Setaria viridis is an emerging model system for C4 grasses. It is closely related to the bioenergy feed stock switchgrass and the grain crop foxtail millet. Recently, the 510 Mb genome of foxtail millet, S. italica, has been sequenced 1,2 and a 25x coverage genome sequence of the weedy relative S. viridis is in progress. S. viridis has a number of characteristics that make it a potentially excellent model genetic system including a rapid generation time, small stature, simple growth requirements, prolific seed production 3 and developed systems for both transient and stable transformation 4. However, the genetics of S. viridis is largely unexplored, in part, due to the lack of detailed methods for performing crosses. To date, no standard protocol has been adopted that will permit rapid production of seeds from controlled crosses.
The protocol presented here is optimized for performing genetic crosses in S. viridis, accession A10.1. We have employed a simple heat treatment with warm water for emasculation after pruning the panicle to retain 20-30 florets and labeling of flowers to eliminate seeds resulting from newly developed flowers after emasculation. After testing a series of heat treatments at permissive temperatures and varying the duration of dipping, we have established an optimum temperature and time range of 48 °C for 3-6 min. By using this method, a minimum of 15 crosses can be performed by a single worker per day and an average of 3-5 outcross progeny per panicle can be recovered. Therefore, an average of 45-75 outcross progeny can be produced by one person in a single day. Broad implementation of this technique will facilitate the development of recombinant inbred line populations of S. viridis X S. viridis or S. viridis X S. italica, mapping mutations through bulk segregant analysis and creating higher order mutants for genetic analysis.
23 Related JoVE Articles!
Mass Spectrometric Analysis of Glycosphingolipid Antigens
Institutions: Rice University , University of Texas MD Anderson Cancer Center , University of Texas MD Anderson Cancer Center , University of Texas Graduate School of Biological Sciences at Houston.
Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition1-2
. They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Galα4Galβ4Glcβ1Cer) and isoglobotriaosylceramide (Galα3Galβ4Glcβ1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions3-4
. In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases5
. The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions6
Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells7-9
. This method is based on extraction and chemical modification (permethylation, see below Figure 5A
, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids10-15
, followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectrometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present9
Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5
). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.
Immunology, Issue 74, Biochemistry, Molecular Biology, Cellular Biology, Structural Biology, Medicine, Genetics, Proteomics, Proteins, Glycomics, Functional glycomics, glycosphingolipids, GSLs, antigens, natural killer T cells, MALDI-TOF mass spectrometry, LTQ ion trap mass spectrometer, mass spectrometry, glycolipids, lipids
Preparation and Use of Samarium Diiodide (SmI2) in Organic Synthesis: The Mechanistic Role of HMPA and Ni(II) Salts in the Samarium Barbier Reaction
Institutions: Lehigh University .
Although initially considered an esoteric reagent, SmI2
has become a common tool for synthetic organic chemists. SmI2
is generated through the addition of molecular iodine to samarium metal in THF.1,2-3
It is a mild and selective single electron reductant and its versatility is a result of its ability to initiate a wide range of reductions including C-C bond-forming and cascade or sequential reactions. SmI2
can reduce a variety of functional groups including sulfoxides and sulfones, phosphine oxides, epoxides, alkyl and aryl halides, carbonyls, and conjugated double bonds.2-12
One of the fascinating features of SmI-2
-mediated reactions is the ability to manipulate the outcome of reactions through the selective use of cosolvents or additives. In most instances, additives are essential in controlling the rate of reduction and the chemo- or stereoselectivity of reactions.13-14
Additives commonly utilized to fine tune the reactivity of SmI2
can be classified into three major groups: (1) Lewis bases (HMPA, other electron-donor ligands, chelating ethers, etc.), (2) proton sources (alcohols, water etc.), and (3) inorganic additives (Ni(acac)2
Understanding the mechanism of SmI2
reactions and the role of the additives enables utilization of the full potential of the reagent in organic synthesis. The Sm-Barbier reaction is chosen to illustrate the synthetic importance and mechanistic role of two common additives: HMPA and Ni(II) in this reaction. The Sm-Barbier reaction is similar to the traditional Grignard reaction with the only difference being that the alkyl halide, carbonyl, and Sm reductant are mixed simultaneously in one pot.1,15
Examples of Sm-mediated Barbier reactions with a range of coupling partners have been reported,1,3,7,10,12
and have been utilized in key steps of the synthesis of large natural products.16,17
Previous studies on the effect of additives on SmI2
reactions have shown that HMPA enhances the reduction potential of SmI2
by coordinating to the samarium metal center, producing a more powerful,13-14,18
sterically encumbered reductant19-21
and in some cases playing an integral role in post electron-transfer steps facilitating subsequent bond-forming events.22
In the Sm-Barbier reaction, HMPA has been shown to additionally activate the alkyl halide by forming a complex in a pre-equilibrium step.23
Ni(II) salts are a catalytic additive used frequently in Sm-mediated transformations.24-27
Though critical for success, the mechanistic role of Ni(II) was not known in these reactions. Recently it has been shown that SmI2
reduces Ni(II) to Ni(0), and the reaction is then carried out through organometallic Ni(0) chemistry.28
These mechanistic studies highlight that although the same Barbier product is obtained, the use of different additives in the SmI2
reaction drastically alters the mechanistic pathway of the reaction. The protocol for running these SmI2
-initiated reactions is described.
Chemistry, Issue 72, Organic Chemistry, Chemical Engineering, Biochemistry, Samarium diiodide, Sml2, Samarium-Barbier Reaction, HMPA, hexamethylphosphoramide, Ni(II), Nickel(II) acetylacetonate, nickel, samarium, iodine, additives, synthesis, catalyst, reaction, synthetic organic chemistry
High-Throughput Measurement and Classification of Organic P in Environmental Samples
Institutions: University of Vermont.
Many types of organic phosphorus (P) molecules exist in environmental samples1
. Traditional P measurements do not detect these organic P compounds since they do not react with colorimetric reagents2,3
. Enzymatic hydrolysis (EH) is an emerging method for accurately characterizing organic P forms in environmental samples4,5
. This method is only trumped in accuracy by Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy (31
P-NMR) -a method that is expensive and requires specialized technical training6
. We have adapted an enzymatic hydrolysis method capable of measuring three classes of phosphorus (monoester P, diester P and inorganic P) to a microplate reader system7
. This method provides researchers with a fast, accurate, affordable and user-friendly means to measure P species in soils, sediments, manures and, if concentrated, aquatic samples. This is the only high-throughput method for measuring the forms and enzyme-lability of organic P that can be performed in a standard laboratory. The resulting data provides insight to scientists studying system nutrient content and eutrophication potential.
Microbiology, Issue 52, phosphorus, enzymatic-hydrolysis, soil, manure, phosphatase, phytic acid, NaOH-EDTA, organophosphates, molybdate, organic P
Concurrent Quantitative Conductivity and Mechanical Properties Measurements of Organic Photovoltaic Materials using AFM
Institutions: Argonne National Laboratory, University of Chicago.
Organic photovoltaic (OPV) materials are inherently inhomogeneous at the nanometer scale. Nanoscale inhomogeneity of OPV materials affects performance of photovoltaic devices. Thus, understanding of spatial variations in composition as well as electrical properties of OPV materials is of paramount importance for moving PV technology forward.1,2
In this paper, we describe a protocol for quantitative measurements of electrical and mechanical properties of OPV materials with sub-100 nm resolution. Currently, materials properties measurements performed using commercially available AFM-based techniques (PeakForce, conductive AFM) generally provide only qualitative information. The values for resistance as well as Young's modulus measured using our method on the prototypical ITO/PEDOT:PSS/P3HT:PC61
BM system correspond well with literature data. The P3HT:PC61
BM blend separates onto PC61
BM-rich and P3HT-rich domains. Mechanical properties of PC61
BM-rich and P3HT-rich domains are different, which allows for domain attribution on the surface of the film. Importantly, combining mechanical and electrical data allows for correlation of the domain structure on the surface of the film with electrical properties variation measured through the thickness of the film.
Materials Science, Issue 71, Nanotechnology, Mechanical Engineering, Electrical Engineering, Computer Science, Physics, electrical transport properties in solids, condensed matter physics, thin films (theory, deposition and growth), conductivity (solid state), AFM, atomic force microscopy, electrical properties, mechanical properties, organic photovoltaics, microengineering, photovoltaics
One-step Metabolomics: Carbohydrates, Organic and Amino Acids Quantified in a Single Procedure
Institutions: Saint Louis University School of Medicine.
Every infant born in the US is now screened for up to 42 rare genetic disorders called "inborn errors of metabolism". The screening method is based on tandem mass spectrometry and quantifies acylcarnitines as a screen for organic acidemias and also measures amino acids. All states also perform enzymatic testing for carbohydrate disorders such as galactosemia. Because the results can be non-specific, follow-up testing of positive results is required using a more definitive method. The present report describes the "urease" method of sample preparation for inborn error screening. Crystalline urease enzyme is used to remove urea from body fluids which permits most other water-soluble metabolites to be dehydrated and derivatized for gas chromatography in a single procedure. Dehydration by evaporation in a nitrogen stream is facilitated by adding acetonitrile and methylene chloride. Then, trimethylsilylation takes place in the presence of a unique catalyst, triethylammonium trifluoroacetate. Automated injection and chromatography is followed by macro-driven custom quantification of 192 metabolites and semi-quantification of every major component using specialized libraries of mass spectra of TMS derivatized biological compounds. The analysis may be performed on the widely-used Chemstation platform using the macros and libraries available from the author. In our laboratory, over 16,000 patient samples have been analyzed using the method with a diagnostic yield of about 17%--that is, 17% of the samples results reveal findings that should be acted upon by the ordering physician. Included in these are over 180 confirmed inborn errors, of which about 38% could not have been diagnosed using previous methods.
Biochemistry, Issue 40, metabolomics, gas chromatography/mass spectrometry, GC/MS, inborn errors, vitamin deficiency, BNA analyses, carbohydrate, amino acid, organic acid, urease
Improved In-gel Reductive β-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry
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
Ultrahigh Density Array of Vertically Aligned Small-molecular Organic Nanowires on Arbitrary Substrates
Institutions: University of Alberta.
In recent years π-conjugated organic semiconductors have emerged as the active material in a number of diverse applications including large-area, low-cost displays, photovoltaics, printable and flexible electronics and organic spin valves. Organics allow (a) low-cost, low-temperature processing and (b) molecular-level design of electronic, optical and spin transport characteristics. Such features are not readily available for mainstream inorganic semiconductors, which have enabled organics to carve a niche in the silicon-dominated electronics market. The first generation of organic-based devices has focused on thin film geometries, grown by physical vapor deposition or solution processing. However, it has been realized that organic nanostructures
can be used to enhance performance of above-mentioned applications and significant effort has been invested in exploring methods for organic nanostructure fabrication.
A particularly interesting class of organic nanostructures is the one in which vertically oriented organic nanowires, nanorods or nanotubes are organized in a well-regimented, high-density array
. Such structures are highly versatile and are ideal morphological architectures for various applications such as chemical sensors, split-dipole nanoantennas, photovoltaic devices with radially heterostructured "core-shell" nanowires, and memory devices with a cross-point geometry. Such architecture is generally realized by a template-directed approach. In the past this method has been used to grow metal and inorganic semiconductor nanowire arrays. More recently π-conjugated polymer nanowires have been grown within nanoporous templates. However, these approaches have had limited success in growing nanowires of technologically important π-conjugated small molecular weight organics
, such as tris-8-hydroxyquinoline aluminum (Alq3
), rubrene and methanofullerenes, which are commonly used in diverse areas including organic displays, photovoltaics, thin film transistors and spintronics.
Recently we have been able to address the above-mentioned issue by employing a novel "centrifugation-assisted" approach. This method therefore broadens the spectrum of organic materials that can be patterned in a vertically ordered nanowire array. Due to the technological importance of Alq3
, rubrene and methanofullerenes, our method can be used to explore how the nanostructuring of these materials affects the performance of aforementioned organic devices. The purpose of this article is to describe the technical details of the above-mentioned protocol, demonstrate how this process can be extended to grow small-molecular organic nanowires on arbitrary substrates
and finally, to discuss the critical steps, limitations, possible modifications, trouble-shooting and future applications.
Physics, Issue 76, Electrical Engineering, Chemistry, Chemical Engineering, Nanotechnology, nanodevices (electronic), semiconductor devices, solid state devices, thin films (theory, deposition and growth), crystal growth (general), Organic semiconductors, small molecular organics, organic nanowires, nanorods and nanotubes, bottom-up nanofabrication, electrochemical self-assembly, anodic aluminum oxide (AAO), template-assisted synthesis of nanostructures, Raman spectrum, field emission scanning electron microscopy, FESEM
Synthesis and Characterization of Functionalized Metal-organic Frameworks
Institutions: Northwestern University, Warsaw University of Technology, King Abdulaziz University.
Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and technological applications. Their signature property is their ultrahigh porosity, which however imparts a series of challenges when it comes to both constructing them and working with them. Securing desired MOF chemical and physical functionality by linker/node assembly into a highly porous framework of choice can pose difficulties, as less porous and more thermodynamically stable congeners (e.g.
, other crystalline polymorphs, catenated analogues) are often preferentially obtained by conventional synthesis methods. Once the desired product is obtained, its characterization often requires specialized techniques that address complications potentially arising from, for example, guest-molecule loss or preferential orientation of microcrystallites. Finally, accessing the large voids inside the MOFs for use in applications that involve gases can be problematic, as frameworks may be subject to collapse during removal of solvent molecules (remnants of solvothermal synthesis). In this paper, we describe synthesis and characterization methods routinely utilized in our lab either to solve or circumvent these issues. The methods include solvent-assisted linker exchange, powder X-ray diffraction in capillaries, and materials activation (cavity evacuation) by supercritical CO2
drying. Finally, we provide a protocol for determining a suitable pressure region for applying the Brunauer-Emmett-Teller analysis to nitrogen isotherms, so as to estimate surface area of MOFs with good accuracy.
Chemistry, Issue 91, Metal-organic frameworks, porous coordination polymers, supercritical CO2 activation, crystallography, solvothermal, sorption, solvent-assisted linker exchange
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
Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm
Institutions: Saint Louis University.
is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA methyltransferases is viable, thus providing a system to study how loss of DNA methylation in a genome affects growth and development. Imprinting refers to differential expression of maternal and paternal alleles and plays an important role in reproduction development in both mammal and plants. DNA methylation is critical for determining whether the maternal or paternal alleles of an imprinted gene is expressed or silenced. In flowering plants, there is a double fertilization event in reproduction: one sperm cell fertilizes the egg cell to form embryo and a second sperm fuses with the central cell to give rise to endosperm. Endosperm is the tissue where imprinting occurs in plants. MEDEA
, a SET domain Polycomb group gene, and FWA
, a transcription factor regulating flowering, are the first two genes shown to be imprinted in endosperm and their expression is controlled by DNA methylation and demethylation in plants. In order to determine imprinting status of a gene and methylation pattern in endosperm, we need to be able to isolate endosperm first. Since seed is tiny in Arabidopsis
, it remains challenging to isolate Arabidopsis
endosperm and examine its methylation. In this video protocol, we report how to conduct a genetic cross, to isolate endosperm tissue from seeds, and to determine the methylation status by bisulfite sequencing.
Plant Biology, Issue 47, DNA methylation, imprinting, bisulfite sequencing, endosperm, Arabidopsis
Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling
Institutions: Colorado State University, USDA-ARS, Colorado State University.
Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2
fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as 13
C with 15
O or 2
H has the potential to reveal even more information about complex stoichiometric relationships during biogeochemical transformations. Isotope labeled plant material has been used in various studies of litter decomposition and soil organic matter formation1-4
. From these and other studies, however, it has become apparent that structural components of plant material behave differently than metabolic components (i.e
. leachable low molecular weight compounds) in terms of microbial utilization and long-term carbon storage5-7
. The ability to study structural and metabolic components separately provides a powerful new tool for advancing the forefront of ecosystem biogeochemical studies. Here we describe a method for producing 13
C and 15
N labeled plant material that is either uniformly labeled throughout the plant or differentially labeled in structural and metabolic plant components.
Here, we present the construction and operation of a continuous 13
C and 15
N labeling chamber that can be modified to meet various research needs. Uniformly labeled plant material is produced by continuous labeling from seedling to harvest, while differential labeling is achieved by removing the growing plants from the chamber weeks prior to harvest. Representative results from growing Andropogon gerardii
Kaw demonstrate the system's ability to efficiently label plant material at the targeted levels. Through this method we have produced plant material with a 4.4 atom%13
C and 6.7 atom%15
N uniform plant label, or material that is differentially labeled by up to 1.29 atom%13
C and 0.56 atom%15
N in its metabolic and structural components (hot water extractable and hot water residual components, respectively). Challenges lie in maintaining proper temperature, humidity, CO2
concentration, and light levels in an airtight 13
atmosphere for successful plant production. This chamber description represents a useful research tool to effectively produce uniformly or differentially multi-isotope labeled plant material for use in experiments on ecosystem biogeochemical cycling.
Environmental Sciences, Issue 83, 13C, 15N, plant, stable isotope labeling, Andropogon gerardii, metabolic compounds, structural compounds, hot water extraction
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
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
Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study
Institutions: RWTH Aachen University, Fraunhofer Gesellschaft.
Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.
Bioengineering, Issue 83, design of experiments (DoE), transient protein expression, plant-derived biopharmaceuticals, promoter, 5'UTR, fluorescent reporter protein, model building, incubation conditions, monoclonal antibody
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
Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology
Institutions: San Diego State University, University of California San Diego.
Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.
Environmental Sciences, Issue 93, dissolved organic carbon, particulate organic matter, nutrients, DAPI, SYBR, microbial metagenomics, viral metagenomics, marine environment
Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy
Institutions: University of Maine.
Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.
Basic Protocol, Issue 82, Microscopy, Super-resolution imaging, Multicolor, single molecule, FPALM, Localization microscopy, fluorescent proteins
High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities
Institutions: Colorado State University, Oak Ridge National Laboratory, University of Colorado.
Microbes in soils and other environments produce extracellular enzymes to depolymerize and hydrolyze organic macromolecules so that they can be assimilated for energy and nutrients. Measuring soil microbial enzyme activity is crucial in understanding soil ecosystem functional dynamics. The general concept of the fluorescence enzyme assay is that synthetic C-, N-, or P-rich substrates bound with a fluorescent dye are added to soil samples. When intact, the labeled substrates do not fluoresce. Enzyme activity is measured as the increase in fluorescence as the fluorescent dyes are cleaved from their substrates, which allows them to fluoresce. Enzyme measurements can be expressed in units of molarity or activity. To perform this assay, soil slurries are prepared by combining soil with a pH buffer. The pH buffer (typically a 50 mM sodium acetate or 50 mM Tris buffer), is chosen for the buffer's particular acid dissociation constant (pKa) to best match the soil sample pH. The soil slurries are inoculated with a nonlimiting amount of fluorescently labeled (i.e.
C-, N-, or P-rich) substrate. Using soil slurries in the assay serves to minimize limitations on enzyme and substrate diffusion. Therefore, this assay controls for differences in substrate limitation, diffusion rates, and soil pH conditions; thus detecting potential enzyme activity rates as a function of the difference in enzyme concentrations (per sample).
Fluorescence enzyme assays are typically more sensitive than spectrophotometric (i.e.
colorimetric) assays, but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light; so caution is required when handling fluorescent substrates. Likewise, this method only assesses potential enzyme activities under laboratory conditions when substrates are not limiting. Caution should be used when interpreting the data representing cross-site comparisons with differing temperatures or soil types, as in situ
soil type and temperature can influence enzyme kinetics.
Environmental Sciences, Issue 81, Ecological and Environmental Phenomena, Environment, Biochemistry, Environmental Microbiology, Soil Microbiology, Ecology, Eukaryota, Archaea, Bacteria, Soil extracellular enzyme activities (EEAs), fluorometric enzyme assays, substrate degradation, 4-methylumbelliferone (MUB), 7-amino-4-methylcoumarin (MUC), enzyme temperature kinetics, soil
Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana
Institutions: Fraunhofer USA Center for Molecular Biotechnology.
-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana
plants with Agrobacteria
carrying launch vectors. Optimization of Agrobacterium
cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana
, N. excelsiana
× N. excelsior
) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium
harboring pBID4-GFP (Tobacco mosaic virus
-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium
laboratory strain GV3101 showed the highest protein production compared to Agrobacteria
laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria
strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana
resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).
Plant Biology, Issue 86, Agroinfiltration, Nicotiana benthamiana, transient protein production, plant-based expression, viral vector, Agrobacteria
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
Choice and No-Choice Assays for Testing the Resistance of A. thaliana to Chewing Insects
Institutions: Cornell University.
Larvae of the small white cabbage butterfly are a pest in agricultural settings. This caterpillar species feeds from plants in the cabbage family, which include many crops such as cabbage, broccoli, Brussel sprouts etc. Rearing of the insects takes place on cabbage plants in the greenhouse. At least two cages are needed for the rearing of Pieris rapae. One for the larvae and the other to contain the adults, the butterflies. In order to investigate the role of plant hormones and toxic plant chemicals in resistance to this insect pest, we demonstrate two experiments. First, determination of the role of jasmonic acid (JA - a plant hormone often indicated in resistance to insects) in resistance to the chewing insect Pieris rapae. Caterpillar growth can be compared on wild-type and mutant plants impaired in production of JA. This experiment is considered "No Choice", because larvae are forced to subsist on a single plant which synthesizes or is deficient in JA. Second, we demonstrate an experiment that investigates the role of glucosinolates, which are used as oviposition (egg-laying) signals. Here, we use WT and mutant Arabidopsis impaired in glucosinolate production in a "Choice" experiment in which female butterflies are allowed to choose to lay their eggs on plants of either genotype. This video demonstrates the experimental setup for both assays as well as representative results.
Plant Biology, Issue 15, Annual Review, Plant Resistance, Herbivory, Arabidopsis thaliana, Pieris rapae, Caterpillars, Butterflies, Jasmonic Acid, Glucosinolates
Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid
Institutions: EMBL Heidelberg.
In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.
Basic Protocols, Issue 30, SDS-PAGE, Coomassie staining, Protein detection, Protein staining
Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example
Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1
. Antixenosis is the degree to which the plant is avoided when the herbivore is able to select other plants2
. Antibiosis is the degree to which the plant affects the fitness of the herbivore feeding on it1
.Tolerance is the degree to which the plant can withstand or repair damage caused by the herbivore, without compromising the herbivore's growth and reproduction1
. The durability of herbivore resistance in an agricultural setting depends to a great extent on the resistance mechanism favored during crop breeding efforts3
We demonstrate a no-choice experiment designed to estimate the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria
spp. Several species of African grasses of the genus Brachiaria
are valuable forage and pasture plants in the Neotropics, but they can be severely challenged by several native species of spittlebugs (Hemiptera: Cercopidae)4
.To assess their resistance to spittlebugs, plants are vegetatively-propagated by stem cuttings and allowed to grow for approximately one month, allowing the growth of superficial roots on which spittlebugs can feed. At that point, each test plant is individually challenged with six spittlebug eggs near hatching. Infestations are allowed to progress for one month before evaluating plant damage and insect survival. Scoring plant damage provides an estimate of tolerance while scoring insect survival provides an estimate of antibiosis. This protocol has facilitated our plant breeding objective to enhance spittlebug resistance in commercial brachiariagrases5
Plant Biology, Issue 52, host plant resistance, antibiosis, antixenosis, tolerance, Brachiaria, spittlebugs