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Hydrogen sulfide is a key gasotransmitter for plants and has been shown to greatly increase their growth and survival in the presence of environmental stressors. Current methods for slowly releasing hydrogen sulfide use chemicals, such as GYY-4137, but these result in the release of chemicals not found in the environment, and chemicals used may lack structures that can be readily tuned to affect the rate of release of hydrogen sulfide. In this article, we describe the synthesis and slow release of hydrogen sulfide from dialkyldithiophosphates, which are a new set of hydrogen sulfide releasing chemicals that can be used in agriculture. The rates of hydrolysis of dibutyldithiophosphate and GYY-4137 were measured in water at 85 °C and compared with each other to investigate their differences. GYY-4137 is widely used as a chemical that slowly releases H 2 S, but its rate of release was not previously quantified. The release of hydrogen sulfide in water at room temperature was measured for a series of dialkyldithiophosphates using a hydrogen sulfide electrode. It was shown that the structure of the dialkyldithiophosphate affected the amount of hydrogen sulfide released. The final degradation products of dibutyldithiophosphate were shown to be phosphoric acid and butanol, which are chemicals found in the environment. This result was notable because it demonstrated that dialkyldithiophosphates degrade to safe, natural chemicals that will not pollute the environment. To demonstrate that dialkyldithiophosphates have potential applications in agriculture, maize was grown for 4.5 weeks after exposure to 1-200 mg of dibutyldithiophosphate, and the weight of corn plants increased by up to 39% at low loadings of dibutyldithiophosphate.

Shiga-like toxins (verotoxins) are a class of AB5 holotoxins that are primarily responsible for the virulence associated with Shiga-like toxin producing Escherichia coli (STEC) infections. The holotoxins are composed of a pentamer of identical subunits (B subunit) responsible for delivering the catalytic subunit (A subunit) to a host cell and facilitating endocytosis of the toxin into the cell. The B subunits are not associated with toxicity. We developed a multiple reaction monitoring method based on analyzing conserved peptides, derived from the tryptic digestion of the B subunits. Stable-isotope-labeled analogues were prepared and used as internal standards to identify and quantify these characteristic peptides. We were able to detect and quantify Shiga toxins (Stx), Shiga-like toxin type 1 (Stx1) and type 2 (Stx2) subtypes, and to distinguish among most of the known subtypes. The limit of detection for digested pure standards was in the low attomole range/injection (~10 attomoles), which corresponded to a concentration of 1.7 femtomol/mL. A matrix effect was observed when dilute samples were digested in the buffer, Luria broth, or mouse plasma (LOD ~ 30 attomol/injection = 5 femtomol/mL). In addition, we determined that the procedures necessary to perform our mass spectrometry-based analysis completely inactivate the toxins present in the sample. This is a safe and effective method of detecting and quantitating Stx, Stx1, and Stx2, since it does not require the use of intact toxins.

We employed a sensitive mass spectrometry-based method to deconstruct, confirm, and quantitate the prions present in elk naturally infected with chronic wasting disease and sheep naturally infected with scrapie. We used this approach to study the oxidation of a methionine at position 216 (Met216), because this oxidation (MetSO216) has been implicated in prion formation. Three polymorphisms (Ile218, Val218, and Thr218) of sheep recombinant prion protein were prepared. Our analysis showed the novel result that the proportion of MetSO216 was highly dependent upon the amino acid residue at position 218 (I > V > T), indicating that Ile218 in sheep and elk prion protein (PrP) renders the Met216 intrinsically more susceptible to oxidation than the Val218 or Thr218 analogue. We were able to quantitate the prions in the attomole range. The presence of prions was verified by the detection of two confirmatory peptides: GENFTETDIK (sheep and elk) and ESQAYYQR (sheep) or ESEAYYQR (elk). This approach required much smaller amounts of tissue (600 μg) than traditional methods of detection (enzyme-linked immunosorbent assay, Western blot, and immunohistochemical analysis) (60 mg). In sheep and elk, a normal cellular prion protein containing MetSO216 is not actively recruited and converted to prions, although we observed that this Met216 is intrinsically more susceptible to oxidation.

Multiple lines of evidence for indicating factors associated with the sources, transport, and fate of chloroform and three other trihalomethanes (THMs) in untreated groundwater were revealed by evaluating low-level analytical results and logistic regression results for THMs. Samples of untreated groundwater from wells used for drinking water were collected from 1996-2007 from 2492 wells across the United States and analyzed for chloroform, bromodichloromethane, dibromochloromethane, and bromoform by a low-level analytical method implemented in April 1996. Using an assessment level of 0.02 μg/L, chloroform was detected in 36.5% of public-well samples and 17.6% of domestic-well samples, with most concentrations less than 1 μg/L. Brominated THMs occurred less frequently than chloroform but more frequently in public-well samples than domestic-well samples. For both public and domestic wells, THMs occurred most frequently in urban areas. Logistic regression analyses showed that the occurrence of THMs was related to nonpoint sources such as urban land use and to point sources like septic systems. The frequent occurrence and concentration distribution pattern of THMs, as well as their frequent co-occurrence with other organic compounds and nitrate, all known to have anthropogenic sources, and the positive associations between THM occurrence and dissolved oxygen and recharge indicate the recycling of water that contains THMs and other anthropogenic contaminants.

We developed a sensitive mass spectrometry-based method of quantitating the prions present in a variety of mammalian species. Calibration curves relating the area ratios of the integrated MRM signals from selected analyte peptides and their oxidized analogues to their homologous stable isotope labeled internal standards were prepared. The limit of detection (LOD) and limit of quantitation (LOQ) for the synthetic peptides from human, sheep, deer, cow, and mouse PrP were determined to be below 100 amol. Nonanalyte peptides that were characteristic of prions were included in the multiple reaction monitoring method, thereby allowing for both the quantitation and confirmation of the presence of prions in the attomole range. This method was used to quantitate the prions present in brains of hamsters or mice 5 weeks after inoculation (ic) with either four hamster-adapted prion strains (139H, drowsy, 22AH, and 22CH) or four mouse-adapted prion strains (Me7, Me7-298, RML, and 79A). The prions from different brain regions of a sheep naturally infected with scrapie were quantitated. All of the rodent-adapted prion strains were detectable in the asymptomatic animals. In sheep, prions were detectable in the obex, anterior portion of the cerebrum, and the nonobex/nonanterior portion of the cerebrum. This mass spectrometry-based approach can be used to quantitate and confirm the presence of prions before detectable pathology.

Prions are infectious proteins that are able to recruit a normal cellular prion protein and convert it into a prion. The mechanism of this conversion is unknown. Detailed analysis of the normal cellular prion protein and a corresponding prion has shown they possess identical post-translational modifications and differ solely in conformation. Recent work has suggested that the oxidized form of the methionine at position 213 (Met213) plays a role in the conversion of the normal cellular prion protein to the prion conformation and is a prion-specific covalent signature. We developed a sensitive method of quantitating the methionine sulfoxide present at position 213 (MetSO213) and used this method to measure the changes in MetSO213 over the time course of an intracranial challenge, using the 263K strain of hamster-adapted scrapie. These results indicate that the proportion of Met213 that is oxidized decreases over the course of the disease. We examined the quantity of MetSO213 in PrP(C) and compared it to the amount found in animals terminally afflicted with the 263K, 139H, and drowsy strains of hamster-adapted scrapie. These strains show only low levels of MetSO213 that is comparable to that of PrP(C). These data suggest that MetSO213 does not appear to be a prion-specific covalent signature.

A cell-free translation (CFT) assay for detecting Shiga toxin (Stx) at different levels of purity has been validated. The limits of detection for pure Stx2 (PStx2) and partially pure Stx2 (PPStx2) in water reached 20 and 3.5 pg/microL, respectively, without the artificial process of proteolytic activation and reduction of the pro-toxin. The specific detection of Stx2 was confirmed by a neutralization test using Stx2-specific mouse monoclonal antibody. This assay can be used for differentiation of Stx-producing Escherichia coli from non-Stx-producing E. coli. Four E. coli O157:H7 strains genotypically different for Stx were tested. The translational inhibition of Stx-producing E. coli was significantly higher than that of non-Stx-producing E. coli when bacterial culture supernatants were used for the analysis. Inhibition occurred even with supernatants diluted 1000-fold. The thermal stability of Stx2 was studied using the CFT assay, and significant differences were observed among three Stx2 preparations heated at 70 degrees C for 60 min. It was concluded that the CFT assay is a rapid, specific, and sensitive method for detecting Stx2 activity.

The castor seed contains ricin, which is one of the most potent biological toxins and is widely considered to be a threat agent for bioterrorism. In this study, a rapid and sensitive PCR method was applied to the detection of castor contamination in milk and liquid egg samples. The targeting gene sequence of the primer set, Ricin-F4/R4, was not found in either the bovine or chicken genome. Primers against a highly conserved sequence from the 18S ribosomal RNA gene were used as a positive control for DNA extraction and PCR reaction efficiency. The quantity and quality of DNA prepared from castor spiked or nonspiked milk and egg samples obtained from three different DNA extraction methods were compared. The cetyl trimethylammonium bromide (CTAB) method yielded the highest quality of DNA and is most suitable for the sensitive detection of castor DNA by real-time PCR in both milk and liquid egg matrixes. However, taking time and cost into consideration, a commercial kit designed for extraction of DNA from stool samples could be used as an alternative method for the routine extraction of DNA from milk for real-time PCR assays. The egg matrix was found to inhibit PCR amplification and interfere with two of the three methods tested for DNA extraction. Egg yolk had a greater negative effect on PCR amplification than the egg white matrix. Our results affirm the necessity of performing individual validations for each food matrix. Both real-time PCR systems used in this study, TaqMan and SYBR Green I dye, were capable of detecting 100 ng of castor acetone powder, corresponding to 5 ng of ricin, in 1 mL of milk or liquid egg, well below the toxic dose for humans. On the basis of these results, the real-time PCR method for detection of intentional castor contamination is applicable to milk and egg matrixes.

Due to the potential for intentional contamination of food with crude preparations containing ricin, a real-time PCR method was developed for the detection of castor plant material in ground beef. One primer pair was identified and confirmed to be castor-specific and efficient for amplification of ricin in DNA extracts from castor or beef matrices. Of three different DNA extraction protocols compared, the hexadecyltrimethylammonium bromide (CTAB) method yielded the highest quality of DNA for QPCR assay. The detection limit for castor contamination in ground beef samples was <0.001% (<10 microg of castor acetone powder per gram of beef, corresponding to 0.5 microg of ricin), indicating excellent sensitivity for the assay, well below the threshold for oral toxicity.

The use of linear peptides as antigens for detection of serum antibodies has been studied using a sequence of the Borrelia burgdorferi protein, flagellin, and Lyme disease sera as a model. It was found that a novel presentation of the peptide as a hapten on the carrier protein, bovine serum albumin, in the enzyme-linked immunosorbent assay format can be successfully applied to distinguish between Lyme disease and control sera.