Optimization of methane gas-liquid mass transfer during biogas-based ectoine production in bubble column bioreactors.

Nowadays, the utilization of biogas for energy generation is hindered by the declining production costs of solar and wind power. A shift towards the valorization of biogas into ectoine, a highly valuable bioproduct priced at 1000 €â¸±kg-1, offers a novel approach to fostering a more competitive biogas market while contributing to carbon neutrality. This study evaluated the optimization of CH4 gas-liquid mass transfer in 10 L bubble column bioreactors for CH4 conversion into ectoine and hydroxyectoine using a mixed methanotrophic culture. The influence of the empty bed residence time (EBRTs of 27, 54, and 104 min) at different membrane diffuser pore sizes (0.3 and 0.6 mm) was investigated. Despite achieving CH4 elimination capacities (CH4-ECs) of 10-12 g⸱m-3⸱h-1, an EBRT of 104 min mediated CH4 limitation within the cultivation broth, resulting in a negligible biomass growth. Reducing the EBRT to 54 min entailed CH4-ECs of 21-24 g⸱m-3⸱h-1, concomitant to a significant increase in biomass growth (up to 0.17 g⸱L⸱d-1) and reaching maximum ectoine and hydroxyectoine accumulation of 79 and 13 mg⸱gVSS-1, respectively. Conversely, process operation at an EBRT of 27 min lead to microbial inhibition, resulting in a reduced biomass growth of 0.09 g⸱L⸱d-1 and an ectoine content of 47 mg⸱gVSS-1. While the influence of diffuser pore size was less pronounced compared to EBRT, the optimal process performance was observed with a diffuser pore size of 0.6 mm.

Effects of the Toxic Non-Protein Amino Acid ß-Methylamino-L-Alanine (BMAA) on Intracellular Amino Acid Levels in Neuroblastoma Cells.

The cyanobacterial non-protein amino acid (AA) ß-Methylamino-L-alanine (BMAA) is considered to be a neurotoxin. BMAA caused histopathological changes in brains and spinal cords of primates consistent with some of those seen in early motor neuron disease; however, supplementation with L-serine protected against some of those changes. We examined the impact of BMAA on AA concentrations in human neuroblastoma cells in vitro. Cells were treated with 1000 µM BMAA and intracellular free AA concentrations in treated and control cells were compared at six time-points over a 48 h culture period. BMAA had a profound effect on intracellular AA levels at specific time points but in most cases, AA homeostasis was re-established in the cell. The most heavily impacted amino acid was serine which was depleted in BMAA-treated cells from 9 h onwards. Correction of serine depletion could be a factor in the observation that supplementation with L-serine protects against BMAA toxicity in vitro and in vivo. AAs that could potentially be involved in protection against BMAA-induced oxidation such as histidine, tyrosine, and phenylalanine were depleted in cells at later time points.

Tandem mass tag-based quantitative proteomics reveals osmotic adaptation mechanisms in Alkalicoccus halolimnae BZ-SZ-XJ29T , a halophilic bacterium with a broad salinity range for optimal growth.

The moderate halophilic bacterium Alkalicoccus halolimnae BZ-SZ-XJ29T exhibits optimum growth over a wide range of NaCl concentrations (8.3-12.3%, w/v; 1.42-2.1 mol L-1 ). However, its adaptive mechanisms to cope with high salt-induced osmotic stress remain unclear. Using TMT-based quantitative proteomics, the cellular proteome was assessed under low (4% NaCl, 0.68 mol L-1 NaCl, control (CK) group), moderate (8% NaCl, 1.37 mol L-1 NaCl), high (12% NaCl, 2.05 mol L-1 NaCl), and extremely high (16% NaCl, 2.74 mol L-1 NaCl) salinity conditions. Digital droplet PCR confirmed the transcription of candidate genes related to salinity. A. halolimnae utilized distinct adaptation strategies to cope with different salinity conditions. Mechanisms such as accumulating different amounts and types of compatible solutes (i.e., ectoine, glycine betaine, glutamate, and glutamine) and the uptake of glycine betaine and glutamate were employed to cope with osmotic stress. Ectoine synthesis and accumulation were critical to the salt adaptation of A. halolimnae. The expression of EctA, EctB, and EctC, as well as the intracellular accumulation of ectoine, significantly and consistently increased with increasing salinity. Glycine betaine and glutamate concentrations remained constant under the four NaCl concentrations. The total content of glutamine and glutamate maintained a dynamic balance and, when exposed to different salinities, may play a role in low salinity-induced osmoadaptation. Moreover, cellular metabolism was severely affected at high salt concentrations, but the synthesis of amino acids, carbohydrate metabolism, and membrane transport related to haloadptation was preserved to maintain cytoplasmic concentration at high salinity. These findings provide insights into the osmoadaptation mechanisms of moderate halophiles and can serve as a theoretical underpinning for industrial production and application of compatible solutes.

The biotoxin BMAA promotes dysfunction via distinct mechanisms in neuroblastoma and glioblastoma cells.

Chronic exposure to the Cyanobacteria biotoxin Beta-methylamino-L-alanine (BMAA) has been associated with development of a sporadic form of ALS called Amyotrophic Lateral Sclerosis/Parkinsonism-Dementia Complex (ALS/PDC), as observed within certain Indigenous populations of Guam and Japan. Studies in primate models and cell culture have supported the association of BMAA with ALS/PDC, yet the pathological mechanisms at play remain incompletely characterized, effectively stalling the development of rationally-designed therapeutics or application of preventative measures for this disease. In this study we demonstrate for the first time that sub-excitotoxic doses of BMAA modulate the canonical Wnt signaling pathway to drive cellular defects in human neuroblastoma cells, suggesting a potential mechanism by which BMAA may promote neurological disease. Further, we demonstrate here that the effects of BMAA can be reversed in cell culture by use of pharmacological modulators of the Wnt pathway, revealing the potential value of targeting this pathway therapeutically. Interestingly, our results suggest the existence of a distinct Wnt-independent mechanism activated by BMAA in glioblastoma cells, highlighting the likelihood that neurological disease may result from the cumulative effects of distinct cell-type specific mechanisms of BMAA toxicity.

Identification and characterization of the key enzyme in the biosynthesis of the neurotoxin ß-ODAP in grass pea.

Grass pea (Lathyrus sativus L.) is a grain legume commonly grown in Asia and Africa for food and forage. It is a highly nutritious and robust crop, capable of surviving both droughts and floods. However, it produces a neurotoxic compound, ß-N-oxalyl-L-α,ß-diaminopropionic acid (ß-ODAP), which can cause a severe neurological disorder when consumed as a primary diet component. While the catalytic activity associated with ß-ODAP formation was demonstrated more than 50 years ago, the enzyme responsible for this activity has not been identified. Here, we report on the identity, activity, 3D structure, and phylogenesis of this enzyme-ß-ODAP synthase (BOS). We show that BOS belongs to the benzylalcohol O-acetyltransferase, anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase, deacetylvindoline 4-O-acetyltransferase superfamily of acyltransferases and is structurally similar to hydroxycinnamoyl transferase. Using molecular docking, we propose a mechanism for its catalytic activity, and using heterologous expression in tobacco leaves (Nicotiana benthamiana), we demonstrate that expression of BOS in the presence of its substrates is sufficient for ß-ODAP production in vivo. The identification of BOS may pave the way toward engineering ß-ODAP-free grass pea cultivars, which are safe for human and animal consumption.

Neuropathological Mechanisms of ß-N-Methylamino-L-Alanine (BMAA) with a Focus on Iron Overload and Ferroptosis.

The incidence of neurodegenerative diseases and cyanobacterial blooms is concomitantly increasing worldwide. The cyanotoxin ß-N-methylamino-L-alanine (BMAA) is produced by most of the Cyanobacteria spp. This cyanotoxin is described as a potential environmental etiology factor for some sporadic neurodegenerative diseases. Climate change and eutrophication significantly increase the frequency and intensity of cyanobacterial bloom in water bodies. This review evaluates different neuropathological mechanisms of BMAA at molecular and cellular levels and compares the related studies to provide some useful recommendations. Additionally, the structure and properties of BMAA as well as its microbial origin, especially by gut bacteria, are also briefly covered. Unlike previous reviews, we hypothesize the possible neurotoxic mechanism of BMAA through iron overload. We also discuss the involvement of BMAA in excitotoxicity, TAR DNA-binding protein 43 (TDP-43) translocation and accumulation, tauopathy, and other protein misincorporation and misfolding.

Cysteine biosynthesis contributes to ß-methylamino-l-alanine tolerance in Escherichia coli.

In contrast to mammalian cells, bacteria such as Escherichia coli have been shown to display tolerance towards the neurotoxin ß-methylamino-l-alanine (BMAA) suggesting that these prokaryotes possess a way to metabolise BMAA or its products, resulting in their export, degradation, or detoxification. Single gene deletion mutants of E. coli K-12 with inactivated amino acid biosynthesis pathways were treated with 500 µg/ml BMAA and the resulting growth was monitored. Wild type E. coli and most of the gene deletion mutants displayed unaltered growth in the presence of BMAA over 12 h. Conversely, deletion of genes in the cysteine biosynthesis pathway, cysE, cysK or cysM resulted in a BMAA dose-dependent growth delay in minimal medium. Through further studies of the ΔcysE strain, we observed increased susceptibility to oxidative stress from H2O2 in minimal medium, and disruptions in glutathione levels and oxidation state. The cysteine biosynthesis pathway is therefore linked to the tolerance of BMAA and oxidative stress in E. coli, which potentially represents a mechanism of BMAA detoxification.

Bradyrhizobium diazoefficiens Requires Chemical Chaperones To Cope with Osmotic Stress during Soybean Infection.

When engaging in symbiosis with legume hosts, rhizobia are confronted with environmental changes, including nutrient availability and stress exposure. Genetic circuits allow responding to these environmental stimuli to optimize physiological adaptations during the switch from the free-living to the symbiotic life style. A pivotal regulatory system of the nitrogen-fixing soybean endosymbiont Bradyrhizobium diazoefficiens for efficient symbiosis is the general stress response (GSR), which relies on the alternative sigma factor σEcfG However, the GSR-controlled process required for symbiosis has not been identified. Here, we demonstrate that biosynthesis of trehalose is under GSR control, and mutants lacking the respective biosynthetic genes otsA and/or otsB phenocopy GSR-deficient mutants under symbiotic and selected free-living stress conditions. The role of trehalose as a cytoplasmic chemical chaperone and stress protectant can be functionally replaced in an otsA or otsB mutant by introducing heterologous genetic pathways for biosynthesis of the chemically unrelated compatible solutes glycine betaine and (hydroxy)ectoine. Alternatively, uptake of exogenously provided trehalose also restores efficient symbiosis and tolerance to hyperosmotic and hyperionic stress of otsA mutants. Hence, elevated cytoplasmic trehalose levels resulting from GSR-controlled biosynthesis are crucial for B. diazoefficiens cells to overcome adverse conditions during early stages of host infection and ensure synchronization with root nodule development.IMPORTANCE The Bradyrhizobium-soybean symbiosis is of great agricultural significance and serves as a model system for fundamental research in bacterium-plant interactions. While detailed molecular insight is available about mutual recognition and early nodule organogenesis, our understanding of the host-imposed conditions and the physiology of infecting rhizobia during the transition from a free-living state in the rhizosphere to endosymbiotic bacteroids is currently limited. In this study, we show that the requirement of the rhizobial general stress response (GSR) during host infection is attributable to GSR-controlled biosynthesis of trehalose. Specifically, trehalose is crucial for an efficient symbiosis by acting as a chemical chaperone to protect rhizobia from osmostress during host infection.

In vitro generation of functional murine heart organoids via FGF4 and extracellular matrix.

Our understanding of the spatiotemporal regulation of cardiogenesis is hindered by the difficulties in modeling this complex organ currently by in vitro models. Here we develop a method to generate heart organoids from mouse embryonic stem cell-derived embryoid bodies. Consecutive morphological changes proceed in a self-organizing manner in the presence of the laminin-entactin (LN/ET) complex and fibroblast growth factor 4 (FGF4), and the resulting in vitro heart organoid possesses atrium- and ventricle-like parts containing cardiac muscle, conducting tissues, smooth muscle and endothelial cells that exhibited myocardial contraction and action potentials. The heart organoids exhibit ultrastructural, histochemical and gene expression characteristics of considerable similarity to those of developmental hearts in vivo. Our results demonstrate that this method not only provides a biomimetic model of the developing heart-like structure with simplified differentiation protocol, but also represents a promising research tool with a broad range of applications, including drug testing.

Investigations of Dimethylglycine, Glycine Betaine, and Ectoine Uptake by a Betaine-Carnitine-Choline Transporter Family Transporter with Diverse Substrate Specificity in Vibrio Species.

Fluctuations in osmolarity are one of the most prevalent stresses to which bacteria must adapt, both hypo- and hyperosmotic conditions. Most bacteria cope with high osmolarity by accumulating compatible solutes (osmolytes) in the cytoplasm to maintain the turgor pressure of the cell. Vibrio parahaemolyticus, a halophile, utilizes at least six compatible solute transporters for the uptake of osmolytes: two ABC family ProU transporters and four betaine-carnitine-choline transporter (BCCT) family transporters. The full range of compatible solutes transported by this species has yet to be determined. Using an osmolyte phenotypic microarray plate for growth analyses, we expanded the known osmolytes used by V. parahaemolyticus to include N,N-dimethylglycine (DMG), among others. Growth pattern analysis of four triple-bccT mutants, possessing only one functional BCCT, indicated that BccT1 (VP1456), BccT2 (VP1723), and BccT3 (VP1905) transported DMG. BccT1 was unusual in that it could take up both compounds with methylated head groups (glycine betaine [GB], choline, and DMG) and cyclic compounds (ectoine and proline). Bioinformatics analysis identified the four coordinating amino acid residues for GB in the BccT1 protein. In silico modeling analysis demonstrated that GB, DMG, and ectoine docked in the same binding pocket in BccT1. Using site-directed mutagenesis, we showed that a strain with all four residues mutated resulted in the loss of uptake of GB, DMG, and ectoine. We showed that three of the four residues were essential for ectoine uptake, whereas only one of the residues was important for GB uptake. Overall, we have demonstrated that DMG is a highly effective compatible solute for Vibrio species and have elucidated the amino acid residues in BccT1 that are important for the coordination of GB, DMG, and ectoine transport.IMPORTANCEVibrio parahaemolyticus possesses at least six osmolyte transporters, which allow the bacterium to adapt to high-salinity conditions. In this study, we identified several additional osmolytes that were utilized by V. parahaemolyticus We demonstrated that the compound DMG, which is present in the marine environment, was a highly effective osmolyte for Vibrio species. We determined that DMG is transported via BCCT family carriers, which have not been shown previously to take up this compound. BccT1 was a carrier for GB, DMG, and ectoine, and we identified the amino acid residues essential for the coordination of these compounds. The data suggest that for BccT1, GB is more easily accommodated than ectoine in the transporter binding pocket.

Halotolerance mechanisms of the methanotroph Methylomicrobium alcaliphilum.

Methylomicrobium alcaliphilum is an alkaliphilic and halotolerant methanotroph. The physiological responses of M. alcaliphilum to high NaCl concentrations, were studied using RNA sequencing and metabolic modeling. This study revealed that M. alcaliphilum possesses an unusual respiratory chain, in which complex I is replaced by a Na+ extruding NQR complex (highly upregulated under high salinity conditions) and a Na+ driven adenosine triphosphate (ATP) synthase coexists with a conventional H+ driven ATP synthase. A thermodynamic and metabolic model showing the interplay between these different components is presented. Ectoine is the main osmoprotector used by the cells. Ectoine synthesis is activated by the transcription of an ect operon that contains five genes, including the ectoine hydroxylase coding ectD gene. Enzymatic tests revealed that the product of ectD does not have catalytic activity. A new Genome Scale Metabolic Model for M. alcaliphilum revealed a higher flux in the oxidative branch of the pentose phosphate pathway leading to NADPH production and contributing to resistance to oxidative stress.

Ectoine interaction with DNA: influence on ultraviolet radiation damage.

Ectoine is a small zwitterionic osmolyte and compatible solute, which does not interfere with cell metabolism even at molar concentrations. Plasmid DNA (pUC19) was irradiated with ultraviolet radiation (UV-C at 266 nm) under quasi physiological conditions (PBS) and in pure water in the presence and absence of ectoine (THP(B)) and hydroxyectoine (THP(A)). Different types of UV induced DNA damage were analysed: DNA single-strand breaks (SSBs), abasic sites and cyclobutane pyrimidine dimers (CPDs). A complex interplay between these factors was observed with respect to the nature and occurrence of DNA damage with 266 nm photons. In PBS, the cosolutes showed efficient protection against base damage, whilst in pure water, a dramatic shift from SSB damage to base damage was observed when cosolutes were added. To test whether these effects are caused by ectoine binding to DNA, further experiments were conducted: small-angle X-ray scattering (SAXS), surface-plasmon resonance (SPR) measurements and Raman spectroscopy. The results show, for the first time, a close interaction between ectoine and DNA. This is in stark contrast to the assumption made by preferential exclusion models, which are often used to interpret the behaviour of compatible solutes within cells and with biomolecules. It is tentatively proposed that the alterations of UV damage to DNA are attributed to ectoine influence on nucleobases through the direct interaction between ectoine and DNA.

Metabolism of the neurotoxic amino acid ß-N-methylamino-L-alanine in human cell culture models.

Human dietary exposure to the environmental neurotoxin ß-N-methylamino-L-alanine (BMAA) has been implicated in an increased risk of developing sporadic neurodegenerative diseases like Alzheimer's and amyotrophic lateral sclerosis. Evidence suggests that humans are exposed to BMAA globally, but very little is known about BMAA metabolism in mammalian systems, let alone in humans. The most plausible, evidence-based mechanisms of BMAA toxicity rely on the metabolic stability of the amino acid and that, following ingestion, it enters the circulatory system unmodified. BMAA crosses from the intestinal lumen into the circulatory system, and the small intestine and liver are the first sites for dietary amino acid metabolism. Both tissues have substantial amino acid metabolic needs, which are largely fulfilled by dietary amino acids. Metabolism of BMAA in these tissues has been largely overlooked, yet is important in gauging the true human exposure risk. Here we investigate the potential for BMAA metabolism by the human liver and small intestine, using in vitro cell systems. Data show that BMAA metabolism via common proteinogenic amino acid metabolic pathways is negligible, and that in the presence of other amino acids cellular uptake of BMAA is substantially reduced. These data suggest that the majority of ingested BMAA remains unmodified following passage through the small intestine and liver. This not only supports oral BMAA exposure as a plausible exposure route to toxic doses of BMAA, but also supports previous notions that protein deficient diets or malnutrition may increase an individual's susceptibility to BMAA absorption and subsequent toxicity.

Glycine Betaine and Ectoine Are the Major Compatible Solutes Used by Four Different Halophilic Heterotrophic Ciliates.

One decisive factor controlling the distribution of organisms in their natural habitats is the cellular response to environmental factors. Compared to prokaryotes, our knowledge about salt adaptation strategies of microbial eukaryotes is very limited. We, here, used a recently introduced approach (implementing proton nuclear magnetic resonance spectroscopy) to investigate the presence of compatible solutes in halophilic, heterotrophic ciliates. Therefore, we isolated four ciliates from solar salterns, which were identified as Cyclidium glaucoma, Euplotes sp., Fabrea salina, and Pseudocohnilembus persalinus based on their 18S rRNA gene signatures and electron microscopy. The results of 1H-NMR spectroscopy revealed that all four ciliates employ the "low-salt-in" strategy by accumulating glycine betaine and ectoine as main osmoprotectants. We recorded a linear increase of these compatible solutes with increasing salinity of the external medium. Ectoine in particular stands out as its use as compatible solute was thought to be exclusive to prokaryotes. However, our findings and those recently made on two other heterotroph species call for a re-evaluation of this notion. The observation of varying relative proportions of compatible solutes within the four ciliates points to slight differences in haloadaptive strategies by regulatory action of the ciliates. Based on this finding, we provide an explanatory hypothesis for the distribution of protistan diversity along salinity gradients.

Development and validation of polar RP-HPLC method for screening for ectoine high-yield strains in marine bacteria with green chemistry.

A novel, green, rapid, and precise polar RP-HPLC method has been successfully developed and screened for ectoine high-yield strain in marine bacteria. Ectoine is a polar and extremely useful solute which allows microorganisms to survive in extreme environmental salinity. This paper describes a polar-HPLC method employed polar RP-C18 (5 µm, 250 × 4.6 mm) using pure water as the mobile phase and a column temperature of 30 °C, coupled with a flow rate at 1.0 mL/min and detected under a UV detector at wavelength of 210 nm. Our method validation demonstrates excellent linearity (R 2 = 0.9993), accuracy (100.55%), and a limit of detection LOQ and LOD of 0.372 and 0.123 µgmL-1, respectively. These results clearly indicate that the developed polar RP-HPLC method for the separation and determination of ectoine is superior to earlier protocols.

Streptomyces albulus yields ε-poly-L-lysine and other products from salt-contaminated glycerol waste.

Actinomycetes are the most important microorganisms for the industrial production of secondary metabolites with antimicrobial and anticancer properties. However, they have not been implicated in biorefineries. Here, we study the ability of the ε-poly-L-lysine producing Streptomyces albulus BCRC 11814 to utilize biodiesel-derived crude glycerol. S. albulus was cultured in a mineral medium supplemented with up to 10% w/v sodium chloride or potassium chloride, and with crude glycerol as the sole carbohydrate source. Under these conditions, the strain produced 0.1 g ε-poly-L-lysine per 1 g of biomass. RNA sequencing revealed upregulation of the ectoine biosynthetic pathway of S. albulus, which provides proof of halotolerance. S. albulus has several silent secondary metabolite biosynthetic clusters predicted within the genome. Based on the results, we conclude that S. albulus BCRC 11814 is a halotolerant microorganism capable of utilizing biodiesel-derived crude glycerol better than other actinomycetes included in the present study. S. albulus has the potential to be established as microbial platform production host for a range of high-value biological products.

With a pinch of extra salt-Did predatory protists steal genes from their food?

The cellular adjustment of Bacteria and Archaea to high-salinity habitats is well studied and has generally been classified into one of two strategies. These are to accumulate high levels either of ions (the "salt-in" strategy) or of physiologically compliant organic osmolytes, the compatible solutes (the "salt-out" strategy). Halophilic protists are ecophysiological important inhabitants of salt-stressed ecosystems because they are not only very abundant but also represent the majority of eukaryotic lineages in nature. However, their cellular osmostress responses have been largely neglected. Recent reports have now shed new light on this issue using the geographically widely distributed halophilic heterotrophic protists Halocafeteria seosinensis, Pharyngomonas kirbyi, and Schmidingerothrix salinarum as model systems. Different approaches led to the joint conclusion that these unicellular Eukarya use the salt-out strategy to cope successfully with the persistent high salinity in their habitat. They accumulate various compatible solutes, e.g., glycine betaine, myo-inositol, and ectoines. The finding of intron-containing biosynthetic genes for ectoine and hydroxyectoine, their salt stress-responsive transcription in H. seosinensis, and the production of ectoine and its import by S. salinarum come as a considerable surprise because ectoines have thus far been considered exclusive prokaryotic compatible solutes. Phylogenetic considerations of the ectoine/hydroxyectoine biosynthetic genes of H. seosinensis suggest that they have been acquired via lateral gene transfer by these bacterivorous Eukarya from ectoine/hydroxyectoine-producing food bacteria that populate the same habitat.

Investigation of the interaction of ß-methylamino-L-alanine with eukaryotic and prokaryotic proteins.

There is a strong body of evidence linking the non-protein amino acid (NPAA) ß-methylamino-L-alanine (BMAA) to the development of a number of neurodegenerative diseases. BMAA has been found globally, is produced by a number of organisms including cyanobacteria, diatoms, and dinoflagellates; and has been shown to biomagnify through trophic levels. The role of BMAA in neurodegenerative disease is highlighted by its presence in the brains of a number of neurodegenerative disease patients, where it was found in a protein-bound form. We have previously shown that BMAA is bound to cell proteins, and results in the upregulation of the unfolded protein response, an endoplasmic reticulum stress response activated by the presence of misfolded proteins within the cell. Structurally aberrant proteins are features of a number of neurodegenerative diseases, and further investigation of how BMAA interacts with proteins is crucial to our understanding of its toxicity. Here we use radiolabelled BMAA to investigate the interaction and binding of BMAA to eukaryotic and prokaryotic proteins. We found differences in the presence and distribution of protein-bound BMAA between E. coli and neuroblastoma cells, with an increase in binding over time only seen in the eukaryotic cells. We also found that BMAA was unable to bind to pure proteins, or cell lysate in native or denaturing conditions, indicating that biological processing is required for BMAA to bind to proteins.

Transport of BMAA into Neurons and Astrocytes by System xc.

The study of the mechanism of ß-N-methylamino-L-alanine (BMAA) neurotoxicity originally focused on its effects at the N-methyl-D-aspartate (NMDA) receptor. In recent years, it has become clear that its mechanism of action is more complicated. First, there are certain cell types, such as motor neurons and cholinergic neurons, where the dominate mechanism of toxicity is through action at AMPA receptors. Second, even in cortical neurons where the primary mechanism of toxicity appears to be activation of NMDA receptors, there are other mechanisms involved. We found that along with NMDA receptors, activation of mGLuR5 receptors and effects on the cystine/glutamate antiporter (system xc-) were involved in the toxicity. The effects on system xc- are of particular interest. System xc- mediates the transport of cystine into the cell in exchange for releasing glutamate into the extracellular fluid. By releasing glutamate, system xc- can potentially cause excitotoxicity. However, through providing cystine to the cell, it regulates the levels of cellular glutathione (GSH), the main endogenous intracellular antioxidant, and in this way may protect cells against oxidative stress. We have previously published that BMAA inhibits cystine uptake leading to GSH depletion and had indirect evidence that BMAA is transported into the cells by system xc-. We now present direct evidence that BMAA is transported into both astrocytes and neurons through system xc-. The fact that BMAA is transported by system xc- also provides a mechanism for BMAA to enter brain cells potentially leading to misincorporation into proteins and protein misfolding.

Stationary phase-dependent accumulation of ectoine is an efficient adaptation strategy in Vibrio anguillarum against cold stress.

The capability of cold-adaptation is a prerequisite of microorganisms that survive in an environment with frequent fluctuations in temperature. As a global causative agent of vibriosis in marine fish farming, Vibrio anguillarum can efficiently grow and proliferate under cold-stress conditions, which is 15°C lower than the optimal growth temperatures (25-30°C). Our data showed that V. anguillarum was able to synthesize ectoine de novo and that ectoine was essential for its growth under cold stress. Using 1H nuclear magnetic resonance spectroscopy and mutants lacking ectABC and proVWX (ectoine synthesis and transporter system genes, respectively), we confirmed that accumulation of this compatible solute occurs strictly at low temperatures and that the expression of ectA and proV is highly activated in the stationary growth phase. However, the synthesis of ectoine was repressed by exogenous choline (precursor of glycine betaine), suggesting that ectoine is an alternative compatible solute as a cold-stress protectant in V. anguillarum. Based on these results, we present possible scenarios of the synthesis and uptake of ectoine, which will facilitate the understanding of the molecular mechanism of V. anguillarum adaptation to cold environments and help improve freezing-dry processes for the V. anguillarum live vaccine.