The overall organization of the ARCH Program is divided into an Administrative
and Planning Core and a Research Program Development Core. The Research
Program and Development Core oversee the shared core facility and the
one research and multiple pilot projects. The Administrative and Planning
Core is composed of Internal and External Advisory Committees (IAC and
EAC). This core oversees the workshop and seminar program. Typically three
to four external speakers per year are invited through the ARCH program.
Workshops may be internal or external. The EAC is composed of a distinguished
panel of four scientists that meet with us annually to review our goals
and progress and make recommendations for meeting those goals. They also
provide the first level of scientific merit review for new research proposals.
New Toxins from Algae and Cyanobacteria
PILOT RESEARCH PROJECTS
- Identification of Developmental Toxins from Cyanobacteria
- Molecular Approaches to Monitor Toxic Dinoflagellates
- MD/ NMR Characterization of the Internal Motions of Peptide Toxins
- Impacts of Arsenic from CCA Treated Wood within Marine and Terrestial Environments
- Treatment of Microcystins by Ultrasonic Irradiation
- Cyanobacteria, their Toxins and Recreational Exposure
- An Ion Exchange Approach for the Design of Toxic Metal Ion Ligands and Sensors for Aquatic Environments
RESEARCH PROJECT
New
Toxins from Algae and Cyanobacteria
Project Leaders: Kathleen S. Rein,
Co- PI, Department of Chemistry, Florida International University
Robert Gawley, Departments
of Chemistry, University of Miami and University of Arkansas
The aims of this project
are two-fold. First we shall identify and characterize new toxins, produced
by algae and cyanobacteria, found in Florida's aquatic environments. Florida
waters harbor over sixty toxic or potentially toxic microorganisms. These
toxic microorganisms have significant impacts on public health, recreation,
tourism, wildlife and local economies. Gymnodinium breve blooms annually
on the West Coast of Florida causing closure of shellfish beds to prevent
the occurrence of neurotoxic shellfish poisoning. Cyanobacterial blooms
are becoming increasingly frequent in freshwater lakes, making recreational
use hazardous. In addition to these well-known and well-characterized
toxin-producing organisms, other toxic microorganisms are present for
which toxins have yet to be identified. This proposal will focus on the
identification of these as yet uncharacterized toxins. Prior to mass culturing,
small scale cultures and assemblages will be screened by RT-PCR for the
expression of biosynthetic capability. Specifically, flagellates will
be screened for polyketide synthase gene expression, while cyanobacteria
will be screened for non-ribosomal peptide biosynthesis. New toxins will
be isolated by bioassy guided fractionation using a variety of screening
methods.
Second, the role of associated bacteria in dinoflagellate toxin biosynthesis
will be examined. Since many dinoflagellate cultures have not been successfully
maintained axenically, the ultimate origins of the toxins remains a topic
of ongoing debate. The existence of toxic and-not toxic strains of dinoflagellates
and the sudden unexplained loss of toxicity of dinoflagellates in culture
has led to speculation that associated bacteria are responsible for toxin
biosynthesis. We have isolated bacteria from dinoflagellate cultures that
carry polyketide synthase encoding genes, but express them only in the
presence of dinoflagellates. The role of these bacteria in the biosynthesis
of dinoflagellate derived polyketides will be examined.
Papers Published on this Project
Snyder, R. V.; Guerrero, M. A.; Sinigalliano, C. D.; Winshell, J.; Perez, R.; Lopez, J. V.; and Rein, K. S. 2005. Localization of polyketide synthase encoding genes to the toxic dinoflagellate Karenia brevis. Phytochemistry, 66, 1767-1780.
Berry, J. P.; Gantar, M.; Gawley, R. E.; Wang, M.; Rein, K. S. 2004. Pharmacology and Toxicology of Pahayokolide A, a Bioactive Metabolite from a Freshwater Species of Lyngbya Isolated from the Florida Everglades. Comp. Biochem. Phys. Prt C. 139, 231-238.
Berry, J. P., Gantar, M.; Gawley, R. E.; Rein, K. S. 2004. Isolation of bioactive metabolites from a Lyngbya species isolated from periphyton of the Florida Everglades. pp. 192-194. In K. A. Steidinger, J. H. Landsberg, C. R. Tomas and G. A. Vargo (eds.) Harmful Algae, 2002. Florida Fish and Wildlife Conservation Commission, Florida Institute of Oceanography, and Intergovernmental Oceanographic Commission of UNESCO.
Rein, K. S.; Snyder, R. V. Biosynthesis of Polyketide Metabolites by Dinoflagellates. Adv. Appl. Micro. (Invited review, submitted)
PILOT RESEARCH PROJECTS
Identification
of Developmental Toxins from Cyanobacteria
Project Leaders: Miro Gantar
(Department of Biological Sciences, Florida International University)
John Berry (Department of Chemistry,
University of Miami), Michael
Schmale (Department of Marine Biology and Fisheries, University of
Miami)
This project utilizes the zebrafish embryo as a model of vertebrate development to identify and characterize developmental toxins from Cyanobacteria (or “blue-green algae”). Found in diverse environments worldwide, the Cyanobacteria represent a group of photosynthetic prokaryotes of which many are known to produce potent toxins. Humans and animals can be exposed to such toxins in aquatic environments through various routes including skin contact, ingestion, inhalation and haemodialysis, and increasing eutrophication of surface water bodies may be leading to increased concentrations of these toxins in drinking and recreational waters. A 2001 study, for example, of drinking water sources that supply 185,000 residents in Central Florida revealed the cyanobacterial toxin, microcystin, at levels up to five-times the safe limit recommended by the WHO (Orlando Sentinel, May 27, 2001). Effects of exposure of humans and animals to cyanobacterial toxins, however, are not well understood. Many of the cyanobacterial toxins have recognized cytotoxic, or otherwise inhibitory and/or promotive effects of on cellular proliferation. As such, it is conceivable that the exposure to even low levels of cyanobacterial toxins may lead to dysfunction in development of both human and animal embryos. Specifically, this research utilizes cultures of cyanobacteria from the Everglades and other freshwater sources in South Florida, isolated and maintained as part of the FIU ARCH Algal Toxins/Culture Facility Core. Assessment of the development of zebrafish embryos exposed to extracts from these cultures is used as an indicator to screen Cyanobacterial isolates for those which produce metabolites, including both described and currently undescribed toxins, that inhibit developmental pathways. In the case of previously undescribed toxins from these isolates, active compounds will be isolated by bioassay-guided fractionation (using the zebrafish embryo assay), and chemically characterized. Additionally, the zebrafish model can be used to characterize the targets and modes-of-action of these toxins, and more generally as a predictor of environmental health aspects associated with cyanobacterial toxins. Thus, this research provides an important interface with other research projects, as well as the overall research goals, of the FIU ARCH program as a model of the environmental risks associated with freshwater of cyanobacteria and their toxins.
Papers Published on this Project
K.D. Goodwin, G. Scorzetti, S.A. Cotton, T.L. Kiesling, P.B. Ortner, and J.W. Fell. 2004. Detection of Karenia brevis by a Microtiter Plate Assay. In K. A. Steidinger, J. H. Landsberg, C. R. Tomas and G. A. Vargo (eds.) Harmful Algae, 2002. Florida Fish and Wildlife Conservation Commission, Florida Institute of Oceanography.
Goodwin, K.D.; Cotton, S.A.; Scorzetti, G.; Fell, J.W. 2005. A DNA hybridization assay to identify toxic dinoflagellates in coastal waters: detection of Karenia brevis in the Rookery Bay National Estuarine Research Reserve., Harmful Algae, 4, 411-422.
Molecular Approaches to Monitor Toxic Dinoflagellates
Project Leaders: Christopher
D. Sinigalliano/Maria Guerrero,
Southeast Environmental Research Center, Florida International University
Jack Fell, Department of Marine Biology
and Fisheries, University of Miami
The long term goals of this ARCH pilot project is to develop, optimize, and assess the effectiveness of a set of molecular methods for the detection and monitoring of toxic dinoflagellates among the epiphyton and free-living communities of microorganisms in coastal waters. As a group, these toxic dinoflagellates cause a variety of public health problems, including toxic seafood and shellfish poisoning, marine animal and bird kills, respiratory distress in humans, "red tide" fish kills, etc. Such toxic microorganisms are particularly common in Florida coastal waters. The State of Florida's Harmful Algal Bloom (HAB) Task Force has identified a critical need for the development of new technologies and approaches (particularly molecular probes) to monitor these toxins and the organisms that produce them, and how environmental variables affect their impact on public health and environmental quality. Particularly in the case of ciguatera HABs, there is a need to develop accurate and rapid methods to survey and monitor Florida waters for ciguateric dinoflagellate species and hot spots, and to test for the presence of their toxins. Specifically, this project will involve the development and field trials of assays combining genetic labeling, immunological labeling, and flow cytometry for the enumeration and cell sorting of dinoflagellate cells producing toxins of public health concern, such as ciguatoxins, maitotoxins, gambiertoxins, brevetoxins, and okadaic acid. Particular interest will focus on the detection of Karenia brevis causing "red tide", Gambierdiscus species causing ciguatera food poisoning, and on toxin-producing Prorocentrum species. Molecular assays will be developed to monitor these toxic dinoflagellates and to examine their associated bacteria, by targeting both ribosomal RNA genes and genes for polyketide synthase (PKS). Such molecular probes for Prorocentrum lima have already been developed by our group and successfully utilized with preliminary in vitro studies. The work proposed here may also serve as a model for other toxic polyketide-producing marine microorganisms. This project will take three different approaches to investigate the presence and toxic activity of these marine dinoflagellates: 1) in vitro real-time PCR and probe macroarray assays of nucleic acid extracts from epiphyton and water column microbial communities, 2) flow cytometry (FCM) analysis using diagnostic light scatter and fluorescent properties of targeted dinoflagellate cells, and 3) fluorescent in situ hybridization and in situ PCR assays combined with FCM and fluorescent microscopy to assay probe-labeled cells. Multivariate analysis will be used to correlate this data to the extensive database of monthly biogeochemical data generated by our ongoing Water Quality Monitoring Network to identify the role and effect of environmental and anthropogenic factors influencing dinoflagellate toxicity in this region.
Papers Published on this Project
K.D. Goodwin, G. Scorzetti, S.A. Cotton, T.L. Kiesling, P.B. Ortner, and J.W. Fell. 2004. Detection of Karenia brevis by a Microtiter Plate Assay. In K. A. Steidinger, J. H. Landsberg, C. R. Tomas and G. A. Vargo (eds.) Harmful Algae, 2002. Florida Fish and Wildlife Conservation Commission, Florida Institute of Oceanography.
Goodwin, K.D.; Cotton, S.A.; Scorzetti, G.; Fell, J.W. 2005. A DNA hybridization assay to identify toxic dinoflagellates in coastal waters: detection of Karenia brevis in the Rookery Bay National Estuarine Research Reserve., Harmful Algae, 4, 411-422.
MD/
NMR Characterization of the Internal Motions of Peptide Toxins
Project Leaders: David Chatfield, Department of Chemistry,
Florida International University
TK Harris, Department of Biochemistry,
University of Miami
This project focuses on the internal motions of alpha-conotoxin GI, a 13-residue, cross-linked peptide. The analysis will combine 15N NMR relaxation measurements of the isotopically labeled peptide, from which motional parameters describing the peptide's backbone flexibility will be obtained; and molecular dynamics (MD) simulation, which will provide an atomic-level interpretation of the motions. The work is significant in three respects: (1) It will further our understanding of alpha-conotoxin GI's interaction with the acetylcholine receptor and the consequent toxicity. (2) This small yet conformationally restricted peptide is an excellent model for developing methods for interpreting NMR motional parameters in terms of mechanical and thermodynamic properties of biological molecules.. (3) Characterizing the flexibility of alpha-conotoxin GI may help guide efforts to use it as a drug model. In the first year of the project, the NMR characterization will be carried out. In the second and third years, MD simulations will be performed to address the following issues: What is the quantitative value of the backbone's conformational entropy? Does the pairwise correlation of bond vector motions affect the calculation of the conformational entropy significantly? With what larger-scale motions are backbone bond vector motions correlated? In addition, restrained MD simulations of unfolded conformers with the disulfide bonds severed are proposed in order to quantify selected contributions of the backbone's conformational entropy to the conformational entropy of folding. The methodology developed in the work on alpha-conotoxin GI will be also applied to microcystins, hepatotoxins.
Impacts
of Arsenic from CCA Treated Wood within Marine and Terrestial Environments
Project Leader: Yong Cai,
Department of Chemistry, Florida International University
Helena Solo-Gabriele,
Department of Engineering, University of Miami
Limited information is available about the ultimate fate of the arsenic found in chromated copper arsenate (CCA) during the service life and disposal of the treated wood product. This lack of information coupled with the large quantity of arsenic currently in service associated with treated wood (130,000 tonnes estimated), results in a potential risk of human and ecological exposure. The toxicity of arsenic is strongly a function of the speciation of the metalloid, with the inorganic species being more toxic than the methylated forms. Within the inorganic forms, species that are characterized by a lower valence are the most toxic. The objectives of the current study are to evaluate the toxicity of arsenic in leachates from CCA-treated wood by measuring the species of arsenic that are leached from different environmental samples. A considerable effort will be placed on method development, which will expand the applicability of cartridges designed to preserve samples in the field. Leaching will be evaluated in both laboratory and field settings. Laboratory studies will focus on standardized leaching tests aimed at simulating the impacts of rainfall, seawater, and landfill conditions. Laboratory samples evaluated will include CCA-treated wood at various retention levels. Field work will focus on evaluating the impacts of decks and marine docks constructed of CCA-treated wood on the surrounding environment. Samples will be routinely analyzed for arsenite (As(III)), arsenate (As(V)), monomethylarsonic acid (MMAA), and dimethylarsinic acid (DMAA). Other arsenic species, if any, will be detected and quantified on a periodic basis. Metals species will be routinely measured in the dissolved phase and methods will be developed for measuring arsenic speciation within the particulate phase. Results will be used to estimate the total U.S. arsenic releases from CCA-treated wood structures. These data will be useful as inputs into environmental risk models that evaluate the probability of human disease or other environmental outcome associated with the use or disposal of CCA-treated wood.
Papers Published on this Project
Articles in Peer-reviewed Journals
Georgiadis, M.; Cai, Y.; Solo-Gabriele, H. 2005. Extraction of Arsenate and Arsenite Species from Soils and Sediments. Environmental Pollution.
(In press).
Feng, M.; Schrlau, J.; Snyder, R.; Snyder, G.; Chen, M.; Cisar, J. and Cai, Y. 2005. Arsenic Transport and Transformation Associated with MSMA Application on a Golf Course Green. J. Agric. Food Chem. 53, 3556-3562.
Khan, B. I.; Solo-Gabriele, H. M.; Dubey, B. K.; Townsend, T. G.; Cai, Y. 2004. Arsenic Speciation of Solvent-Extracted Leachate from New and Weathered CCA-Treated Wood. Environ. Sci. Technol. 38, 4527-4534
Khan, B.; Jambeck, J.; Solo-Gabriele, H.; Townsend, T.; and Cai, Y. 2005. Release of Arsenic to the Environment from CCA-Treated Wood: Part I – Leaching and Speciation during Service. Environ. Sci. Technol. (accepted).
Khan, B.; Jambeck, J.; Solo-Gabriele, H.; Townsend, T.; and Cai, Y. 2005. Release of Arsenic to the Environment from CCA-Treated Wood: Part II – Leaching and Speciation during Disposal. Environ. Sci. Technol. (accepted).
Articles as Book Chapters
Xu, T.; Cai, Y.; Mezyk, S.; O'Shea, K. E. 2005. The Roles of Hydroxyl Radical, Superoxide Anion Radical and Hydrogen Peroxide in the Oxidation of Arsenic by Ultrasonic Irradiation In Advances in Arsenic Research, Intergration of Expereimental and Observational Studies and Implications for Mitigation, O’Day, P.; Vlassopoulos, D.; Meng, X.; Benning, L. G., Eds; Symposium Series 915; American Chemical Society, Washington DC, 2005, Ch 24, 45(1), 335.
Cai, Y.; Solo-Gabriele, H. M.; Townsend, T.; Khan, B.; Georgiadis, M.; Dubey, B. 2005. Elemental speciation and environmental importance associated with CCA-treated wood. Townsend and Solo-Gabriele, Eds. CRC Press, (in press).
Link
to Yong Cai's web site.
http://www.fiu.edu/~ebacg/page7.html
Treatment
of Microcystins by Ultrasonic Irradiation
Project Leader: Kevin
E. O'Shea, Department of Chemistry, Florida International University
Microcystins, a family of peptides produced primarily by freshwater cyanobacteria have lead to animal fatalities worldwide and represent a considerable health threat to humans. Their potential for causing both acute and chronic toxicity has prompted the need for extensive research into their detection, toxicology and removal from potable water. The goal of this research project is to assess the use of ultrasonic irradiation for the degradation of naturally occurring aquatic toxins, specifically microcystins. It is well established that ultrasonic irradiation of aqueous media can lead to the mineralization of a variety of organic substrates. Initial sonolytic experiments will be conducted on cyanobacteria to establish the extent that toxic microcystins are released into the water and/or destroyed under treatment conditions. Microcystins will be isolated from cyanobacteria cultures available through the toxin probes facility core of the ARCH program (director Professor Kathleen Rein). Aqueous matrix samples containing microcystins will be prepared from isolated material and treated with ultrasonic irradiation. The disappearance of microcystins will be monitored as a function of treatment time and initial concentrations. These degradation profiles will allow us to determine the kinetic parameters, which are useful for predictive tools and modeling parameters in the applications of water treatment.
Papers Published on this Project
Weihua Song, Terri Teshiba, Kathleen Rein, and Kevin E. O’Shea, “Ultrasonically induced degradation and detoxification of Microcystin-LR (Cyanobacterial toxin)” Environmental Science and Technology, 2005, 39, 6300-6305.
Cyanobacteria,
their Toxins and Recreational Exposure
Project Leaders: Miroslav
Gantar, Department of Biology and Southeastern Environmental Research
Center, Florida International University
Lora Fleming, NIEHS Marine
& Freshwater Biomedical Sciences Center, University of Miami
Blue green algae (cyanobacteria) represent a diverse group of organisms that are common inhabitants of freshwater lakes and reservoirs, and produce potent natural toxins. Potential routes of exposure for these toxins are dermal, oral, and possibly inhalation; humans can be exposed through drinking water, as well as recreationally and occupationally. There have been case reports of severe morbidity and mortality in domestic animals through drinking contaminated water, but little epidemiologic research on the human health effects of cyanobacteria and their toxins.
Recently in Florida, cyanobacteria and their toxins have been identified in freshwater lakes and reservoirs, with growing media and community concern. This proposed research will be a prospective cohort study of recreational users in Florida freshwater lakes with and without toxic cyanobacteria. Participants will be evaluated by initial and follow up interview as to symptoms and exposures. Water samples taken on the day of exposure will identify organisms and toxins. Reported human symptoms will be used to identify cyanobacteria species of potential environmental health impact for future study by researchers involved in the isolation and cultivation of cyanobacteria. At the end of the study, study participants and recreational lake managers will receive outreach and education materials concerning the cyanobacteria, their toxins, and possible human health effects.
An
Ion Exchange Approach for the Design of Toxic Metal Ion Ligands and Sensors
for Aquatic Environments
Project Leaders: Konstantinos
Kavallieratos, Department of Chemistry, Florida International University
A new strategy for designing synthesizing and screening self-organized hosts for recognition and sensing of toxic metal salts is proposed. Ligand libraries formed from combinations of simple subunits, which may potentially bind toxic metal ion-guests, will be generated using single-pot methods. The most effective ligands will be contacted with aqueous phases containing the metal ion and will be identified by mass spectrometry. Pb(II), will be our primary target with Hg(II) and Cd(II) following. The identified hosts and their complexes will be characterized structurally and spectroscopically and their thermodynamic and kinetic stabilities will be compared to macrocyclic or acyclic systems that have been previously used with the goal to optimize them for ion-exchange separation and sensing applications. Fluorophore groups will be attached to the optimized building blocks and thus generate hosts with the ability for fluorescent sensing. Our strategy may be expanded for other cationic or anionic guests of importance in aquatic environments and offers a powerful tool for identifying simple, and efficient hosts, which may be synthetically available on multigram quantities.
Papers Published on this Project
Kavallieratos, K.; Rosenberg J. M.; Bryan, J. C. Inorg. Chem., 2005, 44, 2573-2575.
Kavallieratos, K.; Rosenberg, J. M.; Chen, W.-Z.; Ren, T. J. Am. Chem. Soc. 2005, 127, 6514-6515.
Kavallieratos, K.; Sabucedo, A. J..; Pau, A. T.; Rodriguez, J. M. J. Am. Soc. Mass Spectrom. 2005, 16, 1377-1385.
R. J. Alvarado, J. M. Rosenberg, A. Andreu, J. C. Bryan, W.-Z. Chen. T. Ren. K. Kavallieratos Inorg. Chem., 2005, 44, in press. Published on the web Sep.30, 2005
W. Zhang, Y. Cai, K. Kavallieratos, Rapid Commun. Mass Spectrom. 2005, submitted.
Copyright
2006. University of Miami & Florida International University,
All Rights Reserved.
The
ARCH Program is funded by the
National Institute of
Environmental Health Sciences
Grant Number: S11 ES11181
