Large outbreak of Jimsonweed (Datura stramonium) poisoning due to consumption of contaminated humanitarian relief food: Uganda, March-April 2019.

BACKGROUND: Jimsonweed (Datura stramonium) contains toxic alkaloids that cause gastrointestinal and central nervous system symptoms when ingested. This can be lethal at high doses. The plant may grow together with leguminous crops, mixing with them during harvesting. On 13 March 2019, more than 200 case-patients were admitted to multiple health centres for acute gastrointestinal and neurologic symptoms. We investigated to determine the cause and magnitude of the outbreak and recommended evidence-based control and prevention measures. METHODS: We defined a suspected case as sudden onset of confusion, dizziness, convulsions, hallucinations, diarrhoea, or vomiting with no other medically plausible explanations in a resident of Napak or Amudat District from 1 March-30 April 2019. We reviewed medical records and canvassed all villages of the eight affected subcounties to identify cases. In a retrospective cohort study conducted in 17 villages that reported the earliest cases, we interviewed 211 residents about dietary history during 11-15 March. We used modified Poisson regression to assess suspected food exposures. Food samples underwent chemical (heavy metals, chemical contaminants, and toxins), proteomic, DNA, and microbiological testing in one national and three international laboratories. RESULTS: We identified 293 suspected cases; five (1.7%) died. Symptoms included confusion (62%), dizziness (38%), diarrhoea (22%), nausea/vomiting (18%), convulsions (12%), and hallucinations (8%). The outbreak started on 12 March, 2-12 h after Batch X of fortified corn-soy blend (CSB +) was distributed. In the retrospective cohort study, 66% of 134 persons who ate CSB + , compared with 2.2% of 75 who did not developed illness (RRadj = 22, 95% CI = 6.0-81). Samples of Batch X distributed 11-15 March contained 14 tropane alkaloids, including atropine (25-50 ppm) and scopolamine (1-10 ppm). Proteins of Solanaceae seeds and Jimsonweed DNA were identified. No other significant laboratory findings were observed. CONCLUSION: This was the largest documented outbreak caused by food contamination with tropane alkaloids. Implicated food was immediately withdrawn. Routine food safety and quality checks could prevent future outbreaks.

Screening of Processed Foods for Transgenic Proteins from Genetically Engineered Plants Using Targeted Mass Spectrometry.

Screening of food products for the presence of material from genetically engineered (GE) plants is typically done using deoxyribonucleic acid (DNA)-based methods to detect the presence of transgenic DNA. In this study, we have demonstrated the feasibility of using targeted mass spectrometry (MS) to detect a protein expressed by transgenic DNA to confirm the presence of GE plant material in processed foods. Scheduled parallel reaction monitoring (sPRM) was used to detect the enzyme, 5-enolpyruvulshikimate-3-phosphate synthase, from Agrobacterium sp. strain CP4 (CP4 EPSPS), which confers glyphosate tolerance in transgenic crops. Five CP4 EPSPS surrogate peptides and their corresponding retention times identified via data-dependent LC/MS/MS analysis of a glyphosate-tolerant soybean certified reference material, GTS 40-3-2, were used to develop the sPRM assay. The assay was used to screen four soy-based infant formulas, four corn-based cereals, corn tortilla chips, and cornmeal for the presence of CP4 EPSPS. At least four of the five selected surrogate peptides were detected in nine of the products analyzed, suggesting that targeted MS can serve as a complementary analytical method to DNA-based methods for the detection of material from GE plants in processed foods.

Identification of Salmonella Taxon-Specific Peptide Markers to the Serovar Level by Mass Spectrometry.

We present an LC-MS/MS pipeline to identify taxon-specific tryptic peptide markers for the identification of Salmonella at the genus, species, subspecies, and serovar levels of specificity. Salmonella enterica subsp. enterica serovars Typhimurium and its four closest relatives, Saintpaul, Heidelberg, Paratyphi B, and Muenchen, were evaluated. A decision-tree approach was used to identify peptides common to the five Salmonella proteomes for evaluation as genus-, species-, and subspecies-specific markers. Peptides identified for two or fewer Salmonella strains were evaluated as potential serovar markers. Currently, there are approximately 140 000 assembled bacterial genomes publicly available, more than 8500 of which are for Salmonella. Consequently, the specificity of each candidate peptide marker was confirmed across all publicly available protein sequences in the NCBI nonredundant (nr) database. The performance of a subset of candidate taxon-specific peptide markers was evaluated in a targeted mass-spectrometry method. The presented workflow offers a marked improvement in specificity over existing MALDI-TOF-based bacterial identification platforms for the identification of closely related Salmonella serovars.

Influences of simulated gastrointestinal environment on physicochemical properties of gold nanoparticles and their implications on intestinal epithelial permeability.

Gold nanoparticles (Au NPs) hold great promise in food, industrial and biomedical applications due to their unique physicochemical properties. However, influences of the gastrointestinal tract (GIT), a likely route for Au NPs administration, on the physicochemical properties of Au NPs has been rarely evaluated. Here, we investigated the influence of GIT fluids on the physicochemical properties of Au NPs (5, 50, and 100 nm) and their implications on intestinal epithelial permeability in vitro. Au NPs aggregated in fasted gastric fluids and generated hydroxyl radicals in the presence of H2O2. Cell studies showed that GIT fluids incubation of Au NPs affected the cellular uptake of Au NPs but did not induce cytotoxicity or disturb the intestinal epithelial permeability.

Ferroxidase-like and antibacterial activity of PtCu alloy nanoparticles.

Many metal nanoparticles are reported to have intrinsic enzyme-like activities and offer great potential in chemical and biomedical applications. In this study, PtCu alloy nanoparticles (NPs), synthesized through hydrothermal treatment of Cu2+ and Pt2+ in an aqueous solution, were evaluated for ferroxidase-like and antibacterial activity. Electron spin resonance (ESR) spectroscopy and colorimetric methods were used to demonstrate that PtCu NPs exhibited strong ferroxidase-like activity in a weakly acidic environment and that this activity was not affected by the presence of most other ions, except silver. Based on the color reaction of salicylic acid in the presence of Fe3+, we tested the ferroxidase-like activity of PtCu NPs to specifically detect Fe2+ in a solution of an oral iron supplement and compared these results with data acquired from atomic absorption spectroscopy and the phenanthroline colorimetric method. The results showed that the newly developed PtCu NPs detection method was equivalent to or better than the other two methods used for Fe2+ detection. The antibacterial experiments showed that PtCu NPs have strong antibacterial activity against Staphylococcus aureus and Escherichia coli. Herein, we demonstrate that the peroxidase-like activity of PtCu NPs can catalyze H2O2 and generate hydroxyl radicals, which may elucidate the antibacterial activity of the PtCu NPs against S. aureus and E. coli. These results showed that PtCu NPs exhibited both ferroxidase- and peroxidase-like activity and that they may serve as convenient and efficient NPs for the detection of Fe2+ and for antibacterial applications.

Analysis of Gluten in a Wheat-Gluten-Incurred Sorghum Beer Brewed in the Presence of Proline Endopeptidase by LC/MS/MS.

Most gluten-reduced beers are produced using an enzyme called proline endopeptidase (PEP), which proteolyzes the gluten by cleaving at proline residues. However, the gluten content of beers brewed in the presence of PEP cannot be verified since current analytical methods are not able to accurately quantitate gluten in fermented foods. In this work, mass spectrometry was used to qualitatively characterize the gluten in a wheat-gluten-incurred sorghum model beer brewed with and without the addition of PEP. Hydrolyzed gluten peptides and chymotryptic gluten peptides produced from intact gluten proteins were detected in beer brewed in the presence of up to 6 times the manufacturer's recommended dosage of PEP. The observation of chymotryptic gluten peptides indicates that some gluten proteins remained, at least partially, intact after fermentation and enzymatic treatment. Less intact gluten was observed in beer brewed in the presence of PEP, but more hydrolyzed gluten peptides were consequently observed in PEP-containing beer. Gluten peptides that contained immunogenic sequences known to be associated with celiac disease were detected in PEP-containing beer.

Effects of noble metal nanoparticles on the hydroxyl radical scavenging ability of dietary antioxidants.

Noble metal nanoparticles (NPs) have been widely used in many consumer products. Their effects on the antioxidant activity of commercial dietary supplements have not been well evaluated. In this study, we examined the effects of gold (Au NPs), silver (Ag NPs), platinum (Pt NPs), and palladium (Pd NPs) on the hydroxyl radical (·OH) scavenging ability of three dietary supplements vitamin C (L-ascorbic acid, AA), (-)-epigallocatechin gallate (EGCG), and gallic acid (GA). By electron spin resonance (ESR) spin-trapping measurement, the results show that these noble metal NPs can inhibit the hydroxyl radical scavenging ability of these dietary supplements.

Exploring the activities of ruthenium nanomaterials as reactive oxygen species scavengers.

Research on noble metal nanoparticles (NPs) able to scavenge reactive oxygen species (ROS) has undergone a tremendous growth recently. However, the interactions between ruthenium nanoparticles (Ru NPs) and ROS have never been systematically explored thus far. This research focused on the decomposition of hydrogen peroxide (H2O2), scavenging of hydroxyl radicals (•OH), superoxide radical (O2•-), singlet oxygen (1O2), 2,2'-azino-bis(3-ethylbenzenothiazoline- 6-sulfonic acid ion (ABTS•+), and 1,1-diphenyl-2-picrylhydrazyl radical (•DPPH) in the presence of commercial Ru NPs using the electron spin resonance technique. In vitro cell studies demonstrated that Ru NPs have excellent biocompatibility and exert a cytoprotective effect against oxidative stress. These findings may spark fresh enthusiasm for the applications of Ru NPs under relevant physiologically conditions.

Nontargeted Screening of Food Matrices: Development of a Chemometric Software Strategy To Identify Unknowns in Liquid Chromatography-Mass Spectrometry Data.

The ability to identify contaminants or adulterants in diverse, complex sample matrixes is necessary in food safety. Thus, nontargeted screening approaches must be implemented to detect and identify unexpected, unknown hazardous compounds that may be present. Molecular formulas can be generated for detected compounds from high-resolution mass spectrometry data, but analysis can be lengthy when thousands of compounds are detected in a single sample. Efficient data mining methods to analyze these complex data sets are necessary given the inherent chemical diversity and variability of food matrixes. The aim of this work is to determine necessary requirements to successfully apply data analysis strategies to distinguish suspect and control samples. Infant formula and orange juice samples were analyzed with one lot of each matrix containing varying concentrations of a four compound mixture to represent a suspect sample set. Small molecular differences were parsed from the data, where analytes as low as 10 ppb were revealed. This was accomplished, in part, by analyzing a quality control standard, matrix spiked with an analytical standard mixture, technical replicates, a representative number of sample lots, and blanks within the sample sequence; this enabled the development of a data analysis workflow and ensured that the employed method is sufficient for mining relevant molecular features from the data.

Characterization of nanoparticles in silicon dioxide food additive.

Silicon dioxide (SiO2), in its amorphous form, is an approved direct food additive in the United States and has been used as an anticaking agent in powdered food products and as a stabilizer in the production of beer. While SiO2 has been used in food for many years, there is limited information regarding its particle size and size distribution. In recent years, the use of SiO2 food additive has raised attention because of the possible presence of nanoparticles. Characterization of SiO2 food additive and understanding their physicochemical properties utilizing modern analytical tools are important in the safety evaluation of this additive. Herein, we present analytical techniques to characterize some SiO2 food additives, which were obtained directly from manufacturers and distributors. Characterization of these additives was performed using dynamic light scattering (DLS), transmission electron microscopy (TEM), field emission scanning electron microscopy (FE-SEM), and single-particle inductively coupled plasma mass spectrometry (spICP-MS) after the food additive materials underwent different experimental conditions. The data obtained from DLS, spICP-MS, and electron microscopy confirmed the presence of nanosized (1-100 nm) primary particles, as well as aggregates and agglomerates of aggregates with sizes greater than 100 nm. SEM images demonstrated that most of the SiO2 food additives procured from different distributors showed similar morphology. The results provide a foundation for evaluating the nanomaterial content of regulated food additives and will help the FDA address current knowledge gaps in analyzing nanosized particles in commercial food additives.

A robust high-throughput sample preparation and liquid chromatography/tandem mass spectrometry method for the quantitation of ß-lyase metabolites of sulfur mustard as 1,1'-sulfonylbis-[2-(methylthio)ethane] in human urine.

RATIONALE: Sulfur mustard (HD) is a major chemical warfare agent threat to humans. Since World War I, several incidents of human exposure to sulfur mustard have been reported. In order to assist health professionals during an exposure event and support biological monitoring, a rapid analytical method is required to measure the exposure of humans to HD. METHOD: The ß-lyase metabolites of HD, 1-methylsulfinyl-2-[2-(methylthio)ethylsulfonyl]ethane (MSMTESE) and 1,1'-sulfonylbis[2-(methylsulfinyl)ethane] (SBMSE) were reduced to the single biomarker, 1,1'-sulfonylbis-[2-(methylthio)ethane] (SBMTE), using titanium(III) chloride. High-throughput sample preparation was performed on a Tecan Freedom EVO liquid handler and analysis was performed by electrospray ionization liquid chromatography and tandem mass spectrometry (LC/MS/MS) in the multiple-reaction monitoring mode. RESULTS: Each analytical run consisted of a matrix blank, calibration standards (0.1-100 ng/mL), low quality controls (QCs), 2.5 ng/mL, and high QCs, 25.0 ng/mL, of SBMTE in human urine. The method was validated with 20 analytical runs performed by four analysts. The mean calculated concentrations of the low and high QCs were 2.52 and 25.5 ng/mL with relative standard deviations of 3.6% and 2.3%, respectively. CONCLUSION: This semi-automated method has few manual transfer steps, thus minimizing common manual errors and saving time. Therefore, this method would be very helpful to responding laboratories in a large-scale exposure event related to HD.

Combining targeted and nontargeted data analysis for liquid chromatography/high-resolution mass spectrometric analyses.

Increasing importation of food and the diversity of potential contaminants have necessitated more analytical testing of these foods. Historically, mass spectrometric methods for testing foods were confined to monitoring selected ions (SIM or MRM), achieving sensitivity by focusing on targeted ion signals. A limiting factor in this approach is that any contaminants not included on the target list are not typically identified and retrospective data mining is limited. A potential solution is to utilize high-resolution MS to acquire accurate mass full-scan data. Based on the instrumental resolution, these data can be correlated to the actual mass of a contaminant, which would allow for identification of both target compounds and compounds that are not on a target list (nontargets). The focus of this research was to develop software algorithms to provide rapid and accurate data processing of LC/MS data to identify both targeted and nontargeted analytes. Software from a commercial vendor was developed to process LC/MS data and the results were compared to an alternate, vendor-supplied solution. The commercial software performed well and demonstrated the potential for a fully automated processing solution.

The Utility of Genomic and Transcriptomic Data in the Construction of Proxy Protein Sequence Databases for Unsequenced Tree Nuts.

As the apparent incidence of tree nut allergies rises, the development of MS methods that accurately identify tree nuts in food is critical. However, analyses are limited by few available tree nut protein sequences. We assess the utility of translated genomic and transcriptomic data for library construction with Juglans regia, walnut, as a model. Extracted walnuts were subjected to nano-liquid chromatography-mass spectrometry (n-LC-MS/MS), and spectra were searched against databases made from a six-frame translation of the genome (6FT), a transcriptome, and three proteomes. Searches against proteomic databases yielded a variable number of peptides (1156-1275), and only ten additional unique peptides were identified in the 6FT database. Searches against a transcriptomic database yielded results similar to those of the National Center for Biotechnology Information (NCBI) proteome (1200 and 1275 peptides, respectively). Performance of the transcriptomic database was improved via the adjustment of RNA-Seq read processing methods, which increased the number of identified peptides which align to seed allergen proteins by ~20%. Together, these findings establish a path towards the construction of robust proxy protein databases for tree nut species and other non-model organisms.

Optimized chemical coverage and data quality for non-targeted screening applications using liquid chromatography/high-resolution mass spectrometry.

Non-targeted small molecule screening methods are used to analyze samples for potential compounds of interest without focusing on specific molecular species. There is great interest in these methods for metabolomic, environmental, forensic, and food safety applications, among others, to determine compounds that are responsible for a particular disease state or the presence of a harmful compound. In order for non-targeted analyses to become standardized and routine, best practices for sample preparation, data collection, and data analysis must be determined. This work focuses on optimizing specific aspects of a non-targeted workflow that utilizes high-resolution mass spectrometry using an Orbitrap instrument coupled to liquid chromatography. Sample preparation, liquid chromatography gradient length, and mass spectrometry resolving power and ionization modes were investigated to determine optimal conditions for detecting and extracting compounds from the data that cover broad molecular and polarity ranges. Infant rice cereal, orange juice, and yogurt with spiked standards were analyzed; food is inherently challenging to analyze due in part to sample complexity and diversity. Of the conditions tested, optimal conditions included a generic sample extraction using acetonitrile, water, and formic acid, a 25 min chromatographic gradient, collecting data in both positive and negative ion modes, and using 70 k resolving power. There are of course tradeoffs associated with each of these options that will be described in detail so that the appropriate conditions can be chosen for the desired application.

Liquid chromatography/mass spectrometry characterization of Escherichia coli and Shigella species.

Liquid chromatography/quadrupole time of flight mass spectrometry (LC/QTOF MS) utilizing electrospray ionization was employed to monitor protein expression in Escherichia coli and Shigella organisms. Comparison with MALDI/TOF-MS revealed more proteins, particularly above 15 kDa. A combination of automated charge state deconvolution, spectral mirroring, and spectral subtraction was used to reveal subtle differences in the LC/MS data. Reproducible intact protein biomarker candidates were discovered based on their unique mass, retention time, and relative intensity. These marker candidates were implemented to differentiate closely related strain types, (e.g., two distinct isolates of E. coli O157:H7) and to correctly identify unknown pathogens. This LC/MS approach is less labor-intensive than pulsed-field gel electrophoresis, affords greater specificity than real-time PCR, and requires no primers or antibodies. Additionally, this approach would be beneficial during outbreaks of foodborne disease or bioterrorism investigations by complementing methods typically used in diagnostic microbiology laboratories.

Ultra-performance liquid chromatography/mass spectrometry of intact proteins.

Given that numerous small molecule applications of ultra-performance liquid chromatography (UPLC) have been published, efforts were made to examine the potential of UPLC to enhance the separation of intact proteins. Beginning with typically employed conditions, column temperature and organic solvent were optimized followed by an HPLC vs. UPLC comparison. When applied to a mixture of 10 protein standards, the optimized method yielded improved chromatographic resolution, enhanced sensitivity, and a threefold increase in throughput. Subsequent cell lysate analysis demonstrated no compromise in chromatographic or mass spectral data quality at 1/3 of the original run time.

Liquid chromatography/quadrupole ion trap/time-of-flight determination of the efficacy of drug test kits for rapid screening of food.

The goal of this study was to evaluate the plausibility and accuracy of commercially available on-site immunoassay urinalysis kits for the screening of compounds of interest within food matrices. In conjunction with this study, a sensitive, robust, and reproducible analytical method, utilizing solid-phase extraction liquid chromatography/quadrupole ion trap/time-of-flight mass spectrometry for confirmation analysis, was developed. The food matrices analyzed were tomato juice, apple juice, milk, beer, white wine, ground beef, powdered milk, and all-purpose flour. Compounds fortified into the food matrices included heroin, phencyclidine, cocaine, benzoylecgonine, methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine, imipramine, doxepin, nitrazepam, diazepam, oxazepam, temazepam, alprazolam, flunitrazepam, clonazepam, and lorazepam. Standard curves were prepared for each matrix from 10 to 500 ng/ml for each analyzed compound. All liquid chromatography/tandem mass spectrometry samples were fortified with 20 microl of deuterated internal standard at 90 ng/ml. Quality control standards were prepared at 20 and 400 ng/ml, and > 90% were within 2 SD of the mean for each analyte. The test kits were found to produce up to 85% of the expected results based on concentration levels of adulterants (i-Screen in milk). This study shows that lateral-flow immunoassay test kits are plausible as a rapid, accurate, and reliable screening method in the event of adulteration of the food supply.

Bactericidal Effects of Silver Nanoparticles on Lactobacilli and the Underlying Mechanism.

While the antibacterial properties of silver nanoparticles (AgNPs) have been demonstrated across a spectrum of bacterial pathogens, the effects of AgNPs on the beneficial bacteria are less clear. To address this issue, we compared the antibacterial activity of AgNPs against two beneficial lactobacilli ( Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus casei) and two common opportunistic pathogens ( Escherichia coli and Staphylococcus aureus). Our results demonstrate that those lactobacilli are highly susceptible to AgNPs, while the opportunistic pathogens are not. Acidic environment caused by the lactobacilli is associated with the bactericidal effects of AgNPs. Our mechanistic study suggests that the acidic growth environment of lactobacilli promotes AgNP dissolution and hydroxyl radical (•OH) overproduction. Furthermore, increases in silver ions (Ag+) and •OH deplete the glutathione pool inside the cell, which is associated with the increase in cellular reactive oxygen species (ROS). High levels of ROS may further induce DNA damage and lead to cell death. When E. coli and S. aureus are placed in a similar acidic environment, they also become more susceptible to AgNPs. This study provides a mechanistic description of a pH-Ag+-•OH bactericidal pathway and will contribute to the responsible development of products containing AgNPs.

Platinum nanoparticles: an avenue for enhancing the release of nitric oxide from S-nitroso-N-acetylpenicillamine and S-nitrosoglutathione.

Nitric oxide (NO) is an endogenous bioregulator with established roles in diverse fields. The difficulty in the modulation of NO release is still a significant obstacle to achieving successful clinical applications. We report herein our initial work using electron spin resonance (ESR) spectroscopy to detect NO generated from S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione (GSNO) donors catalyzed by platinum nanoparticles (Pt NPs, 3 nm) under physiological conditions. With ESR spectroscopy coupled with spin trapping and spin labeling techniques, we identified that Pt NPs can significantly promote the generation of NO from SNAP and GSNO under physiological conditions. A classic NO colorimetric detection kit was also employed to verify that Pt NPs truly triggered the release of NO from its donors. Pt NPs can act as promising delivery vehicles for on-demand NO delivery based on time and dosage. These results, along with the detection of the resulting disulfide product, were confirmed with mass spectrometry. In addition, cellular experiments provided a convincing demonstration that the triggered release of NO from its donors by Pt NPs is efficient in killing human cancer cells in vitro. The catalytic mechanism was elucidated by X-ray photo-electron spectroscopy (XPS) and ultra-high performance liquid chromatography/high-resolution mass spectrometry (UHPLC-HRMS), which suggested that Pt-S bond formation occurs in the solution of Pt NPs and NO donors. Identification of Pt NPs capable of generating NO from S-nitrosothiols (RSNOs) is an important step in harnessing NO for investigations into its clinical applications and therapies.

Isotope dilution LC/MS/MS for the detection of nerve agent exposure in urine.

Organophosphorus nerve agents (OPNA), chemically related to and derived from organophosphate insecticides, constitute a clear and present threat to both military and civilian targets. Military regimes and terrorist organizations have demonstrated the will and ability to produce mass casualties by dispersing organophosphorus nerve agents, which, in turn could terrorize populations and overwhelm healthcare systems. A high throughput, robust and sensitive analytical protocol has been developed for the quantitation of the urinary metabolites of sarin (GB), soman (GD), VX, Russian VX (RVX) and cyclohexylsarin (GF) utilizing solid phase extraction (SPE) followed by High Performance Liquid Chromatography (HPLC)-isotope dilution tandem mass spectrometry (LC/MS/MS). The method has demonstrated linearity and reproducibility (1-200 ng/mL) for all analytes and has a Limit of Quantitation (LOQ)< or =0.5 ng/mL for all analytes (S/N> or =10/1). The method was validated by performing 20 individual analyses over 10 days by five scientists with all values falling within two standard deviations of the mean.