Metabolite diversity among representatives of divergent Prochlorococcus ecotypes.

IMPORTANCE: Approximately half of the annual carbon fixation on Earth occurs in the surface ocean through the photosynthetic activities of phytoplankton such as the ubiquitous picocyanobacterium Prochlorococcus. Ecologically distinct subpopulations (or ecotypes) of Prochlorococcus are central conduits of organic substrates into the ocean microbiome, thus playing important roles in surface ocean production. We measured the chemical profile of three cultured ecotype strains, observing striking differences among them that have implications for the likely chemical impact of Prochlorococcus subpopulations on their surroundings in the wild. Subpopulations differ in abundance along gradients of temperature, light, and nutrient concentrations, suggesting that these chemical differences could affect carbon cycling in different ocean strata and should be considered in models of Prochlorococcus physiology and marine carbon dynamics.

Xenoprotein engineering via synthetic libraries.

Chemical methods have enabled the total synthesis of protein molecules of ever-increasing size and complexity. However, methods to engineer synthetic proteins comprising noncanonical amino acids have not kept pace, even though this capability would be a distinct advantage of the total synthesis approach to protein science. In this work, we report a platform for protein engineering based on the screening of synthetic one-bead one-compound protein libraries. Screening throughput approaching that of cell surface display was achieved by a combination of magnetic bead enrichment, flow cytometry analysis of on-bead screens, and high-throughput MS/MS-based sequencing of identified active compounds. Direct screening of a synthetic protein library by these methods resulted in the de novo discovery of mirror-image miniprotein-based binders to a ∼150-kDa protein target, a task that would be difficult or impossible by other means.

Library Design-Facilitated High-Throughput Sequencing of Synthetic Peptide Libraries.

A methodology to achieve high-throughput de novo sequencing of synthetic peptide mixtures is reported. The approach leverages shotgun nanoliquid chromatography coupled with tandem mass spectrometry-based de novo sequencing of library mixtures (up to 2000 peptides) as well as automated data analysis protocols to filter away incorrect assignments, noise, and synthetic side-products. For increasing the confidence in the sequencing results, mass spectrometry-friendly library designs were developed that enabled unambiguous decoding of up to 600 peptide sequences per hour while maintaining greater than 85% sequence identification rates in most cases. The reliability of the reported decoding strategy was additionally confirmed by matching fragmentation spectra for select authentic peptides identified from library sequencing samples. The methods reported here are directly applicable to screening techniques that yield mixtures of active compounds, including particle sorting of one-bead one-compound libraries and affinity enrichment of synthetic library mixtures performed in solution.

Toxicity of pulp and paper solid organic waste constituents to soil organisms.

This study examined the potential biological hazard of pulp and paper waste solids. The solids examined were chosen on the basis of the range of wood-related organic extractives and were either primary solids screened from the effluent stream before secondary treatment, or biosolids from aerated stabilisation lagoons. Acute effects were tested at the level of plants, invertebrates and soil microbes using an oat germination and growth test, earthworm survival and reproduction test, an enchytraeid worm survival and reproduction test, and standard measures of microbial respiration. This was further benchmarked against a marine bacteria toxicity test using extract of the waste solids. Resin acids and resin acid neutrals made up the greatest proportion of organic extractives measured in biosolids whereas resin acids and fatty acids were the main constituents detected in primary solids. Examination of the tissue of earthworms from the tests revealed no net bioconcentration of the organic extractives. The waste solids were not acutely toxic to any of the soil organisms as tested without any dilution. Conversely, extracts of the waste solids demonstrated toxicity in the marine bacteria. In some cases, the solid waste material enhanced the growth of plants, earthworm reproduction and microbial respiration. The only adverse affect was that reproduction of enchytraeids was reduced by some of the waste solid treatments. However these effects did not appear to be associated with concentrations of resin acid neutrals and resin acids in these materials. Overall pulp and paper wastes were relatively benign in terms of toxicity to the soil organisms tested.

Response of a New Zealand mayfly (Deleatidium spp.) to acid mine drainage: implications for mine remediation.

Investigating the toxicity of acid mine drainage (AMD) on benthic communities in receiving waters can be highly challenging because of the difficulty in unraveling the effects of acidity, dissolved metals, and precipitates. Furthermore, the survival of different species may vary depending on any natural adaptation they may have acquired to low pH, metals, or sedimentation. We investigated the effect of different pHs and AMD on the survival of a common New Zealand leptophlebiid mayfly (Deleatidium spp.) in 96-h laboratory trials. Our results indicate that the primary driver of toxicity in AMD was pH, although some mortality could be attributable to the presence of dissolved heavy metals at low pH (

Hepatic and immune biological effect assays in C57BL/6 mice to measure polycyclic aromatic hydrocarbon bioavailability under laboratory exposures with increasing environmental relevance.

BACKGROUND: Monitoring biological responses that are mediated via the aryl-hydrocarbon receptor (AhR) in animals exposed to environmental contaminants can indicate both the presence of chemicals that act through this biochemical pathway and whether these chemicals are bioavailable. OBJECTIVES: The use of an ex-situ method that incorporated biological responsiveness monitoring in mice for determining the presence of 'biologically active' hydrocarbons in contaminated soils was investigated. METHODS: The use of C57BL/6 as a test organism was validated by determining hepatic and immune responsiveness to two polyaromatic hydrocarbons (PAHs): 3,4 benz[a]pyrene (B[a]P) and 1,2 benz (a)anthracene (BA) administered via intraperitoneal (i.p.) injection. The responsiveness of mice exposed to soils spiked with hydrocarbons or ex situ exposures to soil removed from two contaminated sites was also investigated. RESULTS AND DISCUSSION: Mice that were exposed to B[a]P via i.p. injections showed a 14-fold increase in liver microsomal ethoxyresorufin O-deethylase (EROD) activity compared to the control group. In contrast EROD activity following BA exposure at the same level was not significantly enhanced. Mouse immune response was significantly inhibited in a dose-dependent manner by i.p. injections of B[a]P. No significant inhibition occurred with the same doses of BA. Following i.p. exposure, the retention of B[a]P in mouse carcasses was greater than BA. Mice exposed to clean soils spiked with environmentally relevant concentrations of B[a]P and BA failed to show any significantly different hepatic or immune responses. Carcass residue data indicated a limited uptake of PAH from the soil. In contrast, EROD activity in mice exposed (ex situ) to hydrocarbon-contaminated soils removed from a fuel-loading depot and decommissioned gas works was significantly enhanced (4- and 2-fold respectively). However, this increase in EROD activity did not appear to correlate with either soil or carcass PAH concentrations. CONCLUSIONS AND OUTLOOK: These results support the assumption that B[a]P has a higher affinity for the aryl hydrocarbon receptor (AhR) compared to BA. Soil parameters such as organic carbon content, structure and particle size distribution can modulate the bioavailability of contaminants to biological receptors. These factors are implicated in the lack of responsiveness demonstrated in the spiked soil experiments. However the responsiveness of EROD activity in mice exposed (ex situ) to soil contaminated with complex mixtures of hydrocarbon compounds confirms the potential usefulness of this model to determine the presence of 'biologically active' compounds in aged soils removed from contaminated sites.

Effect of in vitro and in vivo organotin exposures on the immune functions of murray cod (Maccullochella peelii peelii).

Murray cod (Maccullochella peelii peelii) is an iconic native Australian freshwater fish and an ideal species for ecotoxicological testing of environmental pollutants. The species is indigenous to the Murray-Darling basin, which is the largest river system in Australia but also the ultimate sink for many environmental pollutants. The organotins tributyltin (TBT) and dibutyltin (DBT) are common pollutants of both freshwater and marine environments and are also known for their immunotoxicity in both mammals and aquatic organisms. In this study, TBT and DBT were used as exemplar immunotoxins to assess the efficiency of immune function assays (i.e., mitogen-stimulated lymphoproliferation, phagocytosis in head kidney tissue, and serum lysozyme activity) and to compare the sensitivity of Murray cod to other fish species. The organotins were lethal to Murray cod at concentrations previously reported as sublethal in rainbow trout (i.e., intraperitoneal [i.p.] lethal dose to 75% of the Murray cod [LD75] = 2.5 mg/kg DBT and i.p. lethal dose to 100% of the Murray cod [LD100] = 12.5 mg/kg TBT and DBT). In vivo TBT exposure at 0.1 and 0.5 mg/kg stimulated the phagocytic function of Murray cod (F = 6.89, df = 18, p = 0.004), while the highest concentration of 2.5 mg/kg TBT decreased lymphocyte numbers (F = 7.92, df = 18, p = 0.02) and mitogenesis (F = 3.66, df = 18, p = 0.035). Dibutyltin was the more potent immunosuppressant in Murray cod, causing significant reductions in phagocytic activity (F = 5.34, df = 16, p = 0.013) and lymphocyte numbers (F = 10.63, df = 16, p = 0.001).

Flow cytometric analysis and optimisation for measuring phagocytosis in three Australian freshwater fish.

The phagocytic activity of fish immunocytes has been measured by a wide range of methods, and has been used as a bio-indicator to assess the immunotoxicity of environmental pollutants and the efficiency of immunostimulants used in aquaculture. This study demonstrates the utilisation of a flow cytometric technique for measuring phagocytosis as an alternative to manual evaluations by light microscopy. Optimal conditions for the phagocytosis of latex beads were ascertained, including incubation period, cell:bead ratio and media components, for head kidney cells isolated from three native Australian fish that inhabit the Murray-Darling basin, i.e. silver perch (Bidyanus bidyanus), golden perch (Macquaria ambigua) and crimson-spotted rainbowfish (Melanotaenia fluviatilis). Thus, standardised protocols have now been established for future use in the immunotoxicity testing of xenobiotics in native Australian freshwater fish.

The effects of in vitro pesticide exposures on the phagocytic function of four native Australian freshwater fish.

There are limited data concerning the lethal and sublethal effects of environmental pollutants on Australian freshwater fish and consequently many of the Australian water quality guidelines are based on data from exotic fish species. This study used a flow cytometric assay to assess the effect of in vitro exposures to commonly used pesticides, on the phagocytic function and cellular composition of head kidney cells from four Australian native fish, i.e. crimson-spotted rainbowfish (Melanotaenia fluviatilis), silver perch (Bidyanus bidyanus), golden perch (Macquaria ambigua) and Murray cod (Maccullochella peelii). Head kidney immune cells were isolated from the four native fish and incubated (1 x 10(6) cells/mL) under previously optimised conditions to measure the phagocytosis of fluorescent-latex beads. These cell cultures were exposed to three classes of pesticides, i.e. the organotins, tributyltin and dibutyltin, the organochlorine endosulfan and the organophosphate chlorpyrifos. The in vitro organotin exposures were highly immunotoxic to head kidney cells from the Australian fish, although there were some differences in immunotoxic responses between species. At the highest concentration (i.e. 10 mg/L), endosulfan exposure resulted in the modulation of phagocytic responses in all species except for silver perch. Chlorpyrifos displayed little immunotoxicity, although there was a dose-dependent reduction in Murray cod lymphocytes. These studies describe the first investigation of the phagocytic response of Australian freshwater fish immunocytes in the presence of environmental pollutants, and will help to determine appropriate ecotoxicity testing for Australian freshwater environments.

Ecotoxicity of sodium fluoroacetate (compound 1080) to soil organisms.

Sodium fluoroacetate (compound 1080) is applied as a vertebrate pesticide in New Zealand for control of introduced mammalian pests. Despite its widespread use, little is known about the soil ecotoxicity of 1080. Therefore, the hazard of 1080 to soil invertebrates, plants, and soil microorganisms was evaluated in a series of controlled laboratory tests. No earthworm (Eisenia fetida) mortality was reported with 1080 exposures up to 865 mg/kg soil. The lowest-observable-effect concentration and the median effect level for earthworm reproduction were 100 and 90 mg/kg soil, respectively, for cocoon production and 100 and 160 mg/kg soil, respectively, for juvenile production. Lettuce (Lactuca sativa) was more sensitive than oats (Avena sativa) to 1080. Lettuce seedling emergence and seedling shoot growth were adversely affected at a soil concentration of 7 mg/kg. The presence of 1080 in soil at concentrations up to 1 g/kg soil did not affect the ability of soil microorganisms to mineralize nitrogen. Furthermore, nitrate mineralization was not inhibited in soil treated with the urine of 1080-poisoned possums (Trichosurus vulpecula). The data collectively indicate that 1080-related effects on soil organisms occur at levels well above those that have been measured in soil (<0.1 mg/kg) following application of 1080 in baits for vertebrate pest control in New Zealand.

Biomarkers in toxicology versus ecological risk assessment.

Toxicity testing of drugs, pesticides, and hazardous compounds has evolved into a battery of standardized tests conducted in a range of surrogate test organisms. The toxicity of these xenobiotics in terms of their LD(50) and LC(50) (Dose or concentration lethal to 50% of the test population), ED(50) and EC(50) (Dose or concentration producing a specified response in 50% of the test population), MATC (Maximum acceptable toxicant concentration), LOEL (Lowest observable effects level), LOEC (Lowest observable effects concentration), NOEL (No observable effects level) or NOEC (No observable effects concentration) is extrapolated to humans and wildlife. Historical failures in the risk assessment process have been largely due to over reliance on regulatory toxicology and an 'assembly line' mentality to toxicology. The importance of toxicokinetics, receptor studies and biomarkers are reviewed, firstly, with reference to toxicological incidences in drug development programmes, and secondly, with reference to improved environmental risk assessment of pesticides and other contaminants. Ecological risk assessments also require multidisciplinary skills to study the entry, distribution, and biological effect and fate of chemicals to fully characterise and understand the potential adverse implications of contamination. Optimum integration of chemical measurements and biomarker responses is a challenge that will lead to an improved understanding of adverse effects and their significance in both human and ecological risk assessment.