Download or read book Single cell Detection of Antimicrobial Peptide s Attack on Live E Coli by Super resolution Fluorescence Microscopy written by Yanyu Zhu and published by . This book was released on 2020 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: The era of multidrug-resistant bacterial infections necessitates the development of new antimicrobial agents. Evolutionarily conserved antimicrobial peptides (AMPs) are promising prototypes for designing new antimicrobial agents in this context. We combined single-cell, real-time fluorescence microscopy and super-resolution, single-particle tracking to study the novel effects of the cationic human AMP LL-37 on the E. coli cytoplasm. After membrane permeabilization, approximately 108 cationic LL-37 copies flood the cell and the chromosomal DNA becomes rigidified on a length scale of ~30 nm. The diffusive motion of ribosomes, DNA binding protein HU and the non-endogenous protein Kaede also decreased. Much of the LL-37 remains bound within the cell after extensive rinsing with fresh growth medium, and growth never recovers. Our results suggest that the high concentration of absorbed polycationic peptides form a dense network of noncovalent, electrostatic "pseudo-crosslinks" within the chromosomal DNA and among 70S-polysomes, thus rigidifying bacterial cytoplasm. The results indicate that the damage of AMPs to bacterial cells goes far beyond the membrane permeabilization and might help explain why bacteria develop resistance very slowly to AMPs. We further extended the method to study other AMPs and synthetic polymers. We also applied super-resolution fluorescence microscopy to study the spatial distributions and diffusive properties of key components of the transcription-translation machinery in stationary phase E. coli cells. Compared to exponential phase, the chromosomal DNA in stationary phase is locally more rigid but remains sufficiently permeable to RNAP and to ribosomal subunits. There appears to be no need to postulate migration of actively transcribed genes to the nucleoid periphery. This description contrasts with the usual picture of a rigid stationary phase cytoplasm with DNA highly condensed.
Download or read book Time lapse Fluorescence Microscopy of the Effects of Antimicrobial Peptides on Single Live Escherichia Coli Cells written by Nambirajan Rangarajan and published by . This book was released on 2015 with total page 179 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Live Single Cell Fluorescence Microscopy written by Alexandria Ray and published by . This book was released on 2020 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Single-cell fluorescence microscopy is a powerful tool which can be used to investigate the nature of cellular responses to external stimuli. The application of single-cell fluorescence microscopy to the growing problem of antimicrobial susceptibility detection stands to improve and refine our ability to address bacterial resistance in human infections. In cases of severe bacterial infection, determining the correct antibiotic to use to combat an infection is a race against the patient's dwindling lifespan. By combining the RedoxSensorTM Green fluorescent dye with live single-cell imaging, we have developed a method for antimicrobial susceptibility testing which can identify susceptibility to a given antibiotic within 100 minutes of treatment. We show that this method is reproducible and can identify susceptibility to several bacterial cell wall targets and bacterial cell membrane targeted antibiotics. Our methodology has considerable applicability within the sphere of rational antibiotic drug design as well in its ability to identify antibiotic efficacy as a function of time instead of antibiotic concentration. We use our method to compare the efficacy of 4 recently synthesized polyurethane antimicrobials. Our work lays the framework for expansion upon our method into microfluidics systems and use in screening candidate antimicrobial drugs.Mitochondrial morphological analysis within living eukaryotic cells represents another challenge which requires careful application of single-cell fluorescence microscopy. The cuprizone mouse treatment model for multiple sclerosis is known to generate enlarged mitochondria within oligodendrocytes, but much remains unknown about the dynamics of their formation. Cultured MO3.13 oligodendrocyte cells treated with cuprizone are shown to undergo mitochondrial enlargement within 8 hours of direct cuprizone exposure via single-cell mitochondrial size determination with MitoTracker Red fluorescent dye. Cuprizone treatment is also shown to increase oxygen consumption rapidly following oligodendrocyte exposure using porphyrin-based fluorescence lifetime oxygen consumption measurement.
Download or read book Fluorescence Microscopy written by Anda Cornea and published by Elsevier. This book was released on 2014-02-24 with total page 261 pages. Available in PDF, EPUB and Kindle. Book excerpt: Fluorescence Microscopy: Super-Resolution and other Novel Techniques delivers a comprehensive review of current advances in fluorescence microscopy methods as applied to biological and biomedical science. With contributions selected for clarity, utility, and reproducibility, the work provides practical tools for investigating these ground-breaking developments. Emphasizing super-resolution techniques, light sheet microscopy, sample preparation, new labels, and analysis techniques, this work keeps pace with the innovative technical advances that are increasingly vital to biological and biomedical researchers. With its extensive graphics, inter-method comparisons, and tricks and approaches not revealed in primary publications, Fluorescence Microscopy encourages readers to both understand these methods, and to adapt them to other systems. It also offers instruction on the best visualization to derive quantitative information about cell biological structure and function, delivering crucial guidance on best practices in related laboratory research. - Presents a timely and comprehensive review of novel techniques in fluorescence imaging as applied to biological and biomedical research - Offers insight into common challenges in implementing techniques, as well as effective solutions
Download or read book Single molecule and Super resolution Microscopy of Bacterial Cells written by Marissa Kim Lee and published by . This book was released on 2015 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Single molecules were first detected at low temperatures twenty-six years ago in the laboratory of W.E. Moerner. Subsequent technological advances have allowed researchers to study single molecules at room temperatures and within living cells, providing novel biological insight about underlying spatial and dynamical heterogeneity. By combining single molecule detection with the ability to control the emissive state of the fluorescent label (also called "active control"), a suite of super-resolution imaging techniques has been developed. These single-molecule-based super-resolution imaging strategies leverage the fluorescence microscope's ability to non-invasively study multiple targets within living cells, while bridging the resolution gap between optical and electron microscopies. In large part, future advances to improve single molecule and super-resolution imaging require better fluorophore and labeling technologies. Utilizing fluorophore with higher photon yields will increase the resolution of super-resolution images and the data acquisition speed. Additionally, a greater library fluorophores with different of colors and sensing capabilities will enable application to more imaging targets. Currently, many single molecule and super-resolution experiments within living systems use fluorescent proteins because the labeling of target proteins is more straightforward. However, the limited photon yield of fluorescent proteins often results in tantalizingly fuzzy super-resolution images. Imaging the same targets, labeled instead with brighter organic emitters, could provide more image detail, but better fluorogenic and genetically encoded labeling schemes must be developed and discovered. The first chapter of this dissertation will introduce and discuss the historical context and basic principles of single molecule and super-resolution imaging. Chapter 2 will then describe the general experimental procedures necessary for quantitative single molecule and super-resolution imaging, including quantifying the number of photons detected (and emitted) from a single molecule, as well as the preparation of bacterial samples for fluorescence microscopy. Later chapters apply these fundamental experimental measurements to study bacterial biology and fluorophore photophysics. Chapters 3 and 4 concern the development and characterization of organic emitters suitable for single molecule or super-resolution imaging, work achieved with the synthetic collaboration of organic chemists in the laboratory of Professor Robert J. Twieg at Kent State University. Chapter 3 discusses the optimization of rhodamine spirolactam photoswitching such that activation could occur at visible wavelengths. The optimized rhodamine spirolactams were then covalently attached to the surface of bacterial cells and imaged with three-dimensional super-resolution. Images of the bacterial cell surface demonstrates a marked improvement in labeling uniformity, specificity, and density compared to previous methods which labeled the surface with the transient binding of a membrane sensitive dye. Chapter 4 introduces a novel enzyme-based strategy to control the fluorescence from nitro-aryl fluorogens. A proof-of-principle experiment demonstrated that endogenous nitroreductase enzymes within bacterial cells could catalyze the fluorescence-activating reaction, thus generating free fluorophores, which were detectable on the single-molecule-level within the cell. Lastly, chapter 5 summarizes three-dimensional imaging experiments (performed in collaboration with the laboratory of Professor Lucy Shapiro in the Department of Developmental Biology at Stanford University) of components of the bacterial gene expression machinery labeled with fluorescent proteins. Super-resolution imaging is ideally suited to the small size scale of bacterial cells, and a wealth of biological insights remains to be discovered. Simultaneously improving fluorophore photon yield, specificity, and active control strategies will have a profound impact on super-resolution precision and speed.
Download or read book Quanititative Fluorescence Microscopy on the Escherichia Coli Envelope written by Kem A. Sochacki and published by . This book was released on 2011 with total page 138 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Antimicrobial Peptides written by Katsumi Matsuzaki and published by Springer. This book was released on 2019-04-12 with total page 300 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book presents an overview of antimicrobial peptides (AMPs), their mechanisms of antimicrobial action, other activities, and various problems that must still be overcome regarding their clinical application. Divided into four major parts, the book begins with a general overview of AMPs (Part I), and subsequently discusses the various mechanisms of antimicrobial action and methods for researching them (Part 2). It then addresses a range of activities other than antimicrobial action, such as cell penetration, antisepsis, anticancer, and immunomodulatory activities (Part 3), and explores the prospects of clinical application from various standpoints such as the selective toxicity, design, and discovery of AMPs (Part 4). A huge number of AMPs have been discovered in plants, insects, and vertebrates including humans, and constitute host defense systems against invading pathogenic microorganisms. Consequently, many attempts have been made to utilize AMPs as antibiotics. AMPs could help to solve the urgent problem of drug-resistant bacteria, and are also promising with regard to sepsis and cancer therapy. Gathering a wealth of information, this book will be a bible for all those seeking to develop antibiotics, anti-sepsis, or anticancer agents based on AMPs.
Download or read book Three dimensional Super resolution Microscopy and Single particle Tracking of Bacterial Proteins written by Camille Bayas and published by . This book was released on 2019 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: The first optical detection of a single molecule (SM) at cryogenic temperatures 30 years ago laid the groundwork for the routine detection of SMs today at biologically relevant temperatures, thus uncovering hidden heterogeneity that might be obscured by ensemble techniques. In addition to enabling studies of the intricate photochemistry and photophysics of fluorescent labels at the SM level, SM fluorescence has also proven useful for the imaging and tracking of cellular structures and biomolecules in a non-invasive manner with high sensitivity. The ability to genetically express fluorescent protein fusions in live cells has allowed specific labeling, and thus imaging and tracking, of dynamic processes and structures in cells. This dissertation describes applications of SM-based single-particle tracking (SPT) and super-resolution (SR) microscopy for the study of spatial organization and dynamics of bacterial proteins in two and three spatial dimensions. In an SPT experiment, the position of a SM emitter at very low concentration is measured over time to generate a trajectory, allowing for observation and quantification of labeled biomolecule dynamics at the SM level. In a SR microscopy experiment, the diffraction-limited (DL) resolution of a conventional fluorescence microscope (~200 nm in xy) is circumvented by temporally separating the emission of many SM emitters decorating a structure through control of their emissive state. A "super-resolved" image, with a factor of ~5-10 resolution improvement over a conventional DL fluorescence image, is generated by estimating the positions of many non-moving SM emitters over many frames and building up an image reconstruction in a pointillist manner. Chapter 1 of this dissertation provides an introduction to fluorescence, SM imaging, SM-based SR microscopy, and SPT. Chapter 1 also gives a brief introduction to Caulobacter crescentus, the bacterium used as the model organism in the SM studies in Chapters 4 and 5. Chapter 2 describes the experimental methods used to perform quantitative SM imaging of bacterial cells. The combination of SM imaging with point spread function (PSF) engineering has enabled the accurate and precise localization of SMs in three dimensions (3D) by the intentional introduction of specifically chosen aberrations in the emission path of an SM microscope. Throughout this dissertation, the double-helix (DH) PSF, a rotating PSF composed of two lobes whose angle encodes axial position, was used to estimate 3D SM positions. Chapter 2 describes the implementation of the DH-PSF via optical Fourier processing, and Chapter 3 describes the robust and comprehensible two-color Easy-DHPSF v2 software for localizing molecules in 3D and for registering localizations from two spectral channels into the same coordinate system with nanoscale accuracy. The resolution improvement gained from SM-based techniques is particularly useful for bacteria, the sizes of which are on the order of the DL. 3D SM-based SR and SPT have enabled the observation of structures and dynamics at length scales below the DL. Caulobacter is a useful biological target where understanding of the mechanisms for asymmetric cell division need to be explored and quantified. Central to Caulobacter's asymmetric division is the dynamic spatiotemporal regulation of gene expression and protein localization. Chapters 4 and 5 describes research performed in collaboration with Prof. Lucy Shapiro's laboratory (Department of Developmental Biology, Stanford School of Medicine) to study gene expression and signaling proteins in Caulobacter. Chapter 4 describes work studying the spatial organization and dynamics of ribosomes and a RNA-degrading enzyme RNase E using 3D SR microscopy and SPT. Results showed that the organization and dynamics of RNase E and ribosomes are closely related to the transcriptional activity of the cell. Finally, Chapter 5 describes SPT studies of the membrane-bound histidine kinase and stalked cell fate determinant DivJ in an effort to probe the physical properties of the Caulobacter stalked pole. Preliminary SPT results suggest that disrupting the physical properties and interactions at the stalked pole has an influence on DivJ diffusion and signaling.
Download or read book Stimulated Emission Depletion Super resolution Fluorescence Microscopy written by Colin James Comerci and published by . This book was released on 2019 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Fluorescence microscopy is a widely used tool in biological studies, allowing specific labeling and imaging of proteins and structures within living cells. As reviewed in Chapter 1, conventional fluorescence microscopy is unable to distinguish structures below ~250 nm in size due to the diffraction limit of light. Over the last two decades, the field of super-resolution microscopy has overcome this diffraction limit, allowing fluorescence imaging of structures just a few tens of nanometers in size. This thesis discusses one super-resolution technique, stimulated emission depletion (STED) microscopy, and its application to various cellular and molecular biophysics questions. In Chapter 2, the bespoke STED microscope used for much of this thesis is described in detail. Some guiding principles behind STED microscopes and their design are discussed. We next discuss the major improvements to the STED microscope: fast scanning and two-color. Fast scanning protects fluorophores from photobleaching during imaging, improving image quality and expanding the range of available fluorophores for STED imaging. Two-color imaging allows the examination of two different protein species, a universal requirement in many biological studies. In Chapter 3, we apply STED microscopy to study how the bacteria Caulobacter crescentus assembles a 2D paracrystalline surface layer (S-layer) composed of the protein RsaA. We show that S-layer assembly is localized to highly curved regions of the cell, and that this localized assembly depends upon the presence of a pre-existing crystalline S-layer. Together, our results suggest the topology of the cell surface combined with continuous protein crystallization localizes S-layer assembly. In Chapter 5, 2-color STED microscopy is used to examine protein interactions important for the 3D organization of the genome in eukaryotic cells. We quantify clustering of the DNA binding protein CCCTC-binding factor (CTCF), as well as the molecular coupling between CTCF and cohesin, two proteins hypothesized to play a major role in organizing topologically associating domains (TADs). We show that cohesin plays a regulatory role in CTCF clustering by perturbing its activity and monitoring cluster size. Finally, we discuss several applications of STED imaging to support larger studies. Chapter 4 discusses applying a genetically encoded fluoromodule for STED imaging in live bacteria cells. In the first half of Chapter 6, STED reveals the localization of the centriole protein Cby1 to the transition zone, supporting a role for Cby1 in recruiting Ahi1, another transition zone protein. Finally, the second half of Chapter 6 uses two-color STED microscopy to demonstrate that deletion of the intracellular cargo transport protein IFT25 causes simultaneous defects in the cilium cytoskeleton as well as ciliary membrane morphology.
Download or read book Lipid Domains written by and published by Academic Press. This book was released on 2015-06-08 with total page 393 pages. Available in PDF, EPUB and Kindle. Book excerpt: Current Topics in Membranes is targeted toward scientists and researchers in biochemistry and molecular and cellular biology, providing the necessary membrane research to assist them in discovering the current state of a particular field and in learning where that field is heading. This volume offers an up to date presentation of current knowledge in the field of Lipid Domains. - Written by leading experts - Contains original material, both textual and illustrative, that should become a very relevant reference material - The material is presented in a very comprehensive manner - Both researchers in the field and general readers should find relevant and up-to-date information
Download or read book Flow Cytometry and Cell Sorting written by Andreas Radbruch and published by Springer Science & Business Media. This book was released on 2013-03-14 with total page 365 pages. Available in PDF, EPUB and Kindle. Book excerpt: The analysis and sorting of large numbers of cells with a fluorescence-activated cell sorter (FACS) was first achieved some 30 years ago. Since then, this technology has been rapidly developed and is used today in many laboratories. A Springer Lab Manual Review of the First Edition: "This is a most useful volume which will be a welcome addition for personal use and also for laboratories in a wide range of disciplines. Highly recommended." CYTOBIOS
Download or read book The Perfect Slime written by Hans-Curt Flemming and published by IWA Publishing. This book was released on 2016-09-15 with total page 336 pages. Available in PDF, EPUB and Kindle. Book excerpt: The Perfect Slime presents the latest state of knowledge and all aspects of the Extracellular Polymeric Substances, (EPS) matrix – from the ecological and health to the antifouling perspectives. The book brings together all the current material in order to expand our understanding of the functions, properties and characteristics of the matrix as well as the possibilities to strengthen or weaken it. The EPS matrix represents the immediate environment in which biofilm organisms live. From their point of view, this matrix has paramount advantages. It allows them to stay together for extended periods and form synergistic microconsortia, it retains extracellular enzymes and turns the matrix into an external digestion system and it is a universal recycling yard, it protects them against desiccation, it allows for intense communication and represents a huge genetic archive. They can remodel their matrix, break free and eventually, they can use it as a nutrient source. The EPS matrix can be considered as one of the emergent properties of biofilms and are a major reason for the success of this form of life. Nevertheless, they have been termed the “black matter of biofilms” for good reasons. First of all: the isolation methods define the results. In most cases, only water soluble EPS components are investigated; insoluble ones such as cellulose or amyloids are much less included. In particular in environmental biofilms with many species, it is difficult to impossible isolate, separate the various EPS molecules they are encased in and to define which species produced which EPS. The regulation and the factors which trigger or inhibit EPS production are still very poorly understood. Furthermore: bacteria are not the only microorganisms to produce EPS. Archaea, Fungi and algae can also form EPS. This book investigates the questions, What is their composition, function, dynamics and regulation? What do they all have in common?
Download or read book Metal Nanoparticles in Microbiology written by Mahendra Rai and published by Springer Science & Business Media. This book was released on 2011-04-02 with total page 306 pages. Available in PDF, EPUB and Kindle. Book excerpt: Following an introduction to biogenic metal nanoparticles, this book presents how they can be biosynthesized using bacteria, fungi and yeast, as well as their potential applications in biomedicine. It is shown that the synthesis of nanoparticles using microbes is eco-friendly and results in reproducible metal nanoparticles of well-defined sizes, shapes and structures. This biotechnological approach based on the process of biomineralization exploits the effectiveness and flexibility of biological systems. Chapters include practical protocols for microbial synthesis of nanoparticles and microbial screening methods for isolating a specific nanoparticle producer as well as reviews on process optimization, industrial scale production, biomolecule-nanoparticle interactions, magnetosomes, silver nanoparticles and their numerous applications in medicine, and the application of gold nanoparticles in developing sensitive biosensors.
Download or read book MCQs in Microbiology written by G. Vidya Sagar and published by New Age International. This book was released on 2008 with total page 21 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Peptide Self Assembly written by Bradley L. Nilsson and published by Humana. This book was released on 2018-05-10 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: This volume details methods and protocols on b-sheet assemblies and collagen. Divided into three parts chapters focus on expanding use of solid-state NMR as a powerful method to enhance structural understanding of self-assembled peptide materials, methods for the design, synthesis, and application of self-assembled peptide materials, and structural and mechanistic analyses of pathological amyloid systems that provide novel ways to assess function of the various possible aggregates as well to determine how the structure of these materials correlates to function/dysfunction in the biological context. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Peptide Self-Assembly: Methods and Protocols aims to capture modern methods that span the breadth of the exciting and expanding field of peptide self-assembly.
Download or read book Lactoferrin written by T. William Hutchens and published by Springer Science & Business Media. This book was released on 2012-12-06 with total page 295 pages. Available in PDF, EPUB and Kindle. Book excerpt: Lactoferrin is an intriguing protein with an interesting structure and several known or suggested biological activities. We feel that attention on this protein has been too limited and diffuse, partly because it has been "hidden" among other well-known iron-binding proteins such as hemoglobin, ferritin and transferrin, but also perhaps because its biological functions are so diverse. Investigators that focus on lactoferrin represent a wide variety of medical and scientific disciplines that do not usually come together. It was our intention to improve that situation with this symposium. In this book, experts from a variety of disciplines describe the present knowledge of the structural features of lactoferrin, its carbohydrate side chains and its capacity to bind different metal ions and anions. Several of the possible physiological functions of lactoferrin are described in detail, including the role of lactoferrin in bacterial killing, its involvement in growth and proliferation, in immune function and in iron absorption. Aspects of the molecular biology of lactoferrin and its specific interactions with different cell types are also included. Finally, as lactoferrin now has become commercially available in larger quantities, possible industrial applications are discussed. The book should give the interested reader a thorough insight into our present knowledge of lactoferrin.
Download or read book Polymyxin Antibiotics written by Jian Li (Pharmacologist) and published by . This book was released on 2019 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: It is our wish that readers discover the importance of polymyxin structure in relation to the mechanisms of activity, resistance and toxicity. We emphasized that reliable analytic methods for polymyxins are critical when investigating their pharmacokinetics (PK) and pharmacodynamics (PD). The complicated dose definitions and different pharmacopoeial standards have already compromised the safe use of polymyxins in patients. Therefore, informed by the latest pharmacological information, scientifically-based dosing recommendations have been proposed for intravenous polymyxins. Considering the PK/PD limitations and potential development of resistance, polymyxin combinations are encouraged; however, the current literature has not shown definite microbiological benefits, possibly because most clinical studies to date overlooked key PK/PD principles. Nephrotoxicity is the major dose-limiting factor and it is imperative to elucidate the mechanisms and develop novel approaches to minimize polymyxin-associated toxicities. In addition, the anti-endotoxin effect of polymyxins supports their clinical use to treat Gram-negative sepsis. Fortunately, the discovery of new-generation polymyxins with wider therapeutic windows has benefited from the latest achievements in polymyxin research.
