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Cellular Imaging

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https://www.readbyqxmd.com/read/28449134/characterizing-exogenous-mrna-delivery-trafficking-cytoplasmic-release-and-rna-protein-correlations-at-the-level-of-single-cells
#1
Jonathan L Kirschman, Sushma Bhosle, Daryll Vanover, Emmeline L Blanchard, Kristin H Loomis, Chiara Zurla, Kathryn Murray, Blaine C Lam, Philip J Santangelo
The use of synthetic messenger ribonucleic acid (mRNA) to express specific proteins is a highly promising therapeutic and vaccine approach that avoids many safety issues associated with viral or DNA-based systems. However, in order to optimize mRNA designs and delivery, technology advancements are required to study fundamental mechanisms of mRNA uptake and localization at the single-cell and tissue level. Here, we present a single RNA sensitive fluorescent labeling method which allows us to label and visualize synthetic mRNA without significantly affecting function...
April 26, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28448055/transient-expression-and-cellular-localization-of-recombinant-proteins-in-cultured-insect-cells
#2
Jeffrey A Fabrick, J Joe Hull
Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized...
April 20, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28448052/an-in-vitro-organ-culture-model-of-the-murine-intervertebral-disc
#3
Jennifer W Liu, Kevin H Lin, Christian Weber, Sameer Bhalla, Sean Kelso, Kaixi Wang, Simon Y Tang
Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices...
April 11, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28448036/using-microfluidic-devices-to-measure-lifespan-and-cellular-phenotypes-in-single-budding-yeast-cells
#4
Ke Zou, Diana S Ren, Qi Ou-Yang, Hao Li, Jiashun Zheng
Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells...
March 30, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28448021/detection-of-ligand-activated-g-protein-coupled-receptor-internalization-by-confocal-microscopy
#5
Jingwen Yang, Yunjun Yan, Xiaowei Xiang, Yuchao Xu, Naiming Zhou, Tianming Wang
Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent fusion proteins, such as green fluorescent protein (GFP), to determine their expression, localization, and function. The subcellular localization of target proteins is important for identification, characterization, and functional analyses. Internalization is one of the predominant mechanisms controlling G protein-coupled receptor (GPCR) signaling to ensure the appropriate cellular responses to stimuli...
April 9, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28448004/live-confocal-imaging-of-developing-arabidopsis-flowers
#6
Nathanaël Prunet
The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. Recent advances in confocal microscopy, combined with the development of numerous fluorophores, have overcome these issues and opened the possibility to study the expression of several genes simultaneously, with a good cellular resolution, in live samples. Live confocal imaging provides plant biologists with a powerful tool to study development, and has been extensively used to study root growth and the formation of lateral organs on the flanks of the shoot apical meristem...
April 1, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28448000/high-resolution-imaging-and-analysis-of-individual-astral-microtubule-dynamics-in-budding-yeast
#7
Colby P Fees, Cassi Estrem, Jeffrey K Moore
Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells...
April 20, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28447612/distinct-molecular-cues-ensure-a-robust-microtubule-dependent-nuclear-positioning-in-the-drosophila-oocyte
#8
Nicolas Tissot, Jean-Antoine Lepesant, Fred Bernard, Kevin Legent, Floris Bosveld, Charlotte Martin, Orestis Faklaris, Yohanns Bellaïche, Maïté Coppey, Antoine Guichet
Controlling nucleus localization is crucial for a variety of cellular functions. In the Drosophila oocyte, nuclear asymmetric positioning is essential for the reorganization of the microtubule (MT) network that controls the polarized transport of axis determinants. A combination of quantitative three-dimensional live imaging and laser ablation-mediated force analysis reveal that nuclear positioning is ensured with an unexpected level of robustness. We show that the nucleus is pushed to the oocyte antero-dorsal cortex by MTs and that its migration can proceed through distinct tracks...
April 27, 2017: Nature Communications
https://www.readbyqxmd.com/read/28446707/deterministic-versus-stochastic-model-of-reprogramming-new-evidence-from-cellular-barcoding-technique
#9
Anastasia M Yunusova, Veniamin S Fishman, Gennady V Vasiliev, Nariman R Battulin
Factor-mediated reprogramming of somatic cells towards pluripotency is a low-efficiency process during which only small subsets of cells are successfully reprogrammed. Previous analyses of the determinants of the reprogramming potential are based on average measurements across a large population of cells or on monitoring a relatively small number of single cells with live imaging. Here, we applied lentiviral genetic barcoding, a powerful tool enabling the identification of familiar relationships in thousands of cells...
April 2017: Open Biology
https://www.readbyqxmd.com/read/28446689/bone-clarity-clearing-imaging-and-computational-analysis-of-osteoprogenitors-within-intact-bone-marrow
#10
Alon Greenbaum, Ken Y Chan, Tatyana Dobreva, David Brown, Deepak H Balani, Rogely Boyce, Henry M Kronenberg, Helen J McBride, Viviana Gradinaru
Bone tissue harbors unique and essential physiological processes, such as hematopoiesis, bone growth, and bone remodeling. To enable visualization of these processes at the cellular level in an intact environment, we developed "Bone CLARITY," a bone tissue clearing method. We used Bone CLARITY and a custom-built light-sheet fluorescence microscope to detect the endogenous fluorescence of Sox9-tdTomato(+) osteoprogenitor cells in the tibia, femur, and vertebral column of adult transgenic mice. To obtain a complete distribution map of these osteoprogenitor cells, we developed a computational pipeline that semiautomatically detects individual Sox9-tdTomato(+) cells in their native three-dimensional environment...
April 26, 2017: Science Translational Medicine
https://www.readbyqxmd.com/read/28446216/the-effect-of-adipose-tissue-derived-stem-cells-in-a-middle-cerebral-artery-occlusion-stroke-model-depends-on-their-engraftment-rate
#11
Saskia Grudzenski, Sebastian Baier, Anne Ebert, Pim Pullens, Andreas Lemke, Karen Bieback, Rick M Dijkhuizen, Lothar R Schad, Angelika Alonso, Michael G Hennerici, Marc Fatar
BACKGROUND: In the field of experimental stem cell therapy, intra-arterial (IA) delivery yields the best results concerning, for example, migrated cell number at the targeted site. However, IA application also appears to be associated with increased mortality rates and infarction. Since many rodent studies systemically apply 1 × 10(6) cells, this could also be a consequence of engrafted cell number. The aim of this study was therefore to investigate the effect of different doses of adipose tissue-derived stem cells (ASCs) on engraftment rates and stroke outcome measured in vivo using 9...
April 26, 2017: Stem Cell Research & Therapy
https://www.readbyqxmd.com/read/28445743/egfr-targeted-micelles-containing-near-infrared-dye-for-enhanced-photothermal-therapy-in-colorectal-cancer
#12
Ying-Hsia Shih, Tsai-Yueh Luo, Ping-Fang Chiang, Cheng-Jung Yao, Wuu-Jyh Lin, Cheng-Liang Peng, Ming-Jium Shieh
The purpose of this research was to investigate the effectiveness of epidermal growth factor receptor (EGFR) targeted micelles loaded with IR-780 (Cetuximab/IR-780/micelles) for generating tumor targeting, multimodal images, and photothermal therapy (PTT). We initially studied the cellular uptake of these micelles using the HCT-116 and SW-620 cell lines. HCT-116 (high expression of EGFR) and SW-620 (low expression of EGFR) cell lines were used to examine biodistribution and antitumor effects of Cetuximab/IR-780/micelles...
April 23, 2017: Journal of Controlled Release: Official Journal of the Controlled Release Society
https://www.readbyqxmd.com/read/28445739/autophagy-associated-cytotoxicity-and-cellular-uptake-mechanisms-of-bismuth-nanoparticles-in-human-kidney-cells
#13
Yongming Liu, Jing Zhuang, Xihui Zhang, Cong Le, Ning Zhu, Liecheng Yang, Yong Wang, Tao Chen, Yangyun Wang, Leshuai W Zhang
Bismuth compounds have been used for treatment of bacterial infection, and recently bismuth nanoparticles (BiNP) were synthesized for imaging and diagnostic purpose, while safety concern of bismuth cannot be ignored. Here, we prepared ultrasmall BiNP and showed an enhanced tumor imaging, but BiNP revealed a differentiated cytotoxicity in human embryonic kidney 293 cells (HEK293) compared to other cell types. For the first time, we found that BiNP can induce autophagy, shown as the increase of monodansylcadaverine fluorescence staining and the amount of LC3II that can be inhibited by 3-MA...
April 23, 2017: Toxicology Letters
https://www.readbyqxmd.com/read/28444369/three-dimensional-intracellular-structure-of-a-whole-rice-mesophyll-cell-observed-with-fib-sem
#14
Takao Oi, Sakiko Enomoto, Tomoyo Nakao, Shigeo Arai, Koji Yamane, Mitsutaka Taniguchi
Background and Aims: Ultrathin sections of rice leaf blades observed two-dimensionally using a transmission electron microscope (TEM) show that the chlorenchyma is composed of lobed mesophyll cells, with intricate cell boundaries, and lined with chloroplasts. The lobed cell shape and chloroplast positioning are believed to enhance the area available for the gas exchange surface for photosynthesis in rice leaves. However, a cell image revealing the three-dimensional (3-D) ultrastructure of rice mesophyll cells has not been visualized...
April 21, 2017: Annals of Botany
https://www.readbyqxmd.com/read/28443896/an-msn-peg-ip-drug-delivery-system-and-il13r%C3%AE-2-as-targeted-therapy-for-glioma
#15
Jinlong Shi, Shiqiang Hou, Jianfei Huang, Shanshan Wang, Wei Huan, Chuanjun Huang, Xiaojiang Liu, Rui Jiang, Wenbo Qian, Jingjing Lu, Xiubing Wang, Wei Shi, Rongqin Huang, Jian Chen
A combination of gene therapy and chemotherapy has recently received interest as a targeted therapy for glioma. A mesoporous silica nanoparticle (MSN)-based vehicle coated with IL13Rα2-targeted peptide (IP) using polyethylene glycol (PEG), MSN-PEG-IP (MPI), was constructed and confirmed as a potential glioma-targeted drug delivery system in vitro. In this work, tissue microarray (TMA) results revealed that IL13Rα2 was over-expressed in human glioma tissues and that high expression of IL13Rα2 in patients was associated with poor survival...
April 26, 2017: Nanoscale
https://www.readbyqxmd.com/read/28443626/nanodiamond-enhanced-mri-via-in-situ-hyperpolarization
#16
David E J Waddington, Mathieu Sarracanie, Huiliang Zhang, Najat Salameh, David R Glenn, Ewa Rej, Torsten Gaebel, Thomas Boele, Ronald L Walsworth, David J Reilly, Matthew S Rosen
Nanodiamonds are of interest as nontoxic substrates for targeted drug delivery and as highly biostable fluorescent markers for cellular tracking. Beyond optical techniques, however, options for noninvasive imaging of nanodiamonds in vivo are severely limited. Here, we demonstrate that the Overhauser effect, a proton-electron polarization transfer technique, can enable high-contrast magnetic resonance imaging (MRI) of nanodiamonds in water at room temperature and ultra-low magnetic field. The technique transfers spin polarization from paramagnetic impurities at nanodiamond surfaces to (1)H spins in the surrounding water solution, creating MRI contrast on-demand...
April 26, 2017: Nature Communications
https://www.readbyqxmd.com/read/28443327/acidity-triggered-tumor-retention-internalization-of-chimeric-peptide-for-enhanced-photodynamic-therapy-and-real-time-monitoring-of-therapeutic-effects
#17
Kai Han, Wei-Yun Zhang, Zhao-Yu Ma, Shi-Bo Wang, Lu-Ming Xu, Jia Liu, Xianzheng Zhang, Heyou Han
Photodynamic therapy (PDT) holds great promise in tumor treatment. Nevertheless, it remains highly desirable to develop easy-to-fabricated PDT systems with improved tumor accumulation/internalization and timely therapeutic feedback. Here, we report a tumor acidity responsive chimeric peptide for enhanced PDT and noninvasively real-time apoptosis imaging. Both in vitro and in vivo studies revealed that tumor mildly acidic microenvironment could trigger rapid protonation of carboxylate anion in chimeric peptide, which led to increased zeta potential, improved hydrophobicity, controlled size enlargement and precise morphology switch from sphere to spherocylinder shape of chimeric peptide...
April 26, 2017: ACS Applied Materials & Interfaces
https://www.readbyqxmd.com/read/28443093/a-novel-cervical-spinal-cord-window-preparation-allows-for-two-photon-imaging-of-t-cell-interactions-with-the-cervical-spinal-cord-microvasculature-during-experimental-autoimmune-encephalomyelitis
#18
Neda Haghayegh Jahromi, Heidi Tardent, Gaby Enzmann, Urban Deutsch, Naoto Kawakami, Stefan Bittner, Dietmar Vestweber, Frauke Zipp, Jens V Stein, Britta Engelhardt
T-cell migration across the blood-brain barrier (BBB) is a crucial step in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Two-photon intravital microscopy (2P-IVM) has been established as a powerful tool to study cell-cell interactions in inflammatory EAE lesions in living animals. In EAE, central nervous system inflammation is strongly pronounced in the spinal cord, an organ in which 2P-IVM imaging is technically very challenging and has been limited to the lumbar spinal cord...
2017: Frontiers in Immunology
https://www.readbyqxmd.com/read/28442431/reducing-isoform-complexity-of-human-tetraspanins-by-optimized-expression-in-dictyostelium-discoideum-enables-high-throughput-functional-read-out
#19
Tineke Scheltz, Julia von Bülow, Eric Beitz
The human tetraspanin family of scaffold proteins comprises 33 isoforms. Being integral membrane proteins, they organize a so-called tetraspanin web via homomeric and heteromeric protein-protein interactions with integrins, immunoglobulins, growth factors, receptor tyrosine kinases, proteases, signaling proteins, and viral capsid proteins. Tetraspanins promote cellular effects, such as adhesion, migration, invasion, signaling, membrane fusion, protein trafficking, cancer progression, and infections. The ubiquitous expression of multiple tetraspanin isoforms and partner proteins hampers specific interaction studies...
April 22, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28442408/plasma-membrane-activatable-polymeric-nanotheranostics-with-self-enhanced-light-triggered-photosensitizer-cell-influx-for-photodynamic-cancer-therapy
#20
Hao-Ran Jia, Yao-Wen Jiang, Ya-Xuan Zhu, Yan-Hong Li, Hong-Yin Wang, Xiaofeng Han, Zhi-Wu Yu, Ning Gu, Peidang Liu, Zhan Chen, Fu-Gen Wu
To address the issue of low cellular uptake of photosensitizers by cancer cells in photodynamic therapy (PDT), we designed a smart plasma membrane-activatable polymeric nanodrug by conjugating the photosensitizer protoporphyrin IX (PpIX) and polyethylene glycol (PEG) with glycol chitosan (GC). The as-prepared GC-PEG-PpIX can self-assemble into core-shell nanoparticles (NPs) in aqueous solution and the fluorescence of PpIX moieties in the inner core is highly quenched due to strong π-π stacking. Interestingly, when encountering plasma membranes, the GC-PEG-PpIX NPs can disassemble and stably attach to plasma membranes due to the membrane affinity of PpIX moieties, which effectively suppresses the self-quenching of PpIX, leading to significantly enhanced fluorescence and singlet oxygen ((1)O2) production upon laser irradiation...
April 22, 2017: Journal of Controlled Release: Official Journal of the Controlled Release Society
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