SMARCA4 mutation causes human otosclerosis and a similar phenotype in mice.

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.

The mechanosensitive ion channel ASIC2 mediates both proprioceptive sensing and spinal alignment.

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.

Factors Controlling Complex Morphologies of Isomorphous Metal-Organic Frameworks.

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.

Structural organization of xanthine crystals in the median ocellus of a member of the ancestral insect group Archaeognatha.

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.

Microstructural heterogeneity of the collagenous network in the loaded and unloaded periodontal ligament and its biomechanical implications.

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.

Optically functional isoxanthopterin crystals in the mirrored eyes of decapod crustaceans.

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.

Unique three-dimensional structure of a fish pharyngeal jaw subjected to unusually high mechanical loads.

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.

Characterization and possible function of an enigmatic reflector in the eye of the shrimp Litopenaeus vannamei.

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.

Response of the tooth-periodontal ligament-bone complex to load: A microCT study of the minipig molar.

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.

Guanine and 7,8-Dihydroxanthopterin Reflecting Crystals in the Zander Fish Eye: Crystal Locations, Compositions, and Structures.

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.

Mineral Deposits in Ficus Leaves: Morphologies and Locations in Relation to Function.

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 (Ficusmicrocarpa) or only abaxially (Ficuscarica). 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.

Mechanisms of biofilm inhibition and degradation by antimicrobial peptides.

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.

Stress-induced condensation of bacterial genomes results in re-pairing of sister chromosomes: implications for double strand DNA break repair.

Genome condensation is increasingly recognized as a generic stress response in bacteria. To better understand the physiological implications of this response, we used fluorescent markers to locate specific sites on Escherichia coli chromosomes following exposure to cytotoxic stress. We find that stress-induced condensation proceeds through a nonrandom, zipper-like convergence of sister chromosomes, which is proposed to rely on the recently demonstrated intrinsic ability of identical double-stranded DNA molecules to specifically identify each other. We further show that this convergence culminates in spatial proximity of homologous sites throughout chromosome arms. We suggest that the resulting apposition of homologous sites can explain how repair of double strand DNA breaks might occur in a mechanism that is independent of the widely accepted yet physiologically improbable genome-wide search for homologous templates. We claim that by inducing genome condensation and orderly convergence of sister chromosomes, diverse stress conditions prime bacteria to effectively cope with severe DNA lesions such as double strand DNA breaks.

Direct microCT imaging of non-mineralized connective tissues at high resolution.

The 3D imaging of soft tissues in their native state is challenging, especially when high resolution is required. An X-ray-based microCT is, to date, the best choice for high resolution 3D imaging of soft tissues. However, since X-ray attenuation of soft tissues is very low, contrasting enhancement using different staining materials is needed. The staining procedure, which also usually involves tissue fixation, causes unwanted and to some extent unknown tissue alterations. Here, we demonstrate that a method that enables 3D imaging of soft tissues without fixing and staining using an X-ray-based bench-top microCT can be applied to a variety of different tissues. With the sample mounted in a custom-made loading device inside a humidity chamber, we obtained soft tissue contrast and generated 3D images of fresh, soft tissues with a resolution of 1 micron voxel size. We identified three critical conditions which make it possible to image soft tissues: humidified environment, mechanical stabilization of the sample and phase enhancement. We demonstrate the capability of the technique using different specimens: an intervertebral disc, the non-mineralized growth plate, stingray tessellated radials (calcified cartilage) and the collagenous network of the periodontal ligament. Since the scanned specimen is fresh an interesting advantage of this technique is the ability to scan a specimen under load and track the changes of the different structures. This method offers a unique opportunity for obtaining valuable insights into 3D structure-function relationships of soft tissues.

Three-dimensional correlative microscopy of the Drosophila female reproductive tract reveals modes of communication in seminal receptacle sperm storage.

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.

Tooth periodontal ligament: Direct 3D microCT visualization of the collagen network and how the network changes when the tooth is loaded.

The periodontal ligament (PDL), a soft tissue connecting the tooth and the bone, is essential for tooth movement, bone remodeling and force dissipation. A collagenous network that connects the tooth root surface to the alveolar jaw bone is one of the major components of the PDL. The organization of the collagenous component and how it changes under load is still poorly understood. Here using a state-of-the-art custom-made loading apparatus and a humidified environment inside a microCT, we visualize the PDL collagenous network of a fresh rat molar in 3D at 1 µm voxel size without any fixation or contrasting agents. We demonstrate that the PDL collagen network is organized in sheets. The spaces between sheets vary thus creating dense and sparse networks. Upon vertical loading, the sheets in both networks are stretched into well aligned arrays. The sparse network is located mainly in areas which undergo compressive loading as the tooth moves towards the bone, whereas the dense network functions mostly in tension as the tooth moves further from the bone. This new visualization method can be used to study other non-mineralized or partially mineralized tissues, and in particular those that are subjected to mechanical loads. The method will also be valuable for characterizing diseased tissues, as well as better understanding the phenotypic expressions of genetic mutants.

Tooth movements are guided by specific contact areas between the tooth root and the jaw bone: A dynamic 3D microCT study of the rat molar.

Teeth sustain high loads over a lifetime and yet intact tooth failure is rare. The different structures of the tooth, jaw bone and the intervening soft periodontal ligament enable the tooth to endure repeated loading during mastication. Although mechanical and functional properties of the different components are thoroughly investigated, the manner in which the whole tooth functions under load is still enigmatic. A custom-made loading system inside a microCT scanner was used to directly visualize the root movements in relation to the jaw bone as the rat molar tooth was loaded. At low loads no contact was observed between the root surface and the bone, whereas at higher loads three specific contact areas between the root surface and the jaw bone were observed. These contact areas restrict tooth movement in the buccal-lingual direction, but enable the tooth to rock in a "seesaw" like manner in the distal-mesial direction. The contact areas appear to play a role in determining tooth motion and in turn define the manner in which the whole tooth moves when loaded. These observations are important for understanding basic structure-function relations of the tooth-PDL-bone system, and have direct implications for better understanding pathological and therapeutic processes in orthodontics, periodontics and jaw bone regeneration.

Osteodentin in the Atlantic wolffish (Anarhichas lupus): Dentin or bone?

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.

The petrous bone contains high concentrations of osteocytes: One possible reason why ancient DNA is better preserved in this bone.

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.

Calcium carbonate mineralization is essential for biofilm formation and lung colonization.

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.