933 research outputs found

    An efficient strategy for cell-based antibody library selection using an integrated vector system

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    BACKGROUND: Cell panning of phage-displayed antibody library is a powerful tool for the development of therapeutic and imaging agents since disease-related cell surface proteins in native complex conformation can be directly targeted. Here, we employed a strategy taking advantage of an integrated vector system which allows rapid conversion of scFv-displaying phage into scFv-Fc format for efficient cell-based scFv library selection on a tetraspanin protein, CD9. RESULTS: A mouse scFv library constructed by using a phagemid vector, pDR-D1 was subjected to cell panning against stable CD9 transfectant, and the scFv repertoire from the enriched phage pool was directly transferred to a mammalian cassette vector, pDR-OriP-Fc1. The resulting constructs enabled transient expression of enough amounts of scFv-Fcs in HEK293E cells, and flow cytometric screening of binders for CD9 transfectant could be performed simply by using the culture supernatants. All three clones selected from the screening showed correct CD9-specificity. They could immunoprecipitate CD9 molecules out of the transfectant cell lysate and correctly stain endogenous CD9 expression on cancer cell membrane. Furthermore, competition assay with a known anti-CD9 monoclonal antibody (mAb) suggested that the binding epitopes of some of them overlap with that of the mAb which resides within the large extracellular loop of CD9. CONCLUSIONS: This study demonstrates that scFv-Fc from mammalian transient expression can be chosen as a reliable format for rapid screening and validation in cell-based scFv library selection, and the strategy described here will be applicable to efficient discovery of antibodies to diverse cell-surface targets

    Measurement of the Background Activities of a 100Mo-enriched powder sample for AMoRE crystal material using a single high purity germanium detector

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    The Advanced Molybdenum-based Rare process Experiment (AMoRE) searches for neutrino-less double-beta (0{\nu}\b{eta}\b{eta}) decay of 100Mo in enriched molybdate crystals. The AMoRE crystals must have low levels of radioactive contamination to achieve low background signals with energies near the Q-value of the 100Mo 0{\nu}\b{eta}\b{eta} decay. To produce low-activity crystals, radioactive contaminants in the raw materials used to form the crystals must be controlled and quantified. 100EnrMoO3 powder, which is enriched in the 100Mo isotope, is of particular interest as it is the source of 100Mo in the crystals. A high-purity germanium detector having 100% relative efficiency, named CC1, is being operated in the Yangyang underground laboratory. Using CC1, we collected a gamma spectrum from a 1.6-kg 100EnrMoO3 powder sample enriched to 96.4% in 100Mo. Activities were analyzed for the isotopes 228Ac, 228Th, 226Ra, and 40K. They are long-lived naturally occurring isotopes that can produce background signals in the region of interest for AMoRE. Activities of both 228Ac and 228Th were < 1.0 mBq/kg at 90% confidence level (C.L.). The activity of 226Ra was measured to be 5.1 \pm 0.4 (stat) \pm 2.2 (syst) mBq/kg. The 40K activity was found as < 16.4 mBq/kg at 90% C.L.Comment: 20 pages, 6 figures, 5 table

    Unleashing the full potential of Hsp90 inhibitors as cancer therapeutics through simultaneous inactivation of Hsp90, Grp94, and TRAP1

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    Cancer therapeutics: Extending a drug&apos;s reach A new drug that blocks heat shock proteins (HSPs), helper proteins that are co-opted by cancer cells to promote tumor growth, shows promise for cancer treatment. Several drugs have targeted HSPs, since cancer cells are known to hijack these helper proteins to shield themselves from destruction by the body. However, the drugs have had limited success. Hye-Kyung Park and Byoung Heon Kang at Ulsan National Institutes of Science and Technology in South Korea and coworkers noticed that the drugs were not absorbed into mitochondria, a key cellular compartment, and HSPs in this compartment were therefore not being blocked. They identified a new HSP inhibitor that can reach every cellular compartment and inhibit all HSPs. Testing in mice showed that this inhibitor effectively triggered death of tumor cells, and therefore shows promise for anti-cancer therapy. The Hsp90 family proteins Hsp90, Grp94, and TRAP1 are present in the cell cytoplasm, endoplasmic reticulum, and mitochondria, respectively; all play important roles in tumorigenesis by regulating protein homeostasis in response to stress. Thus, simultaneous inhibition of all Hsp90 paralogs is a reasonable strategy for cancer therapy. However, since the existing pan-Hsp90 inhibitor does not accumulate in mitochondria, the potential anticancer activity of pan-Hsp90 inhibition has not yet been fully examined in vivo. Analysis of The Cancer Genome Atlas database revealed that all Hsp90 paralogs were upregulated in prostate cancer. Inactivation of all Hsp90 paralogs induced mitochondrial dysfunction, increased cytosolic calcium, and activated calcineurin. Active calcineurin blocked prosurvival heat shock responses upon Hsp90 inhibition by preventing nuclear translocation of HSF1. The purine scaffold derivative DN401 inhibited all Hsp90 paralogs simultaneously and showed stronger anticancer activity than other Hsp90 inhibitors. Pan-Hsp90 inhibition increased cytotoxicity and suppressed mechanisms that protect cancer cells, suggesting that it is a feasible strategy for the development of potent anticancer drugs. The mitochondria-permeable drug DN401 is a newly identified in vivo pan-Hsp90 inhibitor with potent anticancer activity

    Evaluation of the transporter-mediated herb-drug interaction potential of DA-9801, a standardized dioscorea extract for diabetic neuropathy, in human in vitro and rat in vivo

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    BACKGROUND: Drug transporters play important roles in the absorption, distribution, and elimination of drugs and thereby, modulate drug efficacy and toxicity. With a growing use of poly pharmacy, concurrent administration of herbal extracts that modulate transporter activities with drugs can cause serious adverse reactions. Therefore, prediction and evaluation of drug-drug interaction potential is important in the clinic and in the drug development process. DA-9801, comprising a mixed extract of Dioscoreae rhizoma and Dioscorea nipponica Makino, is a new standardized extract currently being evaluated for diabetic peripheral neuropathy in a phase II clinical study. METHOD: The inhibitory effects of DA-9801 on the transport functions of organic cation transporter (OCT)1, OCT2, organic anion transporter (OAT)1, OAT3, organic anion transporting polypeptide (OATP)1B1, OATP1B3, P-glycoprotein (P-gp), and breast cancer resistance protein (BCRP) were investigated in HEK293 or LLC-PK1 cells. The effects of DA-9801 on the pharmacokinetics of relevant substrate drugs of these transporters were also examined in vivo in rats. RESULTS: DA-9801 inhibited the in vitro transport activities of OCT1, OCT2, OAT3, and OATP1B1, with IC(50) values of 106, 174, 48.1, and 273 μg/mL, respectively, while the other transporters were not inhibited by 300 μg/mL DA-9801. To investigate whether this inhibitory effect of DA-9801 on OCT1, OCT2, and OAT3 could change the pharmacokinetics of their substrates in vivo, we measured the pharmacokinetics of cimetidine, a substrate for OCT1, OCT2, and OAT3, and of furosemide, a substrate for OAT1 and OAT3, by co-administration of DA-9801 at a single oral dose of 1,000 mg/kg. Pre-dose of DA-9801 5 min or 2 h prior to cimetidine administration decreased the C(max) of cimetidine in rats. However, DA-9801 did not affect the elimination parameters such as half-life, clearance, or amount excreted in the urine, suggesting that it did not inhibit elimination process of cimetidine, which is governed by OCT1, OCT2, and OAT3. Moreover, DA-9801 did not affect the pharmacokinetic characteristics of furosemide, as evidenced by its unchanged pharmacokinetic parameters. CONCLUSION: Inhibitory effects of DA-9801 on OCT1, OCT2, and OAT3 observed in vitro may not necessarily translate into in vivo herb-drug interactions in rats even at its maximum effective dose

    VGLL3 expression is associated with macrophage infiltration and predicts poor prognosis in epithelial ovarian cancer

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    Background/objectiveHigh-grade serous ovarian carcinoma (HGSOC) is the most common histologic type of epithelial ovarian cancer (EOC). Due to its poor survival outcomes, it is essential to identify novel biomarkers and therapeutic targets. The hippo pathway is crucial in various cancers, including gynaecological cancers. Herein, we examined the expression of the key genes of the hippo pathway and their relationship with clinicopathological significance, immune cells infiltration and the prognosis of HGSOC.MethodsThe Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) data were curated to analyse the mRNA expression as well as the clinicopathological association and correlation with immune cell infiltration in HGSOC. The protein levels of significant genes in the HGSOC tissue were analysed using Tissue Microarray (TMA)-based immunohistochemistry. Finally, DEGs pathway analysis was performed to find the signalling pathways associated with VGLL3.ResultsVGLL3 mRNA expression was significantly correlated with both advanced tumor stage and poor overall survival (OS) (p=0.046 and p=0.003, respectively). The result of IHC analysis also supported the association of VGLL3 protein with poor OS. Further, VGLL3 expression was significantly associated with tumor infiltrating macrophages. VGLL3 expression and macrophages infiltration were both found to be independent prognostic factors (p=0.003 and p=0.024, respectively) for HGSOC. VGLL3 was associated with four known and three novel cancer-related signalling pathways, thus implying that VGLL3 is involved in the deregulation of many genes and pathways.ConclusionOur study revealed that VGLL3 may play a distinct role in clinical outcomes and immune cell infiltration in patients with HGSOC and that it could potentially be a prognostic marker of EOC

    Titanium Oxide Nanotube Surface Topography and MicroRNA-488 Contribute to Modulating Osteogenesis

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    Understanding the biocomplexity of cell behavior in relation to the topographical characteristics of implants is essential for successful osseointegration with good longevity and minimum failure. Here, we investigated whether culture on titanium oxide (TiO2) nanotubes of various diameters could affect the behavior and differentiation of MC3T3-E1 cells. Among the tested nanotubes, those of 50 nm in diameter were found to trigger the expression of the osteoblast-specific transcription factors, sp7 and Dlx5, and upregulate the expression of alkaline phosphatase (ALP). Here, we report that miR-488 was significantly induced in osteoblasts cultured on 50 nm nanotubes and continued to increase with the progression of osteoblast differentiation. Furthermore, downregulation of miR-488 suppressed the expression levels of ALP and matrix metalloprotease-2 (MMP-2). This suppression of ALP transcription was overcome by treatment with the MMP-2 activator, bafilomycin A1. Collectively, these results suggest that 50 nm is the optimum TiO2 nanotube diameter for implants, and that modulation of miR-488 can change the differentiation activity of cells on TiO2 nanotubes. This emphasizes that we must fully understand the physicochemical properties of TiO2 nanotubes and the endogenous biomolecules that interact with such surfaces, in order to fully support their clinical application

    Measurement of the Background Activities of a 100Mo-enriched Powder Sample for an AMoRE Crystal Material by using Fourteen High-Purity Germanium Detectors

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    The Advanced Molybdenum-based Rare process Experiment in its second phase (AMoRE-II) will search for neutrinoless double-beta (0{\nu}\b{eta}\b{eta}) decay of 100Mo in 200 kg of molybdate crystals. To achieve the zero-background level in the energy range of the double-beta decay Q-value of 100Mo, the radioactive contamination levels in AMoRE crystals should be low. 100EnrMoO3 powder, which is enriched in the 100Mo isotope, is used to grow the AMoRE crystals. A shielded array of fourteen high-purity germanium detectors with 70% relative efficiency each was used for the measurement of background activities in a sample of 9.6-kg powder. The detector system named CAGe located at the Yangyang underground laboratory was designed for measuring low levels of radioactivity from natural radioisotopes or cosmogenic nuclides such as 228Ac, 228Th, 226Ra, 88Y, and 40K. The activities of 228Ac and 228Th in the powder sample were 0.88 \pm 0.12 mBq/kg and 0.669 \pm 0.087 mBq/kg, respectively. The activity of 226Ra was measured to be 1.50 \pm 0.23 mBq/kg. The activity of 88Y was 0.101 \pm 0.016 mBq/kg. The activity of 40K was found as 36.0 \pm 4.1 mBq/kg.Comment: 24 pages, 10 figures, 5 table
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