Organogenesis and foetal haemodynamics during the normal gestation of healthy black-rumped agoutis (Dasyprocta prymnolopha, Wagler, 1831) bred in captivity.

The objective of this study was to define the patterns of organogenesis and foetal haemodynamics during the normal gestation of healthy agoutis (Dasyprocta prymnolopha) kept in captivity. Thirty pregnant agoutis that ranged in size from small to medium and weighed between 2.5 and 3 kg underwent B-mode and Doppler ultrasonography for the biometric evaluation of the foetal organs. The foetal aortic blood flow proved to be predominantly systolic, and the measured flow velocity was 78.89 ± 2.95 cm/s, with a maximum pressure gradient of 2.12 ± 0.27 mmHg. The liver was characterized by its large volume, occupying the entire cranial aspect of the abdominal cavity, and it was associated cranially with the diaphragm and caudally with the stomach. The flow velocity in the portal vein was estimated to equal 12.17 ± 2.37 cm/s, with a resistivity index of 0.82 ± 0.05. The gallbladder was centrally located and protruded cranially towards the diaphragm. The spleen was visualized as an elongated structure with tapered cranial and caudal extremities, and the foetal kidneys were visualized bilaterally in the retroperitoneal region, with the right kidney positioned slightly more cranially than the left. The morphological characterization and hemodynamic analysis of the foetal organs of black-rumped agoutis via B-mode and Doppler ultrasonography allow determination of the vascular network and of reference values for the blood flow required for perfusing the anatomical elements essential for maintaining the viability of foetuses at different gestational ages.

Acoustic property reconstruction of a pygmy sperm whale (Kogia breviceps) forehead based on computed tomography imaging.

Computed tomography (CT) imaging and sound experimental measurements were used to reconstruct the acoustic properties (density, velocity, and impedance) of the forehead tissues of a deceased pygmy sperm whale (Kogia breviceps). The forehead was segmented along the body axis and sectioned into cross section slices, which were further cut into sample pieces for measurements. Hounsfield units (HUs) of the corresponding measured pieces were obtained from CT scans, and regression analyses were conducted to investigate the linear relationships between the tissues' HUs and velocity, and HUs and density. The distributions of the acoustic properties of the head at axial, coronal, and sagittal cross sections were reconstructed, revealing that the nasal passage system was asymmetric and the cornucopia-shaped spermaceti organ was in the right nasal passage, surrounded by tissues and airsacs. A distinct dense theca was discovered in the posterior-dorsal area of the melon, which was characterized by low velocity in the inner core and high velocity in the outer region. Statistical analyses revealed significant differences in density, velocity, and acoustic impedance between all four structures, melon, spermaceti organ, muscle, and connective tissue (p < 0.001). The obtained acoustic properties of the forehead tissues provide important information for understanding the species' bioacoustic characteristics.

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.

Deep learning-enabled multi-organ segmentation in whole-body mouse scans.

Whole-body imaging of mice is a key source of information for research. Organ segmentation is a prerequisite for quantitative analysis but is a tedious and error-prone task if done manually. Here, we present a deep learning solution called AIMOS that automatically segments major organs (brain, lungs, heart, liver, kidneys, spleen, bladder, stomach, intestine) and the skeleton in less than a second, orders of magnitude faster than prior algorithms. AIMOS matches or exceeds the segmentation quality of state-of-the-art approaches and of human experts. We exemplify direct applicability for biomedical research for localizing cancer metastases. Furthermore, we show that expert annotations are subject to human error and bias. As a consequence, we show that at least two independently created annotations are needed to assess model performance. Importantly, AIMOS addresses the issue of human bias by identifying the regions where humans are most likely to disagree, and thereby localizes and quantifies this uncertainty for improved downstream analysis. In summary, AIMOS is a powerful open-source tool to increase scalability, reduce bias, and foster reproducibility in many areas of biomedical research.

In vivo imaging of Zika virus reveals dynamics of viral invasion in immune-sheltered tissues and vertical propagation during pregnancy.

Rationale: Zika virus (ZIKV) is a pathogenic virus known to cause a wide range of congenital abnormalities, including microcephaly, Guillain-Barre syndrome, meningoencephalitis, and other neurological complications, in humans. This study investigated the noninvasive detection of ZIKV infection in vivo, which is necessary for elucidating the virus's mechanisms of viral replication and pathogenesis, as well as to accelerate the development of anti-ZIKV therapeutic strategies. Methods: In this study, a recombinant ZIKV harbouring Nluc gene (ZIKV-Nluc) was designed, recovered, and purified. The levels of bioluminescence were directly correlated with viral loads in vitro and in vivo. The dynamics of ZIKV infection in A129 (interferon (IFN)-α/ß receptor deficient), AG6 (IFN-α/ß and IFN-γ receptor deficient), and C57BL/6 mice were characterized. Pregnant dams were infected with ZIKV-Nluc at E10 via intra footpad injection. Then, the pooled immune sera (anti-ZIKV neutralizing antibodies) #22-1 in ZIKV-Nluc virus-infected mice were visualized. Results: ZIKV-Nluc showed a high genetic stability and replicated well in cells with similar properties to the wild-type ZIKV (ZIKVwt). Striking bioluminescence signals were consistently observed in animal organs, including spleen, intestine, testis, uterus/ovary, and kidney. The ileocecal junction was found to be the crucial visceral target. Infection of pregnant dams with ZIKV-Nluc showed that ZIKV was capable of crossing the maternal-fetal barrier to infect the fetuses via vertical transmission. Furthermore, it was visualized that treatment with the pooled immune sera was found to greatly restrict the spread of the ZIKV-Nluc virus in mice. Conclusions: This study is the first to report the real-time noninvasive tracking of the progression of ZIKV invading immune-sheltered tissues and propagating vertically during pregnancy. The results demonstrate that ZIKV-Nluc represents a powerful tool for the study of the replication, dissemination, pathogenesis, and treatment of ZIKV in vitro and in vivo.

Plasticity of mycangia in Xylosandrus ambrosia beetles.

Insects that depend on microbial mutualists evolved a variety of organs to transport the microsymbionts while dispersing. The ontogeny and variability of such organs is rarely studied, and the microsymbiont's effects on the animal tissue development remain unknown in most cases. Ambrosia beetles (Coleoptera: Curculionidae: Scolytinae or Platypodinae) and their mutualistic fungi are an ideal system to study the animal-fungus interactions. While the interspecific diversity of their fungus transport organ-mycangia-is well-known, their developmental plasticity has been poorly described. To determine the ontogeny of the mycangium and the influence of the symbiotic fungus on the tissue development, we dissected by hand or scanned with micro-CT the mycangia in various developmental stages in five Xylosandrus ambrosia beetle species that possess a large, mesonotal mycangium: Xylosandrus amputatus, Xylosandrus compactus, Xylosandrus crassiusculus, Xylosandrus discolor, and Xylosandrus germanus. We processed 181 beetle samples from the United States and China. All five species displayed three stages of the mycangium development: (1) young teneral adults had an empty, deflated and cryptic mycangium without fungal mass; (2) in fully mature adults during dispersal, the pro-mesonotal membrane was inflated, and most individuals developed a mycangium mostly filled with the symbiont, though size and symmetry varied; and (3) after successful establishment of their new galleries, most females discharged the bulk of the fungal inoculum and deflated the mycangium. Experimental aposymbiotic individuals demonstrated that the pronotal membrane invaginated independently of the presence of the fungus, but the fungus was required for inflation. Mycangia are more dynamic than previously thought, and their morphological changes correspond to the phases of the symbiosis. Importantly, studies of the fungal symbionts or plant pathogen transmission in ambrosia beetles need to consider which developmental stage to sample. We provide illustrations of the different stages, including microphotography of dissections and micro-CT scans.

Tooth development and replacement in the Atlantic Cutlassfish, Trichiurus lepturus, with comparisons to other Scombroidei.

Atlantic Cutlassfish, Trichiurus lepturus, have large, barbed, premaxillary and dentary fangs, and sharp dagger-shaped teeth in their oral jaws. Functional teeth firmly ankylose to the dentigerous bones. We used dry skeletons, histology, SEM, and micro-CT scanning to study 92 specimens of T. lepturus from the western North Atlantic to describe its dentition and tooth replacement. We identified three modes of intraosseous tooth replacement in T. lepturus depending on the location of the tooth in the jaw. Mode 1 relates to replacement of premaxillary fangs, in which new tooth germs enter the lingual surface of the premaxilla, develop horizontally, and rotate into position. We suggest that growth of large fangs in the premaxilla is accommodated by this horizontal development. Mode 2 occurs for dentary fangs: new tooth germs enter the labial surface of the dentary, develop vertically, and erupt into position. Mode 3 describes replacement of lateral teeth, in which new tooth germs enter a trench along the crest of the dentigerous bone, develop vertically, and erupt into position. Such distinct modes of tooth replacement in a teleostean species are unknown. We compared modes of replacement in T. lepturus to 20 species of scombroids to explore the phylogenetic distribution of these three replacement modes. Alternate tooth replacement (in which new teeth erupt between two functional teeth), ankylosis, and intraosseous tooth development are plesiomorphic to Bluefish + other Scombroidei. Our study highlights the complexity and variability of intraosseous tooth replacement. Within tooth replacement systems, key variables include sites of formation of tooth germs, points of entry of tooth germs into dentigerous bones, coupling of tooth germ migration and bone erosion, whether teeth develop horizontally or immediately beneath the tooth to be replaced, and how tooth eruption and ankylosis occur. Developmentally different tooth replacement processes can yield remarkably similar dentitions.

Sensory system plasticity in a visually specialized, nocturnal spider.

The interplay between an animal's environmental niche and its behavior can influence the evolutionary form and function of its sensory systems. While intraspecific variation in sensory systems has been documented across distant taxa, fewer studies have investigated how changes in behavior might relate to plasticity in sensory systems across developmental time. To investigate the relationships among behavior, peripheral sensory structures, and central processing regions in the brain, we take advantage of a dramatic within-species shift of behavior in a nocturnal, net-casting spider (Deinopis spinosa), where males cease visually-mediated foraging upon maturation. We compared eye diameters and brain region volumes across sex and life stage, the latter through micro-computed X-ray tomography. We show that mature males possess altered peripheral visual morphology when compared to their juvenile counterparts, as well as juvenile and mature females. Matching peripheral sensory structure modifications, we uncovered differences in relative investment in both lower-order and higher-order processing regions in the brain responsible for visual processing. Our study provides evidence for sensory system plasticity when individuals dramatically change behavior across life stages, uncovering new avenues of inquiry focusing on altered reliance of specific sensory information when entering a new behavioral niche.

Structural analysis of the tongue and hyoid apparatus in a woodpecker.

UNLABELLED: Woodpeckers avoid brain injury while they peck at trees up to 20Hz with speeds up to 7m/s, undergoing decelerations up to 1200g. Along with the head, beak and neck, the hyoid apparatus (tongue bone and associated soft tissues) is subjected to these high impact forces. The shape of the hyoid apparatus is unusual in woodpeckers and its structure and mechanical properties have not been reported in detail. High-resolution X-ray micro-computed tomography and scanning electron microscopy with energy dispersive X-ray spectroscopy were performed and correlated with nanoindentation mapping. The hyoid apparatus has four distinct bone sections, with three joints between these sections. Nanoindentation results on cross-sectional regions of each bone reveal a previously unreported structure consisting of a stiff core and outer, more compliant shell with moduli of up to 27.4GPa and 8.5GPa, respectively. The bending resistance is low at the posterior section of the hyoid bones, indicating that this region has a high degree of flexibility to absorb impact. These new structural findings can be applied to further studies on the energy dissipation of the woodpecker during its drumming behavior, and may have implications for the design of engineered impact-absorbing structures. STATEMENT OF SIGNIFICANCE: Woodpeckers avoid brain injury while they peck at trees, which results in extreme impact conditions. One common adaptation in woodpeckers is the unusual shape of the elongated tongue, also called the hyoid apparatus. The relationship between the structure and mechanical properties of the bony part of the hyoid apparatus has not been previously reported. A three dimensional model of the bony tongue was developed, and the hardness and stiffness were evaluated. A new type of bone structure, which is opposite of typical skeletal bone structure was found. The combined microstructural and mechanical property analysis indicate possible energy absorption routes for the hyoid apparatus and are applicable to the design of engineered structures.

Thorns and dermal denticles of skates Atlantoraja cyclophora and A. castelnaui: Microscopic features and functional implications.

Some batoid species are covered with dermal denticles (or placoid scales) that occasionally develop into thorns. In sexually mature males, sharp teeth and alar thorns found on the apex of the lateral disc are used to hold the female during copulation. This study set out to analyze microscopic features of modified dermal denticles and thorns and to investigate sexual dimorphism in Atlantoraja cyclophora and A. castelnaui species. Skin samples collected from areas covered with thorns were fixed in 10% formaldehyde, processed and analyzed using scanning electron microscopy. Alar thorn morphology varied within species, while caudal thorn, rostral and caudal dermal denticle morphology varied within and between species. These structures play an important role in the protection and reproduction of the species studied and constitute important taxonomic information, given they are often the only elements preserved in archaeological sites and fossil records.

Multimodal Correlative Preclinical Whole Body Imaging and Segmentation.

Segmentation of anatomical structures and particularly abdominal organs is a fundamental problem for quantitative image analysis in preclinical research. This paper presents a novel approach for whole body segmentation of small animals in a multimodal setting of MR, CT and optical imaging. The algorithm integrates multiple imaging sequences into a machine learning framework, which generates supervoxels by an efficient hierarchical agglomerative strategy and utilizes multiple SVM-kNN classifiers each constrained by a heatmap prior region to compose the segmentation. We demonstrate results showing segmentation of mice images into several structures including the heart, lungs, liver, kidneys, stomach, vena cava, bladder, tumor, and skeleton structures. Experimental validation on a large set of mice and organs, indicated that our system outperforms alternative state of the art approaches. The system proposed can be generalized to various tissues and imaging modalities to produce automatic atlas-free segmentation, thereby enabling a wide range of applications in preclinical studies of small animal imaging.

Skeletal microstructure of Galaxea fascicularis exsert septa: a high-resolution SEM study.

The deposition of four crystal types at the growth surface of the septa of several color morphs of the coral Galaxea fascicularis was investigated over a 24-h period. Results suggest that nanocrystals, on denticles at the apices of exsert septa, may be the surface manifestation of centers of calcification. These crystals were also found on the septa of the axial corallite of Acropora formosa. The deposition of nanocrystals appears to be independent of diurnal rhythms. Internally and proximal to the septal apices, distinct clusters of polycrystalline fibers originate from centers of calcification and form fanlike fascicles. Upon these fascicles, acicular crystals grow and extend to form the visible fasciculi at the skeletal surface. Deposition of aragonitic fusiform crystals in both G. fascicularis and A. formosa occurs without diurnal rhythm. Nucleation of fusiform crystals appears to be independent of centers of calcification and may occur by secondary nucleation. Formation of semi-solid masses by fusiform crystals suggests that the crystals may play a structural role in septal extension. Lamellar crystals, which have not been reported as a component of scleractinian coral skeletons before, possess distinct layers of polyhedral plates, although these layers also do not appear to be associated with daily growth increments. The relationship of lamellar crystals to other components of the scleractinian coral skeleton and their involvement in skeletal growth is unknown.

Normal echoanatomy of the red-eared slider terrapin (Trachemys scripta elegans).

Thirty red-eared slider terrapins (Trachemys scripta elegans) were examined by ultrasound to establish the normal ultrasonographic appearance of their coelomic structures. They were not sedated, and owing to their small size they were examined through the inguinal window of the carapace. High resolution transducers (7.5 and 11 MHz) enhanced the ultrasonographic imaging of the bowel, urinary bladder, liver, gall bladder, heart, kidney and gonads, but the pancreas, adrenal glands, thyroid glands and spleen could not be visualised.

Mechanical properties and structure of the biological multilayered material system, Atractosteus spatula scales.

During recent decades, research on biological systems such as abalone shell and fish armor has revealed that these biological systems employ carefully arranged hierarchical multilayered structures to achieve properties of high strength, high ductility and light weight. Knowledge of such structures may enable pathways to design bio-inspired materials for various applications. This study was conducted to investigate the spatial distribution of structure, chemical composition and mechanical properties in mineralized fish scales of the species Atractosteus spatula. Microindentation tests were conducted, and cracking patterns and damage sites in the scales were examined to investigate the underlying protective mechanisms of fish scales under impact and penetration loads. A difference in nanomechanical properties was observed, with a thinner, stiffer and harder outer layer (indentation modulus ∼69 GPa and hardness ∼3.3 GPa) on a more compliant and thicker inner layer (indentation modulus ∼14.3 GPa and hardness ∼0.5 GPa). High-resolution scanning electron microscopy imaging of a fracture surface revealed that the outer layer contained oriented nanorods embedded in a matrix, and that the nanostructure of the inner layer contained fiber-like structures organized in a complex layered pattern. Damage patterns formed during microindentation show complex deformation mechanisms. Images of cracks identify growth through the outer layer, then deflection along the interface before growing and arresting in the inner layer. High-magnification images of the crack tip in the inner layer show void-linking and fiber-bridging exhibiting inelastic behavior. The observed difference in mechanical properties and unique nanostructures of different layers may have contributed to the resistance of fish scales to failure by impact and penetration loading.

Optimized siRNA-PEG conjugates for extended blood circulation and reduced urine excretion in mice.

Some of the main concerns with in vivo application of naked small interfering RNA are rapid degradation and urinary excretion resulting in a short plasma half-life. In this study we investigated how conjugation of polyethylene glycol (PEG) with variable chain length affects siRNA pharmacokinetics and biodistribution. The PEG chains were conjugated to chemically stabilized siRNA at the 5' terminal end of the passenger strand using click chemistry. The siRNA conjugate remained functionally active and showed significantly prolonged circulation in the blood stream after intravenous injection. siRNA conjugated with 20kDa PEG (PEG20k-siRNA) was most persistent, approximately 50% PEG20k-siRNA remained 1h post-injection, while the uncoupled siRNA was rapidly removed >90% at 15min. In vivo fluorescent imaging of the living animal showed increased concentration of siRNA in peripheral tissue and delayed urine excretion when coupled to PEG 20k. Biodistribution studies by northern blotting revealed equal distribution of conjugated siRNA in liver, kidney, spleen and lung without significant degradation 24 h post-injection. Our study demonstrates that PEG conjugated siRNA can be applied as a delivery system to improve siRNA bioavailability in vivo and may potentially increase the efficiency of siRNA in therapeutic applications.

Structural design and mechanical behavior of alligator (Alligator mississippiensis) osteoderms.

Alligator is a well-adapted living fossil covered with dorsal armor. This dermal shield consists of bony plates, called osteoderms, interconnected by sutures and non-mineralized collagen fibers, providing a dual function of protection and flexibility. Osteoderm features a sandwich structure, combining an inner porous core and an outer dense cortex, to offer enhancements for stiffness and energy absorbance. In this study, we investigated the multi-scale structure and mechanical behaviors of the American alligator (Alligator mississippiensis) osteoderm. Microcomputed tomography was applied to reveal the complex neurovascular network. Through the observation under optical and scanning electron microscopes, the osteoderm was found to consist of woven bone in the dorsal region and lamellar-zonal bone in the ventral region. Nanoindentation and compressive tests were performed to evaluate the mechanical properties of osteoderms. The varying mineral contents and porosity result in a graded mechanical property: a hard and stiff dorsal cortex gradually transform to a more compliant ventral base. Three protective mechanisms optimized for alligator osteoderms were proposed and elucidated.

Micromechanics of Sea Urchin spines.

The endoskeletal structure of the Sea Urchin, Centrostephanus rodgersii, has numerous long spines whose known functions include locomotion, sensing, and protection against predators. These spines have a remarkable internal microstructure and are made of single-crystal calcite. A finite-element model of the spine's unique porous structure, based on micro-computed tomography (microCT) and incorporating anisotropic material properties, was developed to study its response to mechanical loading. Simulations show that high stress concentrations occur at certain points in the spine's architecture; brittle cracking would likely initiate in these regions. These analyses demonstrate that the organization of single-crystal calcite in the unique, intricate morphology of the sea urchin spine results in a strong, stiff and lightweight structure that enhances its strength despite the brittleness of its constituent material.

Synchrotron X-ray microscopic computed tomography of the pump system of a female mosquito.

The pumping organ of blood-sucking female mosquitoes has a three-dimensional (3D) structure. However, conventional two-dimensional imaging methods are insufficient for visualizing the 3D structure in detail. Furthermore, their 3D imaging tasks are highly time consuming and sample preparation process requires elaborate skill. Among 3D imaging techniques, synchrotron X-ray microscopic computed tomography (SR-µCT) is especially suitable for small insects with opaque cuticles, such as mosquitoes. In this study, the 3D morphological structure of the pump system of a female mosquito was visualized using SR-µCT. Expandable volume capacities of two pump chambers were measured for several mosquito samples of similar size. To verify the cross-sectional images acquired by SR-µCT, complementary paraffin-sectioning data were compared.

Physiological characterization of stolon regression in a colonial hydroid.

As with many colonial animals, hydractiniid hydroids display a range of morphological variation. Sheet-like forms exhibit feeding polyps close together with short connecting stolons, whereas runner-like forms have more distant polyps and longer connecting stolons. These morphological patterns are thought to derive from rates of stolon growth and polyp formation. Here, stolon regression is identified and characterized as a potential process underlying this variation. Typically, regression can be observed in a few stolons of a normally growing colony. For detailed studies, many stolons of a colony can be induced to regress by pharmacological manipulations of reactive oxygen species (e.g. hydrogen peroxide) or reactive nitrogen species (e.g. nitric oxide). The regression process begins with a cessation of gastrovascular flow to the distal part of the stolon. High levels of endogenous H(2)O(2) and NO then accumulate in the regressing stolon. Remarkably, exogenous treatments with either H(2)O(2) or an NO donor equivalently trigger endogenous formation of both H(2)O(2) and NO. Cell death during regression is suggested by both morphological features, detected by transmission electron microscopy, and DNA fragmentation, detected by TUNEL. Stolon regression may occur when colonies detect environmental signals that favor continued growth in the same location rather than outward growth.