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.
25 Related JoVE Articles!
Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography
Institutions: The University of Texas at Austin.
Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo
microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time.
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.
Neuroscience, Issue 67, Microdialysis, operant ethanol self-administration, gas chromatography, appetitive, consummatory, sterotaxic surgery
Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe
Institutions: University of Washington.
All organisms inhabit a world full of sensory stimuli that determine their behavioral and physiological response to their environment. Olfaction is especially important in insects, which use their olfactory systems to respond to, and discriminate amongst, complex odor stimuli. These odors elicit behaviors that mediate processes such as reproduction and habitat selection1-3
. Additionally, chemical sensing by insects mediates behaviors that are highly significant for agriculture and human health, including pollination4-6
, herbivory of food crops7
, and transmission of disease8,9
. Identification of olfactory signals and their role in insect behavior is thus important for understanding both ecological processes and human food resources and well-being.
To date, the identification of volatiles that drive insect behavior has been difficult and often tedious. Current techniques include gas chromatography-coupled electroantennogram recording (GC-EAG), and gas chromatography-coupled single sensillum recordings (GC-SSR)10-12
. These techniques proved to be vital in the identification of bioactive compounds. We have developed a method that uses gas chromatography coupled to multi-channel electrophysiological recordings (termed 'GCMR') from neurons in the antennal lobe (AL; the insect's primary olfactory center)13,14
. This state-of-the-art technique allows us to probe how odor information is represented in the insect brain. Moreover, because neural responses to odors at this level of olfactory processing are highly sensitive owing to the degree of convergence of the antenna's receptor neurons into AL neurons, AL recordings will allow the detection of active constituents of natural odors efficiently and with high sensitivity. Here we describe GCMR and give an example of its use.
Several general steps are involved in the detection of bioactive volatiles and insect response. Volatiles first need to be collected from sources of interest (in this example we use flowers from the genus Mimulus
(Phyrmaceae)) and characterized as needed using standard GC-MS techniques14-16
. Insects are prepared for study using minimal dissection, after which a recording electrode is inserted into the antennal lobe and multi-channel neural recording begins. Post-processing of the neural data then reveals which particular odorants cause significant neural responses by the insect nervous system.
Although the example we present here is specific to pollination studies, GCMR can be expanded to a wide range of study organisms and volatile sources. For instance, this method can be used in the identification of odorants attracting or repelling vector insects and crop pests. Moreover, GCMR can also be used to identify attractants for beneficial insects, such as pollinators. The technique may be expanded to non-insect subjects as well.
Neuroscience, Issue 72, Neurobiology, Physiology, Biochemistry, Chemistry, Entomlogy, Behavior, electrophysiology, olfaction, olfactory system, insect, multi-channel recording, gas chromatography, pollination, bees, Bombus impatiens, antennae, brain, animal model
Herbivore-induced Blueberry Volatiles and Intra-plant Signaling
Institutions: Rutgers University .
Herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack1,2
. These HIPVs are mainly regulated by the defensive plant hormone jasmonic acid (JA) and its volatile derivative methyl jasmonate (MeJA)3,4,5
. Over the past 3 decades researchers have documented that HIPVs can repel or attract herbivores, attract the natural enemies of herbivores, and in some cases they can induce or prime plant defenses prior to herbivore attack. In a recent paper6
, I reported that feeding by gypsy moth caterpillars, exogenous MeJA application, and mechanical damage induce the emissions of volatiles from blueberry plants, albeit differently. In addition, blueberry branches respond to HIPVs emitted from neighboring branches of the same plant by increasing the levels of JA and resistance to herbivores (i.e., direct plant defenses), and by priming volatile emissions (i.e., indirect plant defenses). Similar findings have been reported recently for sagebrush7
, and lima beans9
Here, I describe a push-pull method for collecting blueberry volatiles induced by herbivore (gypsy moth) feeding, exogenous MeJA application, and mechanical damage. The volatile collection unit consists of a 4 L volatile collection chamber, a 2-piece guillotine, an air delivery system that purifies incoming air, and a vacuum system connected to a trap filled with Super-Q adsorbent to collect volatiles5,6,10
. Volatiles collected in Super-Q traps are eluted with dichloromethane and then separated and quantified using Gas Chromatography (GC). This volatile collection method was used n my study6
to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants. These methods are described here. Briefly, undamaged blueberry branches are exposed to HIPVs from neighboring branches within the same plant. Using the same techniques described above, volatiles emitted from branches after exposure to HIPVs are collected and analyzed.
Plant Biology, Issue 58, herbivore-induced plant volatiles, HIPV, eavesdropping, plant defense, priming
Closed System Cell Culture Protocol Using HYPERStack Vessels with Gas Permeable Material Technology
Institutions: Corning Life Science, Corning Life Science, Corning Life Science.
Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice. HYPERStack vessels allow closed system scale up from the current stacked plate products and delivers >2.5X more cells in the same volumetric footprint. The HYPERStack vessels function via gas permeable material which allows gas exchange to occur, therefore eliminating the need for internal headspace within a vessel. The elimination of headspace allows the compartment where cell growth occurs to be minimized to reduce space, allowing more layers of cell growth surface area within the same volumetric footprint.
For many applications such as cell therapy or vaccine production, a closed system is required for cell growth and harvesting. The HYPERStack vessel allows cell and reagent addition and removal via tubing from media bags or other methods.
This protocol will explain the technology behind the gas permeable material used in the HYPERStack vessels, gas diffusion results to meet the metabolic needs of cells, closed system cell growth protocols, and various harvesting methods.
Cellular Biology, Issue 45, cell culture, bioprocess, adherent, primary cell, HYPERStack, closed system, gas permeable, cell therapy, vaccine, scale up
Characterization of Recombination Effects in a Liquid Ionization Chamber Used for the Dosimetry of a Radiosurgical Accelerator
Institutions: Centre Oscar Lambret.
Most modern radiation therapy devices allow the use of very small fields, either through beamlets in Intensity-Modulated Radiation Therapy (IMRT) or via stereotactic radiotherapy where positioning accuracy allows delivering very high doses per fraction in a small volume of the patient. Dosimetric measurements on medical accelerators are conventionally realized using air-filled ionization chambers. However, in small beams these are subject to nonnegligible perturbation effects. This study focuses on liquid ionization chambers, which offer advantages in terms of spatial resolution and low fluence perturbation. Ion recombination effects are investigated for the microLion detector (PTW) used with the Cyberknife system (Accuray). The method consists of performing a series of water tank measurements at different source-surface distances, and applying corrections to the liquid detector readings based on simultaneous gaseous detector measurements. This approach facilitates isolating the recombination effects arising from the high density of the liquid sensitive medium and obtaining correction factors to apply to the detector readings. The main difficulty resides in achieving a sufficient level of accuracy in the setup to be able to detect small changes in the chamber response.
Physics, Issue 87, Radiation therapy, dosimetry, small fields, Cyberknife, liquid ionization, recombination effects
Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy
Institutions: Max-Planck-Institute for Molecular Biomedicine and Institute of Cell Biology, Yale Cardiovascular Research Center and Section of Cardiovascular Medicine.
Primary rat neonatal cardiomyocytes are useful in basic in vitro
cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope’s autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators.
Cellular Biology, Issue 88, live cell imaging, cardiomyocyte, primary cell culture, adenovirus, lentivirus, confocal spinning disk microscopy
Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy
Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.
optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (>120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.
Bioengineering, Issue 82, biophysics, protein unfolding, atomic force microscopy, surface immobilization
Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish
Like many aquatic animals, zebrafish (Danio rerio
) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc
. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals.
Behavior, Issue 82, neuroscience, Zebrafish, Danio rerio, anxiety, Shoaling, Pharmacology, 3D-tracking, MK801
Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus
Institutions: Karolinska Institutet.
A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the anterior hypothalamus. The clock is directly synchronized by light via the retina and optic nerve. Circadian oscillations are generated by interacting negative feedback loops of a number of so called "clock genes" and their protein products, including the Period
) genes. The core clock is also dependent on membrane depolarization, calcium and cAMP 1
. The SCN shows daily oscillations in clock gene expression, metabolic activity and spontaneous electrical activity. Remarkably, this endogenous cyclic activity persists in adult tissue slices of the SCN 2-4
. In this way, the biological clock can easily be studied in vitro
, allowing molecular, electrophysiological and metabolic investigations of the pacemaker function.
The SCN is a small, well-defined bilateral structure located right above the optic chiasm 5
. In the rat it contains ~8.000 neurons in each nucleus and has dimensions of approximately 947 μm (length, rostrocaudal axis) x 424 μm (width) x 390 μm (height) 6
. To dissect out the SCN it is necessary to cut a brain slice at the specific level of the brain where the SCN can be identified. Here, we describe the dissecting and slicing procedure of the SCN, which is similar for mouse and rat brains. Further, we show how to culture the dissected tissue organotypically on a membrane 7
, a technique developed for SCN tissue culture by Yamazaki et al. 8
. Finally, we demonstrate how transgenic tissue can be used for measuring expression of clock genes/proteins using dynamic luciferase reporter technology, a method that originally was used for circadian measurements by Geusz et al. 9
. We here use SCN tissues from the transgenic knock-in PERIOD2::LUCIFERASE mice produced by Yoo et al. 10
. The mice contain a fusion protein of PERIOD (PER) 2 and the firefly enzyme LUCIFERASE. When PER2 is translated in the presence of the substrate for luciferase, i.e. luciferin, the PER2 expression can be monitored as bioluminescence when luciferase catalyzes the oxidation of luciferin. The number of emitted photons positively correlates to the amount of produced PER2 protein, and the bioluminescence rhythms match the PER2 protein rhythm in vivo 10
. In this way the cyclic variation in PER2 expression can be continuously monitored real time during many days. The protocol we follow for tissue culturing and real-time bioluminescence recording has been thoroughly described by Yamazaki and Takahashi 11
Neuroscience, Issue 48, suprachiasmatic nucleus, mice, organotypic tissue culture, circadian rhythm, clock gene, Period 2, luciferase
Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles
Institutions: Heart Research Center Goettingen, University Medical Center Goettingen, German Center for Cardiovascular Research (DZHK) partner site Goettingen, University of Maryland School of Medicine.
In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+
release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.
Bioengineering, Issue 92, cardiac myocyte, atria, ventricle, heart, primary cell isolation, fluorescence microscopy, membrane tubule, transverse-axial tubule system, image analysis, image processing, T-tubule, collagenase
In-situ Tapering of Chalcogenide Fiber for Mid-infrared Supercontinuum Generation
Institutions: Stanford University .
Supercontinuum generation (SCG) in a tapered chalcogenide fiber is desirable for broadening mid-infrared (or mid-IR, roughly the 2-20 μm wavelength range) frequency combs1, 2
for applications such as molecular fingerprinting, 3
trace gas detection, 4
laser-driven particle acceleration, 5
and x-ray production via high harmonic generation. 6
Achieving efficient SCG in a tapered optical fiber requires precise control of the group velocity dispersion (GVD) and the temporal properties of the optical pulses at the beginning of the fiber, 7
which depend strongly on the geometry of the taper. 8
Due to variations in the tapering setup and procedure for successive SCG experiments-such as fiber length, tapering environment temperature, or power coupled into the fiber, in-situ
spectral monitoring of the SCG is necessary to optimize the output spectrum for a single experiment.
fiber tapering for SCG consists of coupling the pump source through the fiber to be tapered to a spectral measurement device. The fiber is then tapered while the spectral measurement signal is observed in real-time. When the signal reaches its peak, the tapering is stopped. The in-situ
tapering procedure allows for generation of a stable, octave-spanning, mid-IR frequency comb from the sub harmonic of a commercially available near-IR frequency comb. 9
This method lowers cost due to the reduction in time and materials required to fabricate an optimal taper with a waist length of only 2 mm.
tapering technique can be extended to optimizing microstructured optical fiber (MOF) for SCG10
or tuning of the passband of MOFs, 11
optimizing tapered fiber pairs for fused fiber couplers12
and wavelength division multiplexers (WDMs), 13
or modifying dispersion compensation for compression or stretching of optical pulses.14-16
Physics, Issue 75, Engineering, Photonics, Optics, infrared spectra, nonlinear optics, optical fibers, optical waveguides, wave propagation (optics), fiber optics, infrared optics, fiber tapering, chalcogenide, supercontinuum generation, mid-infrared, in-situ, frequency comb, scanning electron microscopy, SEM
A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats
Institutions: Concordia University.
Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate drug-seeking behavior and prompt relapse in abstinent drug users. We have developed a procedure to study the behavioral and neural processes that mediate the impact of context on alcohol-seeking behavior in rats. Following acclimation to the taste and pharmacological effects of 15% ethanol in the home cage, male Long-Evans rats receive Pavlovian discrimination training (PDT) in conditioning chambers. In each daily (Mon-Fri) PDT session, 16 trials each of two different 10 sec auditory conditioned stimuli occur. During one stimulus, the CS+, 0.2 ml of 15% ethanol is delivered into a fluid port for oral consumption. The second stimulus, the CS-, is not paired with ethanol. Across sessions, entries into the fluid port during the CS+ increase, whereas entries during the CS- stabilize at a lower level, indicating that a predictive association between the CS+ and ethanol is acquired. During PDT each chamber is equipped with a specific configuration of visual, olfactory and tactile contextual stimuli. Following PDT, extinction training is conducted in the same chamber that is now equipped with a different configuration of contextual stimuli. The CS+ and CS- are presented as before, but ethanol is withheld, which causes a gradual decline in port entries during the CS+. At test, rats are placed back into the PDT context and presented with the CS+ and CS- as before, but without ethanol. This manipulation triggers a robust and selective increase in the number of port entries made during the alcohol predictive CS+, with no change in responding during the CS-. This effect, referred to as context-induced renewal, illustrates the powerful capacity of contexts associated with alcohol consumption to stimulate alcohol-seeking behavior in response to Pavlovian alcohol cues.
Behavior, Issue 91, Behavioral neuroscience, alcoholism, relapse, addiction, Pavlovian conditioning, ethanol, reinstatement, discrimination, conditioned approach
Fruit Volatile Analysis Using an Electronic Nose
Institutions: University of California, Davis, University of California, Davis, University of California, Davis.
Numerous and diverse physiological changes occur during fruit ripening, including the development of a specific volatile blend that characterizes fruit aroma. Maturity at harvest is one of the key factors influencing the flavor quality of fruits and vegetables1
. The validation of robust methods that rapidly assess fruit maturity and aroma quality would allow improved management of advanced breeding programs, production practices and postharvest handling.
Over the last three decades, much research has been conducted to develop so-called electronic noses, which are devices able to rapidly detect odors and flavors2-4
. Currently there are several commercially available electronic noses able to perform volatile analysis, based on different technologies. The electronic nose used in our work (zNose, EST, Newbury Park, CA, USA), consists of ultra-fast gas chromatography coupled with a surface acoustic wave sensor (UFGC-SAW). This technology has already been tested for its ability to monitor quality of various commodities, including detection of deterioration in apple5
; ripeness and rot evaluation in mango6
; aroma profiling of thymus
volatile compounds in grape berries8
; characterization of vegetable oil9
and detection of adulterants in virgin coconut oil10
This system can perform the three major steps of aroma analysis: headspace sampling, separation of volatile compounds, and detection. In about one minute, the output, a chromatogram, is produced and, after a purging cycle, the instrument is ready for further analysis. The results obtained with the zNose can be compared to those of other gas-chromatographic systems by calculation of Kovats Indices (KI). Once the instrument has been tuned with an alkane standard solution, the retention times are automatically converted into KIs. However, slight changes in temperature and flow rate are expected to occur over time, causing retention times to drift. Also, depending on the polarity of the column stationary phase, the reproducibility of KI calculations can vary by several index units11
. A series of programs and graphical interfaces were therefore developed to compare calculated KIs among samples in a semi-automated fashion. These programs reduce the time required for chromatogram analysis of large data sets and minimize the potential for misinterpretation of the data when chromatograms are not perfectly aligned.
We present a method for rapid volatile compound analysis in fruit. Sample preparation, data acquisition and handling procedures are also discussed.
Plant Biology, Issue 61, zNose, volatile profiling, aroma, Kovats Index, electronic nose, gas chromatography, retention time shift
In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions
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
(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
Videomorphometric Analysis of Hypoxic Pulmonary Vasoconstriction of Intra-pulmonary Arteries Using Murine Precision Cut Lung Slices
Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ventilation. Up to now, the underlying mechanisms are not fully understood. The major vascular segment contributing to HPV is the intra-acinar artery. This vessel section is responsible for the blood supply of an individual acinus, which is defined as the portion of lung distal to a terminal bronchiole. Intra-acinar arteries are mostly located in that part of the lung that cannot be selectively reached by a number of commonly used techniques such as measurement of the pulmonary artery pressure in isolated perfused lungs or force recordings from dissected proximal pulmonary artery segments1,2
. The analysis of subpleural vessels by real-time confocal laser scanning luminescence microscopy is limited to vessels with up to 50 µm in diameter3
We provide a technique to study HPV of murine intra-pulmonary arteries in the range of 20-100 µm inner diameters. It is based on the videomorphometric analysis of cross-sectioned arteries in precision cut lung slices (PCLS). This method allows the quantitative measurement of vasoreactivity of small intra-acinar
arteries with inner diameter between 20-40 µm which are located at gussets of alveolar septa next to alveolar ducts and of larger pre-acinar
arteries with inner diameters between 40-100 µm which run adjacent to bronchi and bronchioles. In contrast to real-time imaging of subpleural vessels in anesthetized and ventilated mice, videomorphometric analysis of PCLS occurs under conditions free of shear stress. In our experimental model both arterial segments exhibit a monophasic HPV when exposed to medium gassed with 1% O2
and the response fades after 30-40 min at hypoxia.
Medicine, Issue 83, Hypoxic pulmonary vasoconstriction, murine lungs, precision cut lung slices, intra-pulmonary, pre- and intra-acinar arteries, videomorphometry
Physical, Chemical and Biological Characterization of Six Biochars Produced for the Remediation of Contaminated Sites
Institutions: Royal Military College of Canada, Queen's University.
The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with specific functions (e.g.
carbon sequestration, soil quality improvements, or contaminant sorption). In 2013, the International Biochar Initiative (IBI) made publically available their Standardized Product Definition and Product Testing Guidelines (Version 1.1) which set standards for physical and chemical characteristics for biochar. Six biochars made from three different feedstocks and at two temperatures were analyzed for characteristics related to their use as a soil amendment. The protocol describes analyses of the feedstocks and biochars and includes: cation exchange capacity (CEC), specific surface area (SSA), organic carbon (OC) and moisture percentage, pH, particle size distribution, and proximate and ultimate analysis. Also described in the protocol are the analyses of the feedstocks and biochars for contaminants including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), metals and mercury as well as nutrients (phosphorous, nitrite and nitrate and ammonium as nitrogen). The protocol also includes the biological testing procedures, earthworm avoidance and germination assays. Based on the quality assurance / quality control (QA/QC) results of blanks, duplicates, standards and reference materials, all methods were determined adequate for use with biochar and feedstock materials. All biochars and feedstocks were well within the criterion set by the IBI and there were little differences among biochars, except in the case of the biochar produced from construction waste materials. This biochar (referred to as Old biochar) was determined to have elevated levels of arsenic, chromium, copper, and lead, and failed the earthworm avoidance and germination assays. Based on these results, Old biochar would not be appropriate for use as a soil amendment for carbon sequestration, substrate quality improvements or remediation.
Environmental Sciences, Issue 93, biochar, characterization, carbon sequestration, remediation, International Biochar Initiative (IBI), soil amendment
Quantitative Detection of Trace Explosive Vapors by Programmed Temperature Desorption Gas Chromatography-Electron Capture Detector
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
A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent
Institutions: University of Maine, Orono, University of Maine, Orono.
Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g.
by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5
. Mast cell granule component and the allergic mediator β-hexosaminidase, which is released linearly in tandem with histamine from mast cells6
, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1
. Originally published by Naal et al.1
, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11
, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2
. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using ε280
= 4,200 L/M/cm)12
. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.
Immunology, Issue 81, mast cell, basophil, degranulation, RBL-2H3, triclosan, irgasan, antibacterial, β-hexosaminidase, allergy, Asthma, toxicants, ionophore, antigen, fluorescence, microplate, UV-Vis
Preparation and Use of Photocatalytically Active Segmented Ag|ZnO and Coaxial TiO2-Ag Nanowires Made by Templated Electrodeposition
Institutions: University of Twente.
Photocatalytically active nanostructures require a large specific surface area with the presence of many catalytically active sites for the oxidation and reduction half reactions, and fast electron (hole) diffusion and charge separation. Nanowires present suitable architectures to meet these requirements. Axially segmented Ag|ZnO and radially segmented (coaxial) TiO2
-Ag nanowires with a diameter of 200 nm and a length of 6-20 µm were made by templated electrodeposition within the pores of polycarbonate track-etched (PCTE) or anodized aluminum oxide (AAO) membranes, respectively. In the photocatalytic experiments, the ZnO and TiO2
phases acted as photoanodes, and Ag as cathode. No external circuit is needed to connect both electrodes, which is a key advantage over conventional photo-electrochemical cells. For making segmented Ag|ZnO nanowires, the Ag salt electrolyte was replaced after formation of the Ag segment to form a ZnO segment attached to the Ag segment. For making coaxial TiO2
-Ag nanowires, a TiO2
gel was first formed by the electrochemically induced sol-gel method. Drying and thermal annealing of the as-formed TiO2
gel resulted in the formation of crystalline TiO2
nanotubes. A subsequent Ag electrodeposition step inside the TiO2
nanotubes resulted in formation of coaxial TiO2
-Ag nanowires. Due to the combination of an n
-type semiconductor (ZnO or TiO2
) and a metal (Ag) within the same nanowire, a Schottky barrier was created at the interface between the phases. To demonstrate the photocatalytic activity of these nanowires, the Ag|ZnO nanowires were used in a photocatalytic experiment in which H2
gas was detected upon UV illumination of the nanowires dispersed in a methanol/water mixture. After 17 min of illumination, approximately 0.2 vol% H2
gas was detected from a suspension of ~0.1 g of Ag|ZnO nanowires in a 50 ml 80 vol% aqueous methanol solution.
Physics, Issue 87, Multicomponent nanowires, electrochemistry, sol-gel processes, photocatalysis, photochemistry, H2 evolution
Multi-step Preparation Technique to Recover Multiple Metabolite Compound Classes for In-depth and Informative Metabolomic Analysis
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
Continuously-stirred Anaerobic Digester to Convert Organic Wastes into Biogas: System Setup and Basic Operation
Institutions: Cornell University.
Anaerobic digestion (AD) is a bioprocess that is commonly used to convert complex organic wastes into a useful biogas with methane as the energy carrier 1-3
. Increasingly, AD is being used in industrial, agricultural, and municipal waste(water) treatment applications 4,5
. The use of AD technology allows plant operators to reduce waste disposal costs and offset energy utility expenses. In addition to treating organic wastes, energy crops are being converted into the energy carrier methane 6,7
. As the application of AD technology broadens for the treatment of new substrates and co-substrate mixtures 8
, so does the demand for a reliable testing methodology at the pilot- and laboratory-scale.
Anaerobic digestion systems have a variety of configurations, including the continuously stirred tank reactor (CSTR), plug flow (PF), and anaerobic sequencing batch reactor (ASBR) configurations 9
. The CSTR is frequently used in research due to its simplicity in design and operation, but also for its advantages in experimentation. Compared to other configurations, the CSTR provides greater uniformity of system parameters, such as temperature, mixing, chemical concentration, and substrate concentration. Ultimately, when designing a full-scale reactor, the optimum reactor configuration will depend on the character of a given substrate among many other nontechnical considerations. However, all configurations share fundamental design features and operating parameters that render the CSTR appropriate for most preliminary assessments. If researchers and engineers use an influent stream with relatively high concentrations of solids, then lab-scale bioreactor configurations cannot be fed continuously due to plugging problems of lab-scale pumps with solids or settling of solids in tubing. For that scenario with continuous mixing requirements, lab-scale bioreactors are fed periodically and we refer to such configurations as continuously stirred anaerobic digesters (CSADs).
This article presents a general methodology for constructing, inoculating, operating, and monitoring a CSAD system for the purpose of testing the suitability of a given organic substrate for long-term anaerobic digestion. The construction section of this article will cover building the lab-scale reactor system. The inoculation section will explain how to create an anaerobic environment suitable for seeding with an active methanogenic inoculum. The operating section will cover operation, maintenance, and troubleshooting. The monitoring section will introduce testing protocols using standard analyses. The use of these measures is necessary for reliable experimental assessments of substrate suitability for AD. This protocol should provide greater protection against a common mistake made in AD studies, which is to conclude that reactor failure was caused by the substrate in use, when really it was improper user operation 10
Bioengineering, Issue 65, Environmental Engineering, Chemistry, Anaerobic Digestion, Bioenergy, Biogas, Methane, Organic Waste, Methanogenesis, Energy Crops
Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies
Institutions: University of California, Los Angeles .
In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of microbiology was transformed with the advent of Anton van Leeuwenhoek's microscope, which allowed scientists to visualize prokaryotes for the first time. The development of the polymerase chain reaction (PCR) is one of those innovations that changed the course of molecular science with its impact spanning countless subdisciplines in biology. The theoretical process was outlined by Keppe and coworkers in 1971; however, it was another 14 years until the complete PCR procedure was described and experimentally applied by Kary Mullis while at Cetus Corporation in 1985. Automation and refinement of this technique progressed with the introduction of a thermal stable DNA polymerase from the bacterium Thermus aquaticus
, consequently the name Taq
PCR is a powerful amplification technique that can generate an ample supply of a specific segment of DNA (i.e., an amplicon) from only a small amount of starting material (i.e., DNA template or target sequence). While straightforward and generally trouble-free, there are pitfalls that complicate the reaction producing spurious results. When PCR fails it can lead to many non-specific DNA products of varying sizes that appear as a ladder or smear of bands on agarose gels. Sometimes no products form at all. Another potential problem occurs when mutations are unintentionally introduced in the amplicons, resulting in a heterogeneous population of PCR products. PCR failures can become frustrating unless patience and careful troubleshooting are employed to sort out and solve the problem(s). This protocol outlines the basic principles of PCR, provides a methodology that will result in amplification of most target sequences, and presents strategies for optimizing a reaction. By following this PCR guide, students should be able to:
● Set up reactions and thermal cycling conditions for a conventional PCR experiment
● Understand the function of various reaction components and their overall effect on a PCR experiment
● Design and optimize a PCR experiment for any DNA template
● Troubleshoot failed PCR experiments
Basic Protocols, Issue 63, PCR, optimization, primer design, melting temperature, Tm, troubleshooting, additives, enhancers, template DNA quantification, thermal cycler, molecular biology, genetics
Analysis of Volatile and Oxidation Sensitive Compounds Using a Cold Inlet System and Electron Impact Mass Spectrometry
Institutions: Bielefeld University.
This video presents a protocol for the mass spectrometrical analysis of volatile and oxidation sensitive compounds using electron impact ionization. The analysis of volatile and oxidation sensitive compounds by mass spectrometry is not easily achieved, as all state-of-the-art mass spectrometric methods require at least one sample preparation step, e.g.
, dissolution and dilution of the analyte (electrospray ionization), co-crystallization of the analyte with a matrix compound (matrix-assisted laser desorption/ionization), or transfer of the prepared samples into the ionization source of the mass spectrometer, to be conducted under atmospheric conditions. Here, the use of a sample inlet system is described which enables the analysis of volatile metal organyls, silanes, and phosphanes using a sector field mass spectrometer equipped with an electron impact ionization source. All sample preparation steps and the sample introduction into the ion source of the mass spectrometer take place either under air-free conditions or under vacuum, enabling the analysis of compounds highly susceptible to oxidation. The presented technique is especially of interest for inorganic chemists, working with metal organyls, silanes, or phosphanes, which have to be handled using inert conditions, such as the Schlenk technique. The principle of operation is presented in this video.
Chemistry, Issue 91, mass spectrometry, electron impact, inlet system, volatile, air sensitive
Monitoring Plant Hormones During Stress Responses
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
MALDI Sample Preparation: the Ultra Thin Layer Method
Institutions: Rockefeller University.
This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2
. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g.
dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2
. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3
. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.
Cellular Biology, Issue 3, mass-spectrometry, ultra-thin layer, MALDI, MS, proteins