Mutations in the human enamelin gene cause autosomal dominant hypoplastic amelogenesis imperfecta... more Mutations in the human enamelin gene cause autosomal dominant hypoplastic amelogenesis imperfecta in which the affected enamel is thin or absent. Study of enamelin knockout NLS-lacZ knockin mice revealed that mineralization along the distal membrane of ameloblast is deficient, resulting in no true enamel formation. To determine the function of enamelin during enamel formation, we characterized the developing teeth of the Enam2/2 mice, generated amelogenin-driven enamelin transgenic mouse models, and then introduced enamelin transgenes into the Enam2/2 mice to rescue enamel defects. Mice at specific stages of development were subjected to morphologic and structural analysis using b-galactosidase staining, immunohistochemistry, and transmission and scanning electron microscopy. Enamelin expression was ameloblast-specific. In the absence of enamelin, ameloblasts pathology became evident at the onset of the secretory stage. Although the aggregated ameloblasts generated matrix-containing...
Serine protease inhibitor SerpinB2 is one of the most upregulated proteins following cellular str... more Serine protease inhibitor SerpinB2 is one of the most upregulated proteins following cellular stress. This multifunctional serpin has been attributed a number of pleiotropic activities, including roles in cell survival, proliferation, differentiation, immunity and extracellular matrix (ECM) remodeling. Studies of cancer cells demonstrated that expression of SerpinB2 is directly repressed by the Trps1 transcription factor, which is a regulator of skeletal and dental tissues mineralization. In our previous studies, we identified SerpinB2 as one of the novel genes highly upregulated by phosphate (Pi) at the initiation of the mineralization process, however SerpinB2 has never been implicated in formation nor homeostasis of mineralized tissues. The aim of this study was to establish, if SerpinB2 is involved in function of cells producing mineralized ECM and to determine the interplay between Pi signaling and Trps1 in the regulation of SerpinB2 expression specifically in cells producing mineralized ECM. Analyses of the SerpinB2 expression pattern in mouse skeletal and dental tissues detected high SerpinB2 protein levels specifically in cells producing mineralized ECM. qRT-PCR and Western blot analyses demonstrated that SerpinB2 expression is activated by elevated Pi specifically in osteogenic cells. However, the Pi-induced SerpinB2 expression was diminished by overexpression of Trps1. Decreased SerpinB2 levels were also detected in osteoblasts and odontoblasts of 2.3Col1a1-Trps1 transgenic mice. Chromatin immunoprecipitation assay (ChIP) revealed that the occupancy of Trps1 on regulatory elements in the SerpinB2 gene changes in response to Pi. In vitro functional assessment of the consequences of SerpinB2 deficiency in cells producing mineralized ECM detected impaired mineralization in SerpinB2-deficient cells in comparison with controls. In conclusion, high and specific expression of SerpinB2 in cells producing mineralized ECM, the impaired mineralization of SerpinB2-deficient cells and regulation of SerpinB2 expression by two molecules regulating formation of mineralized tissues suggest involvement of SerpinB2 in physiological mineralization.
INTRODUCTION In the current study, we investigate the effect of the inflammation occupying the ap... more INTRODUCTION In the current study, we investigate the effect of the inflammation occupying the apical foramen-a phenomenon we refer to as "inflammatory plug"-on the regenerative potential of a root canal therapy. METHODS We performed root canal treatment (RCT) in 12 canine root canals while aseptically instrumenting the apex to a 0.5-mm-wide foramen and obturating the canals with the following materials: collagen sponge, platelet-rich fibrin, and blood clot (no material introduced). RESULTS We were successful in maintaining the integrity of the periapical tissue in 8 of 12 RCTs. Injury to the periapical tissue occurred during the remaining 4 RCTs, which initiated inflammation accompanied by bone and dentin resorption. Our histologic analyses showed that the resulting inflammatory plug contained abundant M1 macrophages and was associated with an absence of intracanal cellular infiltration. On the contrary, noninflamed samples showed signs of repair, as indicated by the migration of periapical cells throughout the root canal. CONCLUSIONS We conclude that controlling periapical inflammation is key while attempting to achieve dental pulp regeneration.
A highly specialized cytoskeletal protein, keratin 75 (K75), expressed primarily in hair follicle... more A highly specialized cytoskeletal protein, keratin 75 (K75), expressed primarily in hair follicles, nail beds, and lingual papillae, was recently discovered in dental enamel, the most highly mineralized hard tissue in the human body. Among many questions this discovery poses, the fundamental question regarding the trafficking and secretion of this protein, which lacks a signal peptide, is of an utmost importance. Here, we present evidence that K75 is expressed during the secretory stage of enamel formation and is present in the forming enamel matrix. We further show that K75 is secreted together with major enamel matrix proteins amelogenin and ameloblastin, and it was detected in Golgi and the endoplasmic reticulum (ER)–Golgi intermediate compartment (ERGIC) but not in rough ER (rER). Inhibition of ER–Golgi transport by brefeldin A did not affect the association of K75 with Golgi, whereas ameloblastin accumulated in rER, and its transport from rER into Golgi was disrupted. Together, these results indicate that K75, a cytosolic protein lacking a signal sequence, is secreted into the forming enamel matrix utilizing portions of the conventional ER–Golgi secretory pathway. To the best of our knowledge, this is the first study providing insights into mechanisms of keratin secretion.
Enamel is the hardest and most resilient tissue in the human body. Enamel includes morphologicall... more Enamel is the hardest and most resilient tissue in the human body. Enamel includes morphologically aligned, parallel, ∼50 nm wide, microns-long nanocrystals, bundled either into 5-μm-wide rods or their space-filling interrod. The orientation of enamel crystals, however, is poorly understood. Here we show that the crystalline c-axes are homogenously oriented in interrod crystals across most of the enamel layer thickness. Within each rod crystals are not co-oriented with one another or with the long axis of the rod, as previously assumed: the c-axes of adjacent nanocrystals are most frequently mis-oriented by 1°–30°, and this orientation within each rod gradually changes, with an overall angle spread that is never zero, but varies between 30°–90° within one rod. Molecular dynamics simulations demonstrate that the observed mis-orientations of adjacent crystals induce crack deflection. This toughening mechanism contributes to the unique resilience of enamel, which lasts a lifetime under...
Magnesium (Mg) alloys are embraced for their biodegradability and biocompatibility. However, Mg d... more Magnesium (Mg) alloys are embraced for their biodegradability and biocompatibility. However, Mg degrades spontaneously in the biological environment in vivo and in vitro, triggering deposition of calcium phosphate on the metal. Upon complete metal absorption, minerals remain in the tissue, which could lead to pathogenic calcification. Hence, our aims are to test the feasibility of matrix GLA protein (MGP) to locally inhibit Mg mineralization that is induced by metal alloy degradation. MGP is a small secretory protein that has been shown to inhibit soft tissue calcification. We exposed Mg to MGP, stably transfected into mammalian cells. Results showed that less calcium and phosphorous deposition on the Mg surface when MGP was present relative to when it was not. In the in vivo mouse intramuscular model conducted for 4 and 6 weeks, Mg rods were embedded in collagen scaffolds, seeded with cells overexpressing MGP. Microtomography, electron dispersive x-ray spectroscopy, and histology assessments revealed lower deposited mineral volume around Mg rods from the MGP group. Compared to other groups, higher volume loss after implantation was observed from the MGP group at both time points, indicating a higher corrosion rate without the protective mineral layer. This study is the first to our knowledge to demonstrate that local exposure to a biomolecule, such as MGP, can modulate the corrosion of Mg-based implants. These findings may have important implications for the future design of endovascular stents and orthopedic devices.
Journal of the American Chemical Society, Jan 19, 2018
The recent observation in parrotfish teeth of X-ray linear dichroism motivated an in-depth invest... more The recent observation in parrotfish teeth of X-ray linear dichroism motivated an in-depth investigation into this spectroscopic effect in various apatite crystals, including geologic hydroxyapatite (Ca(PO)OH), fluorapatite (Ca(PO)F), and their biogenic counterparts in human bone, mouse enamel, and in parrotfish bone, dentin, and enameloid, the equivalent of dental enamel in certain fish. These data are important because they now enable visualization of the nano- to microscale structure of apatite crystals in teeth and bone. Polarization-dependent imaging contrast (PIC) maps of lamellar bone, obtained with a new method that minimizes space-charge and charging effects, show the expected rotating apatite crystal orientations. PIC maps of mouse enamel reveal a complex arrangement of hydroxyapatite crystals perpendicular to the dentin-enamel junction, with rods arranged in a decussation pattern in inner enamel and nearly parallel to one another in outer enamel. In both inner and outer e...
Species of Ostreidae family are key ecosystem engineers and many of them (including and ) are com... more Species of Ostreidae family are key ecosystem engineers and many of them (including and ) are commercially important aquaculture species. Despite similarities in their morphology and ecology, these two species differ in their ability to defend against pathogens potentially reflecting species-specific differential specialization of hemocytes on immune defense vs. biomineralization. To test this hypothesis, we investigated the expression levels of immune and biomineralization-related genes as well as mineralogical and mechanical properties of the shells and the calcium sequestration ability of the hemocytes of and The expression of biomineralization related genes was higher in than in in multiple tissues including mantle edge and hemocytes, while the expression of immune genes was higher in the hemocytes of Hemocytes of contained more calcium (stored intracellularly as calcium carbonate mineral) compared with those of Analysis of the adult shells showed that the crystallinity of calci...
Molluscan exoskeleton (shell) plays multiple important roles including structural support, protec... more Molluscan exoskeleton (shell) plays multiple important roles including structural support, protection from predators and stressors, and physiological homeostasis. Shell formation is a tightly regulated biological process that allows mollusks to build their shells even in environments unfavorable for mineral precipitation. Outer mantle edge epithelial cells (OME) and hemocytes were implicated in this process; however, the exact functions of these cell types in biomineralization are not clear. The Pacific oysters Crassostrea gigas were used to study differences in the expression profiles of selected biomineralization-related genes in hemocytes and mantle cells, and the functional characteristics of hemocytes such as adhesion, motility and phagocytosis. The specialized role of OME in shell formation was supported by high expression levels of the extracellular matrix (ECM) related and cell-cell interaction genes. Density gradient separation of hemocytes revealed four distinct phenotypes...
Magnesium (Mg) and its alloys are promising candidates for use as resorbable materials for biomed... more Magnesium (Mg) and its alloys are promising candidates for use as resorbable materials for biomedical devices that can degrade in situ following healing of the defect, eliminating the need for a second surgery to remove the device. Hydrogen gas is the main product of magnesium corrosion, and one of the limitations for use of Mg devices in clinic is the formation of gas pockets around them. One potential solution to this problem is reducing the rate of corrosion to the levels at which H2 can diffuse through the body fluids. The study's aim was to evaluate the potential of hybrid alkylsilane self-assembled multilayer coatings to reduce Mg corrosion and to modify physicochemical properties of the coatings using surface functionalization. The coating was made by copolymerization of n-Decyltriethoxysilane and Tetramethoxysilane followed by dip coating of metal discs. This resulted in a formation of homogeneous, micron thick, and defect free coating. The coated surface was more hydrophobic than bare Mg, however functionalization of the coating with 3-aminopropyltriethoxysilane reduced the hydrophobicity of the coating. The coatings reduced several fold the rate of Mg corrosion based on the H2 evolution and other assessment methods, and effectively prevented the initial corrosion burst over the first 24 h. In vitro tissue culture studies demonstrated cytocompatibility of the coatings. These results reveal excellent anticorrosive properties and good cytocompatibility of the hybrid alkylsilane coatings and suggest great potential for use of these coatings on resorbable Mg devices.
Non-specific fluorescence from demineralized enamel matrix can significantly compromise the immun... more Non-specific fluorescence from demineralized enamel matrix can significantly compromise the immunofluorescence studies and lead to false positives. Our goal was to assess degrees of non-specific binding under different conditions and try to optimize procedures for immunofluorescence studies of forming enamel. Firstly, we compared two methods for background fluorescence elimination, i.e., sodium borohydride and Sudan Black B treatments. The results demonstrated that Sudan Black B is far superior to sodium borohydride in reducing the background fluorescence in dental tissues. We also studied the extent of non-specific binding of normal sera and purified polyclonal immunoglobulins (IgG) from five mammalian species, guinea pig, rat, rabbit, goat, and sheep, over a broad range of dilutions. For all sera tested fluorescence signals increased exponentially from 1:1000 to 1:100. Interestingly, the non-specific binding of sera from rodent species was below that of positive control in the who...
Mutations in the human enamelin gene cause autosomal dominant hypoplastic amelogenesis imperfecta... more Mutations in the human enamelin gene cause autosomal dominant hypoplastic amelogenesis imperfecta in which the affected enamel is thin or absent. Study of enamelin knockout NLS-lacZ knockin mice revealed that mineralization along the distal membrane of ameloblast is deficient, resulting in no true enamel formation. To determine the function of enamelin during enamel formation, we characterized the developing teeth of the Enam2/2 mice, generated amelogenin-driven enamelin transgenic mouse models, and then introduced enamelin transgenes into the Enam2/2 mice to rescue enamel defects. Mice at specific stages of development were subjected to morphologic and structural analysis using b-galactosidase staining, immunohistochemistry, and transmission and scanning electron microscopy. Enamelin expression was ameloblast-specific. In the absence of enamelin, ameloblasts pathology became evident at the onset of the secretory stage. Although the aggregated ameloblasts generated matrix-containing...
Serine protease inhibitor SerpinB2 is one of the most upregulated proteins following cellular str... more Serine protease inhibitor SerpinB2 is one of the most upregulated proteins following cellular stress. This multifunctional serpin has been attributed a number of pleiotropic activities, including roles in cell survival, proliferation, differentiation, immunity and extracellular matrix (ECM) remodeling. Studies of cancer cells demonstrated that expression of SerpinB2 is directly repressed by the Trps1 transcription factor, which is a regulator of skeletal and dental tissues mineralization. In our previous studies, we identified SerpinB2 as one of the novel genes highly upregulated by phosphate (Pi) at the initiation of the mineralization process, however SerpinB2 has never been implicated in formation nor homeostasis of mineralized tissues. The aim of this study was to establish, if SerpinB2 is involved in function of cells producing mineralized ECM and to determine the interplay between Pi signaling and Trps1 in the regulation of SerpinB2 expression specifically in cells producing mineralized ECM. Analyses of the SerpinB2 expression pattern in mouse skeletal and dental tissues detected high SerpinB2 protein levels specifically in cells producing mineralized ECM. qRT-PCR and Western blot analyses demonstrated that SerpinB2 expression is activated by elevated Pi specifically in osteogenic cells. However, the Pi-induced SerpinB2 expression was diminished by overexpression of Trps1. Decreased SerpinB2 levels were also detected in osteoblasts and odontoblasts of 2.3Col1a1-Trps1 transgenic mice. Chromatin immunoprecipitation assay (ChIP) revealed that the occupancy of Trps1 on regulatory elements in the SerpinB2 gene changes in response to Pi. In vitro functional assessment of the consequences of SerpinB2 deficiency in cells producing mineralized ECM detected impaired mineralization in SerpinB2-deficient cells in comparison with controls. In conclusion, high and specific expression of SerpinB2 in cells producing mineralized ECM, the impaired mineralization of SerpinB2-deficient cells and regulation of SerpinB2 expression by two molecules regulating formation of mineralized tissues suggest involvement of SerpinB2 in physiological mineralization.
INTRODUCTION In the current study, we investigate the effect of the inflammation occupying the ap... more INTRODUCTION In the current study, we investigate the effect of the inflammation occupying the apical foramen-a phenomenon we refer to as "inflammatory plug"-on the regenerative potential of a root canal therapy. METHODS We performed root canal treatment (RCT) in 12 canine root canals while aseptically instrumenting the apex to a 0.5-mm-wide foramen and obturating the canals with the following materials: collagen sponge, platelet-rich fibrin, and blood clot (no material introduced). RESULTS We were successful in maintaining the integrity of the periapical tissue in 8 of 12 RCTs. Injury to the periapical tissue occurred during the remaining 4 RCTs, which initiated inflammation accompanied by bone and dentin resorption. Our histologic analyses showed that the resulting inflammatory plug contained abundant M1 macrophages and was associated with an absence of intracanal cellular infiltration. On the contrary, noninflamed samples showed signs of repair, as indicated by the migration of periapical cells throughout the root canal. CONCLUSIONS We conclude that controlling periapical inflammation is key while attempting to achieve dental pulp regeneration.
A highly specialized cytoskeletal protein, keratin 75 (K75), expressed primarily in hair follicle... more A highly specialized cytoskeletal protein, keratin 75 (K75), expressed primarily in hair follicles, nail beds, and lingual papillae, was recently discovered in dental enamel, the most highly mineralized hard tissue in the human body. Among many questions this discovery poses, the fundamental question regarding the trafficking and secretion of this protein, which lacks a signal peptide, is of an utmost importance. Here, we present evidence that K75 is expressed during the secretory stage of enamel formation and is present in the forming enamel matrix. We further show that K75 is secreted together with major enamel matrix proteins amelogenin and ameloblastin, and it was detected in Golgi and the endoplasmic reticulum (ER)–Golgi intermediate compartment (ERGIC) but not in rough ER (rER). Inhibition of ER–Golgi transport by brefeldin A did not affect the association of K75 with Golgi, whereas ameloblastin accumulated in rER, and its transport from rER into Golgi was disrupted. Together, these results indicate that K75, a cytosolic protein lacking a signal sequence, is secreted into the forming enamel matrix utilizing portions of the conventional ER–Golgi secretory pathway. To the best of our knowledge, this is the first study providing insights into mechanisms of keratin secretion.
Enamel is the hardest and most resilient tissue in the human body. Enamel includes morphologicall... more Enamel is the hardest and most resilient tissue in the human body. Enamel includes morphologically aligned, parallel, ∼50 nm wide, microns-long nanocrystals, bundled either into 5-μm-wide rods or their space-filling interrod. The orientation of enamel crystals, however, is poorly understood. Here we show that the crystalline c-axes are homogenously oriented in interrod crystals across most of the enamel layer thickness. Within each rod crystals are not co-oriented with one another or with the long axis of the rod, as previously assumed: the c-axes of adjacent nanocrystals are most frequently mis-oriented by 1°–30°, and this orientation within each rod gradually changes, with an overall angle spread that is never zero, but varies between 30°–90° within one rod. Molecular dynamics simulations demonstrate that the observed mis-orientations of adjacent crystals induce crack deflection. This toughening mechanism contributes to the unique resilience of enamel, which lasts a lifetime under...
Magnesium (Mg) alloys are embraced for their biodegradability and biocompatibility. However, Mg d... more Magnesium (Mg) alloys are embraced for their biodegradability and biocompatibility. However, Mg degrades spontaneously in the biological environment in vivo and in vitro, triggering deposition of calcium phosphate on the metal. Upon complete metal absorption, minerals remain in the tissue, which could lead to pathogenic calcification. Hence, our aims are to test the feasibility of matrix GLA protein (MGP) to locally inhibit Mg mineralization that is induced by metal alloy degradation. MGP is a small secretory protein that has been shown to inhibit soft tissue calcification. We exposed Mg to MGP, stably transfected into mammalian cells. Results showed that less calcium and phosphorous deposition on the Mg surface when MGP was present relative to when it was not. In the in vivo mouse intramuscular model conducted for 4 and 6 weeks, Mg rods were embedded in collagen scaffolds, seeded with cells overexpressing MGP. Microtomography, electron dispersive x-ray spectroscopy, and histology assessments revealed lower deposited mineral volume around Mg rods from the MGP group. Compared to other groups, higher volume loss after implantation was observed from the MGP group at both time points, indicating a higher corrosion rate without the protective mineral layer. This study is the first to our knowledge to demonstrate that local exposure to a biomolecule, such as MGP, can modulate the corrosion of Mg-based implants. These findings may have important implications for the future design of endovascular stents and orthopedic devices.
Journal of the American Chemical Society, Jan 19, 2018
The recent observation in parrotfish teeth of X-ray linear dichroism motivated an in-depth invest... more The recent observation in parrotfish teeth of X-ray linear dichroism motivated an in-depth investigation into this spectroscopic effect in various apatite crystals, including geologic hydroxyapatite (Ca(PO)OH), fluorapatite (Ca(PO)F), and their biogenic counterparts in human bone, mouse enamel, and in parrotfish bone, dentin, and enameloid, the equivalent of dental enamel in certain fish. These data are important because they now enable visualization of the nano- to microscale structure of apatite crystals in teeth and bone. Polarization-dependent imaging contrast (PIC) maps of lamellar bone, obtained with a new method that minimizes space-charge and charging effects, show the expected rotating apatite crystal orientations. PIC maps of mouse enamel reveal a complex arrangement of hydroxyapatite crystals perpendicular to the dentin-enamel junction, with rods arranged in a decussation pattern in inner enamel and nearly parallel to one another in outer enamel. In both inner and outer e...
Species of Ostreidae family are key ecosystem engineers and many of them (including and ) are com... more Species of Ostreidae family are key ecosystem engineers and many of them (including and ) are commercially important aquaculture species. Despite similarities in their morphology and ecology, these two species differ in their ability to defend against pathogens potentially reflecting species-specific differential specialization of hemocytes on immune defense vs. biomineralization. To test this hypothesis, we investigated the expression levels of immune and biomineralization-related genes as well as mineralogical and mechanical properties of the shells and the calcium sequestration ability of the hemocytes of and The expression of biomineralization related genes was higher in than in in multiple tissues including mantle edge and hemocytes, while the expression of immune genes was higher in the hemocytes of Hemocytes of contained more calcium (stored intracellularly as calcium carbonate mineral) compared with those of Analysis of the adult shells showed that the crystallinity of calci...
Molluscan exoskeleton (shell) plays multiple important roles including structural support, protec... more Molluscan exoskeleton (shell) plays multiple important roles including structural support, protection from predators and stressors, and physiological homeostasis. Shell formation is a tightly regulated biological process that allows mollusks to build their shells even in environments unfavorable for mineral precipitation. Outer mantle edge epithelial cells (OME) and hemocytes were implicated in this process; however, the exact functions of these cell types in biomineralization are not clear. The Pacific oysters Crassostrea gigas were used to study differences in the expression profiles of selected biomineralization-related genes in hemocytes and mantle cells, and the functional characteristics of hemocytes such as adhesion, motility and phagocytosis. The specialized role of OME in shell formation was supported by high expression levels of the extracellular matrix (ECM) related and cell-cell interaction genes. Density gradient separation of hemocytes revealed four distinct phenotypes...
Magnesium (Mg) and its alloys are promising candidates for use as resorbable materials for biomed... more Magnesium (Mg) and its alloys are promising candidates for use as resorbable materials for biomedical devices that can degrade in situ following healing of the defect, eliminating the need for a second surgery to remove the device. Hydrogen gas is the main product of magnesium corrosion, and one of the limitations for use of Mg devices in clinic is the formation of gas pockets around them. One potential solution to this problem is reducing the rate of corrosion to the levels at which H2 can diffuse through the body fluids. The study's aim was to evaluate the potential of hybrid alkylsilane self-assembled multilayer coatings to reduce Mg corrosion and to modify physicochemical properties of the coatings using surface functionalization. The coating was made by copolymerization of n-Decyltriethoxysilane and Tetramethoxysilane followed by dip coating of metal discs. This resulted in a formation of homogeneous, micron thick, and defect free coating. The coated surface was more hydrophobic than bare Mg, however functionalization of the coating with 3-aminopropyltriethoxysilane reduced the hydrophobicity of the coating. The coatings reduced several fold the rate of Mg corrosion based on the H2 evolution and other assessment methods, and effectively prevented the initial corrosion burst over the first 24 h. In vitro tissue culture studies demonstrated cytocompatibility of the coatings. These results reveal excellent anticorrosive properties and good cytocompatibility of the hybrid alkylsilane coatings and suggest great potential for use of these coatings on resorbable Mg devices.
Non-specific fluorescence from demineralized enamel matrix can significantly compromise the immun... more Non-specific fluorescence from demineralized enamel matrix can significantly compromise the immunofluorescence studies and lead to false positives. Our goal was to assess degrees of non-specific binding under different conditions and try to optimize procedures for immunofluorescence studies of forming enamel. Firstly, we compared two methods for background fluorescence elimination, i.e., sodium borohydride and Sudan Black B treatments. The results demonstrated that Sudan Black B is far superior to sodium borohydride in reducing the background fluorescence in dental tissues. We also studied the extent of non-specific binding of normal sera and purified polyclonal immunoglobulins (IgG) from five mammalian species, guinea pig, rat, rabbit, goat, and sheep, over a broad range of dilutions. For all sera tested fluorescence signals increased exponentially from 1:1000 to 1:100. Interestingly, the non-specific binding of sera from rodent species was below that of positive control in the who...
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Papers by Elia Beniash