A Randomized, Double-Blind, Placebo-Controlled, Phase 1 Study to Evaluate the Safety, Reactogenicity, and Immunogenicity of Single Vaccination of Ad26.RSV.preF-Based Regimen in Japanese Adults Aged 60 Years and Older.

BACKGROUND: Respiratory syncytial virus (RSV) is increasingly recognized as a significant cause of lower respiratory tract disease (LRTD) in older adults. The Ad26.RSV.preF/RSV preF protein vaccine demonstrated protective efficacy against RSV related LRTD in a Phase 2b study in the United States. Hence, Ad26.RSV.preF/RSV preF protein vaccine candidate was evaluated in the Japanese older adult population. METHODS: This Phase 1 study evaluated safety, reactogenicity, and immunogenicity of Ad26.RSV.preF/RSV preF protein vaccine at dose level of 1 × 1011 vp/150 µg in Japanese healthy adult aged ≥60 years. The study included a screening Phase, vaccination, 28-day follow up Phase, a 182-day follow-up period, and final visit on Day 183. A total of 36 participants were randomized in a 2:1 ratio to receive Ad26.RSV.preF/RSV preF protein vaccine (n = 24) or placebo (n = 12). After study intervention administration, the safety and immunogenicity analysis were performed as per planned schedule. Immune responses including virus-neutralizing and preF-specific binding antibodies were measured on Days 1, 15, 29, and 183. RESULTS: There were no deaths, SAEs, or AEs leading to discontinuation reported during the study. The Ad26.RSV.preF/RSV preF protein vaccine had acceptable safety and tolerability profile with no safety concern in Japanese older adults. The Ad26.RSV.preF/RSV preF protein vaccine induced RSV-specific humoral immunity, with increase in antibody titers on Days 15 and 29 compared with baseline which was well maintained until Day 183. CONCLUSIONS: A single dose of Ad26.RSV.preF/RSV preF protein vaccine had an acceptable safety and tolerability profile and induced RSV-specific humoral immunity in Japanese healthy adults. TRIAL REGISTRATION: NCT number: NCT04354480; Clinical Registry number: CR108768.

Neutron phase imaging by a Talbot-Lau interferometer at Kyoto University Reactor.

We have developed a new neutron phase imaging system with a Talbot-Lau interferometer for utilization at the CN-3 port of the Kyoto University Reactor. To achieve efficient differential-phase imaging and visibility (dark-field) imaging at this beamline, we adopted a relatively shorter design wavelength of 2.7 Å. By fabricating neutron absorption gratings with thick gadolinium absorbers, we were able to obtain clear moiré fringes with a high visibility of 55% for thermal neutrons. As a demonstration of its imaging capabilities for expanded actual utilization in the medium-sized sources, we observed additively manufactured rods of Inconel 718. Using visibility imaging, we successfully examined variations in the size of defects in the rods caused by hot isostatic pressing process. In addition, we conducted tomography measurements of the rods, which allowed us to reveal the spatial distribution of defects at sub-micrometer scales.

Parabolic gratings enhance the X-ray sensitivity of Talbot interferograms.

In grating-based X-ray Talbot interferometry, the wave nature of X-ray radiation is exploited to generate phase contrast images of objects that do not generate sufficient contrast in conventional X-ray imaging relying on X-ray absorption. The phase sensitivity of this interferometric technique is proportional to the interferometer length and inversely proportional to the period of gratings. However, the limited spatial coherency of X-rays limits the maximum interferometer length, and the ability to obtain smaller-period gratings is limited by the manufacturing process. Here, we propose a new optical configuration that employs a combination of a converging parabolic micro-lens array and a diverging micro-lens array, instead of a binary phase grating. Without changing the grating period or the interferometer length, the phase signal is enhanced because the beam deflection by a sample is amplified through the array of converging-diverging micro-lens pairs. We demonstrate that the differential phase signal detected by our proposed set-up is twice that of a Talbot interferometer, using the same binary absorption grating, and with the same overall inter-grating distance.

Distribution of extraintestinal pathogenic Escherichia coli O-serotypes and antibiotic resistance in blood isolates collected from patients in a surveillance study in Japan.

INTRODUCTION: Invasive extraintestinal pathogenic Escherichia coli (ExPEC) disease (IED), characterised by sepsis and bacteraemia, is a major global healthcare concern worsened by emerging multidrug resistant (MDR) strains. The development of multivalent prophylactic vaccines targeting E. coli strains of IED-associated O-serotypes could address this. A better understanding of O-serotype distribution is required for this purpose. Here, we characterised O-serotype prevalence and drug resistance among ExPEC bacteraemia isolates in Japan. METHODS: E. coli blood isolates from patients aged ≥60 years with bacteraemia were obtained from a retrospective surveillance study in Japan (2015-2017). O-serotyping was performed by agglutination; for isolates non-typeable by agglutination, O-genotyping was performed. Antimicrobial susceptibility was evaluated by broth microdilution using a 21-antibiotic panel. The frequency of drug resistant (DR) isolates was evaluated by antimicrobial susceptibility testing. RESULTS: Of 401 ExPEC bacteraemia isolates evaluated, the most prevalent O-serotype (≥1%) was O25 (28.7% [n = 115]), followed by O1 (14.2% [n = 57]), O2 (8.5% n = 34]), O6 (5.5% [n = 22]), O75, O18, O13, O16, O15, O4, O46/O134, O86, O8 and O83 (each <5% prevalence). These 14 O-serotypes accounted for 81.5% of isolates collected. In total, 19% (n = 77) of isolates were DR ≥ 3, of which 59.7% were O25. Fluoroquinolone-resistance among all and O25 isolates was most prevalent (35.7% and 84.3%, respectively). Almost all (98%) isolates identified as O25 were of subtype O25B. CONCLUSIONS: E. coli serotype O25B showed the highest prevalence and highest multidrug resistance among ExPEC bacteraemia isolates from patients ≥60 years in Japan. Our data may inform development of multivalent glycoconjugate vaccines to prevent IED.

Bilaterally Asymmetric Helical Myofibrils in Ascidian Tadpole Larvae.

The locomotor system is highly bilateral at the macroscopic level. Homochirality of biological molecules is fully compatible with the bilateral body. However, whether and how single-handed cells contribute to the bilateral locomotor system is obscure. Here, exploiting the small number of cells in the swimming tadpole larva of the ascidian Ciona, we analyzed morphology of the tail at cellular and subcellular scales. Quantitative phase-contrast X-ray tomographic microscopy revealed a high-density midline structure ventral to the notochord in the tail. Muscle cell nuclei on each side of the notochord were roughly bilaterally aligned. However, fluorescence microscopy detected left-right asymmetry of myofibril inclination relative to the longitudinal axis of the tail. Zernike phase-contrast X-ray tomographic microscopy revealed the presence of left-handed helices of myofibrils in muscle cells on both sides. Therefore, the locomotor system of ascidian larvae harbors symmetry-breaking left-handed helical cells, while maintaining bilaterally symmetrical cell alignment. These results suggest that bilateral animals can override cellular homochirality to generate the bilateral locomotor systems at the supracellular scale.

Time-resolved x-ray stroboscopic phase tomography using Talbot interferometer for dynamic deformation measurements.

Time-resolved x-ray phase tomography using a Talbot interferometer and white synchrotron radiation can provide a three-dimensional movie for visualizing the structural change of materials consisting of light elements. In this study, time-resolved x-ray stroboscopic phase tomography using a Talbot interferometer is demonstrated for a vibrating object under 24 Hz compression-stretch fatigue loading. Moiré patterns are recorded by synchronizing drivers for a shutter, grating displacement, and sample rotation with an x-ray camera with a 200 µs exposure, and phase tomograms are reconstructed at specific motion phases of the vibration. The measurement lasts for a few minutes and the δ value changes before breaking, which is considered due to plastic deformation of soft materials under external vibration are depicted three-dimensionally.

Staircase array of inclined refractive multi-lenses for large field of view pixel super-resolution scanning transmission hard X-ray microscopy.

Owing to the development of X-ray focusing optics during the past decades, synchrotron-based X-ray microscopy techniques allow the study of specimens with unprecedented spatial resolution, down to 10 nm, using soft and medium X-ray photon energies, though at the expense of the field of view (FOV). One of the approaches to increase the FOV to square millimetres is raster-scanning of the specimen using a single nanoprobe; however, this results in a long data acquisition time. This work employs an array of inclined biconcave parabolic refractive multi-lenses (RMLs), fabricated by deep X-ray lithography and electroplating to generate a large number of long X-ray foci. Since the FOV is limited by the pattern height if a single RML is used by impinging X-rays parallel to the substrate, many RMLs at regular intervals in the orthogonal direction were fabricated by tilted exposure. By inclining the substrate correspondingly to the tilted exposure, 378000 X-ray line foci were generated with a length in the centimetre range and constant intervals in the sub-micrometre range. The capability of this new X-ray focusing device was first confirmed using ray-tracing simulations and then using synchrotron radiation at BL20B2 of SPring-8, Japan. Taking account of the fact that the refractive lens is effective for focusing high-energy X-rays, the experiment was performed with 35 keV X-rays. Next, by scanning a specimen through the line foci, this device was used to perform large FOV pixel super-resolution scanning transmission hard X-ray microscopy (PSR-STHXM) with a 780 ± 40 nm spatial resolution within an FOV of 1.64 cm × 1.64 cm (limited by the detector area) and a total scanning time of 4 min. Biomedical implant abutments fabricated via selective laser melting using Ti-6Al-4V medical alloy were measured by PSR-STHXM, suggesting its unique potential for studying extended and thick specimens. Although the super-resolution function was realized in one dimension in this study, it can be expanded to two dimensions by aligning a pair of presented devices orthogonally.

Hypermineralization of Hearing-Related Bones by a Specific Osteoblast Subtype.

Auditory ossicles in the middle ear and bony labyrinth of the inner ear are highly mineralized in adult mammals. Cellular mechanisms underlying formation of dense bone during development are unknown. Here, we found that osteoblast-like cells synthesizing highly mineralized hearing-related bones produce both type I and type II collagens as the bone matrix, while conventional osteoblasts and chondrocytes primarily produce type I and type II collagens, respectively. Furthermore, these osteoblast-like cells were not labeled in a "conventional osteoblast"-specific green fluorescent protein (GFP) mouse line. Type II collagen-producing osteoblast-like cells were not chondrocytes as they express osteocalcin, localize along alizarin-labeled osteoid, and form osteocyte lacunae and canaliculi, as do conventional osteoblasts. Auditory ossicles and the bony labyrinth exhibit not only higher bone matrix mineralization but also a higher degree of apatite orientation than do long bones. Therefore, we conclude that these type II collagen-producing hypermineralizing osteoblasts (termed here auditory osteoblasts) represent a new osteoblast subtype. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Development of X-ray phase CT with a hybrid configuration of Lau and Talbot-Lau interferometers.

X-ray phase computed tomography (CT) is used to observe the inside of light materials. In this paper, we report a new study to develop and test a laboratory assembled X-ray phase CT system that comprises an X-ray Lau interferometer, a rotating Mo anode X-ray tube, and a detector with high spatial resolution. The system has a high spatial resolution lower than 10 µm, which is evaluated by differentiating neighbouring carbon fibres in a polymer composite material. The density resolution is approximately 0.035 g/cm3, which enables to successfully distinguish the high-density polyethylene (HDPE, 0.93 g/cm3) from the ultra-low-density polyethylene (ULDPE, 0.88 g/cm3) in the sample. Moreover, the system can be switched to operate on another mode based on a Talbot-Lau interferometer that provides a wider field of view with a moderate spatial resolution (approximately 100 µm). By analyzing sample images of the biological, this study demonstrates the feasibility and advantages of using hybrid configuration of this X-ray phase CT system.

X-ray phase imaging reaching clinical uses.

X-ray phase imaging that uses the phenomena of X-ray refraction and scattering to generate image contrast has the potential to overcome the drawback of conventional X-ray radiography in observing biological soft tissues. After its dawn at synchrotron radiation facilities 30 years ago, the development of X-ray phase imaging is expanding to hospitals by grating-based phase-imaging approaches available with a conventional X-ray tube. In this review, after introducing the physical advantages and methodological details of X-ray phase imaging, recent trials of instrumentation in hospitals for diagnoses of rheumatoid arthritis and chronic obstructive pulmonary disease are introduced.

Enhancement of low-spatial-frequency components by a new phase-contrast STEM using a probe formed with an amplitude Fresnel zone plate.

Electron microscopy is a powerful tool for visualizing the shapes of sub-nanometer objects. However, Contrast Transfer Function (CTF) principally restricts lower frequency components in the image. To overcome this problem, phase-plate techniques have been proposed and currently Hole Free Phase Plate (HFPP) and Volta Phase Plate (VPP) are widely used especially for biological specimens to retrieve low frequency information of the sample's potential distributions. In this report, we have developed a new phase-contrast scanning transmission electron microscope (STEM) in which a probe beam including side robes is formed with an amplitude Fresnel zone plate (FZP) and the interference patterns produced by the zero and first order diffracted waves generated by the FZP are detected. We name it FZP Phase Contrast STEM (FZP-PC-STEM) hereinafter. The amplitude FZP was manufactured by using focused ion beam (FIB) equipment, and the diffraction data were collected by using diffraction imaging technique. The validity of our proposed optical model was confirmed by comparing experimental and simulated images. Observations of carbon nanotube (CNT) bundles by using this method showed that the contrast of low-spatial-frequency components in the CNT image was significantly enhanced. This method does not, in principle, require the post-image processing used in the diffraction imaging method, and it can be easily introduced into pre-existing equipment without major modifications. The stability and robustness of the FZP inserted in condenser system were also confirmed during long-time operation. We expect that the FZP-PC-STEM will be widely applicable to high-contrast observations of low-Z samples with simple and easy operation.

Imaging evaluation of the cartilage in rheumatoid arthritis patients with an x-ray phase imaging apparatus based on Talbot-Lau interferometry.

X-ray Talbot-Lau interferometry is one of the x-ray phase imaging methods that has high sensitivity in depicting soft tissues. Unlike earlier x-ray phase imaging methods that required particular types of x-ray sources, such as a synchrotron or a micro-focus x-ray tube, x-ray Talbot-Lau interferometry enables to perform clinical x-ray phase imaging using a conventional x-ray source with a relatively compact configuration. We developed an apparatus to depict cartilage in the metacarpophalangeal joints of the hands. In addition, we examined the apparatus performance by applying it to healthy volunteers and patients with rheumatoid arthritis (RA). Cartilage deformation, which is thought to be a precursor of destruction of the joints, was successfully depicted by the apparatus, suggesting a potential early diagnosis of RA.

Development of pink-beam 4D phase CT for in-situ observation of polymers under infrared laser irradiation.

Four-dimensional phase computed tomography (4D phase CT) by an X-ray Talbot interferometer (XTI) with white synchrotron radiation has ever been demonstrated at a temporal resolution of about 1 s for soft-matter samples. However, the radiation damage to samples caused by white synchrotron radiation occasionally hampers our understanding of the sample dynamical properties. Based on the fact that XTI functions with X-rays of a bandwidth up to ca. 10% with performance comparable to that by monochromatic X-rays, filtering white synchrotron radiation to generate a 'pink-beam' of a 10% bandwidth is effective to reduce radiation damage without degrading the image quality and temporal resolution. We have therefore developed pink-beam 4D phase CT at SPring-8, Japan by installing a multilayer mirror with a 10% bandwidth and a 25 keV central photon energy. XTI optimal at this photon energy was built downstream, and a CMOS-based X-ray detector was used to achieve fast image acquisitions with an exposure time of 1 ms (or 0.5 ms) per moiré image. The resultant temporal resolution of pink-beam 4D phase CT was 2 s (1 s). We applied the pink-beam 4D phase CT to in-situ observation of polypropylene, poly(methyl methacrylate), and polycarbonate under infrared laser irradiation (1064 nm). The dynamics of melting, bubbling, and ashing were successfully visualized in 3D movies without problematic radiation damage by synchrotron radiation.

Preliminary research on body composition measurement using X-ray phase contrast imaging.

Body composition measurement is of cardinal significance for medical and clinical applications. Currently, the dual-energy X-ray absorptiometry (DEXA) technique is widely applied for this measurement. In this study, we present a novel measurement method using the absorption and phase information obtained simultaneously from the X-ray grating-based interferometer (XGI). Rather than requiring two projection data sets with different X-ray energy spectra, with the proposed method, both the areal densities of the bone and the surrounding soft tissue can be acquired utilizing one projection data set. By using a human body phantom constructed to validate the proposed method, experimental results have shown that the compositions can be calculated with an improved accuracy comparing to the dual energy method, especially for the soft tissue measurement. Since the proposed method can be easily implemented on current XGI setup, it will greatly extend the applications of the XGI, and meanwhile has the potential to be an alternative to DEXA for human body composition measurement.

Laboratory-based X-ray phase-imaging scanner using Talbot-Lau interferometer for non-destructive testing.

An X-ray Talbot-Lau interferometer scanning setup consisting of three transmission gratings, a laboratory-based X-ray source that emits X-rays vertically, and an image detector on the top has been developed for the application of X-ray phase imaging to moving objects that cannot be tested clearly with conventional absorption contrast. The grating-based X-ray phase imaging method usually employs a phase-stepping (or fringe-scanning) technique by displacing one of the gratings step-by-step while the object stays still. Since this approach is not compatible with a scanner-type application for moving objects, we have developed a new algorithm for achieving the function of phase-stepping without grating displacement. By analyzing the movie of the moiré pattern as the object moves across the field of view, we obtain the absorption, differential phase, and visibility images. The feasibility of the X-ray phase imaging scanner has been successfully demonstrated for a long sample moving at 5 mm/s. This achievement is a breakthrough for the practical industrial application of X-ray phase imaging for screening objects carried on belt-conveyers such as those in factories.

Development toward high-resolution X-ray phase imaging.

Since the 1990s, the use of X-ray phase contrast has been extensively studied for imaging weakly absorbing objects consisting of low-Z elements such as biological soft tissues and polymers. The development of X-ray microscopy was also progressing during this time, although absorption contrast was only available. It was straightforward and important to develop phase-contrast X-ray microscopy. One characteristic in the development is that quantitative phase measurement is possible through the acquisition of phase-contrast images under a specific procedure, thanks to digital X-ray image detectors. Therefore, such a technique is called 'phase imaging' rather than phase-contrast imaging in this review. Highly sensitive three-dimensional phase imaging is feasible in combination with tomography. This article reviews the progress in X-ray phase imaging, especially with regards to X-ray microscopy.

Osteocyte-directed bone demineralization along canaliculi.

The mammalian skeleton stores calcium and phosphate ions in bone matrix. Osteocytes in osteocyte lacunae extend numerous dendrites into canaliculi less than a micron in diameter and which are distributed throughout bone matrix. Although osteoclasts are the primary bone-resorbing cells, osteocytes also reportedly dissolve hydroxyapatite at peri-lacunar bone matrix. However, robust three-dimensional evidence for peri-canalicular bone mineral dissolution has been lacking. Here we applied a previously reported Talbot-defocus multiscan tomography method for synchrotron X-ray microscopy and analyzed the degree of bone mineralization in mouse cortical bone around the lacuno-canalicular network, which is connected both to blood vessels and the peri- and endosteum. We detected cylindrical low mineral density regions spreading around canaliculi derived from a subset of osteocytes. Transmission electron microscopy revealed both intact and demineralized bone matrix around the canaliculus. Peri-canalicular low mineral density regions were also observed in osteopetrotic mice lacking osteoclasts, indicating that osteoclasts are dispensable for peri-canalicular demineralization. These data suggest demineralization can occur from within bone through the canalicular system, and that peri-canalicular demineralization occurs not uniformly but directed by individual osteocytes. Blockade of peri-canalicular demineralization may be a therapeutic strategy to increase bone mass and quality.

Osteogenic capillaries orchestrate growth plate-independent ossification of the malleus.

Endochondral ossification is a developmental process by which cartilage is replaced by bone. Terminally differentiated hypertrophic chondrocytes are calcified, vascularized, and removed by chondroclasts before bone matrix is laid down by osteoblasts. In mammals, the malleus is one of three auditory ossicles that transmit vibrations of the tympanic membrane to the inner ear. The malleus is formed from a cartilaginous precursor without growth plate involvement, but little is known about how bones of this type undergo endochondral ossification. Here, we demonstrate that in the processus brevis of the malleus, clusters of osteoblasts surrounding the capillary loop produce bone matrix, causing the volume of the capillary lumen to decrease rapidly in post-weaning mice. Synchrotron X-ray tomographic microscopy revealed a concentric, cylindrical arrangement of osteocyte lacunae along capillaries, indicative of pericapillary bone formation. Moreover, we report that overexpression of Fosl1, which encodes a component of the AP-1 transcription factor complex, in osteoblasts significantly blocked malleal capillary narrowing. These data suggest that osteoblast/endothelial cell interactions control growth plate-free endochondral ossification through 'osteogenic capillaries' in a Fosl1-regulated manner.

Effect of beam hardening on a visibility-contrast image obtained by X-ray grating interferometry.

X-ray grating interferometry has been highlighted in the last decade as a multi-modal X-ray phase-imaging technique for providing absorption, differential phase, and visibility-contrast images. It has been mainly reported that the visibility contrast in the visibility-contrast image originates from unresolvable random microstructures. In this paper, we show that the visibility contrast is even reduced by a uniform sample with flat surfaces due to the so-called "beam-hardening effect", which has to be taken into account when X-rays with a continuous spectrum is used. We drive a criterion for determining whether the beam-hardening effect occurs or not, and propose a method for correcting the effect of beam hardening on a visibility-contrast image.

Effects of unresolvable edges in grating-based X-ray differential phase imaging.

We investigated effects of unresolvable sharp edges on images obtained in a grating-based X-ray differential phase imaging technique. Results of numerical calculations for monochromatic X-rays show that an unresolvable sharp edge generates not only differential-phase contrast but also visibility contrast. The latter shows that the visibility contrast has another major origin other than ultra-small-angle X-ray scattering (USAXS) from randomly distributed unresolvable microstructures, which has been considered the main origin for the contrast. The effects were experimentally confirmed using a synchrotron X-ray source.