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 Implicated food was immediately withdrawn.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.

Identification and Structural Characterization of Naturally-Occurring Broad-Spectrum Cyclic Antibiotics Isolated from Paenibacillus.

The rise of antimicrobial resistance necessitates the discovery and/or production of novel antibiotics. Isolated strains of Paenibacillus alvei were previously shown to exhibit antimicrobial activity against a number of pathogens, such as E. coli, Salmonella, and methicillin-resistant Staphylococcus aureus (MRSA). The responsible antimicrobial compounds were isolated from these Paenibacillus strains and a combination of low and high resolution mass spectrometry with multiple-stage tandem mass spectrometry was used for identification. A group of closely related cyclic lipopeptides was identified, differing primarily by fatty acid chain length and one of two possible amino acid substitutions. Variation in the fatty acid length resulted in mass differences of 14 Da and yielded groups of related MS(n) spectra. Despite the inherent complexity of MS/MS spectra of cyclic compounds, straightforward analysis of these spectra was accomplished by determining differences in complementary product ion series between compounds that differ in molecular weight by 14 Da. The primary peptide sequence assignment was confirmed through genome mining; the combination of these analytical tools represents a workflow that can be used for the identification of complex antibiotics. The compounds also share amino acid sequence similarity to a previously identified broad-spectrum antibiotic isolated from Paenibacillus. The presence of such a wide distribution of related compounds produced by the same organism represents a novel class of broad-spectrum antibiotic compounds.

Platform for identification of Salmonella serovar differentiating bacterial proteins by top-down mass spectrometry: S. Typhimurium vs S. Heidelberg.

Intact protein expression profiling has proven to be a powerful tool for bacterial subspecies differentiation. To facilitate typing, epidemiology, and trace-back of Salmonella contamination in the food supply, a minimum of serovar level differentiation is required. Subsequent identification and validation of marker proteins is integral to rapid screening development and to determining which proteins are subject to environmental pressure. Bacterial sequencing efforts have expanded the number of sequenced genomes available for single-nucleotide polymorphism (SNP) analyses, but annotation is often missing, start site errors are not uncommon, and the likelihood of expression is not known. In this work we show that the combination of intact protein expression profiles and top-down liquid chromatography-mass spectrometry (LC-MS/MS) facilitates the identification of proteins that result from expressed serovar specific nonsynonymous SNPs. Combinations of these marker proteins can be used in assays for rapid differentiation of bacteria. LC-MS generated intact protein expression profiles establish which bacterial protein masses differ across samples and can be determined without prior knowledge of the sample. Subsequent top-down LC-MS/MS is used to identify expressed proteins and their post-translational modifications (PTM), identify serovar specific markers, and validate genomic predicted orthologues as expressed biomarkers.

Characterization of grain-specific peptide markers for the detection of gluten by mass spectrometry.

Global and targeted mass spectrometry-based proteomic approaches were developed to discover, evaluate, and apply gluten peptide markers to detect low parts per million (ppm) wheat contamination of oats. Prolamins were extracted from wheat, barley, rye, and oat flours and then reduced, alkylated, and digested with chymotrypsin. The resulting peptides were subjected to LC-MS/MS analysis and database matching. No peptide markers common to wheat, barley, and rye were identified that could be used for global gluten detection. However, many grain-specific peptide markers were identified, and a set of these markers was selected for gluten detection and grain differentiation. Wheat flour was spiked into gluten-free oat flour at concentrations of 1-100,000 ppm and analyzed to determine the lowest concentration at which the wheat "contaminant" could be confidently detected in the mixture. The same 2D ion trap instrument that was used for the global proteomics approach was used for the targeted proteomics approach, providing a seamless transition from target discovery to application. A powerful, targeted MS/MS method enabled detection of two wheat peptide markers at the 10 ppm wheat flour-in-oat flour concentration. Because gluten comprises approximately 10% of wheat flour protein, the reported wheat gluten-specific peptides can enable detection of approximately 1 ppm of wheat gluten in oats.

Mass accuracy and isotopic abundance measurements for HR-MS instrumentation: capabilities for non-targeted analyses.

The development of automated non-targeted workflows for small molecule analyses is highly desirable in many areas of research and diagnostics. Sufficient mass and chromatographic resolution is necessary for the detectability of compounds and subsequent componentization and interpretation of ions. The mass accuracy and relative isotopic abundance are critical in correct molecular formulae generation for unknown compounds. While high-resolution instrumentation provides accurate mass information, sample complexity can greatly influence data quality and the measurement of compounds of interest. Two high-resolution instruments, an Orbitrap and a Q-TOF, were evaluated for mass accuracy and relative isotopic abundance with various concentrations of a standard mixture in four complex sample matrices. The overall average ± standard deviation of the mass accuracy was 1.06 ± 0.76 ppm and 1.62 ± 1.88 ppm for the Orbitrap and the Q-TOF, respectively; however, individual measurements were ± 5 ppm for the Orbitrap and greater than 10 ppm for the Q-TOF. Relative isotopic abundance measurements for A + 1 were within 5% of the theoretical value if the intensity of the monoisotopic peak was greater than 1E7 for the Orbitrap and 1E5 for the Q-TOF, where an increase in error is observed with a decrease in intensity. Furthermore, complicating factors were found in the data that would impact automated data analysis strategies, including coeluting species that interfere with detectability and relative isotopic abundance measurements. The implications of these findings will be discussed with an emphasis on reasonable expectations from these instruments, guidelines for experimental workflows, data analysis considerations, and software design for non-targeted analyses.

Photogenerated charge carriers and reactive oxygen species in ZnO/Au hybrid nanostructures with enhanced photocatalytic and antibacterial activity.

Semiconductor nanostructures with photocatalytic activity have the potential for many applications including remediation of environmental pollutants and use in antibacterial products. An effective way for promoting photocatalytic activity is depositing noble metal nanoparticles (NPs) on a semiconductor. In this paper, we demonstrated the successful deposition of Au NPs, having sizes smaller than 3 nm, onto ZnO NPs. ZnO/Au hybrid nanostructures having different molar ratios of Au to ZnO were synthesized. It was found that Au nanocomponents even at a very low Au/ZnO molar ratio of 0.2% can greatly enhance the photocatalytic and antibacterial activity of ZnO. Electron spin resonance spectroscopy with spin trapping and spin labeling was used to investigate the enhancing effect of Au NPs on the generation of reactive oxygen species and photoinduced charge carriers. Deposition of Au NPs onto ZnO resulted in a dramatic increase in light-induced generation of hydroxyl radical, superoxide and singlet oxygen, and production of holes and electrons. The enhancing effect of Au was dependent on the molar ratio of Au present in the ZnO/Au nanostructures. Consistent with these results from ESR measurements, ZnO/Au nanostructures also exhibited enhanced photocatalytic and antibacterial activity. These results unveiled the enhanced mechanism of Au on ZnO and these materials have great potential for use in water purification and antibacterial products.

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.

Global proteomic screening of protein allergens and advanced glycation endproducts in thermally processed peanuts.

Peanuts (Arachis hypogaea) are the cause of one of the most prevalent food allergies worldwide. Thermal processing (e.g., roasting) of peanuts and peanut-containing foods results in complex chemical reactions that alter structural conformations of peanut proteins, preventing accurate detection of allergens by most immunochemical and targeted screening methodologies. To improve food allergen detection and support more accurate food labeling, traditional methods for peanut protein extraction were modified to include protein denaturants and solubilization agents. Qualitative characterization by SDS-PAGE and Western blot analyses of raw and variably roasted peanut extracts confirmed improvements in total protein recovery and provided evidence for the incorporation of Ara h 1, Ara h 3, and, to a lesser extent, Ara h 2 into high molecular weight protein complexes upon roasting. Relative quantification of allergens in peanut lysates was accomplished by label-free spectral feature (MS1) LC-MS/MS methodologies, by which peanut allergen peptides exhibiting a differential MS response in raw versus roasted peanuts were considered to be candidate targets of thermal modification. Identification of lysine-modified Maillard advanced glycation endproducts (AGE) by LC-MS/MS confirmed the formation of (carboxymethyl)lysine (CML), (carboxyethyl)lysine (CEL), and pyrraline (Pyr) protein modifications on Ara h 1 and Ara h 3 tryptic peptides in roasted peanut varieties. These results suggest that complex processed food matrices require initial analysis by an untargeted LC-MS/MS approach to determine optimum analytes for subsequent targeted allergen analyses.

Size-selective fractionation and visual mapping of allergen protein chemistry in Arachis hypogaea.

Peanuts (Arachis hypogaea) in addition to milk, eggs, fish, crustaceans, wheat, tree nuts, and soybean are commonly referred to as the "big eight" foods that contribute to the majority of food allergies worldwide. Despite the severity of allergic reactions and growing prevalence in children and adults, there is no cure for peanut allergy, leaving avoidance as the primary mode of treatment. To improve analytical methods for peanut allergen detection, researchers must overcome obstacles involved in handling complex food matrices while attempting to decipher the chemistry that underlies allergen protein interactions. To address such challenges, we conducted a global proteome characterization of raw peanuts using a sophisticated GELFrEE-PAGE-LC-MS/MS platform consisting of gel-based protein fractionation followed by mass spectrometric identification. The in-solution mass-selective protein fractionation: (1) enhances the number of unique peptide identifications, (2) provides a visual map of protein isoforms, and (3) aids in the identification of disulfide-linked protein complexes. GELFrEE-PAGE-LC-MS/MS not only overcomes many of the challenges involved in the study of plant proteomics, but enriches the understanding of peanut protein chemistry, which is typically unattainable in a traditional bottom-up proteomic analysis. A global understanding of protein chemistry in Arachis hypogaea ultimately will aid the development of improved methods for allergen detection in food.

The chromatographic role in high resolution mass spectrometry for non-targeted analysis.

Resolution improvements in time-of-flight instrumentation and the emergence of the Orbitrap mass spectrometer have researchers using high resolution mass spectrometry to determine elemental compositions and performing screening methods based on the full-scan data from these instruments. This work is focused on examining instrument performance of both a QTOF and a bench-top Orbitrap. In this study, the impact of chromatographic resolution on mass measurement accuracy, mass measurement precision, and ion suppression is examined at a fundamental level. This work was extended to a mixture of over 200 pesticides to determine how well two different software algorithms componentized and correctly identified these compounds under different sets of chromatographic conditions, where co-elution was expected to vary markedly.

Evaluation of protein expression in bovine bronchoalveolar fluid following challenge with Mannheimia haemolytica.

Proteomics analysis of bovine bronchoalveolar fluid (BAF) following induction of pneumonia with Mannheimia haemolytica using nanoflow liquid chromatography coupled with tandem mass spectrometry (nanoLC-MS/MS) resulted in the identification of 88 unique proteins. Proteins detected in BAF included antimicrobial peptides (AMPs), complement factors, acute-phase proteins, protease inhibitors, and proteins involved in oxidation-reduction. Notwithstanding biological variation, differences in relative protein abundance, determined using normalized peptide counts, were detected for select proteins in BAF from genuinely infected versus sham-infected animals. To demonstrate the applicability of using normalized peptide counts to assess protein expression trends, LC-MS/MS data for the acute-phase protein haptoglobin (HPT) were compared with ELISA data, and statistical evaluation of the relationship between the data revealed a strong measure of association. Differences were detected between sham- and genuinely infected animals for haptoglobin, as well as the AMPs cathelicidin-1 and cathelicidin-4, and inter-α-trypsin inhibitor heavy chain-4, a fairly novel protein involved in the acute phase response. Though the small sample size limited the scope of the inferences, the results indicate the likely importance of AMPs and acute-phase proteins during respiratory infection, and provide additional information regarding potential mechanisms involved in the bovine mucosal barrier defense.

A discriminant based charge deconvolution analysis pipeline for protein profiling of whole cell extracts using liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry.

A discriminant based charge deconvolution analysis pipeline is proposed. The molecular weight determination (MoWeD) charge deconvolution method was applied directly to the discrimination rules obtained by the fuzzy rule-building expert system (FuRES) pattern classifier. This approach was demonstrated with synthetic electrospray ionization-mass spectra. Identification of the tentative protein biomarkers by bacterial cell extracts of Salmonella enterica serovar typhimurium strains A1 and A19 by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) was also demonstrated. The data analysis time was reduced by applying this approach. In addition, this method was less affected by noise and baseline drift.

Cucurbitane-type triterpenoids from Momordica charantia.

One new cucurbitane-type triterpenoid glycoside, momordicoside U (1), together with five known cucurbitane-type triterpenoids and related glycosides, 3ß,7 ß,25-trihydroxycucurbita-5,23 (E)-dien-19-al (2), momordicine I (3), momordicine II (4), 3-hydroxycucurbita-5,24-dien-19-al-7,23-di-O-ß-glucopyranoside (5), and kuguaglycoside G (6), were isolated from the whole plant of Momordica charantia. Their structures were determined by chemical and spectroscopic methods. Momordicoside U (1) was evaluated for insulin secretion activity in an in vitro insulin secretion assay and displayed moderate activity.

Confirmation of peanut protein using peptide markers in dark chocolate using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Detection of peptides from the peanut allergen Ara h 1 by liquid chromatography-mass spectrometry (LC-MS) was used to identify and estimate total peanut protein levels in dark chocolate. A comparison of enzymatic digestion subsequent to and following extraction of Ara h 1 from the food matrix revealed better limits of detection (LOD) for the pre-extraction digestion (20 ppm) than for the postextraction digestion (50 ppm). Evaluation of LC-MS instruments and scan modes showed the LOD could be further reduced to 10 ppm via a triple-quadrupole and multiple-reaction monitoring. Improvements in extraction techniques combined with an increase in the amount of chocolate extracted (1 g) improved the LOD to 2 ppm of peanut protein. This method provides an unambiguous means of confirming the presence of the peanut protein in foods using peptide markers from a major allergen, Ara h 1, and can easily be modified to detect other food allergens.

Detection, confirmation, and quantification of staphylococcal enterotoxin B in food matrixes using liquid chromatography--mass spectrometry.

Although immunoassay-based methods are sensitive and widely used for measuring protein toxins in food matrixes, there is a need for methods that can directly confirm the molecular identity of the toxin in situations where immunoassay tests yield a positive result. A method has been developed that uses mass spectrometry to identify a protein toxin, staphylococcal enterotoxin B (SEB), in a model food matrix, apple juice. The approach employs ultrafiltration to remove low molecular weight components from the sample, after which the remaining high molecular weight fraction, containing the protein, is digested with trypsin. The tryptic fragments are separated from residual biopolymers and analyzed by liquid chromatography-electrospray mass spectrometry. The background is still sufficiently complex that tandem mass spectrometry (MS/MS) is used to confirm the identity of target peptides. Limits of detection are 80 ng of SEB for MS and 100 ng for full scan MS/MS, using a tryptic fragment as the analytical target. Lower detection limits can be obtained using selected ion monitoring and multiple reaction monitoring. The presence of SEB can be confirmed at concentrations as low as 5 parts-per-billion by increasing the size of the sample to 10 mL. The method is applicable to the detection of SEB in other water-soluble food matrixes.

Relative quantitation of intact proteins of bacterial cell extracts using coextracted proteins as internal standards.

A method for quantitating protein expression using LC/MS of whole proteins is described. This method is based on the fact that some proteins present in cells are abundant universal proteins whose expression levels exhibit little variation. This method demonstrates that these coextracted proteins can be used as internal standards to which the other proteins in the sample can be compared. By comparing the intensities of a selected protein to marker proteins, or internal standards, a relative ratio is obtained. This ratio can then be used to determine the relative amount of protein expression between cellular extracts. The validity of this approach is described for a standard protein mixture, as well as, E. coli cells that were known to differentially express green fluorescent protein.

Treatment of methyl tert-butyl ether contaminated water using a dense medium plasma reactor: a mechanistic and kinetic investigation.

Plasma treatment of contaminated water appears to be a promising alternative for the oxidation of aqueous organic pollutants. This study examines the kinetic and oxidation mechanisms of methyl tert-butyl ether (MTBE) in a dense medium plasma (DMP) reactor utilizing gas chromatography-mass spectrometry and gas chromatography-thermal conductivity techniques. A rate law is developed for the removal of MTBE from an aqueous solution in the DMP reactor. Rate constants are also derived for three reactor configurations and two pin array spin rates. The oxidation products from the treatment of MTBE-contaminated water in the DMP reactor were found to be predominately carbon dioxide, with smaller amounts of acetone, tert-butyl formate, and formaldehyde. The lack of stable intermediate products suggests that the MTBE is, to some extent, oxidized directly to carbon dioxide, making the DMP reactor a promising tool in the future remediation of water. Chemical and physical mechanisms together with carbon balances are used to describe the formation of the oxidation products and the important aspects of the plasma discharge.

Atmospheric pressure matrix-assisted laser desorption/ionization in transmission geometry.

In both atmospheric pressure matrix-assisted laser desorption/ionization (AP MALDI) and vacuum MALDI, the laser typically illuminates the analyte on the front side of an opaque surface (reflection geometry). Another configuration consisting of laser illumination through the sample backside (transmission geometry) has been used in conventional MALDI; however, its use and the number of reports in the literature are limited. The viability of transmission geometry with AP MALDI is demonstrated here. Such a geometry is simple to implement, eliminates the restriction for a metallic sample holder, and allows for the potential analysis of samples on their native transparent surfaces, e.g., cells or tissue sections on slides.

Capillary electrophoresis as a method for ozone determination in atmospheres.

Capillary electrophoresis (CE) is used to quantify nitrate and nitrite extracted from nitrite-impregnated glass fiber filters (IGFF) that are used to monitor ozone in atmospheres. The amount of nitrate produced from conversion of nitrite in the filters is directly related to the amount of ozone passed over the filter. The limit of detection for ozone using the CE method is 1.17 ppml and the method is linear over two orders of magnitude. The effect of the excess nitrite in the IGFF on the limits of detection is discussed. Results from CE analyses of both active and passive filters are presented. The active filter results are compared to ion chromatographic analyses.