Search

Background: Huntington’s disease (HD) is a fatal neurodegenerative autosomal dominant disorder with prevalence of 1 : 20000 that has no effective treatment to date. Translatability of candidate therapeutics could be enhanced by additional testing in large animal models because of similarities in brain anatomy, size, and immunophysiology. These features enable realistic pre-clinical studies of biodistribution, efficacy, and toxicity. Objective and Methods: Here we non-invasively characterized alterations in brain white matter microstructure, neurochemistry, neurological status, and mutant Huntingtin protein (mHTT) levels in cerebrospinal fluid (CSF) of aged OVT73 HD sheep. Results: Similar to HD patients, CSF mHTT differentiates HD from normal sheep. Our results are indicative of a decline in neurological status, and alterations in brain white matter diffusion and spectroscopy metric that are more severe in aged female HD sheep. Longitudinal analysis of aged female HD sheep suggests that the decline is detectable over the course of a year. In line with reports of HD human studies, white matter alterations in corpus callosum correlates with a decline in gait of HD sheep. Moreover, alterations in the occipital cortex white matter correlates with a decline in clinical rating score. In addition, the marker of energy metabolism in striatum of aged HD sheep, shows a correlation with decline of clinical rating score and eye coordination. Conclusion: This data suggests that OVT73 HD sheep can serve as a pre-manifest large animal model of HD providing a platform for pre-clinical testing of HD therapeutics and non-invasive tracking of the efficacy of the therapy.

Gene therapy continues to be a promising contender for the treatment of cystic fibrosis (CF) airway disease. We have previously demonstrated that airway conditioning with lysophosphatidylcholine (L...

Gene therapy continues to be a promising contender for the treatment of cystic fibrosis (CF) airway disease. We have previously demonstrated that airway conditioning with lysophosphatidylcholine (LPC) followed by delivery of a HIV-1-based lentiviral (LV) vector functionally corrects the CF transmembrane conductance regulator (CFTR) defect in the nasal airways of CF mice. In our earlier pilot study we showed that our technique can transduce marmoset lungs acutely; this study extends that work to examine gene expression in this nonhuman primate (NHP) 1 month after gene vector treatment. A mixture of three separate HIV-1 vesicular stomatitis virus G (VSV-G)-pseudotyped LV vectors containing the

Preclinical imaging studies of fetal hemodynamics require anesthesia to immobilize the animal. This may induce cardiovascular depression and confound measures under investigation. We compared the impact of four anesthetic regimes upon maternal and fetal blood gas and hemodynamics during baseline periods of normoxia, and in response to an acute hypoxic challenge in pregnant sheep. Merino ewes were surgically prepared with maternal and fetal vascular catheters and a fetal femoral artery flow probe at 105–109 days gestation. At 110–120 days gestation, ewes were anesthetized with either isoflurane (1.6%), isoflurane (0.8%) plus ketamine (3.6 mg·kg−1·h−1), ketamine (12.6 mg·kg−1·h−1) plus midazolam (0.78 mg·kg−1·h−1), propofol (30 mg·kg−1·h−1), or remained conscious. Following 60 min of baseline recording, nitrogen was administered directly into the maternal trachea to displace oxygen and induce maternal and thus fetal hypoxemia. During normoxia, maternal PaO2 was ~30 mmHg lower in anesthetized ewes compared to conscious controls, regardless of the type of anesthesia (p .05), but heart rate was 32 ± 8 bpm lower in fetuses from ewes administered isoflurane (p = .044). During maternal hypoxia, fetal MAP increased, and peripheral blood flow decreased in all fetuses except those administered propofol (p, Preclinical imaging studies of fetal haemodynamics require anaesthesia to immobilise the animal. In the presence of isoflurane, isoflurane/ketamine and ketamine/midazolam, but not propofol anaesthesia, maternal and fetal hypoxaemia increased fetal mean arterial pressure and decreased peripheral blood flow. Acute fetal hypoxaemia induced fetal tachycardia regardless of anaesthetic type. These results have implications for the design and interpretation of preclinical imaging studies where anaesthesia is required.

Pre-existing neutralizing antibody (NAb) against adeno-associated virus (AAV) commonly found in primates is a major host barrier that can severely compromise in vivo gene transfer by AAV vectors. To achieve proof-of-concept success in clinical development of recombinant AAV (rAAV)-based in vivo gene therapy, it is crucial to consider the potential interference of NAb and to enroll serologically compatible study subjects. In this study, we report a large AAV NAb dataset comprising multiple large animal species and AAV serotypes and compare two NAb assays based on in vitro or in vivo transduction inhibition, respectively. Together with previously published AAV seroepidemiology studies, these data can serve as a reference for selecting suitable serotypes, study subjects of large animal species, and potentially human patients for rAAV treatment. In addition, we modeled the intrathalamus rAAV9 delivery in the presence of circulating anti-AAV9 NAb generated by either pre-immunization or passive transfer of NAb-positive large animal serum to mice. The data showed that circulating NAb may not be the sole determinant to inhibit brain transduction. Other aspects of pre-existing AAV immunity following natural infection or rAAV administration may be further studied to establish a more accurate inclusion criterion for clinical studies employing intraparenchymal rAAV9 injections. Keywords: adeno-associated virus, CNS gene transfer, gene therapy, large animals, neutralizing antibody

The primary metabolic pathway required to produce ATP differs as a result of tissue type, developmental stage and substrate availability.We utilized molecular and histological techniques to define the metabolic status in foetal and adult, adipose and skeletal muscle tissues. Redox ratios of these tissues were also determined optically by two-photon microscopy.Adult perirenal adipose tissue had a higher optical redox ratio than fetal perirenal adipose tissue, which aligned with glycolysis being used for ATP production;whereas adult skeletal muscle had a lower optical redox ratio than fetal skeletal muscle, which aligned with oxygen demanding oxidative phosphorylation activity being utilized for ATP production. We have compared traditional molecular and microscopy techniques of metabolic tissue characterization with optical redox ratios to provide a more comprehensive report on the dynamics of tissue metabolism. Refereed/Peer-reviewed

Sepsis remains a significant health burden and a major clinical need exists for therapeutics to dampen the excessive and uncontrolled immune activation. Nuclear protein high mobility group box protein 1 (HMGB1) is released following cell death and is a late mediator in sepsis pathogenesis. While approaches targeting HMGB1 have demonstrated reduced mortality in pre-clinical models of sepsis, the impact of HMGB1 blockade on the complex septic inflammatory milieu and the development of subsequent immunosuppression remain enigmatic. Analysis of plasma samples obtained from septic shock patients established an association between increased HMGB1 and non-survival, higher APACHE II scores, and increased pro-inflammatory cytokine responses. Pre-clinically, administration of neutralising ovine anti-HMGB1 polyclonal antibodies improved survival in murine endotoxaemia and caecal ligation and puncture-induced sepsis models, and altered early cytokine profiles to one which corresponded to patterns observed in the surviving patient cohort. Additionally, anti-HMGB1 treated murine sepsis survivors were significantly more resistant to secondary bacterial infection and exhibited altered innate immune cell phenotypes and cytokine responses. These findings demonstrate that anti-HMGB1 antibodies alter inflammation in murine sepsis models and reduce sepsis mortality without potentiating immunosuppression.

This study investigates the effect of contaminant zinc (Zn) on lead (Pb) bioavailability and bioaccessibility in six contrasting soils spiked with 1500 mg Pb/kg and aged 12 months under laboratory conditions. Zn was added to the soils (7500 mgZn/kg soil) and aged for a further two weeks. In vivo studies were conducted using juvenile swine as a surrogate model for young children. Two compartment pharmacokinetic models were used to analyze the biological response produced by Pb oral solution and spiked soils. Absolute and relative bioavailability of Pb in soils (oral dose of 100 μ g Pb/kg body weight/day) were estimated by comparing them with intravenously administered soluble Pb salt (25 μ g Pb/kg/day) and orally administered the same Pb salt [Pb acetate =(CH3COO)2Pb⋅3H2O] administered to 3 juvenile pigs per treatment. Lead bioaccessibility was calculated using the in vitro RBALP (i.e. relative bioaccessibility leaching procedure) method. The in vitro results of RBALP were compared to in vivo relative Pb bioavailability to ascertain whether the changes in bioaccessibility correlated with the in vivo data. Although the in vivo Pb relative bioavailability (RB) in all soils except in MLA (Mount Lofty Acidic) revealed an increase (18%–159%) in the presence of Zn, the in vitro RBALP bioaccessibility results indicated otherwise (1%–38% decrease). In vivo RB of Pb in MLA declined by 37% in the presence of Zn. However, the RBALP in vitro bioaccessible Pb did not correlate with the relative bioavailabilities of Pb in the juvenile swine dosing experiment. Caution is therefore needed when predicting Pb bioavailability/bioaccessibility in the presence of metal mixtures. The literature contains much information on the correlation of metal and metalloid bioaccessibility with their bioavailability. There is, however, a paucity of studies investigating the effects of other metals on Pb and their IVIVC (in vitro and in vivo correlations). The current study addresses this knowledge gap by assessing in vivoand in vitro bioavailability of Pb in the presence of Zn.

Sepsis is a highly complex and often fatal syndrome which varies widely in its clinical manifestations, and therapies that target the underlying uncontrolled immune status in sepsis are needed. The failure of preclinical approaches to provide significant sepsis survival benefit in the clinic is often attributed to inappropriate animal disease models. It has been demonstrated that high mobility group box protein 1 (HMGB1) blockade can reduce inflammation, mortality and morbidity in experimental sepsis without promoting immunosuppression. Within this study, we explored the use of ovine anti-HMGB1 antibodies in a model of ovine septic shock incorporating intensive care supports (OSSICS). Results: Septic sheep exhibited elevated levels of HMGB1 within 12 h after the induction of sepsis. In this study, sepsis was induced in six anaesthetized adult Border Leicester × Merino ewes via intravenous instillation of E. coli and sheep monitored according to intensive care unit standard protocols for 26 h, with the requirement for noradrenaline as the primary endpoint. Septic sheep exhibited a hyperdynamic circulation, renal dysfunction, deranged coagulation profile and severe metabolic acidosis. Sheep were assigned a severity of illness score, which increased over time. While a therapeutic effect of intravenous anti-HMGB1 antibody could not be observed in this model due to limited animal numbers, a reduced bacterial dose induced a septic syndrome of much lower severity. With modifications including a reduced bacterial dose, a longer timeframe and broad spectrum antibiotics, the OSSICS model may become a robust tool for preclinical assessment of sepsis therapeutics.

Global gene delivery to the CNS has therapeutic importance for the treatment of neurological disorders that affect the entire CNS. Due to direct contact with the CNS, cerebrospinal fluid (CSF) is an attractive route for CNS gene delivery. A safe and effective route to achieve global gene distribution in the CNS is needed, and administration of genes through the cisterna magna (CM) via a suboccipital puncture results in broad distribution in the brain and spinal cord. However, translation of this technique to clinical practice is challenging due to the risk of serious and potentially fatal complications in patients. Herein, we report development of a gene therapy delivery method to the CM through adaptation of an intravascular microcatheter, which can be safely navigated intrathecally under fluoroscopic guidance. We examined the safety, reproducibility, and distribution/transduction of this method in sheep using a self-complementary adeno-associated virus 9 (scAAV9)-GFP vector. This technique was used to treat two Tay-Sachs disease patients (30 months old and 7 months old) with AAV gene therapy. No adverse effects were observed during infusion or post-treatment. This delivery technique is a safe and minimally invasive alternative to direct infusion into the CM, achieving broad distribution of AAV gene transfer to the CNS.

Background: There are many uncertainties concerning variations in benzo[a]pyrene (B[a]P) soil guidelines protecting human health based on carcinogenic data obtained in animal studies. Although swine is recognised as being much more representative of the human child in terms of body size, gut physiology and genetic profile the rat/mice model is commonly used in practice. Objectives: We compare B[a]P bioavailability using a rat model to that estimated in a swine model, to investigate the correlation between these two animal models. This may help reduce uncertainty in applying bioavailability to human health risk assessment. Methods: Twelve spiked soil samples and a spiked silica sand (reference material) were dosed to rats in parallel with a swine study. B[a]P bioavailability was estimated by the area under the plasma B[a]P concentration-time curve (AUC) and faecal excretion as well in the rats. Direct comparison between the two animal models was made for: firstly, relative bioavailability (RB) using AUC assay; and secondly, the two assays in the rat model. Results: Both AUC and faecal excretion assays showed linear dose-response for the reference material. However, absolute bioavailability was significantly higher when using faecal excretion assay (p

Huntington's disease (HD) is a fatal neurodegenerative disease caused by a genetic expansion of the CAG repeat region in the huntingtin (HTT) gene. Studies in HD mouse models have shown that artificial miRNAs can reduce mutant HTT, but evidence for their effectiveness and safety in larger animals is lacking. HD transgenic sheep express the full-length human HTT with 73 CAG repeats. AAV9 was used to deliver unilaterally to HD sheep striatum an artificial miRNA targeting exon 48 of the human HTT mRNA under control of two alternative promoters: U6 or CβA. The treatment reduced human mutant (m) HTT mRNA and protein 50-80% in the striatum at 1 and 6 months post injection. Silencing was detectable in both the caudate and putamen. Levels of endogenous sheep HTT protein were not affected. There was no significant loss of neurons labeled by DARPP32 or NeuN at 6 months after treatment, and Iba1-positive microglia were detected at control levels. It is concluded that safe and effective silencing of human mHTT protein can be achieved and sustained in a large-animal brain by direct delivery of an AAV carrying an artificial miRNA.

The heart has high metabolic demand to maintain function. The primary source of energy supply to support correct contractile muscle function differs between a fetus and an adult. In fetal life, ATP is primarily generated by glycolysis and lactate oxidation, whereas following birth, there is a shift towards a reliance on mitochondrial metabolism and fatty acid oxidation. This change in metabolic status is an adaptation to different fuel availability, oxygenation and growth patterns. In this study, we have employed 2-photon excitation fluorescence microscopy to define the relationship between two critical metabolic cofactors nicotinamide adenine dinucleotide(P)H and flavin adenine dinucleotide, effectively utilizing a redox ratio to differentiate between the metabolic status in fetal (proliferative) and adult (quiescent/hypertrophic) hearts. Two-photon imaging was also used to visually confirm the known increase in collagen deposition in the adult heart. The changes observed were consistent with a hypertrophic growth profile and greater availability of fatty acids in the adult heart, compared to the proliferative fetal heart. Two-photon excitation fluorescence microscopy is therefore a convenient imaging technology that enables the monitoring of striated muscle architecture and the metabolic status of heart tissue. This imaging technology can potentially be employed to visualize cardiac and other muscle pathologies. Refereed/Peer-reviewed

Soil plays a significant role in controlling the potential bioavailability of contaminants in the environment. In this study, eleven soils were used to investigate the relationship between soil properties and relative bioavailability (RB) of lead (Pb). To minimise the effect of source of Pb on in vivo bioavailability, uncontaminated study soils were spiked with 1500 mg Pb/kg soil and aged for 10-12 months prior to investigating the relationships between soil properties and in vivo RB of Pb using swine model. The biological responses to oral administration of Pb in aqueous phase or as spiked soils were compared by applying a two-compartment pharmacokinetic model to blood Pb concentration. The study revealed that RB of Pb from aged soils ranged from 30±9% to 83±7%. The very different RB of Pb in these soils was attributed to variations in the soils' physico-chemical properties. This was established using sorption studies showing: firstly, Freundlich partition coefficients that ranged from 21 to 234; and secondly, a strongly significant (R(2)=0.94, P

Background Multidrug resistance-associated protein 1 (MRP1) overexpression plays a major role in chemoresistance in glioblastoma multiforme (GBM) contributing to its notorious deadly nature. Although MRP1-siRNA transfection to GBM in vitro has been shown to sensitise the cells to drug, MRP1 silencing in vivo and the phenotypic influence on the tumour and normal tissues upon MRP1 down-regulation have not been established. Here, porous silicon nanoparticles (pSiNPs) that enable high-capacity loading and delivery of siRNA are applied in vitro and in vivo. Result We established pSiNPs with polyethyleneimine (PEI) capping that enables high-capacity loading of siRNA (92 µg of siRNA/mg PEI-pSiNPs), and optimised release profile (70% released between 24 and 48 h). These pSiNPs are biocompatible, and demonstrate cellular uptake and effective knockdown of MRP1 expression in GBM by 30%. Also, siRNA delivery was found to significantly reduce GBM proliferation as an associated effect. This effect is likely mediated by the attenuation of MRP1 transmembrane transport, followed by cell cycle arrest. MRP1 silencing in GBM tumour using MRP1-siRNA loaded pSiNPs was demonstrated in mice (82% reduction at the protein level 48 h post-injection), and it also produced antiproliferative effect in GBM by reducing the population of proliferative cells. These results indicate that in vitro observations are translatable in vivo. No histopathological signs of acute damage were observed in other MRP1-expressing organs despite collateral downregulations. Conclusions This study proposes the potential of efficient MRP1-siRNA delivery by using PEI-capped pSiNPs in achieving a dual therapeutic role of directly attenuating the growth of GBM while sensitising residual tumour cells to the effects of chemotherapy post-resection. Electronic supplementary material The online version of this article (10.1186/s12951-018-0365-y) contains supplementary material, which is available to authorized users.

Taipans are highly venomous Australo-Papuan elapids. A new species of taipan, the Western Desert Taipan (Oxyuranus temporalis), has been discovered with two specimens housed in captivity at the Adelaide Zoo. This study is the first investigation of O. temporalis venom and seeks to characterise and compare the neurotoxicity, lethality and biochemical properties of O. temporalis venom with other taipan venoms. Analysis of O. temporalis venom using size-exclusion and reverse-phase HPLC indicated a markedly simplified “profile” compared to other taipan venoms. SDS-PAGE and agarose gel electrophoresis analysis also indicated a relatively simple composition. Murine LD50 studies showed that O. temporalis venom is less lethal than O. microlepidotus venom. Venoms were tested in vitro, using the chick biventer cervicis nerve-muscle preparation. Based on t90 values, O. temporalis venom is highly neurotoxic abolishing indirect twitches far more rapidly than other taipan venoms. O. temporalis venom also abolished responses to exogenous acetylcholine and carbachol, indicating the presence of postsynaptic neurotoxins. Prior administration of CSL Taipan antivenom (CSL Limited) neutralised the inhibitory effects of all taipan venoms. The results of this study suggest that the venom of the O. temporalis is highly neurotoxic in vitro and may contain procoagulant toxins, making this snake potentially dangerous to humans.

Porous silicon (pSi) is a nanomaterial with salient properties for optical biosensor applications. For example, the material displays strong and tunable optical reflectance, and is highly biocompatible and biodegradable. The material is compatible with microfabrication and can be produced in the form of films, membranes and particles. Whilst pSi has been used extensively as a biosensor to accept analytes in or purified from body fluids, it has never been used as an implantable biosensor. In this paper, we advance the understanding of the requirements for pSi-based optical biosensors when incorporated under skin. Reflectance spectra from several different pSi thin film architectures were compared. The optical properties of pSi films were optimised and pSi microcavities with resonances at 850 nm were found to be the most suitable for implantable biosensors. This work will provide useful cues for researchers interested in developing implantable biosensors for human medical and veterinary applications. Refereed/Peer-reviewed

In this study, lead (Pb) bioaccessibility was assessed in peri-urban contaminated soils using a variety of established in vitro assays. Bioaccessibility data was then used to predict Pb relative bioavailability (RBA) using published in vivo-in vitro regression models in order to compare calculated estimates and measured values. Lead bioaccessibility varied depending on the in vitro methodology employed with the relative bioavailability leaching procedure (RBALP) and in vitro gastrointestinal (IVG) assays providing more conservative Pb bioaccessibility values compared to those determined using PBET, UBM and Rel-SBRC-I assays. When Pb RBA was calculated, predicted values using PBET-G and UBM-G data were similar to measured Pb RBA values. However, Pb RBA was over-estimated by 1.6-5.5- and 2.6-6.6-fold when data and regression models from RBALP and IVG-G assays were employed. Refereed/Peer-reviewed

It is unclear how septic shock causes acute kidney injury (AKI) and whether this is associated with histological change.We aimed to determine the nature and extent of changes in renal structure and function over time in an ovine model of septic shock.Fifteen sheep were instrumented with a renal artery flow probe and renal vein cannula. Ten were given intravenous Escherichia coli to induce septic shock, and five acted as controls. Animals were mechanically ventilated for 48 hours, while receiving protocol-guided parenteral fluids and a norepinephrine infusion to maintain mean arterial pressure. Renal biopsies were taken every 24 hours or whenever animals were oliguric for 2 hours. A renal pathologist, blinded to tissue source, systematically quantified histological appearance by light and electron microscopy for 31 prespecified structural changes.Sheep given E. coli developed septic shock, oliguria, increased serum creatinine, and reduced creatinine clearance (AKI), but there were no changes over time in renal blood flow between groups (P 0.30) or over time within groups (P 0.50). Renal oxygen consumption increased only in nonseptic animals (P = 0.01), but there was no between-group difference in renal lactate flux (P 0.50). There was little structural disturbance in all biopsies and, although some cellular appearances changed over time, the only difference between septic and nonseptic animals was mesangial expansion on electron microscopy.In an intensive care-supported model of gram-negative septic shock, early AKI was not associated with changes in renal blood flow, oxygen delivery, or histological appearance. Other mechanisms must contribute to septic AKI.

Gaucher disease arises from mutations in the beta-glucocerebrosidase gene which encodes an enzyme required for the lysosomal catabolism of glucosylceramide. We have identified a naturally occurring mutation in the beta-glucocerebrosidase gene in sheep that leads to Gaucher disease with acute neurological symptoms. Here we have examined the clinical phenotype at birth and subsequently quantified lipids in Gaucher lamb brain, in order to characterise the disorder. Enzyme activity assessments showed that a reduction in beta-glucocerebrosidase activity to 1-5% of wild-type occurs consistently across newborn Gaucher lamb brain regions. We analyzed glucosylceramide, glucosylsphingosine, bis(monoacylglycero)phosphate and ganglioside profiles in brain, liver, and spleen, and observed 30- to 130-fold higher glucosylceramide, and 500- to 2000-fold higher glucosylsphingosine concentrations in Gaucher diseased lambs compared to wild-type. Significant increases of bis(monoacylglycero)phosphate and gangliosides [GM1, GM2, GM3] concentrations were also detected in the brain. As these glycosphingolipids are involved in many cellular events, an imbalance or disruption of the cell membrane lipid homeostasis would be expected to impair normal neuronal function. To our knowledge, this is the first detailed analysis of glycosphingolipids in various brain regions in a large animal model of neuronal disease, which permits the mechanistic investigation of lipid deregulation and their contribution to neurodegenerative process. Refereed/Peer-reviewed

Cardiac fibrosis plays an important prognostic role in nonischemic cardiomyopathy (NICM), making it a potential therapeutic target. Although electromechanical mapping has been used to identify myocardial scar and facilitate intramyocardial intervention in the setting of ischemic heart disease, its application has not been described in NICM. We assessed the detection of myocardial fibrosis by endoventricular electromechanical mapping in an experimental model of NICM. The NOGA® XP system was used to perform left ventricular mapping in twelve sheep that had undergone intracoronary doxorubicin dosing to induce NICM and in six healthy control animals. Results for endocardial voltage and mechanical shortening were evaluated against myocardial fibrosis burden, as determined by delayed-enhancement cardiac magnetic resonance and quantitative histomorphometry. Doxorubicin treatment resulted in dilated cardiomyopathy with moderate-severe impairment of left ventricular ejection fraction. Late gadolinium uptake was present in 9/12 doxorubicin animals, while histological fibrosis was approximately doubled compared to controls and was distributed multisegmentally throughout the left ventricle. Cardiomyopathy was associated with widespread reductions in unipolar and bipolar voltage amplitude and endocardial shortening. Each parameter showed an inverse relationship with the burden of fibrosis. Moreover, unipolar voltage and linear local shortening ratio displayed moderate accuracy for identifying myocardial segments with delayed contrast enhancement or increased fibrosis content, with optimal discriminatory thresholds of 7.5 mV and 11.5%, respectively. In this model of NICM, electromechanical mapping shows potential for delineating segmental differences in fibrosis. Pending clinical evaluation, it may therefore have applicability for directing targeted intramyocardial interventions in nonischemic heart disease. Refereed/Peer-reviewed

Study Overview The incidence of adverse pregnancy outcomes is higher in pregnancies where the fetus is male. Sex specific differences in feto-placental perfusion indices identified by Doppler assessment have recently been associated with placental insufficiency and fetal growth restriction. This study aims to investigate sex specific differences in placental perfusion and to correlate these changes with fetal growth. It represents the largest comprehensive study under field conditions of uterine hemodynamics in a monotocous species, with a similar long gestation period to the human. Primiparous 14mo heifers in Australia (n=360) and UK (n=180) were either individually or group fed, respectively, diets with differing protein content (18, 14, 10 or 7% crude protein (CP)) from 60d prior to 98 days post conception (dpc). Fetuses and placentae were excised at 98dpc (n = 48). Fetal development an median uterine artery blood flow were assessed monthly from 36dpc until term using B-mode and Doppler ultrasonography. MUA blood flow to the male feto-placental unit increased in early pregnancy associated with increased fetal growth. Protein restriction before and shortly after conception (-60d up to 23dpc) increased MUA diameter and indices of velocity during late pregnancy, reduced fetal heart weight in the female fetus and increased heart rate at birth, but decreased systolic blood pressure at six months of age. Conclusion and Significance Sex specific differences both in feto-placental Doppler perfusion indices and response of these indices to dietary perturbations were observed. Further, maternal diet affected development of fetal cardiovascular system associated with altered fetal haemodynamics in utero, with such effects having a sex bias. The results from this study provide further insight into the gender specific circulatory differences present in the fetal period and developing cardiovascular system.

In this study, lead (Pb) bioaccessibility in contaminated soils was assessed using an in vitro method (SBRC) encompassing gastric (SBRC-G) and intestinal (SBRC-I) phases. Initially, bioaccessibility studies were performed with a Pb reference material (Pb acetate, 1-10 mg L⁻¹) in order to determine the influence of pH on Pb solubility. In the gastric phase (pH 1.5), Pb solubility was 100% (100 ± 2.9%, n = 16) irrespective of the Pb concentration added, however, when the pH of the intestinal phase was increased to near neutral, Pb solubility decreased to 14.3 ± 7.2%. In contaminated soils, Pb bioaccessibility varied from 35.7 to 64.1% and 1.2 to 2.7% for SBRC-G and SBRC-Iphases, respectively.When relative bioaccessibility (Rel-SBRCI) was calculated by adjusting the dissolution of Pb from contaminated soils by the solubility of Pb acetate at pH 6.5(intestinal phase pH), Rel-SBRC-I values ranged from 11.7-26.1%. A stepwise regression model based on Pearson correlation factors was used to determine the suitability of in vitro assays for predicting in vivo(swine assay) relative Pb bioavailability. Rel-SBRC-I provided the best estimate of in vivo relative Pb bioavailability for soils used in this study (in vivo relative Pb bioavailability [%] = Rel-SBRC-I [pH 6.5%] × 0.58 + 1.98, r² = 0.53). The versatility of Rel-SBRC-I was demonstrated by accurately predicting relative Pb bioavailability from otherreported in vivo studies. Refereed/Peer-reviewed

Arsenic (As) bioaccessibility in contaminated soils (n=50) was assessed using the simplified bioaccessibility extraction test (SBET). Soils used in the study were collected from sites where As was used as an herbicide (railway corridor) or pesticide (cattle dip sites), from former gold mines and from highly mineralised locations containing geogenic As sources (gossans). In all but three soils, As bioaccessibility was less than 50% indicating that a significant proportion of the total As concentration may not be available for absorption in the gastrointestinal tract following incidental soil ingestion. When regression models were developed based on soil properties, the descriptive variables best able to describe As bioaccessibility in railway corridor, dip site and mine site soils were total As and total or dithionite-citrate extractable (free) iron (Fe). While As bioaccessibility could be predicted (r(2)=0.955, n=50) in these contaminated soils, As bioaccessibility for gossan soils was a poor fit using linear or multivariate regression analysis.

Advanced biosensors in future medicine hinge on the evolvement of biomaterials. Porous silicon (pSi), a generally biodegradable and biocompatible material that can be fabricated to include environment-responsive optical characteristics, is an excellent candidate for in vivo biosensors. However, the feasibility of using this material as a subcutaneously implanted optical biosensor has never been demonstrated. Here, we investigated the stability and biocompatibility of a thermally-hydrocarbonised (THC) pSi optical rugate filter, and demonstrated its optical functionality in vitro and in vivo. We first compared pSi films with different surface chemistries and observed that the material was cytotoxic despite the outstanding stability of the THC pSi films. We then showed that the cytotoxicity correlates with reactive oxygen species levels, which could be mitigated by pre-incubation of THC pSi (PITHC pSi). PITHC pSi facilitates normal cellular phenotypes and is biocompatible in vivo. Importantly, the material also possesses optical properties capable of responding to microenvironmental changes that are readable non-invasively in cell culture and subcutaneous settings. Collectively, we demonstrate, for the first time, that PITHC pSi rugate filters are both biocompatible and optically functional for lab-on-a-chip and subcutaneous biosensing scenarios. We believe that this study will deepen our understanding of cell-pSi interactions and foster the development of implantable biosensors. Refereed/Peer-reviewed

Brown snakes, Pseudonaja genus, cause more bites and deaths to animals and humans in Australia than any other terrestrial snake genus. Some aspects of treatment of brown snakebites with antivenom remain poorly managed due to the apparent inability of the antivenom to counter the prothrombin activator in the venom. We present evidence of a new novel antivenom (Antiven Pty Ltd Brown snake antivenom (ABSAV)), which shows high efficacy in reversing the hemostatic abnormality caused by brown snake envenomation. It is also more efficient at reversing overall toxicity. In clotting tests, it is substantially more potent than Commonwealth Serum Laboratories Ltd (CSL) Brown snake antivenom and is twice as effective in reversing overall toxicity than CSL Brown snake antivenom.Antiven Pty. Ltd. Brown snake antivenom is currently being assessed by the National Registration Authority (veterinary regulative body in Australia).

Background The safety and efficiency of gene therapies for cystic fibrosis (CF) need to be assessed in pre-clinical models. Using the normal ferret, this study sought to determine whether ferret airway epithelia could be transduced with a lysophosphatidylcholine (LPC) pre-treatment followed by a VSV-G pseudotyped HIV-1 based lentiviral (LV) vector, in preparation for future studies in CF ferrets. Methods Six normal ferrets (7 -8 weeks old) were treated with a 150 μL LPC pre-treatment, followed one hour later by a 500 μL LV vector dose containing the LacZ transgene. LacZ gene expression in the conducting airways and lung was assessed by X-gal staining after 7 days. The presence of transduction in the lung, as well as off-target transduction in the liver, spleen and gonads, were assessed by qPCR. The levels of LV vector p24 protein bio-distribution in blood sera were assessed by ELISA at 0, 1, 3, 5 and 7 days. Results The dosing protocol was well tolerated. LacZ gene expression was observed en face in the trachea of all animals. Histology showed that ciliated and basal cells were transduced in the trachea, with rare LacZ transduced single cells noted in lung. p24 levels was not detectable in the sera of 5 of the 6 animals. The LacZ gene was not detected in the lung tissue and no off-target transduction was detected by qPCR. Conclusions This study shows that ferret airway epithelia are transducible using our unique two-step pre-treatment and LV vector dosing protocol. We have identified a number of unusual anatomical factors that are likely to influence the level of transduction that can be achieved in ferret airways. The ability to transduce ferret airway epithelium is a promising step towards therapeutic LV-CFTR testing in a CF ferret model.

Oral bioavailability of benzo[a]pyrene (B[a]P) was studied in a swine model using eight spiked soil samples after incubation for 50 and/or 90 days. Silica sand was used as a reference material and the relative bioavailability (RB) of B[a]P in soils was calculated as the quotient of the area under the plasma B[a]P curve (AUC) for soil and AUC for the silica sand. Significantly reduced RB was observed in all study soils after 90 days ageing, ranging from 22.1 ± 0.4% to 62.7 ± 10.1%, except for one very sandy soil (sand content 87.6%) where RB was unchanged (108.1 ± 8.0%). Apart from this, bioavailability decreased during ageing with the decrease (from day 50 to day 90) being only significant for a clayey soil containing expandable clay minerals. Statistical analyses of B[a]P RB at day 90 (eight soils) and soil properties showed no direct correlation between RB and specific soil properties such as total organic carbon (TOC) and clay content which were commonly linked to organic contaminant sequestration. However, strongly significant relationships (p

Objective To determine the extent of the snake bite problem in domestic animals, its regional significance and the effects of antivenom treatment. Design A questionnaire was designed seeking information on the number and type of domestic animals referred, whether treated or untreated, type of snakes and management of the bite. Procedure The survey form was sent to 10% of veterinary surgeons, selected at random throughout Australia. Results The response of 106 veterinary surgeons revealed that snake bite in domestic animals is frequent, with an estimated 6200 cases reported annually. Bites were more prominent in rural (78%) than urban areas (22%) with brown, tiger and black snakes accounting for 76%, 13% and 6% of cases, respectively. Cats and dogs were the most frequently reported victims. Ninety-one percent of cats and 75% of dogs survived following the administration of antivenom whereas 66% of cats and 31% of dogs survived without antivenom. Overall, in 33% of cases antivenom was not used, and venom detection kits were used in only 1% of cases. A number of drugs were used in various combinations with or without antivenom and intravenous fluids in the treatment of animals with snake bite, but their role in reducing the severity of envenomations was not assessed. Clinical implications Antivenom significantly improves the chances of survival of domestic animals bitten by snakes.

Currently, a number of in vitro methods are in use worldwide to assess arsenic (As) bioaccessibility in soils. However, a dearth of research has been undertaken to compare the efficacy of the in vitromethodsfor estimating in vivo relativeAsbioavailability. In this study, As bioaccessibility in contaminated soils (n ) 12) was assessed using four in vitro assays (SBRC, IVG, PBET, DIN). In vitro results were compared to in vivo relative As bioavailabilitydata(swineassay)toascertainwhichmethodologies best correlate with in vivo data. Arsenic bioaccessibility in contaminated soils varied depending on the in vitro method employed. For the SBRC and IVG methods, As bioaccessibility generally decreased when gastric-phase values were compared to the intestinal phase. In contrast, extending the PBET and DIN assays from the gastric to the intestinal phase resulted in an increase in As bioaccessibility for some soils tested. Comparison of in vitro and in vivo results demonstrated that the in vitro assay encompassing the SBRC gastric phase provided the best prediction of in vivo relative As bioavailability (R2) 0.75, Pearson correlation) 0.87). However, relative As bioavailability could also be predicted using gastric or intestinal phases of IVG, PBET, and DIN assays but with varying degrees of confidence (R2) 0.53-0.67, Pearson correlation) 0.73-0.82). Refereed/Peer-reviewed

Considerable information is available in the literature regarding the uptake of arsenic (As) from contaminated soil and irrigation water by vegetables. However, few studies have investigated As speciation in these crops while a dearth of information is available on As bioavailability following their consumption. In this study, the concentration and speciation of As in chard, radish, lettuce and mung beans was determined following hydroponic growth of the vegetables using As-contaminated water. In addition, As bioavailability was assessed using an in vivo swine feeding assay. While As concentrations ranged from 3.0 to 84.2 mg As kg−1 (dry weight), only inorganic As (arsenite and arsenate) was detected in the edible portions of the vegetables. When As bioavailability was assessed through monitoring blood plasma As concentrations following swine consumption of As-contaminated vegetables, between 50% and 100% of the administered As dose was absorbed and entered systemic circulation. Arsenic bioavailability decreased in the order mung beans > radish > lettuce = chard.

Arsenic (As) bioavailability in spiked soils aged for up to 12 months was assessed using in vitro and in vivo methodologies. Ageing (natural attenuation) of spiked soils resulted in a decline in in vivo As bioavailability (swine assay) of over 75% in soil A (Red Ferrosol) but had no significant effect on in vivo As bioavailability even after 12 months of ageing in soil B (Brown Chromosol). Sequential fractionation, however, indicated that there was repartitioning of As within the soil fractions extracted during the time course investigated. In soil A, the As fraction associated with the more weakly bound soil fractions decreased while the residual fraction increased from 12% to 35%. In contrast, little repartitioning of As was observed in soil B indicating that natural attenuation may be only applicable for As in soils containing specific mineralogical properties.

The assessment of arsenic (As) bioavailability from contaminated matrices is a crucial parameter for reducing the uncertainty when estimating exposure for human health risk assessment. In vivo assessment of As utilising swine is considered an appropriate model for human health risk assessment applications as swine are remarkably similar to humans in terms of physiology and As metabolism. While limited in vivo As bioavailability data is available in the literature, few details have been provided regarding technical considerations for performing in vivo assays. This paper describes, with examples, surgical, experimental design and analytical issues associated with performing chronic and acute in vivo swine assays to determine As bioavailability in contaminated soil and food. Refereed/Peer-reviewed

An in vivo swine assay was utilised for the determination of arsenic (As) bioavailability in contaminated soils. Arsenic bioavailability was assessed using pharmacokinetic analysis encompassing area under the blood plasma-As concentration time curve following zero correction and dose normalisation. In contaminated soil studies, As uptake into systemic circulation was compared to an arsenate oral dose and expressed as relative As bioavailability. Arsenic bioavailability ranged from 6.9+/-5.0% to 74.7+/-11.2% in 12 contaminated soils collected from former railway corridors, dip sites, mine sites and naturally elevated gossan soils. Arsenic bioavailability was generally low in the gossan soils and highest in the railway soils, ranging from 12.1+/-8.5% to 16.4+/-9.1% and 11.2+/-4.7% to 74.7+/-11.2%, respectively. Comparison of in vivo and in vitro (Simplified Bioaccessibility Extraction Test [SBET]) data from the 12 contaminated soils and bioavailability data collected from an As spiked soil study demonstrated that As bioavailability and As bioaccessibility were linearly correlated (in vivo As bioavailability (mgkg(-1))=14.19+0.93.SBET As bioaccessibility (mgkg(-1)); r(2)=0.92). The correlation between the two methods indicates that As bioavailability (in vivo) may be estimated using the less expensive, rapid in vitro chemical extraction method (SBET) to predict As exposure in human health risk assessment.

Study Overview: The incidence of adverse pregnancy outcomes is higher in pregnancies where the fetus is male. Sex specific differences in feto-placental perfusion indices identified by Doppler assessment have recently been associated with placental insufficiency and fetal growth restriction. This study aims to investigate sex specific differences in placental perfusion and to correlate these changes with fetal growth. It represents the largest comprehensive study under field conditions of uterine hemodynamics in a monotocous species, with a similar long gestation period to the human. Primiparous 14mo heifers in Australia (n=360) and UK (n=180) were either individually or group fed, respectively, diets with differing protein content (18, 14, 10 or 7% crude protein (CP)) from 60d prior to 98 days post conception (dpc). Fetuses and placentae were excised at 98dpc (n = 48). Fetal development an median uterine artery blood flow were assessed monthly from 36dpc until term using B-mode and Doppler ultrasonography. MUA blood flow to the male feto-placental unit increased in early pregnancy associated with increased fetal growth. Protein restriction before and shortly after conception (-60d up to 23dpc) increased MUA diameter and indices of velocity during late pregnancy, reduced fetal heart weight in the female fetus and increased heart rate at birth, but decreased systolic blood pressure at six months of age. Conclusion and Significance: Sex specific differences both in feto-placental Doppler perfusion indices and response of these indices to dietary perturbations were observed. Further, maternal diet affected development of fetal cardiovascular system associated with altered fetal haemodynamics in utero, with such effects having a sex bias. The results from this study provide further insight into the gender specific circulatory differences present in the fetal period and developing cardiovascular system.

Arsenic contamination of groundwater has been reported in many countries throughout the world, most notably in Southeast Asia. In recent years, much attention has focused on the As calamity in Bangladesh and West Bengal, India, following the highly publicized reports of vast populations being exposed to As-contaminated groundwater. Recently, Chakraborti et al. (2004) reported that As levels in groundwater from 50 districts in Bangladesh (representing ~ 2,000 villages) exceeded the Bangladesh drinking water guidelines for As of 50 μ with As concentrations in some cases > 1,500 μ(Tondel et al. 1999). The issue in Bangladesh has been described as “the largest poisoning of a population in history” (Smith et al. 2000), with an estimated 35–70 million inhabitants being at risk of drinking As-contaminated water (Khan et al. 1997). Chronic exposure to As causes significant human health effects including various cancers (skin, lungs, bladder, and kidneys), skin disorders (hyperkeratosis and pigment changes), vascular disease, and diabetes mellitus (Guha Mazumder et al. 1998; Guo et al. 1997; Lein et al. 2001; Mandal and Suzuki 2002; Rahman et al. 2001). The increased incidence of these health effects have been observed in Bangladesh and West Bengal populations exposed to As (Ahmed et al. 2006; Mukherjee et al. 2006). In addition to drinking water, consumption of As-contaminated food is another major source of As exposure. In Bangladesh, As-contaminated water is also used for irrigating crops, particularly rice (Oryza sativa L.), which represents approximately 83% of the total irrigated area in Bangladesh (Dey et al. 1996). As a consequence of irrigating with As-contaminated water, rice may contain elevated levels of As. Arsenic concentrations ranging from 160 to 580 μ have been reported in rice from the Jessore district in Bangladesh (Alam et al. 2002), whereas Meharg and Rahman (2003) reported As concentrations > 1,830 μg/kg in rice from other regions of Bangladesh. Because rice is a staple food in Bangladesh, providing > 70% of the daily calorific intake (Ninno and Dorosh 2001), consumption of contaminated rice may represent a significant As exposure pathway. In fact, Meharg (2004) estimated that consumption of As-contaminated rice may contribute as much as 60% of the daily Bangladeshi dietary As intake based on conservative As concentrations in rice. In addition, absorption of As-contaminated water during the rice cooking process may significantly increase the amount of As in cooked rice (Ackerman et al. 2005; Bae et al. 2002), which is often overlooked when calculating As daily intake values. A number of studies have reported the presence of As in rice, ranging in concentration from 32 to 1,830 μg As/kg (Abedin et al. 2002a, 2002b; Alam et al. 2002; D’Amato et al. 2004; Heitkemper et al. 2001; Kohlmeyer et al. 2003; Meharg 2004; Meharg and Rahman 2003; Schoof et al. 1998; Williams et al. 2005); however, few studies have determined the proportion of inorganic to organic As in rice. In terms of human health risk assessment, As speciation is important because the toxicity of organic, inorganic, trivalent, and pentavalent As species vary greatly (Petrick et al. 2000; Vahter and Concha 2001). In addition, there is a dearth of information on the bioavailability of As in rice after consumption. Bioavailability, in the context of human health risk assessment, refers to the fraction of an administered dose that reaches the central (blood) compartment from the gastrointestinal tract (Ruby et al. 1999). After consumption of As-contaminated rice, it is unclear what proportion of the rice-bound As is absorbed and whether As speciation influences absorption from the gastrointestinal tract. To reduce the uncertainties in estimating exposure and to provide a more accurate estimate of risk, assessment of As bioavailability is critical. In this study, we investigated the concentration and speciation of As in supermarket-bought rice, in rice grown under greenhouse conditions using As-contaminated irrigation water, and in rice cooked in As-contaminated water. In addition, we assessed the bioavailability of As in rice using an in vivo swine assay—an animal model used to predict As uptake for human health risk assessment. We assessed As bioavailability in two different rice preparations to determine whether the mode of As accumulation in the grain (translocation by the plant vs. absorption during cooking) influenced As uptake in the swine model.

The aim of these studies was to assess and quantitate the effects of cyclosporin-A (CyA) on brain APP messenger RNA and neuronal perikaryal APP antigen expression following controlled focal head impact in sheep. Impact results in a significant increase in both APP mRNA and neuronal perikaryal APP antigen expression. Post-traumatic administration of CyA (intrathecal 10 mg/kg) resulted in a reduction in APP mRNA and neuronal perikaryal antigen expression. At 2 h postinjury, CyA treatment caused a statistically significant (p0.05) 1.3 +/- 0.1-fold decrease in APP mRNA in the central gray matter of impacted sheep compared to untreated impacted sheep. A more profound reduction in APP mRNA synthesis (1.6 +/- 0.2 fold) was evident at 6 h (p0.05). The mean percentage brain area with APP immunoreactive neuronal perikarya at 6 h post-injury was 94.5% in untreated impacted animals, 10.0% in CyA-treated impacted animals, 5.5% in untreated nonimpacted animals, and 6% in CyA-treated non-impacted controls. These results demonstrate that CyA has a downregulatory effect on increased APP expression caused by TBI.

A total of 120 E mu-Pim1 heterozygous mice and 120 wild-type mice were exposed for 1 h/day 5 days/week at each of the four exposure levels in "Ferris-wheel" exposure systems for up to 104 weeks to GSM-modulated 898.4 MHz radiation at SARs of 0.25, 1.0, 2.0 and 4.0 W/kg. In addition, 120 heterozygous and 120 wild-type mice were sham-exposed; there was also an unrestrained negative control group. Four exposure levels were used to investigate whether a dose-response effect could be detected. Independent verification confirmed that the exposures in the current study were nonthermal. There was no significant difference in the incidence of lymphomas between exposed and sham-exposed groups at any of the exposure levels. A dose-response effect was not detected. The findings showed that long-term exposures of lymphoma-prone mice to 898.4 MHz GSM radiofrequency (RF) radiation at SARs of 0.25, 1.0, 2.0 and 4.0 W/kg had no significant effects when compared to sham-irradiated animals. A previous study (Repacholi et al., Radiat. Res. 147, 631-640, 1997) reported that long-term exposure of lymphoma-prone mice to one exposure level of 900 MHz RF radiation significantly increased the incidence of non-lymphoblastic lymphomas when compared to sham-irradiated animals.

The amino acid sequence of a previously undescribed toxin from Australian death adder venom (Acanthophis antarcticus) has been elucidated. It appears to exist in two forms which are separated by reverse-phase high-performance liquid chromatography, but which have the same sequence and mol. wt. It has 79 amino acid residues and is therefore longer than other long postsynaptic neurotoxins. It shows homology with the conserved regions of the other long postsynaptic neurotoxins except for three unique substitutions of conserved residues, which are Arg-29 instead of Trp or Phe, Leu-33 instead of Arg and Thr-43 instead of Ala.

exposure to hypoxia and doxorubicin. Cell death, as measured by flow cytometric analysis of Annexin V and Propidium Iodide staining, was reduced in the presence of each type of CM. Conclusions: Conditioned media from MSCs had both mitogenic and anti-apoptotic effects on cardiomyocytes and endothelial cells, with STRO-1Bright CM performing especially well. This further supports the evidence that paracrine actions contribute to the cardiac reparative properties of MSCs. doi:10.1016/j.hlc.2007.06.071