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RNA Imaging

Caitlin M Davis, Martin Gruebele
Qualitative imaging of biomolecular localization and distribution inside cells has revolutionized cell biology. Most of these powerful techniques require modifications to the target biomolecule. Over the past 10 years, these techniques have been extended to quantitative measurements, from in-cell protein folding rates, to complex dissociation constants, to RNA lifetimes. Such measurements can be affected even when a target molecules is just mildly perturbed by its labels. Here, the impact of labeling on protein (and RNA) structure, stability, and function in cells are discussed via practical examples from recent literature...
March 16, 2018: Biochemistry
Andrea Cristine Koishi, Andréia Akemi Suzukawa, Camila Zanluca, Daria Elena Camacho, Guillermo Comach, Claudia Nunes Duarte Dos Santos
Zika virus (ZIKV) is an emerging arbovirus belonging to the genus flavivirus that comprises other important public health viruses, such as dengue (DENV) and yellow fever (YFV). In general, ZIKV infection is a self-limiting disease, however cases of Guillain-Barré syndrome and congenital brain abnormalities in newborn infants have been reported. Diagnosing ZIKV infection remains a challenge, as viral RNA detection is only applicable until a few days after the onset of symptoms. After that, serological tests must be applied, and, as expected, high cross-reactivity between ZIKV and other flavivirus serology is observed...
March 15, 2018: PLoS Neglected Tropical Diseases
Tsutomu Namikawa, Kazune Fujisawa, Eri Munekage, Masaya Munekage, Yusuke Oki, Hiromichi Maeda, Hiroyuki Kitagawa, Hiroshi Ueta, Michiya Kobayashi, Kazuhiro Hanazaki
The present case report presents a rare case of Epstein-Barr virus (EBV)-associated early gastric carcinoma with lymphoid stroma, accompanied by lymph node metastasis. A 61-year-old woman was referred to our hospital following observation of a gastric mass lesion that was initially diagnosed at a medical check-up. Esophagogastroduodenoscopy revealed a slightly elevated lesion with a central irregular depression in the middle third of the stomach. Endoscopic ultrasonography revealed a well-circumscribed hypoechoic mass located predominantly within the submucosa and the mucosa...
April 2018: Molecular and Clinical Oncology
Bo Yang, Nathan Heath Patterson, Tina Tsui, Richard M Caprioli, Jeremy L Norris
It has been widely recognized that individual cells that exist within a large population of cells, even if they are genetically identical, can have divergent molecular makeups resulting from a variety of factors, including local environmental factors and stochastic processes within each cell. Presently, numerous approaches have been described that permit the resolution of these single-cell expression differences for RNA and protein; however, relatively few techniques exist for the study of lipids and metabolites in this manner...
March 13, 2018: Journal of the American Society for Mass Spectrometry
Fei Xiao, Stanley Wang, Rina Barouch-Bentov, Gregory Neveu, Szuyuan Pu, Melanie Beer, Stanford Schor, Sathish Kumar, Vlad Nicolaescu, Brett D Lindenbach, Glenn Randall, Shirit Einav
Hepatitis C virus (HCV) spreads via secreted cell-free particles or direct cell-to-cell transmission. Yet, virus-host determinants governing differential intracellular trafficking of cell-free- and cell-to-cell-transmitted virus remain unknown. The host adaptor proteins (APs) AP-1A, AP-1B, and AP-4 traffic in post-Golgi compartments, and the latter two are implicated in basolateral sorting. We reported that AP-1A mediates HCV trafficking during release, whereas the endocytic adaptor AP-2 mediates entry and assembly...
March 13, 2018: MBio
Mary Carroll Shapiro, Tien Tang, Atreyi Dasgupta, Lyazat Kurenbekova, Ryan Shuck, M Waleed Gaber, Jason T Yustein
PURPOSE: Radiation therapy (RT) is a viable therapeutic option for Ewing sarcoma (ES) patients. However, little progress has been made to elucidate the mechanisms of radioresistance. This study establishes a novel ES irradiation-adapted model designed to assess molecular and18 F fluorodeoxyglucose (FDG) positron emission tomography (PET) alterations secondary to RT. METHODS AND MATERIALS: Radiation-adapted cell lines (RACLs) were created in vitro by exposing ES human cell lines to fractionated doses of radiation...
February 5, 2018: International Journal of Radiation Oncology, Biology, Physics
Kyoung A Jhang, Jin-Sun Park, Hee-Sun Kim, Young Hae Chong
BACKGROUND: Mer tyrosine kinase (MerTK) activity necessary for amyloid-stimulated phagocytosis strongly implicates that MerTK dysregulation might contribute to chronic inflammation implicated in Alzheimer's disease (AD) pathology. However, the precise mechanism involved in the regulation of MerTK expression by amyloid-β (Aβ) in proinflammatory environment has not yet been ascertained. METHODS: The objective of this study was to determine the underlying mechanism involved in Aβ-mediated decrease in MerTK expression through Aβ-mediated regulation of MerTK expression and its modulation by sulforaphane in human THP-1 macrophages challenged with Aβ1-42...
March 12, 2018: Journal of Neuroinflammation
Anas Z Abidin, Adora M DSouza, Mahesh B Nagarajan, Lu Wang, Xing Qiu, Giovanni Schifitto, Axel Wismüller
HIV is capable of invading the brain soon after seroconversion. This ultimately can lead to deficits in multiple cognitive domains commonly referred to as HIV-associated neurocognitive disorders (HAND). Clinical diagnosis of such deficits requires detailed neuropsychological assessment but clinical signs may be difficult to detect during asymptomatic injury of the central nervous system (CNS). Therefore neuroimaging biomarkers are of particular interest in HAND. In this study, we constructed brain connectivity profiles of 40 subjects (20 HIV positive subjects and 20 age-matched seronegative controls) using two different methods: a non-linear mutual connectivity analysis approach and a conventional method based on Pearson's correlation...
2018: NeuroImage: Clinical
Sinae Kim, Eunji Lee, Jaeyun Jung, Jong Won Lee, Hee Jung Kim, Jisun Kim, Hyun Ju Yoo, Hee Jin Lee, Sun Young Chae, Sang Min Jeon, Byung Ho Son, Gyungyup Gong, Shyam K Sharan, Suhwan Chang
MicroRNA is an endogenous, small RNA controlling multiple target genes and playing roles in various biological processes including tumorigenesis. Here, we addressed the function of miR-155 using LC-MS/MS-based metabolic profiling of miR-155 deficient breast cancer cells. Our results revealed the loss of miR-155 hampers glucose uptake and glycolysis, via the down-regulation of glucose transporters and metabolic enzymes including HK2, PKM2, and LDHA. We showed this is due to the down-regulation of cMYC, controlled through phosphoinositide-3-kinase regulatory subunit alpha (PIK3R1)-PDK1/AKT-FOXO3a pathway...
March 12, 2018: Oncogene
Dinesh Pratap Singh, Carlos Eugenio Herrera, Brijesh Singh, Shipra Singh, Rajesh Kumar Singh, Rajesh Kumar
The two-dimensional (2D) derivative of graphite termed graphene has widespread applications in various frontiers areas of nanoscience and nanotechnologies. Graphene in its oxidized form named as graphene oxide (GO) has a mixed structure equipped with various oxygen containing functional groups (epoxy, hydroxyl, carboxylic and carbonyl etc.) provides attachment sites to various biological molecules including protein, deoxyribonucleic acid (DNA), ribonucleic acid (RNA) etc. The attached biological molecules with the help of functional groups make it a promising candidate in research field of biotechnological and biomedical applications...
May 1, 2018: Materials Science & Engineering. C, Materials for Biological Applications
William Beckman, Ilona M Vuist, Hermannus Kempe, Pernette J Verschure
Single-molecule RNA fluorescent in situ hybridization (smRNA FISH) allows for the visualization, localization, and quantification of RNA transcripts within individual cells and tissues using custom-designed fluorescently labeled oligonucleotide probes. Here we describe a protocol for the preparation, imaging, and analysis of a smRNA FISH experiment that can be applied to any RNA of choice. We also provide insights as to how this powerful tool can be used to study epigenetic regulation, for example, following the epigenetic editing of genes...
2018: Methods in Molecular Biology
Taewook Kang, Pia Jensen, Vita Solovyeva, Jonathan R Brewer, Martin R Larsen
Characterization of molecular mechanisms underlying pancreatic β-cell function in relation to glucose-stimulated insulin secretion is incomplete, especially with respect to global response in the nuclear environment. Here, we focus on the characterization of proteins in the nuclear environment of β-cells after brief, high glucose-stimulation. We compared purified nuclei derived from β-cells stimulated with 17mM glucose for 0, 2, and 5 minutes using quantitative proteomics, a time frame that most likely does not result in translation of new protein in the cell...
March 8, 2018: Journal of Proteome Research
Miaolong Lu, Xianquan Zhan
Cancer with heavily economic and social burden is the hot point in the field of medical research. Some remarkable achievements have been made; however, the exact mechanisms of tumor initiation and development remain unclear. Cancer is a complex, whole-body disease that involves multiple abnormalities in the levels of DNA, RNA, protein, metabolite and medical imaging. Biological omics including genomics, transcriptomics, proteomics, metabolomics and radiomics aims to systematically understand carcinogenesis in different biological levels, which is driving the shift of cancer research paradigm from single parameter model to multi-parameter systematical model...
March 2018: EPMA Journal
H M Steinkamp, J D Hathaway-Schrader, M B Chavez, J D Aartun, L Zhang, T Jensen, A Shojaee Bakhtiari, K L Helke, D J Stumpo, A V Alekseyenko, C M Novince, P J Blackshear, K L Kirkwood
Tristetraprolin (TTP) is an RNA-binding protein that targets numerous immunomodulatory mRNA transcripts for degradation. Many TTP targets are key players in the pathogenesis of periodontal bone loss, including tumor necrosis factor-α. To better understand the extent that host immune factors play during periodontal bone loss, we assessed alveolar bone levels, inflammation and osteoclast activity in periodontal tissues, and immune response in draining cervical lymph nodes in TTP-deficient and wild-type (WT) mice in an aging study...
March 1, 2018: Journal of Dental Research
V Siddartha Yerramilli, Kyung Hyuk Kim
RNAs mediate many different processes that are central to cellular function. The ability to quantify or image RNAs in live cells is very useful in elucidating such functions of RNA. RNA aptamer-fluorogen systems have been increasingly used in labeling RNAs in live cells. Here, we use the malachite green aptamer (MGA), an RNA aptamer that can specifically bind to malachite green (MG) dye and induces it to emit far-red fluorescence signals. Previous studies on MGA showed a potential for the use of MGA for genetically tagging other RNA molecules in live cells...
March 7, 2018: ACS Synthetic Biology
Marianna Szemes, Alexander Greenhough, Zsombor Melegh, Sally Malik, Aysen Yuksel, Daniel Catchpoole, Kelli Gallacher, Madhu Kollareddy, Ji Hyun Park, Karim Malik
Neuroblastoma is one of the commonest and deadliest solid tumours of childhood, and is thought to result from disrupted differentiation of the developing sympathoadrenergic lineage of the neural crest. Neuroblastoma exhibits intra- and intertumoural heterogeneity, with high risk tumours characterised by poor differentiation, which can be attributable to MYCN-mediated repression of genes involved in neuronal differentiation. MYCN is known to co-operate with oncogenic signalling pathways such as Alk, Akt and MEK/ERK signalling, and, together with c-MYC has been shown to be activated by Wnt signalling in various tissues...
March 2, 2018: Neoplasia: An International Journal for Oncology Research
Michael Gotrik, Gurpreet Sekhon, Saumya Saurabh, Margaret Nakamoto, Michael Eisenstein, H Tom Soh
RNA aptamers that generate a strong fluorescence signal upon binding a nonfluorescent small-molecule dye offer a powerful means for the selective imaging of individual RNA species. Unfortunately, conventional in vitro discovery methods are not efficient at generating such fluorescence-enhancing aptamers, because they primarily exert selective pressure based on target affinity-a characteristic that correlates poorly with fluorescence enhancement. Thus, only a handful of fluorescence-enhancing aptamers have been reported to date...
March 5, 2018: Journal of the American Chemical Society
Bomyi Lim
Recent advances in imaging techniques have enabled visualizations of nascent transcripts or individual protein molecules at high spatiotemporal resolution, revealing the complex nature of transcriptional regulation. Here, we highlight recent studies that have provided comprehensive insights to transcriptional dynamics using such quantitative imaging techniques. Specifically, they demonstrated that transcriptional activity is stochastic, and such transcriptional bursting is modulated by multiple components like chromatin environments, concentration of transcription factors, and enhancer-promoter interactions...
March 1, 2018: Current Opinion in Biotechnology
Silvia Kocanova, Isabelle Goiffon, Kerstin Bystricky
Fluorescence in situ hybridization (FISH) is a common technique used to label DNA and/or RNA for detection of a genomic region of interest. However, the technique can be challenging, in particular when applied to single genes in human cancer cells. Here, we provide a step-by-step protocol for analysis of short (35kb to 300kb) genomic regions in three dimensions (3D). We discuss the experimental design and provide practical considerations for 3D imaging and data analysis to determine chromatin folding. We demonstrate that 3D FISH using BACs (Bacterial Artificial Chromosomes) or fosmids can provide detailed information of the architecture of gene domains...
February 28, 2018: Methods: a Companion to Methods in Enzymology
Yang Liu, Ran-Xi Zhang, Wei Yuan, Hou-Qing Chen, Dong-Dong Tian, Hao Li, Xiao Jiang, Zhong-Liang Deng, Yang Wang
BACKGROUND/AIMS: Bone morphogenetic proteins (BMPs) and BMP receptors widely participate in osteolytic metastasis of breast cancer, while their role in tumor-stromal interaction is largely unknown. In this study, we investigated whether BMP receptor type 1a (BMPR1a) can alter the interaction between metastatic cancer cells and osteoclast precursors. METHODS: Adenovirus-mediated RNA interference was used to interrupt target genes of human breast cancer cell lines and nude mice were injected intratibially with the cancer cells...
February 23, 2018: Cellular Physiology and Biochemistry
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