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Intracellular Flow Cytometry

Nadine Czekalski, Stefanie Imminger, Elisabeth Salhi, Marjan Veljkovic, Karolin Kleffel, David Drissner, Frederik Hammes, Helmut Bürgmann, Urs von Gunten
Ozone, a strong oxidant and disinfectant, seems ideal to cope with future challenges of water treatment, such as micropollutants, multiresistant bacteria (MRB) and even intracellular antibiotic resistance genes (ARG), but information on the latter is scarce. In ozonation experiments we simultaneously determined kinetics and dose-dependent inactivation of Escherichia coli and its plasmid-encoded sulfonamide resistance gene sul1 in different water matrixes. Effects in E. coli were compared to an autochthonous wastewater community...
October 24, 2016: Environmental Science & Technology
Marie Garraud, Kahina Khacef, Anne-Clémence Vion, Claire Leconte, Min Yin, Jean-Marie Renard, Catherine Marchand-Leroux, Chantal M Boulanger, Isabelle Margaill, Virginie Beray-Berthat
Thrombolysis with recombinant tissue plasminogen activator (rt-PA) is currently the only approved pharmacological strategy for acute ischemic stroke. However, rt-PA exhibits vascular toxicity mainly due to endothelial damage. To investigate the mechanisms underlying rt-PA-induced endothelial alterations, we assessed the role of rt-PA in the generation of endothelial microparticles (EMPs), emerging biological markers and effectors of endothelial dysfunction. The mouse brain-derived endothelial cell line bEnd...
November 15, 2016: Journal of the Neurological Sciences
Mauro C Wesseling, Lisa Wagner-Britz, Duc Bach Nguyen, Salome Asanidze, Judy Mutua, Nagla Mohamed, Benjamin Hanf, Mehrdad Ghashghaeinia, Lars Kaestner, Ingolf Bernhardt
BACKGROUND/AIMS: In previous publications we were able to demonstrate the exposure of phosphatidylserine (PS) in the outer membrane leaflet after activation of red blood cells (RBCs) by lysophosphatidic acid (LPA), phorbol-12 myristate-13acetate (PMA), or 4-bromo-A23187 (A23187). It has been concluded that three different mechanisms are responsible for the PS exposure in human RBCs: (i) Ca2+-stimulated scramblase activation (and flippase inhibition) by A23187, LPA, and PMA; (ii) PKCα activation by LPA and PMA; and (iii) enhanced lipid flip flop caused by LPA...
October 24, 2016: Cellular Physiology and Biochemistry
Gabriele Picco, Consalvo Petti, Francesco Sassi, Katia Grillone, Giorgia Migliardi, Teresa Rossi, Claudio Isella, Federica Di Nicolantonio, Ivana Sarotto, Anna Sapino, Alberto Bardelli, Livio Trusolino, Andrea Bertotti, Enzo Medico
BACKGROUND: The NEDD8 conjugation pathway modulates the ubiquitination and activity of a wide range of intracellular proteins, and its blockade by pevonedistat is emerging as a promising therapeutic approach in various cancer settings. However, systematic characterization of pevonedistat efficacy in specific tumor types and definition of response predictors are still missing. METHODS: We investigated in vitro sensitivity to pevonedistat in 122 colorectal cancer (CRC) cell lines by an ATP-based proliferation assay and evaluated apoptosis and DNA content by flow cytometry...
February 2017: Journal of the National Cancer Institute
Ge Shi, Ting-Ting Wang, Juan-Hua Quan, Shi-Jie Li, Man-Feng Zhang, Pei-Yu Liao, Yi-Ming Fan
BACKGROUND: The transcription factor Sox9 is pivotal in the morphogenesis of hair follicles, but its role in sebocytes is poorly understood. OBJECTIVE: We investigated the effect of Sox9 on human sebocyte proliferation, differentiation and lipogenesis. METHODS: Sox9 expression was detected by immunohistochemistry in normal skin and acne lesion. Primary cultured human sebocytes were transfected with adenovirus expressing GFP-Sox9 or Sox9 microRNA...
October 13, 2016: Journal of Dermatological Science
Zhan Zhou, Yuhui Zheng, Cheng Zhang Cheng, Jiajia Wen, Qianming Wang
Here we developed the first case of pyropheophorbide-a-loaded PEGylated-hybrid carbon nanohorns (CNH-Pyro) to study tumor targeting therapy. During incubation with living cells, CNH-Pyro exhibited very intense red emissions. The intracellular imaging results were carried out by flow cytometry based on four different kinds of cell lines (including three adherent cell lines and one suspension cell line). Compared with free pyropheophorbide-a, CNH-Pyro demonstrated enhanced photodynamic tumor ablation efficiency during in vitro experiments due to improved biocompatibility of the hybrid nanomaterial and the photothermal therapy effect derived from carbon-network structure...
January 1, 2017: Materials Science & Engineering. C, Materials for Biological Applications
Greg Hodge, Eugene Roscioli, Hubertus Jersmann, Hai B Tran, Mark Holmes, Paul N Reynolds, Sandra Hodge
BACKGROUND: Corticosteroid resistance is a major barrier to effective treatment of COPD. We have shown that the resistance is associated with decreased expression of glucocorticoid receptor (GCR) by senescent CD28nullCD8+ pro-inflammatory lymphocytes in peripheral blood of COPD patients. GCR must be bound to molecular chaperones heat shock proteins (Hsp) 70 and Hsp90 to acquire a high-affinity steroid binding conformation, and traffic to the nucleus. We hypothesized a loss of Hsp70/90 from these lymphocytes may further contribute to steroid resistance in COPD...
October 21, 2016: Respiratory Research
Benoîte Bourdin, Emilie Segura, Marie-Philippe Tétreault, Sylvie Lesage, Lucie Parent
Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1...
September 28, 2016: Journal of Visualized Experiments: JoVE
Yukio Ozaki, Shogo Tamura, Katsue Suzuki-Inoue
Platelets play a key role in the pathophysiological processes of hemostasis and thrombus formation. However, platelet functions beyond thrombosis and hemostasis have been increasingly identified in recent years. A large body of evidence now exists which suggests that platelets also play a key role in inflammation, immunity, malignancy, and furthermore in organ development and regeneration, such as the liver. We have recently identified CLEC-2 on the platelet membrane, which induces intracellular activation signals upon interaction of a snake venom, rhodocytin...
2016: Thrombosis Journal
Alice C Wyse-Jackson, Sarah L Roche, Ana M Ruiz-Lopez, Jennifer N Moloney, Ashleigh M Byrne, Thomas G Cotter
Retinitis pigmentosa (RP) is a degenerative retinal disease leading to photoreceptor cell loss. In 2011, our group identified the synthetic progesterone 'Norgestrel' as a potential treatment for RP. Subsequent research showed Norgestrel to work through progesterone receptor membrane component 1 (PGRMC1) activation and upregulation of neuroprotective basic fibroblast growth factor (bFGF). Using trophic factor deprivation of 661W photoreceptor-like cells, we aimed to further elucidate the mechanism leading to Norgestrel-induced neuroprotection...
October 20, 2016: European Journal of Neuroscience
Anja Göder, Nisintha Mahendrarajah, Oliver H Krämer
Autophagy is a lysosome-dependent, intracellular pathway for the recycling of cellular components. It plays a pivotal role in both cancer development and the response to chemotherapy. Here we describe how autophagy can be monitored in living cells by flow cytometry using the cationic amphiphilic tracer dye Cyto-ID(®) Green. The detection of autophagy induction in the human leukemia cell line K562 after the treatment with the HDAC class I inhibitor MS-275 serves as an example for this approach.
2017: Methods in Molecular Biology
Da Zhou, Bing-Hang Li, Jing Wang, Yong-Nian Ding, Yan Dong, Yuan-Wen Chen, Jian-Gao Fan
BACKGROUND: Prolyl oligopeptidase (POP) is a serine endopeptidase that is widely distributed in vivo, particularly in the liver. Significant changes in functional mitochondrial proteins involved with mitochondrial oxidoreductases/transporters and nucleic acid binding proteins were observed after POP inhibition in the liver, which suggested a role of POP in regulating liver energy metabolism. Steatosis in nonalcoholic fatty liver disease (NAFLD) is associated with disturbances in lipid and energy metabolism in hepatocytes...
2016: PloS One
Teresa Lopes da Silva, Cátia Baptista, Alberto Reis, Paula C Passarinho
Lignocellulosic materials have been considered low-cost effective substrates for bioethanol production. However, lignocellulosic pretreatment releases toxic compounds such as 5-hydroxymethylfurfural (HMF) that is known to inhibit the yeast growth and ethanol production. In this work, flow cytometry was used to monitor the physiological response of the yeast Saccharomyces carlsbergensis ATCC 6269 in the presence of different initial HMF concentrations within the range of 0-15 g/L, in terms of cell membrane integrity, potential, and intracellular lipids...
October 18, 2016: Applied Biochemistry and Biotechnology
Gerrit Ahrenstorf, Hui-Zhi Low, Katja Kniesch, David Ordonez, Dirk Meyer-Olson, Fareed Ahmad, Claudia Kücherer, Barbara Gunsenheimer-Bartmeyer, Matthias Stoll, Torsten Matthias, Reinhold E Schmidt, Torsten Witte
OBJECTIVE: To analyze the influence of LILRA3 and the genetic LILRA3 deletion on transmission and clinical course of HIV infection. DESIGN: Case and control study. METHODS: LILRA3 genotypes were determined by polymerase chain reaction. HIV patients were categorized into short-term progressors, normal progressors and long-term non-progressors according to the clinical course. Functional studies were performed using real-time PCR, intracellular flow cytometry and ELISA...
October 14, 2016: AIDS
Singkome Tima, Siriporn Okonogi, Chadarat Ampasavate, Chad Pickens, Cory Berkland, Songyot Anuchapreeda
This study aimed at developing a curcumin (CM) nanoparticle targeted to Feline McDonough Sarcoma (FMS)-like tyrosine kinase 3 (FLT3) protein on the surface of leukemic cells and at evaluating their properties, specificity, cytotoxicity, and inhibitory effect on FLT3 protein level in FLT3-overexpressing leukemic cells, EoL-1, and MV-4-11 cells. FLT3-specific peptides were conjugated onto modified poloxamer 407 using the copper-catalyzed azide-alkyne cycloaddition reaction. The thin film hydration method was performed for FLT3-specific CM-loaded polymeric micelles (FLT3-CM-micelles) preparation...
October 14, 2016: Journal of Pharmaceutical Sciences
Monika Kasztura, Magdalena Dzięgała, Kamil Kobak, Jacek Bania, Grzegorz Mazur, Waldemar Banasiak, Piotr Ponikowski, Ewa Anita Jankowska
: Background Iron is presumed to play an important role in functioning of cardiomyocytes and skeletal myocytes. There is scarcity of direct data characterizing the cells functioning when exposed to iron depletion or iron overload in cellular environment. There is some clinical evidence demonstrating that iron deficiency has serious negative prognostic consequence in heart failure (HF) patients and its correction brought clinical benefit. BACKGROUND: Iron is presumed to play an important role in functioning of cardiomyocytes and skeletal myocytes...
October 17, 2016: Kardiologia Polska
Yong Xie, Zhen-Jie Gu, Mao-Xiong Wu, Tu-Cheng Huang, Jing-Song Ou, Huiping-Son Ni, Mao-Huan Lin, Wo-Liang Yuan, Jing-Feng Wang, Yang-Xin Chen
AIMS: Adverse cardiovascular effects induced by peroxisome proliferator activator receptor-γ (PPAR-γ) activation were observed in clinical setting. But the underlying mechanism is unclear. Now, transgenic mice with cardiac specific peroxisome proliferator activator receptor-γ overexpression (TG-PPAR-γ) were used to explore the possible mechanisms. MATERIALS AND METHODS: Cardiac tissues from TG-PPAR-γ mice, a PPAR-γ over-expressing human cardiomyocyte line AC16 cell, and PPAR-γ agonist-treated primary cardiomyocytes were used to evaluate the expression of cardiac calcium regulatory proteins as sarcoplasmic reticulum Ca(2+) ATPase, Na(+)/Ca(2+)exchanger 1, ryanodine receptor 2 and phospholamban...
October 13, 2016: Life Sciences
Longbin Zheng, Ying Li, Xuesong Li, Jiayuan Kou, Zhaoyu Zhong, Yueqing Jiang, Zhongni Liu, Ye Tian, Liming Yang
BACKGROUND/AIMS: Sonodynamic therapy (SDT) is considered a new approach for the treatment of atherosclerosis. We previously confirmed that hydroxyl acetylated curcumin (HAC) was a sonosensitizer. In this study, we investigated the mechanism of THP-1 macrophage apoptosis and autophagy induced by HAC mediated SDT (HAC-SDT). METHODS: Cell viability was measured using a CCK-8 assay. Laser scanning confocal microscopy was used to measure the levels of intracellular reactive oxygen species (ROS), sub-cellular HAC localization, BAX and cytochrome C translocation, LC3 expression, monodansylcadaverine staining and Dil-labeled oxidized low density lipoprotein (Dil-ox-LDL) uptake...
October 17, 2016: Cellular Physiology and Biochemistry
Yu Li, Min Wang, Shilei Wang
OBJECTIVE: Previous studies have shown that mitochondrial division inhibitor 1 (mdivi-1) protects rat brain from ischemia/reperfusion (I/R) injury, but the precise mechanisms are unclear. This study aims to elucidate the effect of mdivi-1 on energy metabolism and neuronal apoptosis induced by I/R in vitro. METHODS: Cultured hippocampal neurons from Wistar rats were randomly divided into four treatment groups: control (C), vehicle (V), I/R (I), and I/R plus mdivi-1 (M)...
October 14, 2016: Neurological Research
Maggie L Diller, Ragini R Kudchadkar, Keith A Delman, David H Lawson, Mandy L Ford
Th17 cells represent a distinct subset of CD4 effector T cells with potent pathogenic qualities, capable of directly mediating tumor cell destruction. IL-2 has frequently been shown to have a negative effect on Th17 differentiation while supporting regulatory T-cell (FoxP3CD4, TREG) growth and development in both in vitro models and in vivo animal models. We investigated the effect of in vivo IL-2 on both the Th17 and FoxP3CD4 T-cell compartments in a human model of cancer. High-dose IL-2 (HDIL-2) was administered at a dose of 720,000 IU/kg to patients with melanoma (n=7) and peripheral blood was collected at baseline and at 24, 48, 72, and 96 hours posttreatment...
November 2016: Journal of Immunotherapy
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