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  • 1
    Publication Date: 2018-02-21
    Description: Oxidant stress modifies T lymphocyte activation and function. Previous work demonstrated that murine T cell–specific kelch like-ECH-associated protein 1 ( Keap1 ) deletion enhances antioxidant capacity and protects from experimental acute kidney injury. In this study, we used CRISPR technology to develop clinically translatable human T cell–specific KEAP1 deletion. Delivery of KEAP1 exon 2 specific Cas9:guide RNA in Jurkat T cells led to significant (~70%) editing and upregulation of NRF2-regulated antioxidant genes NADPH dehydrogenase quinone 1 ( NQO1 ) (up to 11-fold), heme oxygenase 1 ( HO1 ) (up to 11-fold), and GCLM (up to 2-fold). In primary human T cells, delivery of KEAP1 exon 2 target site 2-specific ATTO 550–labeled Cas9:guide RNA edited KEAP1 in ~40% cells and significantly ( p ≤ 0.04) increased NQO1 (16-fold), HO1 (9-fold), and GCLM (2-fold) expression. To further enrich KEAP1 -edited cells, ATTO 550–positive cells were sorted 24 h after electroporation. Assessment of ATTO 550–positive cells showed KEAP1 editing in ~55% cells. There was no detectable off-target cleavage in the top three predicted genes in the ATTO 550–positive cells. Gene expression analysis found significantly ( p ≤ 0.01) higher expression of NQO1 mRNA in ATTO 550–positive cells compared with control cells. Flow cytometric assessment showed increased ( p ≤ 0.01) frequency of CD4-, CD25-, and CD69-expressing KEAP1 edited cells whereas frequency of CD8- ( p ≤ 0.01) and IL-17– ( p ≤ 0.05) expressing cells was reduced compared with control cells. Similar experimental conditions resulted in significant KEAP1 editing, increased antioxidant gene expression, and frequency of CD69 and IL-10 positive cells in highly enriched KEAP1 -edited regulatory T cells. KEAP1 -edited T cells could potentially be used for treating multiple human diseases.
    Print ISSN: 0022-1767
    Electronic ISSN: 1550-6606
    Topics: Medicine
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  • 2
    Publication Date: 2018-11-16
    Description: Purpose: IL15 induces the activation and proliferation of natural killer (NK) and memory CD8 + T cells and has preclinical antitumor activity. Given the superior activity and favorable kinetics of ALT-803 (IL15N72D:IL15RαSu/IgG1 Fc complex) over recombinant human IL15 (rhIL15) in animal models, we performed this first-in-human phase I trial of ALT-803 in patients with advanced solid tumors. Patients and Methods: Patients with incurable advanced melanoma, renal cell, non–small cell lung, and head and neck cancer were treated with ALT-803 0.3 to 6 μg/kg weekly intravenously or 6 to 20 μg/kg weekly subcutaneously for 4 consecutive weeks, every 6 weeks. Immune correlates included pharmacokinetics, immunogenicity, and lymphocyte expansion and function. Clinical endpoints were toxicity and antitumor activity. Results: Twenty-four patients were enrolled; 11 received intravenous and 13 received subcutaneous ALT-803. Of these patients, nine had melanoma, six renal, three head and neck, and six lung cancer. Although total lymphocyte and CD8 + T-cell expansion were modest, NK cell numbers rose significantly. Neither anti–ALT-803 antibodies nor clinical activity were observed. Overall, ALT-803 was well tolerated, with adverse effects including fatigue and nausea most commonly with intravenous administration, whereas painful injection site wheal was reported most commonly with subcutaneous ALT-803. Conclusions: Subcutaneous ALT-803 produced the expected NK cell expansion and was well tolerated with minimal cytokine toxicities and a strong local inflammatory reaction at injection sites in patients with advanced cancer. These data, together with compelling evidence of synergy in preclinical and clinical studies, provide the rationale for combining ALT-803 with other anticancer agents. Clin Cancer Res; 24(22); 5552–61. ©2018 AACR .
    Print ISSN: 1078-0432
    Electronic ISSN: 1557-3265
    Topics: Medicine
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  • 3
    Publication Date: 2018-01-25
    Description: Background Genetic testing for BRCA1 and BRCA2 is offered typically to selected women based on age of onset and family history of cancer. However, current internationally accepted genetic testing referral guidelines are built mostly on data from cancer genetics clinics in women of European descent. To evaluate the appropriateness of such guidelines in Asians, we have determined the prevalence of germ line variants in an unselected cohort of Asian patients with breast cancer and healthy controls. Methods Germ line DNA from a hospital-based study of 2575 unselected patients with breast cancer and 2809 healthy controls were subjected to amplicon-based targeted sequencing of exonic and proximal splice site junction regions of BRCA1 and BRCA2 using the Fluidigm Access Array system, with sequencing conducted on a Illumina HiSeq2500 platform. Variant calling was performed with GATK UnifiedGenotyper and were validated by Sanger sequencing. Results Fifty-five (2.1%) BRCA1 and 66 (2.6%) BRCA2 deleterious mutations were identified among patients with breast cancer and five (0.18%) BRCA1 and six (0.21%) BRCA2 mutations among controls. One thousand one hundred and eighty-six (46%) patients and 97 (80%) carriers fulfilled the National Comprehensive Cancer Network guidelines for genetic testing. Conclusion Five per cent of unselected Asian patients with breast cancer carry deleterious variants in BRCA1 or BRCA2 . While current referral guidelines identified the majority of carriers, one in two patients would be referred for genetic services. Given that such services are largely unavailable in majority of low-resource settings in Asia, our study highlights the need for more efficient guidelines to identify at-risk individuals in Asia.
    Keywords: Open access
    Print ISSN: 0022-2593
    Electronic ISSN: 1468-6244
    Topics: Medicine
    Published by BMJ Publishing Group
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  • 4
    Publication Date: 2018-03-06
    Description: G-protein-coupled receptors (GPCRs) mediate multiple signaling pathways in the cell, depending on the agonist that activates the receptor and multiple cellular factors. Agonists that show higher potency to specific signaling pathways over others are known as "biased agonists" and have been shown to have better therapeutic index. Although biased agonists are desirable, their design poses several challenges to date. The number of assays to identify biased agonists seems expensive and tedious. Therefore, computational methods that can reliably calculate the possible bias of various ligands ahead of experiments and provide guidance, will be both cost and time effective. In this work, using the mechanism of allosteric communication from the extracellular region to the intracellular transducer protein coupling region in GPCRs, we have developed a computational method to calculate ligand bias ahead of experiments. We have validated the method for several β -arrestin–biased agonists in β 2 -adrenergic receptor ( β 2AR), serotonin receptors 5-HT1B and 5-HT2B and for G-protein–biased agonists in the -opioid receptor. Using this computational method, we also performed a blind prediction followed by experimental testing and showed that the agonist carmoterol is β -arrestin–biased in β 2AR. Additionally, we have identified amino acid residues in the biased agonist binding site in both β 2AR and -opioid receptors that are involved in potentiating the ligand bias. We call these residues functional hotspots, and they can be used to derive pharmacophores to design biased agonists in GPCRs.
    Print ISSN: 0026-895X
    Electronic ISSN: 1521-0111
    Topics: Chemistry and Pharmacology , Medicine
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  • 5
    Publication Date: 2018-03-23
    Description: Objectives Asthma exacerbation, associated with many risks factors, can reflect management failure. However, little is known about how risk factors are associated with exacerbation, according to asthma severity. We aimed to investigate differences in risk factors in patients with different asthma severity and evaluate whether risk factors differed between frequent exacerbators and patients with single exacerbation. Design Nationwide population-based observational study. Setting Korean National Sample Cohort database. Participants We included 22 130 adults with asthma diagnoses more than twice (ICD-10 (International Classification of Diseases, Tenth revision) codes J45 and J46) and one prescription for asthma medication from 2010 to 2011. Outcome measures Asthma exacerbation was defined as having a corticosteroid (CS) burst characterised by a prescription of high-dose oral CS for ≥3 days or one systemic CS injection, hospitalisation or emergency department visit. Results Among severities, history of CS bursts was significantly associated with exacerbation. In mild and moderate asthma, exacerbation was significantly associated with age ≥45 years, being female, gastro-oesophageal reflux disease and chronic rhinitis. High medication possession ratio (MPR≥50%), compared with low MPR (〈20%) showed adjusted ORs of 0.828 (95% CI 0.707 to 0.971) and 0.362 (0.185 to 0.708) in moderate and severe asthma, respectively. In severe asthma, compared with mild asthma, only allergic rhinitis and history of hospitalisation were strongly associated with exacerbation. When comparing frequent exacerbators to patients with single exacerbation, age ≥45 years, atopic dermatitis, anxiety and history of CS burst were significant risk factors in mild and moderate asthma, whereas no risk factors were significant in severe asthma. Conclusions Different associations between risk factors and asthma exacerbations based on asthma severity suggest that patients with mild asthma require greater attention to their age and comorbidities, whereas those with severe asthma require greater attention to hospitalisation history and drug adherence.
    Keywords: Open access, Respiratory medicine
    Electronic ISSN: 2044-6055
    Topics: Medicine
    Published by BMJ Publishing
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  • 6
    Publication Date: 2018-03-06
    Description: High-grade glioma (HGG) is highly angiogenic, but antiangiogenic therapy has transient clinical benefit in only a fraction of patients. Vascular regulators of these heterogeneous responses remain undetermined. We found up-regulation of Sox7 and down-regulation of Sox17 in tumor endothelial cells (tECs) in mouse HGG. Sox7 deletion suppressed VEGFR2 expression, vascular abnormality, hypoxia-driven invasion, regulatory T cell infiltration, and tumor growth. Conversely, Sox17 deletion exacerbated these phenotypes by up-regulating Sox7 in tECs. Anti-VEGFR2 antibody treatment delayed tumor growth by normalizing Sox17 -deficient abnormal vessels with high Sox7 levels but promoted it by regressing Sox7 -deficient vessels, recapitulating variable therapeutic responses to antiangiogenic therapy in HGG patients. Our findings establish that Sox7 promotes tumor growth via vessel abnormalization, and its level determines the therapeutic outcome of VEGFR2 inhibition in HGG. In 189 HGG patients, Sox7 expression was heterogeneous in tumor vessels, and high Sox7 levels correlated with poor survival, early recurrence, and impaired vascular function, emphasizing the clinical relevance of Sox7 in HGG.
    Keywords: Solid Tumors
    Print ISSN: 0022-1007
    Electronic ISSN: 1540-9538
    Topics: Medicine
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  • 7
    Publication Date: 2018-01-09
    Description: Cecilia Lanny Winata, Maciej Łapinski, Leszek Pryszcz, Candida Vaz, Muhammad Hisyam bin Ismail, Srikanth Nama, Hajira Shreen Hajan, Serene Gek Ping Lee, Vladimir Korzh, Prabha Sampath, Vivek Tanavde, and Sinnakaruppan Mathavan In the earliest stages of animal development following fertilization, maternally deposited mRNAs direct biological processes to the point of zygotic genome activation (ZGA). These maternal mRNAs undergo cytoplasmic polyadenylation (CPA), suggesting translational control of their activation. To elucidate the biological role of CPA during embryogenesis, we performed genome-wide polysome profiling at several stages of zebrafish development. Our analysis revealed a correlation between CPA and polysome-association dynamics, demonstrating a coupling of translation to the CPA of maternal mRNAs. Pan-embryonic CPA inhibition disrupted the maternal-to-zygotic transition (MZT), causing a failure of developmental progression beyond the mid-blastula transition and changes in global gene expression that indicated a failure of ZGA and maternal mRNA clearance. Among the genes that were differentially expressed were those encoding chromatin modifiers and key transcription factors involved in ZGA, including nanog , pou5f3 and sox19b , which have distinct CPA dynamics. Our results establish the necessity of CPA for ensuring progression of the MZT. The RNA-seq data generated in this study represent a valuable zebrafish resource for the discovery of novel elements of the early embryonic transcriptome.
    Print ISSN: 0950-1991
    Electronic ISSN: 1477-9129
    Topics: Biology
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  • 8
    Publication Date: 2018-01-03
    Description: Ten-eleven translocation methylcytosine dioxygenase 1 ( Tet1 ) initiates DNA demethylation by converting 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) at CpG-rich regions of genes, which have key roles in adult neurogenesis and memory. In addition, the overexpression of Tet1 with 5-hmC alteration in patients with psychosis has also been reported, for instance in schizophrenia and bipolar disorders. The mechanism underlying Tet1 overexpression in the brain; however, is still elusive. In the present study, we found that Tet1-transgenic (Tet1-TG) mice displayed abnormal behaviors involving elevated anxiety and enhanced fear memories. We confirmed that Tet1 overexpression affected adult neurogenesis with oligodendrocyte differentiation in the hippocampal dentate gyrus of Tet1-TG mice. In addition, Tet1 overexpression induced the elevated expression of immediate early genes, such as Egr1 , c-fos , Arc , and Bdnf , followed by the activation of intracellular calcium signals ( i.e. , CamKII, ERK, and CREB) in prefrontal and hippocampal neurons. The expression of GABA receptor subunits ( Gabra2 and Gabra4 ) fluctuated in the prefrontal cortex and hippocampus. We evaluated the effects of Tet1 overexpression on intracellular calcium-dependent cascades by activating the Egr1 promoter in vitro . Tet1 enhanced Egr1 expression, which may have led to alterations in Gabra2 and Gabra4 expression in neurons. Taken together, we suggest that the Tet1 overexpression in our Tet1-TG mice can be applied as an effective model for studying various stress-related diseases that show hyperactivation of intracellular calcium-dependent cascades in the brain.—Kwon, W., Kim, H.-S., Jeong, J., Sung, Y., Choi, M., Park, S., Lee, J., Jang, S., Kim, S. H., Lee, S., Kim, M. O., Ryoo, Z. Y. Tet1 overexpression leads to anxiety-like behavior and enhanced fear memories via the activation of calcium-dependent cascade through Egr1 expression in mice.
    Print ISSN: 0892-6638
    Electronic ISSN: 1530-6860
    Topics: Biology
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  • 9
    Publication Date: 2018-05-31
    Description: Background/Aim: MicroRNAs (miRNAs) are closely associated with a number of cellular processes, including cell development, differentiation, proliferation, carcinogenesis, and apoptosis. The aim of the present study was to elucidate the molecular mechanisms underlying the tumor suppressor activity of miRNA-203 (miR-203) in YD-38 human oral cancer cells. Materials and Methods: Polymerase chain reaction analysis, MTT assay, DNA fragmentation assay, fluorescence-activated cell-sorting analysis, gene array, immunoblotting, and luciferase assay were carried out in YD-38 cells. Results: miR-203 expression was significantly down-regulated in YD-38 cells compared to expression levels in normal human oral keratinocytes. miR-203 decreased the viability of YD-38 cells in a time- and dose-dependent manner. In addition, over-expression of miR-203 significantly increased not only DNA segmentation, but also the apoptotic population of YD-38 cells. These results indicate that miR-203 overexpression induces apoptosis in YD-38 cells. Target gene array analysis revealed that the expression of the polycomb complex protein gene Bmi-1, a representative oncogene, was significantly down-regulated by miR-203 in YD-38 cells. Moreover, both mRNA and protein levels of Bmi-1 were significantly reduced in YD-38 cells transfected with miR-203. These results indicate that Bmi-1 is a target gene of miR-203. A luciferase reporter assay confirmed that miR-203 suppressed Bmi-1 expression by directly targeting the 3’-untranslated region. Conclusion: miR-203 induces apoptosis in YD-38 cells by directly targeting Bmi-1, which suggests its possible application as an anti-cancer therapeutic.
    Print ISSN: 0250-7005
    Electronic ISSN: 1791-7530
    Topics: Medicine
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  • 10
    Publication Date: 2018-09-05
    Description: In 2017, an estimated 17,000 individuals were diagnosed with esophageal adenocarcinoma (EAC), and less than 20% will survive 5 years. Positron emission tomography avidity is indicative of high glucose utilization and is nearly universal in EAC. TXNIP blocks glucose uptake and exhibits proapoptotic functions. Higher expression in EAC has been associated with improved disease-specific survival, lack of lymph node involvement, reduced perineural invasion, and increased tumor differentiation. We hypothesized that TXNIP may act as a tumor suppressor that sensitizes EAC cells to standard chemotherapeutics. EAC cell lines and a Barrett epithelial cell line were used. qRT-PCR, immunoblot, and immunofluorescence techniques evaluated gene expression. TXNIP was stably overexpressed or knocked down using lentiviral RNA transduction techniques. Murine xenograft methods examined growth following overexpression of TXNIP. Apoptosis and DNA damage were measured by annexin V and H2AX assays. Activation of the intrinsic apoptosis was quantitated with green fluorescence protein-caspase 3 reporter assay. In cultured cells and an esophageal tissue array, TXNIP expression was higher in Barrett epithelia and normal tissue compared with EAC. Constitutive overexpression of TXNIP decreased proliferation, clonogenicity, and tumor xenograft growth. TXNIP overexpression increased, whereas knockdown abrogated, DNA damage and apoptosis following cisplatin treatment. An HDAC inhibitor, entinostat (currently in clinical trials), upregulated TXNIP and synergistically increased cisplatin-mediated DNA damage and apoptosis. TXNIP is a tumor suppressor that is downregulated in EACC. Its reexpression dramatically sensitizes these cells to cisplatin. Our findings support phase I/II evaluation of "priming" strategies to enhance the efficacy of conventional chemotherapeutics in EAC. Mol Cancer Ther; 17(9); 2013–23. ©2018 AACR .
    Print ISSN: 1535-7163
    Electronic ISSN: 1538-8514
    Topics: Medicine
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