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https://www.readbyqxmd.com/read/27911494/determining-functional-aptamer-protein-interaction-by-biolayer-interferometry
#1
Xinhui Lou, Martin Egli, Xianbin Yang
Short single-stranded nucleic acids called aptamers are widely being explored as recognition molecules of high affinity and specificity for binding a wide range of target molecules, particularly protein targets. In biolayer interferometry (BLI), a simple Dip-and-Read approach in which the aptamer-coated biosensors are dipped into microplate wells is used to study the interactions between an aptamer and its target protein. Here we describe the protocol for the analysis of the interaction between a well-characterized anti-thrombin RNA aptamer with thrombin (Basic Protocol)...
December 1, 2016: Current Protocols in Nucleic Acid Chemistry
https://www.readbyqxmd.com/read/27911416/the-indirect-neuron-astrocyte-coculture-assay-an-in-vitro-set-up-for-the-detailed-investigation-of-neuron-glia-interactions
#2
Christine Gottschling, Egor Dzyubenko, Maren Geissler, Andreas Faissner
Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g., Alzheimer's disease and Schizophrenia. The most detailed analysis of neurons can be performed in vitro...
November 14, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/27911414/isolation-and-culture-of-primary-endothelial-cells-from-canine-arteries-and-veins
#3
Loes A Oosterhoff, Hedwig S Kruitwagen, Bart Spee, Frank G van Steenbeek
Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium...
November 18, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/27911400/visualizing-stromule-frequency-with-fluorescence-microscopy
#4
Jacob O Brunkard, Anne M Runkel, Patricia Zambryski
Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics...
November 23, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/27911395/preparation-of-primary-mixed-glial-cultures-from-adult-mouse-spinal-cord-tissue
#5
Jennifer T Malon, Ling Cao
It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice...
November 19, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/27911388/high-throughput-real-time-dual-readout-testing-of-intracellular-antimicrobial-activity-and-eukaryotic-cell-cytotoxicity
#6
Lucius Chiaraviglio, Yoon-Suk Kang, James E Kirby
Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity...
November 16, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/27911371/development-of-stem-cell-derived-antigen-specific-regulatory-t-cells-against-autoimmunity
#7
Mohammad Haque, Kristin Fino, Praneet Sandhu, Jianxun Song
Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4(+) T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i...
November 8, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/27911362/automated-acoustic-dispensing-for-the-serial-dilution-of-peptide-agonists-in-potency-determination-assays
#8
Jacqueline Naylor, Alessandra Rossi, Christopher Brankin, David C Hornigold
As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists(1)...
November 10, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/27911279/microrna-101-inhibits-proliferation-migration-and-invasion-of-human-glioblastoma-by-targeting-sox9
#9
Nan Liu, Lei Zhang, Zhen Wang, Yingduan Cheng, Pengxing Zhang, Xin Wang, Weihong Wen, Hongwei Yang, Hui Liu, Weilin Jin, Yongsheng Zhang, Yanyang Tu
Glioblastoma multiforme (GBM) is the most common primary malignant tumors originating in the brain parenchyma. At present, GBM patients have a poor prognosis despite the continuous progress in therapeutic technologies including surgery, radiotherapy, photodynamic therapy, and chemotherapy. Recent studies revealed that miR-101 was remarkably down-regulated in kinds of human cancers and was associated with aggressive tumor cell proliferation and stem cell self-renewal. Data also showed that miR-101 was down-regulated in primary glioma samples and cell lines, but the underlying molecular mechanism of the deregulation of miR-101 in glioma remained largely unknown...
November 30, 2016: Oncotarget
https://www.readbyqxmd.com/read/27911270/saha-and-or-mg132-reverse-the-aggressive-phenotypes-of-glioma-cells-an-in-vitro-and-vivo-study
#10
Xue-Feng Yang, Zhi-Juan Zhao, Jia-Jie Liu, Xiang-Hong Yang, Yang Gao, Shuang Zhao, Shuai Shi, Ke-Qiang Huang, Hua-Chuan Zheng
To elucidate the anti-tumor effects and molecular mechanisms of SAHA (a histone deacetylase inhibitor) and MG132 (a proteasome inhibitor) on the aggressive phenotypes of glioma cells, we treated U87 and U251 cells with SAHA or/and MG132, and detected phenotypes' assays with phenotype-related molecules examined. It was found that SAHA or/and MG132 treatment suppressed proliferation in both concentration- and time-dependent manners, inhibited energy metabolism, migration, invasion and lamellipodia formation, and induced G2 arrest and apoptosis in the glioma cells...
November 29, 2016: Oncotarget
https://www.readbyqxmd.com/read/27911269/the-matricellular-protein-cyr61-promotes-breast-cancer-lung-metastasis-by-facilitating-tumor-cell-extravasation-and-suppressing-anoikis
#11
Yu-Ting Huang, Qiang Lan, Girieca Lorusso, Nathalie Duffey, Curzio Rüegg
Matricellular proteins play multiple roles in primary tumor growth, local invasion and tumor angiogenesis. However, their contribution to metastasis and the putative mechanisms involved are less well characterized. In ER-negative human breast cancer, elevated expression levels of the matricellular protein Cysteine-rich angiogenic inducer 61 (CYR61) are associated with more aggressive progression. Here, we investigated the role of CYR61 in breast cancer lung metastasis using the triple negative human breast cancer cell lines MDA-MB-231 and SUM159...
November 29, 2016: Oncotarget
https://www.readbyqxmd.com/read/27911267/oncogenic-secretory-clusterin-in-hepatocellular-carcinoma-expression-at-early-staging-and-emerging-molecular-target
#12
Wenjie Zheng, Min Yao, Qi Qian, Wenli Sai, Liwei Qiu, Junling Yang, Wei Wu, Zhizhen Dong, Dengfu Yao
Secretory clusterin (sCLU) is associated with hepatocellular carcinoma (HCC) progression by contributing to angiogenesis, chemoresistance, cell survival, and metastasis. However, the sCLU expression at early stage of HCC progression remains to be clarified. In this study, the alteration of sCLU oncogenicity was firstly evaluated in HCC- and their para-cancerous- tissues. The incidence of sCLU expression in HCC was significantly higher than that in their non-tumorous tissues at message RNA (mRNA) or protein level, gradually increasing with tumor-node-metastasis (TNM) staging...
November 29, 2016: Oncotarget
https://www.readbyqxmd.com/read/27911266/down-regulation-of-salt-inducible-kinase-1-sik1-is-mediated-by-rnf2-in-hepatocarcinogenesis
#13
Chao Qu, Yaqin Qu
Our previous study reported that down-regulation of SIK1 accelerates the growth and invasion of hepatocellular carcinoma (HCC). However, the underlying mechanism leading to SIK1 down-regulation in HCC largely remains to be determined. Herein, we demonstrated that RNF2 expression is negatively correlated with SIK1 levels in HCC tissues. Kaplan-Meier analysis of tumor samples revealed that high RNF2 expression with concurrent low SIK1 expression is associated with poor overall survival. The down-regulation of RNF2 expression in HCC cells significantly reduces tumor cell growth and metastasis, while the simultaneous down-regulation of both RNF2 and SIK1 restores tumor cell growth in vitro and in tumor xenograft models...
November 29, 2016: Oncotarget
https://www.readbyqxmd.com/read/27911265/mirna-543-promotes-osteosarcoma-cell-proliferation-and-glycolysis-by-partially-suppressing-prmt9-and-stabilizing-hif-1%C3%AE-protein
#14
Heng Zhang, Xiaofeng Guo, Xing Feng, Tingting Wang, Zhaohua Hu, Xiangyong Que, Qingsong Tian, Tianbo Zhu, Guixian Guo, Wei Huang, Xinzhi Li
Osteosarcoma (OS) is the most common primary bone tumor, occurring frequently in adolescents and possessing a high malignant severity. MicroRNAs play critical roles during OS development. Thus, elucidation of the involvement of specific microRNAs in the development of OS may provide novel therapeutic targets for OS treatment. Here, we showed that in the OS specimens from patients, the levels of miR-543 were significantly increased whereas the levels of PRMT9 were significantly decreased, compared to the paired normal bone tissue...
November 28, 2016: Oncotarget
https://www.readbyqxmd.com/read/27911090/antitumor-activity-of-portulaca-oleracea-l-polysaccharide-on-hela-cells-through-inducing-tlr4-nf-%C3%AE%C2%BAb-signaling
#15
Rui Zhao, Tao Zhang, Baoling Ma, Xing Li
Abstarct We have previously shown that Portulaca oleracea L. polysaccharide (POL-P3b) possesses the ability to inhibit cervical cancer cell growth in vitro and in vivo. In this study, we explored how toll-like receptor 4 (TLR4) signaling correlated with the antitumor mechanism of POL-P3b. Western blotting was utilized to detect the expression of TLR4 and the downstream signaling pathway. The level of inflammatory mediator was quantified using enzyme-linked immunosorbent assay (ELISA) kits. The effects of POL-P3b on the proliferation and apoptosis in HeLa cells were determined by WST-8 assay and Hoechst 33342/propidium iodide (PI) assay...
December 2, 2016: Nutrition and Cancer
https://www.readbyqxmd.com/read/27911036/properties-of-embryoid-bodies
#16
REVIEW
Joshua M Brickman, Palle Serup
Embryoid bodies (EBs) have been popular in vitro differentiation models for pluripotent stem cells for more than five decades. Initially, defined as aggregates formed by embryonal carcinoma cells, EBs gained more prominence after the derivation of karyotypically normal embryonic stem cells from early mouse blastocysts. In many cases, formation of EBs constitutes an important initial step in directed differentiation protocols aimed at generated specific cell types from undifferentiated stem cells. Indeed state-of-the-art protocols for directed differentiation of cardiomyocytes still rely on this initial EB step...
December 2, 2016: Wiley Interdisciplinary Reviews. Developmental Biology
https://www.readbyqxmd.com/read/27911005/adsorption-as-a-contributor-for-inflammatory-mediators-removal-by-different-hemofiltration-membranes-a-pilot-study
#17
Benjamin Malard, Nans Florens, Corine Lambert, Laurent Juillard
Atherosclerosis is an important predictor of mortality in patients with chronic kidney disease (CKD) and is associated with a wide inflammatory response. The aim of this study is to evaluate in vitro how different membranes can remove mediators associated with this pathology in a closed loop dialysis model. We performed experimental hemofiltration in vitro using three different membrane materials. Human plasma was preliminarily incubated with various inflammatory mediators and filtered in a closed loop circulation model for 240 min...
December 2, 2016: Artificial Organs
https://www.readbyqxmd.com/read/27910992/tug-of-war-between-two-elastically-coupled-molecular-motors-a-case-study-on-force-generation-and-force-balance
#18
Mehmet Can Uçar, Reinhard Lipowsky
Intracellular transport is performed by molecular motors that pull cargos along cytoskeletal filaments. Many cellular cargos are observed to move bidirectionally, with fast transport in both directions. This behaviour can be understood as a stochastic tug-of-war between two teams of antagonistic motors. The first theoretical model for such a tug-of-war, the Müller-Klumpp-Lipowsky (MKL) model, was based on two simplifying assumptions: (i) both motor teams move with the same velocity in the direction of the stronger team, and (ii) this velocity matching and the associated force balance arise immediately after the rebinding of an unbound motor to the filament...
December 2, 2016: Soft Matter
https://www.readbyqxmd.com/read/27910952/protective-effects-of-ginsenoside-rg1-on-intestinal-ischemia-reperfusion-injury-induced-oxidative-stress-and-apoptosis-via-activation-of-the-wnt-%C3%AE-catenin-pathway
#19
Guo Zu, Jing Guo, Ningwei Che, Tingting Zhou, Xiangwen Zhang
Ginsenoside Rg1 (Rg1) is one of the major bioactive ingredients in Panax ginseng, and it attenuates inflammation and apoptosis. The aims of our study were to explore the potential of Rg1 for the treatment of intestinal I/R injury and to determine whether the protective effects of Rg1 were exerted through the Wnt/β-catenin signaling pathway. In this study, Rg1 treatment ameliorated inflammatory factors, ROS and apoptosis that were induced by intestinal I/R injury. Cell viability was increased and cell apoptosis was decreased with Rg1 pretreatment following hypoxia/reoxygenation (H/R) in the in vitro study...
December 2, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27910950/human-antibody-3e1-targets-the-ha-stem-region-of-h1n1-and-h5n6-influenza-a-viruses
#20
Wenshuai Wang, Xiaoyu Sun, Yanbing Li, Jinpeng Su, Zhiyang Ling, Tianlong Zhang, Fang Wang, Hong Zhang, Hualan Chen, Jianping Ding, Bing Sun
As influenza A viruses remain a major threat to human health worldwide, the discovery of broadly neutralizing monoclonal antibodies that recognize conserved epitopes would facilitate the development of antibody-based therapeutic strategies. Here we report that a VH4-4-encoded human mAb named 3E1 could neutralize H1 and H5 subtype viruses in vitro and protect mice against the H1N1 and H5N6 viruses by inhibiting the low pH-induced conformational rearrangement of haemagglutinin (HA), hence blocking membrane fusion...
December 2, 2016: Nature Communications
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