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.

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.

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.

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.

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.

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.

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.

Expression and distribution of acyl-CoA thioesterases in the white adipose tissue of rats.

Acyl-CoA thioesterases (Acots) are enzymes that catalyze the hydrolysis of fatty acyl-CoAs to free fatty acids and coenzyme A, and have the potential to regulate the intracellular levels of these molecules. In this study, we show that a cytosolic isoform, Acot1, is expressed and distributed in immature adipocytes located in the perivascular region of the white adipose tissue (WAT) of rats. Immunoblot analyses detected Acot1 in all of the WATs examined, while immunohistochemistry revealed positively stained layered structures surrounding the adventitia of blood vessels in the subcutaneous WAT. When the subcutaneous WAT was digested with collagenase and centrifuged, Acot1 was recovered in the stromal vascular fraction (SVF), and not in the large mature adipocytes. In the SVF, undigested cells attached to short tubular fragments of blood vessels showed positive immunostaining, as well as a proportion of the dispersed cells. These fibroblast-like cells contained fine particulate lipid droplets, stained by oil-red O dye, in their cytoplasm, or expressed fatty acid-binding protein 4, an adipocyte marker. After induction of adipocyte differentiation following a 15-day preculture without insulin, the dedifferentiated cells showed increased Acot1 expression with a diffuse distribution throughout the cytosol. These findings suggest that Acot1 expression is transiently upregulated at an early stage of adipocyte maturation, possibly to maintain cytosolic acyl-CoAs below a certain level until the cells acquire their full capability for fat storage.

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.

In vivo physiological saline-infused hepatic vessel imaging using a two-crystal-interferometer-based phase-contrast X-ray technique.

Using a two-crystal-interferometer-based phase-contrast X-ray imaging system, the portal vein, capillary vessel area and hepatic vein of live rats were revealed sequentially by injecting physiological saline via the portal vein. Vessels greater than 0.06 mm in diameter were clearly shown with low levels of X-rays (552 µGy). This suggests that in vivo vessel imaging of small animals can be performed as conventional angiography without the side effects of the presently used iodine contrast agents.

Impaired vibration of auditory ossicles in osteopetrotic mice.

In the middle ear, a chain of three tiny bones (ie, malleus, incus, and stapes) vibrates to transmit sound from the tympanic membrane to the inner ear. Little is known about whether and how bone-resorbing osteoclasts play a role in the vibration of auditory ossicles. We analyzed hearing function and morphological features of auditory ossicles in osteopetrotic mice, which lack osteoclasts because of the deficiency of either cytokine RANKL or transcription factor c-Fos. The auditory brainstem response showed that mice of both genotypes experienced hearing loss, and laser Doppler vibrometry revealed that the malleus behind the tympanic membrane failed to vibrate. Histological analysis and X-ray tomographic microscopy using synchrotron radiation showed that auditory ossicles in osteopetrotic mice were thicker and more cartilaginous than those in control mice. Most interestingly, the malleal processus brevis touched the medial wall of the tympanic cavity in osteopetrotic mice, which was also the case for c-Src kinase-deficient mice (with normal numbers of nonresorbing osteoclasts). Osteopetrotic mice showed a smaller volume of the tympanic cavity but had larger auditory ossicles compared with controls. These data suggest that osteoclastic bone resorption is required for thinning of auditory ossicles and enlargement of the tympanic cavity so that auditory ossicles vibrate freely.

Wavefront measurement for a hard-X-ray nanobeam using single-grating interferometry.

Wavefront measurement for a hard-X-ray nanobeam using single-grating interferometry based on the Talbot effect and the Fourier transform method was demonstrated in the 1-km-long beamline of SPring-8. 10 keV X-rays were one-dimensionally focused down to 32 nm using a total-reflection elliptical mirror. An intentionally distorted wavefront was generated using a deformable mirror placed just upstream of the focusing mirror. The wavefront measured by interferometry was cross-checked with the phase retrieval method using intensity profiles around the beam waist. Comparison of the obtained wavefront errors revealed that they are in good agreement with each other and with the wavefront error estimated from the shape of the deformable mirror at a ~0.5 rad level.

Fourier transform-based iterative method for differential phase-contrast computed tomography.

In this Letter, a novel Fourier transform-based iterative method is proposed to reconstruct a refractive index distribution in an object directly from measured differential phase shifts. This reconstruction method offers distinct advantages over the analytic method in cases of noisy and/or few-view data, since the iterative scheme can easily incorporate compressive sensing and maximum likelihood techniques to achieve high image quality. Numerical simulation and biological sample experiments demonstrate the merits of the proposed approach.

Regulated expression of acyl-CoA thioesterases in the differentiation of cultured rat brown adipocytes.

Acyl-CoA thioesterases (ACOTs) are enzymes that catalyze the hydrolysis of fatty acyl-CoAs to free fatty acids and CoA-SH. In this study, we show that the expression profile of the ACOT isoforms changes remarkably during the differentiation of cultured rat brown adipocytes. Immunocytochemistry suggested that cytosolic ACOT1 was present in the preadipocytes, while mitochondrial ACOT2 was additionally expressed as the cells differentiated, concurrent with the accumulation of lipid droplets in the cytoplasm. Western blotting confirmed that, in contrast to ACOT1, the ACOT2 expression level was very low in the preadipocytes. However, after differentiation, the ACOT1 level fell to one-half of the baseline level and ACOT2 increased 18-fold. ACOT2 expression in the differentiated adipocytes was further enhanced by treatment with lipids or troglitazone. These changes in the ACOT2 expression level correlated well with changes in the expression of carnitine palmitoyltransferase 2, a mitochondrial ß-oxidation enzyme. These results indicate that, in differentiating brown adipocytes, cytosolic ACOT1 becomes downregulated as the cellular use of acyl-CoA increases, while mitochondrial ACOT2 is upregulated as the ß-oxidation capacity increases. ACOT isoform expression may be regulated during brown adipocyte differentiation to support the fat storage and combustion characteristics of this cell type.

Neuronal expression, cytosolic localization, and developmental regulation of the organic solute carrier partner 1 in the mouse brain.

Organic solute carrier partner 1 (OSCP1) is a mammalian, transporter-related protein that is able to facilitate the uptake of structurally diverse organic compounds into the cell when expressed in Xenopus laevis oocytes. This protein has been implicated in testicular handling of organic solutes because its mRNA expression is almost exclusive in the testis. However, in this study, we demonstrated significant expression of OSCP1 protein in mouse brain, the level of which was rather higher than that in the testis, although the corresponding mRNA expression was one-tenth of the testicular level. Immunohistochemistry revealed that OSCP1 was broadly distributed throughout the brain, and various neuronal cells were immunostained, including pyramidal cells in the cerebral cortex and hippocampus. However, there was no evidence of OSCP1 expression in glia. In primary cultures of cerebral cortical neurons, double-labeling immunofluorescence localized OSCP1 to the cytosol throughout the cell body and neurites including peri-synaptic regions. This was consistent with the subcellular fractionation of brain homogenates, in which OSCP1 was mainly recovered after centrifugation both in the cytosolic fraction and the particulate fraction containing synaptosomes. Immunoelectron microscopy of brain sections also demonstrated OSCP1 in the cytosol near synapses. In addition, it was revealed that changes in the expression level of OSCP1 correlated with neuronal maturation during postnatal development of mouse brain. These results indicate that OSCP1 may have a role in the brain indirectly mediating substrate uptake into the neurons in adult animals.

Four-dimensional X-ray phase tomography with Talbot interferometry and white synchrotron radiation: dynamic observation of a living worm.

X-ray Talbot interferometry is attractive as a method for X-ray phase imaging and phase tomography for objects that weakly absorb X-rays. Because X-ray Talbot interferometry has the advantage that X-rays of a broad energy bandwidth can be used, high-speed X-ray phase imaging is possible with white synchrotron radiation. In this paper, we demonstrate time-resolved three-dimensional observation with X-ray Talbot interferometry (namely, four-dimensional X-ray phase tomography). Differential phase images, from which a phase tomogram was reconstructed, were obtained through the Fourier-transform method, unlike the phase-stepping method that requires several (at least three) moiré images to be measured sequentially in order to generate one differential phase image. We demonstrate dynamic observation of a living worm in three dimensions with a time resolution of 0.5 s, visualizing a drastic change in the respiratory tract.

Iterative reconstruction in x-ray computed laminography from differential phase measurements.

Phase-contrast X-ray computed laminography is demonstrated for the volume reconstruction of extended flat objects, not suitable to the usual tomographic scan. Using a Talbot interferometer, differential phase measurements are obtained and used to reconstruct the real part of the complex refractive index. The specific geometry of laminography leads to unsampled frequencies in a double cone in the reciprocal space, which degrades the spatial resolution in the direction normal to the object plane. First, the filtered backprojection formula from differential measurements is derived. Then, reconstruction is improved by the use of prior information of compact support and limited range, included in an iterative filtered backprojection algorithm. An implementation on GPU hardware was required to handle the reconstruction of volumes within a reasonable time. A synchrotron radiation experiment on polymer meshes is reported and results of the iterative reconstruction are compared with the simpler filtered backprojection.

Upregulation of fatty acyl-CoA thioesterases in the heart and skeletal muscle of rats fed a high-fat diet.

In rodent models of diet-induced obesity, prolonged high-fat feeding increases the cellular uptake of fatty acids and causes lipotoxicity in the heart and skeletal muscle, where substrate overload to beta-oxidation generates mitochondrial stress. We examined the hypothesis that, because of its catalytic properties, acyl-CoA thioesterase (ACOT) would counteract these detrimental situations by modulating intracellular acyl-CoA levels. Rats were fed a low- or high-fat diet for up to 20 weeks, and the expressions of ACOT isoforms and fatty acid beta-oxidation enzymes were analyzed by western blotting. The expressions of ACOT1, ACOT2 and ACOT7 proteins in the heart and soleus muscle were significantly increased, by 2.0-7.6-fold, in rats fed the high-fat diet as compared with the low-fat diet group. These effects were accompanied by increases in carnitine palmitoyltransferase and acyl-CoA oxidase expression. However, ACOT was not induced in the extensor digitorum longus muscle or the liver. Subcellular fractionation of heart and soleus muscle homogenates confirmed expression of both the cytosolic and mitochondrial ACOT isoforms. These results underscore the functional relationship between ACOT and fatty acid oxidation, and suggest adaptive upregulation of ACOT to protect against fatty acid oversupply in the heart and skeletal muscle.

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.