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Purpose: 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. Methods and Materials: 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. Results: 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. Conclusions: 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.

Abstract Embryonic stem cells (ESCs) and epiblast-like cells (EpiLCs) recapitulate in vitro the epiblast first cell lineage decision, allowing characterization of the molecular mechanisms underlying pluripotent state transition. Here, we performed a comprehensive and comparative analysis of total glycomes of mouse ESCs and EpiLCs, revealing that overall glycosylation undergoes dramatic changes from early stages of development. Remarkably, we showed for the first time the presence of a developmentally regulated network orchestrating glycosylation changes and identified polycomb repressive complex 2 (PRC2) as a key component involved in this process. Collectively, our findings provide novel insights into the naïve-to-primed pluripotent state transition and advance the understanding of glycosylation complex regulation during early mouse embryonic development.

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.

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.

Background and purpose: Carbon ion (C-ion) beams are concentrated to irradiate pancreatic carcinoma in the upper abdomen; however, this radiotherapy potentially causes adverse reactions in the gastrointestinal tract. FGF1 is a candidate radioprotector for radiation-induced intestinal damage, but may promote the malignancy of pancreatic cancer. An FGF1/CPP-C chimeric protein was created to enhance the intracellular signaling mode of FGF1 instead of FGFR signaling. The present study investigated the effects of FGF1/CPP-C on the intestinal adverse reactions of C-ion radiotherapy as well as its influence on the malignancy of pancreatic cancer. Materials and methods: FGF1/CPP-C was administered intraperitoneally to BALB/c mice without heparin 12 h before total body irradiation (TBI) with low-LET C-ion (17 keV/μm) at 6–8 Gy. Several radioprotective effects were examined in the jejunum. The invasion and migration of the human pancreatic carcinoma cell lines MIAPaCa-2 and PANC-1 were assessed using Boyden chambers after cultures with FGF1/CPP-C. Results: The FGF1/CPP-C treatment promoted crypt survival after C-ion irradiation at 7–8 Gy significantly more than the FGF1 treatment. FGF1/CPP-C also inhibited C-ion radiotherapy-induced apoptosis and reduced γH2AX foci in crypt cells more than FGF1. However, FGF1/CPP-C inhibited the downstream signaling pathways of FGFRs and suppressed the activation of cell-cycle regulatory molecules in the intestine until 4 h after TBI. Furthermore, IEC6 cells were arrested in G2M after cultures with FGF1/CPP-C or FGF1, suggesting that DNA repair after irradiation is promoted by FGF1/CPP-C-induced G2M arrest. In contrast, FGF1/CPP-C appeared to be internalized into MIAPaCa-2 and PANC-1 cells more efficiently than FGF1. Therefore, FGF1/CPP-C reduced the in vitro proliferation, invasion, and migration of MIAPaCa-2 and PANC-1 cells significantly more than FGF1 through the cellular internalization of FGF1. Conclusion: These results suggest that the intracellular signaling mode of FGF1/CPP-C attenuates the intestinal adverse effects of C-ion radiotherapy without enhancing the malignancy of pancreatic carcinoma. Keywords: Carbon ion radiotherapy, FGF1, Cellular internalization, Intestinal adverse effects, Radioprotector, Pancreatic carcinoma

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.

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. Keywords: Angiosarcoma, EGFR, FGF1, Metastasis, Radioprotector, VEGFR

Maintenance of self-renewal and pluripotency in mouse embryonic stem cells (mESCs) is regulated by the balance between several extrinsic signaling pathways. Recently, we demonstrated that heparan sulfate (HS) chains play important roles in the maintenance and differentiation of mESCs by regulating extrinsic signaling. Sulfated HS structures are modified by various sulfotransferases during development. However, the significance of specific HS structures during development remains unclear. Here, we show that 3-O-sulfated HS structures synthesized by HS 3-O-sulfotransferases (3OSTs) and recognized by the antibody HS4C3 increase during differentiation of mESCs. Furthermore, expression of Fas on the cell surface of the differentiated cells also increased. Overexpression of the HS4C3-binding epitope in mESCs induced apoptosis and spontaneous differentiation even in the presence of LIF and serum. These data showed that the HS4C3-binding epitope was required for differentiation of mESCs. Up-regulation of the HS4C3-binding epitope resulted in the recruitment of Fas from the cytoplasm to lipid rafts on the cell surface followed by activation of Fas signaling. Indeed, the HS4C3-binding epitope interacted with a region that included the heparin-binding domain (KLRRRVH) of Fas. Reduced self-renewal capability in cells overexpressing 3OST resulted from the degradation of Nanog by activated caspase-3, which is downstream of Fas signaling, and was rescued by the inhibition of Fas signaling. We also found that knockdown of 3OST and inhibition of Fas signaling reduced the potential for differentiation into the three germ layers during embryoid body formation. This is the first demonstration that activation of Fas signaling is mediated by an increase in the HS4C3-binding epitope and indicates a novel signaling pathway for differentiation in mESCs.

Two cases of patients experienced subsyndromal depression after manic or mixed hypomanic and depressive episodes due to bipolar I (case 1) and II (case 2) disorders prior to the use of lamotrigine. Case 1 showed episodes of mood switching induced by antidepressants and seasonal mood instability. Case 2 showed hippocampal atrophy and a persistent dull headache that preceded the use of lamotrigine. Both were successfully treated with add-on lamotrigine therapy, and the dull headache was effectively treated with olanzapine. Both patients improved in social activity and work performance after these add-on treatments. Thus, add-on treatment with lamotrigine alone or in combination with olanzapine was an effective strategy to improve the quality of life in bipolar depression. Subsyndromal depression that present after the disappearance of the manic or mixed state was suggested to be practical indication for the use of lamotrigine.

It is essential to clarify the characteristics of the material activation in cyclotron-type proton therapy facilities for future decommissioning. In particular, concrete occupies the largest volume among object materials, but its activation characteristics of activity level and distribution are unknown. We investigated concrete radioactivation in cyclotron-type proton therapy facilities at the National Cancer Center Hospital East and Aizawa Hospital. The in situ 24Na measurement method, which is easy and high sensitivity, was applied for the investigation. Using the in situ 24Na measurement method, we obtained the thermal neutron fluence rates in concrete with high sensitivity. Based on these fluence rates, the sum of the 152Eu and 60Co specific activities in the concrete were predicted for an operation duration of 30 years. The study aims to predict general 152Eu and 60Co (which are major radionuclides) specific activities for cyclotron-type proton therapy facilities, and we found the following common characteristics in the investigation results. In the accelerator room, neutrons were mainly produced at the degrader and/or objects surrounding it and diffused over the room. As a result, the sum of the ratios of the concrete floor's specific activities of 152Eu and 60Co for their respective clearance level (0.1 Bq/g) was estimated to be close to 1. On the other hand, the production of neutrons both in the treatment and beam transport line rooms was low, and the sum of the ratios of the 152Eu and 60Co specific activities for their respective clearance level was estimated to be below 1. [ABSTRACT FROM AUTHOR]

Recently, various effects of low-temperature atmospheric-pressure plasma irradiation on living cells have been demonstrated, such as tissue sterilization, blood coagulation, angiogenesis, wound healing, and tumor elimination. However, the effect of plasma-irradiation on the differentiation of mouse embryonic stem cells (mESCs) has not yet been clarified. A large number of reactive species are generated by plasma-irradiation in medium, of which hydrogen peroxide (H2O2) is one of the main species generated. Here, we investigated the effect of plasma-irradiation on the differentiation of mESCs using an embryoid body (EB) formation assay with plasma-irradiated medium or H2O2-supplemented non-irradiated medium. Our findings demonstrated that plasma-irradiated medium potently inhibits the differentiation from mESCs to mesoderm and endoderm by inhibiting Wnt signaling as determined by quantitative polymerase chain reaction and immunoblotting analyses. In contrast, both the plasma-irradiated medium and H2O2-supplemented non-irradiated medium enhanced the differentiation to epiblastoid, ectodermal, and neuronal lineages by activation of fibroblast growth factor 4 (FGF4) signaling, suggesting that these effects are caused by the H2O2 generated by plasma-irradiation in medium. However, in each case, the differentiation to glial cells remained unaffected. This study is the first demonstration that plasma-irradiation affects the differentiation of mESCs by the regulation of Wnt and FGF4 signaling pathways. [ABSTRACT FROM AUTHOR]

Proliferation assays of mouse embryonic stem (ES) cells have been performed with cell culture media exposed to atmospheric-pressure plasmas (APPs), which generate reactive species in the media at room temperature. It is found that serum in cell culture media functions as a scavenger of highly reactive species and tends to protect cells in the media against cellular damage. On the other hand, if serum is not present in a cell culture medium when it is exposed to APP, the medium becomes cytotoxic and cannot be detoxified by serum added afterwards. Plasma-induced cytotoxic media hinder proliferation of mouse ES cells and may even cause cell death. It is also shown by nuclear magnetic resonance spectroscopy that organic compounds in cell culture media are in general not significantly modified by plasma exposure. These results indicate that if there is no serum in media when they are exposed to APPs, highly reactive species (such as OH radicals) generated in the media by the APP exposure are immediately converted to less reactive species (such as H2O2), which can no longer readily react with serum that is added to the medium after plasma exposure. This study has clearly shown that it is these less reactive species, rather than highly reactive species, that make the medium cytotoxic to mouse ES cells. [ABSTRACT FROM AUTHOR]