Neutrophil polarisation in plasma differs to that induced by endogenous chemoattractants with regard to frequency of uropod formation and requirement for divalent cations.

Human neutrophils suspended in Hanks' balanced salt solution (37° C, 20 mM Hepes, pH 7.2) produced extensions, elongated and developed a polarised morphology with both a pseudopod and uropod when exposed to C5a (10 nM), leukotriene B4 (10 nM), platelet activating factor (40 nM) or interleukin-8 (12.5 nM). Responses to each mediator were generally enhanced or unaffected by chelators of extracellular Ca²⁺ and Mg²⁺. Neutrophils suspended in heparinised plasma (90-10% v/v in Hanks' balanced salt solution) produced extensions, elongated and developed a pseudopod, but rarely developed a uropod unless additional Mg²⁺ ions (0.5-5 mM) were added. These findings demonstrate that the polarisation of neutrophils in plasma is significantly different to that induced by endogenous chemoattractants with regard to the frequency of uropod formation and requirement for extracellular divalent cations.     

USE OF A PETHIDINE AND MIDAZOLAM COMBINATION FOR THE REVERSIBLE SEDATION OF CRABEATER SEALS (LOBODON CARCINOPHAGUS)

Between October and December of 1996–1999, off eastern Antarctica (60°-150°E), we darted 31 crabeater seals with midazolam and pethidine at estimated dose rates of 0.15–0.4 mg/kg and 1–3 mg/kg, respectively. Maximum sedation was reached at 23 ± 9 min (n = 18) and first signs of recovery were noted at 54 ± 24 min (n = 4). Seals greater than 250 kg body-mass were sedated by administration of approximately 90–100 mg midazolam and 600 mg pethidine, but the degree of sedation was unpredictable and did not permit invasive procedures in some cases. Behavior of the seal and adjacent conspecifics affected the success of procedures and our ability to monitor vital signs. Naloxone and flumazenil reversed sedation, making this combination attractive for use in animals adjacent to water. Additional ketamine was administered to two seals, resulting in improved restraint.     

An Australian Multi‐Regional Waste Supply‐Use Framework

The production of waste creates both direct and indirect environmental impacts. A range of strategies are available to reduce the generation of waste by industry and households, and to select waste treatment approaches that minimize environmental harm. However, evaluating these strategies requires reliable and detailed data on waste production and treatment. Unfortunately, published Australian waste data are typically highly aggregated, published by a variety of entities in different formats, and do not form a complete time‐series. We demonstrate a technique for constructing a multi‐regional waste supply‐use (MRWSU) framework for Australia using information from numerous waste data sources. This is the first MRWSU framework to be constructed (to the authors' knowledge) and the first sub‐national waste input‐output framework to be constructed for Australia. We construct the framework using the Industrial Ecology Virtual Laboratory (IELab), a cloud‐hosted computational platform for building Australian multi‐regional input‐output tables. The structure of the framework complies with the System of Environmental‐Economic Accounting (SEEA). We demonstrate the use of the MRWSU framework by calculating waste footprints that enumerate the full supply chain waste production for Australian consumers.     

Methanosarcina spp. drive vinyl chloride dechlorination via interspecies hydrogen transfer

Two highly enriched cultures containing Dehalococcoides spp. were used to study the effect of aceticlastic methanogens on reductive vinyl chloride (VC) dechlorination. In terms of aceticlastic methanogens, one culture was dominated by Methanosaeta, while the other culture was dominated by Methanosarcina, as determined by fluorescence in situ hybridization. Cultures amended with 2-bromoethanesulfonate (BES), an efficient inhibitor of methanogens, exhibited slow VC dechlorination when grown on acetate and VC. Methanogenic cultures dominated by Methanosaeta had no impact on dechlorination rates, compared to BES-amended controls. In contrast, methanogenic cultures dominated by Methanosarcina displayed up to sevenfold-higher rates of VC dechlorination than their BES-amended counterparts. Methanosarcina-dominated cultures converted a higher percentage of [2-(14)C]acetate to (14)CO(2) when concomitant VC dechlorination took place, compared to nondechlorinating controls. Respiratory indices increased from 0.12 in nondechlorinating cultures to 0.51 in actively dechlorinating cultures. During VC dechlorination, aqueous hydrogen (H(2)) concentrations dropped to 0.3 to 0.5 nM. However, upon complete VC consumption, H(2) levels increased by a factor of 10 to 100, indicating active hydrogen production from acetate oxidation. This process was thermodynamically favorable by means of the extremely low H(2) levels during dechlorination. VC degradation in nonmethanogenic cultures was not inhibited by BES but was limited by the availability of H(2) as electron donor, in cultures both with and without BES. These findings all indicate that Methanosarcina (but not Methanosaeta), while cleaving acetate to methane, simultaneously oxidizes acetate to CO(2) plus H(2), driving hydrogenotrophic dehalorespiration of VC to ethene by Dehalococcoides.     

Seamless site-directed mutagenesis of the <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> genome using CRISPR-Cas9

CRISPR assisted homology directed repair enables the introduction of virtually any modification to the Saccharomyces cerevisiae genome. Of obvious interest is the marker-free and seamless introduction of point mutations. To fulfill this promise, a strategy that effects single nucleotide changes while preventing repeated recognition and cutting by the gRNA/Cas9 complex is needed. We demonstrate a two-step method to introduce point mutations at 17 positions in the S. cerevisiae genome. We show the general applicability of the method, enabling the seamless introduction of single nucleotide changes at any location, including essential genes and non-coding regions. We also show a quantifiable phenotype for a point mutation introduced in gene GSH1. The ease and wide applicability of this general method, combined with the demonstration of its feasibility will enable genome editing at an unprecedented level of detail in yeast and other organisms.     

Study of a lipophilic captopril analogue binding to angiotensin I converting enzyme

Human ACE is a central component of the renin–angiotensin system and a major therapeutic target for cardiovascular diseases. The somatic form of the enzyme (sACE) comprises two homologous metallopeptidase domains (N and C), each bearing a zinc active site with similar but distinct substrate and inhibitor specificities. In this study, we present the biological activity of silacaptopril, a silylated analogue of captopril, and its binding affinity towards ACE. Based on the recently determined crystal structures of both the ACE domains, a series of docking calculations were carried out in order to study the structural characteristics and the binding properties of silacaptopril and its analogues with ACE. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Enzymatic Engineering of Polysialic Acid on Cells in Vitro and in Vivo Using a Purified Bacterial Polysialyltransferase

In vertebrates, polysialic acid (PSA) is typically added to the neural cell adhesion molecule (NCAM) in the Golgi by PST or STX polysialyltransferase. PSA promotes plasticity, and its enhanced expression by viral delivery of the PST or STX gene has been shown to promote cellular processes that are useful for repair of the injured adult nervous system. Here we demonstrate a new strategy for PSA induction on cells involving addition of a purified polysialyltransferase from Neisseria meningitidis (PST_Nm) to the extracellular environment. In the presence of its donor substrate (CMP-Neu5Ac), PST_Nm synthesized PSA directly on surfaces of various cell types in culture, including Chinese hamster ovary cells, chicken DF1 fibroblasts, primary rat Schwann cells, and mouse embryonic stem cells. Similarly, injection of PST_Nm and donor in vivo was able to produce PSA in different adult brain regions, including the cerebral cortex, striatum, and spinal cord. PSA synthesis by PST_Nm requires the presence of the donor CMP-Neu5Ac, and the product could be degraded by the PSA-specific endoneuraminidase-N. Although PST_Nm was able to add PSA to NCAM, most of its product was attached to other cell surface proteins. Nevertheless, the PST_Nm-induced PSA displayed the ability to attenuate cell adhesion, promote neurite outgrowth, and enhance cell migration as has been reported for endogenous PSA-NCAM. Polysialylation by PST_Nm occurred in vivo in less than 2.5 h, persisted in tissues, and then decreased within a few weeks. Together these characteristics suggest that a PST_Nm-based approach may provide a valuable alternative to PST gene therapy.     

Mixed company: a framework for understanding the composition and organization of mixed‐species animal groups

Mixed‐species animal groups (MSGs) are widely acknowledged to increase predator avoidance and foraging efficiency, among other benefits, and thereby increase participants' fitness. Diversity in MSG composition ranges from two to 70 species of very similar or completely different phenotypes. Yet consistency in organization is also observable in that one or a few species usually have disproportionate importance for MSG formation and/or maintenance. We propose a two‐dimensional framework for understanding this diversity and consistency, concentrating on the types of interactions possible between two individuals, usually of different species. One axis represents the similarity of benefit types traded between the individuals, while the second axis expresses asymmetry in the relative amount of benefits/costs accrued. Considering benefit types, one extreme represents the case of single‐species groups wherein all individuals obtain the same supplementary, group‐size‐related benefits, and the other extreme comprises associations of very different, but complementary species (e.g. one partner creates access to food while the other provides vigilance). The relevance of social information and the matching of activities (e.g. speed of movement) are highest for relationships on the supplementary side of this axis, but so is competition; relationships between species will occur at points along this gradient where the benefits outweigh the costs. Considering benefit amounts given or received, extreme asymmetry occurs when one species is exclusively a benefit provider and the other a benefit user. Within this parameter space, some MSG systems are constrained to one kind of interaction, such as shoals of fish of similar species or leader–follower interactions in fish and other taxa. Other MSGs, such as terrestrial bird flocks, can simultaneously include a variety of supplementary and complementary interactions. We review the benefits that species obtain across the diversity of MSG types, and argue that the degree and nature of asymmetry between benefit providers and users should be measured and not just assumed. We then discuss evolutionary shifts in MSG types, focusing on drivers towards similarity in group composition, and selection on benefit providers to enhance the benefits they can receive from other species. Finally, we conclude by considering how individual and collective behaviour in MSGs may influence both the structure and processes of communities.