Characterization of Neowestiellopsis persica A1387 (Hapalosiphonaceae) based on the cpcA, psbA, rpoC1, nifH and nifD gene sequences.

BACKGROUND: Complex descriptions of new strains of cyanobacteria appear very frequently. The main importance of these descriptions concerns potential new substances that they could synthesise, as well as their different properties as a result of their different ecological niches. The main gene used for these descriptions is 16 S with ITS or whole genome sequencing. Neowestiellopsis persica represents a unique example of the influence of ecology on morphological changes, with almost identical 16 S identity. Although our previously described Neowestiellopsis persica strain A1387 was characterized by 16 S analysis, we used different molecular markers to provide a way to separate strains of this genus that are closely related at the genetic level. MATERIALS AND METHODS: In order to conduct an in-depth study, several molecular markers, namely psbA, rpoC1, nifD, nifH and cpcA were sequenced and studied in Neowestiellopsis persica strain A1387. RESULTS: The results of the phylogenetic analysis, based on cpcA, showed that the studied strain A 1387 falls into a separate clade than N. persica, indicating that this signature sequence could be a useful molecular marker for phylogenetic separation of similar strains isolated in the future. CONCLUSIONS: Analysis of strain A1387 based on gene differences confirmed that it is a Neowestiellopsis strain. The morphological changes observed in the previous study could be due to different ecological and cultivation conditions compared to the type species. At the same time, the sequences obtained have increased our understanding of this species and will help in the future to better identify strains belonging to the genus Neowestiellopsis.

A Direct Analysis of ß-N-methylamino-l-alanine Enantiomers and Isomers and Its Application to Cyanobacteria and Marine Mollusks.

Of the wide variety of toxic compounds produced by cyanobacteria, the neurotoxic amino acid ß-N-methylamino-l-alanine (BMAA) has attracted attention as a result of its association with chronic human neurodegenerative diseases such as ALS and Alzheimer's. Consequently, specific detection methods are required to assess the presence of BMAA and its isomers in environmental and clinical materials, including cyanobacteria and mollusks. Although the separation of isomers such as ß-amino-N-methylalanine (BAMA), N-(2-aminoethyl)glycine (AEG) and 2,4-diaminobutyric acid (DAB) from BMAA has been demonstrated during routine analysis, a further compounding factor is the potential presence of enantiomers for some of these isomers. Current analytical methods for BMAA mostly do not discriminate between enantiomers, and the chiral configuration of BMAA in cyanobacteria is still largely unexplored. To understand the potential for the occurrence of D-BMAA in cyanobacteria, a chiral UPLC-MS/MS method was developed to separate BMAA enantiomers and isomers and to determine the enantiomeric configuration of endogenous free BMAA in a marine Lyngbya mat and two mussel reference materials. After extraction, purification and derivatization with N-(4-nitrophenoxycarbonyl)-l-phenylalanine 2-methoxyethyl ester ((S)-NIFE), both L- and D-BMAA were identified as free amino acids in cyanobacterial materials, whereas only L-BMAA was identified in mussel tissues. The finding of D-BMAA in biological environmental materials raises questions concerning the source and role of BMAA enantiomers in neurological disease.

Cyanotoxin Analysis of Air Samples from the Great Salt Lake.

The Great Salt Lake in Utah is the largest saline lake in the Western hemisphere and one of the largest terminal lakes in the world. Situated at the eastern edge of the Great Basin, it is a remnant of the freshwater Lake Bonneville whose water level precipitously lowered about 12,000 years ago due to a natural break in Red Rock pass to the north. It contains a diverse assemblage of cyanobacteria which vary spatially dependent on salinity. In 1984, the waters of the Great Salt Lake occupied 8500 km2. Nearly four decades later, the waters occupy 2500 km2-a reduction in surface area of 71%. With predominantly westerly winds, there is a potential for the adjacent metropolitan residents to the east to be exposed to airborne cyanobacteria- and cyanotoxin-containing dust. During the summer and fall months of 2022, air and dried sediment samples were collected and assessed for the presence of BMAA which has been identified as a risk factor for ALS. Collection of air samples equivalent to a person breathing for 1 h resulted in BMAA and isomers being found in some air samples, along with their presence in exposed lakebed samples. There was no clear relationship between the presence of these toxins in airborne and adjacent lakebed samples, suggesting that airborne toxins may originate from diffuse rather than point sources. These findings confirm that continued low water levels in the Great Salt Lake may constitute an increasing health hazard for the 2.5 million inhabitants of communities along the Wasatch Front.

Antifungal screening of selenium nanoparticles biosynthesized by microcystin-producing Desmonostoc alborizicum.

Metal nanoparticles exhibit excellent antifungal abilities and are seen as a good substitute for controlling different kinds of fungi. Of all known taxa, cyanobacteria have received significant consideration as nanobiofactories, as a result of the cellular assimilation of heavy metals from the environment. The cellular bioactive enzymes, polysaccharides and pigments can be used as reducers and coatings during biosynthesis. The probability of the antifungal activity of selenium nanoparticles (SeNPs) to prevent plant fungi that can affect humans was evaluated and a toxic Iranian cyanobacterial strain of Desmonostoc alborizicum was used to study the biotechnology of SeNP synthesis for the first time. Characterization of nanoparticles with a UV-Vis spectrophotometer showed the formation of SeNPs in the range of 271-275 nm with the appearance of an orange color. Morphological examination of nanoparticles with Transmission Electron Microscopy (TEM), revealed the spherical shape of nanoparticles. The results of X-Ray Diffraction (XRD) showed 7 peaks and a hexagonal structure of average crystal size equal to 58.8 nm. The dispersion index of SeNPs was reported as 0.635, which indicated the homogeneity of the nanoparticle droplet size. The zeta potential of the nanoparticles was + 22.7. Fourier-transform infrared spectroscopy (FTIR) analysis exhibited a sharp and intense peak located at the wave number of 404 cm- 1, related to the SeNPs synthesized in this research. The results of the antifungal activity of SeNPs showed among the investigated fungi, Pythium ultimum had the highest resistance to SeNPs (14.66 ± 0.52 µg/ml), while Alternaria alternata showed the highest sensitivity (9.66 ± 0.51 µg/ml) (p < 0.05). To the best of our knowledge this is the first report concerning the characterization and antifungal screening of SeNPs biosynthesized by Iranian cyanobacteria, which could be used as effective candidates in medical applications.

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.

Detection of ß-N-methylamino-l-alanine in postmortem olfactory bulbs of Alzheimer's disease patients using UHPLC-MS/MS: An autopsy case-series study.

Introduction: Cyanobacterial blooms produce toxins that may become aerosolized, increasing health risks through inhalation exposures. Health related effects on the lower respiratory tract caused by these toxins are becoming better understood. However, nasal exposures to cyanotoxins remain understudied, especially for those with neurotoxic potential. Here, we present a case series study evaluating exposure to ß-N-methylamino-l-alanine (BMAA), a cyanobacterial toxin linked to neurodegenerative disease, in postmortem olfactory tissues of individuals with varying stages of Alzheimer's disease (AD). Methods: Olfactory bulb (Ob) tissues were collected during autopsies performed between 2014 and 2017 from six South Florida brain donors (ages 47-78) with residences less than 140 m from a freshwater body. A triple quadrupole tandem mass spectrometry (UHPLC-MS/MS) method validated according to peer AOAC International guidelines was used to detect BMAA and two BMAA isomers: 2,4-diaminobutyric acid (2,4-DAB) and N-(2-aminoethyl)glycine (AEG). Quantitative PCR was performed on the contralateral Ob to evaluate the relative expression of genes related to proinflammatory cytokines (IL-6 & IL-18), apoptotic pathways (CASP1 & BCL2), and mitochondrial stress (IRF1 & PINK1). Immunohistochemistry was also performed on the adjacent olfactory tract (Ot) to evaluate co-occurring neuropathology with BMAA tissue concentration. Results: BMAA was detected in the Ob of all cases at a median concentration of 30.4 ng/g (Range

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.

A Novel Microcystin-Producing Cyanobacterial Species from the Genus Desmonostoc, Desmonostoc alborizicum sp. nov., Isolated from a Water Supply System of Iran.

A qanat or kariz is a slightly sloping underground aqueduct used to transport water from wells or aquifers to the surface for irrigation and drinking supply. A cyanobacterial strain was isolated from a cyanobacterial mat colonizing the wall of a qanat in Golestan province, Gorgan City, Iran. Fragments of 16S rRNA, mcyG, and mcyD genes were amplified and sequenced, as well as the 16S-23S internal transcribed spacer (ITS). After microscopic examination, the isolate was related to a morphotype of Nostoc sensu lato group, with similar characteristics to Desmonostoc. The 16S rRNA phylogenetic analysis placed the isolate into the typical cluster of the recently proposed genus Desmonostoc. Morphological analysis revealed distinctive characteristic and secondary 16S-23S rRNA structures derived from comparative analysis, which did not match known species of Desmonostoc. These results lead us to propose a novel Desmonostoc species, Desmonostoc alborizicum, which was described and compared with similar taxa. Furthermore, for the first time a potentially toxic species of Desmonostoc was isolated from a water supply, since the mcyD and mcyG genes of the microcystin synthetase (mcy) cluster were successfully sequenced. Using mass spectrometry, detectable amounts of the hepatotoxin microcystin-LR and -RR, along with demethylated variants, were present in cell extracts of the Desmonostoc strain. Our findings contribute to a deeper understanding of the diversity, systematics, and occurrence of the genus Desmonostoc.

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.

In Vivo and In Vitro Toxicity Testing of Cyanobacterial Toxins: A Mini-Review.

Harmful cyanobacterial blooms are increasing and becoming a worldwide concern as many bloom-forming cyanobacterial species can produce toxic metabolites named cyanotoxins. These include microcystins, saxitoxins, anatoxins, nodularins, and cylindrospermopsins, which can adversely affect humans, animals, and the environment. Different methods to assess these classes of compounds in vitro and in vivo include biological, biochemical, molecular, and physicochemical techniques. Furthermore, toxic effects not attributable to known cyanotoxins can be observed when assessing bloom material. In order to determine exposures to cyanotoxins and to monitor compliance with drinking and bathing water guidelines, it is necessary to have reliable and effective methods for the analysis of these compounds. Many relatively simple low-cost methods can be employed to rapidly evaluate the potential hazard. The main objective of this mini-review is to describe the assessment of toxic cyanobacterial samples using in vitro and in vivo bioassays. Newly emerging cyanotoxins, the toxicity of analogs, or the interaction of cyanobacteria and cyanotoxins with other toxicants, among others, still requires bioassay assessment. This review focuses on some biological and biochemical assays (MTT assay, Immunohistochemistry, Micronucleus Assay, Artemia salina assay, Daphnia magna test, Radionuclide recovery, Neutral red cytotoxicity and Comet assay, Enzyme-Linked Immunosorbent Assay (ELISA), Annexin V-FITC assay and Protein Phosphatase Inhibition Assay (PPIA)) for the detection and measurement of cyanotoxins including microcystins, cylindrospermopsins, anatoxin-a, saxitoxins, and nodularins. Although most bioassay analyses often confirm the presence of cyanotoxins at low concentrations, such bioassays can be used to determine whether some strains or blooms of cyanobacteria may produce other, as yet unknown toxic metabolites. This review also aims to identify research needs and data gaps concerning the toxicity assessment of cyanobacteria.

Plant-cyanobacteria interactions: Beneficial and harmful effects of cyanobacterial bioactive compounds on soil-plant systems and subsequent risk to animal and human health.

Plant-cyanobacteria interactions occur in different ways and at many different levels, both beneficial and harmful. Plant-cyanobacteria interactions, as a beneficial symbiosis, have long been demonstrated in rice-growing areas (Poaceae) where the most efficient nitrogen-fixing cyanobacteria are present in paddies. Moreover, cyanobacteria may in turn produce and/or secrete numerous bioactive compounds that have plant growth-promoting abilities or that may make the plant more resistant to abiotic or biotic stress. In recent years, there has been a growing worldwide interest in the use of cyanobacterial biomass as biofertilizers to replace chemical fertilizers, in part to overcome increasing organic-farming demands. However, the potential presence of harmful cyanotoxins has delayed the use of such cyanobacterial biomass, which can be found in large quantities in freshwater ecosystems around the world. In this review, we describe the existing evidence for the positive benefit of plant-cyanobacteria interactions and discuss the use of cyanobacterial biomass as biofertilizers and its growing worldwide interest. Although mass cyanobacterial blooms and scums are a current and emerging threat to the degradation of ecosystems and to animal and human health, they may serve as a source of numerous bioactive compounds with multiple positive effects that could be of use as an alternative to chemical fertilizers in the context of sustainable development.

Cyanotoxins and the Nervous System.

Cyanobacteria are capable of producing a wide range of bioactive compounds with many considered to be toxins. Although there are a number of toxicological outcomes with respect to cyanobacterial exposure, this review aims to examine those which affect the central nervous system (CNS) or have neurotoxicological properties. Such exposures can be acute or chronic, and we detail issues concerning CNS entry, detection and remediation. Exposure can occur through a variety of media but, increasingly, exposure through air via inhalation may have greater significance and requires further investigation. Even though cyanobacterial toxins have traditionally been classified based on their primary mode of toxicity, increasing evidence suggests that some also possess neurotoxic properties and include known cyanotoxins and unknown compounds. Furthermore, chronic long-term exposure to these compounds is increasingly being identified as adversely affecting human health.

Co-Occurrence of Cyanobacteria and Cyanotoxins with Other Environmental Health Hazards: Impacts and Implications.

Toxin-producing cyanobacteria in aquatic, terrestrial, and aerial environments can occur alongside a wide range of additional health hazards including biological agents and synthetic materials. Cases of intoxications involving cyanobacteria and cyanotoxins, with exposure to additional hazards, are discussed. Examples of the co-occurrence of cyanobacteria in such combinations are reviewed, including cyanobacteria and cyanotoxins plus algal toxins, microbial pathogens and fecal indicator bacteria, metals, pesticides, and microplastics. Toxicity assessments of cyanobacteria, cyanotoxins, and these additional agents, where investigated in bioassays and in defined combinations, are discussed and further research needs are identified.

l-Serine Reduces Spinal Cord Pathology in a Vervet Model of Preclinical ALS/MND.

The early neuropathological features of amyotrophic lateral sclerosis/motor neuron disease (ALS/MND) are protein aggregates in motor neurons and microglial activation. Similar pathology characterizes Guamanian ALS/Parkinsonism dementia complex, which may be triggered by the cyanotoxin ß-N-methylamino-l-alanine (BMAA). We report here the occurrence of ALS/MND-type pathological changes in vervets (Chlorocebus sabaeus; n = 8) fed oral doses of a dry powder of BMAA HCl salt (210 mg/kg/day) for 140 days. Spinal cords and brains from toxin-exposed vervets were compared to controls fed rice flour (210 mg/kg/day) and to vervets coadministered equal amounts of BMAA and l-serine (210 mg/kg/day). Immunohistochemistry and quantitative image analysis were used to examine markers of ALS/MND and glial activation. UHPLC-MS/MS was used to confirm BMAA exposures in dosed vervets. Motor neuron degeneration was demonstrated in BMAA-dosed vervets by TDP-43+ proteinopathy in anterior horn cells, by reactive astrogliosis, by activated microglia, and by damage to myelinated axons in the lateral corticospinal tracts. Vervets dosed with BMAA + l-serine displayed reduced neuropathological changes. This study demonstrates that chronic dietary exposure to BMAA causes ALS/MND-type pathological changes in the vervet and coadministration of l-serine reduces the amount of reactive gliosis and the number of protein inclusions in motor neurons.

Analysis of Neurotoxic Amino Acids from Marine Waters, Microbial Mats, and Seafood Destined for Human Consumption in the Arabian Gulf.

Human health risks associated with exposure to algal and cyanobacterial toxins (phycotoxins) have been largely concerned with aquatic habitats. People inhabiting desert environments may be exposed to phycotoxins present in terrestrial environments, where cyanobacterial crusts dominate. Seafood comprises a significant portion of the human diet in desert environments proximal to an ocean or sea. Consequently, in addition to terrestrial exposure to cyanotoxins, the potential exists that seafood may be an important exposure route for cyanotoxins in desert regions. Understanding the possible risk of exposure from seafood will help create cyanotoxin health guidelines for people living in environments that rely on seafood. Commonly-consumed local seafood products destined for human consumption were purchased from a fish market in Doha, Qatar. Organs were excised, extracted, and analyzed for the neurotoxic amino acid ß-N-methylamino-L-alanine (BMAA) and the isomers 2,4-diaminobutyric acid (DAB) and N-2(aminoethyl)glycine (AEG). The presence and concentration of neurotoxic amino acids were investigated in organisms from various trophic levels to examine the potential for biomagnification. Although BMAA and isomers were detected in marine microbial mats, as well as in marine plankton net trawls associated with diatoms and dinoflagellates, in seafood, only AEG and DAB were present at low concentrations in various trophic levels. The findings of this study suggest that exposure to neurotoxic amino acids through seafood in the Arabian Gulf may be minor, yet the presence of BMAA in phytoplankton confirms the need for further monitoring of marine waters and seafood to protect human health.

Traditional Food Items in Ogimi, Okinawa: l-Serine Content and the Potential for Neuroprotection.

PURPOSE OF REVIEW: Ogimi village is renowned for its aging population. We sought to determine if the l-serine content of their diet could account for their neurological health. RECENT FINDINGS: The most frequently consumed food items, including tofu and seaweeds, are rich in the dietary amino acid l-serine. l-serine content of the Ogimi diet >8 grams/day for Ogimi women significantly exceeds the average American dietary intake of 2.5 grams/day for women >70 years old. SUMMARY: Our hypothesis that the high l-serine content of the Ogimi diet is related to the paucity of tangle diseases among villagers is buttressed by in vivo results with non-human primates where dietary l-serine slowed development of neurofibrillary tangles and ß-amyloid plaques by up to 85% and a human clinical trial finding that l-serine at 15 grams/day twice daily slows functional decline in ALS patients. Analysis of the Ogimi diet suggests that l-serine should be evaluated for therapeutic potential as a neuroprotective agent.

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.

Do vervets and macaques respond differently to BMAA?

Vervets with chronic dietary exposure to BMAA develop neurofibrillary tangles (NFT) and sparse ß-amyloid plaque-like deposits in the brain. Macaques dosed via oral gavage with BMAA developed marked neurological signs in the absence of cell death. These differences may result from increased vulnerability of macaques to BMAA, the higher effective dose they received via oral gavage, and the possibility of stable adducts due to the bicarbonate used to neutralize their BMAA dose. Confirmation of chromatolysis and cell death in macaque brains was visualized using toluidine staining. In contrast, immunological staining with AT8 and ß-amyloid (1-42) antibodies and thioflavine-S stain in vervet brains suggests early stage labeling of neurites and NFT and plaque-like formation in the absence of neuronal loss. The lack of neurologic deficits reported in vervets is in keeping with early preclinical pathology observed with these immunohistochemical methods. BMAA toxicity in vervet brains causes the early events that occur in the genesis of neurofibrillary pathology. Taken together, these different studies of vervets and macaques demonstrate BMAA toxicity in the brain due to chronic exposures. The use of more sensitive immunohistochemical methods in the vervet study most likely explains the differences in neuropathology reported for vervets and macaques.