whereas the levels of catalase and heat-shock protein 70 (HSP70) were analyzed to evaluate the response of the antioxidant defense system and molecular chaperones, respectively. Sequestosome 1 (SQSTM1) levels were determined to assess protein aggregation and the functionality of autophagic degradation. Basal epithelial cells of the KC samples showed increased levels of oxidative stress marker 4-HNE and antioxidant enzyme catalase together with elevated levels of HSP70 and accumulation of SQSTM1. Corneal stromal cells and endothelial cells from MCD and FECD samples, respectively, showed similarly increased levels of these markers. All corneal diseases showed the presence of oxidative stress and activation of the molecular chaperone response to sustain protein homeostasis. However, the accumulation of protein aggregates suggests insufficient function of the protective mechanisms to limit the oxidative damage and removal of protein aggregates via autophagy. These results suggest that oxidative stress has a role in KC, MCD, and FECD at the cellular level as a secondary outcome. Thus, antioxidant- and autophagy-targeted therapies could be included as supporting care when treating KC or corneal dystrophies.

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 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.

MicroRNAs in the regulation of autophagy and their possible use in age-related macular degeneration therapy.

Age-related macular degeneration (AMD) is a progressive sight-impairing disease of the elderly. The pathogenic mechanisms of AMD are not well understood although both genetic and many environmental factors have been associated with the development of AMD. One clinical hallmark of AMD is the detrimental aggregation of damaged proteins. Recently, it has been suggested that the weakening of autophagy clearance is an important mechanism in the pathogenesis of AMD. Autophagy is important in the removal of damaged or no longer needed cellular material and its recycling. A considerable number of autophagy-targeting microRNAs (miRNAs), small RNA molecules and epigenetic regulators have been found to be either up- or down-regulated in AMD patients and experimental models. The important role of autophagy-targeting miRNAs is supported by several studies and can open the prospect of the use of these miRNAs in the therapy for AMD.

Mechanisms of mitochondrial dysfunction and their impact on age-related macular degeneration.

Oxidative stress-induced damage to the retinal pigment epithelium (RPE) is considered to be a key factor in age-related macular degeneration (AMD) pathology. RPE cells are constantly exposed to oxidative stress that may lead to the accumulation of damaged cellular proteins, lipids, nucleic acids, and cellular organelles, including mitochondria. The ubiquitin-proteasome and the lysosomal/autophagy pathways are the two major proteolytic systems to remove damaged proteins and organelles. There is increasing evidence that proteostasis is disturbed in RPE as evidenced by lysosomal lipofuscin and extracellular drusen accumulation in AMD. Nuclear factor-erythroid 2-related factor-2 (NFE2L2) and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) are master transcription factors in the regulation of antioxidant enzymes, clearance systems, and biogenesis of mitochondria. The precise cause of RPE degeneration and the onset and progression of AMD are not fully understood. However, mitochondria dysfunction, increased reactive oxygen species (ROS) production, and mitochondrial DNA (mtDNA) damage are observed together with increased protein aggregation and inflammation in AMD. In contrast, functional mitochondria prevent RPE cells damage and suppress inflammation. Here, we will discuss the role of mitochondria in RPE degeneration and AMD pathology focused on mtDNA damage and repair, autophagy/mitophagy signaling, and regulation of inflammation. Mitochondria are putative therapeutic targets to prevent or treat AMD.

Mitophagy in the Retinal Pigment Epithelium of Dry Age-Related Macular Degeneration Investigated in the NFE2L2/PGC-1α-/- Mouse Model.

Increased oxidative stress and mitochondrial damage are observed in protein aggregation diseases, such as age-related macular degeneration (AMD). We have recently reported elevated levels of oxidative stress markers, damaged mitochondria, accumulating lysosomal lipofuscin and extracellular drusen-like structures in the retinal pigment epithelial cells (RPE) of the dry AMD-resembling NFE2L2/PGC1α double knockout (dKO) mouse model. Here, we provide evidence of a disturbance in the autolysosomal machinery handling mitochondrial clearance in the RPE cells of one-year-old NFE2L2/PGC1α-deficient mice. Confocal immunohistochemical analysis revealed an upregulation of autophagosome marker microtubule-associated proteins 1A/1B light chain 3B (LC3B) as well as numerous mitophagy markers, such as PTE-induced putative kinase 1 (PINK1) and E3 ubiquitin ligase (PARKIN) together with damaged mitochondria. However, we detected no evidence of increased autolysosome formation in transmission electron micrographs or of colocalization of lysosomal marker LAMP2 (lysosome-associated membrane protein 2) and the mitochondrial marker ATP synthase ß in confocal micrographs. Interestingly, we observed an upregulation of late autolysosomal fusion Ras-related protein (Rab7) in the perinuclear space of RPE cells together with autofluorescence aggregates. Our results reveal that there is at least a relative decrease of mitophagy in the RPE cells of NFE2L2/PGC1α dKO mice. This further supports the hypothesis that mitophagy is a putative therapy target in AMD-like pathology.

The Regulation of NFE2L2 (NRF2) Signalling and Epithelial-to-Mesenchymal Transition in Age-Related Macular Degeneration Pathology.

Age-related macular degeneration (AMD) is a mounting cause of loss of sight in the elderly in the developed countries, a trend enhanced by the continual ageing of the population. AMD is a multifactorial and only partly understood, malady. Unfortunately, there is no effective treatment for most AMD patients. It is known that oxidative stress (OS) damages the retinal pigment epithelium (RPE) and contributes to the progression of AMD. We review here the potential importance of two OS-related cellular systems in relation to AMD. First, the nuclear factor erythroid 2-related factor 2 (NFE2L2; NRF2)-mediated OS response signalling pathway is important in the prevention of oxidative damage and a failure of this system could be critical in the development of AMD. Second, epithelial-to-mesenchymal transition (EMT) represents a change in the cellular phenotype, which ultimately leads to the fibrosis encountered in RPE, a characteristic of AMD. Many of the pathways triggering EMT are promoted by OS. The possible interconnections between these two signalling routes are discussed here. From a broader perspective, the control of NFE2L2 and EMT as ways of preventing OS-derived cellular damage could be potentially valuable in the therapy of AMD.

Using online game-based platforms to improve student performance and engagement in histology teaching.

BACKGROUND: Human morphology is a critical component of dental and medical graduate training. Innovations in basic science teaching methods are needed to keep up with an ever-changing landscape of technology. The purpose of this study was to investigate whether students in a medical and dental histology course would have better grades if they used gaming software Kahoot® and whether gamification effects on learning and enjoyment. METHODS: In an effort to both evoke students' interest and expand their skill retention, an online competition using Kahoot® was implemented for first-year students in 2018 (n = 215) at the University of Eastern Finland. Additionally, closed (160/215) or open-ended (41/215) feedback questions were collected and analyzed. RESULTS: The Kahoot® gamification program was successful and resulted in learning gains. The overall participant satisfaction using Kahoot® was high, with students (124/160) indicating that gamification increased their motivation to learn. The gaming approach seemed to enable the students to overcome individual difficulties (139/160) and to set up collaboration (107/160); furthermore, gamification promoted interest (109/160), and the respondents found the immediate feedback from senior professionals to be positive (146/160). In the open-ended survey, the students (23/41) viewed collaborative team- and gamification-based learning positively. CONCLUSION: This study lends support to the use of gamification in the teaching of histology and may provide a foundation for designing a gamification-integrated curriculum across healthcare disciplines.

Fatty acids and oxidized lipoproteins contribute to autophagy and innate immunity responses upon the degeneration of retinal pigment epithelium and development of age-related macular degeneration.

Retinal pigment epithelium (RPE) damage is a primary sign in the development of age-related macular degeneration (AMD) the leading cause of blindness in western countries. RPE cells are exposed to chronic oxidative stress due to constant light exposure, active fatty acid metabolism and high oxygen consumption. RPE cells phagocytosize lipid rich photoreceptor outer segment (POS) which is regulated by circadian rhytmn. Docosahexaenoic acid is present in high quantity in POS and increases oxidative stress, while its metabolites have cytoprotective effects in RPE. During RPE aging, reactive oxygen species and oxidized lipoproteins are considered to be major causes of disturbed autophagy clearance that lead to chronic innate immunity response involved in NOD-Like, Toll-Like, Advanced Glycation End product Receptors (NLRP, TLR, RAGE, respectively), pentraxins and complement systems. We discuss role of fatty acids and lipoproteins in the degeneration of RPE and development of AMD.

Loss of NRF-2 and PGC-1α genes leads to retinal pigment epithelium damage resembling dry age-related macular degeneration.

Age-related macular degeneration (AMD) is a multi-factorial disease that is the leading cause of irreversible and severe vision loss in the developed countries. It has been suggested that the pathogenesis of dry AMD involves impaired protein degradation in retinal pigment epithelial cells (RPE). RPE cells are constantly exposed to oxidative stress that may lead to the accumulation of damaged cellular proteins, DNA and lipids and evoke tissue deterioration during the aging process. The ubiquitin-proteasome pathway and the lysosomal/autophagosomal pathway are the two major proteolytic systems in eukaryotic cells. NRF-2 (nuclear factor-erythroid 2-related factor-2) and PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1 alpha) are master transcription factors in the regulation of cellular detoxification. We investigated the role of NRF-2 and PGC-1α in the regulation of RPE cell structure and function by using global double knockout (dKO) mice. The NRF-2/PGC-1α dKO mice exhibited significant age-dependent RPE degeneration, accumulation of the oxidative stress marker, 4-HNE (4-hydroxynonenal), the endoplasmic reticulum stress markers GRP78 (glucose-regulated protein 78) and ATF4 (activating transcription factor 4), and damaged mitochondria. Moreover, levels of protein ubiquitination and autophagy markers p62/SQSTM1 (sequestosome 1), Beclin-1 and LC3B (microtubule associated protein 1 light chain 3 beta) were significantly increased together with the Iba-1 (ionized calcium binding adaptor molecule 1) mononuclear phagocyte marker and an enlargement of RPE size. These histopathological changes of RPE were accompanied by photoreceptor dysmorphology and vision loss as revealed by electroretinography. Consequently, these novel findings suggest that the NRF-2/PGC-1α dKO mouse is a valuable model for investigating the role of proteasomal and autophagy clearance in the RPE and in the development of dry AMD.

Involvement of adenosine A3 receptors in the chemotactic navigation of macrophages towards apoptotic cells.

The first step in the clearance of apoptotic cells is chemotactic migration of macrophages towards the apoptotic cells guided by find-me signals provided by the dying cells. Upon sensing the chemotactic signals, macrophages release ATP. ATP is then degraded to ADP, AMP and adenosine to trigger purinergic receptors concentrated at the leading edge of the cell. Previous studies have shown that in addition to the chemotactic signals, this purinergic autocrine signaling is required to amplify and translate chemotactic signals into directional motility. In the present study the involvement of adenosine A3 receptors (A3R) was studied in the chemotactic migration of macrophages directed by apoptotic thymocyte-derived find-me signals. By taking video images in vitro, we demonstrate 1, by administering apyrase, which degrades ATP and ADP, that the purinergic autocrine signaling is required for maintaining both the velocity and the directionality of macrophage migration towards the apoptotic thymocytes; 2, by readding 5'-N-ethylcarboxamidoadenosine, an adenosine analogue, to apyrase treated cells that the adenosine receptor signaling alone is sufficient to act so; and 3, by studying migration of various adenosine receptor null or adenosine receptor antagonist-treated macrophages, that the individual loss of the A3R signaling leads to the loss of chemotactic navigation. Though loss of A3Rs does not affect the phagocytotic capacity of macrophages, intraperitoneally-injected apoptotic thymocytes were cleared with a delayed kinetics by A3R null macrophages in vivo due to the impaired chemotactic navigation. All together these data demonstrate the involvement of macrophage A3Rs in the proper chemotactic navigation and consequent in vivo clearance of apoptotic cells. Interestingly, loss of A3Rs did not affect the in vivo clearance of apoptotic thymocytes in the dexamethasone-treated thymus.

Student-focused virtual histology education: Do new scenarios and digital technology matter?

This article was migrated. The article was marked as recommended. Innovative changes have become a critical part of teaching when resources are limited. In this study, we examined whether the student-oriented teaching method, when powered by virtual microscopy, improves histology learning compared to traditional microscope-based studies. Anonymous and voluntary post-course surveys were administered to students and essays were processed for content analysis. Google Analytics was used to obtain accurate Internet usage monitoring for WEBMICROSCOPE®. Using SPSS statistics, the examination scores for 2016 were compared to those of previous year, when the course was taught with a traditional-microscope-based model. The results demonstrated that the new teaching scenario was an effective tool, based on the mean examination scores in 2016 compared to the identical groups in 2015. The survey analysis showed that the students benefited more from using WEBMICROSCOPE® and that they frequently gained access to the Web server when they were not in class. The new scenario helped clarify the concept of histology for most of the students and was generally appreciated during teamwork-based histology classes. Students perceived that the use of the digital technology significantly influenced their confidence in learning the fundamentals of histology. In addition, changing to the new teaching scenario powered by WEBMICROSCOPE® improved the students' motivation to participate in discussions and better understand the concept of Histology between the 2015 and 2016 academic years. Finally, these changes all had a positive impact on the students' attention and satisfaction.

Absence of collagen XVIII in mice causes age-related insufficiency in retinal pigment epithelium proteostasis.

Collagen XVIII has the structural properties of both collagen and proteoglycan. It has been found at the basement membrane/stromal interface where it is thought to mediate their attachment. Endostatin, a proteolytic fragment from collagen XVIII C-terminal end has been reported to possess anti-angiogenic properties. Age-related vision loss in collagen XVIII mutant mice has been accompanied with a pathological accumulation of deposits under the retinal pigment epithelium (RPE). We have recently demonstrated that impaired proteasomal and autophagy clearance are associated with the pathogenesis of age-related macular degeneration. This study examined the staining levels of proteasomal and autophagy markers in the RPE of different ages of the Col18a1 (-/-) mice. Eyes from 3, 6-7, 10-13 and 18 months old mice were enucleated and embedded in paraffin according to the routine protocol. Sequential 5 µm-thick parasagittal samples were immunostained for proteasome and autophagy markers ubiquitin (ub), SQSTM1/p62 and beclin-1. The levels of immunopositivity in the RPE cells were evaluated by confocal microscopy. Collagen XVIII knock-out mice had undergone age-related RPE degeneration accompanied by an accumulation of drusen-like deposits. Ub protein conjugate staining was prominent in both RPE cytoplasm and extracellular space whereas SQSTM1/p62 and beclin-1 stainings were clearly present in the basal part of RPE cell cytoplasm in the Col18a1 (-/-) mice. SQSTM1/p62 displayed mild extracellular space staining. Disturbed proteostasis regulated by collagen XVIII might be responsible for the RPE degeneration, increased protein aggregation, ultimately leading to choroidal neovascularization.

Modification of tooth development by heat shock protein 60.

Although several heat shock proteins have been investigated in relation to tooth development, no available information is available about the spatial and temporal expression pattern of heat shock protein 60 (Hsp 60). To characterize Hsp 60 expression in the structures of the developing tooth germ, we used Western blotting, immunohistochemistry and in situ hybridization. Hsp 60 was present in high amounts in the inner and outer enamel epithelia, enamel knot (EK) and stratum intermedium (SI). Hsp 60 also appeared in odontoblasts beginning in the bell stage. To obtain data on the possible effect of Hsp 60 on isolated lower incisors from mice, we performed in vitro culturing. To investigate the effect of exogenous Hsp 60 on the cell cycle during culturing, we used the 5-bromo-2-deoxyuridine (BrdU) incorporation test on dental cells. Exogenously administered Hsp 60 caused bluntness at the apical part of the 16.5-day-old tooth germs, but it did not influence the proliferation rate of dental cells. We identified the expression of Hsp 60 in the developing tooth germ, which was present in high concentrations in the inner and outer enamel epithelia, EK, SI and odontoblasts. High concentration of exogenous Hsp 60 can cause abnormal morphology of the tooth germ, but it did not influence the proliferation rate of the dental cells. Our results suggest that increased levels of Hsp 60 may cause abnormalities in the morphological development of the tooth germ and support the data on the significance of Hsp during the developmental processes.

TLR signalling can modify the mineralization of tooth germ.

OBJECTIVE: The aim of this work is to investigate the possible role of Toll-like receptor 4 (TLR4) during the development of mouse tooth germ. TLR4 is well known to inhibit mineralization and cause inflammation in mature odontoblasts and dental pulp cells. However, unlike these pathological functions of TLR4, little is known about the developmental function(s) of TLR4 during tooth development. MATERIALS AND METHODS: TLR4 expression was studied via Western blot in developing lower mouse incisors from E13.5 to E18.5. To generate functional data about the effects of TLR4, a specific agonist (LPS) was applied to the medium of in vitro tooth germ cultures, followed by Western blot, histochemical staining, ELISA assay, in situ hybridization and RT-qPCR. RESULTS: Increased accumulation of biotin-labelled LPS was detected in the enamel organ and in preodontoblasts. LPS treatment induced degradation of the inhibitor molecule (IκB) of the NF-κB signalling pathway. However, no morphological alterations were detected in cultured tissue after LPS addition at the applied dosage. Activation of TLR4 inhibited the mineralization of enamel and dentin, as demonstrated by alizarin red staining and as decreased levels of collagen type X. mRNA expression of ameloblastin was elevated after LPS administration. CONCLUSION: These results demonstrate that TLR4 may decrease the mineralization of hard tissues of the tooth germ and may trigger the maturation of ameloblasts; it can give valuable information to understand better congenital tooth abnormalities.

A novel proteotoxic stress associated mechanism for macular corneal dystrophy.

Macular corneal dystrophy is a rare autosomal recessive eye disease affecting primarily the corneal stroma. Abnormal accumulation of proteoglycan aggregates has been observed intra- and extracellularly in the stromal layer. In addition to the stromal keratocytes and corneal lamellae, deposits are also present in the basal epithelial cells, endothelial cells and Descemet's membrane. Misfolding of proteins has a tendency to gather into aggregating deposits. We studied interaction of molecular chaperones and proteasomal clearance in macular dystrophy human samples and in human corneal HCE-2 epithelial cells. Seven cases of macular corneal dystrophy and four normal corneal buttons collected during corneal transplantation were examined for their expression patterns of heat shock protein 70, ubiquitin protein conjugates and SQSTM1/p62. In response to proteasome inhibition the same proteins were analyzed by western blotting. Slit-lamp examination, in vivo confocal cornea microscopy and transmission electron microscopy were used for morphological analyses. Heat shock protein 70, ubiquitin protein conjugates and SQSTM1/p62 were upregulated in both the basal corneal epithelial cells and the stromal keratocytes in macular corneal dystrophy samples that coincided with an increased expression of the same molecules under proteasome inhibition in the HCE-2 cells in vitro. We propose a novel regulatory mechanism that connects the molecular chaperone and proteasomal clearance system in the pathogenesis of macular corneal dystrophy.