First investigation of the temporal distribution of neurotoxin ß-N-methylamino-L-alanine (BMAA) and the candidate causative microalgae along the South Sea Coast of Korea.

The neurotoxin ß-N-methylamino-L-alanine (BMAA), produced by cyanobacteria and diatoms, has been implicated as an environmental risk factor for neurodegenerative diseases. This study first investigated the occurrence and monthly distributions of BMAA and its isomers, 2,4-diaminobutyric acid (DAB) and N-2-aminoethylglycine (AEG), in phytoplankton and mussels from 11 sites along the South Sea Coast of Korea throughout 2021. These toxins were quantified using LC-MS/MS, revealing elevated BMAA concentrations from late autumn to spring, with phase lags observed between phytoplankton and mussels. The highest concentration of BMAA in phytoplankton was detected in November (mean: 1490 ng g-1 dry weight (dw)), while in mussels, it peaked in December (mean: 1240 ng g-1 dw). DAB was detected in phytoplankton but was absent in mussels, indicating limited bioaccumulation potential. In February, the peak mean DAB concentration in phytoplankton was 89 ng g-1 dw. AEG was not detected in any samples. Chlorophyll-a concentrations consistently showed an inverse correlation with BMAA concentrations in mussels throughout the year. Through correlation analysis, four diatom genera, Bacillaria, Hemiaulus, Odontella, and Pleurosigma, were identified as potential causative microalgae of BMAA. This study offers insights into identifying the causative microalgae for BMAA and informs future regulatory efforts regarding unmanaged biotoxins.

Ectoine maintains the flavor and nutritional quality of broccoli during postharvest storage.

The bacterial derived osmolyte ectoine has been shown to stabilize cell structure and function, a property that may help to extend the shelf life of broccoli. The impact of ectoine on broccoli stored for 4 d at 20 °C and 90% relative humidity was investigated. Results indicated that 0.20% ectoine treatment maintained the quality of broccoli, by reducing rate of respiration and ethylene generation, while increasing the levels of total phenolics, flavonoids, TSS, soluble protein, and vitamin C, relative to control. Headspace-gas chromatography-mass spectrometry, transcriptomic and metabolomic analyses revealed that ectoine stabilized aroma components in broccoli by maintaining level of volatile compounds and altered the expression of genes and metabolites associated with sulfur metabolism, as well as fatty acid and amino acid biosynthesis pathways. These findings provide a greater insight into how ectoine preserves the flavor and nutritional quality of broccoli, thus, extending its shelf life.

Presence of the neurotoxin ß-N-methylamino-L-alanine in irrigation water and accumulation in cereal grains with human exposure risk.

The present study demonstrates the presence of the neurotoxin ß-N-methylamino-L-alanine and its cyanobacterial producers in irrigation water and grains of some cereal plants from farmlands irrigated with Nile River water in Egypt. BMAA detected by LC-MS/MS in phytoplankton samples was found at higher concentrations of free form (0.84-11.4 µg L-1) than of protein-bound form (0.16-1.6 µg L-1), in association with the dominance of cyanobacteria in irrigation water canals. Dominant cyanobacterial species isolated from these irrigation waters including Aphanocapsa planctonica, Chroococcus minutus, Dolichospermum lemmermanni, Nostoc commune, and Oscillatoria tenuis were found to produce different concentrations of free (4.8-71.1 µg g-1 dry weight) and protein-bound (0.1-11.4 µg g-1 dry weight) BMAA. In the meantime, BMAA was also detected in a protein-bound form only in grains of corn (3.87-4.51 µg g-1 fresh weight) and sorghum (5.1-7.1 µg g-1 fresh weight) plants, but not in wheat grains. The amounts of BMAA accumulated in these grains correlated with BMAA concentrations detected in relevant irrigation water canals. The presence of BMAA in cereal grains would constitute a risk to human and animal health upon consumption of contaminated grains. The study, therefore, suggests continuous monitoring of BMAA and other cyanotoxins in irrigation waters and edible plants to protect the public against exposure to such potent toxins.

Identification of genes associated to ß -N oxalyl- L-α, ß-diaminopropionic acid and their role in mitigating salt stress in a low-neurotoxin cultivar of Lathyrus sativus.

Grass pea has the potential to become a miracle crop if the stigma attached to it as a toxic plant is ignored. In light of the following, we conducted transcriptome analyses on the high and low ODAP-containing cultivars i.e., Nirmal and Bidhan respectively in both normal and salt stress conditions. In this study, genes that work upstream and downstream to ß-ODAP have been found. Among these genes, AAO3 and ACL5 were related to ABA and polyamine biosynthesis, showing the relevance of ABA and polyamines in boosting the ß-ODAP content in Nirmal. Elevated ß-ODAP levels in salt stress-treated Bidhan may have evolved tolerance by positively regulating the expression of genes involved in phenylpropanoid and jasmonic acid biosynthesis. Although the concentration of ß-ODAP in Bidhan increased under salt stress, it was lower than in stress-treated Nirmal. Despite this, the expression of stress-related genes that work downstream to ß-ODAP was found higher in stress-treated Bidhan. This could be because stress-treated Nirmal has lower GSH, proline, and higher H2O2, resulting in the development of severe oxidative stress. Overall, our research not only identified new genes linked with ß-ODAP, but also revealed the molecular mechanism by which a low ß-ODAP-containing cultivar developed tolerance against salinity stress.

Diamine Oxidase as a Therapeutic Enzyme: Study of Germination from Vegetal Sources and Investigation of the Presence of ß-N-Oxalyl-L-α,ß-diaminopropionic Acid (ß-ODAP) Using LC-MS/MS.

Vegetal diamine oxidase (vDAO), an enzyme proposed to relieve symptoms of histaminosis, shows better reactivity with histamine and aliphatic diamines, as well as higher enzymatic activity than DAO of animal origin. The objective of this study was to evaluate the enzyme activity of vDAO from germinating grains from Lathyrus sativus (grass pea) and Pisum sativum (pea), and to verify the presence of a neurotoxin, ß-N-Oxalyl-L-α,ß-diaminopropionic acid (ß-ODAP), in the crude extract obtained from their seedlings. A targeted liquid chromatography-multiple-reaction monitoring mass spectrometry method was developed and used to quantify ß-ODAP in the analysed extracts. An optimized sample preparation procedure, involving protein precipitation with acetonitrile followed by mixed-anion exchange solid-phase extraction, allowed for high sensitivity and good peak shape for ß-ODAP detection. The Lathyrus sativus extract exhibited the highest vDAO enzyme activity of the extracts, followed by the extract from pea cultivar Amarillo from the Crop Development Centre (CDC). The results have also shown that even though ß-ODAP was present in the crude extract from L. sativus, its content was far below the toxicity threshold (300 mg of ß-ODAP/kg body/day). CDC Amarillo showed 5000-fold less ß-ODAP than the undialysed L. sativus extract. It was concluded that both species can be considered as convenient sources of vDAO for potential therapeutic use.

Putative biosynthesis mechanism of the neurotoxin ß-N-methylamino-L-alanine in marine diatoms based on a transcriptomics approach.

The neurotoxin ß-N-methylamino-L-alanine (BMAA) has been presumed as an environmental cause of human neurodegenerative disorders, such as Alzheimer's disease. Marine diatoms Thalassiosira minima are demonstrated here to produce BMAA-containing proteins in axenic culture while the isomer diaminobutyric acid was bacterially produced. In the co-culture with Cyanobacterium aponinum, diatom growth was inhibited but the biosynthesis of BMAA-containing proteins was stimulated up to seven times higher than that of the control group by cell-cell interactions. The stimulation effect was not caused by the cyanobacterial filtrate. Nitrogen deprivation also doubled the BMAA content of T. minima cells. Transcriptome analysis of the diatom in mixed culture revealed that pathways involved in T. minima metabolism and cellular functions were mainly influenced, including KEGG pathways valine and leucine/isoleucine degradation, endocytosis, pantothenate and CoA biosynthesis, and SNARE interactions in vesicular transport. Based on the expression changes of genes related to protein biosynthesis, it was hypothesized that ubiquitination and autophagy suppression, and limited COPII vesicles transport accuracy and efficiency were responsible for biosynthesis of BMAA-containing proteins in T. minima. This study represents a first application of transcriptomics to investigate the biological processes associated with BMAA biosynthesis in diatoms.

Extraction of Non-Protein Amino Acids from Cyanobacteria for Liquid Chromatography-Tandem Mass Spectrometry Analysis.

Non-protein amino acids (NPAAs) are a large class of amino acids (AAs) that are not genetically encoded for translation into proteins. The analysis of NPAAs can provide crucial information about cellular uptake and/or function, metabolic pathways, and potential toxicity. ß-methylamino-L-alanine (BMAA) is a neurotoxic NPAA produced by various algae species and is associated with an increased risk for neurodegenerative diseases, which has led to significant research interest. There are numerous ways to extract AAs for analysis, with liquid chromatography-tandem mass spectrometry being the most common, requiring protein precipitation followed by acid hydrolysis of the protein pellet. Studies on the presence of BMAA in algal species provide contradictory results, with the use of unvalidated sample preparation/extraction and analysis a primary cause. Like most NPAAs, protein precipitation in 10% aqueous TCA and hydrolysis with fuming HCl is the most appropriate form of extraction for BMAA and its isomers aminoethylglycine (AEG) and 2,4-diaminobutyric acid (2,4-DAB). The present protocol describes the steps in a validated NPAA extraction method commonly used in research and teaching laboratories.

Matrix Effect of Diverse Biological Samples Extracted with Different Extraction Ratios on the Detection of ß-N-Methylamino-L-Alanine by Two Common LC-MS/MS Analysis Methods.

Neurotoxin ß-N-methylamino-L-alanine (BMAA) is hypothesized as an important pathogenic factor for neurodegenerative diseases such as amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS-PDC). Comparative study on the accuracy of BMAA analyzed by the regular LC-MS/MS methods is still limited for different biological matrices. In this study, a free-BMAA sample of cyanobacterium and BMAA-containing positive samples of diatom, mussel, scallop, and oyster were extracted with varied extraction ratios (ER) ranging from 1:20 to 1:2000. These extracts were then purified by MCX cartridges. After SPE purification, these different biological samples were analyzed by two common LC-MS/MS analysis methods, a direct analysis without derivatization by a hydrophilic interaction liquid chromatography (HILIC)-MS/MS and pre-column 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatization combined with a C18 column. The results suggested that the recoveries of BMAA spiked in the cyanobacterial sample were close to 100% in the total soluble form extracts with the ER of 1:100 (g/mL) and the precipitated bound form extracts with the ER of 1:500. The recommended ER for the precipitated bound form of BMAA in diatoms and the total soluble form of BMAA in mollusks are 1:500 and 1:50, respectively. The quantitative results determined by the AQC derivatization method were lower than those determined by the direct analysis of the HILIC method in diatom and mollusk samples. The results of the HILIC method without the derivatization process were closer to the true value of BMAA in cyanobacteria. This work contributes to the performance of the solid-phase extraction (SPE) purification protocol and the accuracy of BMAA analysis by LC-MS/MS in diverse biological samples.

Occurrence of BMAA Isomers in Bloom-Impacted Lakes and Reservoirs of Brazil, Canada, France, Mexico, and the United Kingdom.

The neurotoxic alkaloid ß-N-methyl-amino-l-alanine (BMAA) and related isomers, including N-(2-aminoethyl glycine) (AEG), ß-amino-N-methyl alanine (BAMA), and 2,4-diaminobutyric acid (DAB), have been reported previously in cyanobacterial samples. However, there are conflicting reports regarding their occurrence in surface waters. In this study, we evaluated the impact of amending lake water samples with trichloroacetic acid (0.1 M TCA) on the detection of BMAA isomers, compared with pre-existing protocols. A sensitive instrumental method was enlisted for the survey, with limits of detection in the range of 5−10 ng L−1. Higher detection rates and significantly greater levels (paired Wilcoxon's signed-rank tests, p < 0.001) of BMAA isomers were observed in TCA-amended samples (method B) compared to samples without TCA (method A). The overall range of B/A ratios was 0.67−8.25 for AEG (up to +725%) and 0.69−15.5 for DAB (up to +1450%), with absolute concentration increases in TCA-amended samples of up to +15,000 ng L−1 for AEG and +650 ng L−1 for DAB. We also documented the trends in the occurrence of BMAA isomers for a large breadth of field-collected lakes from Brazil, Canada, France, Mexico, and the United Kingdom. Data gathered during this overarching campaign (overall, n = 390 within 45 lake sampling sites) indicated frequent detections of AEG and DAB isomers, with detection rates of 30% and 43% and maximum levels of 19,000 ng L−1 and 1100 ng L−1, respectively. In contrast, BAMA was found in less than 8% of the water samples, and BMAA was not found in any sample. These results support the analyses of free-living cyanobacteria, wherein BMAA was often reported at concentrations of 2−4 orders of magnitude lower than AEG and DAB. Seasonal measurements conducted at two bloom-impacted lakes indicated limited correlations of BMAA isomers with total microcystins or chlorophyll-a, which deserves further investigation.

Contrasting ß-ODAP content correlates with stress gene expression in Lathyrus cultivars.

Lathyrus sativus, commonly known as grass pea, is a nutrient-rich pulse crop with remarkable climate-resilient attributes. However, wide use of this nutritious crop is not adopted owing to the presence of a non-protein amino acid ß-N-oxalyl-l-α,ß-diaminopropionic acid (ß-ODAP), which is neurotoxic if consumed in large quantities. We conducted a de novo transcriptomic profiling of two ODAP contrasting cultivars, Pusa-24 and its somaclonal variant Ratan, to understand the genetic changes leading to and associated with ß-ODAP levels. Differential gene expression analysis showed that a variety of genes are downregulated in low ß-ODAP cultivar Ratan and include genes involved in biotic/abiotic stress tolerance, redox metabolism, hormonal metabolism, and sucrose, and starch metabolism. Several genes related to chromatin remodeling are differentially expressed in cultivar Ratan. ß-ODAP biosynthetic genes in these cultivars showed differential upregulation upon stress. ODAP content of these cultivars varied differentially upon stress and development. Physiological experiments indicate reduced relative water content and perturbed abscisic acid levels in the low ODAP cultivar. Altogether, our results suggest that the low ODAP cultivar may have a reduced stress tolerance. The dataset provides insight into the biological role of ODAP and will be helpful for hypothesis-driven experiments to understand ODAP biosynthesis and regulation.

Microbial production of ectoine and hydroxyectoine as high-value chemicals.

Ectoine and hydroxyectoine as typical representatives of compatible solutes are not only essential for extremophiles to survive in extreme environments, but also widely used in cosmetic and medical industries. Ectoine was traditionally produced by Halomonas elongata through a "bacterial milking" process, of which the marked feature is using a high-salt medium to stimulate ectoine biosynthesis and then excreting ectoine into a low-salt medium by osmotic shock. The optimal hydroxyectoine production was achieved by optimizing the fermentation process of Halomonas salina. However, high-salinity broth exacerbates the corrosion to fermenters, and more importantly, brings a big challenge to the subsequent wastewater treatment. Therefore, increasing attention has been paid to reducing the salinity of the fermentation broth but without a sacrifice of ectoine/hydroxyectoine production. With the fast development of functional genomics and synthetic biology, quite a lot of progress on the bioproduction of ectoine/hydroxyectoine has been achieved in recent years. The importation and expression of an ectoine producing pathway in a non-halophilic chassis has so far achieved the highest titer of ectoine (~ 65 g/L), while rational flux-tuning of halophilic chassis represents a promising strategy for the next-generation of ectoine industrial production. However, efficient conversion of ectoine to hydroxyectoine, which could benefit from a clearer understanding of the ectoine hydroxylase, is still a challenge to date.

A Single Laboratory Validation for the Analysis of Underivatized ß-N-Methylamino-L-Alanine (BMAA).

ß-N-Methylamino-L-alanine (BMAA) is a non-protein amino acid produced by cyanobacteria that can accumulate in ecosystems and food webs. Human exposure to cyanobacterial and algal blooms may be a risk factor for neurodegenerative diseases such as Alzheimer's disease and amyotrophic lateral sclerosis. Analytical chemists have struggled to find reliable methods for BMAA analysis in complex sample matrices. Analysis of BMAA is complicated by at least 3 naturally occurring isomers: N-(2-aminoethyl)glycine (AEG), 2,4-diaminobutyric acid (DAB), and ß-aminomethyl-L-alanine (BAMA). More than 350 publications have reported detection and quantification of BMAA and its isomers, but varying results have led to controversy in the literature. The objective of this study was to perform a single laboratory validation (SLV) of a frequently published method for BMAA analysis using a ZIC-HILIC column. We investigated the selectivity, linearity, accuracy, precision, and sensitivity of the method and our data show that this HILIC method fails many of the criteria for a validated method. The method fails the criterion for selectivity as the chromatography does not separate BMAA from its isomer BAMA. Sensitivity of the method greatly decreased over the experimental period and it demonstrated a higher limit of detection (LOD) (7.5 pg on column) and a higher lower limit of quantification (LLOQ) (30 pg on column) than other published validated methods. The method demonstrated poor precision of repeated injections of standards of BMAA with % relative standard deviation (%RSD) values that ranged from 37 to 107% while HorRat values for BMAA had a fail rate of 80% and BAMA had a fail rate of 73%. No HorRat values between 0.5 and 2 were found for repeated injections of standards of AEG and DAB. Recovery of 13C3,15N2-BMAA in a cyanobacterial matrix was < 10% in experiments and we were also unable to accurately detect other protein amino acids including methionine, cysteine, or alanine, indicating matrix effects. The results of this study demonstrate that the ZIC-HILIC column is not fit for purpose for the analysis of BMAA in cyanobacterial matrices and further provides explanations for the high level of negative results reported by researchers using this method.

Second Laboratory Validation of ß-N-Methylamino-L-Alanine, N-(2aminoethyl)Glycine, and 2,4-Diaminobuytric Acid by Ultra-Performance Liquid Chromatography and Tandem Mass Spectrometry.

The analysis of ß-N-methylamino-L-alanine (BMAA) has been validated according to AOAC international standards by a single laboratory (Glover et al. 2015). Using the same validated method, we add a second laboratory validation optimizing for different equipment. Given publicized concerns about standardizing methods across laboratories and recent reviews indicating superior results using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatization for the separation of BMAA and its isomers N-(2aminoethyl)glycine (AEG), and 2,4-diaminobuytric acid (DAB) (Bishop and Murch 2019), we add a second laboratory validation to this method demonstrating that the method is robust across laboratories using different equipment. Using the US Food and Drug Administration (FDA 2018) method for evaluating instrument parameters, we calculated a limit of detection (LOD) of 10 pg/ml for BMAA, AEG, and DAB and lower limits of quantification (LLOQ) of 37 pg/ml based on reagent blanks. In biological matrices, a higher LLOQ may be warranted for AEG and DAB. We demonstrate that the endogenous BMAA in mussel tissue can be lost by drying the hydrolyzed preparation and suggest sample preparation parameters be evaluated for robustness.

Biocrust-Produced Cyanotoxins Are Found Vertically in the Desert Soil Profile.

The fate and persistence of the neurotoxin ß-N-methylamino-L-alanine (BMAA) and its isomers N-(2aminoethyl)glycine (AEG) and 2,4-diaminobuytric acid (DAB) in soil profiles is poorly understood. In desert environments, these cyanotoxins are commonly found in both terrestrial and adjacent marine ecosystems; they accumulate in biocrusts and groundwater catchments, and have been previously shown to persist in soil as deep as 25 cm. To determine the depth that BMAA and its isomers can be found, samples were incrementally collected every 5 cm from bedrock to surface in triplicate soil cores in a biocrust field in the terrestrial desert of Qatar. Biocrust surface samples were also collected from each core priorly. Toxins were extracted from soil sub-samples, derivatized, and analyzed with UPLC-MS/MS. All toxins were detected in all soil cores at all depths. AEG and DAB were within a quantifiable concentration threshold; however, the low concentration of BMAA was considered below the threshold for quantification. This may have environmental health implications if these toxins are able to infiltrate and contaminate the bedrock aquifer, as well as the sand and gravel aquifers. Human and animal health may also be impacted through exposure to contaminated groundwater wells or through inhalation of aerosolized particles of soil, resuspended during construction or recreational activities.

Transfer of the Neurotoxin ß-N-methylamino-l-alanine (BMAA) in the Agro-Aqua Cycle.

The neurotoxic non-protein amino acid ß-N-methylamino-l-alanine (BMAA) is connected to the development of neurodegenerative diseases. BMAA has been shown to accumulate in aquatic ecosystems, and filter-feeding molluscs seem particularly susceptible to BMAA accumulation. The blue mussels farmed along the Swedish coastline in the Baltic Sea are, due to their small size, exclusively used to produce feed for chicken and fish in the agro-aqua cycle. We have investigated the possible biotransfer of BMAA from mussels, via mussel-based feed, into chickens. Chickens were divided into two groups, the control and the treatment. BMAA was extracted from the muscle, liver, brain, and eye tissues in both chicken groups; a UPLC-MS/MS method was subsequently used to quantify BMAA. The results indicate detectable concentrations of BMAA in both chicken groups. However, the BMAA concentration in chicken was 5.65 times higher in the treatment group than the control group, with the highest concentration found in muscle tissue extracted from the treatment group chickens. These data suggest that there is a BMAA transfer route within the agro-aqua cycle, so further investigation is recommended before using mussel-based feed in the chicken industry.

Usability of the bivalves Dreissena polymorpha and Anodonta anatina for a biosurvey of the neurotoxin BMAA in freshwater ecosystems.

The environmental neurotoxin ß-methylamino-L-alanine (BMAA) may represent a risk for human health in case of chronic exposure or after short-term exposure during embryo development. BMAA accumulates in freshwater and marine organisms consumed by humans. It is produced by marine and freshwater phytoplankton species, but the range of producers remains unknown. Therefore, analysing the phytoplankton composition is not sufficient to inform about the risk of freshwater contamination by BMAA. Filter-feeders mussels have accumulation capacities and therefore appear to be relevant to monitor various pollutants in aquatic ecosystems. We investigated the suitability of the freshwater mussels Dreissena polymorpha and Anodonta anatina for monitoring BMAA in water. Both species were exposed to 1, 10, and 50 µg of dissolved BMAA/L daily for 21 days, followed by 42 days of depuration in clean water. On days 0, 1, 7, 14, and 21 of exposure and 1, 7, 14, 21 and 42 of depuration, whole D. polymorpha and digestive glands of A. anatina were sampled, and the total BMAA concentration was measured. D. polymorpha accumulated BMAA earlier (from day 1 at all concentrations) and at higher tissue concentrations than A. anatina, which accumulated BMAA from day 14 when exposed to 10 µg BMAA/L and from day 7 when exposed to 50 µg BMAA/L. As BMAA accumulation by D. polymorpha was time and concentration-dependent, with a significant elimination during the depuration period, this species may be able to reflect the levels and dynamics of water contamination by dissolved BMAA. The species A. anatina could be used for monitoring water concentrations above 10 µg BMAA/L.

Effects and mechanism on the removal of neurotoxin ß-N-methylamino-l-alanine (BMAA) by chlorination.

ß-N-Methylamino-l-alanine (BMAA), a new cyanobacterial toxin, is found in different aquatic ecosystems worldwide and is to threaten the human nervous system. Therefore, it is important for water plants to develop feasible methods to counter the effects of BMAA. In this study, the removal of BMAA by chlorine, as well as its intermediate products, at different pH values and the mechanism of pH on the removal BMAA were investigated. The results showed that the chlorination of BMAA is in accordance with the second-order kinetics model. The reaction rate of chlorinated BMAA increased with the increase in the concentration of chlorine. The pH of the solution significantly affected the reaction rate. The apparent kinetic constant (kapp) decreased from 6.00 × 103 M-1·min-1 to 35.5 M-1·min-1 when the pH increased from 4.5 to 9 in the chlorine concentration of 32.23 µM. It is probable that the species distribution and proportion of BMAA and chlorine at different pH values were the main causes of this phenomenon. Additionally, the chlorination reaction consisted of four elementary reactions and hydrogen ions were beneficial to the reaction. The temperature also affected the reaction rate and the activation energy of the reaction was 16.6 ± 1.99 kJ·M-1. A variety of degradation products were detected and the path of degradation was speculated. Chlorination, dechlorination, and decarboxylation were the main processes of oxidative degradation. Furthermore, the composition of the degradation products was the same at different pH values.

Genotyping of Low β -ODAP Grass Pea (Lathyrus sativus L. ) Germplasm with EST-SSR Markers

Abstract Grass pea (Lathyrus sativus L.) is an important protein source in arid regions as both human and animal food. Despite its significance, the use of grass pea is limited by the presence of β-N-oxalyl-L-a,b-diaminopropionic acid (β-ODAP) which can cause neurological disorders. Breeding studies in grass pea have therefore focused on developing high-yielding varieties with low β-ODAP content. However, the narrow range of genetic diversity and the restricted genomic tools in grass pea have slowed progress in such breeding. The present investigation was conducted to explore the genetic diversity of low β-ODAP germplasm consisting of 22 accessions with 31 EST-SSR markers. The molecular analyses revealed a total of 133 alleles ranging from 142 to 330 bp with a mean number of alleles per locus of 4.29. The mean polymorphic information content (PIC) value was calculated as 0.49, and the EST-SSRs in loci S5, S6 and S116 were of the most informative PICs. A dendrogram based on Nei's genetic distance matrix revealed that breeding lines were grouped in two main clusters. Genetic distances were higher between GP6/GP11, GP4/GP11 and GP5/GP8 accessions which could be further used in crop improvement studies for developing wider genetic diversity.

A systematic review of analytical methods for the detection and quantification of ß-N-methylamino-l-alanine (BMAA).

Neurodegenerative diseases are influenced by environmental factors such as exposure to toxins including the cyanotoxin ß-N-methylamino-l-alanine (BMAA) that can bioaccumulate in common food sources such as fish, mussels and crabs. Accurate and precise analytical methods are needed to detect and quantify BMAA to minimize human health risks. The objective of this review is to provide a comprehensive overview of the methods used for BMAA analysis from 2003 to 2019 and to evaluate the reported performance characteristics for each method to determine the consensus data for each analytical approach and different sample matrices. Detailed searches of the database Web of Science™ (WoS) were performed between August 21st, 2018 and April 5th, 2019. Eligible studies included analytical methods for the detection and quantification of BMAA in cyanobacteria and bioaccumulated BMAA in higher trophic levels, in phytoplankton and zooplankton and in human tissues and fluids. This systematic review has limitations in that only the English language literature is included and it did not include standard operating protocols nor any method validation data that have not been made public. We identified 148 eligible studies, of which a positive result for BMAA in one or more samples analyzed was reported in 84% (125 out of 148) of total studies, 57% of HILIC studies, 92% of RPLC studies and 71% of other studies. The largest discrepancy between different methods arose from the analysis of cyanobacteria samples, where BMAA was detected in 95% of RPLC studies but only in 25% of HILIC studies. Without sufficient published validation of each method's performance characteristics, it is difficult to establish each method as fit for purpose for each sample matrix. The importance of establishing methods as appropriate for their intended use is evidenced by the inconsistent reporting of BMAA across environmental samples, despite its prevalence in diverse ecosystems and food webs.

Linking a rapid throughput plate-assay with high-sensitivity stable-isotope label LCMS quantification permits the identification and characterisation of low ß-L-ODAP grass pea lines.

BACKGROUND: Grass pea (Lathyrus sativus) is an underutilised crop with high tolerance to drought and flooding stress and potential for maintaining food and nutritional security in the face of climate change. The presence of the neurotoxin ß-L-oxalyl-2,3-diaminopropionic acid (ß-L-ODAP) in tissues of the plant has limited its adoption as a staple crop. To assist in the detection of material with very low neurotoxin toxin levels, we have developed two novel methods to assay ODAP. The first, a version of a widely used spectrophotometric assay, modified for increased throughput, permits rapid screening of large populations of germplasm for low toxin lines and the second is a novel, mass spectrometric procedure to detect very small quantities of ODAP for research purposes and characterisation of new varieties. RESULTS: A plate assay, based on an established spectrophotometric method enabling high-throughput ODAP measurements, is described. In addition, we describe a novel liquid chromatography mass spectrometry (LCMS)-based method for ß-L-ODAP-quantification. This method utilises an internal standard (di-13C-labelled ß-L-ODAP) allowing accurate quantification of ß-L-ODAP in grass pea tissue samples. The synthesis of this standard is also described. The two methods are compared; the spectrophotometric assay lacked sensitivity and detected ODAP-like absorbance in chickpea and pea whereas the LCMS method did not detect any ß-L-ODAP in these species. The LCMS method was also used to quantify ß-L-ODAP accurately in different tissues of grass pea. CONCLUSIONS: The plate-based spectrophotometric assay allows quantification of total ODAP in large numbers of samples, but its low sensitivity and inability to differentiate α- and ß-L-ODAP limit its usefulness for accurate quantification in low-ODAP samples. Coupled to the use of a stable isotope internal standard with LCMS that allows accurate quantification of ß-L-ODAP in grass pea samples with high sensitivity, these methods permit the identification and characterisation of grass pea lines with a very low ODAP content. The LCMS method is offered as a new 'gold standard' for ß-L-ODAP quantification, especially for the validation of existing and novel low- and/or zero-ß-L-ODAP genotypes.