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  • Video: Genome Engineering with CRISPR-Cas9

    Jennifer Doudna tells the story of how studying the way bacteria fight viral infection turned into a genomic engineering technology that has transformed molecular biology research. In 2013, Doudna and her colleagues developed the CRISPR-Cas9 gene expression system that, when introduced into animal cells, makes site-specific changes to intact genomes. CRISPR-Cas9 is more precise, more efficient, and less expensive than other genome editing tools and, as a result, has facilitated a wide range of studies that were previously unachievable.
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  • Video: Genome Editing with CRISPR-Cas9

    Jennifer Doudna tells the story of how studying the way bacteria fight viral infection turned into a genomic engineering technology that has transformed molecular biology research. In 2013, Doudna and her colleagues developed the CRISPR-Cas9 gene expression system that, when introduced into animal cells, makes site-specific changes to intact genomes. CRISPR-Cas9 is more precise, more efficient, and less expensive than other genome editing tools and, as a result, has facilitated a wide range of studies that were previously unachievable.
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  • Correlation of Phenotype and Genotype – microscopic samples and their biomolecular backgrounds

    Seeing is believing, this is what makes microscopes, imaging systems, or box-type imagers so important for researchers in many different fields like pathology research, cancer research, neuroscience, and developmental research. Correlation of phenotype (what you can see) and genotype (genetic background) is highly desired but often biased by whole tissue approaches as no real solution is available to reliably separate the single cells of interest and surrounding tissue. Thus, results of DNA mutation analysis (sequencing), gene expression profiling (quantitative real-time PCR, qPCR, microarray, digital PCR), next generation sequencing (NGS) or mass spectrometry (MS) approaches can give mixed results as no pure starting material for the biomolecular methods of choice is available.
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  • The Molecular Architecture of Hemidesmosomes as Revealed by Super-Resolution Microscopy

    Hemidesmosomes have been extensively studied by immunofluorescence microscopy, but due to its limited resolution, their precise organization remained poorly understood. We studied hemidesmosome organization in cultured keratinocytes by 2- and 3-color super-resolution microscopy. We observed that in the cell periphery, nascent hemidesmosomes are associated with individual keratin filaments and that β4 is distributed along rather than under keratin filaments. By applying innovative methods to quantify molecular distances, we demonstrate that the hemidesmosomal plaque protein plectin interacts simultaneously and asymmetrically with β4 and keratin.
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  • Super-Resolution Optical Microscopy of Lipid Plasma Membrane Dynamics

    Plasma membrane dynamics are an important ruler of cellular activity, particularly through the interaction and diffusion dynamics of membrane-embedded proteins and lipids. FCS (fluorescence correlation spectroscopy) on an optical (confocal) microscope is a popular tool for investigating such dynamics. Unfortunately, its full applicability is constrained by the limited spatial resolution of a conventional optical microscope. The present chapter depicts the combination of optical super-resolution STED (stimulated emission depletion) microscopy with FCS , and why it is an important tool for investigating molecular membrane dynamics in living cells. Compared with conventional FCS , the STED- FCS approach demonstrates an improved possibility to distinguish free from anomalous molecular diffusion, and thus to give new insights into lipid–protein interactions and the traditional lipid ‘raft’ theory.
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  • Gene Editing with CRISPR/Cas9 - Breakthrough in Genome Engineering

    The CRISPR/Cas9 system is one of several different bacterial systems for defense against viral attacks. It consists of two main components. One is a small piece of RNA which binds to the viral target sequence via Watson-Crick base pairing. It serves as a marker for the foreign nucleic acid. The second component is the Cas9 protein. It binds to the marked sequence and cuts it due to its nuclease activity. Because the base pairing RNA can be synthesized easily and then used to determine a target region, researchers have utilized this system in the laboratory for genome editing.
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  • Practical Guide for Excellent GSDIM Super-Resolution Images

    Do you know that most protists and bacteria lack in one feature that each of our body cell has? Our cells are touch and communicate with one another. They send and receive a variety of signals that coordinate their behavior to act together as a functional multicellular organism. Exploring the way of cellular communication and the ways how the cell surface interacts to organize tissues and body structures is of great interest. Kees Jalink and his team of scientists at the Netherlands Cancer Institute (NKI) in Amsterdam obtained new scientific insights into the molecular architecture of hemidesmosomes, cytoskeletal components, cell surface receptors and vesicular proteins with the help of Ground-State-Depletion (GSD)/ dSTORM microscopy. In this interview, Kees Jalink comments on their developments in imaging chambers, buffer conditions and image analysis to get the perfect super resolution image.
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  • Initiation of Lamellipodia and Ruffles Involves Cooperation Between mDia1 and the Arp2/3 Complex

    Protrusion of lamellipodia and ruffles requires polymerization of branched actin filaments by the Arp2/3 complex. Although regulation of Arp2/3 complex activity has been extensively investigated, the mechanism of initiation of lamellipodia and ruffles remains poorly understood. Here, we show that mDia1 acts in concert with the Arp2/3 complex to promote initiation of lamellipodia and ruffles.
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  • Abstracts of the 6th European Super-Resolution User-Club Meeting

    The 6th European Super-Resolution User Club Meeting was held in collaboration with Dr. Timo Zimmermann, CRG, and Dr. Pablo Loza-Alvarez, ICFO, Barcelona. According to the founding principle of the club of keeping close to science, both imaging facilities at the CRG and the ICFO opened their doors to the User Club members, allowing them to explore exciting super-resolution and and nanoscopy applications. The meeting agenda covered highly relevant talks around this year’s central theme “Core Facilities and Super-Resolution Microscopy”, as well as plenty of opportunities to network amongst super-resolution users from different European countries. Here we present the abstracts of the talks held during the meeting.
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  • C. Elegans

    Work Efficiently in Developmental Biology with Stereo and Confocal Microscopy: C. elegans

    For scientists, technicians, and teachers working with the worm C. elegans in the research lab or classroom, this report is intended to give useful information to help improve their daly work. The aim is to make the work steps of worm picking, transgenesis, RNA interference, screening, and functional imaging efficient. It also details the various possibilities for equipping a research worm lab or biology classroom/teaching lab explaining worm methods.
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  • The Apical Ectodermal Ridge of the Mouse Model of Ectrodactyly Dlx5;Dlx6−/− Shows Altered Stratification and Cell Polarity, which are Restored by Exogenous Wnt5a Ligand

    The congenital malformation split hand/foot (SHFM) is characterized by missing central fingers and dysmorphology or fusion of the remaining ones. During limb development, the apical ectodermal ridge (AER) is a key-signaling center responsible for early proximal–distal growth and patterning. In search for the mechanism, we examined the non-canonical Wnt signaling, considering that Dwnt-5 is a target of distalless in Drosophila and the knockout of Wnt5, Ryk, Ror2 and Vangl2 in the mouse causes severe limb malformations.
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  • Video Talk by Roger Tsien: Fluorescent Protein Indicators

    In this talk, Roger Tsien discusses how fluorescent proteins have been turned into indicators for a wide variety of biological molecules, including pH, ions, redox potential, and signaling molecules like phosphoinositides. The talk also covers reporters used to measure the activity of enzymes like kinases, phosphatases, and proteases. It covers both single proteins whose intensity or wavelength change, as well as reporters using resonance energy transfer (FRET).
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  • Video Talk: CRISPR-Cas – From a Bacterial Adaptive Immune System to a Genome Engineering Tool

    The CRISPR-Cas system was originally discovered as an adaptive immune system of bacteria and archaea to protect against viral attack. During this talk, given at Leica Microsystems in Wetzlar, Dr. Lennart Randau, MPI Marburg, presents this simple and fascinating system in detail. Furthermore, he introduces the adaption of the CRISPR-Cas system into a potent molecular biology tool, which is used heavily for genome editing. In addition to its influence on molecular biology, meanwhile the Cas9 nuclease also stimulates microscopy techniques e.g. to fluorescently label genomic loci in living cells.
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  • STED-FLCS: An Advanced Tool to Reveal Spatiotemporal Heterogeneity of Molecular Membrane Dynamics

    Heterogeneous diffusion dynamics of molecules play an important role in many cellular signaling events, such as of lipids in plasma membrane bioactivity. However, these dynamics can often only be visualized by single-molecule and super-resolution optical microscopy techniques. Using fluorescence lifetime correlation spectroscopy (FLCS, an extension of fluorescence correlation spectroscopy, FCS ) on a super-resolution stimulated emission depletion (STED) microscope, we here extend previous observations of nanoscale lipid dynamics in the plasma membrane of living mammalian cells.
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  • Individual Macromolecule Motion in a Crowded Living Cell

    There is solid evidence for analyzing fluorescence correlation and dual color fluorescence crosscorrelation spectroscopy data ( FCS and dual color FCCS) in cellular applications by equations based on anomalous subdiffusion. Using equations based on normal diffusion causes artifacts of the fitted biological system response parameters and of the interpretations of the FCS and dual color FCCS data in the crowded environment of living cells. Equations based on normal diffusion are not valid in living cells. The original article embraces the status of the experimental situation and touches obstacles that still hinder the applications of single molecules in the cellular environment.
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  • The Bimodally Expressed MicroRNA miR‐142 Gates Exit from Pluripotency

    A stem cell's decision to self‐renew or differentiate is thought to critically depend on signaling cues provided by its environment. It is unclear whether stem cells have the intrinsic capacity to control their responsiveness to environmental signals that can be fluctuating and noisy. Using a novel single‐cell microRNA activity reporter, we show that miR‐142 is bimodally expressed in embryonic stem cells, creating two states indistinguishable by pluripotency markers.
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  • Super-Resolution Microscopy of the Synaptic Active Zone

    At the presynaptic active zone (AZ) a variety of specialized proteins are assembled to complex architectures, which set the basis for speed, precision and plasticity of synaptic transmission. Recently, super-resolution microscopy (SRM) techniques have begun to enter the neurosciences. These approaches combine high spatial resolution with the molecular specificity of fluorescence microscopy. Here, we discuss how SRM techniques can be used to obtain information on the organization of AZ proteins.
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  • Workflows & Protocols: Connecting Microscopy and Molecular Biology in Neuroscience

    The main topic during this course was how to apply laser microdissection in neuroscience. Leica specialists demonstrated why laser microdissection is a suitable techniques for brain investigation as it allows to separate distinct brain layers or even to isolate individual neurons.
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  • Video Talk by Roger Tsien: Fluorescent Proteins

    Live cell imaging has been revolutionized by the discovery of the green fluorescent protein (GFP). This lecture covers the history of GFP, how it folds and becomes fluorescent, how it has been mutated to produce additional colors (blue, cyan, yellow), and the discovery of red fluorescent proteins from corals. It also covers novel photoswitchable and photoactivatible fluorescent proteins, whose color can be changed by light, and new infrared fluorescent proteins.
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  • Webinar: Leica Microsystems Laser Microdissection – Dissection Perfection

    Laser Microdissection (LMD) is a microscopic technique for isolating homogeneous, specific and pure targets from heterogeneous samples for downstream analysis (DNA, RNA & proteins). In this webinar you will learn about techniques for precise, contamination-free isolation of specific cell types and obtain an overview of the scientific and practical considerations for obtaining highly pure material for further molecular analysis in the field of Parkinson's disease and plant research.
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  • Webinar: Applications and Methodology of Super-Resolution Microscopy with Leica SR GSD 3D

    This webinar, sponsored by Leica Microsystems, will highlight applications in super-resolution microscopy. Researchers from the University of Washington and the federal University of São Paulo will share their experiences using the Ground State Depletion (GSD) method of super-resolution imaging to address diverse research topics such as the role of calcium signaling and excitation in cardiac muscle, and the structure and functional relationship of biological macromolecules.
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  • Abstracts of the 5th European Super-Resolution User-Club Meeting

    The 5th Super-Resolution User Club Meeting was held in collaboration with Professor Kees Jalink and The Netherlands Cancer Institute (NKI) in Amsterdam. Having the meeting at a location where super-resolution microscopy is used on a daily basis makes a big difference, offering participants the chance to use live cells for workshops and see systems working in their true environments. Thanks also to the scientists that supported the meeting by coming and giving talks. As super-resolution continues to grow in importance in research, we recognize the need to come together to network, share information and experiences. Here we present the abstracts of the talks.
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  • Webinar: Unleashing the Powers of Super-Resolution Microscopy to Solve Immunological Challenges

    In this webinar, Christian Eggeling and Dongfang Liu will discuss their experiences using STED super-resolution microscopy to explore, uncover and define the intricate machinery involved in immunological pathways and infectious disorders.
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  • Defect Transport Mechanism Leads to Shortened Chromosome Ends

    Scientists at the University of Göttingen have deciphered the biogenesis of an enzyme complex whose role is to ensure that the ends of chromosomes are not shortened during the cell division process and that the genetic material is fully maintained. The results were published in Cell Reports magazine.
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  • Abstracts of the 4th European Super-Resolution User-Club Meeting

    The 4th Super-Resolution User Club Meeting was held in collaboration with Christian Eggeling and the Weatherall Institute of Molecular Medicine in Oxford, UK. Here we present the abstracts of the talks and interviews with participants.
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  • Genomic Survey, Gene Expression Analysis and Structural Modeling Suggest Diverse Roles of DNA Methyltransferases in Legumes

    DNA methylation plays a crucial role in development through inheritable gene silencing. Plants possess three types of DNA methyltransferases (MTases), namely Methyltransferase (MET), Chromomethylase (CMT) and Domains Rearranged Methyltransferase (DRM), which maintain methylation at CG, CHG and CHH sites. DNA MTases have not been studied in legumes so far. Here, we report the identification and analysis of putative DNA MTases in five legumes, including chickpea, soybean, pigeonpea, Medicago and Lotus. MTases in legumes could be classified in known MET, CMT, DRM and DNA nucleotide methyltransferases (DNMT2) subfamilies based on their domain organization.
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  • Map the Brain with CLARITY

    Imaging whole brains with CLARITY and multiphoton microscopy. Image a whole brain without sectioning? Investigate neuronal circuits without reconstruction? Perform molecular phenotyping without destroying subcellular structures? Understanding the brain with molecular resolution and global scope has always been challenging. The novel CLARITY method, developed by the Deisseroth laboratory at Stanford University, USA, pushes the barrier of deep tissue imaging a big step ahead.
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  • Sorbin and SH3 Domain‐Containing Protein 2 Is Released from Infarcted Heart in the Very Early Phase: Proteomic Analysis of Cardiac Tissues From Patients

    Many animal experiments have been performed to date to elucidate the mechanism of cardiac ischemic injury, and these studies have enhanced our understanding of cardiac remodeling following hypoxic stress. Clinical studies using samples from admitted patients have also been conducted to discover diagnostic or prognostic biomarkers for acute myocardial infarction (AMI).
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  • The 50 Most Influential Scientists in the World Today

    From neuroscience, biotechnology and digital media to sustainable energy and cloud computing, almost everything today is somehow affected – and sometimes entirely reshaped – by scientific and technological advances.
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  • Nobel Prize 2013 in Physiology or Medicine for Discoveries of the Machinery Regulating Vesicle Traffic

    On October 7th 2013, The Nobel Assembly at Karolinska Institutet has decided to award The Nobel Prize in Physiology or Medicine 2012 jointly to James E. Rothman, Randy W. Schekman and Thomas C. Südhof "for their discoveries of machinery regulating vesicle traffic, a major transport system in our cells".
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