Integrated multi-omics unveil the impact of H-phosphinic analogues of glutamate and α-ketoglutarate on Escherichia coli metabolism.

Desmethylphosphinothricin (L-Glu-γ-PH) is the H-phosphinic analogue of glutamate with carbon-phosphorus-hydrogen (C-P-H) bonds. In L-Glu-γ-PH the phosphinic group acts as a bioisostere of glutamate γ-carboxyl group allowing the molecule to be a substrate of Escherichia coli glutamate decarboxylase, a pyridoxal 5'-phosphate-dependent α-decarboxylase. In addition, the L-Glu-γ-PH decarboxylation product, GABA-PH, is further metabolized by bacterial GABA-transaminase, another pyridoxal 5'-phosphate-dependent enzyme, and succinic semialdehyde dehydrogenase, a NADP+-dependent enzyme. The product of these consecutive reactions, the so-called GABA shunt, is succinate-PH, the H-phosphinic analogue of succinate, a tricarboxylic acid cycle intermediate. Notably, L-Glu-γ-PH displays an antibacterial activity in the same concentration range of well-established antibiotics in E. coli. The dipeptide L-Leu-Glu-γ-PH was shown to display an even higher efficacy, likely as a consequence of an improved penetration into the bacteria. Herein, with the aim of further understanding the intracellular effects of L-Glu-γ-PH, 1H NMR-based metabolomics and LC-MS-based shotgun proteomics were used. This study included also the keto-derivative of L-Glu-γ-PH, α-ketoglutarate-γ-PH (α-KG-γ-PH), which also exhibits antimicrobial activity. L-Glu-γ-PH and α-KG-γ-PH are found to similarly impact the bacterial metabolism, though the overall effect of α-KG-γ-PH is more pervasive. Notably α-KG-γ-PH is converted intracellularly into L-Glu-γ-PH, but the opposite was not found. In general, both molecules impact the pathways where aspartate, glutamate and glutamine are used as precursors for the biosynthesis of related metabolites, activate the acid stress response and deprive cells of nitrogen. This work highlights the multi-target drug potential of L-Glu-γ-PH and α-KG-γ-PH and paves the way for their exploitation as antimicrobials.

Multifunctional FFP2 Face Mask for White Light Disinfection and Pathogens Detection using Hybrid Nanostructures and Optical Metasurfaces.

A new generation of an FFP2 (Filtering Face Piece of type 2) smart face mask is achieved by integrating broadband hybrid nanomaterials and a self-assembled optical metasurface. The multifunctional FFP2 face mask shows simultaneously white light-assisted on-demand disinfection properties and versatile biosensing capabilities. These properties are achieved by a powerful combination of white light thermoplasmonic responsive hybrid nanomaterials, which provide excellent photo-thermal disinfection properties, and optical metasurface-based colorimetric biosensors, with a very low limit of pathogens detection. The realized system is studied in optical, morphological, spectroscopic, and cell viability assay experiments and environmental monitoring of harmful pathogens, thus highlighting the extraordinary properties in reusability and pathogens detection of the innovative face mask.

Synthesis and Biological Activity of Homohypotaurine Obtained by the Enzyme-Based Conversion of Homocysteine Sulfinic Acid Using Recombinant Escherichia Coli Glutamate Decarboxylase.

l-Homocysteine, formed from S-adenosyl methionine following demethylation and adenosine release, accumulates when the methionine recycling pathway and other pathways become impaired, thus leading to hyperhomocysteinemia, a biomarker in cardiovascular diseases, neurological/psychiatric disorders, and cancer. The partial oxidation of the l-homocysteine thiol group and its decarboxylation on C-alpha lead to the formation of l-homocysteinesulfinic acid (l-HCSA) and homohypotaurine (HHT), respectively. Both compounds are not readily available from commercial suppliers, which hinders the investigation of their biological activities. Herein, the chemical synthesis of l-HCSA, from l-homocystine, was the starting point for establishing the bio-based synthesis of HHT using recombinant Escherichia coli glutamate decarboxylase (EcGadB), an enzyme already successfully employed for the bio-based synthesis of GABA and its phosphinic analog. Prior to HHT synthesis, kcat (33.92 ± 1.07) and KM (38.24 ± 3.45 mM) kinetic constants were determined for l-HCSA on EcGadB. The results of our study show that the EcGadB-mediated synthesis of HHT can be achieved with good yields (i.e., 40% following enzymatic synthesis and column chromatography). Purified HHT was tested in vitro on primary human umbilical vein endothelial cells and rat cardiomyoblasts and compared to the fully oxidized analog, homotaurine (OT, also known as tramiprosate), in widespread pharmaceutical use. The results show that both cell lines display statistically significant recovery from the cytotoxic effects induced by H2O2 in the presence of HHT.

Antibacterial Activity of Peptide Derivatives of Phosphinothricin against Multidrug-Resistant Klebsiella pneumoniae.

The fast spread of bacteria that are resistant to many classes of antibiotics (multidrug resistant) is a global threat to human and animal health with a worrisome scenario ahead. Novel therapeutical strategies are of crucial importance to combat this phenomenon. For this purpose, we investigated the antimicrobial properties of the naturally occurring tripeptide Bialaphos and a dipeptide L-leucyl-L-phosphinoithricin, the synthesis and diastereomers separation of which are herein described. We demonstrate that these compounds are effective on clinical isolates of the human pathogen Klebsiella pneumoniae, causing hospital-acquired and community-acquired infections. The tested isolates were remarkable for their resistance to more than 20 commercial antibiotics of different classes. Based on previous literature data and our experiments consisting of glutamine supplementation, we suggest that both compounds release phosphinothricin-a well-known nanomolar inhibitor of glutamine synthetase-after their penetration in the bacterial cells; and, in this way, exert their antibacterial effect by negatively affecting nitrogen assimilation in this pathogen.

Impact of the Gastrointestinal Tract Microbiota on Cardiovascular Health and Pathophysiology.

ABSTRACT: The microbiota of the gastrointestinal tract (GIT) is an extremely diverse community of microorganisms, and their collective genomes (microbiome) provide a vast arsenal of biological activities, particularly enzymatic ones, which are far from being fully elucidated. The study of the microbiota (and the microbiome) is receiving great interest from the biomedical community because it carries the potential to improve risk prediction models, refine primary and secondary prevention efforts, and also design more appropriate and personalized therapies, including pharmacological ones. A growing body of evidence, although sometimes impaired by the limited number of subjects involved in the studies, suggests that GIT dysbiosis, that is, the altered microbial composition, has an important role in causing and/or worsening cardiovascular disease (CVD). Bacterial translocation and the alteration of levels of microbe-derived metabolites can thus be important to monitor and modulate because they may lead to initiation and progression of CVD and to its establishment as chronic state. We hereby aim to provide readers with details on available resources and experimental approaches that are used in this fascinating field of biomedical research and on some novelties on the impact of GIT microbiota on CVD.

Essential oils from Artemisia species inhibit biofilm formation and the virulence of Escherichia coli EPEC 2348/69.

Enteropathogenic Escherichia coli E2346/69 (EPEC) has caused foodborne outbreaks worldwide and the bacterium forms antimicrobial-tolerant biofilms. The anti-biofilm formation of various components of essential oils extracted from selected medicinal plants were investigated and tested on EPEC and wild strains of E. coli. Oils extracted from the family Asteraceae and their major common constituents at 0.031 and 0.062% (V/v) were found to significantly inhibit biofilm formation without affecting the growth of planktonic cells. In addition, three plants belonging to this family (Artemisia herba alba, Artemisia campestris and Artemisia absinthium) played important roles in the antimicrobial activity. Interestingly, their essential oils reduced the ability of E. coli (the EPEC and K12 strains) to form a biofilm. The crystal violet reduction assay showed that the plant extracts tested reduced biofilm formation with the inhibition of bacterial attachment up to 45% for EPEC and 70% for E. coli K-12 after 24 h treatment at 0.62 mg ml-1, demonstrating that Artemisia oils had a high anti-biofilm activity on the bacteria tested. The results indicate that the locus of enterocyte effacement (LEE) acquired by horizontal transfer promotes the formation of the attaching and effacing (A/E) lesion and increases the capacity of the photogen strain (EPEC) to form a biofilm. The chemical composition of the volatile compounds was obtained by gas chromatography-mass spectrometry analysis, which showed that the essential oils consisted of thirty-four compounds. Chamazulene (39.21%), ß-pinene (32.07%), and α-thujone (29.39%) were the main constituents of the essential oils of A. herba alba, A. absinthium and A. campestris, respectively.

Cofactor-dependent conformational heterogeneity of GAD65 and its role in autoimmunity and neurotransmitter homeostasis.

The human neuroendocrine enzyme glutamate decarboxylase (GAD) catalyses the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) using pyridoxal 5'-phosphate as a cofactor. GAD exists as two isoforms named according to their respective molecular weights: GAD65 and GAD67. Although cytosolic GAD67 is typically saturated with the cofactor (holoGAD67) and constitutively active to produce basal levels of GABA, the membrane-associated GAD65 exists mainly as the inactive apo form. GAD65, but not GAD67, is a prevalent autoantigen, with autoantibodies to GAD65 being detected at high frequency in patients with autoimmune (type 1) diabetes and certain other autoimmune disorders. The significance of GAD65 autoinactivation into the apo form for regulation of neurotransmitter levels and autoantibody reactivity is not understood. We have used computational and experimental approaches to decipher the nature of the holo → apo conversion in GAD65 and thus, its mechanism of autoinactivation. Molecular dynamics simulations of GAD65 reveal coupling between the C-terminal domain, catalytic loop, and pyridoxal 5'-phosphate-binding domain that drives structural rearrangement, dimer opening, and autoinactivation, consistent with limited proteolysis fragmentation patterns. Together with small-angle X-ray scattering and fluorescence spectroscopy data, our findings are consistent with apoGAD65 existing as an ensemble of conformations. Antibody-binding kinetics suggest a mechanism of mutually induced conformational changes, implicating the flexibility of apoGAD65 in its autoantigenicity. Although conformational diversity may provide a mechanism for cofactor-controlled regulation of neurotransmitter biosynthesis, it may also come at a cost of insufficient development of immune self-tolerance that favors the production of GAD65 autoantibodies.

Bicyclic γ-amino acids as inhibitors of γ-aminobutyrate aminotransferase.

The γ-aminobutyrate (GABA)-degradative enzyme GABA aminotransferase (GABA-AT) is regarded as an attractive target to control GABA levels in the central nervous system: this has important implications in the treatment of several neurological disorders and drug dependencies. We have investigated the ability of newly synthesized compounds to act as GABA-AT inhibitors. These compounds have a unique bicyclic structure: the carbocyclic ring bears the GABA skeleton, while the fused 3-Br-isoxazoline ring contains an electrophilic warhead susceptible of nucleophilic attack by an active site residue of the target enzyme. Out of the four compounds tested, only the one named (+)-3 was found to significantly inhibit mammalian GABA-AT in vitro. Docking studies, performed on the available structures of GABA-AT, support the experimental findings: out of the four tested compounds, only (+)-3 suitably orients the electrophilic 3-Br-isoxazoline warhead towards the active site nucleophilic residue Lys329, thereby explaining the irreversible inhibition of GABA-AT observed experimentally.

Glutamate decarboxylase-dependent acid resistance in Brucella spp.: distribution and contribution to fitness under extremely acidic conditions.

Brucella is an expanding genus of major zoonotic pathogens, including at least 10 genetically very close species occupying a wide range of niches from soil to wildlife, livestock, and humans. Recently, we have shown that in the new species Brucella microti, the glutamate decarboxylase (Gad)-dependent system (GAD system) contributes to survival at a pH of 2.5 and also to infection in mice by the oral route. In order to study the functionality of the GAD system in the genus Brucella, 47 isolates, representative of all known species and strains of this genus, and 16 strains of the closest neighbor genus, Ochrobactrum, were studied using microbiological, biochemical, and genetic approaches. In agreement with the genome sequences, the GAD system of classical species was not functional, unlike that of most strains of Brucella ceti, Brucella pinnipedialis, and newly described species (B. microti, Brucella inopinata BO1, B. inopinata-like BO2, and Brucella sp. isolated from bullfrogs). In the presence of glutamate, these species were more acid resistant in vitro than classical terrestrial brucellae. Expression in trans of the gad locus from representative Brucella species in the Escherichia coli MG1655 mutant strain lacking the GAD system restored the acid-resistant phenotype. The highly conserved GAD system of the newly described or atypical Brucella species may play an important role in their adaptation to acidic external and host environments. Furthermore, the GAD phenotype was shown to be a useful diagnostic tool to distinguish these latter Brucella strains from Ochrobactrum and from classical terrestrial pathogenic Brucella species, which are GAD negative.

The Escherichia coli Acid Stress Response and Its Significance for Pathogenesis.

Escherichia coli has a remarkable ability to survive low pH and possesses a number of different genetic systems that enable it to do this. These may be expressed constitutively, typically in stationary phase, or induced by growth under a variety of conditions. The activities of these systems have been implicated in the ability of E. coli to pass the acidic barrier of the stomach and to become established in the gastrointestinal tract, something causing serious infections. However, much of the work characterizing these systems has been done on standard laboratory strains of E. coli and under conditions which do not closely resemble those found in the human gut. Here we review what is known about acid resistance in E. coli as a model laboratory organism and in the context of its lifestyle as an inhabitant-sometimes an unwelcome one-of the human gut.

Exploitation of microbial activities at low pH to enhance planetary health.

Awareness is growing that human health cannot be considered in isolation but is inextricably woven with the health of the environment in which we live. It is, however, under-recognized that the sustainability of human activities strongly relies on preserving the equilibrium of the microbial communities living in/on/around us. Microbial metabolic activities are instrumental for production, functionalization, processing, and preservation of food. For circular economy, microbial metabolism would be exploited to produce building blocks for the chemical industry, to achieve effective crop protection, agri-food waste revalorization, or biofuel production, as well as in bioremediation and bioaugmentation of contaminated areas. Low pH is undoubtedly a key physical-chemical parameter that needs to be considered for exploiting the powerful microbial metabolic arsenal. Deviation from optimal pH conditions has profound effects on shaping the microbial communities responsible for carrying out essential processes. Furthermore, novel strategies to combat contaminations and infections by pathogens rely on microbial-derived acidic molecules that suppress/inhibit their growth. Herein, we present the state-of-the-art of the knowledge on the impact of acidic pH in many applied areas and how this knowledge can guide us to use the immense arsenal of microbial metabolic activities for their more impactful exploitation in a Planetary Health perspective.

On the potential role of naturally occurring carboxylic organic acids as anti-infective agents: opportunities and challenges.

Carboxylic organic acids are intermediates of central carbon metabolic pathways (e.g. acetic, propionic, citric, and lactic acid) long known to have potent antimicrobial potential, mainly at acidic pHs. The food industry has been leveraging those properties for years, using many of these acids as preservatives to inhibit the growth of pathogenic and/or spoilage fungal and bacterial species. A few of these molecules (the most prominent being acetic acid) have been used as antiseptics since Hippocratic medicine, mainly to treat infected wounds in patients with burns. With the growth of antibiotic therapy, the use of carboxylic acids (and other chemical antiseptics) in clinical settings lost relevance; however, with the continuous emergence of multi-antibiotic/antifungal resistant strains, the search for alternatives has intensified. This prospective article raises awareness of the potential of carboxylic acids to control infections in clinical settings, considering not only their previous exploitation in this context (which we overview) but also the positive experience of their safe use in food preservation. At a time of great concern with antimicrobial resistance and the slow arrival of new antimicrobial therapeutics to the market, further exploration of organic acids as anti-infective molecules may pave the way to more sustainable prophylactic and therapeutic approaches.

Glutamate decarboxylase-dependent acid resistance in orally acquired bacteria: function, distribution and biomedical implications of the gadBC operon.

For successful colonization of the mammalian host, orally acquired bacteria must overcome the extreme acidic stress (pH < 2.5) encountered during transit through the host stomach. The glutamate-dependent acid resistance (GDAR) system is by far the most potent acid resistance system in commensal and pathogenic Escherichia coli, Shigella flexneri, Listeria monocytogenes and Lactococcus lactis. GDAR requires the activity of glutamate decarboxylase (GadB), an intracellular PLP-dependent enzyme which performs a proton-consuming decarboxylation reaction, and of the cognate antiporter (GadC), which performs the glutamatein /γ-aminobutyrateout (GABA) electrogenic antiport. Herein we review recent findings on the structural determinants responsible for pH-dependent intracellular activation of E. coli GadB and GadC. A survey of genomes of bacteria (pathogenic and non-pathogenic), having in common the ability to colonize or to transit through the host gut, shows that the gadB and gadC genes frequently lie next or near each other. This gene arrangement is likely to be important to ensure timely co-regulation of the decarboxylase and the antiporter. Besides the involvement in acid resistance, GABA production and release were found to occur at very high levels in lactic acid bacteria originally isolated from traditionally fermented foods, supporting the evidence that GABA-enriched foods possess health-promoting properties.

Intracellular NADPH levels affect the oligomeric state of the glucose 6-phosphate dehydrogenase.

In the yeast Kluyveromyces lactis, glucose 6-phosphate dehydrogenase (G6PDH) is detected as two differently migrating forms on native polyacrylamide gels. The pivotal metabolic role of G6PDH in K. lactis led us to investigate the mechanism controlling the two activities in respiratory and fermentative mutant strains. An extensive analysis of these mutants showed that the NAD(+)(H)/NADP(+)(H)-dependent cytosolic alcohol (ADH) and aldehyde (ALD) dehydrogenase balance affects the expression of the G6PDH activity pattern. Under fermentative/ethanol growth conditions, the concomitant activation of ADH and ALD activities led to cytosolic accumulation of NADPH, triggering an alteration in the oligomeric state of the G6PDH caused by displacement/release of the structural NADP(+) bound to each subunit of the enzyme. The new oligomeric G6PDH form with faster-migrating properties increases as a consequence of intracellular redox unbalance/NADPH accumulation, which inhibits G6PDH activity in vivo. The appearance of a new G6PDH-specific activity band, following incubation of Saccharomyces cerevisiae and human cellular extracts with NADP(+), also suggests that a regulatory mechanism of this activity through NADPH accumulation is highly conserved among eukaryotes.

The glutamic acid decarboxylase system of the new species Brucella microti contributes to its acid resistance and to oral infection of mice.

BACKGROUND: Genome analysis indicated that the new species Brucella microti possesses a potentially functional glutamate decarboxylase (GAD) system involved in extreme acid resistance in several foodborne bacteria. The contribution of this system in adaptation of B. microti to an acidic environment, including the intracellular vacuole and stomach, was investigated. RESULTS: B. microti was GAD positive and able to export its product, γ-aminobutyrate, to the extracellular medium. The resistance of B. microti to acid stress (pH 2.5) was glutamate dependent. Mutants affected in the GAD system lost this resistance, demonstrating its direct involvement in survival under these conditions. The reciprocal heterologous complementation of mutants with the GAD systems of Escherichia coli or B. microti confirmed conserved functions in both bacterial species. A gad mutant was not attenuated during infection of macrophages, where Brucella resides in an acidified vacuole at a pH of 4-4.5 during the early phase of macrophage infection, but GAD contributed to the survival of B. microti in a murine model following oral infection. CONCLUSIONS: This work provides first evidence that the GAD system might play an essential role in the resistance of an environment-borne, pathogenic Brucella species to extreme acid shock and during passage through the host stomach following oral infection.

A Desmethylphosphinothricin Dipeptide Derivative Effectively Inhibits Escherichia coli and Bacillus subtilis Growth.

New antibiotics are unquestionably needed to fight the emergence and spread of multidrug-resistant bacteria. To date, antibiotics targeting bacterial central metabolism have been poorly investigated. By determining the minimal inhibitory concentration (MIC) of desmethylphosphinothricin (Glu-γ-PH), an analogue of glutamate with a phosphinic moiety replacing the γ-carboxyl group, we previously showed its promising antibacterial activity on Escherichia coli. Herein, we synthetized and determined the growth inhibition exerted on E. coli by an L-Leu dipeptide derivative of Glu-γ-PH (L-Leu-D,L-Glu-γ-PH). Furthermore, we compared the growth inhibition obtained with this dipeptide with that exerted by the free amino acid, i.e., Glu-γ-PH, and by their phosphonic and non-desmethylated analogues. All the tested compounds were more effective when assayed in a chemically-defined minimal medium. The dipeptide L-Leu-D,L-Glu-γ-PH had a significantly improved antibacterial activity (2 µg/mL), at a concentration between the non-desmethytaled (0.1 µg/mL) and the phosphonic (80 µg/mL) analogues. Also, in Bacillus subtilis, the dipeptide L-Leu-D,L-Glu-γ-PH displayed an activity comparable to that of the antibiotic amoxicillin. This work highlights the antibacterial relevance of the phosphinic pharmacophore and proposes new avenues for the development of novel antimicrobial drugs containing the phosphinic moiety.

White light thermoplasmonic activated gold nanorod arrays enable the photo-thermal disinfection of medical tools from bacterial contamination.

The outspread of bacterial pathogens causing severe infections and spreading rapidly, especially among hospitalized patients, is worrying and represents a global public health issue. Current disinfection techniques are becoming insufficient to counteract the spread of these pathogens because they carry multiple antibiotic-resistance genes. For this reason, a constant need exists for new technological solutions that rely on physical methods rather than chemicals. Nanotechnology support provides novel and unexplored opportunities to boost groundbreaking, next-gen solutions. With the help of plasmonic-assisted nanomaterials, we present and discuss our findings in innovative bacterial disinfection techniques. Gold nanorods (AuNRs) immobilized on rigid substrates are utilized as efficient white light-to-heat transducers (thermoplasmonic effect) for photo-thermal (PT) disinfection. The resulting AuNRs array shows a high sensitivity change in refractive index and an extraordinary capability in converting white light to heat, producing a temperature change greater than 50 °C in a few minute interval illumination time. Results were validated using a theoretical approach based on a diffusive heat transfer model. Experiments performed with a strain of Escherichia coli as a model microorganism confirm the excellent capability of the AuNRs array to reduce the bacteria viability upon white light illumination. Conversely, the E. coli cells remain viable without white light illumination, which also confirms the lack of intrinsic toxicity of the AuNRs array. The PT transduction capability of the AuNRs array is utilized to produce white light heating of medical tools used during surgical treatments, generating a temperature increase that can be controlled and is suitable for disinfection. Our findings are pioneering a new opportunity for healthcare facilities since the reported methodology allows non-hazardous disinfection of medical devices by simply employing a conventional white light lamp.

Physical, chemical, and sensory properties of water kefir produced from Aronia melanocarpa juice and pomace.

Water kefir is widely consumed all over the world due to its potential health benefits. The aim of this current study was to compare non-fermented juice and fermented beverage of water kefir produced from Aronia melanocarpa juice and pomace in terms of chemical, physical and sensory quality as well as valorisation of pomace in the production of water kefir. When compared to water kefir made with aronia juice, less reduction in total phenolic content (TPC), total flavonoid content (TFC) and total anthocyanin content (TAC) was observed in samples made with aronia pomace during the fermentation process. Similarly, greater antioxidant activity was demonstrated in water kefir made with aronia pomace than juice. Based on sensory evaluation, no difference was found in overall acceptability, taste, aroma/odor, and turbidity of water kefir made with aronia pomace before and after fermentation. Results indicated that aronia pomace has potential in water kefir production.

Endangered North Atlantic right whales (Eubalaena glacialis) experience repeated, concurrent exposure to multiple environmental neurotoxins produced by marine algae.

The western North Atlantic population of right whales (Eubalaena glacialis) is one of the most critically endangered of any whale population in the world. Among the factors considered to have potentially adverse effects on the health and reproduction of E. glacialis are biotoxins produced by certain microalgae responsible for causing harmful algal blooms. The worldwide incidence of these events has continued to increase dramatically over the past several decades and is expected to remain problematic under predicted climate change scenarios. Previous investigations have demonstrated that N. Atlantic right whales are being exposed to at least two classes of algal-produced environmental neurotoxins-paralytic shellfish toxins (PSTs) and domoic acid (DA). Our primary aims during this six-year study (2001-2006) were to assess whether the whales' exposure to these algal biotoxins occurred annually over multiple years, and to what extent individual whales were exposed repeatedly and/or concurrently to one or both toxin classes. Approximately 140 right whale fecal samples obtained across multiple habitats in the western N. Atlantic were analyzed for PSTs and DA. About 40% of these samples were attributed to individual whales in the North Atlantic Right Whale Catalog, permitting analysis of biotoxin exposure according to sex, age class, and reproductive status/history. Our findings demonstrate clearly that right whales are being exposed to both of these algal biotoxins on virtually an annual basis in multiple habitats for periods of up to six months (April through September), with similar exposure rates for females and males (PSTs: ∼70-80%; DA: ∼25-30%). Notably, only one of 14 lactating females sampled did not contain either PSTs or DA, suggesting the potential for maternal toxin transfer and possible effects on neonatal animals. Moreover, 22% of the fecal samples tested for PSTs and DA showed concurrent exposure to both neurotoxins, leading to questions of interactive effects. Targeted studies employing both in vivo and in vitro model systems represent the next logical step in assessing how and to what extent these algal biotoxins might compromise the health and reproduction of this endangered population.