Making marinas bivalve friendly for enhanced biodiversity outcomes.

Natural coastlines are being replaced by artificial structures (pilings, pontoons, breakwaters), with negative environmental impacts, particularly in marinas. Ropes seeded with mussels (Mytilus galloprovincialis) were added to artificial structures in a marina, using aquaculture techniques, to reduce the colonisation of invasive taxa. After 6-months, droplines beneath pontoons had the highest seeded mussel survival and growth, richness of native and invasive taxa, and proportion of invasive to native taxa, compared with the other interventions. Mussel ropes on the intertidal structures (pilings and breakwaters) supported higher biomass of native taxa, whereas mussel ropes on subtidal structures (pontoons and breakwaters) had reduced biomass of invasive taxa, relative to the unseeded ropes. Droplines had the greater biomass of mussels, while mussel ropes placed under pontoons, and in subtidal gabion baskets limited the biomass but not the diversity of invasive species. Further study is required to determine whether these interventions can be upscaled to improve both the native biodiversity and functioning of marinas.

Building blue infrastructure: Assessing the key environmental issues and priority areas for ecological engineering initiatives in Australia's metropolitan embayments.

Ecological engineering principles are increasingly being applied to develop multifunctional artificial structures or rehabilitated habitats in coastal areas. Ecological engineering initiatives are primarily driven by marine scientists and coastal managers, but often the views of key user groups, which can strongly influence the success of projects, are not considered. We used an online survey and participatory mapping exercise to investigate differences in priority goals, sites and attitudes towards ecological engineering between marine scientists and coastal managers as compared to other stakeholders. The surveys were conducted across three Australian cities that varied in their level of urbanisation and environmental pressures. We tested the hypotheses that, relative to other stakeholders, marine scientists and coastal managers will: 1) be more supportive of ecological engineering; 2) be more likely to agree that enhancement of biodiversity and remediation of pollution are key priorities for ecological engineering; and 3) identify different priority areas and infrastructure or degraded habitats for ecological engineering. We also tested the hypothesis that 4) perceptions of ecological engineering would vary among locations, due to environmental and socio-economic differences. In all three harbours, marine scientists and coastal managers were more supportive of ecological engineering than other users. There was also greater support for ecological engineering in Sydney and Melbourne than Hobart. Most people identified transport infrastructure, in busy transport hubs (i.e. Circular Quay in Sydney, the Port in Melbourne and the Waterfront in Hobart) as priorities for ecological engineering, irrespective of their stakeholder group or location. There were, however, significant differences among locations in what people perceive as the key priorities for ecological engineering (i.e. biodiversity in Sydney and Melbourne vs. pollution in Hobart). Greater consideration of these location-specific differences is essential for effective management of artificial structures and rehabilitated habitats in urban embayments.

RESPOND: a programme to prevent secondary falls in older people presenting to the emergency department with a fall: protocol for an economic evaluation.

BACKGROUND: Falls remain common for community-dwelling older people and impose a substantial economic burden to the healthcare system. RESPOND is a novel falls prevention programme that aims to reduce secondary falls and fall injuries among older people who present to a hospital emergency department (ED) with a fall. The present protocol describes a prospective economic evaluation examining the incremental cost-effectiveness of the RESPOND programme, compared with usual care practice, from the Australian health system perspective. METHODS AND DESIGN: This economic evaluation will recruit 528 participants from two major tertiary hospital EDs in Australia and will be undertaken alongside a multisite randomised controlled trial. Outcome and costing data will be collected for all participants over the 12-month trial. It will compare the RESPOND falls prevention programme with usual care practice (current community-based falls prevention practices) to determine its incremental cost-effectiveness according to three intermediate clinical outcomes: (1) falls prevented, (2) fall injuries prevented and (3) injurious falls prevented. In addition, utilities will be derived from a generic quality-of-life measure (EQ-5D-5L) and used to calculate the 'incremental cost per quality-adjusted life years gained'. DISCUSSION: The results of this study will provide healthcare decision makers with evidence to assist with setting spending thresholds for preventive health programmes and inform selection of emergency and community service models of care. TRIAL REGISTRATION NUMBER: The protocol for this study is registered with the Australian New Zealand Clinical Trials Registry (ACTRN12614000336684); Pre-results.

RESPOND: a patient-centred programme to prevent secondary falls in older people presenting to the emergency department with a fall-protocol for a mixed methods programme evaluation.

BACKGROUND: Programme evaluations conducted alongside randomised controlled trials (RCTs) have potential to enhance understanding of trial outcomes. This paper describes a multi-level programme evaluation to be conducted alongside an RCT of a falls prevention programme (RESPOND). OBJECTIVES: (1) To conduct a process evaluation in order to identify the degree of implementation fidelity and associated barriers and facilitators. (2) To evaluate the primary intended impact of the programme: participation in fall prevention strategies and the factors influencing participation. (3) To identify the factors influencing RESPOND RCT outcomes: falls, fall injuries and emergency department (ED) re-presentations. METHODS/DESIGN: 528 community-dwelling adults aged 60-90 years presenting to two EDs with a fall will be recruited and randomly assigned to the intervention or standard care group. All RESPOND participants and RESPOND clinicians will be included in the evaluation. A mixed methods design will be used and a programme logic model will frame the evaluation. Data will be sourced from interviews, focus groups, questionnaires, clinician case notes, recruitment records, participant-completed calendars, hospital administrative datasets and audio-recordings of intervention contacts. Quantitative data will be analysed via descriptive and inferential statistics and qualitative data will be interpreted using thematic analysis. DISCUSSION: The RESPOND programme evaluation will provide information about contextual and influencing factors related to the RESPOND RCT outcomes. The results will assist researchers, clinicians and policy makers regarding decisions about future falls prevention interventions. Insights gained may be applicable to a range of chronic conditions where similar preventive intervention approaches are indicated. TRIAL REGISTRATION NUMBER: This programme evaluation is linked to the RESPOND RCT which is registered with the Australian New Zealand Clinical Trials Registry (ACTRN12614000336684).

mcrA Gene abundance correlates with hydrogenotrophic methane production rates in full-scale anaerobic waste treatment systems.

UNLABELLED: Anaerobic treatment is a sustainable and economical technology for waste stabilization and production of methane as a renewable energy. However, the process is under-utilized due to operational challenges. Organic overload or toxicants can stress the microbial community that performs waste degradation, resulting in system failure. In addition, not all methanogenic microbial communities are equally capable of consistent, maximum biogas production. Opinion varies as to which parameters should be used to monitor the fitness of digester biomass. No standard molecular tools are currently in use to monitor and compare full-scale operations. It was hypothesized that determining the number of gene copies of mcrA, a methanogen-specific gene, would positively correlate with specific methanogenic activity (SMA) rates from biomass samples from six full-scale anaerobic digester systems. Positive correlations were observed between mcrA gene copy numbers and methane production rates against H2  : CO2 and propionate (R(2)  = 0·67-0·70, P < 0·05) but not acetate (R(2)  = 0·49, P > 0·05). Results from this study indicate that mcrA gene targeted qPCR can be used as an alternate tool to monitor and compare certain methanogen communities in anaerobic digesters. SIGNIFICANCE AND IMPACT OF THE STUDY: Using quantitative PCR (qPCR), we demonstrate that the abundance of mcrA, a gene specific to methane producing archaea, correlated with specific methanogenic activity (SMA) measurements when H2 and CO2 , or propionate were provided as substrates. However, mcrA abundance did not correlate with SMA with acetate. SMA values are often used as a fitness indicator of anaerobic biomass. Results from qPCR can be obtained within a day while SMA analysis requires days to weeks to complete. Therefore, qPCR for mcrA abundance is a sensitive and fast method to compare and monitor the fitness of certain anaerobic biomass. As a monitoring tool, qPCR of mcrA will help anaerobic digester operators optimize treatment and encourage more widespread use of this valuable technology.

Subcutaneous concentrations following topical iontophoretic delivery of diclofenac.

A self-contained Wearable Electronic Disposable Drug Delivery (WEDD(®)) patch was used to demonstrate that diclofenac levels delivered by iontophoresis are greater than estimated minimal effective concentrations in local subcutaneous tissue and are also greater than either passive transdermal or intravenous delivery using hairless rats. In vitro iontophoretic delivery was evaluated to optimize donor cell formulation using Franz diffusion cells and 1000 NMWL Millipore ultrafiltration membrane. In vivo animal studies were done using patches powered with a 4-volt system, consisting of a 1-volt Zn anode and Ag/AgCl cathode with built in 3-volt lithium battery. Blood and microdialysis samples were collected at different time points after patch application. Current levels increased to 1.0 mA at 30 min, then fell to a steady state of ~ 0.4 mA. Both WEDD(®) and passive patches produced measurable levels of diclofenac in the subcutaneous tissue below the application site (C(max) ± SE = 113.3 ± 61.7 ng/mL and 36.3 ± 15.9 ng/mL, respectively). The dose delivered in six hours was calculated to be 0.226 ± 0.072 mg and 0.430 ± 0.048 mg in passive and iontophoretic delivery, respectively. Diclofenac was not detected in the subcutaneous tissue after intravenous administration of 1.5 mg/kg diclofenac solution. The trend indicates that WEDD(®) can be used to successfully deliver diclofenac to subcutaneous tissue to concentrations higher when compared to either passive delivery or intravenous dosing of 1.5 mg/kg.

Analysis of cytoskeletal and motility proteins in the sea urchin genome assembly.

The sea urchin embryo is a classical model system for studying the role of the cytoskeleton in such events as fertilization, mitosis, cleavage, cell migration and gastrulation. We have conducted an analysis of gene models derived from the Strongylocentrotus purpuratus genome assembly and have gathered strong evidence for the existence of multiple gene families encoding cytoskeletal proteins and their regulators in sea urchin. While many cytoskeletal genes have been cloned from sea urchin with sequences already existing in public databases, genome analysis reveals a significantly higher degree of diversity within certain gene families. Furthermore, genes are described corresponding to homologs of cytoskeletal proteins not previously documented in sea urchins. To illustrate the varying degree of sequence diversity that exists within cytoskeletal gene families, we conducted an analysis of genes encoding actins, specific actin-binding proteins, myosins, tubulins, kinesins, dyneins, specific microtubule-associated proteins, and intermediate filaments. We conducted ontological analysis of select genes to better understand the relatedness of urchin cytoskeletal genes to those of other deuterostomes. We analyzed developmental expression (EST) data to confirm the existence of select gene models and to understand their differential expression during various stages of early development.

Assessment of state and trait anxiety in subjects with anxiety and depressive disorders.

The State-Trait Anxiety Inventory (STAI) is one of the most widely used scales for the evaluation of anxiety in medical and, to a lesser extent, psychiatric patients. Although there is a relatively large amount of STAI data about anxiety for individuals with a variety of psychiatric disorders, the results of many anxiety studies include only state or trait and many studies have been influenced by comorbidity and by variations in diagnostic criteria used. We studied state and trait anxiety and compared the revised form of the STAI (Form Y) with the original (Form X) to evaluate the anticipated improvement in the measure. In addition, we compared the STAI results with those of another self-report measure (the Symptom Checklist-90 anxiety and depression scales) and also with interviewer-rated measures of anxiety (Hamilton Rating Scale for Anxiety) and depression (Hamilton Rating Scale for Depression). Results indicate that the STAI does not clearly differentiate anxiety disorders from depressive disorders and support the use of multiple tests and of both self-report and interviewer ratings in the evaluation of anxiety and depression in psychiatric patients.

The ability of the Symptom Checklist SCL-90 to differentiate various anxiety and depressive disorders.

We studied the use of the Symptom Checklist-90 (SCL-90) to differentiate between specific anxiety and depressive disorders and/or their symptoms in 280 patients with 6 DSM-III-R diagnoses: major depression (MD), panic disorder (PD), generalized anxiety disorder (GAD), social phobia (SP), obsessive-compulsive disorder (OCD), and mixed anxiety and depression (MAD). Using a comparison group, we found specific patterns for some of the diagnostic categories. Both the MD and MAD subjects had significantly high paranoid ideation, interpersonal sensitivity, hostility, and psychoticism, as well as high depression subscale scores; those with PD and GAD has the highest anxiety and somatization scores; and those with SP or OCD had a mixed pattern. When ranking the severity of psychopathology, the disorders ordered from most to least were MAD, MD, PD, GAD, SP, and OCD. Subsyndromal levels of symptoms frequently were associated with the various conditions. Use of the SCL-90 subscale helps to enlarge our understanding of the various anxiety and depressive disorders.

A kinesin-related protein, KRP(180), positions prometaphase spindle poles during early sea urchin embryonic cell division.

We have investigated the intracellular roles of an Xklp2-related kinesin motor, KRP(180), in positioning spindle poles during early sea urchin embryonic cell division using quantitative, real-time analysis. Immunolocalization reveals that KRP(180) concentrates on microtubules in the central spindle, but is absent from centrosomes. Microinjection of inhibitory antibodies and dominant negative constructs suggest that KRP(180) is not required for the initial separation of spindle poles, but instead functions to transiently position spindle poles specifically during prometaphase.

Analysis of lysine-rich histones from the unicellular green alga Chlamydomonas reinhardtii.

The H1 histones of the unicellular green alga Chlamydomonas reinhardtii were extracted from isolated nuclei, fractionated by high performance liquid chromatography, and analyzed by two-dimensional electrophoresis, peptide mapping, and N-terminal sequencing. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of 5% perchloric acid extracts of isolated C. reinhardtii nuclei revealed two H1 proteins (H1A and H1B). Two-dimensional gel analysis did not reveal heterogeneity of either algal H1 protein, but did detect differences in the hydrophobic amino acid content of the C. reinhardtii H1A and H1B. Digestion of H1A and H1B with V8 protease revealed two distinctly different peptide maps. C. reinhardtii H1 peptide maps were not at all similar to those of Pisum H1, but algal and pea H2B peptide maps did show some peptides in common. Seventeen amino acid residues were obtained from C. reinhardtii H1A amino terminal sequencing, while the H1B N-terminus was blocked. A search of protein data bases revealed no sequence homology of the H1A N-terminus with any known protein. Chlamydomonas histones fractionated by high performance liquid chromatography revealed minor components (histone variants) for H2A and H2B. The amino acid composition of Chlamydomonas lysine-rich histones was compared to those of various other unicellular algae.

Sulfonamidopyrrolidinone factor Xa inhibitors: potency and selectivity enhancements via P-1 and P-4 optimization.

Sulfonamidopyrrolidinones were previously disclosed as a selective class of factor Xa (fXa) inhibitors, culminating in the identification of RPR120844 as a potent member with efficacy in vivo. Recognizing the usefulness of the central pyrrolidinone template for the presentation of ligands to the S-1 and S-4 subsites of fXa, studies to optimize the P-1 and P-4 groups were initiated. Sulfonamidopyrrolidinones containing 4-hydroxy- and 4-aminobenzamidines were discovered to be effective inhibitors of fXa. X-ray crystallographic experiments in trypsin and molecular modeling studies suggest that our inhibitors bind by insertion of the 4-hydroxybenzamidine moiety into the S-1 subsite of the fXa active site. Of the P-4 groups examined, the pyridylthienyl sulfonamides were found to confer excellent potency and selectivity especially in combination with 4-hydroxybenzamidine. Compound 20b (RPR130737) was shown to be a potent fXa inhibitor (K(i) = 2 nM) with selectivity against structurally related serine proteinases (>1000 times). Preliminary biological evaluation demonstrates the effectiveness of this inhibitor in common assays of thrombosis in vitro (e.g. activated partial thromboplastin time) and in vivo (e.g. rat FeCl(2)-induced carotid artery thrombosis model).

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Kinesin- and myosin-driven steps of vesicle recruitment for Ca2+-regulated exocytosis.

Kinesin and myosin have been proposed to transport intracellular organelles and vesicles to the cell periphery in several cell systems. However, there has been little direct observation of the role of these motor proteins in the delivery of vesicles during regulated exocytosis in intact cells. Using a confocal microscope, we triggered local bursts of Ca2+-regulated exocytosis by wounding the cell membrane and visualized the resulting individual exocytotic events in real time. Different temporal phases of the exocytosis burst were distinguished by their sensitivities to reagents targeting different motor proteins. The function blocking antikinesin antibody SUK4 as well as the stalk-tail fragment of kinesin heavy chain specifically inhibited a slow phase, while butanedione monoxime, a myosin ATPase inhibitor, inhibited both the slow and fast phases. The blockage of Ca2+/calmodulin-dependent protein kinase II with autoinhibitory peptide also inhibited the slow and fast phases, consistent with disruption of a myosin-actin- dependent step of vesicle recruitment. Membrane resealing after wounding was also inhibited by these reagents. Our direct observations provide evidence that in intact living cells, kinesin and myosin motors may mediate two sequential transport steps that recruit vesicles to the release sites of Ca2+-regulated exocytosis, although the identity of the responsible myosin isoform is not yet known. They also indicate the existence of three semistable vesicular pools along this regulated membrane trafficking pathway. In addition, our results provide in vivo evidence for the cargo-binding function of the kinesin heavy chain tail domain.

Heterotrimeric kinesin-II is required for the assembly of motile 9+2 ciliary axonemes on sea urchin embryos.

Heterotrimeric kinesin-II is a plus end- directed microtubule (MT) motor protein consisting of distinct heterodimerized motor subunits associated with an accessory subunit. To probe the intracellular transport functions of kinesin-II, we microinjected fertilized sea urchin eggs with an anti-kinesin-II monoclonal antibody, and we observed a dramatic inhibition of ciliogenesis at the blastula stage characterized by the assembly of short, paralyzed, 9+0 ciliary axonemes that lack central pair MTs. Control embryos show no such defect and form swimming blastulae with normal, motile, 9+2 cilia that contain kinesin-II as detected by Western blotting. Injection of anti-kinesin-II into one blastomere of a two-cell embryo leads to the development of chimeric blastulae covered on one side with short, paralyzed cilia, and on the other with normal, beating cilia. We observed a unimodal length distribution of short cilia on anti-kinesin-II-injected embryos corresponding to the first mode of the trimodal distribution of ciliary lengths observed for control embryos. This short mode may represent a default ciliary assembly intermediate. We hypothesize that kinesin-II functions during ciliogenesis to deliver ciliary components that are required for elongation of the assembly intermediate and for formation of stable central pair MTs. Thus, kinesin-II plays a critical role in embryonic development by supporting the maturation of nascent cilia to generate long motile organelles capable of producing the propulsive forces required for swimming and feeding.

Axonal transport of mitochondria along microtubules and F-actin in living vertebrate neurons.

A large body of evidence indicates that microtubules (MTs) conduct organelle transport in axons, but recent studies on extruded squid axoplasm have suggested that actin microfilaments (MFs) may also play a role in this process. To investigate the separate contributions to transport of each class of cytoskeletal element in intact vertebrate axons, we have monitored mitochondrial movements in chick sympathetic neurons experimentally manipulated to eliminate MTs, MFs, or both. First, we grew neurons in the continuous presence of: (a) cytochalasin E to create neurites which had never contained MFs; or (b) nocodazole or vinblastine to produce neurites which had never contained MTs. Mitochondria moved bidirectionally at normal velocities along the length of neurites which contained MTs and lacked MFs, but did not even enter neurites grown without MTs but containing MFs. In a second approach, we treated established neuronal cultures with cytoskeletal drugs to disrupt either MTs or MFs in axons already containing mitochondria. In cytochalasin-treated cells, which retained MTs but lacked MFs, average mitochondrial velocity increased in both directions, but net directional transport decreased. In vinblastine-treated cells, which lacked MTs but retained essentially normal levels of MFs, mitochondria continued to move bidirectionally but the average mitochondrial velocity and excursion length were reduced for both directions of movement, and the mitochondria spent threefold as much time moving in the retrograde as in the anterograde direction, resulting in net retrograde transport. Treatment of established cultures with both drugs produced neurites lacking MTs and MFs but still rich in neurofilaments; these showed a striking absence of any mitochondrial motility. These data indicate that axonal organelle transport can occur along both MTs and MFs in vivo, but with different velocities and net transport properties.

The regulation of bidirectional mitochondrial transport is coordinated with axonal outgrowth.

Although small molecules such as ATP diffuse freely in the cytosol, many types of cells nonetheless position their mitochondria in regions of intense ATP consumption. We reasoned that in the highly elongated axonal processes of growing neurons in culture, the active growth cone would form a focus of ATP consumption so distant from the cell body as to require the positioning of mitochondria nearby via regulated axonal transport. To test this hypothesis, we quantified the distribution and transport behavior of mitochondria in live, aerobically respiring chick sympathetic neurons. We found that in the distal region of actively growing axons, the distribution of mitochondria was highly skewed toward the growth cone, with a sevenfold higher density in the region immediately adjacent to the growth cone than in the region 100 microns away. When axonal outgrowth was blocked by substratum-associated barriers or mild cytochalasin E treatment, the gradient of mitochondrial distribution collapsed as mitochondria exited retrogradely from the distal region, becoming uniformly distributed along the axon within one hour. Analysis of individual mitochondrial behaviors revealed that mitochondrial movement everywhere was bidirectional but balanced so that net transport was anterograde in growing axons and retrograde in blocked axons. This reversal in net transport derived from two separate modulations of mitochondrial movement. First, moving mitochondria underwent a transition to a persistently stationary state in the region of active growth cones that was reversed when growth cone activity was halted. Second, the fraction of time that mitochondria spent moving anterogradely was sharply reduced in non-growing axons. Together, these could account for the formation of gradients of mitochondria in growing axons and their dissipation when outgrowth was blocked. This regulated transport behavior was not dependent upon the ability of mitochondria to produce ATP. Our data indicate that mitochondria possess distinct motor activities for both directions of movement and that mitochondrial transport in axons is regulated by both recruitment between stationary and moving states, and direct regulation of the anterograde motor.