Split luciferase complementation assay to detect regulated protein-protein interactions in rice protoplasts in a large-scale format

© 2014 Fujikawa et al. Background: The rice interactome, in which a network of protein-protein interactions has been elucidated in rice, is a useful resource to identify functional modules of rice signal transduction pathways. Protein-protein interactions occur in cells in two ways, constitutive and regulative. While a yeast-based high-throughput method has been widely used to identify the constitutive interactions, a method to detect the regulated interactions is rarely developed for a large-scale analysis. Results: A split luciferase complementation assay was applied to detect the regulated interactions in rice. A transformation method of rice protoplasts in a 96-well plate was first established for a large-scale analysis. In addition, an antibody that specifically recognizes a carboxyl-terminal fragment of Renilla luciferase was newly developed. A pair of antibodies that recognize amino- and carboxyl- terminal fragments of Renilla luciferase, respectively, was then used to monitor quality and quantity of interacting recombinant-proteins accumulated in the cells. For a proof-of-concept, the method was applied to detect the gibberellin-dependent interaction between GIBBERELLIN INSENSITIVE DWARF1 and SLENDER RICE 1. Conclusions: A method to detect regulated protein-protein interactions was developed towards establishment of the rice interactome

A Reversible Defect in Natural Killer T Cell Function Characterizes the Progression of Premalignant to Malignant Multiple Myeloma

We studied the function of antitumor T and natural killer T (NKT) cells from the blood and tumor bed in 23 patients with premalignant gammopathy, nonprogressive myeloma, or progressive multiple myeloma. We show that antitumor killer T cells can be detected in patients with both progressive or nonprogressive myeloma. Vα24+Vβ11+ invariant NKT cells are detectable in the blood and tumor bed of all cohorts. However, freshly isolated NKT cells from both the blood and tumor bed of patients with progressive disease, but not nonprogressive myeloma or premalignant gammopathy, have a marked deficiency of ligand-dependent interferon-γ production. This functional defect can be overcome in vitro using dendritic cells pulsed with the NKT ligand, α-galactosylceramide (α-GalCer). Fresh myeloma cells express CD1d, and can be efficiently killed by autologous NKT cells. We hypothesize that presentation of tumor derived glycolipids by myeloma cells leads to NKT dysfunction in vivo. These data demonstrate that clinical progression in patients with monoclonal gammopathies is associated with an acquired but potentially reversible defect in NKT cell function and support the possibility that these innate lymphocytes play a role in controlling the malignant growth of this incurable B cell tumor in patients

A post-labeling method for multiplexed and multicolored genotyping analysis of SSR, indel and SNP markers in single tube with bar-coded split tag (BStag)

<p>Abstract</p> <p>Background</p> <p>Genotyping analysis using capillary DNA sequencing with fluorescently labeled primer pairs obtained by polymerase chain reaction (PCR) is widely used, but is expensive. The post-PCR labeling method using fluorescently labeled short oligonucleotides and nested PCR of the amplified product obtained from unlabeled primer pairs is a simple and inexpensive alternative. However, previously reported protocols often produced spurious peaks or inconsistent amplification under multiplexed analysis as a result of simultaneous progress of both the amplification and labeling reactions and local homology of the attached tag sequence.</p> <p>Results</p> <p>A set of 16 bp-long oligonucleotide sequences termed bar-coded split tag (BStag), comprising a common basal region, a three-nucleotide 'bar-code' sequence, and a mismatched nucleotide at the middle position were designed for selective post-PCR labeling. The BStag was attached at the 5' end of the forward primer of interest. The melting temperature of the BStag was low enough to separate the labeling reaction from initial PCR amplification, and each sequence was minimally divergent but maintained maximum selectivity. Post-PCR labeling of the amplified product was achieved by extending for three cycles at a lower annealing temperature after the conventional amplification program with the appropriate fluorescently labeled BStag primer. No amplification was confirmed with BStag primers for 12 plant species. The electropherogram of the labeled product obtained using this method was consistent with that of prelabeled primer, except for their apparent size.</p> <p>Conclusions</p> <p>BStag enabled multiplexed post-PCR labeling of simple sequence repeat or insertion/deletion markers with different dyes in a single tube. BStag in conjunction with locus specific oligo and allele specific oligo was also useful for single nucleotide polymorphism analysis. The labeling protocol was simple and no additional operation was required. Single-tube multiplexed post-PCR labeling is useful for a wide variety of genotyping studies with maximal flexibility and minimal costs.</p

Oxygenated Aromatic Compounds are Important Precursors of Secondary Organic Aerosol in Biomass Burning Emissions

Biomass burning is the largest combustion-related source of volatile organic compounds (VOCs) to the atmosphere. We describe the development of a state-of-the-science model to simulate the photochemical formation of secondary organic aerosol (SOA) from biomass-burning emissions observed in dry (RH <20%) environmental chamber experiments. The modeling is supported by (i) new oxidation chamber measurements, (ii) detailed concurrent measurements of SOA precursors in biomass-burning emissions, and (iii) development of SOA parameters for heterocyclic and oxygenated aromatic compounds based on historical chamber experiments. We find that oxygenated aromatic compounds, including phenols and methoxyphenols, account for slightly less than 60% of the SOA formed and help our model explain the variability in the organic aerosol mass (R² = 0.68) and O/C (R² = 0.69) enhancement ratios observed across 11 chamber experiments. Despite abundant emissions, heterocyclic compounds that included furans contribute to ∼20% of the total SOA. The use of pyrolysis-temperature-based or averaged emission profiles to represent SOA precursors, rather than those specific to each fire, provide similar results to within 20%. Our findings demonstrate the necessity of accounting for oxygenated aromatics from biomass-burning emissions and their SOA formation in chemical mechanisms

Plasma Activation of Microplates Optimized for One-Step Reagent-Free Immobilization of DNA and Protein

Activated microplates are widely used in biological assays and cell culture to immobilize biomolecules, either through passive physical adsorption or covalent cross-linking. Covalent attachment gives greater stability in complex biological mixtures. However, current multistep chemical activation methods add complexity and cost, require specific functional groups, and can introduce cytotoxic chemicals that affect downstream cellular applications. Here, we show a method for one-step linker-free activation of microplates by energetic ions from plasma for covalent immobilization of DNA and protein. Two types of energetic ion plasma treatment were shown to be effective: plasma immersion ion implantation (PIII) and plasma-activated coating (PAC). This is the first time that PIII and PAC have been reported in microwell plates with nonflat geometry. We confirm that the plasma treatment generates radical-activated surfaces at the bottom of wells despite potential shadowing from the walls. Comprehensive surface characterization studies were used to compare the PIII and PAC microplate surface composition, wettability, radical density, optical properties, stability, and biomolecule immobilization density. PAC plates were found to have more nitrogen and lower radical density and were more hydrophobic and more stable over 3 months than PIII plates. Optimal conditions were obtained for high-density DNA (PAC, 0 or 21% nitrogen, pH 3–4) and streptavidin (PAC, 21% nitrogen, pH 5–7) binding while retaining optical properties required for typical high-throughput biochemical microplate assays, such as low autofluorescence and high transparency. DNA hybridization and protein activity of immobilized molecules were confirmed. We show that PAC activation allows for high-density covalent immobilization of functional DNA and protein in a single step on both 96- and 384-well plates without specific linker chemistry. These microplates could be used in the future to bind other user-selected ligands in a wide range of applications, for example, for solid phase polymerase chain reaction and stem cell culture and differentiation

The nitrogen budget of laboratory-simulated western US wildfires during the FIREX 2016 Fire Lab study

Reactive nitrogen (Nr, defined as all nitrogencontaining compounds except for N2 and N2O) is one of the most important classes of compounds emitted from wildfire, as Nr impacts both atmospheric oxidation processes and particle formation chemistry. In addition, several Nr compounds can contribute to health impacts from wildfires. Understanding the impacts of wildfire on the atmosphere requires a thorough description of Nr emissions. Total reactive nitrogen was measured by catalytic conversion to NO and detection by NO-O3 chemiluminescence together with individual Nr species during a series of laboratory fires of fuels characteristic of western US wildfires, conducted as part of the FIREX Fire Lab 2016 study. Data from 75 stack fires were analyzed to examine the systematics of nitrogen emissions. The measured Nr = total-carbon ratios averaged 0.37 % for fuels characteristic of western North America, and these gas-phase emissions were compared with fuel and residue N=C ratios and mass to estimate that a mean (±SD) of 0.68 (±0:14) of fuel nitrogen was emitted as N2 and N2O. The Nr detected as speciated individual compounds included the following: nitric oxide (NO), nitrogen dioxide (NO2), nitrous acid (HONO), isocyanic acid (HNCO), hydrogen cyanide (HCN), ammonia (NH3), and 44 nitrogen-containing volatile organic compounds (NVOCs). The sum of these measured individual Nr compounds averaged 84.8 (±9:8) % relative to the total Nr, and much of the 15.2 % unaccounted Nr is expected to be particle-bound species, not included in this analysis. A number of key species, e.g., HNCO, HCN, and HONO, were confirmed not to correlate with only flaming or with only smoldering combustion when using modified combustion efficiency, MCE D CO2=.CO C CO2/, as a rough indicator. However, the systematic variations in the abundance of these species relative to other nitrogen-containing species were successfully modeled using positive matrix factorization (PMF). Three distinct factors were found for the emissions from combined coniferous fuels: a combustion factor (Comb-N) (800-1200 °C) with emissions of the inorganic compounds NO, NO2, and HONO, and a minor contribution from organic nitro compounds (R-NO2); a high-temperature pyrolysis factor (HT-N) (500-800 °C) with emissions of HNCO, HCN, and nitriles; and a low-temperature pyrolysis factor (LT-N) (\u3c 500 °C) with mostly ammonia and NVOCs. The temperature ranges specified are based on known com bustion and pyrolysis chemistry considerations. The mix of emissions in the PMF factors from chaparral fuels (manzanita and chamise) had a slightly different composition: the Comb-N factor was also mostly NO, with small amounts of HNCO, HONO, and NH3; the HT-N factor was dominated by NO2 and had HONO, HCN, and HNCO; and the LT-N factor was mostly NH3 with a slight amount of NO contributing. In both cases, the Comb-N factor correlated best with CO2 emission, while the HT-N factors from coniferous fuels correlated closely with the high-temperature VOC factors recently reported by Sekimoto et al. (2018), and the LT-N had some correspondence to the LT-VOC factors. As a consequence, CO2 is recommended as a marker for combustion Nr emissions, HCN is recommended as a marker for HT-N emissions, and the family NH3 = particle ammonium is recommended as a marker for LT-N emissions

OH chemistry of non-methane organic gases (NMOGs) emitted from laboratory and ambient biomass burning smoke: Evaluating the influence of furans and oxygenated aromatics on ozone and secondary NMOG formation

Chamber oxidation experiments conducted at the Fire Sciences Laboratory in 2016 are evaluated to identify important chemical processes contributing to the hydroxy radical (OH) chemistry of biomass burning non-methane organic gases (NMOGs). Based on the decay of primary carbon measured by proton transfer reaction time-of-flight mass spectrometry (PTR-ToF-MS), it is confirmed that furans and oxygenated aromatics are among the NMOGs emitted from western United States fuel types with the highest reactivities towards OH. The oxidation processes and formation of secondary NMOG masses measured by PTR-ToF-MS and iodide-clustering time-of-flight chemical ionization mass spectrometry (I-CIMS) is interpreted using a box model employing a modified version of the Master Chemical Mechanism (v. 3.3.1) that includes the OH oxidation of furan, 2-methylfuran, 2,5-dimethylfuran, furfural, 5-methylfurfural, and guaiacol. The model supports the assignment of major PTR-ToF-MS and I-CIMS signals to a series of anhydrides and hydroxy furanones formed primarily through furan chemistry. This mechanism is applied to a Lagrangian box model used previously to model a real biomass burning plume. The customized mechanism reproduces the decay of furans and oxygenated aromatics and the formation of secondary NMOGs, such as maleic anhydride. Based on model simulations conducted with and without furans, it is estimated that furans contributed up to 10% of ozone and over 90% of maleic anhydride formed within the first 4h of oxidation. It is shown that maleic anhydride is present in aline page14876 /\u3e biomass burning plume transported over several days, which demonstrates the utility of anhydrides as markers for aged biomass burning plumes

Split luciferase complementation assay to detect regulated protein-protein interactions in rice protoplasts in a large-scale format

BACKGROUND: The rice interactome, in which a network of protein-protein interactions has been elucidated in rice, is a useful resource to identify functional modules of rice signal transduction pathways. Protein-protein interactions occur in cells in two ways, constitutive and regulative. While a yeast-based high-throughput method has been widely used to identify the constitutive interactions, a method to detect the regulated interactions is rarely developed for a large-scale analysis. RESULTS: A split luciferase complementation assay was applied to detect the regulated interactions in rice. A transformation method of rice protoplasts in a 96-well plate was first established for a large-scale analysis. In addition, an antibody that specifically recognizes a carboxyl-terminal fragment of Renilla luciferase was newly developed. A pair of antibodies that recognize amino- and carboxyl- terminal fragments of Renilla luciferase, respectively, was then used to monitor quality and quantity of interacting recombinant-proteins accumulated in the cells. For a proof-of-concept, the method was applied to detect the gibberellin-dependent interaction between GIBBERELLIN INSENSITIVE DWARF1 and SLENDER RICE 1. CONCLUSIONS: A method to detect regulated protein-protein interactions was developed towards establishment of the rice interactome