Key innovations are pivotal for biodiversity and facilitating evolutionary success, enabling organisms' adaptation to various ecological niches through the diversification of phenotypic traits. In mammals, notable adaptations include evolving hypsodonty for grazing on grasses and, for bats, evolving echolocation and wing acquisition. Recent studies have highlighted the overlooked morphological diversity of the bat larynx, a key organ for echolocation capabilities. Tracheal chambers, found on the trachea's first rings, are enigmatic components within the laryngeal complex whose origins and functions remain incompletely understood. We hypothesized that these structures demonstrate evolutionary convergence and represent a key innovation responsible for the diversification of laryngeal echolocation behaviors. By analyzing 51 bat species, their laryngeal cartilages, and tracheal chambers, we explored relationships between body mass, sound frequencies, and chamber volumes. We hypothesized that tracheal chambers likely facilitated laryngeal echolocation capabilities in bats. Ancestral state reconstructions were conducted to understand the evolution of tracheal chambers and echolocation behaviors in bats. We conclude that tracheal chambers might support higher frequency sound production and could have been pivotal for high-duty cycle echolocation specialization during the development of nasally emitted calls, which allowed bats to thrive in diverse environments. We suggest that tracheal chambers are specialized phenotypic adaptations that might have helped in diversifying laryngeal echolocation behaviors and the evolutionary success of bats.

Bioimaging has transformed our understanding of biological processes, yet extracting meaningful information from complex datasets remains a challenge, particularly for biologists without computational expertise. This paper proposes a simple general approach, to help identify which image analysis methods could be relevant for a given image dataset. We first categorise structures commonly observed in bioimage data into different types related to image analysis domains. Based on these types, we provide a list of methods adapted to the quantification of images from each category. Our approach includes illustrative examples and a visual flowchart, to help researchers define analysis objectives clearly. By understanding the diversity of bioimage structures and linking them with appropriate analysis approaches, the framework empowers researchers to navigate bioimage datasets more efficiently. It also aims to foster a common language between researchers and analysts, thereby enhancing mutual understanding and facilitating effective communication.

Among hornbill birds, the critically endangered helmeted hornbill (Rhinoplax vigil) is notable for its casque (a bulbous beak protrusion) being filled with trabeculae and fronted by a very thick keratin layer. Casque function is debated but appears central to aerial jousting, where birds (typically males) collide casques at high speeds in a mid-flight display that is audible for more than 100 m. We characterized the structural relationship between the skull and casque anatomy using X-ray microtomography and quantitative trabecular network analysis to examine how the casque sustains extreme impact. The casque comprises a keratin veneer (rhamphotheca, ∼8× thicker than beak keratin), which slots over the internal bony casque like a tight-fitting sheath. The bony casque's central cavity contains a network of trabeculae-heavily aligned and predominantly rod-like, among the thickest described in vertebrates-forming a massive rostrocaudal strut spanning the casque's length, bridging rostral (impact), and caudal (braincase) surfaces. Quantitative network characterizations indicate no differences between male and female trabecular architectures. This suggests that females may also joust or that casques play other roles. Our results argue that the casque's impact loading demands and shapes a high-safety-factor construction that involves extreme trabecular morphologies among vertebrates, architectures that also have the potential for informing the design of collision-resistant materials.

Hearing loss is common in people with osteogenesis imperfecta (OI or brittle bone disease). Bisphosphonates are widely used to treat long bone fragility in children with OI. However, its impact on the bone quality of the middle ear ossicles and hearing remains unknown. This study determines whether bisphosphonates treatment itself may contribute to hearing loss in OI by evaluating its effects in the oim/oim mouse model of severe OI having normal auditory function. Specifically, this study reports the effects of alendronate (ALN), a nitrogen-containing bisphosphonate, on ossicle morphology, porosity, and elemental composition in 14-week-old oim/oim mice treated weekly, starting at 2 weeks of age. The ossicles were examined using synchrotron microtomography and X-ray fluorescence microscopy (XFM). Hearing was assessed longitudinally until 26 weeks of age by determining auditory brainstem response (ABR) thresholds in another group of mice also treated weekly starting at 2 weeks of age. ALN treatment further reduces in size the already small oim/oim ossicles, specifically in female mice. Porosity, bone composition, and hearing function, however, were generally not affected by the ALN treatment. Furthermore, ALN does not prevent joint fusions, excessive bone formations, or enlarged joint spaces in WT or oim/oim experimental groups. One ALN-treated oim/oim mouse with a bone formation in the interior of the footplate, and one ALN-treated WT mouse with a fixed footplate had frequency-specific hearing loss. Since footplate abnormalities are not observed in PBS-treated mice in this study, it remains unclear whether ALN fails to prevent these changes or contributes to their development. Future studies should investigate the mechanisms of ossicular abnormalities and bisphosphonates modulatory role in the ossicles.

Hearing loss affects approximately 70% of individuals with osteogenesis imperfecta (OI), a genetic connective tissue disorder characterized by bone fragility and deformities. No effective treatments exist for OI hearing loss, and its etiology is unknown limiting the development of new targeted therapies. This work investigates the impact of OI type I collagen mutations on the ossicle bone properties in the homozygous oim mouse model of severe OI, which is known to exhibit hearing loss. The morphology and porosity of the ossicles of 14-week-old oim and wild-type mice were analyzed using high-resolution synchrotron radiation microtomography. Additionally, the collagen fibers structure, bone tissue composition and mechanical properties were evaluated through second harmonic generation microscopy, Raman spectroscopy, and nanoindentation. The results demonstrated that oim ossicles are small, highly porous with an elevated lacunar number density, a flat stapes footplate and a small malleal processus brevis. One-in-two oim ossicles had incudomalleal joint abnormalities, exhibiting either a localized fracture in the incus head or a joint space widening. No differences were observed in collagen fibers structure, bone tissue composition and mechanical properties. These findings suggest that bone fractures observed in the oim incudes may contribute to their reported hearing loss. However, the underlying mechanism for these fractures' development remains to be investigated, as they do not appear to result from changes in bone tissue properties (collagen fibers organization, tissue composition or mechanical properties). Instead, they may be associated with joint space widening, and possibly altered ossicle chain kinematics.

Most of over 1400 extant bat species produce high-frequency pulses with their larynx for echolocation. However, the debate about the evolutionary origin of laryngeal echolocation in bats remains unresolved. The morphology of the larynx is known to reflect vocal adaptation and thus can potentially help in resolving this controversy. However, the morphological variations of the larynx are poorly known in bats, and a complete anatomical study remains to be conducted. Here, we compare the 3D laryngeal morphology of 23 extant bat species of 11 different families reconstructed by using iodine contrast-enhanced X-ray microtomography techniques. We find that, contrary to previously thought, laryngeal muscle hypertrophy is not a characteristic of all bats and presents differential development. The larynges of Pteropodidae are morphologically similar to those of non-bat mammals. Two morphotypes are described among laryngeal echolocating bats, illustrating morphological differences between Rhinolophoidea and Yangochiroptera, with the main variations being the cricothyroid muscle volume and the shape of the cricoid and thyroid cartilages. For the first time we detail functional specialization for constant frequency echolocation among Rhinolophoidea. Lastly, the nasal-emitting taxa representing a polyphyletic group do not share the same laryngeal form, which raises questions about the potential modular nature of the bat larynx.

Resorption within cortices of long bones removes excess mass and damaged tissue, and increases during periods of reduced mechanical loading. Returning to high-intensity exercise may place bones at risk of failure due to increased porosity caused by bone resorption. We used microradiographs of bone slices from highly-loaded (metacarpal, tibia) and minimally-loaded (rib) bones from 12 racehorses, 6 that died during a period of high-intensity exercise and 6 that had a period of intense exercise followed by at least 35 days of rest prior to death, and measured intracortical canal cross-sectional area (Ca.Ar) and number (N.Ca) to infer remodelling activity across sites and exercise groups. Large canals that are the consequence of bone resorption (Ca.Ar > 0.04 mm 2 ) were 1.4× to 18.7× greater in number and area in the third metacarpal bone from rested than exercised animals (p = 0.005– 0.008), but were similar in number and area in ribs from rested and exercised animals (p = 0.575–0.688). An intermediate relationship was present in the tibia, and when large canals and smaller canals that result from partial bony infilling (Ca.Ar > 0.002 mm 2 ) were considered together. The mechanostat may override targeted remodelling during periods of high mechanical load by enhancing bone formation, reducing resorption and suppressing turnover. Both systems may work synergistically in rest periods to remove excess and damaged tissue.

Purpose:To assess the capability of deep convolutional neural networks to classify anatomical location and projection from a series of 48 standard views of racehorse limbs. Materials and methods:Radiographs (N = 9504) of horse limbs from image sets made for veterinary inspections by 10 independent veterinary clinics were used to train, validate and test (116, 40 and 42 radiographs, respectively) six deep learning architectures available as part of the open source machine learning framework PyTorch. The deep learning architectures with the best top-1 accuracy had the batch size further investigated. Results:Top-1 accuracy of six deep learning architectures ranged from 0.737 to 0.841. Top-1 accuracy of the best deep learning architecture (ResNet-34) ranged from 0.809 to 0.878, depending on batch size. ResNet-34 (batch size = 8) achieved the highest top-1 accuracy (0.878) and the majority (91.8%) of misclassification was due to laterality error. Class activation maps indicated that joint morphology, not side markers or other non-anatomical image regions, drove the model decision. Conclusions:Deep convolutional neural networks can classify equine pre-import radiographs into the 48 standard views including moderate discrimination of laterality, independent of side marker presence.

Laryngeal echolocation in bats could have evolved following two scenarios: a single origin from a common ancestor or an independent acquisition inside the two clades Yinpterochiroptera and Yangochiroptera. Later, some members of Yinpterochiroptera possibly lost their ability to echolocate. In bats, the larynx produces vocalizations for communication and, in most species, for echolocation. Here, we describe how comparative chiropteran laryngeal morphology is a novel area of research that could improve the understanding of echolocation and may help resolve the evolutionary history of bats. This review provides morphological descriptions and comparisons of the bat larynx and bioacoustics interpretations. We discuss the importance of understanding: (1) laryngeal sound production so it may be linked with the evolution of the chiropteran auditory system; and (2) the evolution of laryngeal morphology to understand the ecological and behavioural aspects of bat biology. We find that a strong phylogenetic signal is potentially the main source explaining macroevolutionary variation in laryngeal form among bats. We predict that the three parameters of sound production in echolocation (frequency, intensity, and rate of calls) are independently modulated by different laryngeal components, but this hypothesis remains understudied in terms of species diversity.

Lamellae are sheets of mineralized collagen 1–20 µm thick, extending over hundreds of µm in bone tissue, occupying bone's structural hierarchy at a level above collagen fibres and osteocytes, and below osteons and trabeculae. Osteons are tubular arrangements of lamellae surrounding central neurovascular canals. Lamellae in osteons are usually described as concentric cylinders based on their annular appearance in transverse section. In this review, I provide a perspective on current understanding of the relationship between geometry of the bone formation front and the shape of lamellae produced at it, reaching the conclusion that the ‘closing cone’ bone formation front in secondary osteonal remodelling must necessarily result in cone-shaped lamellae in the mature secondary osteon. Secondary osteons replace primary osteons through a tunnelling process of bone turnover, meaning that conical lamellae may become more common in older and damaged bone which is at greatest risk of fracture. Visualization and measurement of three-dimensional lamellar shape over hundreds of microns is needed to provide data for accurate micromechanical simulations. Treating secondary osteonal lamellae as a ‘stack of cones’ rather than ‘nested cylinders’ may have important implications for our appreciation of bone's function as a load-bearing tissue and of its behaviour in fracture.

Modern research in the life sciences is unthinkable without computational methods for extracting, quantifying and visualising information derived from microscopy imaging data of biological samples. In the past decade, we observed a dramatic increase in available software packages for these purposes. As it is increasingly difficult to keep track of the number of available image analysis platforms, tool collections, components and emerging technologies, we provide a conservative overview of software that we use in daily routine and give insights into emerging new tools. We give guidance on which aspects to consider when choosing the platform that best suits the user's needs, including aspects such as image data type, skills of the team, infrastructure and community at the institute and availability of time and budget.

Purpose: To assess the capability of deep convolutional neural networks to classify anatomical location and projection from a series of 48 standard views of racehorse limbs. Materials and Methods: 9504 equine pre-import radiographs were used to train, validate, and test six deep learning architectures available as part of the open source machine learning framework PyTorch. Results: ResNet-34 achieved a top-1 accuracy of 0.8408 and the majority (88%) of misclassification was because of wrong laterality. Class activation maps indicated that joint morphology drove the model decision. Conclusion: Deep convolutional neural networks are capable of classifying equine pre-import radiographs into the 48 standard views including moderate discrimination of laterality independent of side marker presence.

The characterization of developmental phenotypes often relies on the accurate linear measurement of structures that are small and require laborious preparation. This is tedious and prone to errors, especially when repeated for the multiple replicates that are required for statistical analysis, or when multiple distinct structures have to be analyzed. To address this issue, we have developed a pipeline for characterization of long-bone length using X-ray microtomography (XMT) scans. The pipeline involves semi-automated algorithms for automatic thresholding and fast interactive isolation and 3D-model generation of the main limb bones, using either the open-source ImageJ plugin BoneJ or the commercial Mimics Innovation Suite package. The tests showed the appropriate combination of scanning conditions and analysis parameters yields fast and comparable length results, highly correlated with the measurements obtained via ex vivo skeletal preparations. Moreover, since XMT is not destructive, the samples can be used afterward for histology or other applications. Our new pipelines will help developmental biologists and evolutionary researchers to achieve fast, reproducible and non-destructive length measurement of bone samples from multiple animal species.

Changes in trabecular micro-architecture are key to our understanding of osteoporosis. Previous work focusing on structure model index (SMI) measurements have concluded that disease progression entails a shift from plates to rods in trabecular bone, but SMI is heavily biased by bone volume fraction. As an alternative to SMI, we proposed the ellipsoid factor (EF) as a continuous measure of local trabecular shape between plate-like and rod-like extremes. We investigated the relationship between EF distributions, SMI and bone volume fraction of the trabecular geometry in a murine model of disuse osteoporosis as well as from human vertebrae of differing bone volume fraction. We observed a moderate shift in EF median (at later disease stages in mouse tibia) and EF mode (in the vertebral samples with low bone volume fraction) towards a more rod-like geometry, but not in EF maximum and minimum. These results support the notion that the plate to rod transition does not coincide with the onset of bone loss and is considerably more moderate, when it does occur, than SMI suggests. A variety of local shapes not straightforward to categorize as rod or plate exist in all our trabecular bone samples.

Homeopathy is “a therapeutic method of using preparations of substances [...]

Research software is often developed with expedience as a core development objective because experimental results, but not the software, are specified and resourced as a project output. While such code can help find answers to specific research questions, it may lack longevity and flexibility to make it reusable. We reimplemented BoneJ, our software for skeletal biology image analysis, to address design limitations that put it at risk of becoming unusable. We improved the quality of BoneJ code by following contemporary best programming practices. These include separation of concerns, dependency management, thorough testing, continuous integration and deployment, source code management, code reviews, issue and task ticketing, and user and developer documentation. The resulting BoneJ2 represents a generational shift in development technology and integrates with the ImageJ2 plugin ecosystem.

Research software is often developed with expedience as a core development objective because experimental results, but not the software, are specified and resourced as a project output. While such code can help find answers to specific research questions, it may lack longevity and flexibility to make it reusable. We reimplemented BoneJ, our software for skeletal biology image analysis, to address design limitations that put it at risk of becoming unusable. We improved the quality of BoneJ code by following contemporary best programming practices. These include separation of concerns, dependency management, thorough testing, continuous integration and deployment, source code management, code reviews, issue and task ticketing, and user and developer documentation. The resulting BoneJ2 represents a generational shift in development technology and integrates with the ImageJ2 plugin ecosystem.

Research software is often developed with expedience as a core development objective because experimental results, but not the software, are specified and resourced as a project output. While such code can help find answers to specific research questions, it may lack longevity and flexibility to make it reusable. We reimplemented BoneJ, our software for skeletal biology image analysis, to address design limitations that put it at risk of becoming unusable. We improved the quality of BoneJ code by following contemporary best programming practices. These include separation of concerns, dependency management, thorough testing, continuous integration and deployment, source code management, code reviews, issue and task ticketing, and user and developer documentation. The resulting BoneJ2 represents a generational shift in development technology and integrates with the ImageJ2 plugin ecosystem.

Research software is often developed with expedience as a core development objective because experimental results, but not the software, are specified and resourced as a project output. While such code can help find answers to specific research questions, it may lack longevity and flexibility to make it reusable. We reimplemented BoneJ, our software for skeletal biology image analysis, to address design limitations that put it at risk of becoming unusable. We improved the quality of BoneJ code by following contemporary best programming practices. These include separation of concerns, dependency management, thorough testing, continuous integration and deployment, source code management, code reviews, issue and task ticketing, and user and developer documentation. The resulting BoneJ2 represents a generational shift in development technology and integrates with the ImageJ2 plugin ecosystem.