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Preparation and Enantioseparation Characteristics of a Novel ?-Cyclodextrin Derivative Chiral Stationary Phase in High-Performance Liquid Chromatography.
J Chromatogr Sci
PUBLISHED: 11-01-2014
A novel cyclodextrin (CD) derivative mono(6(A)-N-allylamino-6(A)-deoxy)per-3-chlorine-4-methyl-phenylcarbamoylated-?-CD was synthesized and chemically immobilized onto the surface of ?-mercaptopropyl-functionalized silica gel step by step. The products were all purified and characterized and then got a substance with definitional structure. This chiral stationary phase (CSP) of 3-chlorine-4-methyl-phenylcarbamoylated-?-CD bonded on silica gel exhibited excellent separation capability for several chiral compounds in high-performance liquid chromatography. In the enantiomeric separations of five racemates with a mixture of methanol and aqueous KH2PO4 buffer as the mobile phase, this CSP presented good chiral recognition performance.
ABSTRACT
The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. Diiodomethyllithium is prepared by the deprotonation of diiodomethane with LiHMDS, in a THF/diethyl ether mixture, at -78 °C in the dark. These conditions are essential for the stability of the LiCHI2 reagent generated. The subsequent dropwise addition of N-Ts aldimines to the preformed diiodomethyllithium solution affords an amino-diiodide intermediate, which is not isolated. Rapid warming of the reaction mixture to 0 °C promotes cyclization to afford iodoaziridines with exclusive cis-diastereoselectivity. The addition and cyclization stages of the reaction are mediated in one reaction flask by careful temperature control. Due to the sensitivity of the iodoaziridines to purification, assessment of suitable methods of purification is required. A protocol to assess the stability of sensitive compounds to stationary phases for column chromatography is described. This method is suitable to apply to new iodoaziridines, or other potentially sensitive novel compounds. Consequently this method may find application in range of synthetic projects. The procedure involves firstly the assessment of the reaction yield, prior to purification, by 1H NMR spectroscopy with comparison to an internal standard. Portions of impure product mixture are then exposed to slurries of various stationary phases appropriate for chromatography, in a solvent system suitable as the eluent in flash chromatography. After stirring for 30 min to mimic chromatography, followed by filtering, the samples are analyzed by 1H NMR spectroscopy. Calculated yields for each stationary phase are then compared to that initially obtained from the crude reaction mixture. The results obtained provide a quantitative assessment of the stability of the compound to the different stationary phases; hence the optimal can be selected. The choice of basic alumina, modified to activity IV, as a suitable stationary phase has allowed isolation of certain iodoaziridines in excellent yield and purity.
26 Related JoVE Articles!
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Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis
Authors: Stacy L. Gelhaus, A. Clementina Mesaros, Ian A. Blair.
Institutions: University of Pennsylvania , University of Pennsylvania .
The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products. While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13. Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.
Bioengineering, Issue 57, lipids, extraction, stable isotope dilution, chiral chromatography, electron capture, mass spectrometry
3399
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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
50511
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Thin-layer Chromatographic (TLC) Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds
Authors: Isabelle A. Kagan, Michael D. Flythe.
Institutions: United States Department of Agriculture.
A common screen for plant antimicrobial compounds consists of separating plant extracts by paper or thin-layer chromatography (PC or TLC), exposing the chromatograms to microbial suspensions (e.g. fungi or bacteria in broth or agar), allowing time for the microbes to grow in a humid environment, and visualizing zones with no microbial growth. The effectiveness of this screening method, known as bioautography, depends on both the quality of the chromatographic separation and the care taken with microbial culture conditions. This paper describes standard protocols for TLC and contact bioautography with a novel application to amino acid-fermenting bacteria. The extract is separated on flexible (aluminum-backed) silica TLC plates, and bands are visualized under ultraviolet (UV) light. Zones are cut out and incubated face down onto agar inoculated with the test microorganism. Inhibitory bands are visualized by staining the agar plates with tetrazolium red. The method is applied to the separation of red clover (Trifolium pratense cv. Kenland) phenolic compounds and their screening for activity against Clostridium sticklandii, a hyper ammonia-producing bacterium (HAB) that is native to the bovine rumen. The TLC methods apply to many types of plant extracts and other bacterial species (aerobic or anaerobic), as well as fungi, can be used as test organisms if culture conditions are modified to fit the growth requirements of the species.
Chemistry, Issue 85, Thin-layer chromatography, bioautography, anaerobic bacteria, tetrazolium red, phenolic compounds, plant
51411
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Quantitative Detection of Trace Explosive Vapors by Programmed Temperature Desorption Gas Chromatography-Electron Capture Detector
Authors: Christopher R. Field, Adam Lubrano, Morgan Woytowitz, Braden C. Giordano, Susan L. Rose-Pehrsson.
Institutions: U.S. Naval Research Laboratory, NOVA Research, Inc., U.S. Naval Research Laboratory, U.S. Naval Research Laboratory.
The direct liquid deposition of solution standards onto sorbent-filled thermal desorption tubes is used for the quantitative analysis of trace explosive vapor samples. The direct liquid deposition method yields a higher fidelity between the analysis of vapor samples and the analysis of solution standards than using separate injection methods for vapors and solutions, i.e., samples collected on vapor collection tubes and standards prepared in solution vials. Additionally, the method can account for instrumentation losses, which makes it ideal for minimizing variability and quantitative trace chemical detection. Gas chromatography with an electron capture detector is an instrumentation configuration sensitive to nitro-energetics, such as TNT and RDX, due to their relatively high electron affinity. However, vapor quantitation of these compounds is difficult without viable vapor standards. Thus, we eliminate the requirement for vapor standards by combining the sensitivity of the instrumentation with a direct liquid deposition protocol to analyze trace explosive vapor samples.
Chemistry, Issue 89, Gas Chromatography (GC), Electron Capture Detector, Explosives, Quantitation, Thermal Desorption, TNT, RDX
51938
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Detection of Architectural Distortion in Prior Mammograms via Analysis of Oriented Patterns
Authors: Rangaraj M. Rangayyan, Shantanu Banik, J.E. Leo Desautels.
Institutions: University of Calgary , University of Calgary .
We demonstrate methods for the detection of architectural distortion in prior mammograms of interval-cancer cases based on analysis of the orientation of breast tissue patterns in mammograms. We hypothesize that architectural distortion modifies the normal orientation of breast tissue patterns in mammographic images before the formation of masses or tumors. In the initial steps of our methods, the oriented structures in a given mammogram are analyzed using Gabor filters and phase portraits to detect node-like sites of radiating or intersecting tissue patterns. Each detected site is then characterized using the node value, fractal dimension, and a measure of angular dispersion specifically designed to represent spiculating patterns associated with architectural distortion. Our methods were tested with a database of 106 prior mammograms of 56 interval-cancer cases and 52 mammograms of 13 normal cases using the features developed for the characterization of architectural distortion, pattern classification via quadratic discriminant analysis, and validation with the leave-one-patient out procedure. According to the results of free-response receiver operating characteristic analysis, our methods have demonstrated the capability to detect architectural distortion in prior mammograms, taken 15 months (on the average) before clinical diagnosis of breast cancer, with a sensitivity of 80% at about five false positives per patient.
Medicine, Issue 78, Anatomy, Physiology, Cancer Biology, angular spread, architectural distortion, breast cancer, Computer-Assisted Diagnosis, computer-aided diagnosis (CAD), entropy, fractional Brownian motion, fractal dimension, Gabor filters, Image Processing, Medical Informatics, node map, oriented texture, Pattern Recognition, phase portraits, prior mammograms, spectral analysis
50341
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Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies
Authors: Sungsoo Lee, Hui Zheng, Liang Shi, Qiu-Xing Jiang.
Institutions: University of Texas Southwestern Medical Center at Dallas.
To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.
Molecular Biology, Issue 77, Biochemistry, Genetics, Cellular Biology, Structural Biology, Biophysics, Membrane Lipids, Phospholipids, Carrier Proteins, Membrane Proteins, Micelles, Molecular Motor Proteins, life sciences, biochemistry, Amino Acids, Peptides, and Proteins, lipid-protein interaction, channel reconstitution, lipid-dependent gating, voltage-gated ion channel, conformation-specific ligands, lipids
50436
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Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)
Authors: Nathaniel W. Snyder, Maya Khezam, Clementina A. Mesaros, Andrew Worth, Ian A. Blair.
Institutions: University of Pennsylvania .
Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.
Biochemistry, Issue 75, Chemistry, Molecular Biology, Cellular Biology, Physiology, Medicine, Pharmacology, Genetics, Genomics, Mass Spectrometry, MS, Metabolism, Metabolomics, untargeted, extraction, lipids, accurate mass, liquid chromatography, ultraperformance liquid chromatography, UPLC, high resolution mass spectrometry, HRMS, spectrometry
50433
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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)
50594
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Getting to Compliance in Forced Exercise in Rodents: A Critical Standard to Evaluate Exercise Impact in Aging-related Disorders and Disease
Authors: Jennifer C. Arnold, Michael F. Salvatore.
Institutions: Louisiana State University Health Sciences Center.
There is a major increase in the awareness of the positive impact of exercise on improving several disease states with neurobiological basis; these include improving cognitive function and physical performance. As a result, there is an increase in the number of animal studies employing exercise. It is argued that one intrinsic value of forced exercise is that the investigator has control over the factors that can influence the impact of exercise on behavioral outcomes, notably exercise frequency, duration, and intensity of the exercise regimen. However, compliance in forced exercise regimens may be an issue, particularly if potential confounds of employing foot-shock are to be avoided. It is also important to consider that since most cognitive and locomotor impairments strike in the aged individual, determining impact of exercise on these impairments should consider using aged rodents with a highest possible level of compliance to ensure minimal need for test subjects. Here, the pertinent steps and considerations necessary to achieve nearly 100% compliance to treadmill exercise in an aged rodent model will be presented and discussed. Notwithstanding the particular exercise regimen being employed by the investigator, our protocol should be of use to investigators that are particularly interested in the potential impact of forced exercise on aging-related impairments, including aging-related Parkinsonism and Parkinson’s disease.
Behavior, Issue 90, Exercise, locomotor, Parkinson’s disease, aging, treadmill, bradykinesia, Parkinsonism
51827
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Metal-free Synthesis of Ynones from Acyl Chlorides and Potassium Alkynyltrifluoroborate Salts
Authors: Cassandra L. Taylor, Yuri Bolshan.
Institutions: University of Ontario Institute of Technology.
Ynones are a valuable functional group and building block in organic synthesis. Ynones serve as a precursor to many important organic functional groups and scaffolds. Traditional methods for the preparation of ynones are associated with drawbacks including harsh conditions, multiple purification steps, and the presence of unwanted byproducts. An alternative method for the straightforward preparation of ynones from acyl chlorides and potassium alkynyltrifluoroborate salts is described herein. The adoption of organotrifluoroborate salts as an alternative to organometallic reagents for the formation of new carbon-carbon bonds has a number of advantages. Potassium organotrifluoroborate salts are shelf stable, have good functional group tolerance, low toxicity, and a wide variety are straightforward to prepare. The title reaction proceeds rapidly at ambient temperature in the presence of a Lewis acid without the exclusion of air and moisture. Fair to excellent yields may be obtained via reaction of various aryl and alkyl acid chlorides with alkynyltrifluoroborate salts in the presence of boron trichloride.
Chemistry, Issue 96, Organic synthesis, trifluoroborates, ketones, ynones, Lewis acid, boron, acyl chlorides
52401
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Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans
Authors: Arielle M. Bryan, Amir M. Farnoud, Visesato Mor, Maurizio Del Poeta.
Institutions: Stony Brook University.
Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFNγ) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.
Immunology, Issue 94, Infection, phagocytosis, Cryptococcus, cholesterol, cyclodextrin, macrophages
52432
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Preparation of Highly Porous Coordination Polymer Coatings on Macroporous Polymer Monoliths for Enhanced Enrichment of Phosphopeptides
Authors: Alexandros Lamprou, Hongxia Wang, Adeela Saeed, Frantisek Svec, David Britt, Fernando Maya.
Institutions: E. O. Lawrence Berkeley National Laboratory, Bahauddin Zakariya University, University of the Balearic Islands, Beijing University of Chemical Technology.
We describe a protocol for the preparation of hybrid materials based on highly porous coordination polymer coatings on the internal surface of macroporous polymer monoliths. The developed approach is based on the preparation of a macroporous polymer containing carboxylic acid functional groups and the subsequent step-by-step solution-based controlled growth of a layer of a porous coordination polymer on the surface of the pores of the polymer monolith. The prepared metal-organic polymer hybrid has a high specific micropore surface area. The amount of iron(III) sites is enhanced through metal-organic coordination on the surface of the pores of the functional polymer support. The increase of metal sites is related to the number of iterations of the coating process. The developed preparation scheme is easily adapted to a capillary column format. The functional porous polymer is prepared as a self-contained single-block porous monolith within the capillary, yielding a flow-through separation device with excellent flow permeability and modest back-pressure. The metal-organic polymer hybrid column showed excellent performance for the enrichment of phosphopeptides from digested proteins and their subsequent detection using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The presented experimental protocol is highly versatile, and can be easily implemented to different organic polymer supports and coatings with a plethora of porous coordination polymers and metal-organic frameworks for multiple purification and/or separation applications.
Chemistry, Issue 101, Porous materials, hybrid materials, polymer monoliths, porous coordination polymers, flow-through supports, phosphopeptide enrichment, mass spectrometry
52926
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Controlling the Size, Shape and Stability of Supramolecular Polymers in Water
Authors: Pol Besenius, Isja de Feijter, Nico A.J.M. Sommerdijk, Paul H.H. Bomans, Anja R. A. Palmans.
Institutions: Westfälische Wilhelms-Universität Münster, Eindhoven University of Technology, Eindhoven University of Technology.
For aqueous based supramolecular polymers, the simultaneous control over shape, size and stability is very difficult1. At the same time, the ability to do so is highly important in view of a number of applications in functional soft matter including electronics, biomedical engineering, and sensors. In the past, successful strategies to control the size and shape of supramolecular polymers typically focused on the use of templates2,3, end cappers4 or selective solvent techniques5. Here we disclose a strategy based on self-assembling discotic amphiphiles that leads to the control over stack length and shape of ordered, chiral columnar aggregates. By balancing electrostatic repulsive interactions on the hydrophilic rim and attractive non-covalent forces within the hydrophobic core of the polymerizing building block, we manage to create small and discrete spherical objects6,7. Increasing the salt concentration to screen the charges induces a sphere-to-rod transition. Intriguingly, this transition is expressed in an increase of cooperativity in the temperature-dependent self-assembly mechanism, and more stable aggregates are obtained. For our study we select a benzene-1,3,5-tricarboxamide (BTA) core connected to a hydrophilic metal chelate via a hydrophobic, fluorinated L-phenylalanine based spacer (Scheme 1). The metal chelate selected is a Gd(III)-DTPA complex that contains two overall remaining charges per complex and necessarily two counter ions. The one-dimensional growth of the aggregate is directed by π-π stacking and intermolecular hydrogen bonding. However, the electrostatic, repulsive forces that arise from the charges on the Gd(III)-DTPA complex start limiting the one-dimensional growth of the BTA-based discotic once a certain size is reached. At millimolar concentrations the formed aggregate has a spherical shape and a diameter of around 5 nm as inferred from 1H-NMR spectroscopy, small angle X-ray scattering, and cryogenic transmission electron microscopy (cryo-TEM). The strength of the electrostatic repulsive interactions between molecules can be reduced by increasing the salt concentration of the buffered solutions. This screening of the charges induces a transition from spherical aggregates into elongated rods with a length > 25 nm. Cryo-TEM allows to visualise the changes in shape and size. In addition, CD spectroscopy permits to derive the mechanistic details of the self-assembly processes before and after the addition of salt. Importantly, the cooperativity -a key feature that dictates the physical properties of the produced supramolecular polymers- increases dramatically upon screening the electrostatic interactions. This increase in cooperativity results in a significant increase in the molecular weight of the formed supramolecular polymers in water.
Chemical Engineering, Issue 66, Chemistry, Physics, Self-assembly, cryogenic transmission electron microscopy, circular dichroism, controlled architecture, discotic amphiphile
3975
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Conducting Miller-Urey Experiments
Authors: Eric T. Parker, James H. Cleaves, Aaron S. Burton, Daniel P. Glavin, Jason P. Dworkin, Manshui Zhou, Jeffrey L. Bada, Facundo M. Fernández.
Institutions: Georgia Institute of Technology, Tokyo Institute of Technology, Institute for Advanced Study, NASA Johnson Space Center, NASA Goddard Space Flight Center, University of California at San Diego.
In 1953, Stanley Miller reported the production of biomolecules from simple gaseous starting materials, using an apparatus constructed to simulate the primordial Earth's atmosphere-ocean system. Miller introduced 200 ml of water, 100 mmHg of H2, 200 mmHg of CH4, and 200 mmHg of NH3 into the apparatus, then subjected this mixture, under reflux, to an electric discharge for a week, while the water was simultaneously heated. The purpose of this manuscript is to provide the reader with a general experimental protocol that can be used to conduct a Miller-Urey type spark discharge experiment, using a simplified 3 L reaction flask. Since the experiment involves exposing inflammable gases to a high voltage electric discharge, it is worth highlighting important steps that reduce the risk of explosion. The general procedures described in this work can be extrapolated to design and conduct a wide variety of electric discharge experiments simulating primitive planetary environments.
Chemistry, Issue 83, Geosciences (General), Exobiology, Miller-Urey, Prebiotic chemistry, amino acids, spark discharge
51039
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Multi-step Preparation Technique to Recover Multiple Metabolite Compound Classes for In-depth and Informative Metabolomic Analysis
Authors: Charmion Cruickshank-Quinn, Kevin D. Quinn, Roger Powell, Yanhui Yang, Michael Armstrong, Spencer Mahaffey, Richard Reisdorph, Nichole Reisdorph.
Institutions: National Jewish Health, University of Colorado Denver.
Metabolomics is an emerging field which enables profiling of samples from living organisms in order to obtain insight into biological processes. A vital aspect of metabolomics is sample preparation whereby inconsistent techniques generate unreliable results. This technique encompasses protein precipitation, liquid-liquid extraction, and solid-phase extraction as a means of fractionating metabolites into four distinct classes. Improved enrichment of low abundance molecules with a resulting increase in sensitivity is obtained, and ultimately results in more confident identification of molecules. This technique has been applied to plasma, bronchoalveolar lavage fluid, and cerebrospinal fluid samples with volumes as low as 50 µl.  Samples can be used for multiple downstream applications; for example, the pellet resulting from protein precipitation can be stored for later analysis. The supernatant from that step undergoes liquid-liquid extraction using water and strong organic solvent to separate the hydrophilic and hydrophobic compounds. Once fractionated, the hydrophilic layer can be processed for later analysis or discarded if not needed. The hydrophobic fraction is further treated with a series of solvents during three solid-phase extraction steps to separate it into fatty acids, neutral lipids, and phospholipids. This allows the technician the flexibility to choose which class of compounds is preferred for analysis. It also aids in more reliable metabolite identification since some knowledge of chemical class exists.
Bioengineering, Issue 89, plasma, chemistry techniques, analytical, solid phase extraction, mass spectrometry, metabolomics, fluids and secretions, profiling, small molecules, lipids, liquid chromatography, liquid-liquid extraction, cerebrospinal fluid, bronchoalveolar lavage fluid
51670
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In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions
Authors: Grant E. Johnson, K. Don Dasitha Gunaratne, Julia Laskin.
Institutions: Pacific Northwest National Laboratory.
Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using conventional synthesis techniques. Coupling soft landing with in situ characterization using secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS) enables analysis of well-defined surfaces under clean vacuum conditions. The capabilities of three soft-landing instruments constructed in our laboratory are illustrated for the representative system of surface-bound organometallics prepared by soft landing of mass-selected ruthenium tris(bipyridine) dications, [Ru(bpy)3]2+ (bpy = bipyridine), onto carboxylic acid terminated self-assembled monolayer surfaces on gold (COOH-SAMs). In situ time-of-flight (TOF)-SIMS provides insight into the reactivity of the soft-landed ions. In addition, the kinetics of charge reduction, neutralization and desorption occurring on the COOH-SAM both during and after ion soft landing are studied using in situ Fourier transform ion cyclotron resonance (FT-ICR)-SIMS measurements. In situ IRRAS experiments provide insight into how the structure of organic ligands surrounding metal centers is perturbed through immobilization of organometallic ions on COOH-SAM surfaces by soft landing. Collectively, the three instruments provide complementary information about the chemical composition, reactivity and structure of well-defined species supported on surfaces.
Chemistry, Issue 88, soft landing, mass selected ions, electrospray, secondary ion mass spectrometry, infrared spectroscopy, organometallic, catalysis
51344
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The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin
Authors: Monica Soldi, Tiziana Bonaldi.
Institutions: European Institute of Oncology.
Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.
Biochemistry, Issue 86, chromatin, histone post-translational modifications (hPTMs), epigenetics, mass spectrometry, proteomics, SILAC, chromatin immunoprecipitation , histone variants, chromatome, hPTMs cross-talks
51220
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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
51299
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Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria
Authors: Jeremy C. Henderson, John P. O'Brien, Jennifer S. Brodbelt, M. Stephen Trent.
Institutions: The University of Texas at Austin, The University of Texas at Austin, The University of Texas at Austin.
Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.
Chemistry, Issue 79, Membrane Lipids, Toll-Like Receptors, Endotoxins, Glycolipids, Lipopolysaccharides, Lipid A, Microbiology, Lipids, lipid A, Bligh-Dyer, thin layer chromatography (TLC), lipopolysaccharide, mass spectrometry, Collision Induced Dissociation (CID), Photodissociation (PD)
50623
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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
50379
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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
51840
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Retropinacol/Cross-pinacol Coupling Reactions - A Catalytic Access to 1,2-Unsymmetrical Diols
Authors: Ulf Scheffler, Rainer Mahrwald.
Institutions: Humboldt University of Berlin.
Unsymmetrical 1,2-diols are hardly accessible by reductive pinacol coupling processes. A successful execution of such a transformation is bound to a clear recognition and strict differentiation of two similar carbonyl compounds (aldehydes → secondary 1,2-diols or ketones → tertiary 1,2-diols). This fine-tuning is still a challenge and an unsolved problem for an organic chemist. There exist several reports on successful execution of this transformation but they cannot be generalized. Herein we describe a catalytic direct pinacol coupling process which proceeds via a retropinacol/cross-pinacol coupling sequence. Thus, unsymmetrical substituted 1,2-diols can be accessed with almost quantitative yields by means of an operationally simple performance under very mild conditions. Artificial techniques, such as syringe-pump techniques or delayed additions of reactants are not necessary. The procedure we describe provides a very rapid access to cross-pinacol products (1,2-diols, vicinal diols). A further extension of this new process, e.g. an enantioselective performance could provide a very useful tool for the synthesis of unsymmetrical chiral 1,2-diols.
Chemistry, Issue 86, cross-pinacol coupling reactions, unsymmetrical 1,2-diols, catalysis, titanium(IV) alkoxides, mechanism, aldehydes, ketones
51258
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Monitoring Plant Hormones During Stress Responses
Authors: Marie J. Engelberth, Jurgen Engelberth.
Institutions: University of Texas.
Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. Among the most severe stresses are insect herbivory, pathogen infection, and drought stress. For each of these stresses a specific set of hormones and/or combinations thereof are known to fine-tune the responses, thereby ensuring the plant's survival. The major hormones involved in the regulation of these responses are jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA). To better understand the role of individual hormones as well as their potential interaction during these responses it is necessary to monitor changes in their abundance in a temporal as well as in a spatial fashion. For the easy, sensitive, and reproducible quantification of these and other signaling compounds we developed a method based on vapor phase extraction and gas chromatography/mass spectrometry (GC/MS) analysis (1, 2, 3, 4). After extracting these compounds from the plant tissue by acidic aqueous 1-propanol mixed with dichloromethane the carboxylic acid-containing compounds are methylated, volatilized under heat, and collected on a polymeric absorbent. After elution into a sample vial the analytes are separated by gas chromatography and detected by chemical ionization mass spectrometry. The use of appropriate internal standards then allows for the simple quantification by relating the peak areas of analyte and internal standard.
Plant Biology, Issue 28, Jasmonic acid, salicylic acid, abscisic acid, plant hormones, GC/MS, vapor phase extraction
1127
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Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery
Authors: Chuck Witkowski, Jay Harkins.
Institutions: Protein Discovery, Inc..
The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations. This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.
Basic Protocols, Cellular Biology, Issue 34, GELFREE, SDS PAGE, gel electrophoresis, protein fractionation, separation, electrophoresis, proteomics, mass spectrometry
1842
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Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos
Authors: Tadeusz J. Kaczynski, Shermali Gunawardena.
Institutions: SUNY-University at Buffalo.
The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudière et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.
Neuroscience, Issue 46, Drosophila neurobiology, Embryo, Immuno Fluorescence
2150
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Fabrication of the Thermoplastic Microfluidic Channels
Authors: Arpita Bhattacharyya, Dominika Kulinski, Catherine Klapperich.
Institutions: Boston University.
In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using polymer/nanoparticle composite microscale lysis and solid phase extraction columns. The recovered samples are concentrated, small volume samples that are PCRable, without any additional cleanup. Here, we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then, we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns. We demonstrate grafting and polymerization of a carbon nanotube/polymer composite column for bacterial cell lysis. We then show the lysis process followed by solid phase extraction of nucleic acids from the sample on chip using a silica/polymer composite column. The attached protocols contain detailed instructions on how to make both lysis and solid phase extraction columns.
Cellular Biology, Issue 12, bioengineering, purification, microfluidics, DNA, RNA, solid phase, column
664
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