The Effects of Long-term, Low-dose ß-N-methylamino-L-alanine (BMAA) Exposures in Adult SODG93R Transgenic Zebrafish.

ß-N-Methylamino-L-alanine (BMAA) is a non-proteinogenic amino acid produced by cyanobacteria, which has been implicated in several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). It is postulated that chronic exposure to BMAA can lead to formation of protein aggregates, oxidative stress, and/or excitotoxicity, which are mechanisms involved in the etiology of ALS. While specific genetic mutations are identified in some instances of ALS, it is likely that a combination of genetic and environmental factors, such as exposure to the neurotoxin BMAA, contributes to disease. We used a transgenic zebrafish with an ALS-associated mutation, compared with wild-type fish to explore the potential neurotoxic effects of BMAA through chronic long-term exposures. While our results revealed low concentrations of BMAA in the brains of exposed fish, we found no evidence of decreased swim performance or behavioral differences that might be reflective of neurodegenerative disease. Further research is needed to determine if chronic BMAA exposure in adult zebrafish is a suitable model to study neurodegenerative disease initiation and/or progression.

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.

Cyclotides Chemosensitize Glioblastoma Cells to Temozolomide.

Glioblastoma multiforme (GBM) is the most aggressive cancer originating in the brain, with a median survival of 12 months. Most patients do not respond to or develop resistance to the only effective chemotherapeutic drug, temozolomide (TMZ), used to treat gliomas. Novel treatment methods are critically needed. Cyclotides are plant peptides that may be promising adjuvants to TMZ chemotherapy. They exhibit antitumor activity and chemosensitize cells to doxorubicin in breast cancer studies. During this research, we optimized cyclotide isolation techniques, and several cyclotides (CyO2, CyO13, kalata B1, and varv peptide A) exhibited dose-dependent cytotoxicity in MTT assays with IC50 values of 2.15-7.92 µM against human brain astrocytoma cells (U-87 MG) and human bone marrow derived neuroblastoma cells (SH-SY5Y). CyO2 and varv peptide A increased TMZ-induced cell death in U-87 MG cultures alone and when coexposed with CyO2 or varv peptide A plus TMZ. Phase contrast microscopy of glioblastoma cells exposed to cyclotides alone and coexposed to TMZ indicated shrunken, granular cells with blebbing, and the most pronounced effects were observed with coexposure treatments of cyclotides and TMZ. Cumulative results provide the proof-of-concept that cyclotides may enhance TMZ chemotherapy, and in vivo pharmacokinetic investigations of cyclotides are warranted with respect to GBM.

Phase I clinical trial of safety of L-serine for ALS patients.

We performed a randomized, double-blind phase I clinical trial for six months on the effects of oral L-serine in patients with ALS. The protocol called for enrollment of patients with a diagnosis of probable or definite ALS, age 18-85 years, disease duration of less than three years and forced vital capacity (FVC) ≥ 60%. Patients were randomly assigned to four different oral twice-daily dose regimens (0.5, 2.5, 7.5, or 15 g/dose). Blood, urine and CSF samples, ALS Functional Rating Scale-Revised (ALSFRS-R) scores and forced vital capacity (FVC) were obtained throughout the trial. Disease progression was compared with matched historical placebo controls from five previous ALS therapeutic trials. Of 20 patients enrolled, one withdrew before receiving study drug and two withdrew with gastro-intestinal problems. Three patients died during the trial. L-serine was generally well tolerated by the patients and L-serine did not appear to accelerate functional decline of patients as measured by slope of their ALSFRS-R scores. Based on this small study, L-serine appears to be generally safe for patients with ALS.

Cyanobacteria and cyanotoxins are present in drinking water impoundments and groundwater wells in desert environments.

Desert environments and drylands experience a drastic scarcity of water resources. To alleviate dependence on freshwater for drinking water needs, countries have invested in infrastructure development of desalination plants. Collectively, the countries of the Arabian Gulf produce 45% of the world's desalinated water, which is stored in dams, mega-reservoirs and secondary house water tanks to secure drinking water beyond daily needs. Improper storage practices of drinking water in impoundments concomitant with increased temperatures and light penetration may promote the growth of cyanobacteria and accumulation of cyanotoxins. To shed light on this previously unexplored research area in desert environments, we examined drinking and irrigation water of urban and rural environments to determine whether cyanobacteria and cyanotoxins are present, and what are the storage and transportation practices as well as the environmental parameters that best predict their presence. Cyanobacteria were present in 80% of the urban and 33% of the rural water impoundments. Neurotoxins BMAA, DAB and anatoxin-a(S) were not detected in any of the water samples, although they have been found to accumulate in the desert soils, which suggests a bioaccumulation potential if they are leached into the aquifer. A toxic BMAA isomer, AEG, was found in 91.7% of rural but none of the urban water samples and correlated with water-truck transportation, light exposure and chloride ions. The hepatotoxic cyanotoxin microcystin-LR was present in the majority of all sampled impoundments, surpassing the WHO provisional guideline of 1 µg/l in 30% of the urban water tanks. Finally, we discuss possible management strategies to improve storage and transportation practices in order to minimize exposure to cyanobacteria and cyanotoxins, and actions to promote sustainable use of limited water resources.

Grazing livestock are exposed to terrestrial cyanobacteria.

While toxins from aquatic cyanobacteria are a well-recognised cause of disease in birds and animals, exposure of grazing livestock to terrestrial cyanobacteria has not been described. This study identified terrestrial cyanobacteria, predominantly Phormidium spp., in the biofilm of plants from most livestock fields investigated. Lower numbers of other cyanobacteria, microalgae and fungi were present on many plants. Cyanobacterial 16S rDNA, predominantly from Phormidium spp., was detected in all samples tested, including 6 plant washings, 1 soil sample and ileal contents from 2 grazing horses. Further work was performed to test the hypothesis that ingestion of cyanotoxins contributes to the pathogenesis of some currently unexplained diseases of grazing horses, including equine grass sickness (EGS), equine motor neuron disease (EMND) and hepatopathy. Phormidium population density was significantly higher on EGS fields than on control fields. The cyanobacterial neurotoxic amino acid 2,4-diaminobutyric acid (DAB) was detected in plant washings from EGS fields, but worst case scenario estimations suggested the dose would be insufficient to cause disease. Neither DAB nor the cyanobacterial neurotoxins ß-N-methylamino-L-alanine and N-(2-aminoethyl) glycine were detected in neural tissue from 6 EGS horses, 2 EMND horses and 7 control horses. Phormidium was present in low numbers on plants where horses had unexplained hepatopathy. This study did not yield evidence linking known cyanotoxins with disease in grazing horses. However, further study is warranted to identify and quantify toxins produced by cyanobacteria on livestock fields, and determine whether, under appropriate conditions, known or unknown cyanotoxins contribute to currently unexplained diseases in grazing livestock.

Linking ß-methylamino-L-alanine exposure to sporadic amyotrophic lateral sclerosis in Annapolis, MD.

Most amyotrophic lateral sclerosis (ALS) cases occur sporadically. Some environmental triggers have been implicated, including beta-methylamino-L-alanine (BMAA), a cyanobacteria produced neurotoxin. This study aimed to identify environmental risk factors common to three sporadic ALS patients who lived in Annapolis, Maryland, USA and developed the disease within a relatively short time and within close proximity to each other. A questionnaire was used to identify potential risk factors for ALS among the cohort of patients. One common factor among the ALS patients was the frequent consumption of blue crab. Samples of blue crab from the patients' local fish market were tested for BMAA using LC-MS/MS. BMAA was identified in these Chesapeake Bay blue crabs. We conclude that the presence of BMAA in the Chesapeake Bay food web and the lifetime consumption of blue crab contaminated with BMAA may be a common risk factor for sporadic ALS in all three patients.

Cyanobacterial neurotoxin ß-N-methylamino-L-alanine (BMAA) in shark fins.

Sharks are among the most threatened groups of marine species. Populations are declining globally to support the growing demand for shark fin soup. Sharks are known to bioaccumulate toxins that may pose health risks to consumers of shark products. The feeding habits of sharks are varied, including fish, mammals, crustaceans and plankton. The cyanobacterial neurotoxin ß-N-methylamino-L-alanine (BMAA) has been detected in species of free-living marine cyanobacteria and may bioaccumulate in the marine food web. In this study, we sampled fin clips from seven different species of sharks in South Florida to survey the occurrence of BMAA using HPLC-FD and Triple Quadrupole LC/MS/MS methods. BMAA was detected in the fins of all species examined with concentrations ranging from 144 to 1836 ng/mg wet weight. Since BMAA has been linked to neurodegenerative diseases, these results may have important relevance to human health. We suggest that consumption of shark fins may increase the risk for human exposure to the cyanobacterial neurotoxin BMAA.

Presence of the neurotoxic amino acids beta-N-methylamino-L-alanine (BMAA) and 2,4-diamino-butyric acid (DAB) in shallow springs from the Gobi Desert.

The Gobi Desert in Mongolia, home to the critically endangered Gobi bear (Ursus arctos isabellinus), has few water resources for the animals that inhabit this environment. The majority of these water resources are shallow, small bodies of water, from approximately 30 cm to 3 m in diameter. Due to the harsh nature of the Gobi Desert environment, such pools of water are crucial resources for wildlife inhabiting the area and little information is currently available on the presence of organisms, including cyanobacteria, and the toxins they produce within these waters. Drinking water sources and small pools within the Gobi Desert were sampled on two separate occasions in October 2008 and April-May 2009. Samples were assessed for the presence of cyanobacteria; subsamples were taken for the analysis of beta-N-methylamino-L-alanine (BMAA) and 2,4-diaminobutyric acid (DAB). According to LC-MS/MS analyses, both of these neurotoxic amino acids were present in both years and BMAA was present when cyanobacteria were major components of the pools. The results indicate that assessment of cyanotoxins to organisms that live in desert environments is warranted.

Variability in content of the anti-AIDS drug candidate prostratin in Samoan populations of Homalanthus nutans.

Homalanthus nutans, used by Samoan healers to treat hepatitis, produces the antiviral compound 12-deoxyphorbol 13-acetate, prostratin (1). Prostratin is being developed as an adjuvant therapy to clear latent viral reservoirs, the major obstacle to eradication of HIV-AIDS within the human body. A validated reversed-phase HPLC method was developed to assay concentrations of 1 in H. nutans. A survey of four distinct populations on two different Samoan islands revealed significant variability in content. The stem tissue (range 0.2-52.6 microg/g 1), used by healers in indigenous therapies,gave a higher median concentration of prostratin (3.5 microg/g) than root or leaf tissues (2.9 and 2.5 microg/g, respectively).The high variability and skewness of these data indicate that cultivar selection for drug production will be important for this species. The reversed-phase HPLC assay will allow plants to be selected for agricultural development and genetic analysis by identifying those individuals above and below a 95% confidence interval for the median concentration.