Search

Diabetic nephropathy (DN) is one of the common microvascular complications of diabetes. Autotaxin (ATX) is an enzyme with lysophospholipase D activity, producing lysophosphatidic acid (LPA). LPA signaling has been implicated in renal fibrosis, thereby inducing renal dysfunction. BBT-877 is an orally administered small molecule inhibitor of ATX. However, its effect on DN has not been studied so far. In this study, we investigated the effect of BBT-877, a novel inhibitor of ATX, on the pathogenesis of DN in a mouse model of streptozotocin (STZ)-induced diabetes. BBT-877 treatment significantly reduced albuminuria, albumin-to-creatinine ratio (ACR), neutrophil gelatinase-associated lipocalin (NGAL), and glomerular volume compared to the STZ-vehicle group. Interestingly, BBT-877 treatment attenuated hyperglycemia and dyslipidemia in STZ-induced diabetes mice. In the liver, the expression levels of β-oxidation-related genes such as PPAR α and CPT1 were significantly decreased in STZ-induced diabetic mice. However, this effect was reversed by BBT-877 treatment. BBT-877 treatment also suppressed mRNA levels of pro-inflammatory cytokines IL-6, MCP-1, and TNF-α and protein levels of fibrotic factors (TGF-β, fibronectin, CTGF, and collagen type Ι alpha Ι (COL1A1)) in the kidneys of STZ-induced diabetic mice. In conclusion, our results indicate that BBT-877 is effective in preventing the pathogenesis of DN by reducing systemic blood glucose levels and inhibiting inflammation and fibrosis in the renal tissue of diabetes mice. These novel findings suggest that inhibition of ATX may be a potential therapeutic target for DN.

Feline hypertrophic cardiomyopathy (HCM) is a common heart disease affecting 10–15% of all cats. Cats with HCM exhibit breathing difficulties, lethargy, and heart murmur; furthermore, feline HCM can also result in sudden death. Among various methods and indices, radiography and ultrasound are the gold standards in the diagnosis of feline HCM. However, only 75% accuracy has been achieved using radiography alone. Therefore, we trained five residual architectures (ResNet50V2, ResNet152, InceptionResNetV2, MobileNetV2, and Xception) using 231 ventrodorsal radiographic images of cats (143 HCM and 88 normal) and investigated the optimal architecture for diagnosing feline HCM through radiography. To ensure the generalizability of the data, the x-ray images were obtained from 5 independent institutions. In addition, 42 images were used in the test. The test data were divided into two; 22 radiographic images were used in prediction analysis and 20 radiographic images of cats were used in the evaluation of the peeking phenomenon and the voting strategy. As a result, all models showed > 90% accuracy; Resnet50V2: 95.45%; Resnet152: 95.45; InceptionResNetV2: 95.45%; MobileNetV2: 95.45% and Xception: 95.45. In addition, two voting strategies were applied to the five CNN models; softmax and majority voting. As a result, the softmax voting strategy achieved 95% accuracy in combined test data. Our findings demonstrate that an automated deep-learning system using a residual architecture can assist veterinary radiologists in screening HCM.

Myeloid-derived suppressor cells (MDSCs) are a heterogenous group of myeloid cells that are highly suppressive to antitumor lymphocyte function and trafficking to the tumor. The accumulation of MDSCs in the tumor immune microenvironment (TIME) makes immunosuppressive TIME and progressive tumor. In fact, despite notable clinical outcomes in cancer immunotherapy, many patients are refractory or relapsed to immune checkpoint inhibitor therapy, wherein obstacles imposed by TIME contribute to a great extent. Here, we show that translationally controlled tumor protein (TCTP) released by dying tumor cells is an immunomodulator crucial to full-blown MDSC accumulation in the TIME in mouse models. We provide evidence that extracellular TCTP mediates recruitment of the polymorphonuclear MDSC (PMN-MDSC) population in the TIME, which is Toll-like receptor-2 (TLR2) dependent. For human translation, we confirmed that human TCTP binds directly to human TLR2 extracellular domain and stimulates human PBMC to produce multiple cytokines. To explore therapeutic applicability, we generated anti-TCTP antibodies for TCTP inhibition. The antibodies successfully neutralized TCTP in molecular and cellular level. The antibodies also suppressed PMN-MDSC accumulation and tumor growth. The combination of anti-TCTP antibody and immune checkpoint inhibitor showed synergistic effect on tumor growth inhibition. In conclusion, extracellular TCTP is an immunomodulator recruiting PMN-MDSCs to TIME. When TCTP is removed or neutralized, PMN-MDSC counts in TIME decreased. Anti-TCTP therapy may offer a new immunotherapy strategy. Citation Format: Jeong-Hyun Ryou, Gwanghee Lee, Tadatsugu Taniguchi, Hideyuki Yanai, Sho Hangai. TCTP is a target for cancer immunotherapy modulating myeloid-derived suppressor 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 242.

This study systematically investigated the feasibility of the microbubble ozonation process to degrade the 17α-ethinylestradiol, ibuprofen, and atenolol through the comparison with the millibubble ozonation process for elucidating the degradation behavior and mechanisms during the microbubble ozonation processes. The proportions of small microbubbles (diameter 1−25 μm) were increased with increasing the cavity pump frequency (40 Hz: 51.4%; 50 Hz: 57.5%; 60 Hz: 59.9%). The increased concentrations of O3 and OH radicals due to the higher specific area of O3 microbubbles compared to O3 millibubbles could facilitate their mass transfer at the gas−water interface. Furthermore, the elevated reactivity of O3 by increasing the temperature might improve the degradation of the pharmaceutical compounds, which was more pronounced for the microbubble ozonated waters than the millibubble ozonated waters. Although the degradation efficiency of the pharmaceutical compounds during the microbubble ozonation processes was significantly influenced by the existence of humic acids compared to the millibubble ozonation process, the increased solubilization rate of O3 and OH radicals by collapsing O3 microbubbles enhanced the degradation of the pharmaceutical compounds. Overall, these results clearly showed that the microbubble ozonation process could be an alternative option to conventional ozonation processes for the abatement of the pharmaceutical compounds.

Idiopathic Pulmonary Fibrosis (IPF) is a progressive, irreversible and fatal lung disease with unmet medical needs. Autotaxin (ATX) is an extracellular enzyme involved in the generation of lysophosphatidic acid (LPA). Preclinical and clinical data have suggested the ATX – LPA – LPA receptor (LPAR) axis plays a pivotal role in the pathogenesis and the progression of IPF. BBT-877 is an orally available small molecule inhibitor against ATX. In ex vivo enzymatic assays using human plasma, IC50 of BBT-877 was measured 6.5 – 6.9 nM (LPA 18:2) whereas that of GLPG1690 was measured 75 – 132 nM. To determine in vivo anti-fibrotic efficacy of BBT-877, bleomycin was intranasally administrated in mice at day 0, and BBT-877 was administrated orally twice a day from day 7 to 21. The BBT-877 treatment showed anti-fibrotic efficacy as revealed by significantly reduced body weight loss, lung weight and Ashcroft score as well as collagen content compared to the vehicle-treated group. During phase 1 clinical trial with 80 healthy volunteers, in which 50 – 800 mg (SAD) and 200 – 800 mg QD or 100 – 200 mg BID for two weeks (MAD) doses were administrated, only mild adverse events were noted. Pharmacokinetic analysis revealed the dose-proportional increase in systemic exposure with elimination half-life of 12hr. The decrease of plasma LPA level was maintained at 80% or higher for 24hr when 400 mg BBT-877 was administrated. Taken together, nonclinical data suggest BBT-877 is a potent, selective, and potentially best-in-class ATX inhibitor. Phase 1 clinical data demonstrate BBT-877 is a safe and well-tolerated drug with excellent pharmacokinetic-pharmacodynamic profiles.

Deliberate control of titanium surfaces such as crack-free and white-color coatings, is one of the key issues for wide range of titanium applications. A white crack-free BaSO4-TiO2 coating with high density was obtained on titanium surface via electrophoretic deposition (EPD) method using BaSO4 and TiO2 with different particle sizes. The crack-free coating was achieved by reducing the shrinkage stress per unit volume acting coating. The effects of the particle size and the mixed condition of the BaSO4-TiO2 were confirmed by comparing the crack size, density and error of the coatings. Additionally, density of the coating was increased by the effect of bimodal particles. The density and thickness of the superior coating are 1.9 ± 0.1 g/cm3 and 12.72 ± 0.63 μm, respectively, under mixed conditions of TiO2: BaSO4 4 g/L: 11.6 g/L. The coating was further covered with 3-aminopropyltriethoxysilane (APTES) silane with tetrathylorthosilicate (TEOS) and polymethyl-methacrylate (PMMA) to improve the mechanical coating properties. The PMMA and silane molecules bonded between the nano- and micro- particles were confirmed through the scanning electron microscopy (SEM) images and Fourier transform infrared (FTIR) spectroscopy. The hardness of the PMMA/Silane/BaSO4-TiO2 composite coating is 0.221 ± 0.017 GPa. Mechanical properties such as the durability and hardness of the coating were improved by the highly packed bimodal structure and complex network bonding between the nano- and micro- particles, silane and PMMA interfaces.

By combining conventional single cell analysis with flow cytometry and public database searches with bioinformatics tools, we extended the expression profiling of thymic stromal cotransporter (TSCOT), Slc46A2/Ly110, that was shown to be expressed in bipotent precursor and cortical thymic epithelial cells. Genome scale analysis verified TSCOT expression in thymic tissue- and cell type- specific fashion and is also expressed in some other epithelial tissues including skin and lung. Coexpression profiling with genes, Foxn1 and Hoxa3, revealed the role of TSCOT during the organogenesis. TSCOT expression was detected in all thymic epithelial cells (TECs), but not in the CD31(+) endothelial cell lineage in fetal thymus. In addition, ABC transporter-dependent side population and Sca-1(+) fetal TEC populations both contain TSCOT-expressing cells, indicating TEC stem cells express TSCOT. TSCOT expression was identified as early as in differentiating embryonic stem cells. TSCOT expression is not under the control of Foxn1 since TSCOT is present in the thymic rudiment of nude mice. By searching variations in the expression levels, TSCOT is positively associated with Grhl3 and Irf6. Cytokines such as IL1b, IL22 and IL24 are the potential regulators of the TSCOT expression. Surprisingly, we found TSCOT expression in the lung is diminished in lung cancers, suggesting TSCOT may be involved in the suppression of lung tumor development. Based on these results, a model for TEC differentiation from the stem cells was proposed in context of multiple epithelial organ formation.

>> Gas resources captured in the form of gas hydrates are an order of magnitude larger than the resourcesavailable from conventional resources. Focus of this research is to investigate the effect of DME on phase equilibriaof methane hydrate, as well as the possibility of the use of the PRO/II computer simulation to estimate the phaseequilibria. In systems containing water and a gaseous component like, for instance, methane, ethane , and propane,gas hydrates may occur, if conditions in terms of pressure and temperature are satisfied. Mixtures of gases, e.g.LPG or natural gas, are also able to form gas hydrates in the presence of water. The experiments presented herewere performed at temperatures varying between 268.15K and 288.15K and at pressures varying between 1.88MPa and 10.56 MPa. It was found that the phase equilibria of methane hydrate is influenced by the addition of DME to the system. The pressure for the equilibrium hydrate-liquid water-vapor (H - L w - V) in the system water + methane is reduced upon addition of DME. The phase equilibria of methane hydrate can be estimatedby the PRO/II computer simulation, whereas those of methane hydrate containing DME or LPG can't be estimatedproperly.

Summary Cell cycle progression requires the E3 ubiquitin ligase anaphase-promoting complex (APC/C), which uses the substrate adaptors CDC20 and CDH1 to target proteins for proteasomal degradation. The APC CDH1 substrate cyclin A is critical for the G1/S transition and, paradoxically, accumulates even when APC CDH1 is active. We show that the deubiquitinase USP37 binds CDH1 and removes degradative polyubiquitin from cyclin A. USP37 was induced by E2F transcription factors in G1, peaked at G1/S, and was degraded in late mitosis. Phosphorylation of USP37 by CDK2 stimulated its full activity. USP37 overexpression caused premature cyclin A accumulation in G1 and accelerated S phase entry, whereas USP37 knockdown delayed these events. USP37 was inactive in mitosis because it was no longer phosphorylated by CDK2. Indeed, it switched from an antagonist to a substrate of APC CDH1 and was modified with degradative K11-linked polyubiquitin.

The gene LamC2 encoding the γ2 chain of laminin 5, an epithelial cell-specific extracellular matrix protein, was identified in a PCR-based subtracted cDNA library from mouse thymic stromal cells. The mRNA existed in two alternative forms (5.1 and 2.4 kb). The full-length message was highly expressed in SCID thymus and in a nurse cell line, but not in other thymic epithelial cell lines, while the short form was more widely expressed. In situ hybridization and immunohistochemical staining revealed laminin 5 expression mostly in the subcapsular region of the adult thymus. Addition to fetal thymic organ cultures of a cell adhesion-blocking mAb to the α3 chain of laminin 5 interrupted T cell development. There was a 40% reduction in the total yield of thymocytes, and the most profound decrease (75–90%) was seen in the CD25+CD44+ and CD25+CD44−subsets of the CD4−CD8− double negative fraction. Most of the surviving double negative thymocytes expressed Sca-1, and there were significant increases in the number of cells with CD69 expression and in the fraction of annexin V-stained cells. None of these changes were observed with a nonblocking anti-laminin α3 chain mAb. These results suggest that the interaction between double negative thymoctyes and laminin 5 made by subcapsular epithelial cells is required for the survival and differentiation of mouse thymocytes.

Background: The faithful determination of the concentration and viability of yeast cells is important for biological research as well as industry. To this end, it is important to develop an automated cell counting algorithm that can provide not only fast but also accurate and precise measurement of yeast cells. Results: With the proposed method, we measured the precision of yeast cell measurements by using 0%, 25%, 50%, 75% and 100% viability samples. As a result, the actual viability measured with the proposed yeast cell counting algorithm is significantly correlated to the theoretical viability (R-2 = 0.9991). Furthermore, we evaluated the performance of our algorithm in various computing platforms. The results showed that the proposed algorithm could be feasible to use with low-end computing platforms without loss of its performance. Conclusions: Our yeast cell counting algorithm can rapidly provide the total number and the viability of yeast cells with exceptional accuracy and precision. Therefore, we believe that our method can become beneficial for a wide variety of academic field and industries such as biotechnology, pharmaceutical and alcohol production.

In order to investigate the role of thymic epithelial cell (TEC) subsets during T-cell development, we established a new transgenic system, enabling inducible cell-specific ablation as well as marking the TEC subsets using bicistronic bacterial nitroreductase and EGFP genes. Two different lengths of the TSCOT promoter in transgenic mice, named 3.1T-NE and 9.1T-NE, drive EGFP expression into TECs. In adult life, EGFP expression was located in the medulla with a smaller 3.1 kb TSCOT promoter, while it was maintained in the cortex with a 9.1 kb promoter, suggesting putative TEC specific as well as compartment specific cis elements within two promoters. Nitroreductase induced cell death was specific without bystander killing upon the treatment of prodrugs such as nitrofurantoin and metronidazol. The degree of cell death was dependent on the dose of the prodrug in the cell and the fetal thymic organ cultures (FTOCs). Fetal thymic stromal populations were analyzed based on the expression levels of EpCAM, MHCII, CDR1 and/or UEA-1. EGFP expression patterns varied among subsets indicating the differential TSCOT promoter activity in each TEC subset. Prodrug treatment in FTOCs reduced the numbers of total and subsets of thymocytes. A CD4(+)CD8(+) double positive cell population was highly susceptible in both transgenic lines. Surprisingly, there was a distinct reduction in γδ T cell population only in the 9.1T-NE thymus, indicating that they require a NTREGFP expressing TEC population. Therefore, these results support a division of labor within TEC subsets for the αβ and γδ lineage specification.

The CDC25 protein phosphatases (CDC25A, B, and C) drive cell cycle transitions by activating key components of the cell cycle engine. CDC25A and CDC25B are frequently overproduced in human cancers. Disruption of Cdc25B or Cdc25C individually or in combination has no effect on mouse viability. Here we report that CDC25A is the only family member to provide an essential function during early embryonic development, and that other family members compensate for its loss in adult mice. In contrast, conditional disruption of the entire family is lethal in adults due to a loss of small intestinal epithelial cell proliferation in crypts of Lieberkühn. Cdc25 loss induced Wnt signaling, and overall crypt structures were preserved. In the face of continuous Wnt signaling, nearly all crypt epithelial progenitors differentiated into multiple cell lineages, including crypt base columnar cells, a proposed stem cell. A small population of Musashi/Dcamkl-1/nuclear β-catenin–positive epithelial cells was retained in these crypts. These findings have implications for the development of novel, less cytotoxic cancer chemotherapeutic drugs that specifically target the cell cycle.

Although much effort has been directed at dissecting the mechanisms of central tolerance, the role of thymic stromal cells remains elusive. In order to further characterize this event, we developed a mouse model restricting LacZ to thymic stromal cotransporter (TSCOT)-expressing thymic stromal cells (TDLacZ). The thymus of this mouse contains approximately 4,300 TSCOT+ cells, each expressing several thousand molecules of the LacZ antigen. TSCOT+ cells express the cortical marker CDR1, CD40, CD80, CD54, and major histocompatibility complex class II (MHCII). When examining endogenous responses directed against LacZ, we observed significant tolerance. This was evidenced in a diverse T cell repertoire as measured by both a CD4 T cell proliferation assay and an antigen-specific antibody isotype analysis. This tolerance process was at least partially independent of Autoimmune Regulatory Element gene expression. When TDLacZ mice were crossed to a novel CD4 T cell receptor (TCR) transgenic reactive against LacZ (BgII), there was a complete deletion of double-positive thymocytes. Fetal thymic reaggregate culture of CD45- and UEA-depleted thymic stromal cells from TDLacZ and sorted TCR-bearing thymocytes excluded the possibility of cross presentation by thymic dendritic cells and medullary epithelial cells for the deletion. Overall, these results demonstrate that the introduction of a neoantigen into TSCOT-expressing cells can efficiently establish complete tolerance and suggest a possible application for the deletion of antigen-specific T cells by antigen introduction into TSCOT+ cells.