Spatial and Temporal Resolution of Cyanobacterial Bloom Chemistry Reveals an Open-Ocean Trichodesmium thiebautii as a Talented Producer of Specialized Metabolites.

While the ecological role that Trichodesmium sp. play in nitrogen fixation has been widely studied, little information is available on potential specialized metabolites that are associated with blooms and standing stock Trichodesmium colonies. While a collection of biological material from a T. thiebautii bloom event from North Padre Island, Texas, in 2014 indicated that this species was a prolific producer of chlorinated specialized metabolites, additional spatial and temporal resolution was needed. We have completed these metabolite comparison studies, detailed in the current report, utilizing LC-MS/MS-based molecular networking to visualize and annotate the specialized metabolite composition of these Trichodesmium blooms and colonies in the Gulf of Mexico (GoM) and other waters. Our results showed that T. thiebautii blooms and colonies found in the GoM have a remarkably consistent specialized metabolome. Additionally, we isolated and characterized one new macrocyclic compound from T. thiebautii, trichothilone A (1), which was also detected in three independent cultures of T. erythraeum. Genome mining identified genes predicted to synthesize certain functional groups in the T. thiebautii metabolites. These results provoke intriguing questions of how these specialized metabolites affect Trichodesmium ecophysiology, symbioses with marine invertebrates, and niche development in the global oligotrophic ocean.

Temporal Dynamics of Cyanobacterial Bloom Community Composition and Toxin Production from Urban Lakes.

With a long evolutionary history and a need to adapt to a changing environment, cyanobacteria in freshwater systems use specialized metabolites for communication, defense, and physiological processes. However, the role that these metabolites play in differentiating species, maintaining microbial communities, and generating niche persistence and expansion is poorly understood. Furthermore, many cyanobacterial specialized metabolites and toxins present significant human health concerns due to their liver toxicity and their potential impact to drinking water. Gaps in knowledge exist with respect to changes in species diversity and toxin production during a cyanobacterial bloom (cyanoHAB) event; addressing these gaps will improve understanding of impacts to public and ecological health. In the current project, we utilized a multiomics strategy (DNA metabarcoding and metabolomics) to determine the cyanobacterial community composition, toxin profile, and the specialized metabolite pool at three freshwater lakes in Providence, RI during summer-fall cyanoHABs. Species diversity decreased at all study sites over the course of the bloom event, and toxin production reached a maximum at the midpoint of the event. Additionally, LC-MS/MS-based molecular networking identified new toxin congeners. This work provokes intriguing questions with respect to the use of allelopathy by organisms in these systems and the presence of emerging toxic compounds that can impact public health.

Simulated upwelling and marine heatwave events promote similar growth rates but differential domoic acid toxicity in Pseudo-nitzschia australis.

Along the west coast of the United States, highly toxic Pseudo-nitzschia blooms have been associated with two contrasting regional phenomena: seasonal upwelling and marine heatwaves. While upwelling delivers cool water rich in pCO2 and an abundance of macronutrients to the upper water column, marine heatwaves instead lead to warmer surface waters, low pCO2, and reduced nutrient availability. Understanding Pseudo-nitzschia dynamics under these two conditions is important for bloom forecasting and coastal management, yet the mechanisms driving toxic bloom formation during contrasting upwelling vs. heatwave conditions remain poorly understood. To gain a better understanding of what drives Pseudo-nitzschia australis growth and toxicity during these events, multiple-driver scenario or 'cluster' experiments were conducted using temperature, pCO2, and nutrient levels reflecting conditions during upwelling (13 °C, 900 ppm pCO2, replete nutrients) and two intensities of marine heatwaves (19 °C or 20.5 °C, 250 ppm pCO2, reduced macronutrients). While P. australis grew equally well under both heatwave and upwelling conditions, similar to what has been observed in the natural environment, cells were only toxic in the upwelling treatment. We also conducted single-driver experiments to gain a mechanistic understanding of which drivers most impact P. australis growth and toxicity. These experiments indicated that nitrogen concentration and N:P ratio were likely the drivers that most influenced domoic acid production, while the impacts of temperature or pCO2 concentration were less pronounced. Together, these experiments may help to provide both mechanistic and holistic perspectives on toxic P. australis blooms in the dynamic and changing coastal ocean, where cells interact simultaneously with multiple altered environmental variables.

Developing virtual and augmented reality applications for science, technology, engineering and math education.

The Rhode Island IDeA Network of Biomedical Research Excellence Molecular Informatics Core at the University of Rhode Island Information Technology Services Innovative Learning Technologies developed virtual and augmented reality applications to teach concepts in biomedical science, including pharmacology, medicinal chemistry, cell culture and nanotechnology. The apps were developed as full virtual reality/augmented reality and 3D gaming versions, which do not require virtual reality headsets. Development challenges included creating intuitive user interfaces, text-to-voice functionality, visualization of molecules and implementing complex science concepts. In-app quizzes are used to assess the user's understanding of topics, and user feedback was collected for several apps to improve the experience. The apps were positively reviewed by users and are being implemented into the curriculum at the University of Rhode Island.

What Is Authentic Maple Water? A Twelve-Month Shelf-Life Study of the Chemical Composition of Maple Water and Its Biological Activities.

Maple water (maple sap) products are produced from sap tapped directly from maple trees, but there is confusion and lack of industry consensus and consumer knowledge as to what constitutes 'authentic' maple water. With an immense potential for growth in the multi-billion dollar functional beverage market, the market promotion of maple water products hinges on establishing standards of identity (SI), which are currently lacking. Herein, we aim to provide publishable SI and compositional chemistry findings of maple water. The chemical composition (including polyphenols, sugars, amino acids, and organic acids) of a pasteurized maple water was monitored over a 12-month (at 0, 4, 8, and 12 months) shelf-life. Furthermore, LC-MS/MS and molecular networking-based methods were developed to identify the phytochemical profile of a maple water extract (MWX) and to compare it to a previously chemically characterized phenolic-enriched maple syrup extract (MSX). Both MSX and MWX have similar phytochemical profiles and chemical characteristics. In addition, MSX and MWX showed moderate antioxidant capacity (in free radical scavenging and anti-tyrosinase assays) and anti-inflammatory effects (in soluble epoxide hydrolase and cyclooxygenase-2 inhibition assays). Our findings provide critical information on the SI and stability (in chemical composition) of maple water, which will help define, authenticate, and distinguish it from other functional beverages, thereby positioning the maple industry for promotion and growth in this market sector.

Evaluations of Skin Permeability of Cannabidiol and Its Topical Formulations by Skin Membrane-Based Parallel Artificial Membrane Permeability Assay and Franz Cell Diffusion Assay.

Introduction: Cannabinoids including cannabidiol (CBD) have attracted enormous interest as bioactive ingredients for various dermatological and/or cosmeceutical uses. However, topical applications of cannabinoids might be limited without a fundamental understanding of their skin permeability. Herein, we aimed to evaluate the skin permeability of CBD and its topical formulations using artificial skin membrane assays. The solubility and stability of CBD in various surfactants that are commonly used in topical applications were also evaluated. Methods: CBD and two CBD-incorporated topical formulations (cream and gel) were prepared for this study. Computational predictions (SwissADME and DERMWIN™) and the parallel artificial membrane permeability assay (PAMPA) were used to evaluate the skin permeability of CBD isolate. The Franz cell diffusion (in vitro release testing) assay was used to evaluate the skin permeability of CBD formulations. The solubility and stability of CBD in surfactants were assessed by high-performance liquid chromatography and mass spectrometry analysis. Results: CBD isolate showed favorable skin permeability in the SwissADME and DERMWIN™ predictions (-Log Kp of 3.6 and 5.7 cm/s, respectively) and PAMPA (-LogPe value of 5.0 at pH of 6.5 and 7.4). In addition, CBD had higher solubility (378.4 µg/mL) in surfactant Tween 20 as compared to its solubility in polyisobutene. In an acidic environment (pH 5 and 6), Tween 20 maintained the CBD content at 81% and 70% over 30 days, respectively. CBD in the formulations of cream and gel also had moderate skin permeability in the Franz cell diffusion assay. Conclusion: Data from artificial membrane-based assays support that CBD is a skin permeable cannabinoid and the permeability and stability of its formulations may be influenced by several factors such as surfactant and pH environment. Findings from our study suggest that CBD may have suitable skin permeability for the development of dermatological and/or cosmeceutical applications but further studies using in vivo models are warranted to confirm this.

Screening the PRISM Library against Staphylococcus aureus Reveals a Sesquiterpene Lactone from Liriodendron tulipifera with Inhibitory Activity.

Infections caused by the bacterium Staphylococcus aureus continue to pose threats to human health and put a financial burden on the healthcare system. The overuse of antibiotics has contributed to mutations leading to the emergence of methicillin-resistant S. aureus, and there is a critical need for the discovery and development of new antibiotics to evade drug-resistant bacteria. Medicinal plants have shown promise as sources of new small-molecule therapeutics with potential uses against pathogenic infections. The principal Rhode Island secondary metabolite (PRISM) library is a botanical extract library generated from specimens in the URI Youngken Medicinal Garden by upper-division undergraduate students. PRISM extracts were screened for activity against strains of methicillin-susceptible S. aureus (MSSA). An extract generated from the tulip tree (Liriodendron tulipifera) demonstrated growth inhibition against MSSA, and a bioassay-guided approach identified a sesquiterpene lactone, laurenobiolide, as the active constituent. Intriguingly, its isomers, tulipinolide and epi-tulipinolide, lacked potent activity against MSSA. Laurenobiolide also proved to be more potent against MSSA than the structurally similar sesquiterpene lactones, costunolide and dehydrocostus lactone. Laurenobiolide was the most abundant in the twig bark of the tulip tree, supporting the twig bark's historical and cultural usage in poultices and teas.

Analysis of botanicals and botanical supplements by LC-MS/MS-based molecular networking: Approaches for annotating plant metabolites and authentication.

Prior to the advent of modern medicine, humans have used botanicals extensively for their therapeutic potential. With the majority of newly approved drugs having their origins in natural products, plants remain at the forefront of drug discovery. Continued research and discovery necessitate the use of high-throughput analytical methods to screen and identify bioactive components and potential therapeutic molecules from plants. Utilizing a pre-generated plant extract library, we subjected botanicals to LC-MS/MS-based molecular networking to determine their chemical composition and relatively quantify already known metabolites. The LC-MS/MS-based molecular networking approach was also used to authenticate the composition of dietary supplements against their corresponding plant specimens. The networking procedures provided concise visual representations of the chemical space and highly informative assessments of the botanicals. The procedures also proved to define the composition of the botanical supplements quickly and efficiently. This offered an innovative approach to metabolite profiling and authentication practices and additionally allowed for the identification of new, putatively unknown metabolites for future isolation and biological evaluation.

Incorporating LC-MS/MS Analysis and the Dereplication of Natural Product Samples into an Upper-Division Undergraduate Laboratory Course.

Growth in the biomedical and biotechnology sectors requires a highly trained and highly skilled workforce to answer the next great scientific questions. Undergraduate laboratory courses incorporating hands-on training based in authentic research position soon-to-be graduates to learn in environments that mirror that of academic, industrial, and government laboratories. Mass spectrometry is one of the most broadly applied analyses carried out in the biomedical and pharmaceutical sciences and thus it is essential that upper-division students gain hands-on experience in techniques and analytical workflows in mass spectrometry. Our pre-course assessments identified weaknesses in student experience and knowledge in the fundamentals of mass spectrometry, supporting that it was a necessary area for improvement. We incorporated a laboratory experiment focused on tandem mass spectrometry and database searching into a preexisting mini-semester project devoted to identifying metabolites from medicinal plants. Implementation of the experiment allowed students to make more confident metabolite identifications, introduced them to a cutting-edge database analysis platform (GNPS: Global Natural Products Social Molecular Networking), and increased student experience and knowledge of mass spectrometry in addition to the principle of dereplication of samples derived from nature.

Emerging harmful algal blooms caused by distinct seasonal assemblages of a toxic diatom.

Diatoms in the Pseudo-nitzschia genus produce the neurotoxin domoic acid. Domoic acid bioaccumulates in shellfish, causing illness in humans and marine animals upon ingestion. In 2017, high domoic acid levels in shellfish meat closed shellfish harvest in Narragansett Bay, Rhode Island for the first and only time in history, although abundant Pseudo-nitzschia have been observed for over 60 years. To investigate whether an environmental factor altered endemic Pseudo-nitzschia physiology or new domoic acid-producing strain(s) were introduced to Narragansett Bay, we conducted weekly sampling from 2017 to 2019 and compared closure samples. Plankton-associated domoic acid was quantified by LC-MS/MS and Pseudo-nitzschia spp. were identified using a taxonomically improved high-throughput rDNA sequencing approach. Comparison with environmental data revealed a detailed understanding of domoic acid dynamics and seasonal multi-species assemblages. Plankton-associated domoic acid was low throughout 2017-2019, but recurred in fall and early summer maxima. Fall domoic acid maxima contained known toxic species as well as a novel Pseudo-nitzschia genotype. Summer domoic acid maxima included fewer species but also known toxin producers. Most 2017 closure samples contained the particularly concerning toxic species, P. australis, which also appeared infrequently during 2017-2019. Recurring Pseudo-nitzschia assemblages were driven by seasonal temperature changes, and plankton-associated domoic acid correlated with low dissolved inorganic nitrogen. Thus, the Narragansett Bay closures were likely caused by both resident assemblages that become toxic depending on nutrient status as well as the episodic introductions of toxic species from oceanographic and climatic shifts.

Draft Genome Sequence of Salegentibacter sp. Strain BDJ18, a Plankton-Associated Bacterium in the Northeast Atlantic Ocean.

Salegentibacter sp. strain BDJ18 was isolated from a plankton-associated seawater sample from the northeast Atlantic Ocean. We report its draft genome assembly, which includes genes potentially important for microbial interactions in the marine environment.

New Micropeptins with Anti-Neuroinflammatory Activity Isolated from a Cyanobacterial Bloom.

Metabolite mining of environmentally collected aquatic and marine microbiomes offers a platform for the discovery of new therapeutic lead molecules. Combining a prefractionated chromatography library with liquid chromatography tandem mass spectrometry (LC-MS/MS)-based molecular networking and biological assays, we isolated and characterized two new micropeptins (1 and 2) along with the previously characterized micropeptin 996. These metabolites showed potency in anti-neuroinflammatory assays using BV-2 mouse microglial cells, showing a 50% reduction in inflammation in a range from 1 to 10 µM. These results show promise for cyanobacterial peptides in the therapeutic realm apart from their impact on environmental health and provide another example of the utility of large prefractionated natural product libraries for therapeutic hit and lead identification.

Unnarmicin D, an Anti-inflammatory Cyanobacterial Metabolite with δ and µ Opioid Binding Activity Discovered via a Pipeline Approach Designed to Target Neurotherapeutics.

To combat the bottlenecks in drug discovery and development, a pipeline to identify neuropharmacological candidates using in silico, in vitro, and receptor specific assays was devised. The focus of this pipeline was to identify metabolites with the ability to reduce neuroinflammation, due to the implications that chronic neuroinflammation has in chronic pain and neurodegenerative diseases. A library of pure compounds isolated from the cyanobacterium Trichodesmium thiebautii was evaluated using this method. In silico analysis of drug likelihood and in vitro permeability analysis using the parallel artificial membrane permeability assay (PAMPA) highlighted multiple metabolites of interest from the library. Murine BV-2 microglia were used in conjunction with the Griess assay to determine if metabolites could reduce lipopolysaccharide induced neuroinflammation followed by analysis of pro-inflammatory cytokine concentrations in the supernatant of the treated cell cultures. The nontoxic metabolite unnarmicin D was further evaluated due to its moderate permeability in the PAMPA assay, promising ADME data, modulation of all cytokines tested, and prediction as an opioid receptor ligand. Molecular modeling of unnarmicin D to the µ and δ opioid receptors showed strong theoretical binding potential to the µ opioid receptor. In vitro binding assays validated this pipeline showing low micromolar binding affinity for the µ opioid receptor launching the potential for further analysis of unnarmicin D derivatives for the treatment of pain and neuroinflammation related diseases.

Isolation of Isotrichophycin C and Trichophycins G-I from a Collection of Trichodesmium thiebautii.

The trichophycin family of compounds are chlorinated polyketides first discovered from environmental collections of a bloom-forming Trichodesmium sp. cyanobacterium. In an effort to fully capture the chemical space of this group of metabolites, the utilization of MS/MS-based molecular networking of a Trichodesmium thiebautii extract revealed a metabolome replete with halogenated compounds. Subsequent MS-guided isolation resulted in the characterization of isotrichophycin C and trichophycins G-I (1-4). These new metabolites had intriguing structural variations from those trichophycins previously characterized, which allowed for a comparative study to examine structural features that are associated with toxicity to murine neuroblastoma cells. Additionally, we propose the absolute configuration of the previously characterized trichophycin A (5). Overall, the metabolome of the Trichodesmium bloom is hallmarked by an unprecedented amount of chlorinated molecules, many of which remain to be structurally characterized.

Synergism of anisotropic and computational NMR methods reveals the likely configuration of phormidolide A.

Characterization of the complex molecular scaffold of the marine polyketide natural product phormidolide A represents a challenge that has persisted for nearly two decades. In light of discordant results arising from recent synthetic and biosynthetic reports, a rigorous study of the configuration of phormidolide A was necessary. This report outlines a synergistic effort employing computational and anisotropic NMR investigation, that provided orthogonal confirmation of the reassigned side chain, as well as supporting a further correction of the C7 stereocenter.

A joint molecular networking study of a Smenospongia sponge and a cyanobacterial bloom revealed new antiproliferative chlorinated polyketides.

The bloom-forming cyanobacteria Trichodesmium sp. have been recently shown to produce some of the chlorinated peptides/polyketides previously isolated from the marine sponge Smenospongia aurea. A comparative analysis of extracts from S. aurea and Trichodesmium sp. was performed using tandem mass spectrometry-based molecular networking. The analysis, specifically targeted to chlorinated metabolites, showed that many of them are common to the two organisms, but also that some general differences exist between the two metabolomes. Following this analysis, six new chlorinated metabolites were isolated and their structures elucidated: four polyketides, smenolactones A-D (1-4) from S. aurea, and two new conulothiazole analogues, isoconulothiazole B (5) and conulothiazole C (6) from Trichodesmium sp. The absolute configuration of smenolactone C (3) was determined by taking advantage of the conformational rigidity of open 1,3-disubstituted alkyl chains. The antiproliferative activity of smenolactones was evaluated on three tumor cell lines, and they were active at low-micromolar or sub-micromolar concentrations.

Polyphenol Microbial Metabolites Exhibit Gut and Blood⁻Brain Barrier Permeability and Protect Murine Microglia against LPS-Induced Inflammation.

Increasing evidence supports the beneficial effects of polyphenol-rich diets, including the traditional Mediterranean diet, for the management of cardiovascular disease, obesity and neurodegenerative diseases. However, a common concern when discussing the protective effects of polyphenol-rich diets against diseases is whether these compounds are present in systemic circulation in their intact/parent forms in order to exert their beneficial effects in vivo. Here, we explore two common classes of dietary polyphenols, namely isoflavones and lignans, and their gut microbial-derived metabolites for gut and blood-brain barrier predicted permeability, as well as protection against neuroinflammatory stimuli in murine BV-2 microglia. Polyphenol microbial metabolites (PMMs) generally showed greater permeability through artificial gut and blood-brain barriers compared to their parent compounds. The parent polyphenols and their corresponding PMMs were evaluated for protective effects against lipopolysaccharide-induced inflammation in BV-2 microglia. The lignan-derived PMMs, equol and enterolactone, exhibited protective effects against nitric oxide production, as well as against pro-inflammatory cytokines (IL-6 and TNF-α) in BV-2 microglia. Therefore, PMMs may contribute, in large part, to the beneficial effects attributed to polyphenol-rich diets, further supporting the important role of gut microbiota in human health and disease prevention.

Trichophycins B-F, Chlorovinylidene-Containing Polyketides Isolated from a Cyanobacterial Bloom.

NMR-guided isolation (based on 1D 1H and 13C NMR resonances consistent with a chlorovinylidene moiety) resulted in the characterization of five new highly functionalized polyketides, trichophycins B-F (1-5), and one nonchlorinated metabolite tricholactone (6) from a collection of Trichodesmium bloom material from the Gulf of Mexico. The planar structures of 1-6 were determined using 1D and 2D NMR spectroscopy, mass spectrometry, and complementary spectroscopic procedures. Absolute configuration analysis of 1 and 2 were carried out by 1H NMR analysis of diastereomeric Mosher esters in addition to ECD spectroscopy, J-based configuration analysis, and DFT calculations. The absolute configurations of 3-6 were proposed on the basis of comparative analysis of 13C NMR chemical shifts, relative configurations, and optical rotation values to compounds 1 and 2. Compounds 1-5 represent new additions to the trichophycin family and are hallmarked by a chlorovinylidene moiety. These new trichophycins and tricholactone (1-6) feature intriguing variations with respect to putative biosynthetic starting units, halogenation, and terminations, and trichophycin E (4) features a rare alkynyl bromide functionality. The phenyl-containing trichophycins showed low cytotoxicity to neuro-2A cells, while the alkyne-containing trichophycins showed no toxicity.

Cyanobufalins: Cardioactive Toxins from Cyanobacterial Blooms.

Cyanobufalins A-C (1-3), a new series of cardiotoxic steroids, have been discovered from cyanobacterial blooms in Buckeye Lake and Grand Lake St. Marys in Ohio. Compounds 1-3 contain distinctive structural features, including geminal methyl groups at C-4, a 7,8 double bond, and a C-16 chlorine substituent that distinguish them from plant- or animal-derived congeners. Despite these structural differences, the compounds are qualitatively identical to bufalin in their cytotoxic profiles versus cell lines in tissue culture and cardiac activity, as demonstrated in an impedance-based cellular assay conducted with IPSC-derived cardiomyocytes. Cyanobufalins are nonselectively toxic to human cells in the single-digit nanomolar range and show stimulation of contractility in cardiomyocytes at sub-nanomolar concentrations. The estimated combined concentration of 1-3 in the environment is in the same nanomolar range, and consequently more precise quantitative analyses are recommended along with more detailed cardiotoxicity studies. This is the first time that cardioactive steroid toxins have been found associated with microorganisms in an aquatic environment. Several factors point to a microbial biosynthetic origin for the cyanobufalins.