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Job Fermie, Nalan Liv, Corlinda Ten Brink, Elly G van Donselaar, Wally H Müller, Nicole L Schieber, Yannick Schwab, Hans C Gerritsen, Judith Klumperman
Live-cell correlative light-electron microscopy (live-cell-CLEM) integrates live movies with the corresponding EM image, but a major challenge is to relate the dynamic characteristics of single organelles to their 3D ultrastructure. Here we introduce focused ion beam scanning electron microscopy (FIB-SEM) in a modular live-cell CLEM pipeline for single organelle CLEM. We transfected cells with LAMP-1-GFP, analyzed the dynamics of individual GFP-positive spots, and correlated these to their corresponding fine-architecture and immediate cellular environment...
February 16, 2018: Traffic
Marit A de Beer, Jeroen Kuipers, Paul M P van Bergen En Henegouwen, Ben N G Giepmans
Probes are essential to visualize proteins in their cellular environment, both using light microscopy as well as electron microscopy (EM). Correlated light microscopy and electron microscopy (CLEM) requires probes that can be imaged simultaneously by both optical and electron-dense signals. Existing combinatorial probes often have impaired efficiency, need ectopic expression as a fusion protein, or do not target endogenous proteins. Here, we present FLIPPER-bodies to label endogenous proteins for CLEM. Fluorescent Indicator and Peroxidase for Precipitation with EM Resolution (FLIPPER), the combination of a fluorescent protein and a peroxidase, is fused to a nanobody against a target of interest...
January 11, 2018: Histochemistry and Cell Biology
Thomas Burgoyne, Amelia Lane, William E Laughlin, Michael E Cheetham, Clare E Futter
Correlative light-electron microscopy (CLEM) is a powerful technique allowing localisation of specific macromolecules within fluorescence microscopy (FM) images to be mapped onto corresponding high-resolution electron microscopy (EM) images. Existing methods are applicable to limited sample types and are technically challenging. Here we describe novel methods to perform CLEM and immuno-electron microscopy (iEM) on cryostat sections utilising the popular FM embedding solution, optimal cutting temperature (OCT) compound...
2018: PloS One
Elizabeth K Lucas, Roger L Clem
Fear conditioning is a form of associative learning that is fundamental to survival and involves potentiation of activity in excitatory projection neurons (PNs). Current models stipulate that the mechanisms underlying this process involve plasticity of PN synapses, which exhibit strengthening in response to fear conditioning. However, excitatory PNs are extensively modulated by a diverse array of GABAergic interneurons whose contributions to acquisition, storage, and expression of fear memory remain poorly understood...
December 2, 2017: Brain Research Bulletin
Pavel Tchelidze, Aassif Benassarou, Hervé Kaplan, Marie-Françoise O'Donohue, Laurent Lucas, Christine Terryn, Levan Rusishvili, Giorgi Mosidze, Nathalie Lalun, Dominique Ploton
The nucleolus produces the large polycistronic transcript (47S precursor) containing the 18S, 5.8S and 28S rRNA sequences and hosts most of the nuclear steps of pre-rRNA processing. Among numerous components it contains condensed chromatin and active rRNA genes which adopt a more accessible conformation. For this reason, it is a paradigm of chromosome territory organization. Active rRNA genes are clustered within several fibrillar centers (FCs), in which they are maintained in an open configuration by Upstream Binding Factor (UBF) molecules...
2017: PloS One
Neeraj Prabhakar, Anni Määttänen, Jouko Peltonen, Pekka Hänninen, Markus Peurla, Jessica M Rosenholm
Correlative light and electron microscopy (CLEM) allows combining the advantages of fluorescence microscopy and electron microscopy for cell imaging. Rare phenomenon expressing cells can be studied by specifically tagged fluorophores with fluorescence microscopy. Subsequently, cells can be fixed and ultra-structural details can be studied with transmission electron microscopy (TEM) at a higher resolution. However, precise landmarks are necessary to track the same cell throughout the CLEM process. In this technical report, we present a high contrast inkjet-printed gold nanoparticle patterns over commercial glass coverslip to facilitate cell tracking with correlative microscopy...
February 1, 2018: Microscopy
Robert M Kaplan, Suzanne Bennett Johnson, Patricia Clem Kobor
The history of behavioral and social science research funding at the National Institutes of Health (NIH) between 1980 and 2016 is reviewed. Noncommunicable diseases are now the primary cause of death worldwide and most are strongly linked to behavior. Developed under the prevailing zeitgeist of the biomedical model, behavioral and social science has often been underfunded at NIH. In 1990, the Senate Appropriations Committee, recognizing that behavior may contribute to about half of all premature deaths, recommended that funding for behavioral and social sciences research should be about 10% of the NIH budget...
November 2017: American Psychologist
Shuoguo Li, Gang Ji, Yang Shi, Lasse Hyldgaard Klausen, Tongxin Niu, Shengliu Wang, Xiaojun Huang, Wei Ding, Xiang Zhang, Mingdong Dong, Wei Xu, Fei Sun
Cryo-correlative light and electron microscopy (cryo-CLEM) offers a unique way to analyze the high-resolution structural information of cryo-vitrified specimen by cryo-electron microscopy (cryo-EM) with the guide of the search for unique events by cryo-fluorescence microscopy (cryo-FM). To achieve cryo-FM, a trade-off must be made between the temperature and performance of objective lens. The temperature of specimen should be kept below devitrification while the distance between the objective lens and specimen should be short enough for high resolution imaging...
November 4, 2017: Journal of Structural Biology
Hananel Hazan, Noam E Ziv
There is growing need for multichannel electrophysiological systems that record from and interact with neuronal systems in near real-time. Such systems are needed, for example, for closed loop, multichannel electrophysiological/optogenetic experimentation in vivo and in a variety of other neuronal preparations, or for developing and testing neuro-prosthetic devices, to name a few. Furthermore, there is a need for such systems to be inexpensive, reliable, user friendly, easy to set-up, open and expandable, and possess long life cycles in face of rapidly changing computing environments...
2017: Frontiers in Neuroscience
Stephen D Carter, Shrawan K Mageswaran, Zachary J Farino, João I Mamede, Catherine M Oikonomou, Thomas J Hope, Zachary Freyberg, Grant J Jensen
In cryogenic correlated light and electron microscopy (cryo-CLEM), frozen targets of interest are identified and located on EM grids by fluorescence microscopy and then imaged at higher resolution by cryo-EM. Whilst working with these methods, we discovered that a variety of mammalian cells exhibit strong punctate autofluorescence when imaged under cryogenic conditions (80 K). Autofluorescence originated from multilamellar bodies (MLBs) and secretory granules. Here we describe a method to distinguish fluorescent protein tags from these autofluorescent sources based on the narrower emission spectrum of the former...
October 25, 2017: Journal of Structural Biology
Philipp Homan, Qi Lin, James W Murrough, Laili Soleimani, Dominik R Bach, Roger L Clem, Daniela Schiller
The α-1 adrenoreceptor antagonist prazosin has shown promise in the treatment of post-traumatic stress disorder (PTSD) symptoms, but its mechanisms are not well understood. Here we administered prazosin or placebo prior to threat conditioning (day 1) and tested subsequent extinction (day 2) and reextinction (day 3) in healthy human participants. Prazosin did not affect threat conditioning but augmented stimulus discrimination during extinction and reextinction, via lower responding to the safe stimulus. These results suggest that prazosin during threat acquisition may have influenced encoding or consolidation of safety processing in particular, subsequently leading to enhanced discrimination between the safe and threatening stimuli...
November 2017: Learning & Memory
Kristin Wilmoth, Christian LoBue, Matthew A Clem, Rajadhar Reddy, Linda S Hynan, Nyaz Didehbani, Kathleen Bell, Kyle B Womack, John Hart, Hunt Batjer, C Munro Cullum
OBJECTIVE: Medical history information regarding prior traumatic brain injury (TBI) usually relies on self-report, although little is known about the reliability of this information with regard to injuries sustained years or decades earlier. Even less is known about the reliability of self-reported medical history information in older individuals with cognitive impairment. To this end, we assessed the test-retest reliability of self-reported TBI history in a large, national sample. METHODS: Participants (n = 4309) were older adults with intact cognition, mild cognitive impairment (MCI) and Alzheimer's disease (AD) from the National Alzheimer's Coordinating Center...
October 9, 2017: Clinical Neuropsychologist
Errin Johnson, Rainer Kaufmann
Correlative super-resolution light and electron microscopy (super-resolution CLEM) is a powerful and emerging tool in biological research. The practical realization of these two very different microscopy techniques with their individual requirements remains a challenging task. There is a broad range of approaches to choose from, each with their own advantages and limitations. Here, we present a detailed protocol for in-resin super-resolution CLEM of high-pressure frozen and freeze substituted cultured cells...
2017: Methods in Molecular Biology
Milena D Vukic, Nenad L Vukovic, Ana D Obradovic, Suzana Lj Popovic, Milan M Zaric, Predrag M Djurdjevic, Snezana D Markovic, Dejan D Baskic
In the present study, five root extracts of Onosma visianii Clem were investigated for their in vitro cytotoxic activity. On the basis of HPLC-PDA analysis, these extracts have proved to be a rich source of naphthoquinones as natural colourants for food and cosmetic industry. All investigated root extracts contain acetylshikonin, isobutyrylshikonin and α-methylbutyrylshikonin as major compounds. As the most abundant source of active compounds for antitumour therapy, acetone, chloroform and ethyl acetate extracts showed strong cytotoxic activity towards HCT-116 and MDA-MB-231 cancer cell lines...
September 7, 2017: Natural Product Research
Maarten W Tuijtel, Aat A Mulder, Clara C Posthuma, Barbara van der Hoeven, Abraham J Koster, Montserrat Bárcena, Frank G A Faas, Thomas H Sharp
Correlative light-electron microscopy (CLEM) combines the high spatial resolution of transmission electron microscopy (TEM) with the capability of fluorescence light microscopy (FLM) to locate rare or transient cellular events within a large field of view. CLEM is therefore a powerful technique to study cellular processes. Aligning images derived from both imaging modalities is a prerequisite to correlate the two microscopy data sets, and poor alignment can limit interpretability of the data. Here, we describe how uranyl acetate, a commonly-used contrast agent for TEM, can be induced to fluoresce brightly at cryogenic temperatures (-195 °C) and imaged by cryoFLM using standard filter sets...
September 5, 2017: Scientific Reports
Drayton A Hammond, Julia Cullen, Jacob T Painter, Kelsey McCain, Oktawia A Clem, Amy L Brotherton, Divyan Chopra, Nikhil Meena
BACKGROUND: Delays in achievement of target mean arterial pressure (MAP) have been associated with increased mortality in patients with septic shock. Vasopressin may be added to norepinephrine to raise MAP or decrease norepinephrine dosage. The purpose of this study was to determine whether early initiation of vasopressin to norepinephrine resulted in a reduced time to target MAP compared to norepinephrine monotherapy. METHODS: This retrospective cohort study compared early addition of vasopressin within 4 hours of septic shock onset to norepinephrine versus initial norepinephrine monotherapy in medically, critically ill patients with septic shock admitted from May 2014 to October 2015...
January 1, 2017: Journal of Intensive Care Medicine
James R Clem, Jodi Heins
No abstract text is available yet for this article.
April 2017: South Dakota Medicine: the Journal of the South Dakota State Medical Association
Shengliu Wang, Shuoguo Li, Gang Ji, Xiaojun Huang, Fei Sun
Correlative cryo-fluorescence and cryo-electron microscopy (cryo-CLEM) system has been fast becoming a powerful technique with the advantage to allow the fluorescent labeling and direct visualization of the close-to-physiologic ultrastructure in cells at the same time, offering unique insights into the ultrastructure with specific cellular function. There have been various engineered ways to achieve cryo-CLEM including the commercial FEI iCorr system that integrates fluorescence microscope into the column of transmission electron microscope...
2017: Biophysics Reports
P J Silk, E Eveleigh, L Roscoe, K Burgess, S Weatherby, G Leclair, P Mayo, M Brophy
The primary sex pheromone components of the female spruce budworm, Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae), are (E)- and (Z)-11-tetradecenal, produced in 95:5 ratio. However, male flight responses to calling females in a wind tunnel were faster and maintained longer than responses to any synthetic aldehyde blend. Analyses of cuticular extracts from spruce budworm adults revealed series of n-alkanes and n-monoalkenes with predominantly odd numbers of carbon atoms from C23- C29 in both sexes...
August 2017: Journal of Chemical Ecology
Drayton A Hammond, Jelena Stojakovic, Niranjan Kathe, Julie Tran, Oktawia A Clem, Kristina Erbach, Jarrod King
BACKGROUND: "Rules of thumb" for the replacement of electrolytes, including magnesium, in critical care settings are used, despite minimal empirical validation of their ability to achieve a target serum concentration. This study's purpose was to evaluate the effectiveness and safety surrounding magnesium replacement in medically, critically ill patients with mild-to-moderate hypomagnesemia. METHODS: This was a single-center, retrospective, observational evaluation of episodes of intravenous magnesium replacement ordered for patients with mild-to-moderate hypomagnesemia (1...
January 1, 2017: Journal of Intensive Care Medicine
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