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Tristrontium aluminate (SExtracts of SSS

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PDF file - 32KB, Effects of T-3256336 in MDA-MB-231-Luc mice model

PDF file - 61KB, T-3256336 induced TNF-� dependent apoptosis in MDA-MB-231 cancer cells

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Immune checkpoint inhibitors (ICIs) are widely used to treat various malignancies. Although the gut microbiome is known to influence the efficacy of ICIs on epithelial tumors, the functional interactions between gut taxa and colonic mucosa remain poorly understood. Here we performed transcriptomic profiling and 16S rRNA sequencing to investigate the relationships between mucosal gene expression and microbial composition with ICI responses and gastrointestinal immune-related adverse events (GI irAEs). In responders, genes related to DNA repair and cell cycle signatures were enriched in responders whereas signatures related to innate immune response, NFAT and IFN-γ signaling pathways were enriched in nonresponders. Gut microbial composition revealed an association between moderate GI irAE and favorable response to ICI therapy. Favorable therapeutic responses to ICI and GI irAE treatments were associated with taxa classified as Enterobacteriaceae and were related to ribonucleoprotein complex biogenesis, cytokine-mediated signaling pathway, tRNA metabolic process, and ribonucleoprotein complex assembly in the colon. These findings open new perspectives for improving the efficacy and safety of cancer immunotherapy.

Tyrosine kinase inhibitors (TKIs) that target the ROS proto-oncogene 1, receptor tyrosine kinase (ROS1) gene have shown dramatic therapeutic effects in patients with ROS1-rearranged non-small-cell lung cancer (NSCLC). Nevertheless, advanced ROS1-rearranged NSCLC is rarely cured as a portion of the tumor cells can survive the initial stages of ROS1-TKI treatment, even after maximum tumor shrinkage. Therefore, understanding the mechanisms underlying initial cell survival during ROS1-TKI treatment is necessary to prevent cell survival and achieve a cure for ROS1-rearranged NSCLC. In this study, we clarified the initial survival mechanisms during treatment with lorlatinib, a ROS1 TKI. First, we established a patient-derived ezrin gene-ROS1-rearranged NSCLC cell line (KTOR71). Then, following proteomic analysis, we focused on yes-associated protein 1 (YAP1), which is a major mediator of the Hippo pathway, as a candidate factor involved in cell survival during early lorlatinib treatment. Yes-associated protein 1 was activated by short-term lorlatinib treatment both in vitro and in vivo. Genetic inhibition of YAP1 using siRNA, or pharmacological inhibition of YAP1 function by the YAP1-inhibitor verteporfin, enhanced the sensitivity of KTOR71 cells to lorlatinib. In addition, the prosurvival effect of YAP1 was exerted through the reactivation of AKT. Finally, combined therapy with verteporfin and lorlatinib was found to achieve significantly sustained tumor remission compared with lorlatinib monotherapy in vivo. These results suggest that YAP1 could mediate initial cell resistance to lorlatinib in KTOR71 cells. Thus, combined therapy targeting both YAP1 and ROS1 could potentially improve the outcome of ROS1-rearranged NSCLC.

Immune checkpoint therapy (ICT) with nivolumab has been widely used to treat malignant pleural mesothelioma (MPM) since clinical trials confirmed its efficacy. However, only a few clinical trials have been conducted for the treatment of sarcomatoid MPM, which is a rare histological type of MPM. Additionally, clinical reports of sarcomatoid MPM are scarce. Therefore, the benefits and risks of nivolumab treatment for sarcomatoid MPM remain unclear. The present report describes the treatment of 3 cases of sarcomatoid MPM (all 3 were men) with nivolumab monotherapy. In all three cases, nivolumab was effective despite variations in the duration of treatment, although side effects were observed in 2 patients. Programmed death ligand 1 (PD-L1) expression was positive in all 3 cases. In particular, the patient with the highest PD-L1 expression had the most rapid response of the 3 patients, and the effect lasted as long as those of the other 2, despite receiving the smallest number of doses of nivolumab. It has been reported that sarcomatoid MPM tends to respond poorly to chemotherapy and express higher levels of PD-L1 than epithelial MPM; thus, ICT may be necessary in these cases. This case series suggests that ICT with nivolumab is a promising treatment option for sarcomatoid MPM.

Increased expression of the transient receptor potential ankyrin 1 (TRPA1) channel has been detected in carious tooth pulp, suggesting involvement of TRPA1 in defense or repair of this tissue after exogenous noxious stimuli. This study aimed to elucidate the induction mechanism in response to lipopolysaccharide (LPS) stimulation and the function of TRPA1 in dental pulp cells. Stimulation of human dental pulp cells with LPS up-regulated TRPA1 expression, as demonstrated by quantitative RT-PCR and Western blotting. LPS stimulation also promoted nitric oxide (NO) synthesis and p38/mitogen-activated protein kinase (MAPK) phosphorylation. NOR5, an NO donor, up-regulated TRPA1 expression, whereas 1400W, an inhibitor of inducible nitric oxide synthase, and SB202190, a p38/MAPK inhibitor, down-regulated LPS-induced TRPA1 expression. Moreover, JT010, a TRPA1 agonist, increased the intracellular calcium concentration and extracellular signal-regulated kinase 1/2 phosphorylation, and up-regulated alkaline phosphatase mRNA in human dental pulp cells. Icilin-a TRPA1 agonist-up-regulated secreted phosphoprotein 1 mRNA expression and promoted mineralized nodule formation in mouse dental papilla cells. In vivo expression of TRPA1 was detected in odontoblasts along the tertiary dentin of carious teeth. In conclusion, this study demonstrated that LPS stimulation induced TRPA1 via the NO-p38 MAPK signaling pathway and TRPA1 agonists promoted differentiation or mineralization of dental pulp cells.

Hypoxia-inducible factor 1 alpha (HIF1α) is a transcriptional factor that plays a key role in the regulation of various molecules expressed in hypoxic conditions. Ischemic/hypoxic conditions are regarded as a distinct characteristic of dental pulp inflammation due to the encasement of pulp tissue within the rigid tooth structure. This study was performed to examine the role of HIF1α in the regulation of interleukin (IL)-6, a proinflammatory cytokine expressed in inflamed dental pulp, in lipopolysaccharide (LPS)-stimulated human dental pulp cells (hDPCs). LPS stimulation promoted the expression of IL-6 in hDPCs, while HIF1α suppressed the expression of IL-6. Moreover, HIF1α induced suppressor of cytokine signaling 3 (SOCS3) expression in LPS-stimulated hDPCs, and SOCS3 activity led to downregulate expression of CCAAT enhancer-binding protein beta (CEBPβ), an inducer of IL-6. LPS stimulation promoted HIF1α expression in hDPCs and mouse pulp tissue explants cultured under hypoxic conditions. These findings suggest that HIF1α negatively regulates IL-6 synthesis in LPS-stimulated hDPCs via upregulation of SOCS3 and subsequent downregulation of CEBPβ.

In this paper, we present vehicle velocity control based on stochastic model predictive control applied to an actual automobile near other vehicles with uncertain motion. Modern external sensors can measure the pose and velocity of transportation participants, whose motion can be predicted utilizing a model; however, observation and process noise results in uncertainty. Thus, near other vehicles, the velocity of the ego vehicle should be reduced to account for the variance of nearby vehicle motion while suppressing the reduction of speed. In this paper, we utilize stochastic model predictive control to reduce the expectation of relative velocity with respect to nearby vehicles during passing. We evaluate the proposed control using numerical simulation and an experiment with an automobile developed for self-driving and equipped with GNSS, radar, and LiDAR. The results show that the vehicle velocity is automatically reduced based on the expectation of relative velocity calculated from its probabilistic distribution.

microRNAs are small noncoding RNA molecules that regulate RNA silencing and posttranscriptional gene expression, and many microRNAs are involved in inflammatory processes. In particular, microRNA 21 (miR-21) is upregulated in inflammatory environment and reported to induce anti-inflammatory responses. However, the involvement of miR-21 in pulpal inflammation and the precise mechanisms of anti-inflammatory reactions induced by miR-21 remain unclear. We hypothesized that miR-21-5p expression is induced in lipopolysaccharide (LPS)-stimulated human dental pulp cells (hDPCs) and that miR-21-5p downregulates the proinflammatory cytokine expression in LPS-stimulated hDPCs. We found that miR-21-5p was upregulated in LPS-stimulated hDPCs concomitant with elevated proinflammatory cytokine expression and nuclear factor-kappa B (NF-κB) phosphorylation. miR-21-5p and cytokine expression were downregulated by BAY11-7085 and caffeic acid phenylethyl ester (CAPE), specific and potent NF-κB inhibitors. Enforced expression of miR-21-5p downregulated the Toll-like receptor (TLR)/NF-κB signaling via reducing the expression of TNF receptor-associated factor 6 (TRAF6) and programmed cell death 4 (PDCD4), which further induced the decrease of proinflammatory cytokine expression. hDPCs forcibly overexpressing miR-21-5p downregulated the LPS-induced expression of TNF receptor-associated factor 6 (TRAF6; a component of the Toll-like receptor [TLR]/NF-κB signaling pathway), programmed cell death 4 (PDCD4, a positive regulator of the TLR/NF-κB signaling pathway), and proinflammatory cytokines. In contrast, miR-21-5p inhibitor-transfected hDPCs upregulated the expression of TRAF6, PDCD4, and inflammatory cytokines following LPS stimulation. These findings suggest that miR-21-5p expression was induced by the NF-κB signaling pathway, which was in turn negatively regulated by miR-21-5p via downregulation of TRAF6 and PDCD4 expression in LPS-stimulated hDPCs.

Dental pulp stem cells (DPSCs) are a good source for tissue regeneration, however, the number of DPSCs in the pulp tissue is limited. Cell propagation is essential for tissue engineering using DPSCs and the cell culture conditions may affect the properties of DPSCs. The purpose of this study was to analyze the effect of cell culture condition, especially dense culture condition, on the property and differentiation pathway of DPSCs. We cultured DPSCs under sparse (sDPSCs; 5 × 103 cells/cm2) or dense (dDPSCs; 1 × 105 cells/cm2) conditions for 4 days and compared their properties. The populations of CD73+ and CD105+ cells were significantly decreased in dDPSCs. Both groups showed multi-differentiation potential, but mineralized nodule formation was enhanced in dDPSCs. The phosphorylation of focal adhesion kinase (FAK) and phosphoinositide 3-kinase (PI3K) proteins was promoted in dDPSCs, and alkaline phosphatase (ALP) mRNA expression in dDPSCs was abolished in the presence of pan-PI3K and FAK inhibitors. dDPSCs implanted into mouse bone cavities induced more mineralized tissue formation than sDPSCs and control. These findings indicate that dense culture conditions modified the properties of DPSCs and gave rise to osteogenic-lineage commitment via integrin signaling and suggest that dense culture conditions favor the propagation of DPSCs to be used for mineralized tissue regeneration.

This study compared the cytocompatibilities of three methacrylate resin-based root canal sealers [MetaSEAL Soft (MSS), Hybrid Root SEAL (HRS), and Superbond Sealer (SBS)] in either freshly mixed or set conditions using the Kusa A1 osteoblastic cell line. The three sealers and an epoxy resin-based sealer (AH Plus; AHP) were extracted in culture medium; cell growth and osteogenic properties were analyzed. Cell adhesion on set sealers was analyzed with scanning electron microscopy. The respective extents of cell growth were as follows in freshly mixed and set sealer extracts: SBS>MSS>AHP>HRS and SBS=AHP>MSS>HRS. Light irradiation of MSS and HRS increased the cell growth of set sealer extracts. Set SBS, MSS, and AHP did not alter expression of osteogenic genes or formation of mineralized nodules. Attached cells were observed only on SBS. In conclusion, the four sealers exhibited varying degrees of compatibility to osteoblasts; SBS and HRS were the most and least compatible, respectively.

Overcoming the treatment resistance of cancer is a problem to be solved in improving the prognosis of cancer patients. Recently, genome analysis has revealed that multiple clusters exist in one tumor, and it has been reported that intratumor heterogeneity causes treatment resistance even in lung cancer. However, the mechanism of intratumor heterogeneity has not yet been clarified. Previous studies of intratumor heterogeneity have mainly focused on analyzing cancer cells from patients, classifying clusters of different properties, and identifying factors that define their characteristics. Multiple clusters are present in the tumor tissue of a single patient, and analysis of multiple patients classifies them into even more clusters, but it is difficult to research all of them. To solve this problem, an early model of intratumor heterogeneity in which homogeneous cells become heterogeneous is needed.We hypothesized that intratumor heterogeneity cell model could be found in resistant strains.Alectinib was exposed in vitro to an ALK-positive lung cancer cell line (H2228) to create an alectinib-resistant cell line (H2228-AR1S). H2228-AR1S has two subpopulations that look different. Of the two subpopulations, the smaller cells have high CD47 expression (CD47 high subpopulation), and the scattered spindle-shaped cells have low CD47 expression (CD47 low subpopulation). Using flow cytometry, each subpopulation was isolated, and its properties were investigated. There was no difference in sensitivity to alectinib between the CD47 high subpopulation and the CD47 low subpopulation. In the low subpopulation of CD47, epithelial markers were decreased, and mesenchymal markers were increased using immunoblotting. It suggests that CD47 low subpopulation has undergone epithelial-mesenchymal transition (EMT). The CD47 high subpopulation had high sphere formation ability in vitro, and high tumorigenicity using Xenograft model. CD47 gene inhibition using siRNA reduced the sphere formation ability of the CD47 high subpopulation. This suggests that CD47 is involved in sphere formation.There have been no reports of CD47 being involved in the characteristics of intratumor heterogeneity. Furthermore, using intratumor heterogeneity cell model, we are exploring the mechanism that regulate division into two subpopulations. Citation Format: Tomoko Y. Funazo, Hiroaki Ozasa, Kentaro Hashimoto, Hiroshi Yoshida, Tatsuya Ogimoto, Kazutaka Hosoya, Hitomi Ajimizu, Takahiro Tsuji, Hironori Yoshida, Ryo Itotani, Yuichi Sakamori, Toyohiro Hirai. CD47 related to intratumor heterogeneity in alectinib-resistant ALK-rearranged lung cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1601.

Background: EGFR mutation-positive lung cancer has high response rates to EGFR-tyrosine kinase inhibitors (TKIs), but eventually acquires resistance to EGFR-TKIs. Acquired resistance mechanisms are diverse because of tumor heterogeneity, which makes it difficult to overcome acquired resistance. Initial resistance is the resistance mechanism of tumor cells that survive the initial treatment. If we could overcome the initial resistance, we could prevent cancer cells from developing various types of acquired resistance. We are now focusing on YAP1 and cancer stem cells, which have been also reported as acquired resistance mechanisms in various types of cancers. Methods: PC-9 cells and HCC827 cells which were EGFR mutation-positive lung cancer cell lines were mainly utilized in our experiments; they were purchased from ECACC and ATCC, respectively. We mainly utilized osimertinib as an EGFR-TKI and verteporfin as a YAP1 inhibitor. The nuclear translocation of YAP1 was confirmed by fluorescence immunostaining. siRNA was utilized to knock down YAP1. Cell viability assays were performed using CellTiter-Glo reagent. qRT-PCR was performed to confirm the gene expression of cancer stem cells. Results: YAP1 is activated by nuclear translocation. In PC-9 cells, YAP1 was localized in the nucleus after osimertinib exposure. In contrast, YAP1 was localized in the nucleus of HCC827 cells before osimertinib exposure and remained in the nucleus even after osimertinib exposure. These data suggest that YAP1 activation is correlated with initial resistance. Cell viability assay showed that both PC-9 cells and HCC827 cells became more sensitive to osimertinib with the knocking down of YAP1. In addition, cell viability assays using PC-9 cells with verteporfin and osimertinib showed increased sensitivity to osimertinib. The gene expression of ALDH1A1 and SOX2 which are involved in cancer stem cells were increased in PC-9 cells after osimertinib exposure. On the other hand, the gene expression of ALDH1A1 was up-regulated but SOX2 was down-regulated in HCC827 cells after osimertinib exposure. We hypothesized that increased gene expression of cancer stem cells occurred downstream of YAP1 followed by initial resistance, thus we are currently confirming the gene alteration of cancer stem cells associated with osimertinib exposure with the knocking down YAP1. We are also considering the investigation of combination therapy of EGFR-TKIs and YAP1 inhibitors in vivo studies. Conclusions: YAP1 and cancer stem cells may be involved in initial resistance mechanisms to EGFR-TKIs in EGFR mutation-positive lung cancer. Our results suggest that especially YAP1 can be a potential therapeutic target, thus we will continue additional studies of combination therapy of EGFR-TKIs and YAP1 inhibitors. In addition, we will continue to investigate whether the gene for cancer stem cells acts downstream of YAP1. Citation Format: Tatsuya Ogimoto, Hiroaki Ozasa, Kentaro Hashimoto, Hiroshi Yoshida, Kazutaka Hosoya, Masatoshi Yamazoe, Hitomi Ajimizu, Tomoko Funazo, Takahiro Tsuji, Hironori Yoshida, Ryo Itotani, Yuichi Sakamori, Toyohiro Hirai. Elucidation of initial resistance mechanisms in EGFR mutation-positive lung cancer focusing on YAP1 and cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5335.

Streptomyces incarnatus NRRL8089 produces the antiviral, antifungal, antiprotozoal nucleoside antibiotic sinefungin. To enhance sinefungin production, multiple mutations were introduced to the rpoB gene encoding RNA polymerase (RNAP) β-subunit at the target residues, D447, S453, H457, and R460. Sparse regression analysis using elastic-net lasso-ridge penalties on previously reported H457X mutations identified a numeric parameter set, which suggested that H457R/Y/F may cause production enhancement. H457R/R460C mutation successfully enhanced the sinefungin production by 3-fold, while other groups of mutations, such as D447G/R460C or D447G/H457Y, made moderate or even negative effects. To identify why the rif cluster residues have diverse effects on sinefungin production, an RNAP/DNA/mRNA complex model was constructed by homology modeling and molecular dynamics simulation. The 4 residues were located near the mRNA strand. Density functional theory–based calculation suggested that D447, H457, and R460 are in direct contact with ribonucleotide, and partially positive charges are induced by negatively charged chain of mRNA.

AIM To elucidate mechanisms by which mineral trioxide aggregate (MTA) suppresses pro-inflammatory cytokine mRNA expression in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. METHODOLOGY Mineral trioxide aggregate extracts were prepared by immersing set ProRoot MTA in culture medium. RAW264.7 cells were cultured in the presence of LPS and MTA extracts. mRNA expression levels of interleukin (IL)-1α, IL-6, early growth response 2 (Egr2), suppressor of cytokine signalling 3 (Socs3) and IL-10 were quantified with reverse transcription-quantitative polymerase chain reaction. Phosphorylation of nuclear factor-kappa B (NF-κB) p65 in RAW264.7 cells was analysed by Western blotting. Intracellular calcium imaging was performed with Fluo-4 AM. The activity of nuclear factor of activated T cells (NFAT) was determined by luciferase assays. Enforced expression and silencing of Egr2 in RAW264.7 cells were carried out using an expression vector and specific RNAi, respectively. In vivo kinetics of Egr2+ cells in MTA-treated rat molar pulp tissues were examined using immunohistochemistry. Data were analysed by one-way analysis of variance, followed by the Tukey-Kramer test (P

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

AIM To elucidate the role of HIF1α in pro-inflammatory cytokine mRNA expression from lipopolysaccharide (LPS)-stimulated human dental pulp cells (hDPCs). METHODOLOGY mRNA expression of interleukin (IL) 1β and tumour necrosis factor (TNF) α in LPS-stimulated hDPCs was determined by quantitative RT-PCR. Expression of nuclear factor kappa B (NFκB) p65 and phospho-NFκB p65 was analysed by Western blotting. Activation of NFκB signalling was measured by luciferase assay using a reporter vector containing an NFκB response element. Enforced expression of HIF1α was induced by transfection of expression vectors with native or constitutively active forms of HIF1α. Expression of HIF1α protein in hDPCs was evaluated by immunocytochemistry and Western blotting. One-way analysis of variance and the Tukey-Kramer test were performed to determine a significant difference (P

Inhibitors of apoptosis proteins (IAPs) are antiapoptotic regulators that block cell death, and are frequently overexpressed in several human cancers, where they facilitate evasion of apoptosis and promote cell survival. IAP antagonists are also known as second mitochondria-derived activator of caspase (SMAC)-mimetics, and have recently been considered as novel therapeutic agents for inducing apoptosis, alone and in combination with other anticancer drugs. In this study, we showed that T-3256336, the orally available IAP antagonist has synergistically enhances the antiproliferative effects of the NEDD8-activating enzyme (NAE) inhibitor pevonedistat (TAK-924/MLN4924), and these effects were attenuated by a TNFα-neutralizing antibody. In the present mechanistic analyses, pevonedistat induced TNFα mRNA and triggered IAP antagonist-dependent extrinsic apoptotic cell death in cancer cell lines. Furthermore, synergistic effects of the combination of T-3256336 and pevonedistat were demonstrated in a HL-60 mouse xenograft model. Our findings provide mechanistic evidence of the effects of IAP antagonists in combination with NAE inhibitors, and demonstrate the potential of a new combination therapy for cancer.

Cubic silicon carbide (3C-SiC) has attracted much attention since it has a wide bandgap, high dielectric breakdown strength, and high thermal stability. 3C-SiC promises various electronic applications such as power devices and MEMS. Also, 3C-SiC has an advantage for a lower cost among many SiC polytypes, as it can grow epitaxially on Si substrate. Generally, two types of precursor source materials, including Si and C, are necessary to grow 3C-SiC thin films for chemical vapor deposition (CVD) method. In addition, complex processes including carbonization with a temperature as high as about 1200 °C are required for conventional epitaxial growth of 3C-SiC [1]. We focus on vinylsilane as a single precursor material for SiC growth. The Si-C bond in vinylsilane is relatively stable due to the relationship of intramolecular bond energy, that is appreciable for SiC growth. The growth of SiC crystals using vinylsilane can be achieved at a low temperature of 1000 ºC or less and the deposition process will be simplified. Recently, we have achieved the formation of polycrystalline SiC thin films on metal substrate using CVD method only with vinylsilane [2]. In this study, we examine the epitaxial growth of 3C-SiC and also report phosphorous (P) doping for SiC thin films grown on Si substrate. SiC thin films were deposited on Si(001) and Si(111) substrates using CVD method with a vinylsilane precursor (manufactured by Japan Advanced Chemicals Co., Ltd.). The substrate temperature of the growth was ranging from 900 to 1000 °C, the growth pressure was 17 kPa, and the growth time was 60 min. The surface morphology of deposited film was observed using scanning electron microscopy (SEM). We can confirm that the uniform morphology of SiC film was formed on the Si substrate for a sample prepared with Si(111) substrate at 1000 ºC and also that there are no voids or defects at the interface. We also examined X-ray diffraction measurements to characterize the crystalline structure. Out-of-plane XRD measurement (2θ/ω method) shows a sufficiently large diffraction peak related to 3C-SiC(111) for the Si(111) sample prepared at 1000 ºC, which means the growth of a well (111)-oriented 3C-SiC thin film on Si(111) substrate. On the other hand, in the case of the grazing angle XRD measurement (2θ method) for the SiC/Si(001) samples prepared at both 900 and 1000 ºC, we observed many diffraction peaks meaning the formation of polycrystalline 3C-SiC. The analysis of peak intensity and full width at half maximum value (FWHM) revealed that the crystallinity of SiC film improved with increasing the growth temperature. Raman scattering spectroscopy measurement also revealed that the dominant bonding structure in the film is Si-C and not C-Hn or C-C for samples prepared at higher than 900 ºC, that means the formation of a high crystalline quality SiC thin film. In our presentation, we will discuss pros and cons of epitaxial growth of SiC films on Si(111) substrates and also reports P-doping into the SiC films will be discussed. References [1] Z. Zhao, Y. Li, Z. Yin, and Z. Li, J. Mater. Sci: Mater. Electron. 27, 7095 (2016). [2] T. Doi, W. Takeuchi, Y. Jin, H. Kokubun, S. Yasuhara, O. Nakatsuka, and S. Zaima, Jpn. J. Appl. Phys. 57, 01AE08 (2018).

Surface‑reaction‑type prereacted glass-ionomer (S‑PRG) fillers exhibit bioactive properties by the release of multiple ions. This study examined whether a novel endodontic sealer containing S‑PRG fillers (PRG+) has the capacity to induce osteoblast differentiation. Kusa‑A1 osteoblastic cells were cultured with extracts of PRG+, PRG&minus, (an experimental sealer containing S‑PRG‑free silica fillers), AH Plus (an epoxy-resin‑based sealer), and Canals N (a zinc-oxide noneugenol sealer). Cell viability and mineralized nodule formation were determined using WST‑8 assay and Alizarin red staining, respectively. Osteoblastic-marker expression was analyzed with RT‑qPCR and immunofluorescence. Phosphorylation of extracellular signal‑regulated kinase (ERK) and p38 mitogen‑activated protein kinase (MAPK) was determined with Western blotting. Extracts of freshly mixed PRG+, PRG&minus, and AH Plus significantly decreased cell growth, but extracts of the set samples were not significantly cytotoxic. Set PRG+ significantly upregulated mRNAs for alkaline phosphatase and bone sialoprotein (IBSP) compared to set PRG&minus, and upregulation was blocked by NPS2143, a calcium‑sensing receptor antagonist. Set PRG+ significantly accelerated IBSP expression, mineralized nodule formation, and enhanced the phosphorylation of ERK and p38 compared with set PRG&minus, In conclusion, PRG+ induced the differentiation and mineralization of Kusa‑A1 cells via the calcium-sensing receptor-induced activation of ERK and p38 MAPK.

We investigated the impact of defects formed at the SiO2/4H-SiC interface on the Schottky barrier height of metal/4H-SiC(0001) contacts. We found that an ultra-thin SiC x O y layer remains on the 4H-SiC surface after SiO2 sputtering at various powers and its removal by diluted hydro fluoride solution. Ni, Mo, or Al was deposited on 4H-SiC surface without and with a residual SiC x O y layer. It was found that metal/4H-SiC contacts without a residual SiC x O y layer exhibit ideal Schottky property, while Fermi-level pinning (FLP) is caused for metal/4H-SiC contacts with a residual SiC x O y layer, and the degree of FLP increases increasing sputtering power of SiO2. The pinning position was estimated to be ~0.8 eV below the conduction band minimum of 4H-SiC, which does not correspond to the charge neutrality level of 4H-SiC. Finally, we proposed a physical model where a SiC x O y interlayer causes FLP, and the model was experimentally verified by intentionally forming a SiC x O y interlayer.

Different crystal packing of hydrates from anhydrate crystals leads to different physical properties, such as solubility and stability. Investigation of the potential of varied hydrate formation, and understanding the stability in an anhydrous/hydrate system, are crucial to prevent an undesired transition during the manufacturing process and storage. Only one anhydrous form of T-3256336, a novel inhibitor of apoptosis (IAP) protein antagonist, was discovered during synthesis, and no hydrate form has been identified. In this study, we conducted hydrate screening such as dynamic water vapor sorption/desorption (DVS), and the slurry experiment, and characterized the solid-state properties of anhydrous/hydrate forms to determine the most desirable crystalline form for development. New hydrate forms, both mono-hydrate and hemi-hydrate forms, were discovered as a result of this hydrate screening. The characterization of two new hydrate forms was conducted, and the anhydrous form was determined to be the most desirable development form of T-3256336 in terms of solid-state stability. In addition, the stability of the anhydrous form was investigated using the water content and temperature controlled slurry experiment to obtain the desirable crystal form in the crystallization process. The water content regions of the stable phase of the desired form, the anhydrous form, were identified for the cooling crystallization process.

This study examined the effects and mechanisms of strontium ranelate (SrRn)—a drug used to treat osteoporosis—on the proliferation and differentiation/mineralization of cloned dental pulp-like cells (mouse dental papillae cells; MDPs). It also determined whether topical application of SrRn to exposed dental pulp tissue promotes the formation of mineralized tissue in vivo. The MDPs were cultured with or without SrRn, and cell proliferation, odonto-/osteoblastic gene expression, mineralized nodule formation, and Akt phosphorylation were evaluated. The formation of mineralized tissue in SrRn-treated pulp tissue in rat upper first molars was evaluated histologically. The SrRn up-regulated cell proliferation and expression of Alp (alkaline phosphatase), Bsp (bone sialoprotein), Dmp (dentin matrix acidic phosphoprotein)-1, Dspp (dentin sialophosphoprotein), and Oc (osteocalcin) in a dose-dependent manner. Mineralized nodule formation was also enhanced by SrRn. NPS-2143, a calcium-sensing receptor (CaSR) antagonist, and siRNA against the CaSR gene blocked SrRn-induced proliferation, odonto-/osteoblastic gene expression, and mineralized nodule formation. SrRn induced Akt phosphorylation, and this was blocked by NPS-2143. Topical application of SrRn to exposed rat molar pulps induced the formation of osteodentin-like mineralized tissue. Our study revealed for the first time that SrRn promotes proliferation and odonto-/osteogenic differentiation/mineralization of MDPs via PI3K/Akt signaling activated by CaSR in vitro; mineralized tissue forms from the dental pulp in vivo.

The disturbance of cellular attachment to dentin by sodium hypochlorite (NaOCl) may hamper pulp tissue regeneration. The aims of this study were to examine the recovering effect of EDTA on the attachment/differentiation of stemlike cells and to address the mechanisms of EDTA-induced recovery under the hypothesis that attachment to the exposed dentin matrix and the subsequent activation of integrin/phosphatidylinositol 3-kinase (PI3K) signaling play a crucial role.Mouse dental papilla (MDP) cells were cultured on bovine dentin disks treated with NaOCl (0%, 1.5%, or 6%) followed by EDTA (0%, 3%, or 17%). Cell attachment was evaluated by cell density, viability, and scanning and transmission electron microscopy. Odonto-/osteoblastic gene expression in attached MDP cells was analyzed with or without a pan-PI3K inhibitor (LY294002) using real-time polymerase chain reaction.NaOCl treatment (1.5%, 10 minutes) significantly diminished attached MDP cells (P .00001), but EDTA treatment (3% and 17%, ≥10 minutes) of NaOCl-pretreated dentin induced a significant increase in attached cells (P .05). Ultrastructurally, MDP cells on EDTA-treated dentin showed attachment to exposed collagen fibers. MDP cells cultured on EDTA-treated disks (with or without 1.5% NaOCl pretreatment) showed significant up-regulation of alkaline phosphatase, dentin matrix protein 1, and dentin sialophosphoprotein messenger RNAs (P .05). Alkaline phosphatase expression was down-regulated by LY294002 (P .05).Under the present experimental conditions, 10 minutes of EDTA treatment was sufficient to recover attachment/differentiation of MDP cells on 1.5% NaOCl-pretreated dentin. EDTA-induced exposure of collagen fibers and subsequent activation of integrin/PI3K signaling may contribute, at least partly, to the recovery.

Crizotinib is one of the molecularly-targeted agents targeted against anaplastic lymphoma kinase (ALK)-rearranged non-small-cell lung cancer (NSCLC). Although its effects appear to be promising, crizotinib may cause adverse effects in patients with ALK-rearranged NSCLC. Hepatic laboratory abnormalities are frequently observed with crizotinib and treatment discontinuation is occasionally required. We herein report the case of a 51-year-old woman diagnosed with relapsed ALK-rearranged NSCLC, who received crizotinib as second-line systemic chemotherapy. After 17 days of crizotinib therapy, the patient developed grade >3 hepatotoxicity. Treatment discontinuation improved the laboratory abnormalities and fifth-line oral desensitization with crizotinib achieved successful response without hepatotoxicity. Therefore, oral desensitization with crizotinib may be a viable option following crizotinib-induced hepatitis.

We recently reported the discovery of octahydropyrrolo[1,2-a]pyrazine A as a lead compound for an inhibitor of apoptosis proteins (IAP) antagonist. To develop IAP antagonists with favorable PK profiles, we designed novel tri-cyclic compounds, octahydro-1H-cyclopropa[4,5]pyrrolo[1,2-a]pyrazines 1 and 2 based on co-crystal structural analysis of A with cellular IAP-1 (cIAP-1). The additional cyclopropane moiety was used to block the predicted metabolic site of compound A without detriment to the binding affinity for cIAP. Compounds 1 and 2 were stereoselectively synthesized via intermediates 4a and 5b', which were obtained by Simmons-Smith cyclopropanation of ethylester 3a and silyl ether 3b'. Compounds 1 and 2 showed strong growth inhibition in MDA-MB-231 breast cancer cells and improved metabolic stability in comparison to A. Compound 2 exhibited significant in vivo PD effects to increase tumor necrosis factor-alpha mRNA in a dose dependent manner.

Inhibitor of apoptosis proteins (IAP), which are key regulators of apoptosis, are inhibited by second mitochondria-derived activator of caspase (SMAC). Small-molecule IAP antagonists have recently been reported as novel therapeutic treatments for cancer. In this study, we showed that the octahydro-pyrrolo[1,2-a]pyrazine derivative, T-3256336, is a novel and orally available small-molecule IAP antagonist. T-3256336 selectively binds to and antagonizes protein interactions involving cellular IAP-1 (cIAP-1), cIAP-2, and X-linked IAP (XIAP). T-3256336 induced the rapid proteasomal degradation of cIAP-1 and activated TNF-α–dependent extrinsic apoptosis signaling in cultured cells. In a MDA-MB-231-Luc breast cancer xenograft model, T-3256336 induced cIAP-1 degradation, TNF-α production, and caspase activation in tumors, which resulted in strong antitumor activities. T-3256336 induced increases in the plasma levels of TNF-α and fragmented cytokeratin-18, which correlated with the antitumor potency in MDA-MB-231-Luc xenograft models. This study provided further insights into biomarkers of IAP antagonists. Furthermore, our data provided evidence that T-3256336 is a promising new anticancer drug worthy of further evaluation and development. Mol Cancer Ther; 12(2); 230–40. ©2012 AACR.

Inhibitors of apoptosis proteins (IAPs) are a family of antiapoptotic regulators that have attracted attention as potential targets for cancer therapeutics. Although recent studies have revealed that small-molecule IAP antagonists induce tumor selective cell death in an autocrine tumor necrosis factor (TNF)α-dependent manner, the single-agent efficacy of IAP antagonists is restricted to a small subset of cancer cells. In this study, we showed that the single-agent activity of T-3256336 was limited to a few cancer cell lines in vitro, and these cell lines were defined by relatively high levels of TNFα mRNA expression. However, some other cancer cells, including PANC-1 cells, become drastically sensitive to T-3256336 when costimulated with exogenous TNFα. In PANC-1 mouse xenograft models, the administration of T-3256336 increased levels of several cytokines including TNFα and lead to tumor regression as a single agent, which was attenuated by the neutralization of circulating mouse TNFα with an antibody. These results suggest dual roles of IAP antagonists, increase systemic cytokines including TNFα, and sensitization of tumors to IAP antagonist-induced death.

Topographical, geomagnetic and geochemical survey in and around the Iheya small ridge, which is located on the mid Okinawa trough, are investigated by a surface ship, and a deep towed sensor suite. The active plumes are confirmed by WCIs of MBES from the surface ship in the vicinity of the southern foot of the Iheya small ridge. And a lot of whisker-like signals are also recognized on the water column part of SSS record towed at the depth of 1400m. Since it is cleared that these are signals from whole seawater, they are judged as signals caused by plumes from the sea bottom. Some plumes observed by SSS are located in the plume groups by WCIs of MBES, but others exist outside the group. The plume distributions observed by MBES and SSS are compared with the topography, the geological features estimated from the topography, magnetization structure derived from the 3D geomagnetic inversion and a score diagram of geochemistry which is an indicator of hydrothermal activity (low pH, low ORP, high Turbidity). It is found out that the plume distribution is limited to the distribution area of the latest lava field and also corresponds to the low magnetization area. Distributions of scores of 2 and 3 of geochemistry by which all three or two indicate anomalies, are also limited to the area of the latest lava field and the low magnetization area. The scores 2 and 3 do not necessarily coincide with the plume groups by MBES, but the plume distribution by SSS closely corresponds to both score. Chemical components of the plume by MBES are unclear from this survey, but it is estimated to be gas plume associated with submarine volcanic activities because it corresponds to the location of the latest lava field. On the other hand, the plume by SSS indicates the possibility to be hydrothermal plume because they can well correspond to the score of geochemistry. In the area estimated to be covered with the latest or second latest lava, the magnetization in Layer 2(shallow layer) is slightly high, and Layer 3 and deeper (deep layer) have low magnetizations. From above discussions, there is a possibility that hydrothermal alternation has been progressing.