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Summary: A faience head depicting a bearded male was unearthed in a ninth‐century BC context at Tel Abel Beth Maacah, located on the modern Israel‐Lebanese border. During the Iron Age, the site was at the interface between the kingdoms of Israel and Aram‐Damascus and the Phoenician city‐states of Tyre and Sidon. The article discusses the object's iconography, style, composition and manufacturing technology in comparison to other faience heads, as well as its regional and contextual significance. Despite its unique iconography and high quality, we demonstrate its similarity to at least one other head in manufacturing technology and style. It is suggested that it depicts an ideal elite persona, possibly serving as a votive. [ABSTRACT FROM AUTHOR]

In many taxa, females store sperm in specialized storage organs. Most insect sperm storage organs have a tubular structure, typically consisting of a central lumen surrounded by epithelial cells. These specialized tubules perform the essential tasks of transporting sperm through the female reproductive tract and supporting long-term sperm survival and function. Little is known about the way in which female sperm storage organs provide an environment conducive to sperm survival. We address this using a combined light microscopy, micro computed tomography (microCT), and Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) approach for high-resolution correlative three-dimensional imaging to advance our understanding of sperm-female interactions in Drosophila melanogaster. Using this multimodal approach, we were able to scan the lower female reproductive tract and distal portion of the seminal receptacle at low magnification, and to subsequently zoom in for further analysis on an ultrastructural level. Our findings highlight aspects of the way in which the seminal receptacle keeps sperm viable in the lumen, and set the stage for further studies. The methods developed are suitable not only for Drosophila but also for other organisms with soft, delicate tissues., (© 2024. The Author(s).)

Background: Otosclerosis is a common cause of adult-onset progressive hearing loss, affecting 0.3%-0.4% of the population. It results from dysregulation of bone homeostasis in the otic capsule, most commonly leading to fixation of the stapes bone, impairing sound conduction through the middle ear. Otosclerosis has a well-known genetic predisposition including familial cases with apparent autosomal dominant mode of inheritance. While linkage analysis and genome-wide association studies suggested an association with several genomic loci and with genes encoding structural proteins involved in bone formation or metabolism, the molecular genetic pathophysiology of human otosclerosis is yet mostly unknown., Methods: Whole-exome sequencing, linkage analysis, generation of CRISPR mutant mice, hearing tests and micro-CT., Results: Through genetic studies of kindred with seven individuals affected by apparent autosomal dominant otosclerosis, we identified a disease-causing variant in SMARCA4 , encoding a key component of the PBAF chromatin remodelling complex. We generated CRISPR-Cas9 transgenic mice carrying the human mutation in the mouse SMARCA4 orthologue. Mutant Smarca4 +/E1548K mice exhibited marked hearing impairment demonstrated through acoustic startle response and auditory brainstem response tests. Isolated ossicles of the auditory bullae of mutant mice exhibited a highly irregular structure of the incus bone, and their in situ micro-CT studies demonstrated the anomalous structure of the incus bone, causing disruption in the ossicular chain., Conclusion: We demonstrate that otosclerosis can be caused by a variant in SMARCA4 , with a similar phenotype of hearing impairment and abnormal bone formation in the auditory bullae in transgenic mice carrying the human mutation in the mouse SMARCA4 orthologue., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

By translating mechanical forces into molecular signals, proprioceptive neurons provide the CNS with information on muscle length and tension, which is necessary to control posture and movement. However, the identities of the molecular players that mediate proprioceptive sensing are largely unknown. Here, we confirm the expression of the mechanosensitive ion channel ASIC2 in proprioceptive sensory neurons. By combining in vivo proprioception-related functional tests with ex vivo electrophysiological analyses of muscle spindles, we showed that mice lacking Asic2 display impairments in muscle spindle responses to stretch and motor coordination tasks. Finally, analysis of skeletons of Asic2 loss-of-function mice revealed a specific effect on spinal alignment. Overall, we identify ASIC2 as a key component in proprioceptive sensing and a regulator of spine alignment., (© 2023 The Authors. Experimental Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

We demonstrate here how nitrate salts of bivalent copper, nickel, cobalt, and manganese, along with an achiral organic ligand, assemble into various structures such as symmetrical double-decker flowers, smooth elongated hexagonal bipyramids, and hexagonal prisms. Large morphological changes occur in these structures because of different metal cations, although they maintain isomorphous hexagonal crystallographic structures. Metal cations with stronger coordination to ligands (Cu and Ni) tend to form uniform crystals with unusual shapes, whereas weaker coordinating metal cations (Mn and Co) produce crystals with more regular hexagonal morphologies. The unusual flower-like crystals formed with copper nitrate have two pairs of six symmetrical petals with hexagonal convex centers. The texture of the petals indicates dendritic growth. Two different types of morphologies were formed by using different copper nitrate-to-ligand ratios. An excess of the metal salt results in uniform and hexagonal crystals having a narrow size distribution, whereas the use of an excess of ligand results in double-decker morphologies. Mechanistically, an intermediate structure was observed with slightly concave facets and a domed center. Such structures most likely play a key role in the formation of double-decker crystals that can be formed by fusion processes. The coordination chemistry results in isostructural chiral frameworks consisting of two types of continuous helical channels. Four pyridine units from four separate ligands are coordinated to the metal center in a plane having a chiral (propeller-type) arrangement. The individual double-decker flower crystals are homochiral and a batch consists of crystals having both handedness., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

The characterization of ancient DNA in fossil bones is providing invaluable information on the genetics of past human and other animal populations. These studies have been aided enormously by the discovery that ancient DNA is relatively well preserved in the petrous bone compared to most other bones. The reasons for this better preservation are however not well understood. Here we examine the hypothesis that one reason for better DNA preservation in the petrous bone is that fresh petrous bone contains more DNA than other bones. We therefore determined the concentrations of osteocyte cells occluded inside lacunae within the petrous bone and compared these concentrations to other bones from the domestic pig using high resolution microCT. We show that the concentrations of osteocyte lacunae in the inner layer of the pig petrous bone adjacent to the otic chamber are about three times higher (around 95,000 lacunae per mm3) than in the mastoid of the temporal bone (around 28,000 lacunae per mm3), as well as the cortical bone of the femur (around 27,000 lacunae per mm3). The sizes and shapes of the lacuna in the inner layer of the petrous bone are similar to those in the femur. We also show that the pig petrous bone lacunae do contain osteocytes using a histological stain for DNA. We therefore confirm and significantly expand upon previous observations of osteocytic lacuna concentrations in the petrous bone, supporting the notion that one possible reason for better preservation of ancient DNA in the petrous bone is that this bone initially contains at least three times more DNA than other bones. Thus during diagenesis more DNA is likely to be preserved in the petrous bone compared to other bones., Competing Interests: The authors have declared that no competing interests exist.

Biofilms are differentiated microbial communities held together by an extracellular matrix. μCT X-ray revealed structured mineralized areas within biofilms of lung pathogens belonging to two distant phyla - the proteobacteria Pseudomonas aeruginosa and the actinobacteria Mycobacterium abscessus . Furthermore, calcium chelation inhibited the assembly of complex bacterial structures for both organisms with little to no effect on cell growth. The molecular mechanisms promoting calcite scaffold formation were surprisingly conserved between the two pathogens as biofilm development was similarly impaired by genetic and biochemical inhibition of calcium uptake and carbonate accumulation. Moreover, chemical inhibition and mutations targeting mineralization significantly reduced the attachment of P. aeruginosa to the lung, as well as the subsequent damage inflicted by biofilms to lung tissues, and restored their sensitivity to antibiotics. This work offers underexplored druggable targets for antibiotics to combat otherwise untreatable biofilm infections., Competing Interests: The authors have submitted a patent titled “methods of disrupting a biofilm and/or preventing formation of same.” Publication number: 20210015889., (© 2022 The Authors.)

Biogenic purine crystals function in vision as mirrors, multilayer reflectors and light scatterers. We investigated a light sensory organ in a primarily wingless insect, the jumping bristletail Lepismachilis rozsypali (Archaeognatha), an ancestral group. The visual system of this animal comprises two compound eyes, two lateral ocelli, and a median ocellus, which is located on the front of the head, pointing downwards to the ground surface. We determined that the median ocellus contains crystals of xanthine, and we obtained insights into their function. To date, xanthine biocrystals have only been found in the Archaeognatha. We performed a structural analysis, using reflection light microscopy, cryo-FIB-SEM, microCT and cryo-SEM. The xanthine crystals cover the bottom of a bowl-shaped volume in the median ocellus, in analogy to a tapetum, and reflect photons to light-sensitive receptors that are spread in the volume without apparent order or preferential orientation. We infer that the median ocellus operates as an irregular multifocal reflector, which is not capable of forming images. A possible function of this organ is to improve photon capture, and by so doing assess distances from the ground surface when jumping by determining changes in the intensity and contrast of the incident light., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The teeth of actinopterygian fish, like those of mammals, consist of a thin outer hyper-mineralized layer (enamel or enameloid) that surrounds a core of dentin. While all mammalian species have a single type of dentin (called orthodentin), various dentin types have been reported in the teeth of actinopterygian fish. The most common type of actinopterygian fish dentin is orthodentin. However, the second most common type of actinopterygian fish dentin, called osteodentin, found in several teleost species and in many Selachians, is structurally radically different from orthodentin. Osteodentin, comprising denteons and inter-denteonal matrix, is characterized by an appearance that is similar to mammalian osteonal bone, however, it lacks cells and a lacuno-canalicular system. The current consensus is that although osteodentin is morphologically different from orthodentin, it is a true dentinal material, the product of odontoblast cells. We present the results of a study of osteodentin found in the teeth of the Atlantic wolffish, Anarhichas lupus. Using a variety of microscopy techniques, high-resolution microCT scans, and micro-indentation we describe the three-dimensional structure of both its components (denteons and inter-denteonal matrix), as well as their mineral density distribution and mechanical properties, at several length-scales. We show that wolffish osteodentin is remarkably similar to the anosteocytic bone of the swords of several swordfish species. We also describe the three-dimensional network of canals found in mature osteodentin. The high density of these canals in a metabolically inactive, acellular tissue casts doubt upon the accepted paradigm, that the canals house a vascular network., (© 2021 Wiley Periodicals LLC.)

The periodontal ligament (PDL) is a highly heterogeneous fibrous connective tissue and plays a critical role in distributing occlusal forces and regulating tissue remodeling. Its mechanical properties are largely determined by the extracellular matrix, comprising a collagenous fiber network interacting with the capillary system as well as interstitial fluid containing proteoglycans. While the phase-contrast micro-CT technique has portrayed the 3D microscopic heterogeneity of PDL, the topological parameters of its network, which is crucial to understanding the multiscale constitutive behavior of this tissue, has not been characterized quantitatively. This study aimed to provide new understanding of such microscopic heterogeneity of the PDL with quantifications at both tissue and collagen network levels in a spatial manner, by combining phase-contrast micro-CT imaging and a purpose-built image processing algorithm for fiber analysis. Both variations within a PDL and among the PDL with different shapes, i.e. round-shaped and kidney-shaped PDLs, are described in terms of tissue thickness, fiber distribution, local fiber densities, and fiber orientation (namely azimuthal and elevation angles). Furthermore, the tissue and collagen fiber network responses to mechanical loading were evaluated in a similar manner. A 3D helical alignment pattern was observed in the fiber network, which appears to regulate and adapt a screw-like tooth motion under occlusion. The microstructural heterogeneity quantified here allows development of sample-specific constitutive models to characterize the PDL's functional and pathological loading responses, thereby providing a new multiscale framework for advancing our knowledge of this complex limited mobility soft-hard tissue interface., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Bone-resorbing osteoclasts (OCLs) are multinucleated phagocytes, whose central roles in regulating bone formation and homeostasis are critical for normal health and development. OCLs are produced from precursor monocytes in a multistage process that includes initial differentiation, cell-cell fusion, and subsequent functional and morphological maturation; the molecular regulation of osteoclastogenesis is not fully understood. Here, we identify the receptor-type protein tyrosine phosphatase PTPRJ as an essential regulator specifically of OCL maturation. Monocytes from PTPRJ-deficient (JKO) mice differentiate and fuse normally, but their maturation into functional OCLs and their ability to degrade bone are severely inhibited. In agreement, mice lacking PTPRJ throughout their bodies or only in OCLs exhibit increased bone mass due to reduced OCL-mediated bone resorption. We further show that PTPRJ promotes OCL maturation by dephosphorylating the M-CSF receptor (M-CSFR) and Cbl, thus reducing the ubiquitination and degradation of the key osteoclastogenic transcription factor NFATc1. Loss of PTPRJ increases ubiquitination of NFATc1 and reduces its amounts at later stages of osteoclastogenesis, thereby inhibiting OCL maturation. PTPRJ thus fulfills an essential and cell-autonomous role in promoting OCL maturation by balancing between the pro- and anti-osteoclastogenic activities of the M-CSFR and maintaining NFATc1 expression during late osteoclastogenesis., (© 2021 Federation of European Biochemical Societies.)

The coexistence of single-crystallinity with a multidomain morphology is a paradoxical phenomenon occurring in biomineralization. Translating such feature to synthetic materials is a highly challenging process in crystal engineering. We demonstrate the formation of metallo-organic single-crystals with a unique appearance: six-connected half-rods forming a hexagonal-like tube. These uniform objects are formed from unstable, monodomain crystals. The monodomain crystals dissolve from the inner regions, while material is anisotropically added to their shell, resulting in hollow, single-crystals. Regardless of the different morphologies and growth mechanism, the crystallographic structures of the mono- and multidomain crystals are nearly identical. The chiral crystals are formed from achiral components, and belong to a rare space group (P622). Sonication of the solvents generating radical species is essential for forming the multidomain single-crystals. This process reduces the concentration of the active metal salt. Our approach offers opportunities to generate a new class of crystals.

Gaucher disease (GD) is currently the focus of considerable attention due primarily to the association between the gene that causes GD (GBA) and Parkinson's disease. Mouse models exist for the systemic (type 1) and for the acute neuronopathic forms (type 2) of GD. Here we report the generation of a mouse that phenotypically models chronic neuronopathic type 3 GD. Gba -/- ;Gba tg mice, which contain a Gba transgene regulated by doxycycline, accumulate moderate levels of the offending substrate in GD, glucosylceramide, and live for up to 10 months, i.e. significantly longer than mice which model type 2 GD. Gba -/- ;Gba tg mice display behavioral abnormalities at ∼4 months, which deteriorate with age, along with significant neuropathology including loss of Purkinje neurons. Gene expression is altered in the brain and in isolated microglia, although the changes in gene expression are less extensive than in mice modeling type 2 disease. Finally, bone deformities are consistent with the Gba -/- ;Gba tg mice being a genuine type 3 GD model. Together, the Gba -/- ;Gba tg mice share pathological pathways with acute neuronopathic GD mice but also display differences that might help understand the distinct disease course and progression of type 2 and 3 patients., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The vomeronasal organ (VNO) specializes in detection of chemosignals, mainly pheromones, which control social communication and reproduction in many mammals. These pheromones must solubilize with nasal fluids before entering the VNO, and it was suggested that they are delivered to and cleared from the VNO by active pumping. Yet, the details of this pheromone delivery process are unclear. In this study, we first constructed a high-resolution 3D morphological image of the whole adult mouse snout, by using ultra-high-resolution micro-CT. We identified a net of micro tunnels starting from the nostrils and extending around and through the VNO. These micro tunnels connect the nasal cavity with the VNO and the oral cavity via the nasopalatine ducts (NPD). Other micro tunnels connect the nasal cavity to the main olfactory epithelium. We next demonstrated that physical obstruction of the NPD severely impairs the clearance of dissolved compounds from the VNO lumen. Moreover, we found that mice with blocked NPD display alterations in chemosignaling-evoked neuronal activation in brain regions associated with the vomeronasal system. Finally, NPD-blocked male mice exhibit reduced preference for female chemosignals, and impaired social interaction behavior. Taken together, our findings indicate that the NPD in mice are connected to both the nasal and oral cavity, serving an essential role in regulating the flow of soluble chemosignals through the VNO, and are required for proper pheromone-mediated social communication., (Copyright © 2020 Levy, Sofer, Brumfeld, Zilkha and Kimchi.)

Reflective assemblies of high refractive index organic crystals are used to produce striking optical phenomena in organisms based on light reflection and scattering. In aquatic animals, organic crystal-based reflectors are used both for image-formation and to increase photon capture. Here we report the characterization of a poorly-documented reflector in the eye of the shrimp L. vannamei lying 150 μm below the retina, which we term the proximal reflective layer (PR-layer). The PR-layer is made from a dense but disordered array of polycrystalline isoxanthopterin nanoparticles, similar to those recently reported in the tapetum of the same animal. Each spherical nanoparticle is composed of numerous isoxanthopterin single crystal plates arranged in concentric lamellae around an aqueous core. The highly reflective plate faces of the crystals are all aligned tangentially to the particle surface with the optical axes projecting radially outwards, forming a birefringent spherulite which efficiently scatters light. The nanoparticle assemblies form a broadband reflective sheath around the screening pigments of the eye, resulting in pronounced eye-shine when the animal is viewed from a dorsal-posterior direction, rendering the eye pigments inconspicuous. We assess possible functions of the PR-layer and conclude that it likely functions as a camouflage device to conceal the dark eye pigments in an otherwise largely transparent animal.

We examine the structure of the bone of the pharyngeal jaws of a large fish, the black drum (Pogonias cromis), that uses its tooth-jaw complex to crush hard-shelled bivalve mollusks. During mastication huge compressive forces are concentrated in a tiny zone at the tooth-bone interface. We report on the structure of this bone, with emphasis on its contact with the teeth, at different hierarchical levels and in 3D. Micro-CT shows that the molariform teeth do not have roots and are supported by a circular narrow bony rim that surrounds the periphery of the tooth base. The lower pharyngeal jaw is highly porous, as seen by reflected light microscopy and secondary electron microscopy (SE-SEM). Porosity decreases close to the bone-tooth interface and back-scattered electron (BSE-SEM) microscopy shows a slight elevation in mineral density. Focused ion beam - scanning electron microscopy (FIB-SEM) in the serial surface view (SSV) mode reveals a most surprising organization at the nanoscale level: parallel arrays of mineralized collagen fibrils surrounding channels of ~100 nm diameter, both with their long axes oriented along the load direction. The channels are filled with organic matter. These fibril-channel arrays are surrounded by a highly disordered mineralized material. This unusual structure clearly functions efficiently under compression, but the precise way by which this unique arrangement achieves this function is unknown., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In humans, mutations in the PIEZO2 gene, which encodes for a mechanosensitive ion channel, were found to result in skeletal abnormalities including scoliosis and hip dysplasia. Here, we show in mice that loss of Piezo2 expression in the proprioceptive system recapitulates several human skeletal abnormalities. While loss of Piezo2 in chondrogenic or osteogenic lineages does not lead to human-like skeletal abnormalities, its loss in proprioceptive neurons leads to spine malalignment and hip dysplasia. To validate the non-autonomous role of proprioception in hip joint morphogenesis, we studied this process in mice mutant for proprioceptive system regulators Runx3 or Egr3. Loss of Runx3 in the peripheral nervous system, but not in skeletal lineages, leads to similar joint abnormalities, as does Egr3 loss of function. These findings expand the range of known regulatory roles of the proprioception system on the skeleton and provide a central component of the underlying molecular mechanism, namely Piezo2.

WS 2 inorganic nanotubes (WS 2 -NT) have been incorporated into Polylactic Acid (PLA) by melt mixing to create a bio-degradable, mechanically reinforced nanocomposite filament. The filament was then processed by Fused Filament Fabrication (FFF) 3D-printer, and the morphology and characteristics before and after printing were compared. We found that addition of WS 2 -NT to PLA by extrusion mixing increases the elastic modulus, yield strength and strain-at-failure by 20%, 23% and 35%, respectively. Moreover, we found that the printing process itself improves the dispersion of WS 2 -NT within the PLA filament, and does not require changing of the printing parameters compared to pure PLA. The results demonstrate the advantage of WS 2 -NT as reinforcement specifically in 3D-printable polymers, over more traditional nano-reinforcements such as graphene and carbon nanotubes. WS 2 -NT based 3D-printable nanocomposites can be used for variety of applications from custom-made biodegradable scaffold of soft implants such as cartilage-based organs and biodegradable soft stents to the more general easy-to-apply nano-reinforced polymers.

Trees of the genus Prunus produce some of the most widely consumed fruits globally. The combination of climate change-related warming and increased drought stress, scarcity of freshwater resources for irrigation, and increasing demands due to population growth creates a need for increased drought tolerance in these tree species. Recently, we have shown in the field that a native wild pear species performs better under drought than two cultivated pear species. Here, a comparative field study was conducted in Israel to investigate traits associated with drought tolerance in almond (cultivated Prunus dulcis (Mill.) D. A. Webb vs wild Prunus ramonensis Danin) and plum (cultivated Prunus domestica L. vs wild Prunus ursina Kotschy). Measurements of xylem embolism and shoot and root carbon reserves were done along a year, including seasonal drought in the wild and a 35-day drought experiment in the orchards. Synchronous measurements of native xylem embolism and shoot water potential showed that cultivated and wild almond trees lost ~50% of hydraulic conductivity at -2.3 and -3.2 MPa, respectively. Micro-CT images confirmed the higher embolism ratio in cultivated versus wild almond, whereas the two plum species were similar. Dynamics of tissue concentrations of nonstructural carbohydrates were mostly similar across species, with higher levels in cultivated versus wild plum. Our results indicate an advantage for the wild almond over its cultivated relative in terms of xylem resistance to embolism, a major risk factor for trees under drought stress. This result is in line with our previous experiment on pear species. However, the opposite trends observed among the studied plum species mean that these trends cannot be generalized. It is possible that the potential for superior drought tolerance in wild tree species, relative to their cultivated relatives, is limited to wild species from dry and hot habitats., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Naturally occurring single crystals having a multidomain morphology are a counterintuitive phenonomon: the macroscopic appearance is expected to follow the symmetry of the unit cell. Growing such crystals in the lab is a great challenge, especially from organic molecules. We achieve here uniform metallo-organic crystals that exhibit single crystallinity with apparently distinct domains and chirality. The chirality is present at both the molecular and macroscopic levels, although only achiral elements are used. "Yo-yo"-like structures having opposite helical handedness evolve from initially formed seemingly achiral cylinders. This non-polyhedral morphology coexists with a continuous coordination network forming homochiral channels. This work sheds light on the enigmatic aspects of fascinating crystallization processes occurring in biological mineralization. Our findings open up opportunities to generate new porous and hierarchical chiral materials.

Endochondral ossification in the growth plate of long bones involves cartilage mineralization, bone formation and the budding vasculature. Many of these processes take place in a complex and dynamic zone, the provisional ossification zone, of the growth plate. Here we investigate aspects of mineralization in 2D and 3D in the provisional ossification zone at different length scales using samples preserved under cryogenic or fully hydrated conditions. We use confocal light microscopy, cryo-SEM and micro-CT in the phase contrast mode. We show in 9 week old BALB/c mice the presence of vesicles containing mineral particles in the blood serum, as well as mineral particles without membranes integrated with the blood vessel walls. We also observe labeled mineral particles within cells associated with bone formation, but not in the hypertrophic cartilage cells that are involved with cartilage mineralization. High resolution micro-CT images of fresh hydrated tibiae, show that there are open continuous pathways between the blood vessel extremities and the hypertrophic chondrocyte zone. As the blood vessel extremities, the mineralizing cartilage and the forming bone are all closely associated within this narrow zone, we raise the possibility that in addition to ion transport, mineral necessary for both cartilage and bone formation is also transported through the vasculature., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The eyes of many fish contain a reflecting layer of organic crystals partially surrounding the photoreceptors of the retina, which are commonly believed to be composed of guanine. Here we study an unusual fish eye from Stizostedion lucioperca that contains two layers of organic crystals. The crystals in the outer layer are thin plates, whereas the crystals in the inner tapetum layer are block-shaped. We show that the outer layer indeed contains guanine crystals. Analyses of solutions of crystals from the inner layer indicated that the block-shaped crystals are composed of xanthopterin. A model of the structure of the block-shaped crystals was produced using symmetry arguments based on electron diffraction data followed by dispersion-augmented DFT calculations. The resulting crystal structure of xanthopterin included, however, a problematic repulsive interaction between C═O and N of two adjacent molecules. Knowing that dissolved 7,8-dihydroxanthopterin can oxidize to xanthopterin, we replaced xanthopterin with 7,8-dihydroxanthopterin in the model. An excellent fit was obtained with the powder X-ray diffraction pattern of the biogenic crystals. We then analyzed the biogenic block-shaped crystals in their solid state, using MALDI-TOF and Raman spectroscopy. All three methods unequivocally prove that the block-shaped crystals in the eye of S. lucioperca are crystals of 7,8-dihydroxanthopterin. On the basis of the eye anatomy, we deduce that the guanine crystals form a reflective layer producing the silvery color present on part of the eye surface, whereas the block-shaped crystals backscatter light into the retina in order to increase the light sensitivity of the eye.

We investigate the structures and mechanical properties of leaf midribs of Ficus microcarpa and Prunus dulcis, which deposit calcium oxalate crystals, and of Olea europaea midribs which contain no mineral deposits, but do contain lignified fibers. The midrib mechanical performance contributes to the leaf's ability to maintain a flat conformation for light harvesting and to efficiently reconfigure to reduce wind drag. We use a novel approach involving 3D visualization of the vein structure during mechanical load. This involves the use of customized mechanical loading devices that fit inside a microCT chamber. We show that the elastic, compression and torsional moduli of the midribs of leaves from the 3 species examined vary significantly. We also observed different modes of fracture and buckling of the leaves during compression. We assess the contributions of the calcium oxalate crystals to the mechanical and fracture properties. In F. microcarpa midrib linear arrays of calcium oxalate crystals contribute to resisting the bending, in contrast to P. dulcis leaves, where the calcium oxalate crystals do not resist bending. In both F. microcarpa and P. dulcis isolated calcium oxalate crystals enable high torsional compliance. The integrated microCT - mechanical testing approach could be used to investigate the structure-mechanics relationships in other complex biological samples. STATEMENT OF SIGNIFICANCE: Leaves need to maintain a flat conformation for light harvesting, but they also need to efficiently reconfigure to reduce wind drag. The leaf central vein (midrib) is a key structural component for leaf mechanicss. 3D visualization of the vein structure under mechanical loads showed that veins can be stiffened by reinforcement units composed of calcium oxalates crystals and lignin. The stiffening units can influence the bending and fracture properties of the midribs, and can contribute to determine if buckling will occur during folding. Mineral stiffening elements could be a widespread strategy to reinforce leaf veins and other biological structures. This structural-mechanical approach could be used to study other complex biological samples., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

One strategy evolved by teeth to avoid irreversible damage is to move and deform under the loads incurred during mastication. A key component in this regard is the periodontal ligament (PDL). The role of the bone underlying the PDL is less well defined. We study the interplay between the PDL and the underlying alveolar bone when loaded in the minipig. Using an Instron loading device we confirmed that the force-displacement curves of the molars and premolars of relatively fresh minipig intact mandibles are similar to those obtained for humans and other animals. We then used this information to obtain 3D images of the teeth before and after loading the tooth in a microCT such that the load applied is in the third linear part of the force displacement curve. We observed that at many locations there is a complimentary topography of the cementum and alveolar bone surface, strongly suggesting an active interplay between the tooth and the bone during mastication. We also observed that the loaded tooth does not come into direct contact with the underlying bone surface. A highly compressed layer of PDL is present between the tooth and the bone. The structure of the bone in the upper furcation region has a unique appearance with little obvious microstructure, abundant pores that have a large size range and at many locations the bone at the PDL interface has a needle-like shape. We conclude that there is a close interaction between the tooth, the PDL and the underlying alveolar bone during mastication. The highly compressed PDL layer that separates the tooth from the bone may fulfill a key shock absorbing function., (Copyright © 2019. Published by Elsevier Inc.)

Bone resorption by osteoclasts is essential for bone homeostasis. The kinase Src promotes osteoclast activity and is activated in osteoclasts by the receptor-type tyrosine phosphatase PTPROt. In other contexts, however, PTPROt can inhibit Src activity. Through in vivo and in vitro experiments, we show that PTPROt is bifunctional and can dephosphorylate Src both at its inhibitory residue Tyr 527 and its activating residue Tyr 416 Whereas wild-type and PTPROt knockout mice exhibited similar bone masses, mice in which a putative C-terminal phosphorylation site, Tyr 399 , in endogenous PTPROt was replaced with phenylalanine had increased bone mass and reduced osteoclast activity. Osteoclasts from the knock-in mice also showed reduced Src activity. Experiments in cultured cells and in osteoclasts derived from both mouse strains demonstrated that the absence of phosphorylation at Tyr 399 caused PTPROt to dephosphorylate Src at the activating site pTyr 416 In contrast, phosphorylation of PTPROt at Tyr 399 enabled PTPROt to recruit Src through Grb2 and to dephosphorylate Src at the inhibitory site Tyr 527 , thus stimulating Src activity. We conclude that reversible phosphorylation of PTPROt at Tyr 399 is a molecular switch that selects between its opposing activities toward Src and maintains a coherent signaling output, and that blocking this phosphorylation event can induce physiological effects in vivo. Because most receptor-type tyrosine phosphatases contain potential phosphorylation sites at their C termini, we propose that preventing phosphorylation at these sites or its consequences may offer an alternative to inhibiting their catalytic activity to achieve therapeutic benefit., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Many marine organisms have evolved a reflective iris to prevent unfocused light from reaching the retina. The fish iris has a dual function, both to camouflage the eye and serving as a light barrier. Yet, the physical mechanism that enables this dual functionality and the benefits of using a reflective iris have remained unclear. Using synchrotron microfocused diffraction, cryo-scanning electron microscopy imaging, and optical analyses on zebrafish at different stages of development, it is shown that the complex optical response of the iris is facilitated by the development of high-order organization of multilayered guanine-based crystal reflectors and pigments. It is further demonstrated how the efficient light reflector is established during development to allow the optical functionality of the eye, already at early developmental stages.

In nature, bacteria predominantly exist as highly structured biofilms, which are held together by extracellular polymeric substance and protect their residents from environmental insults, such as antibiotics. The mechanisms supporting this phenotypic resistance are poorly understood. Recently, we identified a new mechanism maintaining biofilms - an active production of calcite minerals. In this work, a high-resolution and robust µCT technique is used to study the mineralized areas within intact bacterial biofilms. µCT is a vital tool for visualizing bacterial communities that can provide insights into the relationship between bacterial biofilm structure and function. Our results imply that dense and structured calcium carbonate lamina forms a diffusion barrier sheltering the inner cell mass of the biofilm colony. Therefore, µCT can be employed in clinical settings to predict the permeability of the biofilms. It is demonstrated that chemical interference with urease, a key enzyme in biomineralization, inhibits the assembly of complex bacterial structures, prevents the formation of mineral diffusion barriers and increases biofilm permeability. Therefore, biomineralization enzymes emerge as novel therapeutic targets for highly resistant infections., Competing Interests: The authors declare no competing interests.

The eyes of some aquatic animals form images through reflective optics. Shrimp, lobsters, crayfish, and prawns possess reflecting superposition compound eyes, composed of thousands of square-faceted eye units (ommatidia). Mirrors in the upper part of the eye (the distal mirror) reflect light collected from many ommatidia onto the photosensitive elements of the retina, the rhabdoms. A second reflector, the tapetum, underlying the retina, back-scatters dispersed light onto the rhabdoms. Using microCT and cryo-SEM imaging accompanied by in situ micro-X-ray diffraction and micro-Raman spectroscopy, we investigated the hierarchical organization and materials properties of the reflective systems at high resolution and under close-to-physiological conditions. We show that the distal mirror consists of three or four layers of plate-like nanocrystals. The tapetum is a diffuse reflector composed of hollow nanoparticles constructed from concentric lamellae of crystals. Isoxanthopterin, a pteridine analog of guanine, forms both the reflectors in the distal mirror and in the tapetum. The crystal structure of isoxanthopterin was determined from crystal-structure prediction calculations and verified by comparison with experimental X-ray diffraction. The extended hydrogen-bonded layers of the molecules result in an extremely high calculated refractive index in the H-bonded plane, n = 1.96, which makes isoxanthopterin crystals an ideal reflecting material. The crystal structure of isoxanthopterin, together with a detailed knowledge of the reflector superstructures, provide a rationalization of the reflective optics of the crustacean eye., Competing Interests: The authors declare no conflict of interest.

Ficus trees are adapted to diverse environments and have some of the highest rates of photosynthesis among trees. Ficus leaves can deposit one or more of the three major mineral types found in leaves: amorphous calcium carbonate cystoliths, calcium oxalates, and silica phytoliths. In order to better understand the functions of these minerals and the control that the leaf exerts over mineral deposition, we investigated leaves from 10 Ficus species from vastly different environments (Rehovot, Israel; Bologna, Italy; Issa Valley, Tanzania; and Ngogo, Uganda). We identified the mineral locations in the soft tissues, the relative distributions of the minerals, and mineral volume contents using microcomputed tomography. Each Ficus species is characterized by a unique 3D mineral distribution that is preserved in different environments. The mineral distribution patterns are generally different on the adaxial and abaxial sides of the leaf. All species examined have abundant calcium oxalate deposits around the veins. We used micromodulated fluorimetry to examine the effect of cystoliths on photosynthetic efficiency in two species having cystoliths abaxially and adaxially ( Ficus microcarpa ) or only abaxially ( Ficus carica ). In F. microcarpa , both adaxial and abaxial cystoliths efficiently contributed to light redistribution inside the leaf and, hence, increased photosynthetic efficiency, whereas in F. carica , the abaxial cystoliths did not increase photosynthetic efficiency., (© 2018 American Society of Plant Biologists. All Rights Reserved.)

Although bone is one of the most studied living materials, many questions about the manner in which bones form remain unresolved, including fine details of the skeletal structure during development. In this study, we monitored skeleton development of zebrafish larvae, using calcein fluorescence, high-resolution micro-CT 3D images and FIB-SEM in the block surface serial imaging mode. We compared calcein staining of the skeletons of the wild type and nacre mutants, which are transparent zebrafish, with micro-CT for the first 30 days post fertilization embryos, and identified significant differences. We quantified the bone volumes and mineral contents of bones, including otoliths, during development, and showed that such developmental differences, including otolith development, could be helpful in identifying phenotypes. In addition, high-resolution imaging revealed the presence of mineralized aggregates in the notochord, before the formation of the first bone in the axial skeleton. These structures might play a role in the storage of the mineral. Our results highlight the potential of these high-resolution 3D approaches to characterize the zebrafish skeleton, which in turn could prove invaluable information for better understanding the development and the characterization of skeletal phenotypes.

Scallops possess a visual system comprising up to 200 eyes, each containing a concave mirror rather than a lens to focus light. The hierarchical organization of the multilayered mirror is controlled for image formation, from the component guanine crystals at the nanoscale to the complex three-dimensional morphology at the millimeter level. The layered structure of the mirror is tuned to reflect the wavelengths of light penetrating the scallop's habitat and is tiled with a mosaic of square guanine crystals, which reduces optical aberrations. The mirror forms images on a double-layered retina used for separately imaging the peripheral and central fields of view. The tiled, off-axis mirror of the scallop eye bears a striking resemblance to the segmented mirrors of reflecting telescopes., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Calcium oxalate and silica minerals are common components of a variety of plant leaves. These minerals are found at different locations within the leaf, and there is little conclusive evidence about the functions they perform. Here tools are used from the fields of biology, optics, and imaging to investigate the distributions of calcium oxalate, silica minerals, and chloroplasts in okra leaves, in relation to their functions. A correlative approach is developed to simultaneously visualize calcium oxalates, silica minerals, chloroplasts, and leaf soft tissue in 3D without affecting the minerals or the organic components. This method shows that in okra leaves silica and calcium oxalates, together with chloroplasts, form a complex system with a highly regulated relative distribution. This distribution points to a significant role of oxalate and silica minerals to synergistically optimize the light regime in the leaf. The authors also show directly that the light scattered by the calcium oxalate crystals is utilized for photosynthesis, and that the ultraviolet component of light passing through silica bodies, is absorbed. This study thus demonstrates that calcium oxalates increase the illumination level into the underlying tissue by scattering the incoming light, and silica reduces the amount of UV radiation entering the tissue.

Unlabelled: Trabecular bone is an intricate 3D network of struts and plates. Although the structure-function relations in trabecular bone have been studied since the time of Julius Wolff, controversy still exists regarding the architectural parameters responsible for its stability and resilience. We present a parameter that measures the angle between two connected trabeculae - the Inter-Trabecular Angle (ITA). We studied the ITA values derived from μCT scans of different regions of the proximal femora of 5 individuals of different age and sex. We show that the ITA angle distribution of nodes with 3 connecting trabeculae has a mean close to 120°, nodes with 4 connecting trabeculae has a mean close to 109° and nodes of higher connectivity have mean ITA values around 100°. This tendency to spread the ITAs around geometrically symmetrical motifs is highly conserved. The implication is that the ITAs are optimized such that the smallest amount of material spans the maximal 3D volume, and possibly by so doing trabecular bone might be better adapted to multidirectional loading. We also draw a parallel between trabecular bone and tensegrity structures - where lightweight, resilient and stable tetrahedron-based shapes contribute to strain redistribution amongst all the elements and to collective impact dampening., Statement of Significance: The Inter-Trabecular Angle (ITA) is a new topological parameter of trabecular bone. The ITA characterizes the way trabeculae connect with each other at nodes, regardless of their thickness and shape. The mean ITA value of nodes with 3 trabeculae is close to 120°, of nodes with 4 trabeculae is just below 109°, and the mean ITA of nodes with 5 and more trabeculae is around 100°. Thus the connections of trabeculae trend towards adopting symmetrical shapes. This implies that trabeculae can maximally span 3D space using the minimal amount of material. We draw a parallel between this motif and the concept of tensegrity - an engineering premise to which many living creatures conform at multiple levels of organization., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The Hula Painted Frog (Latonia nigriventer) is a rare frog species endemic to the Hula Valley, Israel. The species is the sole relict of a clade that was widespread mainly in Europe from the Oligocene until the beginning of the Pleistocene. The osteological characteristics of L. nigriventer are described based on X-ray microtomography scans of extant specimens and Pleistocene bones from the Hula Valley, to elucidate the evolutionary history of Alytidae and more specifically of Latonia. Based on the osteological description of L. nigriventer, we now better understand the differences, between Latonia and its sister taxon Discoglossus. They differ mainly in their cranial structure with the reinforced skull of Latonia having powerful jaws. Latonia nigriventer can achieve great force while closing its jaws, due to increased adductor muscle insertion surfaces as expressed by the presence of an additional paracoronoid process and an enlarged upper margin of the postero-lateral wall of the lower jaw. In addition, a wider pterygoid fossa and higher canthus postero-lateralis of the frontoparietal, compared to that of Discoglossus, also suggest the presence of well-developed adductor muscles. Furthermore, L. nigriventer have particularly strong skulls as expressed by: long articulations between different skull elements, interdigitation in the contact area between the nasals and between nasals and the frontoparietals, and fused frontoparietals. Both males and females L. nigriventer have very robust forelimbs, as indicated by well-developed medial crests of the humerus. Based on limited eastern Mediterranean paleontological data, we can only suspect that the dispersal of Latonia into the Levant from Asia Minor occurred at some point during the Miocene or later. The first appearance of L. nigriventer in the Hula Valley, its current habitat, dates to approximately 780 thousand years ago at the archaeological site of Gesher Benot Ya'aqov. J. Morphol. 277:1131-1145, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Historically, multicellular bacterial communities, known as biofilms, have been thought to be held together solely by a self-produced extracellular matrix. Our study identified a novel mechanism maintaining Bacillus subtilis and Mycobacterium smegmatis biofilms-active production of calcite minerals. We studied, for the first time, the effects of mutants defective in biomineralization and calcite formation on biofilm development, resilience and morphology. We demonstrated that an intrinsic rise in carbon dioxide levels within the biofilm is a strong trigger for the initiation of calcite-dependent patterning. The calcite-dependent patterns provide resistance to environmental insults and increase the overall fitness of the microbial community. Our results suggest that it is highly feasible that the formation of mineral scaffolds plays a cardinal and conserved role in bacterial multicellularity., Competing Interests: The authors declare no conflict of interest.

Photothermal characteristics and light-induced structural (volume) changes of carotenoid-containing and noncontaining photosynthetic reaction centers (RCs) were investigated by wide frequency band hydrophone. We found that the presence of carotenoid either does not play considerable role in the light-induced conformational movements, or these rearrangements are too slow for inducing a photoacoustic (PA) signal. The kinetic component with a few tens of microseconds, exhibited by the carotenoid-less RCs, appears to be similar to that of triplet state lifetimes, identified by other methods. The binding of terbutryn to the acceptor side is shown to affect the dynamics of the RC. Our results do not confirm large displacements or volume changes induced by the charge movements and by the charge relaxation processes in the RCs in few hundreds of microseconds time scale that accompanies the electron transfer between the primary and secondary electron acceptor quinones., (© 2015 The American Society of Photobiology.)

Many bacteria live as biofilms to cope with unfavourable surroundings. Biofilms start from (i) a planktonic stage, (ii) initial adhesion to surfaces and (iii) formation of sessile micro-colonies that secrete extracellular polymeric substance (EPS), leading to bacterial resistance to antibiotics. Antimicrobial peptides (AMPs) are extensively studied with regard to planktonic bacteria but much less so with regard to biofilm formation. In the present study, we investigated how the above three steps are affected by the properties of the AMPs using a series of peptides composed of six lysines and nine leucines, which differ in their sequences and hence their biophysical properties. Treatment with bactericidal peptides at non-inhibitory concentrations resulted in reduced biofilm growth, for some starting from 25 nM which is 0.2 and 0.4% of their minimum inhibitory concentration (MIC 6.3 and 12.5 μM, respectively), continuing in a dose-dependent manner. We suggest that reduced bacterial adhesion to surfaces and decreased biofilm growth are due to the peptide's ability to coat either the biomaterial surface or the bacterium itself. Degradation of established biofilms by bactericidal and non-bactericidal peptides, within 1 h of incubation, occurs by either killing of embedded bacteria or detachment of live ones. In addition to shedding light on the mechanism of biofilm inhibition and degradation, these data may assist in the design of anti-biofilm AMPs.

Skeletal integrity is maintained by the co-ordinated activity of osteoblasts, the bone-forming cells, and osteoclasts, the bone-resorbing cells. In this study, we show that mice overexpressing galectin-8, a secreted mammalian lectin of the galectins family, exhibit accelerated osteoclasts activity and bone turnover, which culminates in reduced bone mass, similar to cases of postmenopausal osteoporosis and cancerous osteolysis. This phenotype can be attributed to a direct action of galectin-8 on primary cultures of osteoblasts that secrete the osteoclastogenic factor RANKL upon binding of galectin-8. This results in enhanced differentiation into osteoclasts of the bone marrow cells co-cultured with galectin-8-treated osteoblasts. Secretion of RANKL by galectin-8-treated osteoblasts can be attributed to binding of galectin-8 to receptor complexes that positively (uPAR and MRC2) and negatively (LRP1) regulate galectin-8 function. Our findings identify galectins as new players in osteoclastogenesis and bone remodeling, and highlight a potential regulation of bone mass by animal lectins.

Trabecular bone is morphologically and functionally different from compact bone at the tissue level, but both are composed of lamellae at the micrometer-scale level. We present a three-dimensional study of the collagenous network of human trabecular lamellar bone from the proximal femur using the FIB-SEM serial surface view method. The results are compared to human compact lamellar bone of the femoral shaft, studied by the same method. Both demineralized trabecular and compact lamellar bone display the same overall structural organization, namely the presence of ordered and disordered materials and the confinement of the canalicular network to the disordered material. However, in trabecular bone lamellae a significant proportion of the ordered collagen fibril arrays is aligned with the long axis of the trabecula and, unlike in compact bone, is not related to the anatomical axis of the whole femur. The remaining ordered collagen fibrils are offset from the axis of a trabecula either by about 30° or 70°. Interestingly, at the tissue scale of millimeters, the most abundant angles between any two connected trabeculae - the inter-trabecular angles - center around 30° and 70°. This implies that within a framework of interconnected trabeculae the same lamellar structure will always have a significant component of the fibrils aligned with the long axes of connected trabeculae. This structural complementarity at different hierarchical levels presumably reflects an adaptation of trabecular bone to function., (Copyright © 2014 Elsevier Inc. All rights reserved.)