Investigation of an optimal cell lysis method for the study of the zinc metalloproteome of Histoplasma capsulatum.

This work sought to assess optimal extraction conditions in the study of the metalloproteome of the dimorphic fungus Histoplasma capsulatum. One of the body's responses to H. capsulatum infection is sequestration of zinc within host macrophage (MØ), as reported by Vignesh et al. (Immunity 39:697-710, 2013) and Vignesh et al. (PLOS Pathog 9:E1003815, 2013). Thus, metalloproteins containing zinc were of greatest interest as it plays a critical role in survival of the fungus. One challenge in metalloproteomics is the preservation of the native structure of proteins to retain non-covalently bound metals. Many of the conventional cell lysis, separation, and identification techniques in proteomics are carried out under conditions that could lead to protein denaturation. Various cell lysis techniques were investigated in an effort to both maintain the metalloproteins during lysis and subsequent analysis while, at the same time, serving to be strong enough to break the cell wall, allowing access to cytosolic metalloproteins. The addition of 1% Triton x-100, a non-ionic detergent, to the lysis buffer was also studied. Seven lysis methods were considered and these included: Glass Homogenizer (H), Bead Beater (BB), Sonication Probe (SP), Vortex with 1% Triton x-100 (V, T), Vortex with no Triton x-100 (V, NT), Sonication Bath, Vortex, and 1% Triton x-100 (SB, V, T) and Sonication Bath, Vortex, and no Triton x-100 (SB, V, NT). A Qubit® Assay was used to compare total protein concentration and inductively coupled plasma-mass spectrometry (ICP-MS) was utilized for total metal analysis of cell lysates. Size exclusion chromatography coupled to ICP-MS (SEC-HPLC-ICP-MS) was used for separation of the metalloproteins in the cell lysate and the concentration of Zn over a wide molecular weight range was examined. Additional factors such as potential contamination sources were also considered. A cell lysis method involving vortexing H. capsulatum yeast cells with 500 µm glass beads in a 1% Triton x-100 lysis buffer (V, T) was found to be most advantageous to extract intact zinc metalloproteins as demonstrated by the highest Zn to protein ratio, 1.030 ng Zn/µg protein, and Zn distribution among high, mid, and low molecular weights suggesting the least amount of protein denaturation. Graphical abstract In this work, several cell lysis techniques and two lysis buffers were investigated to evaluate the preservation of the zinc metalloproteome of H. capsulatum while maintaining compatibility with the analytical techniques employed.

Influences on transfer of selected synthetic pyrethroids from treated Formica to foods.

Children's unstructured eating habits and activities may lead to excess dietary exposures not traditionally measured by the US Environmental Protection Agency. Influence of these activities on transfer of pesticides from treated Formica to foods was studied. The objective was to perform simulation experiments using four foods (bread, apple slices, bologna, and sugar cookies) exposed to treated Formica after varied time intervals between surface contamination and contact (1, 6, and 24 h) and frequency of contact with and without recontamination. Pesticides investigated included permethrin, bifenthrin, cyfluthrin, cypermethrin, and deltamethrin. Data will be used as input parameters for transfer efficiencies (TEs) within the Children's Dietary Intake Model (CDIM), which predicts total dietary exposure of a child. Pesticide transfer from surfaces to bologna and apples was more efficient than to bread and cookies. For the bread and cookies, all pyrethroids had a TE that ranged from below detectible levels to ≤ 4%. A combined average of 32-64% and 19-43% was transferred to bologna and apples, respectively, for the three contact times for all pyrethroids. The TEs of the varied time intervals indicated that increased time between contamination and contact showed little difference for bologna, bread, and cookies, but a significant difference for apples. As long as pesticide levels are measureable on surfaces in children's eating environment, it can be concluded that transfer of pesticides to foods will take place. Foods' characteristics had an important function in the transfer of pesticides when multiple contacts occurred. Regardless of recontamination, pesticides were efficiently transferred from the treated surface to bologna. The bologna did not reach a saturation point during the contacts. Pesticides were also efficiently transferred to apples, but reached a maximum TE during the second contact. The distribution of activity factors within CDIM needs to reflect the differences in the characteristics of the foods.

Robust properties of membrane-embedded connector channel of bacterial virus phi29 DNA packaging motor.

Biological systems contain highly-ordered macromolecular structures with diverse functions, inspiring their utilization in nanotechnology. A motor allows linear dsDNA viruses to package their genome into a preformed procapsid. The central component of the motor is the portal connector that acts as a pathway for the translocation of dsDNA. The elegant design of the connector and its channel motivates its application as an artificial nanopore (Nature Nanotechnology, 4, 765-772). Herein, we demonstrate the robust characteristics of the connector of the bacteriophage phi29 DNA packaging motor by single pore electrophysiological assays. The conductance of each pore is almost identical and is perfectly linear with respect to the applied voltage. Numerous transient current blockade events induced by dsDNA are consistent with the dimensions of the channel and dsDNA. Furthermore, the connector channel is stable under a wide range of experimental conditions including high salt and pH 2-12. The robust properties of the connector nanopore made it possible to develop a simple reproducible approach for connector quantification. The precise number of connectors in each sheet of the membrane was simply derived from the slopes of the plot of voltage against current. Such quantifications led to a reliable real time counting of DNA passing through the channel. The fingerprint of DNA translocation in this system has provided a new tool for future biophysical and physicochemical characterizations of DNA transportation, motion, and packaging.

Accessibility of polybrominated diphenyl ether congeners in aging soil.

Polybrominated diphenyl ethers (PBDEs) are considered to be persistent environmental pollutants. Although soil is considered the most likely sink for these contaminants, little is known about the potential adsorption and aging of these compounds to the soil matrix. A previous study performed in our lab suggested that abiotic sorption of PBDEs to soil constituents was the most important determinant of PBDE accessibility. Building on this work, the present paper examined the availability of congeners of a commercial PBDE mixture (DE-71) in soils that varied in organic matter content, clay content, and pH. Both sterile and non-sterile soils were amended with DE-71 and then monitored over eight weeks of aging. Recovery of all congeners from soil by acetone extraction dropped significantly over time. Comparisons between sterile and non-sterile samples, along with the results of a separate phase partitioning study, indicated a strong affinity between soils and monitored DE-71 components. This general phenomenon was so dominant that, in this study, varying soil characteristics had no significant effect on PBDE recovery. Unexpectedly, final recovery was significantly higher in the non-sterile soil. The biological impact on congener accessibility was also observed when zucchini and radish plants were grown in soil that had been aged 8 weeks following DE-71 fortification. After 10 weeks of growth, recovery of congeners was up to five times higher than it had been prior to planting.

Analysis of permethrin isomers in composite diet samples by molecularly imprinted solid-phase extraction and isotope dilution gas chromatography-ion trap mass spectrometry.

Determination of an individual's aggregate dietary ingestion of pesticides entails analysis of a difficult sample matrix. Permethrin-specific molecularly imprinted polymer (MIP) solid-phase extraction cartridges were developed for use as a sample preparation technique for a composite food matrix. Vortexing with acetonitrile and centrifugation were found to provide optimal extraction of the permethrin isomers from the composite foods. The acetonitrile (with 1% acetic acid) was mostly evaporated and the analytes reconstituted in 90:10 water/acetonitrile in preparation for molecularly imprinted solid-phase extraction. Permethrin elution was accomplished with acetonitrile and sample extracts were analyzed by isotope dilution gas chromatography-ion trap mass spectrometry. Quantitation of product ions provided definitive identification of the pesticide isomers. The final method parameters were tested with fortified composite food samples of varying fat content (1%, 5%, and 10%) and recoveries ranged from 99.3% to 126%. Vegetable samples with incurred pesticide levels were also analyzed with the given method and recoveries were acceptable (81.0-95.7%). Method detection limits were demonstrated in the low ppb range. Finally, the applicability of the MIP stationary phase to extract other pyrethroids, specifically cyfluthrin and cypermethrin, was also investigated.

Development of an analytical scheme for the determination of pyrethroid pesticides in composite diet samples.

Analysis of an individual's total daily food intake may be used to determine aggregate dietary ingestion of given compounds. However, the resulting composite sample represents a complex mixture, and measurement of such can often prove to be difficult. In this work, an analytical scheme was developed for the determination of 12 select pyrethroid pesticides in dietary samples. In the first phase of the study, several cleanup steps were investigated for their effectiveness in removing interferences in samples with a range of fat content (1-10%). Food samples were homogenized in the laboratory, and preparatory techniques were evaluated through recoveries from fortified samples. The selected final procedure consisted of a lyophilization step prior to sample extraction. A sequential 2-fold cleanup procedure of the extract included diatomaceous earth for removal of lipid components followed with a combination of deactivated alumina and C(18) for the simultaneous removal of polar and nonpolar interferences. Recoveries from fortified composite diet samples (10 microg kg(-1)) ranged from 50.2 to 147%. In the second phase of this work, three instrumental techniques [gas chromatography-microelectron capture detection (GC-microECD), GC-quadrupole mass spectrometry (GC-quadrupole-MS), and GC-ion trap-MS/MS] were compared for greatest sensitivity. GC-quadrupole-MS operated in selective ion monitoring (SIM) mode proved to be most sensitive, yielding method detection limits of approximately 1 microg kg(-1). The developed extraction/instrumental scheme was applied to samples collected in an exposure measurement field study. The samples were fortified and analyte recoveries were acceptable (75.9-125%); however, compounds coextracted from the food matrix prevented quantitation of four of the pyrethroid analytes in two of the samples considered.

Surface-to-food pesticide transfer as a function of moisture and fat content.

Transfer of pesticides from household surfaces to foods may result in excess dietary exposure in children (i.e., beyond that inherent in foods due to agricultural application). In this study, transfer was evaluated as a function of the moisture and fat content of various foods. Surfaces chosen for investigation were those commonly found in homes and included Formica, ceramic tile, plastic, carpet, and upholstery fabric. Each surface type was sprayed with an aqueous emulsion of organophosphates, fipronil, and synthetic pyrethroids. In the first phase of the study, multiple foods (apples, watermelon, wheat crackers, graham crackers, white bread, flour tortillas, bologna, fat-free bologna, sugar cookies, ham, Fruit Roll-ups, pancakes, and processed American cheese) were categorized with respect to moisture and fat content. All were evaluated for potential removal of applied pesticides from a Formica surface. In the second phase of the study, representative foods from each classification were investigated for their potential for pesticide transfer with an additional four surfaces: ceramic tile, plastic, upholstery, and carpet. Moisture content, not fat, was found to be a determining factor in most transfers. For nearly all surfaces, more efficient transfer occurred with increased hardness (Formica and ceramic tile). Comparatively, the polymer composition of the plastic delivered overall lower transfer efficiencies, presumably due to an attraction between it and the organic pesticides of interest.

Polybrominated diphenyl ethers: causes for concern and knowledge gaps regarding environmental distribution, fate and toxicity.

This manuscript critically considers several areas of study of the polybrominated diphenyl ether compounds. Specifically, a brief review of PBDE toxicity is followed by an in depth discussion of PBDE occurrence in abiotic and biotic environmental matrices. Temporal and geographic trends are examined in conjunction with risk assessment factors. Rather than summarize or tabulate the growing body of literature on PBDEs in the environment, the overall goal of this review paper is to highlight broad patterns that may contribute to a more holistic understanding of PBDE behavior in the environment, as well as to identify critical areas of research that warrant further attention.

An HPLC-ICP-MS technique for determination of cadmium-phytochelatins in genetically modified Arabidopsis thaliana.

A reversed-phase high-performance liquid chromatographic technique was developed to separate cadmium-phytochelatin complexes (Cd-PC2, Cd-PC3, and Cd-PC4) of interest in the plant Arapidopsis thaliana. High-performance liquid chromatography (HPLC) was coupled to an inductively coupled plasma mass spectrometric (ICP-MS) system with some modification to the interface. This was done in order to sustain the plasma with optimum sensitivity for cadmium detection in the presence of the high methanol loads used in the gradient elution of the reversed-phase separation. The detection limits were found to be 91.8 ngl(-1), 77.2 ngl(-1) and 49.2 ngl(-1) for Cd-PC2, Cd-PC3, and Cd-PC4 respectively. The regression coefficients (r2) for Cd-PC2 to Cd-PC4 detection ranged from 0.998 to 0.999. The method was then used to investigate the occurrence and effect of cadmium-phytochelatin complexes in wild-type Arabidopsis and a phytochelatin-deficient mutant cad1-3 that had been genetically modified to ectopically express the wheat TaPCS1 phytochelatin synthase enzyme. The primary complex found in both wild-type and transgenic plants was Cd-PC2. In both lines, higher levels of Cd-PC2 were found in shoots than in roots, showing that phytochelatin synthases contribute to the accumulation of cadmium in shoots, in the Cd-PC2 form. Genetic modification did, however, impact the overall accumulation of Cd. Transgenic plants contained almost two times more cadmium in the form of Cd-PC2 in their roots than did the corresponding wild-type plants. Similarly, the shoot samples of the modified species also contained more (by 1.6 times) cadmium in the form of Cd-PC2 than the wild type. The enhanced role of PC2 in the transgenic Arabidopsis correlates with data showing long-distance transport of Cd in transgenic plants. Targeted transgenic expression of non-native phytochelatin synthases may contribute to improving the efficiency of plants for phytoremediation.

Fate of pentabrominated diphenyl ethers in soil: abiotic sorption, plant uptake, and the impact of interspecific plant interactions.

Polybrominated diphenyl ethers (PBDEs) are potentially harmful and persistent environmental pollutants. Despite evidence that soils are a major sink for PBDEs, little is known regarding their behavior in this medium. An environmentally relevant level of a commercial penta-BDE mixture (75 microg kg(-1)) was added to topsoil, and the extractability of three congeners (BDE-47, -99, and -100) was monitored over 10 weeks in planted and unplanted treatments. The extractability of each congener decreased rapidly in the experimental soil due largely to abiotic sorption to soil particles, which was demonstrated by low PBDE recovery from sterilized and dry soils. Monoculture plantings of zucchini and radish did not affect the recovery of PBDEs from soil. However, PBDE recovery from mixed species plantings was nearly 8 times higher than that of unplanted and monoculture treatments, indicating that interspecific plant interactions may enhance PBDE bioavailablity in soil. Evidence for competitive interactions between the two species was revealed by reduced shoot biomass of zucchini plants in mixed treatments relative to pots containing only zucchini. Both plant species accumulated PBDEs in root and shoot tissue (<5 microg kg(-1) plant tissue). PBDE uptake was higher in zucchini, and translocation of PBDEs to zucchini shoots was congener-specific. Our results suggest that although abiotic sorption may limit the potential for human exposure to PBDEs in soil, plants may increase the exposure risk by taking up and translocating PBDEs into aboveground tissues and by enhancing bioavailability in soil.

Comparing a selenium accumulator plant (Brassica juncea) to a nonaccumulator plant (Helianthus annuus) to investigate selenium-containing proteins.

Selenoproteins have been identified in a diverse range of organisms, including bacteria and animals. Their occurrence and role in the plant kingdom are, however, less well-understood. This work investigated the water-soluble selenium-containing proteins extracted from a selenium-accumulating plant species (Brassica juncea) and a nonaccumulator species (Helianthus annuus) exposed to varying forms and concentrations of selenium. Firstly, protein extracts were analyzed by size exclusion chromatography coupled to inductively coupled plasma mass spectrometry; specific detection was achieved by monitoring characteristic isotopes. Then, proteolytic digests of the plant extracts were analyzed by reversed phase chromatography coupled to ICP-MS in order to investigate selenoamino acid and selenopeptide content. Selenomethionine was observed to be the primary constituent of the proteins of the nonaccumulator plant, while selenocystine and selenomethionine were found in the same proportion in the accumulator extract. One main selenium-containing species was present at higher levels in the root digests than in the leaf digests; levels were greater in the nonaccumulator than in the accumulator plant.

Use of SEC-ICP-MS with a collision cell for determining the interaction of chromium with DNA extracted from metal-contaminated soils.

The potential of chromium to bind to DNA isolated directly from soil microbial communities was investigated in this study. An analytical scheme was developed to distinguish between chromium bound to DNA and its fragments or chromium contained elsewhere in an environmental DNA extract. DNA was extracted from chromium-contaminated soils and purified using DNA clean-up resins. Size-exclusion chromatography was employed due to its advantages in the separation and molecular weight approximation of large biomolecules. It was coupled with two on-line detection systems (spectrophotometric and inductively coupled plasma mass spectrometric) to study the binding of chromium to DNA or other components in a DNA extract. A collision cell was pressurized with helium to remove diatomic and polyatomic interferents resulting from the chosen mobile phase. Chromium peaks were observed in both the large and small molecular weight regions of the chromatogram; to further confirm that the environmentally extracted DNA contained Cr, the subsequently purified DNA was examined for total Cr using flow injection ICP-MS to accommodate small sample volumes. DNA samples isolated from the two soils examined contained 0.5-0.7 ppb Cr, indicating that DNA isolated directly from a chromium-contaminated soil has chromium bound to the nucleic acids.

Investigation of selenium-containing root exudates of Brassica juncea using HPLC-ICP-MS and ESI-qTOF-MS.

Selenium-containing root exudates were investigated in a known selenium accumulator model plant. Indian mustard (Brassica juncea) plants were grown hydroponically and supplemented with selenite (SeO(3)(2-)) in a 25% Hoagland's nutrient solution. Additive concentrations were 0, 1, 5 and 20 microg mL(-1) Se with five replicate plants per treatment level. Plants were exposed to the respective Se solutions for two weeks, then placed in deionized water for two more weeks. The hydroponic solutions were collected for analysis after the first two weeks of selenium supplementation (day 14) and twice during the deionized water period (days 21 and 28). Separation by ion-pairing high performance liquid chromatography was followed by inductively coupled plasma-mass spectrometry (ICP-MS) for selenium specific detection. Chromatographic peaks unable to be identified by retention-time matching were collected for analysis by electrospray ionization mass spectrometry (ESI-MS). Additional chemical experiments were performed for structural elucidation. Several selenium-containing compounds were identified in the exudate-containing solution and two were identified as selenocystine and the selenosulfate (SSeO(3)(2-)) ion. The presence of dimethylselenide (CH(3)SeCH(3)) is also observed but cannot be attributed exclusively to plant exudation because plants were not grown in sterile conditions. Further, the incorporation of fortified selenoamino acids into peptide structures was found to occur under neutral pH conditions, suggesting that exuded enzymes might facilitate such a reaction. Finally, physiological differences resulting from selenium supplementations were noted and discussed.

Analysis of phosphorus herbicides by ion-pairing reversed-phase liquid chromatography coupled to inductively coupled plasma mass spectrometry with octapole reaction cell.

A reversed phase ion-pairing high performance liquid chromatographic (RPIP-HPLC) method is developed for the separation of two phosphorus herbicides, Glufosinate and Glyphosate as well as Aminomethylphosphonic acid (AMPA), the major metabolite of Glyphosate. Tetrabutylammonium hydroxide is used as the ion-pairing reagent in conjunction with an ammonium acetate/acetic acid buffering system at pH 4.7. An inductively coupled plasma mass spectrometer (ICP-MS) is coupled to the chromatographic system to detect the herbicides at m/z = 31P. Historically, phosphorus has been recognized as one of the elements difficult to analyze in argon plasma. This is due to its relatively high ionization potential (10.5 eV) as well as the inherent presence of the polyatomic interferences 14N16O1H+ and 15N16O+ overlapping its only isotope at m/z = 31. An octapole reaction cell is utilized to minimize the isobaric polyatomic interferences and to obtain the highest signal-to-background ratio. Detection limits were found to be in the low ppt range (25-32 ng/l). The developed method is successfully applied to the analysis of water samples collected from the Ohio River and spiked with a standard compounds at a level of 20 microg/l.

Retention of Cr(III) by high-performance chelation ion chromatography interfaced to inductively-coupled plasma mass spectrometric detection with collision cell.

High-performance chelation ion chromatography (HPCIC) was employed to retain cationic Cr(III) on an anion-exchange column and hence allow the separation of the two most prevalent forms of chromium, Cr(II) and Cr(VI). A mobile phase of nitric acid was utilized at pH = 1.5; additionally, 2,6-pyridinedicarboxylic acid was used at a concentration of 6 mM. Additives with different structural characteristics were used in an effort to elucidate retention mechanisms. Inductively-coupled plasma mass spectrometry was used for chromium detection. A collision cell was utilized to reduce chloride-based polyatomic ions that may interfere with the detection of Cr(III), and a detection limit study yielded levels in the low part-per-billion range. The newly developed method was applied to the chromatographic analysis of samples of an incubation medium containing Cr(VI) incubated with cell nuclei.

Studies of selenium-containing volatiles in roasted coffee.

Coffee has been an important and heavily used beverage in many cultures over a long period of time. Although sulfur species have been found to be abundant constituents, no work to date has explored the presence of selenium analogues. Investigation of volatile selenium species from green coffee beans, roasted beans, and brewed coffee drink was performed using solid phase microextraction (SPME) sample preconcentration in conjunction with GC/ICP-MS. Several volatile selenium species at trace levels were detected from roasted coffee beans as well as in the steam from brewed coffee drinks. No detectable selenium (and sulfur) species, however, were found in the headspace of green beans, indicating that selenium-containing volatiles are formed during roasting, as is the case for the sulfur volatiles. Matching standards were prepared and used to identify the compounds found in coffee. Artificial supplementation of the green coffee beans with selenium before roasting was performed to further characterize the selenium-containing volatiles formed during the coffee-roasting process.

Characterization of selenium species in Brazil nuts by HPLC-ICP-MS and ES-MS.

Brazil nuts have been classified as the foodstuffs that contain the highest level of unadulterated selenium, an essential trace element that appears to prevent cancer. To date, characterization of the selenium species in brazil nuts has not yet been investigated. In this work, various sample preparation approaches, including microwave extractions and enzymatic treatments, are examined with the goal of species preservation and subsequent selenium speciation; of these approaches, an enzymatic treatment with Proteinase K proved most effective. High-performance liquid chromatography (HPLC) separation strategies and inductively coupled plasma mass spectrometry (ICP-MS) detection schemes will also be presented. Extracts are evaluated against available standards for the commercially obtainable seleno-amino acids, selenomethionine (SeMet), selenoethionine (SeEt), and selenocystine (SeCys); selenomethionine was demonstrated to be the most abundant of these seleno-amino acids. Further characterization of unidentified selenium-containing peaks is attempted by the employment of several procedures, including electrospray-mass spectrometry (ES-MS). A peptide structure was identified; however, this was considered a tentative proposal due to the large background produced by the extremely complicated brazil nut matrix.

A study of method robustness for arsenic speciation in drinking water samples by anion exchange HPLC-ICP-MS.

Regulating arsenic species in drinking waters is a reasonable objective, since the various species have different toxicological impacts. However, developing robust and sensitive speciation methods is mandatory prior to any such regulations. Numerous arsenic speciation publications exist, but the question of robustness or ruggedness for a regulatory method has not been fully explored. The present work illustrates the use of anion exchange chromatography coupled to ICP-MS with a commercially available "speciation kit" option. The mobile phase containing 2 mM NaH(2)PO(4) and 0.2 mM EDTA at pH 6 allowed adequate separation of four As species (As(III), As(V), MMAA, DMAA) in less than 10 min. The analytical performance characteristics studied, including method detection limits (lower than 100 ng L(-1) for all the species evaluated), proved the suitability of the method to fulfill the current regulation. Other parameters evaluated such as laboratory fortified blanks, spiked recoveries, and reproducibility over a certain period of time produced adequate results. The samples analyzed were taken from water utilities in different areas of the United States and were provided by the U.S. EPA. The data suggests the speciation setup performs to U.S. EPA specifications but sample treatment and chemistry are also important factors for achieving good recoveries for samples spiked with As(III) as arsenite and As(V) as arsenate.

Solid-phase microextraction as a sample preparation strategy for the analysis of seleno amino acids by gas chromatography-inductively coupled plasma mass spectrometry.

Solid-phase microextraction (SPME) is used as a sample preparation strategy for gas chromatographic (GC) analysis of the seleno amino acids, selenomethionine (SeMet), selenoethionine (SeEt) and selenocystine (SeCys). Acylation of the amino group and esterification of the carboxylic group in these compounds was performed with isobutylchloroformate to increase volatility. The amino acid derivatives were then extracted by silica fibers with polydimethylsiloxane (PDMS) coatings prepared by the sol-gel process. Investigations of extraction time, acid and salt addition, and polymer length (for the sol-gel process) were conducted with the goal of procedural optimization. Initial characterizations were conducted using gas chromatography with flame ionization detection (GC-FID). Inductively coupled plasma mass spectrometric detection was employed for final selenium detection. Sub-ppb detection limits were obtained for all analytes although relative standard deviations were higher than those typically obtained in solid-phase microextraction.