Search

Heterogeneous clusters of cancer cells and leukocytes in blood were visualized by combining high-throughput and high-sensitivity fluorescence imaging flow cytometry with 5-aminolevulinic acid stimulation.

To establish systematic guidelines for accelerator decommissioning, a large-scale activation survey was conducted at representative synchrotron radiation facilities in Japan. The neutron flux during accelerator operation was measured with various dosimeters. A Monte Carlo simulation was conducted for some facilities to verify the special neutron distribution and their spectra. Beam loss points, reflected as high-dose-rate areas, were identified by a whole-beamline survey with a survey meter, and the generated radionuclides and their activity were determined with a lanthanum bromide (LaBr3) scintillation spectrometer. In all facilities, the activation level was quite low. Whole-beamline tunnels made of concrete were not activated, and no radionuclides were detected except for natural nuclides. In addition, almost all beamline components were either not or minimally activated. Although the acceleration energy is very high for radiation synchrotron facilities, the generation of radioactive waste would be very low.

Typically, the concentrations of cobalt and europium, which are trace elements in concrete, are difficult to determine by chemical analysis. While data sheets do not usually contain this information, it is important for the decommissioning of accelerators, especially for the decontamination of activated concrete. To understand the variations in trace element concentrations in concrete, we analyzed cobalt and europium concentrations in concrete samples from accelerator facilities in Japan by neutron activation analysis, which is suitable for trace element analysis on the order of parts per million (ppm).

O-GlcNAcylation is the only sugar modification for proteins present in the cytoplasm and nucleus and is thought to be involved in the regulation of protein function and localization. Currently, several methods are known for detecting O-GlcNAcylated proteins using monoclonal antibodies or wheat germ agglutinin, but these methods have some limitations in their sensitivity and quantitative comparison. We developed a new disaccharide-tag method to overcome these problems. This is a method in which a soluble GalNAc transferase is expressed intracellularly, extended to a disaccharide of GalNAc-GlcNAc, and detected using a Wisteria japonica agglutinin specific to this disaccharide. We verified the method using human c-Rel protein and also highly sensitively compared the difference in O-GlcNAc modification of intracellular proteins associated with differentiation from embryonic stem cell (ESC) to epiblast-like cells (EpiLC). As one example of such a modification, a novel O-GlcNAc modification was found in the transcription factor Sox2 at residue Ser263, and the modification site could be identified by nano liquid chromatography-mass spectrometry.

Summary: Mouse embryonic stem cell (ESC) pluripotency is tightly regulated by a complex network composed of extrinsic and intrinsic factors that allow proper organismal development. O-linked β-N-acetylglucosamine (O-GlcNAc) is the sole glycosylation mark found on cytoplasmic and nuclear proteins and plays a pivotal role in regulating fundamental cellular processes; however, its function in ESC pluripotency is still largely unexplored. Here, we identify O-GlcNAcylation of proteasome activator subunit 3 (Psme3) protein as a node of the ESC pluripotency network. Mechanistically, O-GlcNAc modification of serine 111 (S111) of Psme3 promotes degradation of Ddx6, which is essential for processing body (P-body) assembly, resulting in the maintenance of ESC pluripotent state. Conversely, loss of Psme3 S111 O-GlcNAcylation stabilizes Ddx6 and increases P-body levels, culminating in spontaneous exit of ESC from the pluripotent state. Our findings establish O-GlcNAcylation at S111 of Psme3 as a switch that regulates ESC pluripotency via control of P-body homeostasis.

The role of valence electrons for the muon capture process by molecules is experimentally investigated with the aid of cascade calculations. Low-momentum muons are introduced to gas targets of CO, CO2, and COS below atmospheric pressure. The initial states of captured muons are determined from the measured muonic X-ray structure of the Lyman and Balmer series. We propose that the lone pair electrons in the carbon atom of CO significantly contribute to the capture of a muon with large angular momenta.

Per-atom muon capture ratios are precisely determined from characteristic muonic X-ray measurements of three nitrogen oxide samples (NO, N2O, and NO2) at pressures below 100 kPa. A higher density of covalent electrons at the N atoms relative to the O atoms is required if we explain the experimental results using the proposed muon capture models. From molecular orbit calculations, we found that the electron distributions of only the highest occupied molecular orbitals (HOMO) are dominant at the N atoms, and the electrons in the HOMO significantly contribute to the muon capture phenomena in the nitrogen oxide molecules.

Many growth factors, such as fibroblast growth factors (FGFs), are useful for the treatment or prevention of radiation damage after radiation therapy. Although heparin can be supplemented to increase the therapeutic effects of FGFs, it possesses strong anticoagulant effects, which limit its potential for clinical use. Therefore, chemically sulfated hyaluronic acid (HA) was developed as a safe alternative to heparin. This study examined the involvement of sulfated HA in radioprotective and anticoagulant effects.FGF1 was administered intraperitoneally to BALB/c mice with sulfated HA 24 hours before or after total body irradiation with γ-rays. Several radioprotective effects were examined in the jejunum. The blood coagulation time in the presence of sulfated HA was measured using murine whole blood.FGF1 with high-sulfated HA (HA-HS) exhibited almost the same level of in vitro mitogenic activity as heparin, whereas FGF1 with HA or low-sulfated HA exhibited almost no mitogenic activity. Furthermore, HA-HS had high binding capability with FGF1. FGF1 with HA-HS significantly promoted crypt survival to the same level as heparin after total body irradiation and reduced radiation-induced apoptosis in crypt cells. Moreover, pretreatment of HA-HS without FGF1 also increased crypt survival and reduced apoptosis. Crypt survival with FGF1 in the presence of HA depended on the extent of sulfation of HA. Moreover, the blood anticoagulant effects of sulfated HA were weaker than those of heparin. As sulfated HA did not promote the reactivity of antithrombin III to thrombin, it did not increase anticoagulative effects to the same extent as heparin.This study suggested that HA-HS promotes the radioprotective effects of FGF1 without anticoagulant effects. HA-HS has great potential for practical use to promote tissue regeneration after radiation damage.

By virtue of the combined merits of flow cytometry and fluorescence microscopy, imaging flow cytometry (IFC) has become an established tool for cell analysis in diverse biomedical fields such as cancer biology, microbiology, immunology, hematology, and stem cell biology. However, the performance and utility of IFC are severely limited by the fundamental trade-off between throughput, sensitivity, and spatial resolution. Here we present an optomechanical imaging method that overcomes the trade-off by virtually freezing the motion of flowing cells on the image sensor to effectively achieve 1000 times longer exposure time for microscopy-grade fluorescence image acquisition. Consequently, it enables high-throughput IFC of single cells at >10,000 cells s−1 without sacrificing sensitivity and spatial resolution. The availability of numerous information-rich fluorescence cell images allows high-dimensional statistical analysis and accurate classification with deep learning, as evidenced by our demonstration of unique applications in hematology and microbiology., High throughput imaging flow cytometry suffers from trade-offs between throughput, sensitivity and spatial resolution. Here the authors introduce a method to virtually freeze cells in the image acquisition window to enable 1000 times longer signal integration time and improve signal-to-noise ratio.

Summary Mouse embryonic stem cells (ESCs) differentiate into multiple cell types during organismal development. Fibroblast growth factor 4 (FGF4) signaling induces differentiation from ESCs via the phosphorylation of downstream molecules such as mitogen-activated protein kinase/extracellular signal-related kinase (MEK) and extracellular signal-related kinase 1/2 (ERK1/2). The FGF4-MEK-ERK1/2 pathway is inhibited to maintain ESCs in the undifferentiated state. However, the inhibitory mechanism of the FGF4-MEK-ERK1/2 pathway in ESCs is uncharacterized. O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) is a post-translational modification characterized by the attachment of a single N-acetylglucosamine (GlcNAc) to the serine and threonine residues of nuclear or cytoplasmic proteins. Here, we showed that the O-GlcNAc on the phosphorylation site of PKCζ inhibits PKCζ phosphorylation (activation) and, consequently, the FGF4-PKCζ-MEK-ERK1/2 pathway in ESCs. Our results demonstrate the mechanism for the maintenance of the undifferentiated state of ESCs via the inhibition of the FGF4-PKCζ-MEK-ERK1/2 pathway by O-GlcNAcylation on PKCζ., Graphical Abstract, Highlights • PKCζ activates the MEK-ERK1/2 pathway by FGF4 stimulation • O-GlcNAc on the phosphorylation site of PKCζ inhibits PKCζ activation in ESCs • FGF4-PKCζ-MEK-ERK1/2 pathway is inhibited by O-GlcNAc on PKCζ in ESCs, In this article, Nishihara and colleagues show that PKCζ is modified by O-GlcNAc and that O-GlcNAc on PKCζ inhibits PKCζ phosphorylation (activation) in mouse embryonic stem cells (ESCs). Phosphorylated PKCζ phosphorylates MEK-ERK1/2 via FGF4 stimulation, and phosphorylated ERK1/2 induces ESC differentiation. Inhibition of PKCζ phosphorylation by O-GlcNAc resulted in the inhibition of the FGF4 signal in ESCs.

Highlights • Angiosarcoma is associated with a poor prognosis and is treated with radiotherapy. • Highly stable FGF1 mutants exhibit stronger mitogenic activity than wild-type FGF1 and are candidates for radioprotectors. • They were examined as strong signaling agonists to clarify the effects of FGF1 on the murine angiosarcoma cell line ISOS-1. • Strong FGF1 signaling reduced the proliferative, invasive, and migration capabilities of ISOS-1cells. • Their malignancy was reduced through the dual inhibition of EGFR and VEGFR pathways by strong FGF1 signaling., Background and purpose Angiosarcoma is associated with a poor prognosis and is treated with radiotherapy. Although FGF1 is a potential radioprotector, the influence of FGF1 on the malignancy of angiosarcoma remains unknown. Materials and methods Highly stable FGF1 mutants, which exhibit stronger mitogenic activity than wild-type FGF1, were examined as strong radioprotectors and signaling agonists to clarify the effects of FGF1 on the murine angiosarcoma cell line ISOS-1. Results FGF1 mutants reduced colony formation by and the in vitro invasion and migration of ISOS-1 cells, in addition to an increase in radiosensitivity to X-rays. In contrast, an FGFR inhibitor blocked the inhibitory effects of FGF1 mutants on colony formation, invasion, and migration. siRNA targeting the Fgfr1 gene showed that strong FGFR1 signaling reduced colony formation by ISOS-1 cells. However, the FGF1 mutant reduced the activation of VEGFRs and EGFRs in ISOS-1 cells more strongly than wild-type FGF1. Moreover, the inhibition of VEGFRs and EGFRs synergistically reduced colony formation by and invasion and migration of ISOS-1 cells. Conclusion These results suggest that strong FGF1 signaling exerts not only radioprotective effects, but also inhibitory effects on proliferative and metastatic capacities of angiosarcoma through the dual inhibition of EGFR and VEGFR pathways.

Mouse embryonic stem cell (ESC) pluripotency is tightly regulated by a complex network composed of extrinsic and intrinsic factors that allow proper organismal development. O-linked β-N-acetylglucosamine (O-GlcNAc) is the sole glycosylation mark found on cytoplasmic and nuclear proteins and plays a pivotal role in regulating fundamental cellular processes; however, its function in ESC pluripotency is still largely unexplored. Here, we identify O-GlcNAcylation of proteasome activator subunit 3 (Psme3) protein as a node of the ESC pluripotency network. Mechanistically, O-GlcNAc modification of serine 111 (S111) of Psme3 promotes degradation of Ddx6, which is essential for processing body (P-body) assembly, resulting in the maintenance of ESC pluripotent state. Conversely, loss of Psme3 S111 O-GlcNAcylation stabilizes Ddx6 and increases P-body levels, culminating in spontaneous exit of ESC from the pluripotent state. Our findings establish O-GlcNAcylation at S111 of Psme3 as a switch that regulates ESC pluripotency via control of P-body homeostasis.

Three methods to detect neutrons emitted from an electrostatic accelerator were examined. These were gold foil activation, thermoluminescent dosimeters (TLDs), and CR-39. The gold-foil activation method has previously been proven effective in the measurement of neutrons emitted from cyclotron sources. In the case of electrostatic accelerators, a greater amount of fast neutrons and a smaller amount of thermal neutrons are observed compared to a cyclotron accelerator. Therefore, the error in calculating the thermal neutron flux became unacceptably large. By comparison the CR-39 detectors were apparently unable to measure thermal neutrons well, because some tracks were also generated by fast neutrons causing a signal interference. In the case of TLDs, as the material was not sensitive to fast neutrons, it was not affected by them, and hence could adequately measure the thermal neutron flux. However, it was found that in some cases its measurements could be offset by the influence of high dose rate of gamma rays.

Accelerator operations often generate neutrons that may activate concrete in the accelerator room. When accelerators are decommissioned, the concrete will remain as radioactive waste. Not all of concrete in the room will be radioactive wastes, but at place with large amount of neutrons will be radioactive wastes. Therefore, it is very important to determine where and how many neutrons are generated during accelerator operation. We used gold foils to obtain this information in positron emission tomography (PET) cyclotron facilities. We obtained concrete core samples from a PET cyclotron to determine the amount of radioactivity contained in concrete. We obtained the operational parameters (particle, radioactive product, current, and operation time) of the PET cyclotron facility and estimated the radioactivity generated in concrete using the operational parameters and gold foil results. A comparison of the estimated and core sampling measurement results showed good agreement.

Activated accelerator magnets were measured with three different types of commercially available gamma-ray imaging devices (pinhole, Compton scattering, and masked type) and the results were compared to simplify a distinction between activated/non-activated areas in the magnet during the decommissioning of an accelerator facility. In general, an activated magnet includes 60Co and emits gamma rays over 1 MeV; however, current devices are not designed to detect such high-energy photons. Our results showed that two devices could identify the location of highly activated areas, although they could not detect the total absorption peaks of the 1173 and 1333 keV photons from 60Co.

An in situ method to determine the specific activity in concrete structures using a survey meter was established in this study for decommissioning accelerator rooms. This method is much simpler than the conventional sampling method that requires core boring. A survey meter (T-SP2, TAC Inc., Japan) was customised and equipped with a small detector probe, whose size allowed the design of a light-weight Pb shield. For dose rate measurement, a Pb shield thickness of 4.5 cm was enough to shield ambient γ-rays, and its weight (16 kg) was low enough for the device to be carried by a human. A calibration curve was obtained to quickly and easily convert the net contact dose rate on concrete into the specific activity in a PET cyclotron room. The specific activity detection limit for this device was lower than the clearance limit of 152Eu and 60Co in activated materials. Therefore, this method can be used for separating nonactivated concrete from activated concrete during decommissioning. A general correction factor for the slope of the calibration curve was also obtained for use at other accelerator facilities.

Human induced pluripotent stem (hiPS) cells are attracting attention as a tool for regenerative medicine. However, several problems need to be overcome for their widespread and safe use, for example, the high cost of maintaining hiPS cells and the possibility of xenogeneic cell contamination in hiPS cell cultures. One of the main contributors to the high cost of maintaining hiPS cells is basic fibroblast growth factor (bFGF), which is essential for such cultures. Xenogeneic contamination can occur because of the use of mouse-derived feeder cells to culture hiPS cells. To overcome the problems of cell culture cost and xenogeneic contamination, we have developed a novel culture method in which the undifferentiated state and pluripotency of hiPS cells can be maintained under feeder-free and bFGF-free conditions. Our new approach involves the addition to the culture medium of highly sulfated hyaluronic acid (HA-HS), in which the hydroxyl groups of d-glucuronic acid (GlcA) and N-acetyl-d-glucosamine (GlcNAc) are chemically sulfated. HA-HS promotes bFGF signaling and maintains the undifferentiated state and pluripotency of hiPS cells under feeder-free and bFGF-free conditions. By contrast, non-sulfated hyaluronic acid and low sulfated hyaluronic acid do not maintain the undifferentiated state and pluripotency of hiPS cells. These results indicate that the maintenance of hiPS cells under feeder-free and bFGF-free conditions is an HA-HS specific effect. This study is the first to demonstrate the effects of sulfated hyaluronic acid on mammalian pluripotent stem cells, and provides a novel method for maintaining hiPS cells using HA-HS.

Fluorescence imaging flow cytometry offers highly accurate analysis of a large number of cells compared with conventional flow cytometry by virtue of its imaging capability. Unfortunately, the throughput of conventional fluorescence imaging flow cytometers is limited to ~1,000 cells/sec, which is one order of magnitude lower than that of conventional non-imaging flow cytometers. This is due to the low data transfer rate of a CCD image sensor with a time-delay integration technique employed to achieve sufficient sensitivity for fluorescence imaging of fast flowing cells. Replacing the CCD image sensor with a CMOS image sensor can potentially overcome the throughput limitation by virtue of its high data transfer rate, but critically sacrifice the imaging sensitivity because the time-delay integration cannot be employed to current CMOS image sensors. Here we present a fluorescence imaging flow cytometer that achieves comparable throughput and sensitivity with non-imaging flow cytometers. It is enabled by high-energy-density light-sheet excitation of flowing cells on a mirror-embedded PDMS-based microfluidic chip and by fluorescence image acquisition with a CMOS image sensor. The light-sheet excitation allows us obtain fluorescence images of flowing cells at a speed of >1 m/s, corresponding to a high throughput of >10,000 cells/sec. To show its biomedical utility, we use it combined with machine learning to demonstrate accurate screening of white blood cells and real-time identification of cancer cells in blood.

When a negatively charged muon is stopped in a substance, it is captured by an atom of the substance, and the muonic atom is formed. The muon capture process is significantly affected by the chemical environment of the atom and factors such as molecular structure (chemical effect). In this study, we performed muon irradiation for low-pressure CO, CO2, and COS molecules and measured the muonic X-rays emitted immediately after muon capture by an atom. In this paper, we quantitatively discuss the muon capture probability of each type of atom using the LMM model.

"Naive" mouse embryonic stem cells (ESCs) are derived from pre-implantation embryos and possess pluripotency, the ability to differentiate into any cell type of the body. "Primed" mouse epiblast stem cells (EpiSCs) are also pluripotent but are derived from post-implantation embryos. ESC-derived EpiSCs (ESD-EpiSCs) are "primed" pluripotent stem cells and can revert to naive reverted ESCs (rESCs). O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) is a posttranslational modification in the cytoplasm and nucleus. O-GlcNAc is transferred to serine and threonine residues of proteins by O-GlcNAc transferase (Ogt) and removed from them by O-GlcNAcase (Oga). In naive ESCs, O-GlcNAc contributes to maintain the undifferentiated state. In the transition from naive state to primed state, Ogt maintains cell survival, whereas Oga has no function. However, the function of O-GlcNAc in primed ESD-EpiSCs and during the reversion from the primed state to naive rESCs remains unclear. Here, we show that Ogt is required for the survival of primed ESD-EpiSCs. The expression of cytosolic Oga was significantly increased during induction from naive ESCs to primed ESD-EpiSCs. Furthermore, both Ogt and Oga were required for the reversion from primed ESD-EpiSCs to naive rESCs. These findings indicate that O-GlcNAcylation plays an important role in the survival of primed ESD-EpiSCs and in their reversion to naive rESCs.

We present on-chip fluorescence imaging flow cytometry by light-sheet excitation on a mirror-embedded microfluidic chip. The method allows us to obtain microscopy-grade fluorescence images of cells flowing at a high speed of 1 m/s, which is comparable to the flow speed of conventional non-imaging flow cytometers. To implement the light-sheet excitation of flowing cells in a microchannel, we designed and fabricated a mirror-embedded PDMS-based microfluidic chip. To show its broad utility, we used the method to classify large populations of microalgal cells (Euglena gracilis) and human cancer cells (human adenocarcinoma cells). Our method holds promise for large-scale single-cell analysis.

In order to investigate the neutron transportation from a beam-line tunnel to an access maze at a 12-GeV proton accelerator, we measured the spatial distribution of thermal and epithermal neutrons by using the Au activation method in detail. Gold foils were placed at about 70 positions in the maze in the case of the insertion (or extraction) of a copper target of 1 mm thickness into (or from) the beam axis in front of the maze. After the end of accelerator operation, relative activities of the Au foils were simultaneously measured by using an imaging plate technique and the radioactivity of one reference foil was also measured with a HPGe detector to convert to the absolute activities of all foils. It was found that the neutrons reach to the depth of the maze in the case of the insertion of the copper target. This result reflects higher proportion of high-energy particles from the copper target to that from other beam loss points and high-energy particles become the successive source of low-energy neutrons. Furthermore, it was found that several circumstances such as door walls and electric wire cables obviously affect the absorption effect of thermal neutrons. The reaction rates obtained in this study were also used for the benchmark of the Monte Carlo simulation code, MARS15 (version of February 2008). The results of the MARS15 calculations precisely reproduced experimental results and significant effects of the electric wire cables and door walls.

For evaluation of radioactivity induced in the concrete samples from accelerator facilities, the residual radioactivity in concrete sample, collected from seven accelerator facilities, was determined by γ-ray spectrometry. The tritium was extracted by the heating method using an IR furnace, and measured with a liquid scintillation counter. It was found that the major radioisotopes activated mainly by neutrons in the concrete samples were 152Eu, 60Co, 134Cs and 3H. The concentrations of radioactivities induced by thermal neutron capture are the highest at a depth of 10 cm in the concrete wall. The correlation between tritium, 60Co and 152Eu activity was investigated by measuring many concrete samples for seven accelerator facilities. The results indicate that their activities are strongly correlated with each other. So it would also be concluded that the total activity in shielding concrete could be estimated on the basis of the activities of 60Co and 152Eu.