Differential proteomic responses of selectively bred and wild-type Sydney rock oyster populations exposed to elevated CO2.

Previous work suggests that larvae from Sydney rock oysters that have been selectively bred for fast growth and disease resistance are more resilient to the impacts of ocean acidification than nonselected, wild-type oysters. In this study, we used proteomics to investigate the molecular differences between oyster populations in adult Sydney rock oysters and to identify whether these form the basis for observations seen in larvae. Adult oysters from a selective breeding line (B2) and nonselected wild types (WT) were exposed for 4 weeks to elevated pCO2 (856 µatm) before their proteomes were compared to those of oysters held under ambient conditions (375 µatm pCO2 ). Exposure to elevated pCO2 resulted in substantial changes in the proteomes of oysters from both the selectively bred and wild-type populations. When biological functions were assigned, these differential proteins fell into five broad, potentially interrelated categories of subcellular functions, in both oyster populations. These functional categories were energy production, cellular stress responses, the cytoskeleton, protein synthesis and cell signalling. In the wild-type population, proteins were predominantly upregulated. However, unexpectedly, these cellular systems were downregulated in the selectively bred oyster population, indicating cellular dysfunction. We argue that this reflects a trade-off, whereby an adaptive capacity for enhanced mitochondrial energy production in the selectively bred population may help to protect larvae from the effects of elevated CO2 , whilst being deleterious to adult oysters.

Reintroducing a keystone bioturbator can facilitate microbial bioremediation in urban polluted sediments.

Anthropogenic environmental stressors have significantly reduced biodiversity and the capacity of remnant natural habitats to deliver ecosystem functions and services in urban areas. To mitigate these impacts and recover biodiversity and function, ecological restoration strategies are needed. While habitat restoration is proliferating in rural and peri-urban areas, strategies purposely designed to succeed under the environmental, social and political pressures of urban areas are lacking. Here, we propose that ecosystem health in marine urban areas can be improved by restoring biodiversity to the most dominant habitat, unvegetated sediments. We reintroduced a native ecosystem engineer, the sediment bioturbating worm Diopatra aciculata, and assessed their effects on microbial biodiversity and function. Results showed that worms can affect the diversity of microbes, but effects varied between locations. Worms caused shifts in microbial community composition and function at all locations. Specifically, the abundance of microbes capable of chlorophyll production (i.e. benthic microalgae) increased and the abundance of microbes capable of methane production decreased. Moreover, worms increased the abundances of microbes capable of denitrification in the site with lowest sediment oxygenation. Worms also affected microbes capable of degrading the polycyclic aromatic hydrocarbon toluene, although the direction of that effect was site-specific. This study provides evidence that a simple intervention such as the reintroduction of a single species can enhance sediment functions important for the amelioration of contamination and eutrophication, although further studies are needed to understand the variation in outcomes between sites. Nevertheless, restoration strategies targeting unvegetated sediments provide an opportunity to combat anthropogenic stressors in urban ecosystems and may be used for precondition before more traditional forms of habitat restoration such as seagrass, mangrove and shellfish restoration.

Exposure to 17α-ethynylestradiol causes dose and temporally dependent changes in intersex, females and vitellogenin production in the Sydney rock oyster.

Although mounting evidence suggests exposure to estrogenic contaminants increases vitellogenin production in molluscs, demonstration of dose-response relationships and knowledge of the temporal nature of the vitellogenin response with continual exposure is currently lacking for biomarker utility. To address this knowledge gap, adult Sydney rock oysters, Saccostrea glomerata, were exposed to a range of environmentally relevant concentrations of 17α-ethynylestradiol (EE2) (0, 6.25, 12.5, 25 or 50 ng/l) in seawater under laboratory conditions. Vitellogenin induction and gonadal development was assessed following 4, 21 and 49 days exposure to EE2. Vitellogenin was found to increase in a dose dependent manner with EE2 exposure for females (4 and 49 days) and males (4 and 21 days). Histological examination of gonads revealed a number of individuals exhibited intersex (ovotestis) in 50 ng/l EE2 (after 21 days) and in 6.25 and 12.5 ng/l EE2 (after 49 days). Furthermore, a significant shift towards females was observed following 49 days exposure at 50 ng/l EE2 suggesting estrogenic exposure is capable of facilitating a progression for protandric males from male-intersex-female gametal status. Increases in female vitellogenin (4 days) were predictive of later increases in female developmental stages at 21 days and increases in oocyte area following 49 days. Male vitellogenin (4 days) was predictive of decreased male percentages and lower male developmental stages at 49 days. Vitellogenin in S. glomerata is a predictive biomarker of estrogenic exposure and effect if sampled soon after exposure and at the commencement of a gonadal development cycle.

Protein markers of Marteilia sydneyi infection in Sydney rock oysters, Saccostrea glomerata.

Marteilia sydneyi is the causative agent of QX disease in Sydney rock oyster, Saccostrea glomerata. It is responsible for disease outbreaks among oysters that occur during summer and can result in up to 95% mortality. QX disease has significantly decreased S. glomerata production in some areas of Australia's eastern seaboard over the past 30 years. Marteilia sydneyi sporulates in the digestive gland of oysters leading to complete disorganization of the infected tissues. The current study used proteomics to identify potential molecular markers of sporulating M. sydneyi infection during a field trial undertaken in the Georges River, Sydney, between December 2006 and May 2007. Early stages of M. sydneyi infection were detected by polymerase chain reaction, whilst cytological examination was used to identify sporulating M. sydneyi in the gut. Protein expression in oyster haemolymph was assessed during the M. sydneyi infection period by two dimensional electrophoresis. Proteome maps identified significant differences in the expression of four proteins in oysters with sporulating M. sydneyi infections.

Bayesian estimation of synaptic physiology from the spectral responses of neural masses.

We describe a Bayesian inference scheme for quantifying the active physiology of neuronal ensembles using local field recordings of synaptic potentials. This entails the inversion of a generative neural mass model of steady-state spectral activity. The inversion uses Expectation Maximization (EM) to furnish the posterior probability of key synaptic parameters and the marginal likelihood of the model itself. The neural mass model embeds prior knowledge pertaining to both the anatomical [synaptic] circuitry and plausible trajectories of neuronal dynamics. This model comprises a population of excitatory pyramidal cells, under local interneuron inhibition and driving excitation from layer IV stellate cells. Under quasi-stationary assumptions, the model can predict the spectral profile of local field potentials (LFP). This means model parameters can be optimised given real electrophysiological observations. The validity of inferences about synaptic parameters is demonstrated using simulated data and experimental recordings from the medial prefrontal cortex of control and isolation-reared Wistar rats. Specifically, we examined the maximum a posteriori estimates of parameters describing synaptic function in the two groups and tested predictions derived from concomitant microdialysis measures. The modelling of the LFP recordings revealed (i) a sensitization of post-synaptic excitatory responses, particularly marked in pyramidal cells, in the medial prefrontal cortex of socially isolated rats and (ii) increased neuronal adaptation. These inferences were consistent with predictions derived from experimental microdialysis measures of extracellular glutamate levels.

Effects of 4-nonylphenol and 17alpha-ethynylestradiol exposure in the Sydney rock oyster, Saccostrea glomerata: Vitellogenin induction and gonadal development.

Adult Saccostrea glomerata were exposed to environmentally relevant concentrations of 4-nonylphenol (1microg/L and 100microg/L) and 17alpha-ethynylestradiol (5ng/L and 50ng/L) in seawater over 8 weeks. Exposures were performed to assess effects on vitellogenin induction and gonadal development during reproductive conditioning. Chronic direct estrogenicity within gonadal tissue was assessed via an estrogen receptor-mediated, chemical-activated luciferase reporter gene-expression assay (ER-CALUX). Estradiol equivalents (EEQ) were greatest in the 100microg/L 4-nonylphenol exposure (28.7+/-2.3ng/g tissue EEQ) while 17alpha-ethynylestradiol at concentrations of 50ng/L were 2.2+/-1.5ng/g tissue EEQ. Results suggest 4-nonylphenol may be accumulated in tissue and is partly resistant to biotransformation; maintaining its potential for chronic estrogenic action, while 17alpha-ethynylestradiol, although exhibiting greater estrogenic potency on biological endpoints possibly exerts its estrogenic action before being rapidly metabolised and/or excreted. A novel methodology was developed to assess vitellogenin using high-performance liquid chromatography (HPLC). Exposure to both 17alpha-ethynylestradiol (50ng/L) and 4-nonylphenol (100microg/L) produced increases in vitellogenin for females, whereas males exhibited increases in vitellogenin when exposed to 50ng/L 17alpha-ethynylestradiol only. Females exhibited greater vitellogenin responses than males at 50ng/L 17alpha-ethynylestradiol only. Histological examination of gonads revealed a number of individuals exhibiting intersex (ovotestis) in 50ng/L 17alpha-ethynylestradiol exposures. Male individuals in 1microg/L and 100microg/L 4-nonylphenol exposures and 5ng/L 17alpha-ethynylestradiol were at earlier stages of spermatogenic development than corresponding controls.

Validity and reliability of the Moxy oxygen monitor during incremental cycling exercise.

INTRODUCTION: The Moxy is a novel, cutaneously placed muscle oxygen monitor which claims to measure local oxygen saturation (SmO2) and total haemoglobin (THb) using near-infrared spectroscopy. If shown to be reliable, its data storage and telemetric capability will be useful for assessing localised O2 usage during field-based exercise. This study investigated the reliability of the Moxy during cycling and assessed the correlations between its measurements, whole-body O2 consumption (VO2) and heart rate (HR). METHODS: Ten highly trained cyclists performed an incremental, step-wise cycling protocol on two occasions while wearing the Moxy. SmO2, THb, VO2 and HR were recorded in the final minute of each five-minute stage. Data were analysed using Spearman's Order-Rank Coefficient (SROC), Intraclass Correlation (ICC), and Coefficient of Variance (COV). Significance was set at p ≤ .05. RESULTS: SmO2 showed a 'strong' or 'very large' correlation between trials (SROC: r = 0.842-0.993, ICC: r = 0.773-0.992, p ≤ .01) and was moderately correlated with VO2 and HR (r = -0.71-0.73, p ≤ .01). SmO2 showed a moderate to high reliability at low intensities, but this decreased as relative exercise intensity increased. THb showed poor correlations between tests and with the other measured variables, but was highly reliable at all power outputs. CONCLUSIONS: The Moxy is a reliable device to measure SmO2 at low to moderate intensities, but at higher intensities, greater variation in measurements occurs, likely due to tissue ischaemia or increased movement artefacts due to more frequent muscular contractions. THb has low variation during exercise, and does not appear to be a valid indicator of muscle oxygenation.

The Akoya pearl oyster shell as an archival monitor of lead exposure.

The Akoya pearl oyster (Pinctada imbricata) was experimentally exposed to (a) constant levels of lead (Pb) at 180 microg L(-1) for nine weeks, or (b) two short term (pulse) exposures of Pb at 180 microg L(-1) (three weeks each) with an intervening depuration period (three weeks), to assess its utility as an (i) accumulative monitor of Pb contamination and an (ii) archival monitor for discriminating constant versus pulsed Pb exposure events. P. imbricata showed similar reductions in growth (based on shell morphology and wet weight) and Pb accumulation patterns for whole tissue and shell in response to both Pb exposure regimes. Thus the whole oyster was deemed an inappropriate accumulative monitor for assessing short-term temporal variation of Pb exposure and effect. However, using secondary ion mass spectrometry, Pb was shown to accumulate in the successively deposited nacreous layers of the shell of P. imbricata, documenting the exposure history of constant versus pulsed Pb events. Patterns of Pb deposition not only reflected the frequency of Pb exposure events but also their relative durations. Thus, the shell of P. imbricata may be employed as a suitable biological archive of Pb exposure.

Monoclonal antibody to vascular endothelial-cadherin is a potent inhibitor of angiogenesis, tumor growth, and metastasis.

Vascular endothelial-cadherin (VE-cad) is an endothelial cell-specific adhesion molecule that is crucial for proper assembly of vascular tubes. Here we show that a monoclonal antibody (BV13) directed to the extracellular region of VE-cad inhibits formation of adherens junctions and capillary-like structures by endothelial cells and blocks angiogenesis in the mouse cornea and in Matrigel plugs in vivo. Systemic administration of BV13 markedly decreases the growth of s.c. Lewis lung or human A431 epidermoid tumors and strongly suppresses the growth of Lewis lung metastases. These data demonstrate that VE-cad is essential for postnatal angiogenesis and thus validate VE-cad as a novel target for antiangiogenesis agents.

Antivascular endothelial growth factor receptor (fetal liver kinase 1) monoclonal antibody inhibits tumor angiogenesis and growth of several mouse and human tumors.

Tumor angiogenesis is mediated by tumor-secreted angiogenic growth factors that interact with their surface receptors expressed on endothelial cells. Vascular endothelial growth factor (VEGF) and its receptor [fetal liver kinase 1 (Flk-1)/kinase insert domain-containing receptor] play an important role in vascular permeability and tumor angiogenesis. Previously, we reported on the development of anti-Flk-1 and antikinase insert domain-containing receptor monoclonal antibodies (mAbs) that potently inhibit VEGF binding and receptor signaling. Here, we report the effect of anti-Flk-1 mAb (DC101) on angiogenesis and tumor growth. Angiogenesis in vivo was examined using a growth factor supplemented (basic fibroblast growth factor + VEGF) Matrigel plug and an alginate-encapsulated tumor cell (Lewis lung) assay in C57BL/6 mice. Systemic administration of DC101 every 3 days markedly reduced neovascularization of Matrigel plugs and tumor-containing alginate beads in a dose-dependent fashion. Histological analysis of Matrigel plugs showed reduced numbers of endothelial cells and vessel structures. Several mouse tumors and human tumor xenografts in athymic mice were used to examine the effect of anti-Flk-1 mAb treatment on tumor angiogenesis and growth. Anti-Flk-1 mAb treatment significantly suppressed the growth of primary murine Lewis lung, 4T1 mammary, and B16 melanoma tumors and growth of Lewis lung metastases. DC101 also completely inhibited the growth of established epidermoid, glioblastoma, pancreatic, and renal human tumor xenografts. Histological examination of anti-Flk-1 mAb-treated tumors showed evidence of decreased microvessel density, tumor cell apoptosis, decreased tumor cell proliferation, and extensive tumor necrosis. These findings support the conclusion that anti-Flk-1 mAb treatment inhibits tumor growth by suppression of tumor-induced neovascularization and demonstrate the potential for therapeutic application of anti-VEGF receptor antibody in the treatment of angiogenesis-dependent tumors.

Ventriculocoronary connections in hypoplastic right heart syndrome: autopsy serial section study of six cases.

Myocardial sinusoids communicating with the coronary systems occur in pulmonary atresia with intact ventricular septum. To test the hypothesis that the extent of ventriculocoronary connections correlates with the degree of right ventricular outflow obstruction as evidenced by clinical, angiographic and gross anatomic findings, a serial section study of six human autopsy hearts representing a spectrum of hypoplastic right heart was undertaken. Slides were evaluated for the presence and extent of ventriculocoronary connections, associated developmental abnormalities and secondary changes in the ventricular walls. Whereas extensive blind-ended deep sinusoids were a feature of all five cases with unrelieved obstruction, ventriculocoronary connections were identified in three. Changes that suggested ongoing remodeling provide new evidence for the postnatal temporal evolution of these anomalous communications. The regional distribution of myofiber disarray in hypoplastic right heart supports the concept that vascularization parallels myocardial organization in the developing human heart.

Anomalous left coronary artery from the pulmonary artery: significance of associated intracardiac defects.

Two patients with anomalous origin of the left main coronary artery from the pulmonary artery had an associated defect (one, critical pulmonary stenosis; the other, ventricular septal defect). They presented with signs and symptoms of the associated defect and the coronary anomaly was unrecognized. Both cases at autopsy lacked the usual large right coronary artery seen with this anomaly. The pathophysiologic features of the combined defects are described, their differences from the isolated anomaly are noted and their relation to surgery is discussed.

Evidence for a selective prefrontal cortical GABA(B) receptor-mediated inhibition of glutamate release in the ventral tegmental area: a dual probe microdialysis study in the awake rat.

Glutamate-containing pyramidal neurons in the medial prefrontal cortex (mPfc) project to the ventral tegmental area (VTA) where they synapse on mesocorticolimbic dopamine containing cell bodies and GABA interneurons. In the present study we employed dual probe microdialysis in intact conscious rat brain to investigate the effects of intra-mPfc perfusion with a depolarising concentration of potassium chloride (KCl) (100 mM, 20 min) alone and in the presence of local GABA(A) and GABA(B) receptor blockade on VTA glutamate release. Intra-mPfc KCl transiently increased VTA glutamate release (+71.48+/-14.29%, 20 min). Intra-mPfc perfusion with a concentration of the GABA(A) receptor antagonist bicuculline (10 microM, 120 min) did not influence the intra-mPfc KCl-induced increase in VTA glutamate release (+102.35+/-33.61%, 20 min). In contrast, intra-mPfc perfusion with a concentration of the GABA(B) receptor antagonist CGP35348 (100 microM, 120 min) which when given alone did not influence basal glutamate levels in the VTA was associated with an enhanced KCl-induced stimulation of VTA glutamate release (+375.19+/-89.69%, 40 min). Furthermore, this enhancement was reversed in the presence of the selective GABA(B) receptor agonist baclofen (10 microM, 120 min). The present findings suggest a key role for the prefrontal cortex in the regulation of glutamate release in the VTA. Furthermore, we demonstrate a selective cortical GABA(B) receptor-mediated inhibition of glutamate transmission in the VTA. These findings may be important in the context of abnormalities in amino acid neurotransmission at the network level in schizophrenia.

Quantification of in situ nutrient and heavy metal remediation by a small pearl oyster (Pinctada imbricata) farm at Port Stephens, Australia.

The use of pearl oysters has recently been proposed as an environmental remediation tool in coastal ecosystems. This study quantified the nitrogen, phosphorus and heavy metal content of the tissue and shell of pearl oysters harvested from a small pearl oyster farm at Port Stephens, Australia. Each tonne of pearl oyster material harvested resulted in approximately 703 g metals, 7452 g nitrogen, and 545 g phosphorus being removed from the waters of Port Stephens. Increasing current farm production of 9.8 tyr(-1) to 499 tyr(-1) would balance current nitrogen loads entering Port Stephens from a small Sewage Treatment Plant (STP) located on its southern shores. Furthermore, manipulation of harvest dates to coincide with oyster condition would likely remove substantially greater quantities of nutrients. This study demonstrates that pearl aquaculture may be used to assist in the removal of pollutants from coastal waters while producing a commercially profitable commodity.

Sodium excretion in normal conscious dogs.

Bitches maintained on a low Na intake, were given doses of saline (0.125 mol.litre(-1) NaCl, 0.025 mol.litre(-1) NaHCO3, 0.004 mol.litre(-1) KCl) by stomach tube. Doses of 100 and 200 cm3 produced only minor increases in Na excretion; after 300 cm3, Na excretion rose from about 2 to about 60 micromol.min(-1). Plasma protein fell by 1.8 litre(-1) for each 100 cm3 of saline retained. Within normal ranges of Na excretion there is a threshold of plasma protein concentration above which Na is retained and below which Na is excreted. Changes in exogenous creatinine clearance were measured allowing calculation of the filtered load of Na, which shows that the absolute tubular reabsorption of Na and water is increased in volume expansion by isotonic saline. Meat produced large increase in glomerular filtration rate without much increase in Na excretion and mechanisms are discussed by which Na reabsorptin is more effective after meat than after doses of saline. Creatinine cleaerance increased by 0.67 cm3.min(-1) for each fall of 1 g.litre(-1) in plasma protein; this is predicted by a theory that the glomerular capillary blood pressure is 9.3 kPa (70 mmHg) rather than 6.7 kPa (50 mmHg).

Neurocircuitry of the basal ganglia studied by monitoring neurotransmitter release. Effects of intracerebral and perinatal asphyctic lesions.

The neurocircuitries of the basal ganglia are studied with in vivo microdialysis, with special consideration to dopamine transmission and its interaction with other neurotransmitter systems. The aim is to develop experimental models to study the pathophysiology and therapy of neurodegenerative disorders of the basal ganglia, as well as to develop models to study the short- and long-term consequences of perinatal asphyctic lesions. A main goal of these studies is to find and to characterize new treatments for these disorders.

Human ecology and mental illness.

Psychiatrists and others in the mental health field have long been aware that forces within the surrounding social network effect both favorably and unfavorably the individual psyche and the course of mental illness, but the systematic use of such factors in therapy and prevention has awaited a model. The authors identify a beginning yet workable approach to what an APA task force termed "ecopsychiatry"; the model proposed is not that of traditional linear or cause-and-effect perspectives but is based on a systemic model derived originally form biological ecology.

Spinal cord stimulation attenuates augmented dorsal horn release of excitatory amino acids in mononeuropathy via a GABAergic mechanism.

Neuropathic pain may be effectively relieved by electric stimulation of the spinal cord (SCS). However, the underlying mechanisms for the ensuing pain relief are poorly understood. In a rat model of neuropathy displaying hypersensitivity to innocuous tactile stimuli, (allodynia), we have earlier demonstrated that SCS may normalise withdrawal response thresholds. In the present study, using microdialysis, it is shown that SCS induces a decreased release of the dorsal horn excitatory amino acids (EAA), glutamate and aspartate, concomitant with an increase of the GABA release. Local perfusion with a GABA(B)-receptor antagonist in the dorsal horn transiently abolishes the SCS-induced suppression of the EAA release. Thus, the effect of SCS on neuropathic pain and allodynia may be due to an activation of local GABAergic mechanisms inhibiting the EAA release which is chronically elevated in such conditions.

The vigilance promoting drug modafinil increases extracellular glutamate levels in the medial preoptic area and the posterior hypothalamus of the conscious rat: prevention by local GABAA receptor blockade.

The effects of modafinil on glutamatergic and GABAergic transmission in the rat medial preoptic area (MPA) and posterior hypothalamus (PH), are analysed. Modafinil (30-300 mg/kg) increased glutamate and decreased GABA levels in the MPA and PH. Local perfusion with the GABAA agonist muscimol (10 microM), reduced, while the GABAA antagonist bicuculline (1 microM and 10 microM) increased glutamate levels. The modafinil (100 mg/kg)-induced increase of glutamate levels was antagonized by local perfusion with bicuculline (1 microM). When glutamate levels were increased by the local perfusion with the glutamate uptake inhibitor L-trans-PDC (0.5 mM), modafinil produced an additional enhancement of glutamate levels. Modafinil (1-33 microM) failed to affect [3H]glutamate uptake in hypothalamic synaptosomes and slices. These findings show that modafinil increases glutamate and decreases GABA levels in MPA and PH. The evidence that bicuculline counteracts the modafinil-induced increase of glutamate levels strengthens the evidence for an inhibitory GABA/glutamate interaction in the above regions controlling the sleep-wakefulness cycle.

Effect of chronic administration of the 6-aza analogue of mianserin (Org. 3770) and its enantiomers on behaviour and changes in noradrenaline metabolism of olfactory-bulbectomized rats in the "open field" apparatus.

Following its chronic administration, the racemic mianserin analogue of Org. 3770 attenuated the hypermotility of bilaterally olfactory-bulbectomized rats in the "open-field" apparatus, over the dose range of 0.5-2 mg/kg; only the largest dose of the drug was found to reduce the motility of the sham-operated animals. The activity of this compound would appear to reside in its S(+)isomer; the R(-)isomer being inactive. Bulbectomy was associated with a slight decrease in the concentration of noradrenaline and its major metabolite MHPG in the amygdaloid cortex and mid-brain. Following chronic administration of Org. 3770, the concentration of noradrenaline and MHPG returned to control levels. When the enantiomers were tested, it was found that the behaviourally-inactive R(-)isomer was most effective in normalizing the deficit in this neurotransmitter. Thus, no correlation could be found between the behavioural activity of the enantiomers of Org. 3770 and changes in the metabolism of noradrenaline in the amygdaloid cortex and mid-brain.