Direct effects of organic pollutants on the growth and gene expression of the Baltic Sea model bacterium Rheinheimera sp. BAL341

Organic pollutants (OPs) are critically toxic, bioaccumulative and globally widespread. Moreover, several OPs negatively influence aquatic wildlife. Although bacteria are major drivers of the ocean carbon cycle and the turnover of vital elements, there is limited knowledge of OP effects on heterotrophic bacterioplankton. We therefore investigated growth and gene expression responses of the Baltic Sea model bacterium Rheinheimera sp. BAL341 to environmentally relevant concentrations of distinct classes of OPs in 2-h incubation experiments. During exponential growth, exposure to a mix of polycyclic aromatic hydrocarbons, alkanes and organophosphate esters (denoted MIX) resulted in a significant decrease (between 9% and 18%) in bacterial abundance and production compared with controls. In contrast, combined exposure to perfluorooctanesulfonic acids and perfluorooctanoic acids (denoted PFAS) had no significant effect on growth. Nevertheless, MIX and PFAS exposures both induced significant shifts in gene expression profiles compared with controls in exponential growth. This involved several functional metabolism categories (e.g. stress response and fatty acids metabolism), some of which were pollutant-specific (e.g. phosphate acquisition and alkane-1 monooxygenase genes). In stationary phase, only two genes in the MIX treatment were significantly differentially expressed. The substantial direct influence of OPs on metabolism during bacterial growth suggests that widespread OPs could severely alter biogeochemical processes governed by bacterioplankton.     

Integrating human health impact from indoor emissions into an LCA: a case study evaluating the significance of the use stage

Purpose

Indoor emissions of toxic substances from products can have a negative effect on human health. These are typically not considered in a life cycle assessment (LCA), potentially underestimating the importance of the use stage. The purpose of this paper is to develop a method that, based on a set of measured emission rates, calculates the impact on human health during the use stage of products that are used indoors and that emit volatile organic compounds (VOCs).

Methods

Emissions from a product are measured in a test chamber and reported as a set of emission rates (microgrammes per hour) at specific points in time (hour/day). Constrained non-linear regression (CNLR) analysis is then used to determine parameters for three emission models, and a model is selected based on goodness of fit with the measured emission rates (R ² and expert judgement). The emission model is integrated over a defined time period to estimate the total use stage emissions per functional unit (FU). The total emissions are subsequently integrated in a homogeneously mixed one-box model within the USEtox model. Intake fraction (iF) is calculated based on size of residential home, inhalation rate, exposure time, ventilation rate, mixing factor and number of people exposed.

Results and discussion

The method is tested in a case study of a chair, with the results showing that the impacts in the use stage are in most cases significantly higher than from the production and disposal stages combined. The sensitivity to parameter variations is evaluated. Intake fraction (factor of 761), replacement frequency (factor of 70) and emission model (factor of 24) are found to be the most important model parameters. Limiting early exposure (>14 % of emissions may occur in the first month and >50 % in the first year) and replacing furniture less frequently will reduce exposure.

Conclusions

The case study shows that the impact on human health from indoor emissions can be of significance, when compared to the impact on human health from total outdoor emissions. Without specific exposure data (e.g. ventilation rates) the uncertainty will be high. The developed method is applicable to all products that emit VOCs, provided that the emission rate can be modelled using an exponential decay model and that the product amount is related to a meaningful functional unit. It is recommended that when performing an LCA of products that emit VOCs, the indoor use stage is included in the life cycle impact assessment.     

THE EVOLUTION OF BIPEDAL RUNNING IN LIZARDS SUGGESTS A CONSEQUENTIAL ORIGIN MAY BE EXPLOITED IN LATER LINEAGES

The origin of bipedal locomotion in lizards is unclear. Modeling studies have suggested that bipedalism may be an exaptation, a byproduct of features originally designed to increase maneuverability, which were only later exploited. Measurement of the body center of mass (BCOM) in 124 species of lizards confirms a significant rearward shift among bipedal lineages. Further racetrack trials showed a significant acceleration threshold between bipedal and quadrupedal runs. These suggest good general support for a passive bipedal model, in which the combination of these features lead to passive lifting of the front of the body. However, variation in morphology could only account for 56% of the variation in acceleration thresholds, suggesting that dynamics have a significant influence on bipedalism. Deviation from the passive bipedal model was compared with node age, supporting an increase in the influence of dynamics over time. Together, these results show that bipedalism may have first arisen as a consequence of acceleration and a rearward shift in the BCOM, but subsequent linages have exploited this consequence to become bipedal more often, suggesting that bipedalism in lizards may convey some advantage. Exploitation of bipedalism was also associated with increased rates of phenotypic diversity, suggesting exploiting bipedalism may promote adaptive radiation.     

Equine CRISP3 modulates interaction between spermatozoa and polymorphonuclear neutrophils

Equine spermatozoa induce a uterine inflammatory response characterized by a rapid, transient influx of polymorphonuclear neutrophils (PMNs). Seminal plasma proteins have been shown to modulate the interaction between spermatozoa and PMNs, but a specific protein responsible for this function has not been identified. The objective of this study was to isolate and identify a protein in equine seminal plasma that suppresses binding between spermatozoa and PMNs. Seminal plasma was pooled from five stallions, and proteins were precipitated in 60% (w/v) ammonium sulfate and dialyzed (3500 MW cutoff). Proteins were submitted to a Sephacryl S200 column, and fractions were pooled based on the fraction pattern. Each pool was analyzed for protein concentration and tested for its suppressive effect on PMN/sperm binding. Protein pools with biological activity were submitted to ion-exchange chromatography (diethylaminoethyl [DEAE] Sephadex column) with equilibration buffers containing 0.1-0.5M NaCl. Eluants were pooled, analyzed for protein concentration, and tested for suppressive effects on PMN/sperm binding. Protein distribution and purity were determined by one- and two-dimensional SDS-PAGE, and the purified protein was submitted for sequence analysis and identification. This protein was identified as equine CRISP3 and was confirmed by Western blotting. Suppression of PMN/sperm binding by CRISP3 and seminal plasma was confirmed by flow cytometry (22.08% ± 3.05% vs. 2.06% ± 2.02% vs. 63.09% ± 8.67 for equine seminal plasma, CRISP3, and media, respectively; P < 0.0001). It was concluded that CRISP3 in seminal plasma suppresses PMNs/sperm binding, suggesting that CRISP3 regulates sperm elimination from the female reproductive tract.     

Cryopreservation of equine sperm: optimal cooling rates in the presence and absence of cryoprotective agents determined using differential scanning calorimetry.

Optimization of equine sperm cryopreservation protocols requires an understanding of the water permeability characteristics and volumetric shrinkage response during freezing. A cell-shape-independent differential scanning calorimeter (DSC) technique was used to measure the volumetric shrinkage during freezing of equine sperm suspensions at cooling rates of 5 degrees C/min and 20 degrees C/min in the presence and absence of cryoprotective agents (CPAs), i.e., in the Kenney extender and in the lactose-EDTA extender, respectively. The equine sperm was modeled as a cylinder of length 36.5 microm and a radius of 0.66 microm with an osmotically inactive cell volume (V(b)) of 0.6V(o), where V(o) is the isotonic cell volume. Sperm samples were collected using water-insoluble Vaseline in the artificial vagina and slow cooled at < or = 0.3 degrees C/min in an Equitainer-I from 37 degrees C to 4 degrees C. By fitting a model of water transport to the experimentally obtained DSC volumetric shrinkage data, the best-fit membrane permeability parameters (L(pg) and E(Lp)) were determined. The combined best-fit parameters of water transport (at both 5 degrees C/min and 20 degrees C/min) in Kenney extender (absence of CPAs) are L(pg) = 0.02 microm min(-1) atm(-1) and E(Lp) = 32.7 kcal/mol with a goodness-of-fit parameter R(2) = 0.96, and the best-fit parameters in the lactose-EDTA extender (the CPA medium) are L(pg)[cpa] = 0.008 microm min(-1) atm(-1) and E(Lp)[cpa] = 12.1 kcal/mol with R(2) = 0.97. These parameters suggest that the optimal cooling rate for equine sperm is approximately 29 degrees C/min and is approximately 60 degrees C/min in the Kenney extender and in the lactose-EDTA extender. These rates are predicted assuming no intracellular ice formation occurs and that the approximately 5% of initial osmotically active water volume trapped inside the cells at -30 degrees C will form innocuous ice on further cooling. Numerical simulations also showed that in the lactose-EDTA extender, equine sperm trap approximately 3.4% and approximately 7.1% of the intracellular water when cooled at 20 degrees C/min and 100 degrees C/min, respectively. As an independent test of this prediction, the percentage of viable equine sperm was obtained after freezing at 6 different cooling rates (2 degrees C/min, 20 degrees C/min, 50 degrees C/min, 70 degrees C/min, 130 degrees C/min, and 200 degrees C/min) to -80 degrees C in the CPA medium. Sperm viability was essentially constant between 20 degrees C/min and 130 degrees C/min.     

Structural basis for molecular recognition in an affibody:affibody complex

Affibody molecules constitute a class of engineered binding proteins based on the 58-residue three-helix bundle Z domain derived from staphylococcal protein A (SPA). Affibody proteins are selected as binders to target proteins by phage display of combinatorial libraries in which typically 13 side-chains on the surface of helices 1 and 2 in the Z domain have been randomized. The Z(Taq):anti-Z(Taq) affibody-affibody complex, consisting of Z(Taq), originally selected as a binder to Taq DNA polymerase, and anti-Z(Taq), selected as binder to Z(Taq), is formed with a dissociation constant K(d) approximately 100 nM. We have determined high-precision solution structures of free Z(Taq) and anti-Z(Taq), and the Z(Taq):anti-Z(Taq) complex under identical experimental conditions (25 degrees C in 50 mM NaCl with 20 mM potassium phosphate buffer at pH 6.4). The complex is formed with helices 1 and 2 of anti-Z(Taq) in perpendicular contact with helices 1 and 2 of Z(Taq). The interaction surface is large ( approximately 1670 A(2)) and unusually non-polar (70 %) compared to other protein-protein complexes. It involves all varied residues on anti-Z(Taq), most corresponding (Taq DNA polymerase binding) side-chains on Z(Taq), and several additional side-chain and backbone contacts. Other notable features include a substantial rearrangement (induced fit) of aromatic side-chains in Z(Taq) upon binding, a close contact between glycine residues in the two subunits that might involve aliphatic glycine Halpha to backbone carbonyl hydrogen bonds, and four hydrogen bonds made by the two guanidinium N(eta)H(2) groups of an arginine side-chain. Comparisons of the present structure with other data for affibody proteins and the Z domain suggest that intrinsic binding properties of the originating SPA surface might be inherited by the affibody binders. A thermodynamic characterization of Z(Taq) and anti-Z(Taq) is presented in an accompanying paper.     

The Role of Sharks and Longline Fisheries in a Pelagic Ecosystem of the Central Pacific

The increased exploitation of pelagic sharks by longline fisheries raised questions about changes in the food webs that include sharks as apex predators. We used a version of Ecopath/Ecosim models to evaluate changes in trophic interactions due to shark exploitation in the Central North Pacific. Fisheries targeted on blue sharks tend to produce compensatory responses that favor other shark species and billfishes, but they have only modest effects on the majority of food web components. Modest levels of intraguild predation (adult sharks that eat juvenile sharks) produce strong, nonlinear responses in shark populations. In general, analysis of the Central North Pacific model reveals that sharks are not keystone predators, but that increases in longline fisheries can have profound effects on the food webs that support sharks. Received 19 April 2001; accepted 2 October 2001.