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Herein, we propose a spherical shell robot that can roll and move on its legs, and develop a prototype of the robot. Recently, there has been a growing demand for robots that can move freely and gather information on rough terrains, such as disaster sites, which are not accessible to humans. The robot developed here has two types of mobilities: rolling movement using a spherical shape and walking movement using its legs. Because the morphological transformation does not require recombination of parts, it can be reversibly performed via remote control. Therefore, the robot can select the movement method according to the environment, and reach the target point reliably even on uneven terrains, such as a disaster site. We designed a mechanism that enabled the transformation of the form and devised an operation method. Accordingly, a prototype was developed and tested. A rolling test on flat ground confirmed that the robot can roll over 5.0 m and its speed could be controlled using a gyro sensor. The leg locomotion test confirmed that the robot can turn and move straight ahead without turning over. In addition, we also conducted experiments, such as sudden stops and remote morphological deformation, to confirm the operation of the robot during rolling.

In designing thermoelectric and heat transfer devices on the micrometer scale, the accurate thermal conductivity measurement is very important, and a variety of measurement methods have been developed so far. Among them, the 3ω method is one of the best for conductive wires because it can directly measure thermal conductivity without measuring density or specific heat, and also in the same direction as electrical or thermoelectric property. However, previous studies have not sufficiently considered the effects of ambient pressure and the conductive adhesive used to attach the sample to the electrode, which may hinder accurate measurement. In this study, using a thin gold wire as a test sample, the influence of ambient pressure and the length of conductive adhesive along the sample has been investigated quantitatively as major factors of systematic errors in the 3ω method. When the pressure was increased in the transition flow region, the measured apparent thermal conductivity increased. An analytical model for the low-pressure gas heat conduction is proposed to quantitatively explain the pressure dependence. The measured value also increased when the length of the conductive adhesive exceeded 20% of the sample length. This work has revealed that the ambient should be evacuated to the molecular flow region and the length of conductive adhesive be less than 20% of the sample length. The guidelines proposed here will help researchers in various fields to more accurately determine the thermal conductivity of micrometer-scale wires.

A 59-year-old man who had smoked for 23 pack-years was admitted to our hospital because of two-month history of back pain. The chest computed tomography scan demonstrated combined pulmonary fibrosis and emphysema (CPFE) and an irregular shaped nodule in the left lower lobe of the lung. A biopsy obtained from samples from subcarinal lymph nodes revealed non-small cell lung cancer. Anti-aminoacyl-tRNA synthetase (ARS) antibody was elevated up to 166 U/mL, although he had no symptoms suggestive connective tissue diseases. It is well known that most of CPFE patients are current or former heavy smokers, and some researchers described the relationship between CPFE and connective tissue diseases. To our best knowledge, this was the first report of lung cancer in patient with anti-ARS antibody-positive CPFE. In some anti-ARS antibody-positive patients, smoking might have a relationship with development of CPFE and lung cancer.

Platypnea-orthodeoxia syndrome (POS) is a rare disease characterized by dyspnea and hypoxemia in orthostatism that improves in the recumbent position. We herein report an 81-year-old woman with dyspnea in the upright position following thoracic vertebral compression fractures. After the patient's daughter brought a recording showing decreasing SpO

Radiation is an effective cancer treatment, but cancer cells can acquire radioresistance, which is associated with increased DNA damage response and enhanced proliferative capacity, and therefore, it is important to understand the intracellular biochemical responses to γ-irradiation. The transient receptor potential melastatin 8 (TRPM8) channel plays roles in the development and progression of tumors, but it is unclear whether it is involved in the DNA damage response induced by γ-irradiation. Here, we show that a TRPM8 channel inhibitor suppresses the DNA damage response (phosphorylated histone variant H2AX-p53-binding protein 1 (γH2AX-53BP1) focus formation) and colony formation of B16 melanoma cells. Furthermore, the TRPM8 channel-specific agonist WS-12 enhanced the DNA damage response and increased the survival fraction after γ-irradiation. We found that the TRPM8 channel inhibitor enhanced G2/M phase arrest after γ-irradiation. Phosphorylation of ataxia telangiectasia mutated and p53, which both contribute to the DNA damage response was also suppressed after γ-irradiation. In addition, the TRPM8 channel inhibitor enhanced the γ-irradiation-induced suppression of tumor growth in vivo. We conclude that the TRPM8 channel is involved in radiation-induced DNA damage repair and contributes to the radioresistance of B16 melanoma cells. TRPM8 channel inhibitors might be clinically useful as radiosensitizers to enhance radiation therapy of melanoma.

Although disturbed phosphate metabolism frequently accompanies chronic kidney disease (CKD), its causal role in CKD progression remains unclear. It is also not fully understood how excess salt induces organ damage. We here show that urinary phosphate-containing nanoparticles promote kidney injury in salt-sensitive hypertension. In Dahl salt-sensitive rats, salt loading resulted in a significant increase in urinary phosphate excretion without altering serum phosphate levels. An intestinal phosphate binder sucroferric oxyhydroxide attenuated renal inflammation and proteinuria in this model, along with the suppression of phosphaturia. Using cultured proximal tubule cells, we confirmed direct pathogenic roles of phosphate-containing nanoparticles in renal tubules. Finally, transcriptome analysis revealed a potential role of complement C1q in renal inflammation associated with altered phosphate metabolism. These data demonstrate that increased phosphate excretion promotes renal inflammation in salt-sensitive hypertension and suggest a role of disturbed phosphate metabolism in the pathophysiology of hypertensive kidney disease and high salt-induced kidney injury., Qin Wang et al. show that altered phosphate metabolism contributes to the progression of kidney disease using a salt-sensitive rat model. They demonstrate that using the phosphate binder sucroferric oxyhydroxide reduces kidney injury and, using transcriptome sequencing, identify a potential role for complement C1q in kidney inflammation linked to phosphate metabolism defects.

LSD1/KDM1A is a widely conserved lysine-specific demethylase that removes methyl groups from methylated proteins, mainly histone H3. We previously isolated the zebrafish LSD1 gene and demonstrated that it is required for primitive hematopoiesis. Recently, a neuron-specific splicing variant of LSD1 was found in mammals and its specific functions and substrate specificities were reported. To our surprise, zebrafish LSD1 cDNA, which we previously analyzed, was corresponded to the neuron-specific variant in mammals. In this study, we investigated the structures and expression of LSD1 splicing variants in zebrafish and found all 4 types of LSD1 isoforms: LSD1, LSD1+2al, LSD1+8al and LSD1+2al8al. Interestingly, LSD1+8al/LSD1+2al8al, which correspond to mammalian neuron-specific variants, expressed ubiquitously in zebrafish. We also performed phenotypic rescue experiments of a zebrafish LSD1 mutant (kdm1ait627) using human and zebrafish LSD1 variants to identify which variant is involved in primitive hematopoiesis. Unexpectedly, the overexpression of all types of human and zebrafish variants was able to rescue the hematopoietic phenotypes in LSD1 mutants. Furthermore, enzymatic-deficient LSD1K661A (human) and K638A (zebrafish) were also able to rescue the mutant phenotypes. These results suggest that the LSD1 functions in zebrafish primitive hematopoiesis are free from any splicing-dependent regulation or demethylation reaction.

Low birth weight has been reported as a risk factor for non-communicable diseases, which can be caused by metabolic dysfunction. Here, we conducted a retrospective study to define the correlation between birth weight and present physical characteristics/fitness in Japanese university students, by gender. The subjects were from among 1,333 healthy university students. They carried out a self-administered questionnaire on birth weight, physical characteristics, and exercise habits, and 8 types of physical fitness tests. We excluded data from subjects that lacked information in their questionnaire and those who were not a full-term single birth. Finally, data of 378 participants (28.4% of all subjects, 116 males and 262 females) were analyzed. We categorized the participants into lower and higher birth weight groups, based on the overall median value calculated for birth weight. The lower birth weight group of males demonstrated lower grip strength; however, there was no significant association after adjustment for height, weight, and exercise habits. The lower birth weight group of females showed higher scores on the 50-meter running test even after the adjustment for physical characteristics and exercise habits. These results suggest that lower birth weight males had a lower grip strength, which was strongly associated with their physical size. In contrast, higher sprint performance was shown to be independent of physical size in lower birth weight females.

Recipient endogenous memory CD8 T cells expressing reactivity to donor class I MHC infiltrate MHC-mismatched cardiac allografts within 24 hours after reperfusion and express effector functions mediating graft injury. The current study tested the efficacy of Very Late Antigen-4 (VLA-4) blockade to inhibit endogenous memory CD8 T cell infiltration into cardiac allografts and attenuate early posttransplant inflammation. Peritransplant anti-VLA-4 mAb given to C57BL6 (H-2b ) recipients of AJ (H-2a ) heart allografts completely inhibited endogenous memory CD4 and CD8 T cell infiltration with significant decrease in macrophage, but not neutrophil, infiltration into allografts subjected to either minimal or prolonged cold ischemic storage (CIS) prior to transplant, reduced intra-allograft IFN-γ-induced gene expression and prolonged survival of allografts subjected to prolonged CIS in CTLA-4Ig treated recipients. Anti-VLA-4 mAb also inhibited priming of donor-specific T cells producing IFN-γ until at least day 7 posttransplant. Peritransplant anti-VLA plus anti-CD154 mAb treatment similarly prolonged survival of allografts subjected to minimal or increased CIS prior to transplant. Overall, these data indicate that peritransplant anti-VLA-4 mAb inhibits early infiltration memory CD8 T cell infiltration into allografts with a marked reduction in early graft inflammation suggesting an effective strategy to attenuate negative effects of heterologous alloimmunity in recipients of higher risk grafts.

FMS-like tyrosine kinase 3 (FLT3) in hematopoietic cells binds to its ligand at the plasma membrane (PM), then transduces growth signals. FLT3 gene alterations that lead the kinase to assume its permanently active form, such as internal tandem duplication (ITD) and D835Y substitution, are found in 30–40% of acute myelogenous leukemia (AML) patients. Thus, drugs for molecular targeting of FLT3 mutants have been developed for the treatment of AML. Several groups have reported that compared with wild-type FLT3 (FLT3-wt), FLT3 mutants are retained in organelles, resulting in low levels of PM localization of the receptor. However, the precise subcellular localization of mutant FLT3 remains unclear, and the relationship between oncogenic signaling and the mislocalization is not completely understood. In this study, we show that in cell lines established from leukemia patients, endogenous FLT3-ITD but not FLT3-wt clearly accumulates in the perinuclear region. Our co-immunofluorescence assays demonstrate that Golgi markers are co-localized with the perinuclear region, indicating that FLT3-ITD mainly localizes to the Golgi region in AML cells. FLT3-ITD biosynthetically traffics to the Golgi apparatus and remains there in a manner dependent on its tyrosine kinase activity. Tyrosine kinase inhibitors, such as quizartinib (AC220) and midostaurin (PKC412), markedly decrease FLT3-ITD retention and increase PM levels of the mutant. FLT3-ITD activates downstream in the endoplasmic reticulum (ER) and the Golgi apparatus during its biosynthetic trafficking. Results of our trafficking inhibitor treatment assays show that FLT3-ITD in the ER activates STAT5, whereas that in the Golgi can cause the activation of AKT and ERK. We provide evidence that FLT3-ITD signals from the early secretory compartments before reaching the PM in AML cells.

Resistance training adaptively increases the muscle strength associated with protein anabolism. Previously, we showed that the combined intake of astaxanthin, &beta, carotene, and resveratrol can accelerate protein anabolism in the skeletal muscle of mice. The purpose of this study was to investigate the effect of anabolic nutrient-rich foods on muscle adaptation induced by resistance training. Twenty-six healthy men were divided into control and intervention groups. All participants underwent a resistance training program twice a week for 10 weeks. Astaxanthin-, &beta, carotene-, and resveratrol-rich foods were provided to the intervention group. Body composition, nutrient intake, maximal voluntary contraction of leg extension, oxygen consumption, and serum carbonylated protein level were measured before and after training. The skeletal muscle mass was higher after training than before training in both groups (p &lt, 0.05). Maximal voluntary contraction was increased after training in the intervention group (p &lt, 0.05), but not significantly increased in the control group. Resting oxygen consumption was higher after training in the intervention group only (p &lt, 0.05). As an oxidative stress marker, serum carbonylated protein level tended to be lower immediately after exercise than before exercise in the intervention group only (p = 0.056). Intake of astaxanthin-, &beta, carotene-, and resveratrol-rich foods supported resistance training-induced strength and metabolic adaptations.

Background: Silica nanoparticles (SiO2 NPs) are widely used in industrial products as additives for rubber and plastics or as filler strengthening concrete, as well as being used in the biomedical field for drug delivery and theranostic purposes. The present study investigated the effects of amino or carboxyl functionalization of rhodamine-labeled SiO2 NPs on cellular uptake and cytotoxicity. Methods: Male mice were randomly divided into seven groups (n=6, each) and exposed to non-functionalized (plain), carboxyl or amino-functionalized rhodamine-labeled SiO2 NPs at 2 or 10 mg/kg bw, or endotoxin-free water, by pharyngeal aspiration. At 24 hours after administration, the mice were euthanized and bronchoalveolar lavage fluid (BALF) was collected for differential cell count and identification of silica nanoparticle uptake using confocal microscopy. In the in vitro studies, murine RAW264.7 macrophages were exposed to non-functionalized, amino- or carboxyl-functionalized Rhodamine-labeled SiO2 NPs. Nonspecific caspase inhibitor and necrostatin-1 were used to determine the involvement of caspase or receptor-interacting protein 1 kinase domain in the cytotoxicity. Results: The in vivo study demonstrated that the neutrophil and macrophage counts and the percentage of macrophages with internalized particles was highest in the order of carboxyl >= amino- > > non-functionalized particles. The in vitro study demonstrated greater cytotoxicity for non-functionalized silica nanoparticles, compared to the others. Treatment with non-specific caspase or necroptosis inhibitor did not attenuate MTS cytotoxicity of non-functionalized silica nanoparticles. Conclusion: We conclude that carboxyl-functionalzed SiO2 NPs are internalized by macrophages more efficiently but less cytotoxic than plain SiO2 NPs. The cytotoxic effect of plain SiO2 NPs, which cannot be explained by apoptosis or necroptosis, can be avoided by carboxyl- or amino- functionalization.

// Shoma Tamori 1, 6 , Yuka Nozaki 1, 6 , Hitomi Motomura 1, 6 , Hiromi Nakane 1 , Reika Katayama 1 , Chotaro Onaga 1 , Eriko Kikuchi 1 , Nami Shimada 2 , Yuhei Suzuki 1 , Mei Noike 1 , Yasushi Hara 3 , Keiko Sato 4, 6 , Tsugumichi Sato 2, 6 , Kouji Yamamoto 5, 6 , Takehisa Hanawa 2, 6 , Misa Imai 8, 9 , Ryo Abe 3, 11 , Atsushi Yoshimori 10 , Ryoko Takasawa 2 , Sei-Ichi Tanuma 2, 7 and Kazunori Akimoto 1, 6 1 Department of Medicinal and Life Science, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba, Japan 2 Department of Pharmacy, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba, Japan 3 Research Institute for Biochemical Sciences, Tokyo University of Science, Chiba, Japan 4 Department of Information Sciences, Faculty of Science and Technology, Tokyo University of Science, Chiba, Japan 5 Department of Biostatistics, Yokohama City University, School of Medicine, Yokohama, Japan 6 Translational Research Center, Research Institute for Science and Technology, Tokyo University of Science, Chiba, Japan 7 Laboratory of Genomic Medicinal Science, Research Institute for Science and Technology, Tokyo University of Science, Chiba, Japan 8 Department of Hematology, Juntendo University School of Medicine, Tokyo, Japan 9 Leading Center for the Development and Research of Cancer Medicine, Juntendo University School of Medicine, Tokyo, Japan 10 Institute for Theoretical Medicine, Inc., Kanagawa, Japan 11 Strategic Innovation and Research Center, Teikyo University, Tokyo, Japan Correspondence to: Kazunori Akimoto, email: akimoto@rs.tus.ac.jp Keywords: cancer stem cell; glyoxalase 1; breast cancer; the Warburg effect; target therapy Received: July 04, 2018 Accepted: November 01, 2018 Published: November 23, 2018 ABSTRACT Glyoxalase 1 (GLO1) is a ubiquitous enzyme involved in the detoxification of methylglyoxal, a cytotoxic byproduct of glycolysis that induces apoptosis. In this study, we found that GLO1 gene expression correlates with neoplasm histologic grade ( χ 2 test , p = 0.002) and is elevated in human basal-like breast cancer tissues. Approximately 90% of basal-like cancers were grade 3 tumors highly expressing both GLO1 and the cancer stem cell marker ALDH1A3 . ALDH1 high cells derived from the MDA-MB 157 and MDA-MB 468 human basal-like breast cancer cell lines showed elevated GLO1 activity. GLO1 inhibition using TLSC702 suppressed ALDH1 high cell viability as well as the formation of tumor-spheres by ALDH1 high cells. GLO1 knockdown using specific siRNAs also suppressed ALDH1 high cell viability, and both TLSC702 and GLO1 siRNA induced apoptosis in ALDH1 high cells. These results suggest GLO1 is essential for the survival of ALDH1-positive breast cancer stem cells. We therefore conclude that GLO1 is a potential therapeutic target for treatment of basal-like breast cancers.

Most gastrointestinal stromal tumours (GISTs) are caused by constitutively active mutations in Kit tyrosine kinase. The drug imatinib, a specific Kit inhibitor, improves the prognosis of metastatic GIST patients, but these patients become resistant to the drug by acquiring secondary mutations in the Kit kinase domain. We recently reported that a Kit mutant causes oncogenic signals only on the Golgi apparatus in GISTs. In this study, we show that in GIST, 2-methylcoprophilinamide (M-COPA, also known as "AMF-26"), an inhibitor of biosynthetic protein trafficking from the endoplasmic reticulum (ER) to the Golgi, suppresses Kit autophosphorylation at Y703/Y721/Y730/Y936, resulting in blockade of oncogenic signalling. Results of our M-COPA treatment assay show that Kit Y703/Y730/Y936 in the ER are dephosphorylated by protein tyrosine phosphatases (PTPs), thus the ER-retained Kit is unable to activate downstream molecules. ER-localized Kit Y721 is not phosphorylated, but not due to PTPs. Importantly, M-COPA can inhibit the activation of the Kit kinase domain mutant, resulting in suppression of imatinib-resistant GIST proliferation. Our study demonstrates that Kit autophosphorylation is spatio-temporally regulated and may offer a new strategy for treating imatinib-resistant GISTs.

Detection of anomalous cells such as cancer cells from normal blood cells has the potential to contribute greatly to cancer diagnosis and therapy. Conventional methods for the detection of cancer cells are usually tedious and cumbersome. Herein, we report on the use of a particle size analyzer for the convenient size-based differentiation of cancer cells from normal cells. Measurements made using a particle size analyzer revealed that size parameters for cancer cells are significantly greater (e.g., inner diameter and width) than the corresponding values for normal cells (white blood cells (WBC), lymphocytes and splenocytes), with no significant difference in shape parameters (e.g., circularity and convexity). The inner diameter of many cancer cell lines is greater than 10 µm, in contrast to normal cells. For the detection of WBC having similar size to that of cancer cells, we developed a PC software "Cancer Cell Finder" that differentiates them from cancer cells based on brightness stationary points on a cell surface. Furthermore, the aforementioned method was validated for cancer cell/clusters detection in spiked mouse blood samples (a B16 melanoma mouse xenograft model) and circulating tumor cell cluster-like particles in the cat and dog (diagnosed with cancer) blood samples. These results provide insights into the possible applicability of the use of a particle size analyzer in conjunction with PC software for the convenient detection of cancer cells in experimental and clinical samples for theranostics.

Astaxanthin, a natural antioxidant, exists in non-esterified and esterified forms. Although it is known that astaxanthin can improve exercise endurance and cause metabolic improvement in skeletal muscle, the effects of the two different forms are unclear. We investigated the effects of the different forms of astaxanthin on endurance in mice. Eight-week-old ICR mice were divided into four groups: control; astaxanthin extracted from Haematococcus pluvialis in an esterified form; astaxanthin extracted from Phaffia rhodozyma in a non-esterified form; and astaxanthin synthesized chemically in a non-esterified form. After 5 weeks of treatment, each group was divided into sedentary and exercise groups. In the group fed astaxanthin from Haematococcus, the running time to exhaustion was longest, and the plasma and tissue concentrations of astaxanthin were significantly higher than those in the other groups. Astaxanthin from Haematococcus increased 5'-adenosine monophosphate-activated protein kinase levels in the skeletal muscle. Although the mice in the Haematococcus group ran for longer, hexanoyl lysine adduct levels in the skeletal muscle mitochondria were similar in the control and Haematococcus groups. Our results suggested that esterified astaxanthin promoted energy production and protected tissues from oxidative damage during exercise owing to its favorable absorption properties, leading to a longer running time.

c-Met is a receptor-type tyrosine kinase, which is involved in a wide range of cellular responses such as proliferation, motility, migration and invasion. It has been reported to be overexpressed in various cancers. However, the role of c-Met in breast cancer stem cells (CSCs) still remains unclear. We herein, show that c-Met expression is significantly elevated in Basal-like type of breast cancer in comparison with other subtypes. High expression of c-Met strongly correlated with the expression of two CSC markers, ALDH1A3 and CD133 in breast cancers. In addition, breast cancers at tumor stage III-IV expressing both c-Methigh and ALDH1A3high had poor prognosis. Furthermore, treatment with c-Met inhibitors (Crizotinib, Foretinib, PHA-665752 and Tivantinib) in MDA-MB157 cells with high c-Met protein expression resulted in significant suppression in cell viability, contrary to MDA-MB468 cells with low c-Met protein expression. These c-Met inhibitors also suppressed cell viability and tumor-sphere formation of ALDH1high breast cancer cells with high c-Met expression. These results suggest that c-Met in ALDH1 positive CSCs seems to play an important role in breast cancer repopulation. Therefore, we conclude that c-Met is a potential therapeutic target in ALDH1 positive breast CSCs.

Gastrointestinal stromal tumors (GISTs) are caused by gain-of-function mutations in the Kit receptor tyrosine kinase. Most primary GIST patients respond to the Kit inhibitor imatinib, but this drug often becomes ineffective because of secondary mutations in the Kit kinase domain. The characteristic intracellular accumulation of imatinib-sensitive and -resistant Kit protein is well documented, but its relationship to oncogenic signaling remains unknown. Here, we show that in cancer tissue from primary GIST patients as well as in cell lines, mutant Kit accumulates on the Golgi apparatus, whereas normal Kit localizes to the plasma membrane (PM). In imatinib-resistant GIST with a secondary Kit mutation, Kit localizes predominantly on the Golgi apparatus. Both imatinib-sensitive and imatinib-resistant Kit (Kit(mut)) become fully auto-phosphorylated only on the Golgi and only if in a complex-glycosylated form. Kit(mut) accumulates on the Golgi during the early secretory pathway, but not after endocytosis. The aberrant kinase activity of Kit(mut) prevents its export from the Golgi to the PM. Furthermore, Kit(mut) on the Golgi signals and activates the phosphatidylinositol 3-kinase–Akt (PI3K–Akt) pathway, signal transducer and activator of transcription 5 (STAT5), and the Mek–Erk pathway. Blocking the biosynthetic transport of Kit(mut) to the Golgi from the endoplasmic reticulum inhibits oncogenic signaling. PM localization of Kit(mut) is not required for its signaling. Activation of Src-family tyrosine kinases on the Golgi is essential for oncogenic Kit signaling. These results suggest that the Golgi apparatus serves as a platform for oncogenic Kit signaling. Our study demonstrates that Kit(mut)’s pathogenicity is related to its mis-localization, and may offer a new strategy for treating imatinib-resistant GISTs.

Full activation of T cells and differentiation into effector T cells are essential for many immune responses and require co-stimulatory signaling via the CD28 receptor. Extracellular ligand binding to CD28 recruits protein-tyrosine kinases to its cytoplasmic tail, which contains a YMNM motif. Following phosphorylation of the tyrosine, the proteins growth factor receptor-bound protein 2 (Grb2), Grb2-related adaptor downstream of Shc (Gads), and p85 subunit of phosphoinositide 3-kinase may bind to pYMNM (where pY is phosphotyrosine) via their Src homology 2 (SH2) domains, leading to downstream signaling to distinct immune pathways. These three adaptor proteins bind to the same site on CD28 with variable affinity, and all are important for CD28-mediated co-stimulatory function. However, the mechanism of how these proteins recognize and compete for CD28 is unclear. To visualize their interactions with CD28, we have determined the crystal structures of Gads SH2 and two p85 SH2 domains in complex with a CD28-derived phosphopeptide. The high resolution structures obtained revealed that, whereas the CD28 phosphopeptide bound to Gads SH2 is in a bent conformation similar to that when bound to Grb2 SH2, it adopts a more extended conformation when bound to the N- and C-terminal SH2 domains of p85. These differences observed in the peptide-protein interactions correlated well with the affinity and other thermodynamic parameters for each interaction determined by isothermal titration calorimetry. The detailed insight into these interactions reported here may inform the development of compounds that specifically inhibit the association of CD28 with these adaptor proteins to suppress excessive T cell responses, such as in allergies and autoimmune diseases.

It is therapeutically important to elucidate the factors involved in the radiation resistance of tumors. We previously showed that ATP is released from mouse melanoma B16 cells in response to γ-irradiation, but the role of adenosine, a metabolite of ATP, is still unclear. Here, we show that the adenosine A2B receptor is involved in DNA damage repair and radioresistance in mouse melanoma B16 cells. The DNA damage response after γ-irradiation was attenuated by pretreatment with A2B receptor antagonists, such as PSB603, while it was enhanced by pretreatment with A2B receptor agonists, such as BAY60-6583. γ-Irradiation decreased the cell survival rate, and pretreatment with PSB603 further reduced the survival rate. On the other hand, pretreatment with BAY60-6583 increased the cell survival rate after irradiation. The DNA damage response and the cell survival rate after γ-irradiation were both decreased in A2B-knockdown cells. In vivo experiments in mice confirmed that tumor growth was suppressed and delayed in the irradiated group pretreated with PSB603, compared with the irradiation-alone group. Our results indicate that adenosine A2B receptor contributes to radioresistance, and could be a new target for the development of agents to increase the efficacy of radiotherapy.

Reperfusion of organ allografts induces a potent inflammatory response that directs rapid memory T cell, neutrophil, and macrophage graft infiltration and their activation to express functions mediating graft tissue injury. The role of cardiac allograft IL-1 receptor (IL-1R) signaling in this early inflammation and the downstream primary alloimmune response was investigated. When compared with complete MHC-mismatched wild-type cardiac allografts, IL-1R−/− allografts had marked decreases in endogenous memory CD8 T cell and neutrophil infiltration and expression of proinflammatory mediators at early times after transplant, whereas endogenous memory CD4 T cell and macrophage infiltration was not decreased. IL-1R−/− allograft recipients also had marked decreases in de novo donor-reactive CD8, but not CD4, T cell development to IFN-γ–producing cells. CD8 T cell–mediated rejection of IL-1R−/− cardiac allografts took 3 wk longer than wild-type allografts. Cardiac allografts from reciprocal bone marrow reconstituted IL-1R−/−/wild-type chimeric donors indicated that IL-1R signaling on graft nonhematopoietic-derived, but not bone marrow–derived, cells is required for the potent donor-reactive memory and primary CD8 T cell alloimmune responses observed in response to wild-type allografts. These studies implicate IL-1R–mediated signals by allograft parenchymal cells in generating the stimuli-provoking development and elicitation of optimal alloimmune responses to the grafts.

Hepatitis C virus (HCV) infection and propagation in cultured cells have mainly been investigated using the infectious clinical clone JFH1. However, its infectivity is not high enough for infection to be detected easily. In this study, we attempted to isolate HCV-JFH1 variants adapted to human hepatoma Huh7.5.1 cells. By performing serial passages of the wild-type HCV-JFH1 in Huh7.5.1 cells, we obtained a variant that was capable of inducing severe cytopathic effects and showed approximately 700-fold higher infectivity than the wild-type HCV-JFH1. Further, when highly permissive Huh7.5.1–8 cells were infected with this variant, viral particles were produced at >1011 copies ml−1, making this variant one of the most efficient HCV production systems. Two adaptive mutations were noted in the variant genome: a1994c (K74T) in the core protein region and t3014c (I414T) in the E2 protein region. Both mutations contributed to enhanced infectivity and their combination showed synergistic effects in this regard. An examination of recombinant viruses carrying K74T, I414T and K74T/I414T mutations revealed that none of the mutations had an effect on the steps after viral entry (genome replication, particle assembly and egress), but led to the viral infection becoming less dependent on scavenger receptor class B type I, changes of the infectious particles to a broader and lower range of densities, and enhanced thermal stability of the infectious viruses. Thus, this Huh7.5.1-adapted HCV-JFH1 variant with higher and stable infectivity should be a valuable tool for studying the molecular mechanisms behind the life cycle of HCV and for antiviral screening.

p53 mutations are frequently detected in malignant gastric cancers. However, the molecular mechanisms by which loss of p53 function promotes gastric cancer are not clear. We utilized Gan mice (K19-Wnt1/C2mE), which have functional p53 and develop intestinal-type gastric tumors, to investigate the role of p53 in gastric cancer progression by knocking out p53. We found that gastric epithelial cells acquire tumorigenicity in the subcutis of C57BL/6 mice as a result of Wnt activation, COX-2 activation and p53 deficiency. With repeated allograft transfers, these gastric epithelial cells gradually acquired the properties of malignant gastric cancer. Loss of p53 conferred cell stemness and induced epithelial to mesenchymal transition (EMT) in gastric epithelial cells, and these properties were further enhanced by the in vivo microenvironment, ultimately leading to gastric cancer formation and metastasis. We also found that the in vivo microenvironment enhanced activation of the COX-2 pathway, which further contributed to cancer progression. With this system, we have succeeded in recapitulating the development of malignant gastric cancer from gastric epithelial cells in a normal immune environment.

The IL-7R plays critical roles in lymphocyte development and homeostasis. Although IL-7R expression is strictly regulated during lymphocyte differentiation and the immune response, little is known regarding its in vivo regulation. To address this issue, we established a mouse line with targeted deletion of the conserved non-coding sequence 1 (CNS1) element found 3.6 kb upstream of the IL-7Rα promoter. We report that IL-7Rα is expressed normally on T and B cells in thymus and bone marrow of CNS1−/− mice except for in regulatory T cells. In contrast, these mice show reduced IL-7Rα expression in conventional CD4 and CD8 T cells as well as regulatory T, NKT, and γδ T cells in the periphery. CD4 T cells of CNS1−/− mice showed IL-7Rα upregulation in the absence of growth factors and IL-7Rα downregulation by IL-7 or TCR stimulation, although the expression levels were lower than those in control mice. Naive CD4 and CD8 T cells of CNS1−/− mice show attenuated survival by culture with IL-7 and reduced homeostatic proliferation after transfer into lymphopenic hosts. CNS1−/− mice exhibit impaired maintenance of Ag-stimulated T cells. Furthermore, IL-7Rα upregulation by glucocorticoids and TNF-α was abrogated in CNS1−/− mice. This work demonstrates that the CNS1 element controls IL-7Rα expression and maintenance of peripheral T cells, suggesting differential regulation of IL-7Rα expression between central and peripheral lymphoid organs.

Background: EGFR signaling maintains aerobic glycolysis, but the molecular mechanism is still undefined. Results: Drug inhibition studies reveal that downstream signaling via the PI3K pathway is critical for glucose transport and metabolism. Conclusion: The PI3K signaling regulates key metabolic activities in EGFR-mutant lung adenocarcinoma. Significance: These data may guide the development of chemotherapeutic options, including targeting of the PI3K pathway and glucose transporter machinery., Oncogenic epidermal growth factor receptor (EGFR) signaling plays an important role in regulating global metabolic pathways, including aerobic glycolysis, the pentose phosphate pathway (PPP), and pyrimidine biosynthesis. However, the molecular mechanism by which EGFR signaling regulates cancer cell metabolism is still unclear. To elucidate how EGFR signaling is linked to metabolic activity, we investigated the involvement of the RAS/MEK/ERK and PI3K/AKT/mammalian target of rapamycin (mTOR) pathways on metabolic alteration in lung adenocarcinoma (LAD) cell lines with activating EGFR mutations. Although MEK inhibition did not alter lactate production and the extracellular acidification rate, PI3K/mTOR inhibitors significantly suppressed glycolysis in EGFR-mutant LAD cells. Moreover, a comprehensive metabolomics analysis revealed that the levels of glucose 6-phosphate and 6-phosphogluconate as early metabolites in glycolysis and PPP were decreased after inhibition of the PI3K/AKT/mTOR pathway, suggesting a link between PI3K signaling and the proper function of glucose transporters or hexokinases in glycolysis. Indeed, PI3K/mTOR inhibition effectively suppressed membrane localization of facilitative glucose transporter 1 (GLUT1), which, instead, accumulated in the cytoplasm. Finally, aerobic glycolysis and cell proliferation were down-regulated when GLUT1 gene expression was suppressed by RNAi. Taken together, these results suggest that PI3K/AKT/mTOR signaling is indispensable for the regulation of aerobic glycolysis in EGFR-mutated LAD cells.

An efficient cell culture and infection system for hepatitis C virus (HCV) facilitates analyses of the complete virus life cycle. Human hepatic Huh7.5.1 cells and an HCV-JFH1 strain have been widely employed in infection experiments. In the present study, cultured Huh7.5.1 cells exhibited heterogeneous phenotypes of HCV infection. Using single-cell cloning of Huh7.5.1 cells, we isolated a clone highly permissive to HCV (Huh7.5.1-8) and a CD81-defective clone nonpermissive to HCV (Huh7.5.1-5). Expression of CD81 in Huh7.5.1-5 cells restored permissiveness to HCV, indicating that CD81 is essential for HCV infection and a defect in CD81 causes nonpermissiveness to HCV in Huh7.5.1-5 cells. Huh7.5.1-8 cells had approximately 10-fold higher HCV replication rates, with cellular HCV RNA copy numbers of >10(9) copies/μg of cellular RNA and viral titers of >10(6) infectious units/ml of culture supernatant. Permissiveness of Huh7.5.1-8 cells to HCV infection was phenotypically very stable because there was no difference in permissiveness after more than 100 passages (1-year culture). This efficient cell culture system for HCV using Huh7.5.1-8 cell provides a powerful tool for studying the HCV life cycle and constructing antiviral strategies.

It is well-established that CD28 co-stimulation is required for the development and the proliferation of thymus-derived regulatory T cells (tTregs). Meanwhile, the role of CD28 co-stimulation in the homeostasis of peripherally derived Tregs (pTregs) remains unclear. To clarify this issue, we analyzed Tregs in small and large intestines (SI and LI), the principle sites of pTreg development. Interestingly, and different from in the thymus, Tregs were abundant in the intestines of CD28−/− mice, and most of them were phenotypically pTregs. We showed that CD28−/− naive T cells differentiated into pTregs in the LI after oral exposure to antigens and that CD28−/− pTregs in the LI had the same highly proliferative activity as CD28+/− cells. CD28−/− pTregs acquired these Treg-specific features at transcriptional and epigenetics levels. On the other hand, some immune suppressive molecules were down-regulated in CD28−/− pTregs. Correspondingly, the suppressive activity of CD28−/− pTregs was weaker than CD28+/+ cells. These results indicate that the homeostasis of pTregs in the intestines is maintained even in the absence of CD28, whereas CD28 is required for the maximal suppressive activity of intestinal pTregs.

Melanoma is highly malignant, and generally exhibits radioresistance, responding poorly to radiation therapy. We previously reported that activation of P2X7, P2Y6, and P2Y12 receptors is involved in the DNA damage response after γ-irradiation of human lung adenocarcinoma A549 cells. However, it is not clear whether these receptors are also involved in the case of melanoma cells, although P2X7 receptor is highly expressed in various cancers, including melanoma. Here, we show that P2X7 receptor antagonist enhances radiation-induced cytotoxicity in B16 melanoma cells in vitro and in vivo. We confirmed that these cells express P2X7 receptor mRNA and exhibit P2X7 receptor-mediated activities, such as ATP-induced pore formation and cytotoxicity. We further examined the radiosensitizing effect of P2X7 receptor antagonist Brilliant Blue G (BBG) in vitro by colony formation assay of B16 cells. γ-Irradiation dose-dependently reduced cell survival, and pretreatment with BBG enhanced the radiation-induced cytotoxicity. BBG pretreatment also decreased the number of DNA repair foci in nuclei, supporting involvement of P2X7 receptor in the DNA damage response. Finally, we investigated the radiosensitizing effect of BBG on B16 melanoma cells inoculated into the hind footpad of C57BL/6 mice. Neither 1 Gy γ-irradiation alone nor BBG alone suppressed the increase of tumor volume, but the combination of irradiation and BBG significantly suppressed tumor growth. Our results suggest that P2X7 receptor antagonist BBG has a radiosensitizing effect in melanoma in vitro and in vivo. BBG, which is used as a food coloring agent, appears to be a promising candidate as a radiosensitizer.

The aim of this study was to investigate the anticarcinogenic effects of silymarin in diethylnitrosamine (DEN)-induced hepatocarcinogenic rat models. Severe and mild models of hepatocellular carcinoma (HCC) were generated by the intraperitoneal administration of 40 mg/kg DEN once a week for 18 weeks and 100 mg/kg DEN every 2 weeks for 6 weeks in male Wistar rats, respectively. In the severe and mild models of HCC, the rats were treated with 0.1 and 0.5% silymarin for 18 weeks and with 0.1% silymarin for 5 weeks, respectively. Serum transaminase levels were not significantly decreased by the silymarin treatment in either model. Macroscopic and microscopic features indicated that the silymarin-containing formulations did not significantly inhibit the hepatic tumor formation induced by DEN. Furthermore, immunohistochemical and western blot analyses demonstrated that the expression levels of proliferating cell nuclear antigen and glutathione S-transferase P, which are hepatocarcinogenic markers, were not significantly modified by the silymarin treatment. These results indicate that silymarin may not be considered as a candidate agent against hepatocarcinogenesis.