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Pubmed Article
Synthesis, structural and antioxidant studies of some novel N-ethyl phthalimide esters.
PUBLISHED: 03-06-2015
A series of N-ethyl phthalimide esters 4(a-n) were synthesized and characterized by spectroscopic studies. Further, the molecular structure of majority of compounds were analysed by single crystal X-ray diffraction studies. The X-ray analysis revealed the importance of substituents on the crystal stability and molecular packing. All the synthesized compounds were tested for in vitro antioxidant activity by DPPH radical scavenging, FRAP and CUPRAC methods. Few of them have shown good antioxidant activity.
Authors: Lacey Samuels, Allan DeBono, Patricia Lam, Miao Wen, Reinhard Jetter, Ljerka Kunst.
Published: 05-31-2008
The plant cuticle is a waxy outer covering on plants that has a primary role in water conservation, but is also an important barrier against the entry of pathogenic microorganisms. The cuticle is made up of a tough crosslinked polymer called "cutin" and a protective wax layer that seals the plant surface. The waxy layer of the cuticle is obvious on many plants, appearing as a shiny film on the ivy leaf or as a dusty outer covering on the surface of a grape or a cabbage leaf thanks to light scattering crystals present in the wax. Because the cuticle is an essential adaptation of plants to a terrestrial environment, understanding the genes involved in plant cuticle formation has applications in both agriculture and forestry. Today, we'll show the analysis of plant cuticle mutants identified by forward and reverse genetics approaches.
23 Related JoVE Articles!
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Analysis of Fatty Acid Content and Composition in Microalgae
Authors: Guido Breuer, Wendy A. C. Evers, Jeroen H. de Vree, Dorinde M. M. Kleinegris, Dirk E. Martens, René H. Wijffels, Packo P. Lamers.
Institutions: Wageningen University and Research Center, Wageningen University and Research Center, Wageningen University and Research Center.
A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of microalgal biomass. These fatty acids can be present in different acyl-lipid classes. Especially the fatty acids present in triacylglycerol (TAG) are of commercial interest, because they can be used for production of transportation fuels, bulk chemicals, nutraceuticals (ω-3 fatty acids), and food commodities. To develop commercial applications, reliable analytical methods for quantification of fatty acid content and composition are needed. Microalgae are single cells surrounded by a rigid cell wall. A fatty acid analysis method should provide sufficient cell disruption to liberate all acyl lipids and the extraction procedure used should be able to extract all acyl lipid classes. With the method presented here all fatty acids present in microalgae can be accurately and reproducibly identified and quantified using small amounts of sample (5 mg) independent of their chain length, degree of unsaturation, or the lipid class they are part of. This method does not provide information about the relative abundance of different lipid classes, but can be extended to separate lipid classes from each other. The method is based on a sequence of mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to fatty acid methyl esters (FAMEs), and quantification and identification of FAMEs using gas chromatography (GC-FID). A TAG internal standard (tripentadecanoin) is added prior to the analytical procedure to correct for losses during extraction and incomplete transesterification.
Environmental Sciences, Issue 80, chemical analysis techniques, Microalgae, fatty acid, triacylglycerol, lipid, gas chromatography, cell disruption
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Seeded Synthesis of CdSe/CdS Rod and Tetrapod Nanocrystals
Authors: Karthish Manthiram, Brandon J. Beberwyck, Dmitri V. Talapin, A. Paul Alivisatos.
Institutions: UC Berkeley, UC Berkeley, UC Berkeley, Lawrence Berkeley National Laboratory, University of Chicago, Argonne National Laboratory.
We demonstrate a method for the synthesis of multicomponent nanostructures consisting of CdS and CdSe with rod and tetrapod morphologies. A seeded synthesis strategy is used in which spherical seeds of CdSe are prepared first using a hot-injection technique. By controlling the crystal structure of the seed to be either wurtzite or zinc-blende, the subsequent hot-injection growth of CdS off of the seed results in either a rod-shaped or tetrapod-shaped nanocrystal, respectively. The phase and morphology of the synthesized nanocrystals are confirmed using X-ray diffraction and transmission electron microscopy, demonstrating that the nanocrystals are phase-pure and have a consistent morphology. The extinction coefficient and quantum yield of the synthesized nanocrystals are calculated using UV-Vis absorption spectroscopy and photoluminescence spectroscopy. The rods and tetrapods exhibit extinction coefficients and quantum yields that are higher than that of the bare seeds. This synthesis demonstrates the precise arrangement of materials that can be achieved at the nanoscale by using a seeded synthetic approach.
Chemistry, Issue 82, nanostructures, synthesis, nanocrystals, seeded rods, tetrapods, nanoheterostructures
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Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation
Authors: Amy H. Van Hove, Brandon D. Wilson, Danielle S. W. Benoit.
Institutions: University of Rochester, University of Rochester, University of Rochester Medical Center.
One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young’s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g. primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.
Chemistry, Issue 80, Poly(ethylene glycol), peptides, polymerization, polymers, methacrylation, peptide functionalization, 1H-NMR, MALDI-ToF, hydrogels, macromer synthesis
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Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library
Authors: Yu Gao, Thomas Kodadek.
Institutions: The Scripps Research Institute.
Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid phase by using combinatorial chemistry. One of the most well studied classes of peptidomimetics is peptoids. Peptoids are easy to synthesize and have been shown to be proteolysis-resistant and cell-permeable. Over the past decade, many useful protein ligands have been identified through screening of peptoid libraries. However, most of the ligands identified from peptoid libraries do not display high affinity, with rare exceptions. This may be due, in part, to the lack of chiral centers and conformational constraints in peptoid molecules. Recently, we described a new synthetic route to access peptide tertiary amides (PTAs). PTAs are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. With side chains on both α-carbon and main chain nitrogen atoms, the conformation of these molecules are greatly constrained by sterical hindrance and allylic 1,3 strain. (Figure 1) Our study suggests that these PTA molecules are highly structured in solution and can be used to identify protein ligands. We believe that these molecules can be a future source of high-affinity protein ligands. Here we describe the synthetic method combining the power of both split-and-pool and sub-monomer strategies to synthesize a sample one-bead one-compound (OBOC) library of PTAs.
Chemistry, Issue 88, Split-and-pool synthesis, peptide tertiary amide, PTA, peptoid, high-throughput screening, combinatorial library, solid phase, triphosgene (BTC), one-bead one-compound, OBOC
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Analysis of Oxidative Stress in Zebrafish Embryos
Authors: Vera Mugoni, Annalisa Camporeale, Massimo M. Santoro.
Institutions: University of Torino, Vesalius Research Center, VIB.
High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a “whole embryo ROS-detection method” for qualitative measurement of oxidative stress and ii) a “single-cell ROS detection method” for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.
Developmental Biology, Issue 89, Danio rerio, zebrafish embryos, endothelial cells, redox state analysis, oxidative stress detection, in vivo ROS measurements, FACS (fluorescence activated cell sorter), molecular probes
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The Use of Gas Chromatography to Analyze Compositional Changes of Fatty Acids in Rat Liver Tissue during Pregnancy
Authors: Helena L. Fisk, Annette L. West, Caroline E. Childs, Graham C. Burdge, Philip C. Calder.
Institutions: University of Southampton.
Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, yielding results with high accuracy and high reproducibility. In metabolic and nutrition studies GC allows assessment of changes in fatty acid concentrations following interventions or during changes in physiological state such as pregnancy. Solid phase extraction (SPE) using aminopropyl silica cartridges allows separation of the major lipid classes including triacylglycerols, different phospholipids, and cholesteryl esters (CE). GC combined with SPE was used to analyze the changes in fatty acid composition of the CE fraction in the livers of virgin and pregnant rats that had been fed various high and low fat diets. There are significant diet/pregnancy interaction effects upon the omega-3 and omega-6 fatty acid content of liver CE, indicating that pregnant females have a different response to dietary manipulation than is seen among virgin females.
Chemistry, Issue 85, gas chromatography, fatty acid, pregnancy, cholesteryl ester, solid phase extraction, polyunsaturated fatty acids
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Improved In-gel Reductive β-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry
Authors: David B. Nix, Tadahiro Kumagai, Toshihiko Katoh, Michael Tiemeyer, Kazuhiro Aoki.
Institutions: University of Georgia, University of Georgia, Ishikawa Prefectural University.
Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive β-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.
Chemistry, Issue 93, glycoprotein, glycosylation, in-gel reductive β-elimination, O-linked glycan, sulfated glycan, mass spectrometry, protein ID, SDS-PAGE, glycomics, sulfoglycomics
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Synthesis and Characterization of Functionalized Metal-organic Frameworks
Authors: Olga Karagiaridi, Wojciech Bury, Amy A. Sarjeant, Joseph T. Hupp, Omar K. Farha.
Institutions: Northwestern University, Warsaw University of Technology, King Abdulaziz University.
Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and technological applications. Their signature property is their ultrahigh porosity, which however imparts a series of challenges when it comes to both constructing them and working with them. Securing desired MOF chemical and physical functionality by linker/node assembly into a highly porous framework of choice can pose difficulties, as less porous and more thermodynamically stable congeners (e.g., other crystalline polymorphs, catenated analogues) are often preferentially obtained by conventional synthesis methods. Once the desired product is obtained, its characterization often requires specialized techniques that address complications potentially arising from, for example, guest-molecule loss or preferential orientation of microcrystallites. Finally, accessing the large voids inside the MOFs for use in applications that involve gases can be problematic, as frameworks may be subject to collapse during removal of solvent molecules (remnants of solvothermal synthesis). In this paper, we describe synthesis and characterization methods routinely utilized in our lab either to solve or circumvent these issues. The methods include solvent-assisted linker exchange, powder X-ray diffraction in capillaries, and materials activation (cavity evacuation) by supercritical CO2 drying. Finally, we provide a protocol for determining a suitable pressure region for applying the Brunauer-Emmett-Teller analysis to nitrogen isotherms, so as to estimate surface area of MOFs with good accuracy.
Chemistry, Issue 91, Metal-organic frameworks, porous coordination polymers, supercritical CO2 activation, crystallography, solvothermal, sorption, solvent-assisted linker exchange
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In Situ Neutron Powder Diffraction Using Custom-made Lithium-ion Batteries
Authors: William R. Brant, Siegbert Schmid, Guodong Du, Helen E. A. Brand, Wei Kong Pang, Vanessa K. Peterson, Zaiping Guo, Neeraj Sharma.
Institutions: University of Sydney, University of Wollongong, Australian Synchrotron, Australian Nuclear Science and Technology Organisation, University of Wollongong, University of New South Wales.
Li-ion batteries are widely used in portable electronic devices and are considered as promising candidates for higher-energy applications such as electric vehicles.1,2 However, many challenges, such as energy density and battery lifetimes, need to be overcome before this particular battery technology can be widely implemented in such applications.3 This research is challenging, and we outline a method to address these challenges using in situ NPD to probe the crystal structure of electrodes undergoing electrochemical cycling (charge/discharge) in a battery. NPD data help determine the underlying structural mechanism responsible for a range of electrode properties, and this information can direct the development of better electrodes and batteries. We briefly review six types of battery designs custom-made for NPD experiments and detail the method to construct the ‘roll-over’ cell that we have successfully used on the high-intensity NPD instrument, WOMBAT, at the Australian Nuclear Science and Technology Organisation (ANSTO). The design considerations and materials used for cell construction are discussed in conjunction with aspects of the actual in situ NPD experiment and initial directions are presented on how to analyze such complex in situ data.
Physics, Issue 93, In operando, structure-property relationships, electrochemical cycling, electrochemical cells, crystallography, battery performance
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Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities
Authors: Stephan Böttcher, Joachim Thiem.
Institutions: University of Hamburg.
Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are rapidly oxidized, for example by atmospheric oxygen, to indigo type dyes. This reaction enables fast and easy screening in vivo without isolation or purification of enzymes, as well as rapid tests on agar plates or in solution (e.g., blue-white screening, micro-wells) and is used in biochemistry, histochemistry, bacteriology and molecular biology. Unfortunately the synthesis of such substrates proved to be difficult, due to various side reactions and the low reactivity of the indoxyl hydroxyl function. Especially for glucose type structures low yields were observed. Our novel approach employs indoxylic acid ester as key intermediates. Indoxylic acid esters with varied substitution patterns were prepared on scalable pathways. Phase transfer glycosylations with those acceptors and peracetylated glycosyl halides can be performed under common conditions in high yields. Ester cleavage and subsequent mild silver mediated glycosylation yields the peracetylated indoxyl glycosides in high yields. Finally deprotection is performed according to Zemplén.
Chemistry, Issue 99, Carbohydrates, Indoxyl-Glycosides, Indoxylic Acid Esters, Decarboxylation, Dyes/Pigments, Enzyme Activity, Nitrogen Heterocycles
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Synthesis and Microdiffraction at Extreme Pressures and Temperatures
Authors: Barbara Lavina, Przemyslaw Dera, Yue Meng.
Institutions: University of Nevada, Las Vegas, University of Chicago, Carnegie Institution of Washington.
High pressure compounds and polymorphs are investigated for a broad range of purposes such as determine structures and processes of deep planetary interiors, design materials with novel properties, understand the mechanical behavior of materials exposed to very high stresses as in explosions or impacts. Synthesis and structural analysis of materials at extreme conditions of pressure and temperature entails remarkable technical challenges. In the laser heated diamond anvil cell (LH-DAC), very high pressure is generated between the tips of two opposing diamond anvils forced against each other; focused infrared laser beams, shined through the diamonds, allow to reach very high temperatures on samples absorbing the laser radiation. When the LH-DAC is installed in a synchrotron beamline that provides extremely brilliant x-ray radiation, the structure of materials under extreme conditions can be probed in situ. LH-DAC samples, although very small, can show highly variable grain size, phase and chemical composition. In order to obtain the high resolution structural analysis and the most comprehensive characterization of a sample, we collect diffraction data in 2D grids and combine powder, single crystal and multigrain diffraction techniques. Representative results obtained in the synthesis of a new iron oxide, Fe4O5 1 will be shown.
Physics, Issue 80, x-ray diffraction, geochemistry, geophysics, solid-state physics, high-pressure, high-temperature, Diamond anvil cell, micro-diffraction, novel materials, iron oxides, mantle mineralogy
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Characterization of Electrode Materials for Lithium Ion and Sodium Ion Batteries Using Synchrotron Radiation Techniques
Authors: Marca M. Doeff, Guoying Chen, Jordi Cabana, Thomas J. Richardson, Apurva Mehta, Mona Shirpour, Hugues Duncan, Chunjoong Kim, Kinson C. Kam, Thomas Conry.
Institutions: Lawrence Berkeley National Laboratory, University of Illinois at Chicago, Stanford Synchrotron Radiation Lightsource, Haldor Topsøe A/S, PolyPlus Battery Company.
Intercalation compounds such as transition metal oxides or phosphates are the most commonly used electrode materials in Li-ion and Na-ion batteries. During insertion or removal of alkali metal ions, the redox states of transition metals in the compounds change and structural transformations such as phase transitions and/or lattice parameter increases or decreases occur. These behaviors in turn determine important characteristics of the batteries such as the potential profiles, rate capabilities, and cycle lives. The extremely bright and tunable x-rays produced by synchrotron radiation allow rapid acquisition of high-resolution data that provide information about these processes. Transformations in the bulk materials, such as phase transitions, can be directly observed using X-ray diffraction (XRD), while X-ray absorption spectroscopy (XAS) gives information about the local electronic and geometric structures (e.g. changes in redox states and bond lengths). In situ experiments carried out on operating cells are particularly useful because they allow direct correlation between the electrochemical and structural properties of the materials. These experiments are time-consuming and can be challenging to design due to the reactivity and air-sensitivity of the alkali metal anodes used in the half-cell configurations, and/or the possibility of signal interference from other cell components and hardware. For these reasons, it is appropriate to carry out ex situ experiments (e.g. on electrodes harvested from partially charged or cycled cells) in some cases. Here, we present detailed protocols for the preparation of both ex situ and in situ samples for experiments involving synchrotron radiation and demonstrate how these experiments are done.
Physics, Issue 81, X-Ray Absorption Spectroscopy, X-Ray Diffraction, inorganic chemistry, electric batteries (applications), energy storage, Electrode materials, Li-ion battery, Na-ion battery, X-ray Absorption Spectroscopy (XAS), in situ X-ray diffraction (XRD)
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Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography
Authors: Michael Heethoff, Lukas Helfen, Peter Cloetens.
Institutions: University of Tubingen, European Synchrotron Radiation Facility.
Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning. We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7µm. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.
Developmental Biology, Issue 15, Synchrotron X-ray tomography, Acari, Oribatida, micro-arthropods, non-invasive investigation
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Crystallization of Membrane Proteins in Lipidic Mesophases
Authors: Wei Liu, Vadim Cherezov.
Institutions: The Scripps Research Institute.
Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.
Structural Biology, Issue 49, membrane protein, lipidic cubic phase, crystallization, Fluorescence recovery after photobleaching (FRAP) , G protein-coupled receptors
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Clonogenic Assay: Adherent Cells
Authors: Haloom Rafehi, Christian Orlowski, George T. Georgiadis, Katherine Ververis, Assam El-Osta, Tom C. Karagiannis.
Institutions: The Alfred Medical Research and Education Precinct, The University of Melbourne, The Alfred Medical Research and Education Precinct, The University of Melbourne.
The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.
Cellular Biology, Issue 49, clonogenic assay, clonogenic survival, colony staining, colony counting, radiation sensitivity, radiation modification
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A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke
Authors: Long-Xi Yu, Boris G. Dzikovski, Jack H. Freed.
Institutions: CDCF-AOX Lab, Cornell University.
Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12. Although tobacco tars and related products in the particle phase of cigarette smoke are major causes of carcinogenic and mutagenic related diseases, cigarette smoke contains significant amounts of free radicals that are also considered as an important group of carcinogens9,10. Free radicals attack cell constituents by damaging protein structure, lipids and DNA sequences and increase the risks of developing various types of cancers. Inhaled radicals produce adducts that contribute to many of the negative health effects of tobacco smoke in the lung3. Studies have been conducted to reduce free radicals in cigarette smoke to decrease risks of the smoking-induced damage. It has been reported that haemoglobin and heme-containing compounds could partially scavenge nitric oxide, reactive oxidants and carcinogenic volatile nitrosocompounds of cigarette smoke4. A 'bio-filter' consisted of haemoglobin and activated carbon was used to scavenge the free radicals and to remove up to 90% of the free radicals from cigarette smoke14. However, due to the cost-ineffectiveness, it has not been successfully commercialized. Another study showed good scavenging efficiency of shikonin, a component of Chinese herbal medicine8. In the present study, we report a protocol for introducing common natural antioxidant extracts into the cigarette filter for scavenging gas phase free radicals in cigarette smoke and measurement of the scavenge effect on gas phase free radicals in mainstream cigarette smoke (MCS) using spin-trapping Electron Spin Resonance (ESR) Spectroscopy1,2,14. We showed high scavenging capacity of lycopene and grape seed extract which could point to their future application in cigarette filters. An important advantage of these prospective scavengers is that they can be obtained in large quantities from byproducts of tomato or wine industry respectively11,13
Bioengineering, Issue 59, Cigarette smoke, free radical, spin-trap, ESR
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Synthesis of Nine-atom Deltahedral Zintl Ions of Germanium and their Functionalization with Organic Groups
Authors: Miriam M. Gillett-Kunnath, Slavi C. Sevov.
Institutions: University of Notre Dame .
Although the first studies of Zintl ions date between the late 1890's and early 1930's they were not structurally characterized until many years later.1,2 Their redox chemistry is even younger, just about ten years old, but despite this short history these deltahedral clusters ions E9n- (E = Si, Ge, Sn, Pb; n = 2, 3, 4) have already shown interesting and diverse reactivity and have been at the forefront of rapidly developing and exciting new chemistry.3-6 Notable milestones are the oxidative coupling of Ge94- clusters to oligomers and infinite chains,7-19 their metallation,14-16,20-25 capping by transition-metal organometallic fragments,26-34 insertion of a transition-metal atom at the center of the cluster which is sometimes combined with capping and oligomerization,35-47 addition of main-group organometallic fragments as exo-bonded substituents,48-50 and functionalization with various organic residues by reactions with organic halides and alkynes.51-58 This latter development of attaching organic fragments directly to the clusters has opened up a new field, namely organo-Zintl chemistry, that is potentially fertile for further synthetic explorations, and it is the step-by-step procedure for the synthesis of germanium-divinyl clusters described herein. The initial steps outline the synthesis of an intermetallic precursor of K4Ge9 from which the Ge94- clusters are extracted later in solution. This involves fused-silica glass blowing, arc-welding of niobium containers, and handling of highly air-sensitive materials in a glove box. The air-sensitive K4Ge9 is then dissolved in ethylenediamine in the box and then alkenylated by a reaction with Me3SiC≡CSiMe3. The reaction is followed by electrospray mass spectrometry while the resulting solution is used for obtaining single crystals containing the functionalized clusters [H2C=CH-Ge9-CH=CH2]2-. For this purpose the solution is centrifuged, filtered, and carefully layered with a toluene solution of 18-crown-6. Left undisturbed for a few days, the so-layered solutions produced orange crystalline blocks of [K(18-crown-6)]2[Ge9(HCCH2)2]•en which were characterized by single-crystal X-ray diffraction. The process highlights standard reaction techniques, work-up, and analysis towards functionalized deltahedral Zintl clusters. It is hoped that it will help towards further development and understanding of these compounds in the community at large.
Biochemistry, Issue 60, Zintl ions, deltahedral clusters, germanium, intermetallics, alkali metals
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Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes
Authors: Yu Seon Chung, Alba Guarné.
Institutions: McMaster University .
Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters within the origin of replication1. Beyond the origin, SeqA is found at the replication forks, where it organizes newly replicated DNA into higher ordered structures2. SeqA associates only weakly with single GATC sequences, but it forms high affinity complexes with DNA duplexes containing multiple GATC sites. The minimal functional and structural unit of SeqA is a dimer, thereby explaining the requirement of at least two GATC sequences to form a high-affinity complex with hemimethylated DNA3. Additionally, the SeqA architecture, with the oligomerization and DNA-binding domains separated by a flexible linker, allows binding to GATC repeats separated by up to three helical turns. Therefore, understanding the function of SeqA at a molecular level requires the structural analysis of SeqA bound to multiple GATC sequences. In protein-DNA crystallization, DNA can have none to an exceptional effect on the packing interactions depending on the relative sizes and architecture of the protein and the DNA. If the protein is larger than the DNA or footprints most of the DNA, the crystal packing is primarily mediated by protein-protein interactions. Conversely, when the protein is the same size or smaller than the DNA or it only covers a fraction of the DNA, DNA-DNA and DNA-protein interactions dominate crystal packing. Therefore, crystallization of protein-DNA complexes requires the systematic screening of DNA length4 and DNA ends (blunt or overhang)5-7. In this report, we describe how to design, optimize, purify and crystallize hemimethylated DNA duplexes containing tandem GATC repeats in complex with a dimeric variant of SeqA (SeqAΔ(41-59)-A25R) to obtain crystals suitable for structure determination.
Structural Biology, Issue 69, SeqA, DNA replication, DNA purification, protein-DNA complexes, protein-DNA cocrystallization, X-ray crystallography
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Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans
Authors: Elizabeth C. Pino, Christopher M. Webster, Christopher E. Carr, Alexander A. Soukas.
Institutions: Massachusetts General Hospital and Harvard Medical School, Massachusetts Institute of Technology.
The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.
Genetics, Issue 73, Biochemistry, Cellular Biology, Molecular Biology, Developmental Biology, Physiology, Anatomy, Caenorhabditis elegans, Obesity, Energy Metabolism, Lipid Metabolism, C. elegans, fluorescent lipid staining, lipids, Nile red, fat, high throughput screening, obesity, gas chromatography, mass spectrometry, GC/MS, animal model
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Free Radicals in Chemical Biology: from Chemical Behavior to Biomarker Development
Authors: Chryssostomos Chatgilialoglu, Carla Ferreri, Annalisa Masi, Michele Melchiorre, Anna Sansone, Michael A. Terzidis, Armida Torreggiani.
Institutions: Consiglio Nazionale delle Ricerche.
The involvement of free radicals in life sciences has constantly increased with time and has been connected to several physiological and pathological processes. This subject embraces diverse scientific areas, spanning from physical, biological and bioorganic chemistry to biology and medicine, with applications to the amelioration of quality of life, health and aging. Multidisciplinary skills are required for the full investigation of the many facets of radical processes in the biological environment and chemical knowledge plays a crucial role in unveiling basic processes and mechanisms. We developed a chemical biology approach able to connect free radical chemical reactivity with biological processes, providing information on the mechanistic pathways and products. The core of this approach is the design of biomimetic models to study biomolecule behavior (lipids, nucleic acids and proteins) in aqueous systems, obtaining insights of the reaction pathways as well as building up molecular libraries of the free radical reaction products. This context can be successfully used for biomarker discovery and examples are provided with two classes of compounds: mono-trans isomers of cholesteryl esters, which are synthesized and used as references for detection in human plasma, and purine 5',8-cyclo-2'-deoxyribonucleosides, prepared and used as reference in the protocol for detection of such lesions in DNA samples, after ionizing radiations or obtained from different health conditions.
Chemistry, Issue 74, Biochemistry, Chemical Engineering, Chemical Biology, chemical analysis techniques, chemistry (general), life sciences, radiation effects (biological, animal and plant), biomarker, biomimetic chemistry, free radicals, trans lipids, cyclopurine lesions, DNA, chromatography, spectroscopy, synthesis
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Microwave-assisted Intramolecular Dehydrogenative Diels-Alder Reactions for the Synthesis of Functionalized Naphthalenes/Solvatochromic Dyes
Authors: Laura S. Kocsis, Erica Benedetti, Kay M. Brummond.
Institutions: University of Pittsburgh.
Functionalized naphthalenes have applications in a variety of research fields ranging from the synthesis of natural or biologically active molecules to the preparation of new organic dyes. Although numerous strategies have been reported to access naphthalene scaffolds, many procedures still present limitations in terms of incorporating functionality, which in turn narrows the range of available substrates. The development of versatile methods for direct access to substituted naphthalenes is therefore highly desirable. The Diels-Alder (DA) cycloaddition reaction is a powerful and attractive method for the formation of saturated and unsaturated ring systems from readily available starting materials. A new microwave-assisted intramolecular dehydrogenative DA reaction of styrenyl derivatives described herein generates a variety of functionalized cyclopenta[b]naphthalenes that could not be prepared using existing synthetic methods. When compared to conventional heating, microwave irradiation accelerates reaction rates, enhances yields, and limits the formation of undesired byproducts. The utility of this protocol is further demonstrated by the conversion of a DA cycloadduct into a novel solvatochromic fluorescent dye via a Buchwald-Hartwig palladium-catalyzed cross-coupling reaction. Fluorescence spectroscopy, as an informative and sensitive analytical technique, plays a key role in research fields including environmental science, medicine, pharmacology, and cellular biology. Access to a variety of new organic fluorophores provided by the microwave-assisted dehydrogenative DA reaction allows for further advancement in these fields.
Chemistry, Issue 74, Chemical Engineering, Physical Chemistry, Microwave-assisted synthesis, dehydrogenative Diels-Alder reactions, naphthalenes, fluorescent dyes, solvatochromism, catalyst
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Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening
Authors: Marisa Till, Alice Robson, Matthew J. Byrne, Asha V. Nair, Stefan A. Kolek, Patrick D. Shaw Stewart, Paul R. Race.
Institutions: University of Bristol, Douglas Instruments.
Random microseed matrix screening (rMMS) is a protein crystallization technique in which seed crystals are added to random screens. By increasing the likelihood that crystals will grow in the metastable zone of a protein's phase diagram, extra crystallization leads are often obtained, the quality of crystals produced may be increased, and a good supply of crystals for data collection and soaking experiments is provided. Here we describe a general method for rMMS that may be applied to either sitting drop or hanging drop vapor diffusion experiments, established either by hand or using liquid handling robotics, in 96-well or 24-well tray format.
Structural Biology, Issue 78, Crystallography, X-Ray, Biochemical Phenomena, Molecular Structure, Molecular Conformation, protein crystallization, seeding, protein structure
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The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphosPh Complexes
Authors: Andreas Phanopoulos, Nicholas Long, Philip Miller.
Institutions: Imperial College London.
Herein we report the synthesis of a tridentate phosphine ligand N(CH2PPh2)3 (N-triphosPh) (1) via a phosphorus based Mannich reaction of the hydroxylmethylene phosphine precursor with ammonia in methanol under a nitrogen atmosphere. The N-triphosPh ligand precipitates from the solution after approximately 1 hr of reflux and can be isolated analytically pure via simple cannula filtration procedure under nitrogen. Reaction of the N-triphosPh ligand with [Ru3(CO)12] under reflux affords a deep red solution that show evolution of CO gas on ligand complexation. Orange crystals of the complex [Ru(CO)2{N(CH2PPh2)3}-κ3P] (2) were isolated on cooling to RT. The 31P{1H} NMR spectrum showed a characteristic single peak at lower frequency compared to the free ligand. Reaction of a toluene solution of complex 2 with oxygen resulted in the instantaneous precipitation of the carbonate complex [Ru(CO3)(CO){N(CH2PPh2)3}-κ3P] (3) as an air stable orange solid. Subsequent hydrogenation of 3 under 15 bar of hydrogen in a high-pressure reactor gave the dihydride complex [RuH2(CO){N(CH2PPh2)3}-κ3P] (4), which was fully characterized by X-ray crystallography and NMR spectroscopy. Complexes 3 and 4 are potentially useful catalyst precursors for a range of hydrogenation reactions, including biomass-derived products such as levulinic acid (LA). Complex 4 was found to cleanly react with LA in the presence of the proton source additive NH4PF6 to give [Ru(CO){N(CH2PPh2)3}-κ3P{CH3CO(CH2)2CO2H}-κ2O](PF6) (6).
Chemistry, Issue 98, ligand, phosphine, coordination, complex, catalysis, ruthenium, biomass, levulinic acid
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