Trace Metal Sensors for Coastal Monitoring, Seaside, California, April 11-13, 2005: workshop proceedings

The Alliance for Coastal Technologies (ACT) Workshop on Trace Metal Sensors for Coastal Monitoring was convened April 11-13, 2005 at the Embassy Suites in Seaside, California with partnership from Moss Landing Marine Laboratories (MLML) and the Monterey Bay Aquarium Research Institute (MBARI). Trace metals play many important roles in marine ecosystems. Due to their extreme toxicity, the effects of copper, cadmium and certain organo-metallinc compounds (such as tributyltin and methylmercury) have received much attention. Lately, the sublethal effects of metals on phytoplankton biochemistry, and in some cases the expression of neurotoxins (Domoic acid), have been shown to be important environmental forcing functions determining the composition and gene expression in some groups. More recently the role of iron in controlling phytoplankton growth has led to an understanding of trace metal limitation in coastal systems. Although metals play an important role at many different levels, few technologies exist to provide rapid assessment of metal concentrations or metal speciation in the coastal zone where metal-induced toxicity or potential stimulation of harmful algal blooms, can have major economic impacts. This workshop focused on the state of on-site and in situ trace element detection technologies, in terms of what is currently working well and what is needed to effectively inform coastal zone managers, as well as guide adaptive scientific sampling of the coastal zone. Specifically the goals of this workshop were to: 1) summarize current regional requirements and future targets for metal monitoring in freshwater, estuarine and coastal environments; 2) evaluate the current status of metal sensors and possibilities for leveraging emerging technologies for expanding detection limits and target elements; and 3) help identify critical steps needed for and limits to operational deployment of metal sensors as part of routine water quality monitoring efforts. Following a series of breakout group discussions and overview talks on metal monitoring regulatory issues, analytical techniques and market requirements, workshop participants made several recommendations for steps needed to foster development of in situ metal monitoring capacities: 1. Increase scientific and public awareness of metals of environmental and biological concern and their impacts in aquatic environments. Inform scientific and public communities regarding actual levels of trace metals in natural and perturbed systems. 2. Identify multiple use applications (e.g., industrial waste steam and drinking water quality monitoring) to support investments in metal sensor development. (pdf contains 27 pages)

Single cell proteomics microchip to profile immune function, with applications in stem cell biology, translational disease mechanism study and clinical therapeutics monitoring

In response to infection or tissue dysfunction, immune cells develop into highly heterogeneous repertoires with diverse functions. Capturing the full spectrum of these functions requires analysis of large numbers of effector molecules from single cells. However, currently only 3-5 functional proteins can be measured from single cells. We developed a single cell functional proteomics approach that integrates a microchip platform with multiplex cell purification. This approach can quantitate 20 proteins from >5,000 phenotypically pure single cells simultaneously. With a 1-million fold miniaturization, the system can detect down to ~100 molecules and requires only ~104 cells. Single cell functional proteomic analysis finds broad applications in basic, translational and clinical studies. In the three studies conducted, it yielded critical insights for understanding clinical cancer immunotherapy, inflammatory bowel disease (IBD) mechanism and hematopoietic stem cell (HSC) biology.

To study phenotypically defined cell populations, single cell barcode microchips were coupled with upstream multiplex cell purification based on up to 11 parameters. Statistical algorithms were developed to process and model the high dimensional readouts. This analysis evaluates rare cells and is versatile for various cells and proteins. (1) We conducted an immune monitoring study of a phase 2 cancer cellular immunotherapy clinical trial that used T-cell receptor (TCR) transgenic T cells as major therapeutics to treat metastatic melanoma. We evaluated the functional proteome of 4 antigen-specific, phenotypically defined T cell populations from peripheral blood of 3 patients across 8 time points. (2) Natural killer (NK) cells can play a protective role in chronic inflammation and their surface receptor – killer immunoglobulin-like receptor (KIR) – has been identified as a risk factor of IBD. We compared the functional behavior of NK cells that had differential KIR expressions. These NK cells were retrieved from the blood of 12 patients with different genetic backgrounds. (3) HSCs are the progenitors of immune cells and are thought to have no immediate functional capacity against pathogen. However, recent studies identified expression of Toll-like receptors (TLRs) on HSCs. We studied the functional capacity of HSCs upon TLR activation. The comparison of HSCs from wild-type mice against those from genetics knock-out mouse models elucidates the responding signaling pathway.

In all three cases, we observed profound functional heterogeneity within phenotypically defined cells. Polyfunctional cells that conduct multiple functions also produce those proteins in large amounts. They dominate the immune response. In the cancer immunotherapy, the strong cytotoxic and antitumor functions from transgenic TCR T cells contributed to a ~30% tumor reduction immediately after the therapy. However, this infused immune response disappeared within 2-3 weeks. Later on, some patients gained a second antitumor response, consisted of the emergence of endogenous antitumor cytotoxic T cells and their production of multiple antitumor functions. These patients showed more effective long-term tumor control. In the IBD mechanism study, we noticed that, compared with others, NK cells expressing KIR2DL3 receptor secreted a large array of effector proteins, such as TNF-α, CCLs and CXCLs. The functions from these cells regulated disease-contributing cells and protected host tissues. Their existence correlated with IBD disease susceptibility. In the HSC study, the HSCs exhibited functional capacity by producing TNF-α, IL-6 and GM-CSF. TLR stimulation activated the NF-κB signaling in HSCs. Single cell functional proteome contains rich information that is independent from the genome and transcriptome. In all three cases, functional proteomic evaluation uncovered critical biological insights that would not be resolved otherwise. The integrated single cell functional proteomic analysis constructed a detail kinetic picture of the immune response that took place during the clinical cancer immunotherapy. It revealed concrete functional evidence that connected genetics to IBD disease susceptibility. Further, it provided predictors that correlated with clinical responses and pathogenic outcomes.

Watershed monitoring and the TMDL modeling to assess bacterial loading in estuarine environments to improve management

Shellfish bed closures along the North Carolina coast have increased over the years seemingly concurrent with increases in population (Mallin 2000). More and faster flowing storm water has come to mean more bacteria, and fecal indicator bacterial (FIB) standards for shellfish harvesting are often exceeded when no source of contamination is readily apparent (Kator and Rhodes, 1994). Could management reduce bacterial loads if the source of the bacteria where known? Several potentially useful methods for differentiating human versus animal pollution sources have emerged including Ribotyping and Multiple Antibiotic Resistance (MAR) (US EPA, 2005). Total Maximum Daily Load (TMDL) studies on bacterial sources have been conducted for streams in NC mountain and Piedmont areas (U.S. EPA, 1991 and 2005) and are likely to be mandated for coastal waters. TMDL analysis estimates allowable pollutant loads and allocates them to known sources so management actions may be taken to restore water to its intended uses (U.S. EPA, 1991 and 2005). This project sought first to quantify and compare fecal contamination levels for three different types of land use on the coast, and second, to apply MAR and ribotyping techniques and assess their effectiveness for indentifying bacterial sources. Third, results from these studies would be applied to one watershed to develop a case study coastal TMDL. All three watershed study areas are within Carteret County, North Carolina. Jumping Run Creek and Pettiford Creek are within the White Oak River Basin management unit whereas the South River falls within the Neuse River Basin. Jumping Run Creek watershed encompasses approximately 320 ha. Its watershed was a dense, coastal pocosin on sandy, relic dune ridges, but current land uses are primarily medium density residential. Pettiford Creek is in the Croatan National Forest, is 1133 ha. and is basically undeveloped. The third study area is on Open Grounds Farm in the South River watershed. Half of the 630 ha. watershed is under cultivation with most under active water control (flashboard risers). The remaining portion is forested silviculture.(PDF contains 4 pages)

Design and analysis of nucleic acid reaction pathways

Nucleic acids are a useful substrate for engineering at the molecular level. Designing the detailed energetics and kinetics of interactions between nucleic acid strands remains a challenge. Building on previous algorithms to characterize the ensemble of dilute solutions of nucleic acids, we present a design algorithm that allows optimization of structural features and binding energetics of a test tube of interacting nucleic acid strands. We extend this formulation to handle multiple thermodynamic states and combinatorial constraints to allow optimization of pathways of interacting nucleic acids. In both design strategies, low-cost estimates to thermodynamic properties are calculated using hierarchical ensemble decomposition and test tube ensemble focusing. These algorithms are tested on randomized test sets and on example pathways drawn from the molecular programming literature. To analyze the kinetic properties of designed sequences, we describe algorithms to identify dominant species and kinetic rates using coarse-graining at the scale of a small box containing several strands or a large box containing a dilute solution of strands.

Programming chemical kinetics: engineering dynamic reaction networks with DNA strand displacement

Over the last century, the silicon revolution has enabled us to build faster, smaller and more sophisticated computers. Today, these computers control phones, cars, satellites, assembly lines, and other electromechanical devices. Just as electrical wiring controls electromechanical devices, living organisms employ "chemical wiring" to make decisions about their environment and control physical processes. Currently, the big difference between these two substrates is that while we have the abstractions, design principles, verification and fabrication techniques in place for programming with silicon, we have no comparable understanding or expertise for programming chemistry.

In this thesis we take a small step towards the goal of learning how to systematically engineer prescribed non-equilibrium dynamical behaviors in chemical systems. We use the formalism of chemical reaction networks (CRNs), combined with mass-action kinetics, as our programming language for specifying dynamical behaviors. Leveraging the tools of nucleic acid nanotechnology (introduced in Chapter 1), we employ synthetic DNA molecules as our molecular architecture and toehold-mediated DNA strand displacement as our reaction primitive.

Abstraction, modular design and systematic fabrication can work only with well-understood and quantitatively characterized tools. Therefore, we embark on a detailed study of the "device physics" of DNA strand displacement (Chapter 2). We present a unified view of strand displacement biophysics and kinetics by studying the process at multiple levels of detail, using an intuitive model of a random walk on a 1-dimensional energy landscape, a secondary structure kinetics model with single base-pair steps, and a coarse-grained molecular model that incorporates three-dimensional geometric and steric effects. Further, we experimentally investigate the thermodynamics of three-way branch migration. Our findings are consistent with previously measured or inferred rates for hybridization, fraying, and branch migration, and provide a biophysical explanation of strand displacement kinetics. Our work paves the way for accurate modeling of strand displacement cascades, which would facilitate the simulation and construction of more complex molecular systems.

In Chapters 3 and 4, we identify and overcome the crucial experimental challenges involved in using our general DNA-based technology for engineering dynamical behaviors in the test tube. In this process, we identify important design rules that inform our choice of molecular motifs and our algorithms for designing and verifying DNA sequences for our molecular implementation. We also develop flexible molecular strategies for "tuning" our reaction rates and stoichiometries in order to compensate for unavoidable non-idealities in the molecular implementation, such as imperfectly synthesized molecules and spurious "leak" pathways that compete with desired pathways.

We successfully implement three distinct autocatalytic reactions, which we then combine into a de novo chemical oscillator. Unlike biological networks, which use sophisticated evolved molecules (like proteins) to realize such behavior, our test tube realization is the first to demonstrate that Watson-Crick base pairing interactions alone suffice for oscillatory dynamics. Since our design pipeline is general and applicable to any CRN, our experimental demonstration of a de novo chemical oscillator could enable the systematic construction of CRNs with other dynamic behaviors.

Tratamento Homeopático: buscando estratégias para avaliação de seus resultados e efetividade

Face à crescente procura por outras modalidades terapêuticas que abordam o ser humano de forma holística e a introdução das mesmas no SUS, torna-se muito importante a avaliação da efetividade e segurança dessas formas de cuidado. A Homeopatia faz parte deste conjunto de terapêuticas e, para sua avaliação, pode existir a necessidade de se valer de múltiplos instrumentos para abarcar os vários aspectos de uma resposta integral ao tratamento. Este trabalho teve como objetivo identificar e elaborar categorias de análises e instrumentos que permitam avaliar e mensurar a efetividade deste tratamento, bem como testá-los, considerando-se as características desta racionalidade. Foram levantados, na literatura nacional e internacional, trabalhos sobre efetividade do tratamento homeopático, em busca da definição do estado da arte mas também dos principais problemas, limitações e possibilidades dessas avaliações tendo em vista seu resultado integral. Finda esta etapa, a pesquisa destinou-se a elaboração, proposição e testagem de uma metodologia considerada mais adequada a avaliar o tratamento homeopático nesta perspectiva. Um estudo observacional foi realizado em serviço público homeopático no município de Juiz de Fora, com tratamento individualizado, no qual foi utilizada uma estratégia de avaliação composta por três componentes: (1) avaliação de qualidade de vida pelo instrumento SF-36; (2) análises em busca de objetivar e quantificar queixas clínicas e outros atributos de natureza subjetiva (sensação de bem-estar, sono, estado cognitivo e memória, vida sexual, sensação de felicidade) por meio da utilização de uma escala visual analógica (EVA), na mensuração da intensidade e de opções fechadas, a exemplo do SF-36, na estimativa da frequência desses aspectos e (3) entrevistas qualitativas por intermédio de questionário semiestruturado, com a finalidade de abordar questões relacionadas a biopatografia e mudança da atitude vital (como pacientes enfrentam os problemas do cotidiano, fatores deflagradores das queixas, como se sentem e como reagem, além de indagar seus projetos de vida e felicidade). A aplicação do questionário SF-36 apresentou algumas dificuldades de compreensão pelos participantes, talvez devido à baixa escolaridade dos entrevistados, mas mostrou-se útil à pesquisa, embora demonstre limitações na avaliação do aspecto integral do resultado da terapêutica analisada. O acompanhamento das queixas clínicas, sensação de bem-estar, sono e estado cognitivo e memória foram captados e mensurados de forma satisfatória tanto pela EVA (intensidade dos sintomas) quanto pelas respostas fechadas para medir a frequência. Situações como as avaliações da biopatografia e da sexualidade foram insuficientes para serem adequadamente avaliadas pelo pesquisador e o paciente somente. A participação do médico assistente poderia contribuir nestes casos. Questões mais abrangentes na avaliação da mudança na atitude vital, como reação diante de fatores desequilibrantes e projeto de vida e felicidade, necessitam de metodologia qualitativa até que se possa avançar nas pesquisas à espera de soluções futuras. A combinação dessas estratégias em estudos controlados, randomizados, com amostras de magnitude satisfatória, preferencialmente em rede e que explicitem as condições nas quais o atendimento homeopático ocorreu e como se chegou a cada prescrição, podem ter utilidade para a avaliação da efetividade da dimensão integral do tratamento homeopático.

Monitoring well-being and changing environmental conditions in coastal communities: development of an assessment method

The intersection of social and environmental forces is complex in coastal communities. The well-being of a coastal community is caught up in the health of its environment, the stability of its economy, the provision of services to its residents, and a multitude of other factors. With this in mind, the project investigators sought to develop an approach that would enable researchers to measure these social and environmental interactions. The concept of well-being proved extremely useful for this purpose. Using the Gulf of Mexico as a regional case study, the research team developed a set of composite indicators to be used for monitoring well-being at the county-level. The indicators selected for the study were: Social Connectedness, Economic Security, Basic Needs, Health, Access to Social Services, Education, Safety, Governance, and Environmental Condition. For each of the 37 sample counties included in the study region, investigators collected and consolidated existing, secondary data representing multiple aspects of objective well-being. To conduct a longitudinal assessment of changing wellbeing and environmental conditions, data were collected for the period of 2000 to 2010. The team focused on the Gulf of Mexico because the development of a baseline of well-being would allow NOAA and other agencies to better understand progress made toward recovery in communities affected by the Deepwater Horizon oil spill. However, the broader purpose of the project was to conceptualize and develop an approach that could be adapted to monitor how coastal communities are doing in relation to a variety of ecosystem disruptions and associated interventions across all coastal regions in the U.S. and its Territories. The method and models developed provide substantial insight into the structure and significance of relationships between community well-being and environmental conditions. Further, this project has laid the groundwork for future investigation, providing a clear path forward for integrated monitoring of our nation’s coasts. The research and monitoring capability described in this document will substantially help counties, local organizations, as well state and federal agencies that are striving to improve all facets of community well-being.

Monitoring Hawaii’s marine protected areas: examining spatial and temporal trends using a seascape approach

Hawaii’s coastal marine resources have declined dramatically over the past 100 years due to multiple anthropogenic stressors including overfishing, coastal development, pollution, overuse, invasive species and climate change. It is now becoming evident that ecosystem-based management, in the form of marine protected areas (MPAs), is necessary to conserve biodiversity, maintain viable fisheries, and deliver a broad suite of ecosystem services. Over the past four decades, Hawaii has developed a system of MPAs to conserve and replenish marine resources around the state. These Marine Life Conservation Districts (MLCDs) vary in size, habitat quality, and management regimes, providing an excellent opportunity to test hypotheses concerning MPA design and function using multiple discreet sampling units. NOAA/NOS/NCCOS/Center for Coastal Monitoring and Assessment’s Biogeography Branch used digital benthic habitat maps coupled with comprehensive ecological studies between 2002 and 2004 to evaluate the efficacy of all existing MLCDs using a spatially-explicit stratified random sampling design. The results from this work have shown that areas fully protected from fishing had higher fish biomass, larger overall fish size, and higher biodiversity than adjacent areas of similar habitat quality. Other key findings demonstrated that top predators and other important fisheries species were more abundant and larger in the MPAs, illustrating the effectiveness of these closures in conserving these populations. Habitat complexity, protected area size and habitat diversity were the major factors in determining effectiveness among MPAs.

Parasitic reaction and its effect on the growth rate of AlN by metalorganic chemical vapor deposition

The Al composition of metalorganic chemical vapor deposition (MOCVD)-grown AlGaN alloy layers is found to be greatly influenced by the parasitic reaction between ammonia (NH3) and trimethylaluminum (TMAI). The growth process of AlN is carefully investigated by monitoring the in situ optical reflection. The abnormal dependencies of growth rate on growth temperature, reactor pressure, and flux of NH3 are observed and can be well explained by the effect of parasitic reaction. The increase of growth rate with increasing flux of TMAI is found to depend on the growth temperature and reactor pressure due to the presence of parasitic effect. A relatively low growth temperature and a reduced reactor pressure are suggested for the effective decrease of parasitic reaction during the MOCVD growth of AlN and probably lead to a more effective incorporation of Al into the AlGaN layers. (c) 2005 Elsevier B.V. All rights reserved.

Electrochemical surface plasmon resonance detection of enzymatic reaction in bilayer lipid

In this paper, electrochemical surface plasmon resonance (SPR) method was first used to detect enzymatic reaction in bilayer lipid membrane (BLM) based on immobilizing horseradish peroxidase (HRP) in the BLMs supported by the redox polyaniline (PAn) film. By SPR kinetic curve in situ monitoring the redox transformation of PAn film resulted from the reaction between HRP and PAn, the enzymatic reaction of HRP with H2O2, was successfully analyzed by electrochemical SPR spectroscopy.

Monitoring catalytic degradation of dye molecules on silver-coated ZnO nanowire arrays by surface-enhanced Raman spectroscopy

Catalytic degradation of organic dye molecules has attracted extensive attention due to their high toxicity to water resources. In this paper, we propose a novel method for the fabrication of uniform silver-coated ZnO nanowire arrays. The degradation of typical dye molecule rhodamine 6G (R6G), as an example, is investigated in the presence of the as-prepared silver-coated ZnO nanowire arrays. The experimental results show that such composite nanostructures exhibit high catalytic activity, and the reaction follows pseudo-first-order kinetics. Furthermore, these nanowire arrays are desirable SERS substrates for monitoring the catalytic degradation of dye molecules. Compared with traditional UV-visible spectroscopy, SERS technology can reflect more truly the catalytic degradation process occurring on the surface of the catalysts.

Studies on principal components and antioxidant activity of different Radix Astragali samples using high-performance liquid chromatography/electrospray ionization multiple-stage tandem mass spectrometry

The principal components, isoflavonoids and astragalosides, in the extract of Radix Astragali were detected by a high-performance liquid chromatography Couple to electrospray ionization ion trap multiple-stage tandem mass spectrometry (HPLC-ESI-IT-MSn) method. By comparing the retention time (t(R)) of HPLC, the ESI-MSn data and the structures of analyzed Compounds with the data of reference compounds and in the literature, 17 isoflavonoids and 12 astragalosides have been identified or tentatively deduced. By Virtue of the extracted ion chromatogram (EIC) mode, simultaneous determination of isoflavonoids and astragalosides could be achieved when the different components formed overlapped peaks. And this method has been utilized to analyze the constituents in extracts of Radix Astragali from Helong City and of different growth years. Then the antioxidant activity of different samples has been Successfully investigated by HPLC-ESI-MS method in multiple selected ion monitoring(MIM) mode, applying the spin trapping technology, and the Ferric Reducing Antioxidant Power (FRAP) assay was applied to support the result.

Bronsted guanidine acid-base ionic liquids: Novel reaction media for the palladium-catalyzed Heck reaction

Bronsted acid-base ionic liquids (GILs) based on guanidine and acetic acid are efficient reaction media for palladium-catalyzed Heck reactions. They offer the advantages of high activity and reusability. GIL2 plays multiple roles in the reaction: it could act as solvent, as a strong base to facilitate beta-hydride elimination, and as a ligand to stabilize activated Pd species.

Catalytic mechanisms of triphenyl bismuth, dibutyltin dilaurate, and their combination in polyurethane-forming reaction

The catalytic mechanisms of triphenyl bismuth (TPB), dibutyltin dilaurate (DBTDL) and their combination have been studied in a model polyurethane reaction system consisting of copolyether (tetrahydrofuran-ethyleneoxide) and N-100; NMR spectroscopy was used to detect the associations between reactants and catalysts. A relatively stable complex was shown to be formed between hydroxyl and isocyanate; the catalysts showed different effects on the isocyanate-hydroxyl complex, therefore resulting in different curing characteristics. The formation of hydrogen bonding between the complexed hydroxyl and other hydroxyl or the resulting urethane provided an ''auto-catalysis'' to urethane formation. DBTDL destroyed the isocyanate-hydroxyl complex before catalyzing the reaction through the formation of a ternary complex, whereas TPB was able to activate the isocyanate-hydroxyl complex directly to form urethane. The reaction catalyzed by the combination of TPB and DBTDL gained advantages from the multiple catalytic entities, i.e., TPB, DBTDL, and a TPB-DBTDL complex. (C) 1997 John Wiley & Sons, Inc.

Product monitoring and quantitation of oligosaccharides composition in agar hydrolysates by precolumn labeling HPLC

Since the discovery of multiple bioactivities for agarobiose oligomers, a quantitative method has been in great need to monitor the agarobiose oligomers. This report demonstrates that agarobiose oligomers can be separated with high resolution in HPLC after introducing a-naphthylamine into compounds. Agarobiose oligomers ranged from biose to decaose were isolated by Sephadex column. HPLC analysis indicated that each oliomer could be quantified with good linearity and a low detection limit of 0.1-4 mug/ml. The chromatographic profiles of agaro-oligosaccharides with different hydrolysis modes (hydrochloride, citric acid, solid acid, and hydroxyl radical degradation) showed that agarobiose could be obtained more than 57.8% using solid acid mediated hydrolysis, while hydrochloride acid could degrade agar into a series of agaro-oligosaccharides from biose to decaose. The yield of oligosaccharides was low if hydrolyzed by citric acid. The Fenton degradation can increase the speed of hydrolysis, but the product was complex. (C) 2004 Elsevier B.V. All rights reserved.