RESUMO
Wild and managed bees are critical for the stability of trophic webs, angiosperm reproduction, and agricultural productivity. Unfortunately, as many as 40% of crop pollinators are in a steep decline due to habitat loss and exposure to agrochemicals. Pyrethroids, neonicotinoids, and macrocyclic lactones are among the many agrochemicals toxic to pollinating insects that are used extensively in industrial beef cattle feeding operations throughout the world. Fugitive feedyard particulate matter (PM) transports agrochemicals into the surrounding environs. To determine the impact of agrochemical-laden feedyard particulate matter on bee pollinators, we conducted in situ experiments wherein honeybees and mason bees were placed downwind and upwind of feedyards (N = 40). Concurrent, colocated total suspended particulate matter samples contained multiple insecticides and parasiticides including pyrethroids, neonicotinoids, and macrocyclic lactones, in significantly higher concentrations downwind of feedyards (bifenthrin, 8.45 ± 4.92; permethrin, 1032.34 ± 740.76; clothianidin, 3.61 ± 1.48; imidacloprid, 73.32 ± 47.52; thiamethoxam, 5.81 ± 3.16; abamectin, 0.45 ± 0.29; ivermectin, 8.88 ± 5.06 ng/g). Honeybees and mason bees sited downwind of feedyards always experienced higher mortality than those correspondingly sited upwind, and male mason bees experienced significantly higher mortality compared to females when both were sited downwind. Bees occurring downwind of beef cattle feedyards for 1 h are 232-260% more likely to die than those occurring upwind. Thus, agrochemicals used on and emitted from beef cattle feedyards are significant threats to bee pollinators.
Assuntos
Inseticidas , Praguicidas , Piretrinas , Masculino , Abelhas , Animais , Bovinos , Praguicidas/toxicidade , Praguicidas/análise , Material Particulado/análise , Neonicotinoides , Inseticidas/toxicidade , Agroquímicos , LactonasRESUMO
Environmental transport and deposition of particulate matter (PM) associated with toxic chemicals has begun to receive attention as a source of risk to pollinators. For example, dust arising from manipulations of insecticide-treated seed has potential to exert toxic effects among non-target insects. Similarly, synthetic steroid growth promoters, antibiotics and multiple insecticides and parasiticides detected in fugitive beef cattle feedyard PM may also negatively impact pollinators since many of these chemicals have been detected on wildflowers and pollinators collected near beef cattle feedyards. Therefore, there is a need to assess risk to pollinators posed by deposition of agrochemical-laden PM, both in the field and the laboratory. Unfortunately, established laboratory methods for simulating PM exposure or toxicity associated with contaminated PM are few and highly situation-specific. Herein we describe development and use of a PM circulation system that can be employed to evaluate toxicity of agrochemical-contaminated PM in the laboratory under controlled conditions. Two model organisms (honeybees (Apis mellifera) and mason bees (Osmia lignaria)) were exposed to agrochemical-free PM in the circulator system, and post-exposure mortality was compared with controls. No significant differences in mortality between exposed and control bees were observed. Next, honeybees and mason bees were exposed to PM spiked with an insecticide known to exert toxic effects to pollinators (thiamethoxam). Bees experienced significantly higher mortality when exposed to thiamethoxam-laden PM at environmentally relevant concentrations as compared to bees exposed to agrochemical-free PM. These results confirm the validity of these methods for use in controlled laboratory PM toxicity tests and offer a source of positive and negative control groups for laboratory and field experiments examining exposure of pollinators to potentially toxic agrochemical-laden PM. This method facilitates generation of more realistic toxicity data than standard contact toxicity tests when pollinator exposure scenarios involve particulate-based agrochemicals or other toxic chemicals.
Assuntos
Inseticidas , Bovinos , Abelhas , Animais , Inseticidas/toxicidade , Tiametoxam , Agroquímicos/toxicidade , Material Particulado/toxicidade , Insetos , NeonicotinoidesRESUMO
Characterization of DNA at solid/liquid interfaces remains a challenge because most surface-sensitive techniques are unable to provide quantitative insight into the base content, length, or structure. Surface-enhanced Raman scattering measurements of DNA hybridization on plasmonic-metal substrates have been used to overcome small Raman-scattering cross-sections; however, surface-enhanced Raman spectroscopy measurements are not generally quantitative due to the fall-off in the scattering signal with the decay of the electric field enhancement from the surface, which also limits the length of oligonucleotides that can be investigated. In this work, we introduce an experimental methodology in which confocal Raman microscopy is used to characterize hybridization reactions of ssDNA immobilized at the solid/liquid interface of porous silica particles. By focusing the femtoliter confocal probe volume within a single porous particle, signal enhancement arises from the â¼1500-times greater surface area detected compared to a planar substrate. Because the porous support is a purely dielectric material, the scattering signal is independent of the proximity of the oligonucleotide to the silica surface. With this technique, we characterize a 19-mer capture strand and determine its hybridization efficiency with 9-mer and 16-mer target sequences from the scattering of a structurally insensitive phosphate-stretching mode. Changes in polarizability and frequency of scattering from DNA bases were observed, which are consistent with Watson-Crick base pairing. Quantification of base content from their duplex scattering intensities allows us to discriminate between hybridization of two target strands of equivalent length but with different recognition sequences. A duplex having a single-nucleotide polymorphism could be distinguished from hybridization of a fully complementary strand based on differences in base content and duplex conformation.
Assuntos
DNA , Dióxido de Silício , DNA/genética , Hibridização de Ácido Nucleico , Porosidade , Análise Espectral RamanRESUMO
Analysis of particulate matter originating from beef cattle feed yards on the High Plains of the United States has revealed occurrence of multiple pesticides believed to potentially impact non-Apis pollinators. Among these pesticides are those that are highly toxic to Apis mellifera (honey bees). However, little non-Apis bee species toxicity data exist; especially pertaining to beef cattle feed yard-derived pesticides. Therefore, we conducted a series of 96-h contact toxicity tests with blue orchard mason bees (Osmia lignaria) using three neonicotinoids, two pyrethroids, and two macrocyclic lactones. Neonicotinoids (thiamethoxam, imidacloprid, and clothianidin) were most toxic with LD50 values ranging from 2.88 to 26.35 ng/bee, respectively. Macrocyclic lactones (abamectin and ivermectin) were also highly toxic to O. lignaria with LD50 estimates of 5.51-32.86 ng/bee. Pyrethroids (permethrin and bifenthrin) were relatively less toxic with LD50 values greater than 33 ng/bee. Sensitivity ratios for each pesticide were calculated to relate O. lignaria LD50 values to existing honey bee toxicity data. All three neonicotinoids were more toxic to O. lignaria than A. mellifera, but pyrethroids and abamectin were relatively less toxic. Additionally, three of seven pesticides (43%) resulted in significantly different mass normalized LD50 values for male and female O. lignaria. These results indicate that non-Apis pollinators may be highly susceptible to pesticides originating from beef cattle feed yards, necessitating consideration of more stringent regulatory protections than those based on A. mellifera pesticide sensitivity.
Assuntos
Abelhas/efeitos dos fármacos , Lactonas/toxicidade , Compostos Macrocíclicos/toxicidade , Neonicotinoides/toxicidade , Praguicidas/toxicidade , Piretrinas/toxicidade , Animais , Relação Dose-Resposta a Droga , Feminino , Dose Letal Mediana , Masculino , Modelos Teóricos , Polinização/efeitos dos fármacos , Testes de Toxicidade AgudaRESUMO
Oligonucleotide aptamers can be converted into structure-switching biosensors by incorporating a short, typically labeled oligonucleotide that is complementary to the analyte-binding region. Binding of a target analyte can disrupt the hybridization equilibrium between the aptamer and the labeled-complementary oligo producing a concentration-dependent signal for target-analyte sensing. Despite its importance in the performance of a biosensor, the mechanism of analyte-response of most structure-switching aptamers is not well understood. In this work, we employ single-molecule fluorescence imaging to investigate the competitive kinetics of association of a labeled complementary oligonucleotide and a target analyte, l-tyrosinamide (L-Tym), interacting with an L-Tym-binding aptamer. The complementary readout strand is fluorescently labeled, allowing us to measure its hybridization kinetics with individual aptamers immobilized on a surface and located with super-resolution techniques; the small-molecule L-Tym analyte is not labeled in order to avoid having an attached dye molecule impact its interactions with the aptamer. We measure the association kinetics of unlabeled L-Tym by detecting its influence on the hybridization of the labeled complementary strand. We find that L-Tym slows the association rate of the complementary strand with the aptamer but does not impact its dissociation rate, suggesting an SN1-like mechanism where the complementary strand must dissociate before L-Tym can bind. The competitive model revealed a slow association rate between L-Tym and the aptamer, producing a long-lived L-Tym-aptamer complex that blocks hybridization with the labeled complementary strand. These results provide insight about the kinetics and mechanism of analyte recognition in this structure-switching aptamer, and the methodology provides a general means of measuring the rates of unlabeled-analyte binding kinetics in aptamer-based biosensors.
Assuntos
Aptâmeros de Nucleotídeos/química , DNA/química , Tirosina/análogos & derivados , Sítios de Ligação , Corantes Fluorescentes/química , Imagem Óptica , Tirosina/análiseRESUMO
Considering the recent discovery of veterinary pharmaceutical aerial transport from industrial cattle feeding operations via particulate matter, the objective of this study is to determine the extent to which insecticides are also transported into the environment by total suspended particulates emanating from beef cattle feed yards. Of 16 different pesticides quantified in particulate matter samples collected from beef cattle feed yards, permethrin was detected most frequently at >67% of particulate matter samples and at a mean concentration of 1211.7 ± 781.0 (SE) ng/m3. Imidacloprid was detected at a mean concentration of 62.8 ± 38.2 (SE) ng/m3 or equivalent to published concentrations in dust from treated seed planting activities. When insecticide concentrations observed in this study are projected to all United States of America feed yards, the resulting particulate matter (669,000 kg) could contain enough insecticides (active ingredient mass basis) to kill over a billion honeybees daily. Furthermore, a novel transport pathway for macrocyclic lactone entry into the environment was identified. These data raise concern that nontarget organisms may be exposed to potentially toxic levels of pesticides from beef cattle feed yards.
Assuntos
Inseticidas , Praguicidas , Ração Animal/análise , Animais , Abelhas , Bovinos , Poeira/análise , Material Particulado/análise , Praguicidas/análiseRESUMO
Single-molecule fluorescence methods can count molecules without calibration, measure kinetics at equilibrium, and observe rare events that cannot be detected in an ensemble measurement. We employ total internal reflection fluorescence microscopy to monitor hybridization kinetics between individual spatially resolved target DNA molecules immobilized at a glass interface and fluorescently labeled complementary probe DNA in free solution. Using super-resolution imaging, immobilized target DNA molecules are located with 36 nm precision, and their individual duplex formation and dissociation kinetics with labeled DNA probe strands are measured at site densities much greater than the diffraction limit. The purpose of this study is to evaluate uncertainties in identifying these individual target molecules based on their duplex dissociation kinetics, which can be used to distinguish target molecule sequences randomly immobilized in mixed-target samples. Hybridization kinetics of individual target molecules are determined from maximum likelihood estimation of their dissociation times determined from a sample of hybridization events at each target molecule. The dissociation time distributions thus estimated are sufficiently narrow to allow kinetic discrimination of different target sequences. For example, a single-base thymine-to-guanine substitution on immobilized strands produces a 2.5-fold difference in dissociation rates of complementary probes, allowing for the identification of individual target DNA molecules by their dissociation rates with 95% accuracy. This methodology represents a step toward high-density single-molecule DNA microarray sensors and a powerful tool to investigate the kinetics of hybridization at surfaces at the molecular level, providing information that cannot be acquired in ensemble measurements.
Assuntos
DNA/química , Ácidos Nucleicos Imobilizados/análise , Hibridização de Ácido Nucleico/métodos , DNA/metabolismo , Sondas de DNA/química , Sondas de DNA/metabolismo , Corantes Fluorescentes/química , Ácidos Nucleicos Imobilizados/metabolismo , CinéticaRESUMO
Aptamers are short nucleic-acid biopolymers selected to have high affinity and specificity for protein or small-molecule target analytes. Aptamers can be engineered into split-aptamer biosensors comprising two nucleic acid strands that coassemble as they bind to a target, resulting in a large signal change from attached molecular probes (e.g., molecular beacons). The kinetics of split-aptamer assembly and their dependence on target recognition are largely unknown; knowledge of these kinetics could help in design and optimization of split-aptamer biosensors. In this work, we measure assembly kinetics of cocaine-dependent split-aptamer molecules using single-molecule fluorescence imaging. Assembly is monitored between a DNA strand tethered to a glass substrate and solutions containing the other strand tagged with a fluorescent label, with varying concentrations of the cocaine analyte. Dissociation rates are measured by tracking individual molecules and measuring their bound lifetimes. Dissociation-time distributions are biexponential, possibly indicating different folded states of the aptamer. The dissociation rate of only the longer-lived complex decreases with cocaine concentration, suggesting that cocaine stabilizes the long-lived aptamer complex. The variation in the slow dissociation rate with cocaine concentration is well described with an equilibrium-binding model, where the dissociation rate approaches a saturation limit consistent with the dissociation-equilibrium constant for cocaine-binding to the split aptamer. This single-molecule methodology provides a sensitive readout of cocaine-binding based on the dissociation kinetics of the split aptamer, allowing one to distinguish target-dependent aptamer assembly from background assembly. This methodology could be used to study other systems where target association affects the stability of aptamer duplexes.
Assuntos
Aptâmeros de Nucleotídeos/química , Cocaína/química , Sondas de DNA/química , Aptâmeros de Nucleotídeos/genética , Carbocianinas/química , Sondas de DNA/genética , Fluorescência , Corantes Fluorescentes/química , Cinética , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Imagem Individual de Molécula/métodosRESUMO
Single-molecule imaging of fluorescently labeled biomolecules is a powerful technique for measuring association interactions; however, care must be taken to ensure that the fluorescent labels do not influence the system being probed. Label-free techniques are needed to understand biomolecule interactions free from the influence of an attached label, but these techniques often lack sensitivity and specificity. To solve these challenges, we have developed a competitive assay that uses single-molecule detection to track the population of unlabeled target single-stranded DNA (ssDNA) hybridized with probe DNA immobilized at a glass interface by detecting individual duplexes with a fluorescently labeled "tracer" ssDNA. By labeling a small fraction (<0.2%) of target molecules, the "tracer" DNA tracks the available probe DNA sites without significant competition with the unlabeled target population. Single-molecule fluorescence imaging is a good read-out scheme for competitive assays, as it is sufficiently sensitive to detect tracer DNA on substrates with relatively low densities of probe DNA, â¼10(-3) of a monolayer, so that steric interactions do not hinder DNA hybridization. Competitive assays are used to measure the association constant of complementary strand DNA hybridization of 9- and 10-base pair targets, where the tracer assay predicts the same association constant as a traditional displacement competitive assay. This methodology was used to compare the Ka of hybridization for identical DNA strands differing only by the presence of a fluorescent label tethered to the 5' end of the solution-phase target. The addition of the fluorescent label significantly stabilizes the DNA duplex by 3.6 kJmol(-1), adding more stability than an additional adenine-thymine base-pairing interaction, 2.7 kJmol(-1). This competitive tracer assay could be used to screen a number of labeled and unlabeled target DNA strands to measure the impact of fluorescent labeling on duplex stability. This single-molecule competitive hybridization scheme could be easily adapted into a sensitive assay, where competition between tracer and target oligonucleotides for probe sites could be used to measure concentrations of unlabeled DNA or RNA.
Assuntos
Sondas de DNA/química , DNA/análise , Corantes Fluorescentes/química , Ácidos Nucleicos Imobilizados/química , Hibridização de Ácido Nucleico/métodos , Imagem Óptica/métodos , DNA de Cadeia Simples/química , TermodinâmicaRESUMO
Accurate knowledge of the kinetics of complementary oligonucleotide hybridization is integral to the design and understanding of DNA-based biosensors. In this work, single-molecule fluorescence imaging is applied to measuring rates of hybridization between fluorescently labeled target ssDNA and unlabeled probe ssDNA immobilized on glass surfaces. In the absence of probe site labeling, the capture surface must be highly selective to avoid the influence of nonspecific adsorption on the interpretation of single-molecule imaging results. This is accomplished by increasing the probe molecule site densities by a factor of â¼100 compared to optically resolvable sites so that nonspecific interactions compete with a much greater number of capture sites and by immobilizing sulfonate groups to passivate the surface between probe strands. The resulting substrates exhibit very low nonspecific adsorption, and the selectivity for binding a complementary target sequence exceeds that of a scrambled sequence by nearly 3 orders of magnitude. The population of immobilized DNA probe sites is quantified by counting individual DNA duplexes at low target concentrations, and those results are used to calibrate fluorescence intensities on the same sample at much higher target concentrations to measure a full binding isotherm. Dissociation rates are determined from interfacial residence times of individual DNA duplexes. Equilibrium and rate constants of hybridization, K(a) = 38 (±1) µM(-1), k(on) = 1.64 (±0.06) × 10(6) M(-1) s(-1), and k(off) = 4.3 (±0.1) × 10(-2) s(-1), were found not to change with surface density of immobilized probe DNA, indicating that hybridization events at neighboring probe sites are independent. To test the influence of probe-strand immobilization on hybridization, the kinetics of the probe target reaction at the surface were compared with the same reaction in free solution, and the equilibrium constants and dissociation and association rates were found to be nearly equivalent. The selectivity of these capture surfaces should facilitate sensitive investigations of DNA hybridization at the limit of counting molecules. Because the immobilized probe DNA on these surfaces is unlabeled, photobleaching of a probe label is not an issue, allowing capture substrates to be used for long periods of time or even reused in multiple experiments.
Assuntos
DNA de Cadeia Simples/análise , Imagem Óptica/métodos , Adsorção , Técnicas Biossensoriais , Vidro/química , Cinética , Hibridização de Ácido Nucleico , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Super-resolution imaging was used to quantify organizational changes in the plasma membrane after treatment with hybrid nanoconjugates. The nanoconjugates crosslinked CD20 on the surface of malignant B cells, thereby inducing apoptosis. Super-resolution images were analyzed by using pair-correlation analysis to determine cluster size and to count the average number of molecules in the clusters. The role of lipid rafts was investigated by pre-treating cells with a cholesterol chelator and actin destabilizer to prevent lipid raft formation. Lipid raft cluster size correlated with apoptosis induction after treatment with the nanoconjugates. Lipid raft clusters had radii of â¼ 200 nm in cells treated with the hybrid nanoconjugates. Super-resolution images provided precise molecule location coordinates that could be used to determine density of bound conjugates, cluster size, and number of molecules per cluster.
Assuntos
Proteínas de Membrana/metabolismo , Imagem Molecular , Nanoconjugados/química , Apoptose , Membrana Celular/metabolismo , Humanos , Modelos BiológicosRESUMO
Simultaneous multipass resistive-pulse sensing and fluorescence imaging have been used to correlate the size and fluorescence intensity of individual E. coli lipid liposomes composed of E. coli polar lipid extracts labeled with membrane-bound 3,3-dioctadecyloxacarbocyanine (DiO) fluorescent molecules. Here, a nanopipet serves as a waveguide to direct excitation light to the resistive-pulse sensing zone at the end of the nanopipet tip. Individual DiO-labeled liposomes (>50 nm radius) were multipassed back and forth through the orifices of glass nanopipets' 110-150 nm radius via potential switching to obtain subnanometer sizing precision, while recording the fluorescence intensity of the membrane-bound DiO molecules. Fluorescence was measured as a function of liposome radius and found to be approximately proportional to the total membrane surface area. The observed relationship between liposome size and fluorescence intensity suggests that multivesicle liposomes emit greater fluorescence compared to unilamellar liposomes, consistent with all lipid membranes of the multivesicle liposomes containing DiO. Fluorescent and nonfluorescent liposomes are readily distinguished from each other in the same solution using simultaneous multipass resistive-pulse sensing and fluorescence imaging. A fluorescence "dead zone" of â¼1 µm thickness just outside of the nanopipet orifice was observed during resistive-pulse sensing, resulting in "on/off" fluorescent behavior during liposome multipassing.
Assuntos
Escherichia coli , Lipossomos , Lipídeos , Imagem ÓpticaRESUMO
Due to its high specific surface area and chemical stability, porous silica is used as a support structure in numerous applications, including heterogeneous catalysis, biomolecule immobilization, sensors, and liquid chromatography. Reversed-phase liquid chromatography (RPLC), which uses porous silica support particles, has become an indispensable separations tool in quality control, pharmaceutics, and environmental analysis requiring identification of compounds in mixtures. For complex samples, the need for higher resolution separations requires an understanding of the time scale of processes responsible for analyte retention in the stationary phase. In the present work, single-molecule fluorescence imaging is used to observe transport of individual molecules within RPLC porous silica particles. This technique allows direct measurement of intraparticle molecular residence times, intraparticle diffusion rates, and the spatial distribution of molecules within the particle. On the basis of the localization uncertainty and characteristic measured diffusion rates, statistical criteria were developed to resolve the frame-to-frame behavior of molecules into moving and stuck events. The measured diffusion coefficient of moving molecules was used in a Monte Carlo simulation of a random-walk model within the cylindrical geometry of the particle diameter and microscope depth-of-field. The simulated molecular transport is in good agreement with the experimental data, indicating transport of moving molecules in the porous particle is described by a random-walk. Histograms of stuck-molecule event times, locations, and their contributions to intraparticle residence times were also characterized.
Assuntos
Cromatografia de Fase Reversa , Fluorescência , Dióxido de Silício/química , Método de Monte Carlo , Tamanho da Partícula , Porosidade , Espectrometria de Fluorescência , Propriedades de SuperfícieRESUMO
Optically transparent semiconductors allow simultaneous control of interfacial electrical potential and spectroscopic observation of chemistry near the electrode surface. Care must be taken, however, to avoid unwanted photoexcitation-induced charging of the semiconductor electrode that could influence the results. In this work, we investigate the in situ surface charging by photoexcitation well below the band gap of an optically transparent semiconductor, indium-tin oxide (ITO) electrode. Using total-internal-reflection fluorescence microscopy, the population of ~100-nm negatively charged carboxylate-polystyrene fluorescent nanoparticles at an ITO-aqueous solution interface could be monitored in situ. At positive applied potentials (~0.7 V versus Ag/AgCl), nanoparticles accumulate reversibly in the electrical double-layer of the ITO surface, and the interfacial nanoparticle populations increase with 488-nm excitation intensity. The potential sensitivity of nanoparticle population exhibited no dependence on excitation intensity, varied from 0.1 to 10 W cm(-2), while the onset potential for particle accumulation shifted by as much as 0.3 V. This shift in surface potential appears to be due to photoexcitation-induced charging of the ITO, even though the excitation radiation photon energy, ~2.4 eV, is well below the primary band gap of ITO, >3.5 eV. A kinetic model was developed to determine the photon order of electron-hole generation relative to the electron-hole recombination. The photoexcitation process was found to be first-order in photon flux, suggesting one-photon excitation of an indirect band gap or defect sites, rather than two-photon excitation into the direct band gap. A control experiment was conducted with red-fluorescent carboxylate-polystyrene particles that were counted using 647-nm excitation, where the photon energy is below the indirect band gap or defect site energy and where the optical absorption of the film vanishes. Red illumination between 1 and 15 W cm(-2) produced no detectable shifts in the onset accumulation potential, which is consistent with the negligible optical absorption of the ITO film at this longer wavelength.
RESUMO
Many interfacial chemical phenomena are governed in part by electrostatic interactions between polyelectrolytes and charged surfaces; these phenomena can influence the performance of biosensors, adsorption of natural polyelectrolytes (humic substances) on soils, and production of polyelectrolyte multilayer films. In order to understand electrostatic interactions that govern these phenomena, we have investigated the behavior of a model polyelectrolyte, 15 kbp fluorescently labeled plasmid DNA, near a polarized indium tin oxide (ITO) electrode surface. The interfacial population of DNA was monitored in situ by imaging individual molecules through the transparent electrode using total-internal-reflection fluorescence microscopy. At applied potentials of +0.8 V versus Ag/AgCl, the DNA interfacial population near the ITO surface can be increased by 2 orders of magnitude relative to bulk solution. The DNA molecules attracted to the interface do not adsorb to ITO, but rather they remain mobile with a diffusion coefficient comparable to free solution. Ionic strength strongly influences the sensitivity of the interfacial population to applied potential, where the increase in the interfacial population over a +300 mV change in potential varies from 20% in 30 mM ionic strength to over 25-fold in 300 µM electrolyte. The DNA accumulation with applied potential was interpreted using a simple Boltzmann model to predict average ion concentrations in the electrical double layer and the fraction of interfacial detection volume that is influenced by applied potential. A Gouy-Chapman model was also applied to the data to account for the dependence of the ion population on distance from the electrode surface, which indicates that the net charge on DNA responsible for interactions with the polarized surface is low, on the order of one excess electron. The results are consistent with a small fraction of the DNA plasmid being resident in the double-layer and with counterions screening much of the DNA excess charge.
Assuntos
DNA/química , Eletrólitos/química , Imagem Molecular/métodos , Plasmídeos/química , Adsorção , Difusão , Eletrodos , Microscopia de Fluorescência , Concentração Osmolar , Soluções , Eletricidade Estática , Propriedades de Superfície , Compostos de EstanhoRESUMO
The binding of glucagon-like peptide-1 (GLP-1) to a planar phospholipid bilayer is measured using single-molecule total internal reflection fluorescence microscopy. From several reports in the literature, GLP-1 has been shown to be a random coil in free solution, adopting a folded, α-helix conformation when intercalated into membrane environments. Single-molecule fluorescence measurements of GLP-1 binding to supported lipid bilayers show evidence of two populations of membrane-associated molecules having different residence times, suggesting weakly adsorbed peptides and strongly bound peptides in the lipid bilayer. The path to and from a strongly bound (folded, intercalated) state would likely include an adsorbed state as an intermediate, so that the resulting kinetics would correspond to a consecutive first-order reversible three-state model. In this work, the relationships between measured single-molecule residence times and the microscopic rates in a three-state kinetic model are derived and used to interpret the binding of GLP-1 to a supported lipid bilayer. The system of differential equations associated with the proposed consecutive-three state kinetics scheme is solved, and the solution is applied to interpret histograms of single-molecule, GLP-1 residence times in terms of the microscopic rates in the sequential two-step model. These microscopic rates are used to estimate the free energy barrier to adsorption, the fraction of peptides adsorbing to the membrane surface that successfully intercalate in the bilayer, the lifetime of inserted peptides in the membrane, and the free energy change of insertion into the lipid bilayer from the adsorbed state. The transition from a random coil in solution to a folded state in a membrane has been recognized as a common motif for insertion of membrane active peptides. Therefore, the relationships developed here could have wide application to the kinetic analysis of peptide-membrane interactions.
Assuntos
Peptídeo 1 Semelhante ao Glucagon/química , Bicamadas Lipídicas/química , Modelos Moleculares , Peptídeos/química , Fosfolipídeos/química , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Cinética , Bicamadas Lipídicas/metabolismo , Microscopia de Fluorescência , Peptídeos/metabolismo , Fosfolipídeos/metabolismo , Ligação ProteicaRESUMO
It is well established that agrochemicals can pose significant threats to native pollinators; however, relatively little is known about pollinator risks associated with agrochemicals that are used on beef cattle feed yards. Recently, feed yard-derived agrochemicals and those from row crop agriculture were quantified on wildflowers growing on the High Plains, USA. To better characterize pollinator risks on the High Plains, we collected colocated wildflowers and foraging bees across three field seasons for analytical determination of residual agrochemicals. Agrochemicals were detected and quantified on the majority of wildflowers (85%) and nearly half of bees (49%). Permethrin was the most frequently detected analyte on wildflowers (32%) and bees (17%). Flower hazard quotients and flower hazard indices were calculated to deterministically evaluate risk to foraging pollinators. Mean flower hazard quotients exceeded one for 5/16 analytes (31%), and flower hazard quotients calculated for 30% of wildflowers were greater than 50. Flower hazard quotients for clothianidin exceeded 400 for 14% of wildflowers, which portends conditions conducive to frequent bee mortalities. Flower hazard indices were greater on wildflowers from mid-July to mid-September as compared with wildflowers collected earlier in the summer, which coincides with row crop planting and increased prevalence of feed yard flies. Hazard quotients and hazard index values calculated from agrochemical residue data suggest that pollinators frequenting wildflowers near beef cattle feed yards and row crops on the High Plains are at risk from both individual sources, and more so when considered in combination. Integr Environ Assess Manag 2022;18:163-173. © 2021 SETAC.
Assuntos
Agroquímicos , Produtos Agrícolas , Agricultura , Agroquímicos/toxicidade , Ração Animal , Animais , Abelhas , Bovinos , Polinização , Estações do AnoRESUMO
Encapsulation of molecules in phospholipid vesicles provides unique opportunities to study chemical reactions in small volumes as well as the behavior of individual proteins, enzymes, and ribozymes in a confined region without requiring a tether to immobilize the molecule to a surface. These experiments generally depend on generating a predictable loading of vesicles with small numbers of target molecules and thus raise a significant measurement challenge, namely, to quantify molecular occupancy of vesicles at the single-molecule level. In this work, we describe an imaging experiment to measure the time-dependent fluorescence from individual dye molecules encapsulated in ~130 nm vesicles that are adhered to a glass surface. For determining a fit of the molecular occupancy data to a Poisson model, it is critical to count empty vesicles in the population since these dominate the sample when the mean occupancy is small, λ ≤ ~1. Counting empty vesicles was accomplished by subsequently labeling all the vesicles with a lipophilic dye and reimaging the sample. By counting both the empty vesicles and those containing fluors, and quantifying the number of fluors present, we demonstrate a self-consistent Poisson distribution of molecular occupancy for well-solvated molecules, as well as anomalies due to aggregation of dye, which can arise even at very low solution concentrations. By observation of many vesicles in parallel in an image, this approach provides quantitative information about the distribution of molecular occupancy in a population of vesicles.
Assuntos
Microscopia de Fluorescência/métodos , Fosfolipídeos/químicaRESUMO
Cavity nesting bees are proficient and important pollinators that can augment or replace honey bee pollination services for some crops. Relatively little is known about specific pesticide concentrations present in cavity nesting insect reed matrices and associated potential risks to cavity nesting bees. Nesting substrates (Phragmites australis reeds in bundles) were deployed in an agriculturally intensive landscape to evaluate colonization and agrochemical exposure among cavity nesting pollinators over two consecutive field seasons. Composition of insect species colonizing reeds within nest bundles varied considerably; those placed near beef cattle feed yards were dominated by wasps (93% of the total number of individuals occupying reed nest bundles), whereas nest bundles deployed in cropland-dominated landscapes were colonized primarily by leaf cutter bees (71%). All nesting/brood matrices in reeds (mud, leaves, brood, pollen) contained agrochemicals. Mud used in brood chamber construction at feed yard sites contained 21 of 23 agrochemicals included in analysis and >70% of leaf substrate stored in reeds contained at least one agrochemical. Moxidectin was most frequently detected across all reed matrices from feed yard sites, and moxidectin concentrations in nonviable larvae were more than four times higher than those quantified in viable larvae. Agrochemical concentrations in leaf material and pollen were also quantified at levels that may have induced toxic effects among developing larvae. To our knowledge, this is the first study to characterize agrochemical concentrations in multiple reed matrices provisioned by cavity-nesting insects. Use of nest bundles revealed that cavity nesting pollinators in agriculturally intensive regions are exposed to agrochemicals during all life stages, at relatively high frequencies, and at potentially lethal concentrations. These results demonstrate the utility of nest bundles for characterizing risks to cavity nesting insects inhabiting agriculturally intensive regions.
Assuntos
Agroquímicos , Polinização , Animais , Abelhas , Bovinos , Produtos Agrícolas , Insetos , Larva , Comportamento de NidaçãoRESUMO
Peptide nucleic acid (PNA) is a unique synthetic nucleic acid analog that has been adopted for use in many biological applications. These applications rely upon the robust Franklin-Watson-Crick base pairing provided by PNA, particularly at lower ionic strengths. However, our understanding of the relationship between the kinetics of PNA:DNA hybridization and ionic strength is incomplete. Here we measured the kinetics of association and dissociation of PNA with DNA across a range of ionic strengths and temperatures at single-molecule resolution using total internal reflection fluorescence imaging. Unlike DNA:DNA duplexes, PNA:DNA duplexes are more stable at lower ionic strength, and we demonstrate that this is due to a higher association rate. While the dissociation rate of PNA:DNA duplexes is largely insensitive to ionic strength, it is significantly lower than that of DNA:DNA duplexes having the same number and sequence of base pairing interactions. The temperature dependence of PNA:DNA kinetic rate constants indicate a significant enthalpy barrier to duplex dissociation, and to a lesser extent, duplex formation. This investigation into the kinetics of PNA:DNA hybridization provides a framework towards better understanding and design of PNA sequences for future applications.