Anatomical insights into fish terrestrial locomotion: A study of barred mudskipper (Periophthalmus argentilineatus) fins based on µCT 3D reconstructions.

Mudskippers are a group of extant ray-finned fishes with an amphibious lifestyle and serve as exemplars for understanding the evolution of amphibious capabilities in teleosts. A comprehensive anatomical profile of both the soft and hard tissues within their propulsive fins is essential for advancing our understanding of terrestrial locomotor adaptations in fish. Despite the ecological significance of mudskippers, detailed data on their musculoskeletal anatomy remains limited. In the present research, we utilized contrast-enhanced high-resolution microcomputed tomography (µCT) imaging to investigate the barred mudskipper, Periophthalmus argentilineatus. This technique enabled detailed reconstruction and quantification of the morphological details of the pectoral, pelvic, and caudal fins of this terrestrial mudskipper, facilitating comparison with its aquatic relatives. Our findings reveal that P. argentilineatus has undergone complex musculoskeletal adaptations for terrestrial movement, including an increase in muscle complexity and muscle volume, as well as the development of specialized structures like aponeuroses for pectoral fin extension. Skeletal modifications are also evident, with features such as a reinforced shoulder-pelvic joint and thickened fin rays. These evolutionary modifications suggest biomechanically advanced fins capable of overcoming the gravitational challenges of terrestrial habitats, indicating a strong selective advantage for these features in land-based environments. The unique musculoskeletal modifications in the fins of mudskippers like P. argentilineatus, compared with their aquatic counterparts, mark a critical evolutionary shift toward terrestrial adaptations. This study not only sheds light on the specific anatomical changes facilitating this transition but also offers broader insights into the early evolutionary mechanisms of terrestrial locomotion, potentially mirroring the transformative journey from aquatic to terrestrial life in the lineage leading to tetrapods.

Metabolic and Skeletal Characterization of the KK/Ay Mouse Model-A Polygenic Mutation Model of Obese Type 2 Diabetes.

Type 2 diabetes (T2D) increases fracture incidence and fracture-related mortality rates (KK.Cg-Ay/J. The Jackson Laboratory; Available from: https://www.jax.org/strain/002468 ). While numerous mouse models for T2D exist, few effectively stimulate persistent hyperglycemia in both sexes, and even fewer are suitable for bone studies. Commonly used models like db/db and ob/ob have altered leptin pathways, confounding bone-related findings since leptin regulates bone properties (Fajardo et al. in Journal of Bone and Mineral Research 29(5): 1025-1040, 2014). The Yellow Kuo Kondo (KK/Ay) mouse, a polygenic mutation model of T2D, is able to produce a consistent diabetic state in both sexes and addresses the lack of a suitable model of T2D for bone studies. The diabetic state of KK/Ay stems from a mutation in the agouti gene, responsible for coat color in mice. This mutation induces ectopic gene expression across various tissue types, resulting in diabetic mice with yellow fur coats (Moussa and Claycombe in Obesity Research 7(5): 506-514, 1999). Male and female KK/Ay mice exhibited persistent hyperglycemia, defining them as diabetic with blood glucose (BG) levels consistently exceeding 300 mg/dL. Notably, male control mice in this study were also diabetic, presenting a significant limitation. Nevertheless, male and female KK/Ay mice showed significantly elevated BG levels, HbA1c, and serum insulin concentration when compared to the non-diabetic female control mice. Early stages of T2D are characterized by hyperglycemia and hyperinsulinemia resulting from cellular insulin resistance, whereas later stages may feature hypoinsulinemia due to ß-cell apoptosis (Banday et al. Avicenna Journal of Medicine 10(04): 174-188, 2020 and Klein et al. Cell Metabolism 34(1): 11-20, 2022). The observed hyperglycemia, hyperinsulinemia, and the absence of differences in ß-cell mass suggest that KK/Ay mice in this study are modeling the earlier stages of T2D. While compromised bone microarchitecture was observed in this study, older KK/Ay mice, representing more advanced stages of T2D, might exhibit more pronounced skeletal manifestations. Compared to the control group, the femora of KK/Ay mice had higher cortical area and cortical thickness, and improved trabecular properties which would typically be indicative of greater bone strength. However, KK/Ay mice displayed lower cortical tissue mineral density in both sexes and increased cortical porosity in females. Fracture instability toughness of the femora was lower in KK/Ay mice overall compared to controls. These findings indicate that decreased mechanical integrity noted in the femora of KK/Ay mice was likely due to overall bone quality being compromised.

Xylem-dwelling pathogen unaffected by local xylem vessel network properties in grapevines (Vitis spp.).

BACKGROUND AND AIMS: Xylella fastidiosa (Xf) is the xylem-dwelling bacterium associated with Pierce's disease (PD), which causes mortality in agriculturally important species, such as grapevine (Vitis vinifera). The development of PD symptoms in grapevines depends on the ability of Xf to produce cell-wall-degrading enzymes to break up intervessel pit membranes and systematically spread through the xylem vessel network. Our objective here was to investigate whether PD resistance could be mechanistically linked to xylem vessel network local connectivity. METHODS: We used high-resolution X-ray micro-computed tomography (microCT) imaging to identify and describe the type, area and spatial distribution of intervessel connections for six different grapevine genotypes from three genetic backgrounds, with varying resistance to PD (four PD resistant and two PD susceptible). KEY RESULTS: Our results suggest that PD resistance is unlikely to derive from local xylem network connectivity. The intervessel pit area (Ai) varied from 0.07 ±â€…0.01 mm2 mm-3 in Lenoir to 0.17 ±â€…0.03 mm2 mm-3 in Blanc do Bois, both PD resistant. Intervessel contact fraction (Cp) was not statically significant, but the two PD-susceptible genotypes, Syrah (0.056 ±â€…0.015) and Chardonnay (0.041 ±â€…0.013), were among the most highly connected vessel networks. Neither Ai nor Cp explained differences in PD resistance among the six genotypes. Bayesian re-analysis of our data shows moderate evidence against the effects of the traits analysed: Ai (BF01 = 4.88), mean vessel density (4.86), relay diameter (4.30), relay density (3.31) and solitary vessel proportion (3.19). CONCLUSIONS: Our results show that radial and tangential xylem network connectivity is highly conserved within the six different Vitis genotypes we sampled. The way that Xf traverses the vessel network may limit the importance of local network properties to its spread and may confer greater importance on host biochemical responses.

Reconstruction of an enigmatic Pennsylvanian cone reveals a relationship to Sphenophyllales.

PREMISE: We studied the 3D morphology of a small, well-preserved cone from the Pennsylvanian Mazon Creek Lagerstätte to characterize its structure and determine its systematic affinity. Previously tentatively assigned to the enigmatic Tetraphyllostrobus, we show that it differs in key respects from that genus as described. METHODS: We systematically compared the new fossil with relevant Paleozoic cone genera and employed advanced imaging techniques, including scanning electron microscopy, Airyscan confocal super-resolution microscopy, optical microscopy, and X-ray microcomputed tomography to visualize and reconstruct the fossil cone in 3D. RESULTS: The analyses demonstrate unequivocally that the sporophylls of the new Mazon Creek cone are arranged in whorls of six and have characters typical of Sphenophyllales, including epidermal cells with undulatory margins and in situ spores assignable to Columinisporites. The combination of characters, including sporophyll arrangement, anatomy, and spore type, supports the establishment of Hexaphyllostrobus kostorhysii gen. et sp. nov. within Sphenophyllales. Furthermore, we show that Tetraphyllostrobus, although originally described as possessing smooth monolete spores, actually possesses Columinisporites-type spores, indicating that it, too, was most likely a sphenophyll. CONCLUSIONS: The recognition of Hexaphyllostrobus contributes to our knowledge of Pennsylvanian sphenophyll diversity, and in particular increases the number of species with in situ Columinisporites-type spores. Attribution of Hexaphyllostrobus to Sphenophyllales calls into question current interpretations of Tetraphyllostrobus suggesting that future research on better-preserved macrofossil material may demonstrate a sphenophyllalean relationship.

Pulmonary vasculature development in congenital diaphragmatic hernia: a novel automated quantitative imaging analysis.

PURPOSE: Impaired fetal lung vasculature determines the degree of pulmonary hypertension in the congenital diaphragmatic hernia (CDH). This study aims to demonstrate the morphometric measurements that differ in pulmonary vessels of fetuses with CDH. METHODS: Nitrofen-induced CDH Sprague-Dawley rat fetuses were scanned with microcomputed tomography. The analysis of the pulmonary vascular tree was performed with artificial intelligence. RESULTS: The number of segments in CDH was significantly lower than that in the control group on the left (U = 2.5, p = 0.004) and right (U = 0, p = 0.001) sides for order 1(O1), whereas there was a significant difference only on the right side for O2 and O3. The pooled element numbers in the control group obeyed Horton's law (R2 = 0.996 left and R2 = 0.811 right lungs), while the CDH group broke it. Connectivity matrices showed that the average number of elements of O1 springing from elements of O1 on the left side and the number of elements of O1 springing from elements of O3 on the right side were significantly lower in CDH samples. CONCLUSION: According to these findings, CDH not only reduced the amount of small order elements, but also destroyed the fractal structure of the pulmonary arterial trees.

Bone Marrow Adipose Tissue Is Not Required for Reconstitution of the Immune System Following Irradiation in Male Mice.

Bone marrow adipose tissue (BMAT) is hypothesized to serve as an expandable/contractible fat depot which functions, in part, to minimize energy requirements for sustaining optimal hematopoiesis. We investigated whether BMAT is required for immune reconstitution following injury. Male wild type (WBB6F1, WT) and BMAT-deficient WBB6F1/J-KitW/KitW-v/J (KitW/W-v) mice were lethally irradiated. Irradiation was followed by adoptive transfer of 1000 purified WT hematopoietic stem cells (HSCs). The extent of immune reconstitution in blood, bone marrow, and lymph nodes in the irradiated mice was determined using HSCs from green fluorescent protein (GFP)-expressing mice. We also evaluated skeletal response to treatment. Detection of GFP-positive B and T cells in peripheral blood at 4 and 9 weeks following adoptive transfer and in bone marrow and lymph nodes following necropsy revealed excellent immune reconstitution in both WT and BMAT-deficient mice. Adipocytes were numerous in the distal femur of WT mice but absent or rare in KitW/W-v mice. Bone parameters, including length, mass, density, bone volume, microarchitecture, and turnover balance, exhibited few differences between WT and BMAT-deficient mice. The minimal differences suggest that BMAT is not required for reconstitution of the immune system following lethal radiation and is not a major contributor to the skeletal phenotypes of kit signaling-deficient mice.

A method of characterising the complex anatomy of vascular occlusions and 3D printing biomimetic analogues.

Chronic total occlusions (CTOs) occur in approximately 40% of individuals with symptomatic peripheral arterial disease and are indicative of critical limb ischaemia. Currently, few medical devices can effectively treat CTOs long-term, with amputation often required. This is due to a lack of knowledge of CTO anatomy, making device design and testing difficult. This study is a proof-of-concept study, which aimed to develop a workflow for further characterising the complex multi-material anatomy of CTOs and creating 3D models of CTO components, which may be useful in producing a vascular CTO biomimetic for device testing. Here, we establish such a workflow using samples of atheromatous plaques. We focus on a high-resolution, non-destructive microcomputed tomography (µCT) technique which enables visualisation of occlusion anatomy at a greater resolution than computed tomography angiography (CTA), which is the typical modality used for CTO clinical visualisation. Four arteries (n = 2 superficial femoral; n = 2 popliteal) with evidence of atheromatous plaques were cut into 8 cm segments, which were then stained with iodine and scanned at low resolution, with calcified regions rescanned at high resolution. Resulting files were manually segmented to generate 3D models, which were then 3D printed in resin using a stereolithography printer to produce parts suitable for creating a biomimetic. In total, µCT files from three arterial segments (n = 2 high resolution, n = 1 low resolution) were deemed suitably calcified for segmentation, and thus were segmented to produce 3D models. 3D models of the arterial wall, intima and atheromatous calcium deposits from a high-resolution popliteal artery scan were successfully 3D printed at several scales. While this research is at an early stage, it holds great promise. The workflow for segmentation and 3D printing various components of an atheromatous plaque established here is replicable and uses software and equipment which are accessible to research laboratories in both academia and industry. The ability to print detailed models on a desktop 3D printer is unprecedented and can be improved further, which is promising for future development of biomimetics with multi-material detail of both soft tissue and calcified components of a vascular occlusion. Indeed, this workflow provides a solid foundation for future studies of CTO anatomy and the creation of true, multi-material CTO biomimetics. Such biomimetics may enable the development of improved interventional devices, as they would mimic the general in vivo CTO environment. As this method cannot be applied in vivo, we cannot yet produce patient-specific biomimetics, however, these analogues would still be important in device development, which would improve patient outcomes in critical limb ischaemia.

Combined Thermoneutral Housing and Raloxifene Treatment Improves Trabecular Bone Microarchitecture and Strength in Growing Female Mice.

Thermoneutral housing and Raloxifene (RAL) treatment both have potential for improving mechanical and architectural properties of bone. Housing mice within a 30 to 32 °C range improves bone quality by reducing the consequences of cold stress, such as shivering and metabolic energy consumption (Chevalier et al. in Cell Metab 32(4):575-590.e7, 2020; Martin et al. in Endocr Connect 8(11):1455-1467, 2019; Hankenson et al. in Comp Med 68(6):425-438, 2018). Previous work suggests that Raloxifene can enhance bone strength and geometry (Ettinger et al. in Jama 282(7):637-645, 1999; Powell et al. in Bone Rep 12:100246, 2020). An earlier study in our lab utilized long bones to examine the effect of thermoneutral housing and Raloxifene treatment in mice, but no significant interactive effects were found. The lack of an impact is hypothesized to be connected to the short 6-week duration of the study and the type of bone analyzed. This study will examine the same question within the axial skeleton, which has a higher proportion of trabecular bone. After 6 weeks of treatment with RAL, vertebrae from female C57BL/6 J mice underwent microcomputed tomography (µCT), architectural analysis, and compression testing. Most of the tested geometric properties (bone volume/tissue volume percent, trabecular thickness, trabecular number, trabecular spacing) improved with both the housing and RAL treatment. The effect sizes suggested an additive effect when treating mice housed under thermoneutral conditions. While ultimate force was enhanced with the treatment and housing, force normalized by bone volume fraction was not significantly different between groups. For longer pre-clinical trials, it may be important to consider the impacts of temperature on mice to improve the accuracy of these models.

Microarchitectural Changes of Cardiovascular Calcification in Response to In Vivo Interventions Using Deep-Learning Segmentation and Computed Tomography Radiomics.

BACKGROUND: Coronary calcification associates closely with cardiovascular risk, but its progress is accelerated in response to some interventions widely used to reduce risk. This paradox suggests that qualitative, not just quantitative, changes in calcification may affect plaque stability. To determine if the microarchitecture of calcification varies with aging, Western diet, statin therapy, and high intensity, progressive exercise, we assessed changes in a priori selected computed tomography radiomic features (intensity, size, shape, and texture). METHODS: Longitudinal computed tomography scans of mice (Apoe-/-) exposed to each of these conditions were autosegmented by deep learning segmentation, and radiomic features of the largest deposits were analyzed. RESULTS: Over 20 weeks of aging, intensity and most size parameters increased, but surface-area-to-volume ratio (a measure of porosity) decreased, suggesting stabilization. However, texture features (coarseness, cluster tendency, and nonuniformity) increased, suggesting heterogeneity and likely destabilization. Shape parameters showed no significant changes, except sphericity, which showed a decrease. The Western diet had significant effects on radiomic features related to size and texture, but not intensity or shape. In mice undergoing either pravastatin treatment or exercise, the selected radiomic features of their computed tomography scans were not significantly different from those of their respective controls. Interestingly, the total number of calcific deposits increased significantly less in the 2 intervention groups compared with the respective controls, suggesting more coalescence and/or fewer de novo deposits. CONCLUSIONS: Thus, aging and standard interventions alter the microarchitectural features of vascular calcium deposits in ways that may alter plaque biomechanical stability.

Bone microstructure and volumetric bone mineral density in patients with global sagittal malalignment.

PURPOSE: Sagittal spinal malalignment often leads to surgical realignment, which is associated with major complications. Low bone mineral density (BMD) and impaired bone microstructure are risk factors for instrumentation failure. This study aims to demonstrate differences in volumetric BMD and bone microstructure between normal and pathological sagittal alignment and to determine the relationships among vBMD, microstructure, sagittal spinal and spinopelvic alignment. METHODS: A retrospective, cross-sectional study of patients who underwent lumbar fusion for degeneration was conducted. The vBMD of the lumbar spine was assessed by quantitative computed tomography. Bone biopsies were evaluated using microcomputed tomography (µCT). C7-S1 sagittal vertical axis (SVA; ≥ 50 mm malalignment) and spinopelvic alignment were measured. Univariate and multivariable linear regression analysis evaluated associations among the alignment, vBMD and µCT parameters. RESULTS: A total of 172 patients (55.8% female, 63.3 years, BMI 29.7 kg/m2, 43.0% with malalignment) including N = 106 bone biopsies were analyzed. The vBMD at levels L1, L2, L3 and L4 and the trabecular bone (BV) and total volume (TV) were significantly lower in the malalignment group. SVA was significantly correlated with vBMD at L1-L4 (ρ = -0.300, p < 0.001), BV (ρ = - 0.319, p = 0.006) and TV (ρ = - 0.276, p = 0.018). Significant associations were found between PT and L1-L4 vBMD (ρ = - 0.171, p = 0.029), PT and trabecular number (ρ = - 0.249, p = 0.032), PT and trabecular separation (ρ = 0.291, p = 0.012), and LL and trabecular thickness (ρ = 0.240, p = 0.017). In the multivariable analysis, a higher SVA was associated with lower vBMD (ß = - 0.269; p = 0.002). CONCLUSION: Sagittal malalignment is associated with lower lumbar vBMD and trabecular microstructure. Lumbar vBMD was significantly lower in patients with malalignment. These findings warrant attention, as malalignment patients may be at a higher risk of surgery-related complications due to impaired bone. Standardized preoperative assessment of vBMD may be advisable.

Evolution of hyperossification expands skull diversity in frogs.

Frogs (Anura) are one of the most diverse vertebrate orders, comprising more than 7,000 species with a worldwide distribution and extensive ecological diversity. In contrast to other tetrapods, frogs have a highly derived body plan and simplified skull. In many lineages of anurans, increased mineralization has led to hyperossified skulls, but the function of this trait and its relationship with other aspects of head morphology are largely unexplored. Using three-dimensional morphological data from 158 species representing all frog families, we assessed wide-scale patterns of shape variation across all major lineages, reconstructed the evolutionary history of cranial hyperossification across the anuran phylogeny, and tested for relationships between ecology, skull shape, and hyperossification. Although many frogs share a conserved skull shape, several extreme forms have repeatedly evolved that commonly are associated with hyperossification, which has evolved independently more than 25 times. Variation in cranial shape is not explained by phylogenetic relatedness but is correlated with shifts in body size and ecology. The species with highly divergent, hyperossified skulls often have a specialized diet or a unique predator defense mechanism. Thus, the evolution of hyperossification has repeatedly facilitated the expansion of the head into multiple new shapes and functions.

Characterization of Channeling Effects Applied to Extended-Release Matrix Tablets Containing Pirfenidone.

Pirfenidone (PRF) is an anti-fibrotic agent that has been approved by the Food and Drug Administration (FDA) for the treatment of mild to moderate idiopathic pulmonary fibrosis. However, the current oral administration dosing regimen of PRF is complex and requires high doses. Patients are instructed to take PRF three times daily, with each dose consisting of up to three capsules or tablets (600 mg/d or 1.8 g/d of PRF) taken with food. To improve the dosing regimen, efforts are being made to develop an extended-release tablet with a zero-order release pattern. In this study, two types of extended-release matrix tablets were compared: non-channeled extended-release matrix tablets (NChMT) and channeled extended-release matrix tablets (ChMT). In vitro release tests, swelling and erosion index, rheology studies, and X-ray microcomputed tomography (XRCT), were conducted. The results indicated that ChMT maintained a zero-order release pattern with a constant release rate, while NChMT exhibited a decreased release rate in the latter half of the dissolution. ChMT exhibited accelerated swelling and erosion compared to other formulations, and this was made possible by the presence of channels within the tablet. These channels allowed for thorough wetting and swelling throughout the entire depth of the tablet. The formation of channels was confirmed through XRCT images. In conclusion, the presence of channels in ChMT tablets increased the rate of swelling and erosion, resulting in a zero-order release pattern. This development offers the potential to improve the dosage of PRF and reduce its associated side effects.

Comparison of obturation quality in natural and replica teeth root-filled using different sealers and techniques.

OBJECTIVES: This study aims to assess the obturation efficacy of sealers placed with different techniques using microcomputed tomography (µCT) and assess the influence of µCT testing parameters on the obturation data obtained. MATERIALS AND METHODS: Incisors and mesial roots of lower molars with standardized root length were scanned using µCT, and one tooth of each type was 3D printed in acrylic. Two obturation techniques (warm vertical and single cone) and 4 sealer types (AH Plus, BioRoot RCS, Totalfill BC, and Bio-C Sealers) were assessed following storage in Hank's balanced salt solution for 3 and 6 months by assessing gap and void volume percentages on both natural and replica incisor and molar roots. The storage solution was analysed to assess calcium ion leaching. The influence of temperature, tooth positioning, and moisture content of the teeth while µCT scanning was also investigated. RESULTS: The obturation quality in the incisor group was the same using both natural teeth and replicas (p > 0.05). No changes in void volume were identified when comparing the same sealer using different obturation techniques. The premixed sealers used in single-cone obturation exhibited high void volume in the 3D printed replicas in the long term. The temperature, positioning, and moisture content of the teeth did not affect the outcome of µCT testing. CONCLUSIONS: BioRoot RCS, Totalfill BC, and Bio-C Sealers are suitable for obturation of both complex and simple root canal systems using different obturation techniques with BioRoot RCS exhibiting the highest calcium ion release. 3D printed acrylic teeth can be used to assess the obturation quality in uncomplicated root canal systems. µCT parameters had no significant effect on the µCT measurement. CLINICAL RELEVANCE: The single-cone obturation technique with hydraulic sealer is a simple technique that can be used for obturation of all root canal systems.

Definition of the Region of Interest for the Assessment of Alveolar Bone Repair Using Micro-computed Tomography.

The objective of this study was to evaluate the influence of the extraction socket (distal or lingual root) and the type of region of interest (ROI) definition (manual or predefined) on the assessment of alveolar repair following tooth extraction using micro-computed tomography (micro-CT). The software package used for scanning, reconstruction, reorientation, and analysis of images (NRecon®, DataViewer®, CT-Analyzer®) was acquired through Bruker < https://www.bruker.com > . The sample comprised the micro-CT volumes of seven Wistar rat mandibles, in which the right first molar was extracted. The reconstructed images were analyzed using the extraction sockets, i.e., the distal and intermediate lingual root and the method of ROI definition: manual (MA), central round (CR), and peripheral round (PR). The bone volume fraction (BV/TV) values obtained were analyzed by two-way ANOVA with Tukey's post hoc test (α = 5%). The distal extraction socket resulted in significantly lower BV/TV values than the intermediate lingual socket for MA (P = 0.001), CR (P < 0.001), and PR (P < 0.001). Regarding the ROI, when evaluating the distal extraction socket, the BV/TV was significantly higher (P < 0.001) for MA than for CR and PR, with a lower BV/TV for CR. However, no significant difference was observed for MA (P = 0.855), CR (P = 0.769), or PR (P = 0.453) in the intermediate lingual extraction socket. The bone neoformation outcome (BV/TV) for alveolar bone repair after tooth extraction is significantly influenced by the ROI and the extraction socket. Using the predefined method with a standardized ROI in the central region of the distal extraction socket resulted in the assessment of bone volume, demonstrating the most critical region of the bone neoformation process.

Combining Raman Microspectroscopy and X-ray Microcomputed Tomography for the Study of Bone Quality in Apolipoprotein-Deficient Animal Models.

Raman microspectroscopy and X-ray microcomputed tomography (micro-CT) were used for assessment of the quality of the femur and tibia bones in apolipoprotein-deficient mice compared to control littermates. The cortical and trabecular bone was investigated separately. Raman spectra revealed no differences in the bioapatite-to-collagenous matrix ratio of the cortical bone. The quantities of calcium and collagen, which were measured using atomic absorption spectrometry and thermogravimetric analysis, respectively, were also found to be equal in the two groups. Density and morphometric parameters, which were measured using micro-CT, verified the cortical mineral stability. Bone quality indices were measured using Raman spectra. A decreased collagen crosslink (trivalent-to-divalent) ratio revealed delayed maturation of the collagen network. Such a decrease has been reported in the literature to be connected to decreased bone strength. For the trabecular bone, micro-CT revealed severe osteoporosis in the knock-out group, which was evident from a decreased mineral density, trabecular thickness and increased bone surface/volume ratio. The trabecular bone was not accessible for Raman spectroscopy. According to these results, the cortical and trabecular femur bone is expected to exhibit proneness to fracturing, each for a different reason. A combination of the two techniques was regarded as necessary for an overall assessment of bone quality.

Shark centra microanatomy and mineral density variation studied with laboratory microComputed Tomography.

Centra of shark vertebrae from three species of Lamniformes (Alopias vulpinus, Carcharodon carcharias and Isurus oxyrinchus) and three species of Carcharhiniformes (Carcharhinus plumbeus, Carcharhinus obscurus and Prionace glauca) were imaged with laboratory microcomputed Tomography (microCT) using volume element (voxel) sizes between 16 and 24 µm. Linear attenuation coefficients were the same in the corpus calcarea (hour-glass-shaped cone) and intermedialia of the lamniforms but were smaller in the intermedialia than in the corpus calcarea of the carcharhiniforms. All centra contained growth bands which were visible as small changes in linear attenuation coefficient. In all six cases, the cross-sections of the cones were close to circular, and the cone angles matched those reported in the literature. Cartilage canals were a prominent structure in the intermedialia of all species, 3D renderings of centra of C. obscurus and I. oxyrinchus diameters showed these canals ran radially outward from the cone walls, and canal diameters were consistent with the limited numerical values in the literature. Somewhat higher calcification levels around the periphery of cartilage canals and of outer surfaces of the intermedialia and corpus calcerea suggest microstructural variation exists at scale below that which can be resolved in the present data sets.

Loss of O-GlcNAcase catalytic activity leads to defects in mouse embryogenesis.

O-GlcNAcylation is an essential post-translational modification that has been implicated in neurodevelopmental and neurodegenerative disorders. O-GlcNAcase (OGA), the sole enzyme catalyzing the removal of O-GlcNAc from proteins, has emerged as a potential drug target. OGA consists of an N-terminal OGA catalytic domain and a C-terminal pseudo histone acetyltransferase (HAT) domain with unknown function. To investigate phenotypes specific to loss of OGA catalytic activity and dissect the role of the HAT domain, we generated a constitutive knock-in mouse line, carrying a mutation of a catalytic aspartic acid to alanine. These mice showed perinatal lethality and abnormal embryonic growth with skewed Mendelian ratios after day E18.5. We observed tissue-specific changes in O-GlcNAc homeostasis regulation to compensate for loss of OGA activity. Using X-ray microcomputed tomography on late gestation embryos, we identified defects in the kidney, brain, liver, and stomach. Taken together, our data suggest that developmental defects during gestation may arise upon prolonged OGA inhibition specifically because of loss of OGA catalytic activity and independent of the function of the HAT domain.

Desiccation of the leaf mesophyll and its implications for CO2 diffusion and light processing.

Leaves balance CO2 and radiative absorption while maintaining water transport to maximise photosynthesis. Related species with contrasting leaf anatomy can provide insights into inherent and stress-induced links between structure and function for commonly measured leaf traits for important crops. We used two walnut species with contrasting mesophyll anatomy to evaluate these integrated exchange processes under non-stressed and drought conditions using a combination of light microscopy, X-ray microCT, gas exchange, hydraulic conductance, and chlorophyll distribution profiles through leaves. Juglans regia had thicker palisade mesophyll, higher fluorescence in the palisade, and greater low-mesophyll porosity that were associated with greater gas-phase diffusion (gIAS ), stomatal and mesophyll (gm ) conductances and carboxylation capacity. More and highly-packed mesophyll cells and bundle sheath extensions (BSEs) in Juglans microcarpa led to higher fluorescence in the spongy and in proximity to the BSEs. Both species exhibited drought-induced reductions in mesophyll cell volume, yet the associated increases in porosity and gIAS were obscured by declines in biochemical activity that decreased gm . Inherent differences in leaf anatomy between the species were linked to differences in gas exchange, light absorption and photosynthetic capacity, and drought-induced changes in leaf structure impacted performance via imposing species-specific limitations to light absorption, gas exchange and hydraulics.

Evaluation of neural innervation in the human conventional outflow pathway distal to Schlemm's canal.

The distal outflow pathway of the human eye consists of the outer wall of Schlemm's canal, collector channels, and the deep-scleral, mid-scleral and episcleral vessels. It is the last region of transit for aqueous humor before returning to the venous system. While the trabecular meshwork, scleral spur, and inner wall of Schlemm's canal have been extensively analyzed to define their contributions to aqueous outflow, the role of the distal outflow pathway is not completely understood. Collector channels, emanating from Schlemm's canal were previously thought to be passive conduits for aqueous humor. However, recent studies have shown many collector channels contain flap-like appendages which move with changes in pressure. These findings, along with studies demonstrating innervation of episcleral vessels, have led to questions regarding whether other structures in the distal outflow pathway are under neural regulation and how this may influence aqueous humor outflow. This study evaluates the innervation of the outer wall of Schlemm's canal and collector channels, along with the deep-scleral, mid-scleral and episcleral vasculature with microcomputed tomography and 3-dimensional reconstruction, correlative light microscopy, immunohistochemistry, and transmission electron microscopy. Peripheral, autonomic, and sensory nerve fibers were found to be present adjacent to Schlemm's canal outer wall endothelium, collector channel endothelium, and in the different regions of the distal outflow vasculature. Nerves were more commonly identified in regions that contained collector channels when compared to regions without collector channels. These findings regarding the neural anatomy suggest an active neural regulation of aqueous humor outflow throughout the proximal and distal regions of the conventional outflow pathway.

Female terminalia morphology and cladistic relations among Tok-Tok beetles (Tenebrionidae: Sepidiini).

Tok-tokkies are one of the most iconic lineages within Tenebrionidae. In addition to containing some of the largest darkling beetles, this tribe is recognized for its remarkable form of sexual communication known as substrate tapping. Nevertheless, the phylogenetic relationships within the group remain poorly understood. This study investigates the usefulness of female terminalia morphology for delimiting Sepidiini and reconstructing relationships among it. Data on the structure of the ovipositors, genital tubes and spicula ventrali have been generated for >200 species representing 28 Pimeliinae tribes. This dataset was used in a comparative analysis at the subfamilial level, which resulted in recognition of several unique features of tok-tokkie terminalia. Additionally, new features linking phenotypically challenging tribes also were recovered (Cryptochilini + Idisiini + Pimeliini). Secondly, 23 characters linked to the structure of female terminalia were defined for tok-tok beetles. Cladistic analysis demonstrates the nonmonophyletic nature of most of the recognized subtribes. The morphological dataset was analysed separately and in combination with available molecular data (CAD, Wg, cox1, cox2, 28S). All obtained topologies were largely congruent, supporting the following changes: Palpomodina Kaminski & Gearner subtr.n. is erected to accommodate the genera Namibomodes and Palpomodes; Argenticrinis and Bombocnodulus are transferred from Hypomelina to Molurina; 153 species and subspecies previously classified within Psammodes are distributed over three separate genera (Mariazofia Kaminski nom.n., Piesomera stat.r., Psammodes sens.n.). Psammodes sklodowskae Kaminski & Gearner sp.n. is described. Preliminary investigation of the ovipositor of Mariazofia basuto (Koch) comb.n. was carried out with the application of microcomputed tomography, illuminating the muscular system as a reliable reference point for recognizing homologous elements in highly modified ovipositors.