Search

Background: The gene encoding the transcription factor, Grainyhead-like 3 (Grhl3), plays critical roles in mammalian development and homeostasis. Grhl3-null embryos exhibit thoraco-lumbo-sacral spina bifida and soft-tissue syndactyly. Additional studies reveal that these embryos also exhibit an epidermal proliferation/differentiation imbalance. This manifests as skin barrier defects resulting in peri-natal lethality and defective wound repair. Despite these extensive analyses of Grhl3 loss-of-function models, the consequences of gain-of-function of this gene have been difficult to achieve., Results: In this study, we generated a novel mouse model that expresses Grhl3 from a transgene integrated in the Rosa26 locus on an endogenous Grhl3-null background. Expression of the transgene rescues both the neurulation and skin barrier defects of the knockout mice, allowing survival into adulthood. Despite this, the mice are not normal, exhibiting a range of phenotypes attributable to dysregulated Grhl3 expression. In mice homozygous for the transgene, we observe a severe Shaker-Waltzer phenotype associated with hearing impairment. Micro-CT scanning of the inner ear revealed profound structural alterations underlying these phenotypes. In addition, these mice exhibit other developmental anomalies including hair loss, digit defects, and epidermal dysmorphogenesis., Conclusion: Taken together, these findings indicate that diverse developmental processes display low tolerance to dysregulation of Grhl3., (© 2022 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Breast microcalcifications are an important primary radiological indicator of breast cancer. However, microcalcification classification and diagnosis may be still challenging for radiologists due to limitations of the standard 2D mammography technique, including spatial and contrast resolution. In this study, we propose an approach to improve the detection of microcalcifications in propagation-based phase-contrast X-ray computed tomography of breast tissues. Five fresh mastectomies containing microcalcifications were scanned at different X-ray energies and radiation doses using synchrotron radiation. Both bright-field (i.e. conventional phase-retrieved images) and dark-field images were extracted from the same data sets using different image processing methods. A quantitative analysis was performed in terms of visibility and contrast-to-noise ratio of microcalcifications. The results show that while the signal-to-noise and the contrast-to-noise ratios are lower, the visibility of the microcalcifications is more than two times higher in the dark-field images compared to the bright-field images. Dark-field images have also provided more accurate information about the size and shape of the microcalcifications.

Purpose: Breast cancer is the most common cancer in women in developing and developed countries and is responsible for 15% of women's cancer deaths worldwide. Conventional absorption-based breast imaging techniques lack sufficient contrast for comprehensive diagnosis. Propagation-based phase-contrast computed tomography (PB-CT) is a developing technique that exploits a more contrast-sensitive property of x-rays: x-ray refraction. X-ray absorption, refraction, and contrast-to-noise in the corresponding images depend on the x-ray energy used, for the same/fixed radiation dose. The aim of this paper is to explore the relationship between x-ray energy and radiological image quality in PB-CT imaging. Approach: Thirty-nine mastectomy samples were scanned at the imaging and medical beamline at the Australian Synchrotron. Samples were scanned at various x-ray energies of 26, 28, 30, 32, 34, and 60 keV using a Hamamatsu Flat Panel detector at the same object-to-detector distance of 6 m and mean glandular dose of 4 mGy. A total of 132 image sets were produced for analysis. Seven observers rated PB-CT images against absorption-based CT (AB-CT) images of the same samples on a five-point scale. A visual grading characteristics (VGC) study was used to determine the difference in image quality. Results: PB-CT images produced at 28, 30, 32, and 34 keV x-ray energies demonstrated statistically significant higher image quality than reference AB-CT images. The optimum x-ray energy, 30 keV, displayed the largest area under the curve ( AUC VGC ) of 0.754 ( p = 0.009 ). This was followed by 32 keV ( AUC VGC = 0.731 , p ≤ 0.001 ), 34 keV ( AUC VGC = 0.723 , p ≤ 0.001 ), and 28 keV ( AUC VGC = 0.654 , p = 0.015 ). Conclusions: An optimum energy range (around 30 keV) in the PB-CT technique allows for higher image quality at a dose comparable to conventional mammographic techniques. This results in improved radiological image quality compared with conventional techniques, which may ultimately lead to higher diagnostic efficacy and a reduction in breast cancer mortalities., (© 2021 Society of Photo-Optical Instrumentation Engineers (SPIE).)

Rationale and Objectives: Propagation-based phase-contrast CT (PB-CT) is an advanced X-ray imaging technology that exploits both refraction and absorption of the transmitted X-ray beam. This study was aimed at optimizing the experimental conditions of PB-CT for breast cancer imaging and examined its performance relative to conventional absorption-based CT (AB-CT) in terms of image quality and radiation dose., Materials and Methods: Surgically excised breast mastectomy specimens (n = 12) were scanned using both PB-CT and AB-CT techniques under varying imaging conditions. To evaluate the radiological image quality, visual grading characteristics (VGC) analysis was used in which 11 breast specialist radiologists compared the overall image quality of PB-CT images with respect to the corresponding AB-CT images. The area under the VGC curve was calculated to measure the differences between PB-CT and AB-CT images., Results: The highest radiological quality was obtained for PB-CT images using a 32 keV energy X-ray beam and by applying the Homogeneous Transport of Intensity Equation phase retrieval with the value of its parameter γ set to one-half of the theoretically optimal value for the given materials. Using these optimized conditions, the image quality of PB-CT images obtained at 4 mGy and 2 mGy mean glandular dose was significantly higher than AB-CT images at 4 mGy (AUC VGC  = 0.901, p = 0.001 and AUC VGC  = 0.819, p = 0.011, respectively)., Conclusion: PB-CT achieves a higher radiological image quality compared to AB-CT even at a considerably lower mean glandular dose. Successful translation of the PB-CT technique for breast cancer imaging can potentially result in improved breast cancer diagnosis., (Crown Copyright © 2020. Published by Elsevier Inc. All rights reserved.)

In this study, we have examined ceramic matrix composites with silicon carbide fibers in a melt-infiltrated silicon carbide matrix (SiC/SiC). We subjected samples to tensile loads while collecting micro X-ray computed tomography images. The results showed the expected crack slowing mechanisms and lower resistance to crack propagation where the fibers ran parallel and perpendicular to the applied load respectively. Cracking was shown to initiate not only from the surface but also from silicon inclusions. Post heat-treated samples showed longer fiber pull-out than the pristine samples, which was incompatible with previously proposed mechanisms. Evidence for oxidation was identified and new mechanisms based on oxidation or an oxidation assisted boron nitride phase transformation was therefore proposed to explain the long pull-out. The role of oxidation emphasizes the necessity of applying oxidation resistant coatings on SiC/SiC.

Alternate splicing is a critical regulator of gene expression in eukaryotes, however genetic mutations can cause erroneous splicing and disease. Most recorded splicing disorders are caused by mutations of splice donor/acceptor sites, however intronic mutations can affect splicing. Clinical exome analyses largely ignore intronic sequence, limiting the detection of mutations to within coding regions. We describe 'Trooper', a novel mouse model of CHARGE syndrome harbouring a pathogenic point mutation in Chd7. The mutation is 18 nucleotides upstream of exon 10 and creates a cryptic acceptor site, causing exon skipping and partial intron retention. This mutation, though detectable in exome sequence, was initially dismissed by computational filtering due to its intronic location. The Trooper strain exhibited many of the previously described CHARGE-like anomalies of CHD7 deficient mouse lines; including hearing impairment, vestibular hypoplasia and growth retardation. However, more common features such as facial asymmetry and circling were rarely observed. Recognition of these characteristic features prompted manual reexamination of Chd7 sequence and subsequent validation of the intronic mutation, highlighting the importance of phenotyping alongside exome analyses. The Trooper mouse serves as a valuable model of atypical CHARGE syndrome and reveals a molecular mechanism that may underpin milder clinical presentation of the syndrome.

Ceramic matrix composites (CMCs) are structural materials, which have useful properties that combine high strength at high temperatures with moderate toughness. Carbon fibers within a matrix of carbon and silicon carbide, called C/C-SiC, are a particular class of CMC noted for their high oxidation resistance. Here we use a combination of four-point bending and X-ray radiography, to study the mechanical failure of C/C-SiC CMCs. Correlating X-ray radiographic and load/displacement curve data reveals that the fiber bundles act to slow down crack propagation during four-point bending tests. We attribute this to the fact that strain energy is expended in breaking these fibers and in pulling fiber bundles out of the surrounding matrix material. In addition, we find that the local distribution and concentration of SiC plays an important role in reducing the toughness of the material.

The majority of current laboratory based X-ray sources are polychromatic and are not tuneable. This lack of monochromaticity limits the range of applications for these sources and in particular it reduces the elemental specificity of laboratory based X-ray imaging experiments. Here we present a solution to this problem based on the use of Ross filter pairs. Although such Ross filter arrangements have been applied in proof-of-principle spectroscopy experiments, to date there have been no reports of this approach used for full-field X-ray imaging. Here we report on the experimental demonstration of Ross filter pairs being used for quasi-monochromatic, full-field imaging. This arrangement has several important benefits for laboratory based X-ray imaging including, as we demonstrate, elemental contrast enhancement. The method is demonstrated both for two-dimensional radiography and for three-dimensional X-ray tomography.

Background: Increased apposition of the frontal and parietal bones of the skull during embryogenesis may be a risk factor for the subsequent development of premature skull fusion, or craniosynostosis. Human craniosynostosis is a prevalent, and often serious embryological and neonatal pathology. Other than known mutations in a small number of contributing genes, the aetiology of craniosynostosis is largely unknown. Therefore, the identification of novel genes which contribute to normal skull patterning, morphology and premature suture apposition is imperative, in order to fully understand the genetic regulation of cranial development., Results: Using advanced imaging techniques and quantitative measurement, we show that genetic deletion of the highly-conserved transcription factor Grainyhead-like 3 (Grhl3) in mice (Grhl3 -/- ) leads to decreased skull size, aberrant skull morphology and premature apposition of the coronal sutures during embryogenesis. Furthermore, Grhl3 -/- mice also present with premature collagen deposition and osteoblast alignment at the sutures, and the physical interaction between the developing skull, and outermost covering of the brain (the dura mater), as well as the overlying dermis and subcutaneous tissue, appears compromised in embryos lacking Grhl3. Although Grhl3 -/- mice die at birth, we investigated skull morphology and size in adult animals lacking one Grhl3 allele (heterozygous; Grhl3 +/- ), which are viable and fertile. We found that these adult mice also present with a smaller cranial cavity, suggestive of post-natal haploinsufficiency in the context of cranial development., Conclusions: Our findings show that our Grhl3 mice present with increased apposition of the frontal and parietal bones, suggesting that Grhl3 may be involved in the developmental pathogenesis of craniosynostosis.

Aim: To explore morphological or electrophysiological evidence for the presence of endolymphatic hydrops (EH) in guinea pig cochleae in the first 3 months after cochlear implantation., Methods: Dummy silastic electrodes were implanted atraumatically into the basal turn of scala tympani via a cochleostomy. Round window electrocochleography (ECochG) was undertaken prior to and after implantation. Animals survived for 1, 7, 28 or 72 days prior to a terminal experiment, when ECochG was repeated. The cochleae were imaged using micro-CT after post-fixing with osmium tetroxide to reveal the inner ear soft tissue structure. EH was assessed by visual inspection at a series of frequency specific places along the length of the cochlea, and the extent to which Reissner's membrane departed from its neutral position was quantified. Tissue response volumes were calculated. Using ECochG, the ratio of the summating potential to the action potential (SP/AP ratio) was calculated in response to frequencies between 2 and 32 kHz., Results: There was minimal evidence of electrode trauma from cochlear implantation on micro-CT imaging. Tissue response volumes did not change over time. EH was most prevalent 7 days after surgery in implanted ears, as determined by visual inspection. Scala media areas were increased, as expected in cases of EH, over the first month after cochlear implantation. SP/AP ratios decreased immediately after surgery, but were elevated 1 and 7 days after implantation., Conclusions: EH is prevalent in the first weeks after implant surgery, even in the absence of significant electrode insertion trauma., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Rheumatoid arthritis (RA) and ankylosing spondylitis (AS) are chronic inflammatory diseases that together affect 2-3% of the population. RA and AS predominantly involve joints, but heart disease is also a common feature in RA and AS patients. Here we have studied a new spontaneous mutation that causes severe polyarthritis in bone phenotype spontaneous mutation 1 (BPSM1) mice. In addition to joint destruction, mutant mice also develop aortic root aneurism and aorto-mitral valve disease that can be fatal depending on the genetic background. The cause of the disease is the spontaneous insertion of a retrotransposon into the 3' untranslated region (3'UTR) of the tumor necrosis factor (TNF), which triggers its strong overexpression in myeloid cells. We found that several members of a family of RNA-binding, CCCH-containing zinc-finger proteins control TNF expression through its 3'UTR, and we identified a previously unidentified regulatory element in the UTR. The disease in BPSM1 mice is independent of the adaptive immune system and does not appear to involve inflammatory cytokines other than TNF. To our knowledge, this is the first animal model showing both polyarthritis and heart disease as a direct result of TNF deregulation. These results emphasize the therapeutic potential of anti-TNF drugs for the treatment of heart valve disease and identify potential therapeutic targets to control TNF expression and inflammation.

E26 transformation-specific 1 (ETS1) and friend leukemia integration 1 (FLI1) are members of the ETS family of transcription factors, of which there are 28 in humans. Both genes are hemizygous in Jacobsen syndrome, an 11q contiguous gene deletion disorder involving thrombocytopenia, facial dysmorphism, growth and mental retardation, malformation of the heart and other organs, and hearing impairment associated with recurrent ear infections. To determine whether any of these defects are because of hemizygosity for ETS1 and FLI1, we characterized the phenotype of mice heterozygous for mutant alleles of Ets1 and Fli1. Fli1(+/-) mice displayed mild thrombocytopenia, as did Ets1(+/-)Fli1(+/-) animals. Fli1(+/-) and Ets1(+/-)Fli1(+/-) mice also displayed craniofacial abnormalities, including a small middle ear cavity, short nasal bone, and malformed interface between the nasal bone process and cartilaginous nasal septum. They exhibited hearing impairment, otitis media, fusions of ossicles to the middle ear wall, and deformed stapes. Hearing impairment was more penetrant and stapes malformations were more severe in Ets1(+/-)Fli1(+/-) mice than in Fli1(+/-) mice, indicating partial functional redundancy of these transcription factors during auditory development. Our findings indicate that the short nose, otitis media, and hearing impairment in Jacobsen syndrome are likely because of hemizygosity for ETS1 and FLI1., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

CHARGE syndrome is a rare human disorder caused by mutations in the gene encoding chromodomain helicase DNA binding protein 7 (CHD7). Characteristics of CHARGE are varied and include developmental ear and hearing anomalies. Here we report a novel mouse model of CHD7 dysfunction, termed Looper. The Looper strain harbours a nonsense mutation (c.5690C>A, p.S1897X) within the Chd7 gene. Looper mice exhibit many of the clinical features of the human syndrome, consistent with previously reported CHARGE models, including growth retardation, facial asymmetry, vestibular defects, eye anomalies, hyperactivity, ossicle malformation, hearing loss and vestibular dysfunction. Looper mice display an otosclerosis-like fusion of the stapes footplate to the cochlear oval window and blepharoconjunctivitis but not coloboma. Looper mice are hyperactive and have vestibular dysfunction but do not display motor impairment.

Background: Most tooth cavities start to develop inside pits and fissures where brushing, saliva, fluoride toothpaste or mouthwashes have no access. Therefore, 3D morphology of pits and fissures is an important subject to be studied in relation to maintaining proper oral hygiene., Objective: This study aimed to present high resolution images of pits and fissures in human teeth using a laboratory-based micro X-ray Computed Tomography (mXCT) and also to present the basic structure evaluation that could be obtained., Methods: Three human wisdom teeth were examined. Two different set-ups were used in this study achieving resolution of 14.59 μm (Field of View of 14.9 mm) and resolution of 4.43 μm (FOV of 4.5 mm) respectively. Automated segmentation was performed for further evaluation to distinguish between empty space (pits and fissures) and the filled space (enamel)., Results: The 3D tomography results demonstrate detailed morphology with accurate dimensions and the locations of the pits and fissures, which is important to investigate the relationship with tooth decay that mostly starts deep inside pits and fissures., Conclusions: Segmentation from the mXCT imaging of pits and fissures provides obvious visual evidence to help in promotion of oral health and to improve personal tooth care in preventive treatment protocols.

This study realizes the concept of simultaneous micro-X-ray computed tomography and X-ray powder diffraction using a synchrotron beamline. A thin zinc metal foil was placed in the primary, monochromatic synchrotron beam to generate a divergent wave to propagate through the samples of interest onto a CCD detector for tomographic imaging, thus removing the need for large beam illumination and high spatial resolution detection. Both low density materials (kapton tubing and a piece of plant) and higher density materials (Egyptian faience) were investigated, and elemental contrast was explored for the example of Cu and Ni meshes. The viability of parallel powder diffraction using the direct beam transmitted through the foil was demonstrated. The outcomes of this study enable further development of the technique towards in situ tomography∕diffraction studies combining micrometer and crystallographic length scales, and towards elemental contrast imaging and reconstruction methods using well defined fluorescence outputs from combinations of known fluorescence targets (elements).

X-ray tomography can provide structural information of whole cells in close to their native state. Radiation-induced damage, however, imposes a practical limit to image resolution, and as such, a choice between damage, image contrast, and image resolution must be made. New coherent diffractive imaging techniques, such Fresnel Coherent Diffractive Imaging (FCDI), allows quantitative phase information with exceptional dose efficiency, high contrast, and nano-scale resolution. Here we present three-dimensional quantitative images of a whole eukaryotic cell by FCDI at a spatial resolution below 70 nm with sufficient phase contrast to distinguish major cellular components. From our data, we estimate that the minimum dose required for a similar resolution is close to that predicted by the Rose criterion, considerably below accepted estimates of the maximum dose a frozen-hydrated cell can tolerate. Based on the dose efficiency, contrast, and resolution achieved, we expect this technique will find immediate applications in tomographic cellular characterisation.

We describe a direct quantitative phase reconstruction approach using an X-ray laboratory-based source. Using a single phase-contrast image from each tomographic projection we show that it is possible to modify the filter term in a filtered back projection reconstruction to take account of the broad spectrum from a laboratory source. The accessibility of conventional X-ray laboratory sources makes this method very useful for quantitative phase imaging of homogeneous and weakly absorbing objects.

In this paper we describe novel and specific roles for the apoptotic regulators Bcl2 and Bim in hearing and stapes development. Bcl2 is anti-apoptotic while Bim is pro-apoptotic. Characterization of the auditory systems of mice deficient for these molecules revealed that Bcl2⁻/⁻ mice suffered severe hearing loss. This was conductive in nature and did not affect sensory cells of the inner ear, with cochlear hair cells and neurons present and functional. Bcl2⁻/⁻ mice were found to have a malformed, often monocrural, porous stapes (the small stirrup-shaped bone of the middle ear), but a normally shaped malleus and incus. The deformed stapes was discontinuous with the incus and sometimes fused to the temporal bones. The defect was completely rescued in Bcl2⁻/⁻Bim⁻/⁻ mice and partially rescued in Bcl2⁻/⁻Bim⁺/⁻ mice, which displayed high-frequency hearing loss and thickening of the stapes anterior crus. The Bcl2⁻/⁻ defect arose in utero before or during the cartilage stage of stapes development. These results implicate Bcl2 and Bim in regulating survival of second pharyngeal arch or neural crest cells that give rise to the stapes during embryonic development.

By applying a phase-retrieval step before carrying out standard filtered back-projection reconstructions in tomographic imaging, we were able to resolve structures with small differences in density within a densely absorbing sample. This phase-retrieval tomography is particularly suited for the three-dimensional segmentation of secondary osteons (roughly cylindrical structures) which are superimposed upon an existing cortical bone structure through the process of turnover known as remodelling. The resulting images make possible the analysis of the secondary osteon structure and the relationship between an osteon and the surrounding tissue. Our observations have revealed many different and complex 3D structures of osteons that could not be studied using previous methods. This work was carried out using a laboratory-based x-ray source, which makes obtaining these sorts of images readily accessible.

We introduce theoretically and demonstrate experimentally a contrast transfer function based phase retrieval algorithm that reconstructs the projected thickness of an homogeneous sample using a polychromatic x-ray source. We show excellent quantitative recovery of test samples in 2D using a synchrotron source with significant harmonic contamination, and in 3D using a laboratory source.

Certain phase retrieval methods use knowledge about the free space propagation of a wave to phase a paraxial beam passing through one or more measurement planes. This approach has been widely applied and has been shown to quantitatively retrieve the refractive index profile of a sample. The quality of the phase retrieval will depend on a range of factors including sample feature size, propagation distance, measurement plane separation, wavelength and noise. Here we describe an optimisation study for two-plane phase retrieval using a laboratory-based X-ray source that considers all of these factors. We discuss our results in the context of a three-dimensional reconstruction of a sample refractive index profile.

We report an experimental investigation of the optical transfer functions for an X-ray microscope operated in defocus phase-contrast mode. The results are compared with a theoretical model of partially coherent image formation and are found to be in excellent agreement.

We describe a quantitative phase imaging process using an x-ray laboratory-based source with an extremely broad bandwidth spectrum. The thickness of a homogeneous object can be retrieved by using separately spectrally weighted values for the attenuation coefficient and the decrement of the real part of the refractive index. This method is valid for a wide range of object types, including objects with an absorption edge in the used energy range. The accessibility of conventional x-ray laboratory sources makes this method very useful for quantitative phase retrieval of homogeneous objects. We demonstrate the application of this method for quantitative phase retrieval imaging in tomographic measurements.

Optical coherence theory is used to describe image formation in a telecentric optical system. By assuming a weakly interacting object and by considering points that are not too far from the optical axis, an optical transfer function description is obtained for imaging both the phase and the amplitude components of the object. A dimensionless coordinate system is identified to allow the transfer functions to be expressed independently of the details of the imaging system. Phase-contrast imaging is found to have an essentially coherent behaviour when the coherence length is a factor of 15 larger than the system resolution, and that the coherent region of the illumination therefore does not need to encompass the object.

We examine a direct filtered back projection approach that is suitable for the reconstruction of weakly absorbing homogeneous phase objects. Like recent similar approaches this method needs only one intensity image in each projection without the requirement for an intermediate step of phase retrieval. We tested the method using simulation and experimental results. Simulation results show good quantitative reconstruction which includes the correct refractive index value and distribution of the sample. However, experimental result still indicates the presence of artifacts.