Haematology and Plasma Biochemistry of Wild Black Flying-Foxes, (Pteropus alecto) in Queensland, Australia

This paper establishes reference ranges for hematologic and plasma biochemistry values in wild Black flying-foxes (Pteropus alecto) captured in South East Queensland, Australia. Values were found to be consistent with those of other Pteropus species. Four hundred and forty-seven animals were sampled over 12 months and significant differences were found between age, sex, reproductive and body condition cohorts in the sample population. Mean values for each cohort fell within the determined normal adult reference range, with the exception of elevated levels of alkaline phosphatase in juvenile animals. Hematologic and biochemistry parameters of injured animals showed little or no deviation from the normal reference values for minor injuries, while two animals with more severe injury or abscessation showed leucocytosis, anaemia, thrombocytosis, hyperglobulinemia and hypoalbuminemia.

Inheritance and Characterization of Strong Resistance to Phosphine in Sitophilus oryzae(L.)

Sitophilus oryzae (Linnaeus) is a major pest of stored grain across Southeast Asia and is of increasing concern in other regions due to the advent of strong resistance to phosphine, the fumigant used to protect stored grain from pest insects. We investigated the inheritance of genes controlling resistance to phosphine in a strongly resistant S. oryzae strain (NNSO7525) collected in Australia and find that the trait is autosomally inherited and incompletely recessive with a degree of dominance of -0.66. The strongly resistant strain has an LC50 52 times greater than a susceptible reference strain (LS2) and 9 times greater than a weakly resistant strain (QSO335). Analysis of F2 and backcross progeny indicates that two or more genes are responsible for strong resistance, and that one of these genes, designated Sorph1, not only contributes to strong resistance, but is also responsible for the weak resistance phenotype of strain QSO335. These results demonstrate that the genetic mechanism of phosphine resistance in Soryzae is similar to that of other stored product insect pests. A unique observation is that a subset of the progeny of an F1 backcross generation are more strongly resistant to phosphine than the parental strongly resistant strain, which may be caused by multiple alleles of one of the resistance genes.

Flying-Fox Roost Disturbance and Hendra Virus Spillover Risk

Bats of the genus Pteropus (flying-foxes) are the natural host of Hendra virus (HeV) which periodically causes fatal disease in horses and humans in Australia. The increased urban presence of flying-foxes often provokes negative community sentiments because of reduced social amenity and concerns of HeV exposure risk, and has resulted in calls for the dispersal of urban flying-fox roosts. However, it has been hypothesised that disturbance of urban roosts may result in a stress-mediated increase in HeV infection in flying-foxes, and an increased spillover risk. We sought to examine the impact of roost modification and dispersal on HeV infection dynamics and cortisol concentration dynamics in flying-foxes. The data were analysed in generalised linear mixed models using restricted maximum likelihood (REML). The difference in mean HeV prevalence in samples collected before (4.9%), during (4.7%) and after (3.4%) roost disturbance was small and non-significant (P = 0.440). Similarly, the difference in mean urine specific gravity-corrected urinary cortisol concentrations was small and non-significant (before = 22.71 ng/mL, during = 27.17, after = 18.39) (P= 0.550). We did find an underlying association between cortisol concentration and season, and cortisol concentration and region, suggesting that other (plausibly biological or environmental) variables play a role in cortisol concentration dynamics. The effect of roost disturbance on cortisol concentration approached statistical significance for region, suggesting that the relationship is not fixed, and plausibly reflecting the nature and timing of disturbance. We also found a small positive statistical association between HeV excretion status and urinary cortisol concentration. Finally, we found that the level of flying-fox distress associated with roost disturbance reflected the nature and timing of the activity, highlighting the need for a ‘best practice’ approach to dispersal or roost modification activities. The findings usefully inform public discussion and policy development in relation to Hendra virus and flying-fox management.

Routes of Hendra Virus Excretion in Naturally-Infected Flying-Foxes: Implications for Viral Transmission and Spillover Risk

Pteropid bats or flying-foxes (Chiroptera: Pteropodidae) are the natural host of Hendra virus (HeV) which sporadically causes fatal disease in horses and humans in eastern Australia. While there is strong evidence that urine is an important infectious medium that likely drives bat to bat transmission and bat to horse transmission, there is uncertainty about the relative importance of alternative routes of excretion such as nasal and oral secretions, and faeces. Identifying the potential routes of HeV excretion in flying-foxes is important to effectively mitigate equine exposure risk at the bat-horse interface, and in determining transmission rates in host-pathogen models. The aim of this study was to identify the major routes of HeV excretion in naturally infected flying-foxes, and secondarily, to identify between-species variation in excretion prevalence. A total of 2840 flying-foxes from three of the four Australian mainland species (Pteropus alecto, P. poliocephalus and P. scapulatus) were captured and sampled at multiple roost locations in the eastern states of Queensland and New South Wales between 2012 and 2014. A range of biological samples (urine and serum, and urogenital, nasal, oral and rectal swabs) were collected from anaesthetized bats, and tested for HeV RNA using a qRT-PCR assay targeting the M gene. Forty-two P. alecto (n = 1410) had HeV RNA detected in at least one sample, and yielded a total of 78 positive samples, at an overall detection rate of 1.76% across all samples tested in this species (78/4436). The rate of detection, and the amount of viral RNA, was highest in urine samples (>serum, packed haemocytes >faecal >nasal >oral), identifying urine as the most plausible source of infection for flying-foxes and for horses. Detection in a urine sample was more efficient than detection in urogenital swabs, identifying the former as the preferred diagnostic sample. The detection of HeV RNA in serum is consistent with haematogenous spread, and with hypothesised latency and recrudesence in flying-foxes. There were no detections in P. poliocephalus (n = 1168 animals; n = 2958 samples) or P. scapulatus (n = 262 animals; n = 985 samples), suggesting (consistent with other recent studies) that these species are epidemiologically less important than P. alecto in HeV infection dynamics. The study is unprecedented in terms of the individual animal approach, the large sample size, and the use of a molecular assay to directly determine infection status. These features provide a high level of confidence in the veracity of our findings, and a sound basis from which to more precisely target equine risk mitigation strategies.

Cost-effectiveness of a national initiative to improve hand hygiene compliance using the outcome of healthcare associated staphylococcus aureus bacteraemia

Background The objective is to estimate the incremental cost-effectiveness of the Australian National Hand Hygiene Inititiave implemented between 2009 and 2012 using healthcare associated Staphylococcus aureus bacteraemia as the outcome. Baseline comparators are the eight existing state and territory hand hygiene programmes. The setting is the Australian public healthcare system and 1,294,656 admissions from the 50 largest Australian hospitals are included. Methods The design is a cost-effectiveness modelling study using a before and after quasi-experimental design. The primary outcome is cost per life year saved from reduced cases of healthcare associated Staphylococcus aureus bacteraemia, with cost estimated by the annual on-going maintenance costs less the costs saved from fewer infections. Data were harvested from existing sources or were collected prospectively and the time horizon for the model was 12 months, 2011–2012. Findings No useable pre-implementation Staphylococcus aureus bacteraemia data were made available from the 11 study hospitals in Victoria or the single hospital in Northern Territory leaving 38 hospitals among six states and territories available for cost-effectiveness analyses. Total annual costs increased by $2,851,475 for a return of 96 years of life giving an incremental cost-effectiveness ratio (ICER) of $29,700 per life year gained. Probabilistic sensitivity analysis revealed a 100% chance the initiative was cost effective in the Australian Capital Territory and Queensland, with ICERs of $1,030 and $8,988 respectively. There was an 81% chance it was cost effective in New South Wales with an ICER of $33,353, a 26% chance for South Australia with an ICER of $64,729 and a 1% chance for Tasmania and Western Australia. The 12 hospitals in Victoria and the Northern Territory incur annual on-going maintenance costs of $1.51M; no information was available to describe cost savings or health benefits. Conclusions The Australian National Hand Hygiene Initiative was cost-effective against an Australian threshold of $42,000 per life year gained. The return on investment varied among the states and territories of Australia.

Natural Hendra Virus Infection in Flying-Foxes - Tissue Tropism and Risk Factors

Hendra virus (HeV) is a lethal zoonotic agent that emerged in 1994 in Australia. Pteropid bats (flying-foxes) are the natural reservoir. To date, HeV has spilled over from flying-foxes to horses on 51 known occasions, and from infected horses to close-contact humans on seven occasions. We undertook screening of archived bat tissues for HeV by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Tissues were tested from 310 bats including 295 Pteropodiformes and 15 Vespertilioniformes. HeV was detected in 20 individual flying-foxes (6.4%) from various tissues including spleen, kidney, liver, lung, placenta and blood components. Detection was significantly higher in Pteropus Alecto and Pconspicillatus, identifying species as a risk factor for infection. Further, our findings indicate that HeV has a predilection for the spleen, suggesting this organ plays an important role in HeV infection. The lack of detections in the foetal tissues of HeV-positive females suggests that vertical transmission is not a regular mode of transmission in naturally infected flying-foxes, and that placental and foetal tissues are not a major source of infection for horses. A better understanding of HeV tissue tropism will strengthen management of the risk of spillover from flying-foxes to horses and ultimately humans.

Silica Vesicle Nanovaccine Formulations Stimulate Long-Term Immune Responses to the Bovine Viral Diarrhoea Virus E2 Protein

Bovine Viral Diarrhoea Virus (BVDV) is one of the most serious pathogen, which causes tremendous economic loss to the cattle industry worldwide, meriting the development of improved subunit vaccines. Structural glycoprotein E2 is reported to be a major immunogenic determinant of BVDV virion. We have developed a novel hollow silica vesicles (SV) based platform to administer BVDV-1 Escherichia coli-expressed optimised E2 (oE2) antigen as a nanovaccine formulation. The SV-140 vesicles (diameter 50 nm, wall thickness 6 nm, perforated by pores of entrance size 16 nm and total pore volume of 0.934 cm(3)g(-1)) have proven to be ideal candidates to load oE2 antigen and generate immune response. The current study for the first time demonstrates the ability of freeze-dried (FD) as well as non-FD oE2/SV140 nanovaccine formulation to induce long-term balanced antibody and cell mediated memory responses for at least 6 months with a shortened dosing regimen of two doses in small animal model. The in vivo ability of oE2 (100 mu g)/SV-140 (500 mu g) and FD oE2 (100 mu g)/SV-140 (500 mu g) to induce long-term immunity was compared to immunisation with oE2 (100 mu g) together with the conventional adjuvant Quil-A from the Quillaja saponira (10 mu g) in mice. The oE2/SV-140 as well as the FD oE2/SV-140 nanovaccine generated oE2-specific antibody and cell mediated responses for up to six months post the final second immunisation. Significantly, the cell-mediated responses were consistently high in mice immunised with oE2/SV-140 (1,500 SFU/million cells) at the six-month time point. Histopathology studies showed no morphological changes at the site of injection or in the different organs harvested from the mice immunised with 500 mu g SV-140 nanovaccine compared to the unimmunised control. The platform has the potential for developing single dose vaccines without the requirement of cold chain storage for veterinary and human applications.

Spatiotemporal aspects of Hendra Virus infection in Pteropid Bats (Flying-Foxes) in Eastern Australia

Hendra virus (HeV) causes highly lethal disease in horses and humans in the eastern Australian states of Queensland (QLD) and New South Wales (NSW), with multiple equine cases now reported on an annual basis. Infection and excretion dynamics in pteropid bats (flying-foxes), the recognised natural reservoir, are incompletely understood. We sought to identify key spatial and temporal factors associated with excretion in flying-foxes over a 2300 km latitudinal gradient from northern QLD to southern NSW which encompassed all known equine case locations. The aim was to strengthen knowledge of Hendra virus ecology in flying-foxes to improve spillover risk prediction and exposure risk mitigation strategies, and thus better protect horses and humans. Monthly pooled urine samples were collected from under roosting flying-foxes over a three-year period and screened for HeV RNA by quantitative RT-PCR. A generalised linear model was employed to investigate spatiotemporal associations with HeV detection in 13,968 samples from 27 roosts. There was a non-linear relationship between mean HeV excretion prevalence and five latitudinal regions, with excretion moderate in northern and central QLD, highest in southern QLD/northern NSW, moderate in central NSW, and negligible in southern NSW. Highest HeV positivity occurred where black or spectacled flying-foxes were present; nil or very low positivity rates occurred in exclusive grey-headed flying-fox roosts. Similarly, little red flying-foxes are evidently not a significant source of virus, as their periodic extreme increase in numbers at some roosts was not associated with any concurrent increase in HeV detection. There was a consistent, strong winter seasonality to excretion in the southern QLD/northern NSW and central NSW regions. This new information allows risk management strategies to be refined and targeted, mindful of the potential for spatial risk profiles to shift over time with changes in flying-fox species distribution.

Head motion and Inattention/Hyperactivity share common genetic influences: Implications for fMRI studies of ADHD

Head motion (HM) is a well known confound in analyses of functional MRI (fMRI) data. Neuroimaging researchers therefore typically treat HM as a nuisance covariate in their analyses. Even so, it is possible that HM shares a common genetic influence with the trait of interest. Here we investigate the extent to which this relationship is due to shared genetic factors, using HM extracted from resting-state fMRI and maternal and self report measures of Inattention and Hyperactivity-Impulsivity from the Strengths and Weaknesses of ADHD Symptoms and Normal Behaviour (SWAN) scales. Our sample consisted of healthy young adult twins (N = 627 (63% females) including 95 MZ and 144 DZ twin pairs, mean age 22, who had mother-reported SWAN; N = 725 (58% females) including 101 MZ and 156 DZ pairs, mean age 25, with self reported SWAN). This design enabled us to distinguish genetic from environmental factors in the association between head movement and ADHD scales. HM was moderately correlated with maternal reports of Inattention (r = 0.17, p-value = 7.4E-5) and Hyperactivity-Impulsivity (r = 0.16, p-value = 2.9E-4), and these associations were mainly due to pleiotropic genetic factors with genetic correlations [95% CIs] of rg = 0.24 [0.02, 0.43] and rg = 0.23 [0.07, 0.39]. Correlations between self-reports and HM were not significant, due largely to increased measurement error. These results indicate that treating HM as a nuisance covariate in neuroimaging studies of ADHD will likely reduce power to detect between-group effects, as the implicit assumption of independence between HM and Inattention or Hyperactivity-Impulsivity is not warranted. The implications of this finding are problematic for fMRI studies of ADHD, as failing to apply HM correction is known to increase the likelihood of false positives. We discuss two ways to circumvent this problem: censoring the motion contaminated frames of the RS-fMRI scan or explicitly modeling the relationship between HM and Inattention or Hyperactivity-Impulsivity

A comparative structural bioinformatics analysis of the insulin receptor family ectodomain based on phylogenetic information

The insulin receptor (IR), the insulin-like growth factor 1 receptor (IGF1R) and the insulin receptor-related receptor (IRR) are covalently-linked homodimers made up of several structural domains. The molecular mechanism of ligand binding to the ectodomain of these receptors and the resulting activation of their tyrosine kinase domain is still not well understood. We have carried out an amino acid residue conservation analysis in order to reconstruct the phylogeny of the IR Family. We have confirmed the location of ligand binding site 1 of the IGF1R and IR. Importantly, we have also predicted the likely location of the insulin binding site 2 on the surface of the fibronectin type III domains of the IR. An evolutionary conserved surface on the second leucine-rich domain that may interact with the ligand could not be detected. We suggest a possible mechanical trigger of the activation of the IR that involves a slight ‘twist’ rotation of the last two fibronectin type III domains in order to face the likely location of insulin. Finally, a strong selective pressure was found amongst the IRR orthologous sequences, suggesting that this orphan receptor has a yet unknown physiological role which may be conserved from amphibians to mammals.

Trehalose accumulation triggers autophagy during plant desiccation

Global climate change, increasingly erratic weather and a burgeoning global population are significant threats to the sustainability of future crop production. There is an urgent need for the development of robust measures that enable crops to withstand the uncertainty of climate change whilst still producing maximum yields. Resurrection plants possess the unique ability to withstand desiccation for prolonged periods, can be restored upon watering and represent great potential for the development of stress tolerant crops. Here, we describe the remarkable stress characteristics of Tripogon loliiformis, an uncharacterised resurrection grass and close relative of the economically important cereals, rice, sorghum, and maize. We show that T. loliiformis survives extreme environmental stress by implementing autophagy to prevent Programmed Cell Death. Notably, we identified a novel role for trehalose in the regulation of autophagy in T.loliiformis. Transcriptome, Gas Chromatography Mass Spectrometry, immunoblotting and confocal microscopy analyses directly linked the accumulation of trehalose with the onset of autophagy in dehydrating and desiccated T. loliiformis shoots. These results were supported in vitro with the observation of autophagosomes in trehalose treated T. loliiformis leaves; autophagosomes were not detected in untreated samples. Presumably, once induced, autophagy promotes desiccation tolerance in T.loliiformis , by removal of cellular toxins to suppress programmed cell death and the recycling of nutrients to delay the onset of senescence. These findings illustrate how resurrection plants manipulate sugar metabolism to promote desiccation tolerance and may provide candidate genes that are potentially useful for the development of stress tolerant crops.

Blockade of PD-1/PD-L1 promotes adoptive T-Cell immunotherapy in a tolerogenic environment

Adoptive cellular immunotherapy using in vitro expanded CD8+ T cells shows promise for tumour immunotherapy but is limited by eventual loss of function of the transferred T cells through factors that likely include inactivation by tolerogenic dendritic cells (DC). The coinhibitory receptor programmed death-1 (PD-1), in addition to controlling T-cell responsiveness at effector sites in malignancies and chronic viral diseases is an important modulator of dendritic cell-induced tolerance in naive T cell populations. The most potent therapeutic capacity amongst CD8+ T cells appears to lie within Tcm or Tcm-like cells but memory T cells express elevated levels of PD-1. Based on established trafficking patterns for Tcm it is likely Tcm-like cells interact with lymphoid-tissue DC that present tumour-derived antigens and may be inherently tolerogenic to develop therapeutic effector function. As little is understood of the effect of PD-1/PD-L1 blockade on Tcm-like CD8+ T cells, particularly in relation to inactivation by DC, we explored the effects of PD-1/PD-L1 blockade in a mouse model where resting DC tolerise effector and memory CD8+ T cells. Blockade of PD-1/PDL1 promoted effector differentiation of adoptively-transferred Tcm-phenotype cells interacting with tolerising DC. In tumour-bearing mice with tolerising DC, effector activity was increased in both lymphoid tissues and the tumour-site and anti-tumour activity was promoted. Our findings suggest PD-1/PD-L1 blockade may be a useful adjunct for adoptive immunotherapy by promoting effector differentiation in the host of transferred Tcmlike cells. © 2015 Blake et al.

Whole-exome re-sequencing in a family quartet identifies POP1 mutations as the cause of a novel skeletal dysplasia

Recent advances in DNA sequencing have enabled mapping of genes for monogenic traits in families with small pedigrees and even in unrelated cases. We report the identification of disease-causing mutations in a rare, severe, skeletal dysplasia, studying a family of two healthy unrelated parents and two affected children using whole-exome sequencing. The two affected daughters have clinical and radiographic features suggestive of anauxetic dysplasia (OMIM 607095), a rare form of dwarfism caused by mutations of RMRP. However, mutations of RMRP were excluded in this family by direct sequencing. Our studies identified two novel compound heterozygous loss-of-function mutations in POP1, which encodes a core component of the RNase mitochondrial RNA processing (RNase MRP) complex that directly interacts with the RMRP RNA domains that are affected in anauxetic dysplasia. We demonstrate that these mutations impair the integrity and activity of this complex and that they impair cell proliferation, providing likely molecular and cellular mechanisms by which POP1 mutations cause this severe skeletal dysplasia. © 2011 Glazov et al.

The Mycobacterial MsDps2 Protein Is a Nucleoid-Forming DNA Binding Protein Regulated by Sigma Factors sigma(A) and sigma(B)

The Dps (DNA-binding protein from starved cells) proteins from Mycobacterium smegmatis MsDps1 and MsDps2 are both DNA-binding proteins with some differences. While MsDps1 has two oligomeric states, with one of them responsible for DNA binding, MsDps2 has only one DNA-binding oligomeric state. Both the proteins however, show iron-binding activity. The MsDps1 protein has been shown previously to be induced under conditions of starvation and osmotic stress and is regulated by the extra cellular sigma factors sigma(H) and sigma(F). We show here, that the second Dps homologue in M. smegmatis, namely MsDps2, is purified in a DNA-bound form and exhibits nucleoid-like structures under the atomic force microscope. It appears that the N-terminal sequence of Dps2 plays a role in nucleoid formation. MsDps2, unlike MsDps1, does not show elevated expression in nutritionally starved or stationary phase conditions; rather its promoter is recognized by RNA polymerase containing sigma(A) or sigma(B), under in vitro conditions. We propose that due to the nucleoid-condensing ability, the expression of MsDps2 is tightly regulated inside the cells.

Toward the Discovery of Vaccine Adjuvants: Coupling In Silico Screening and In Vitro Analysis of Antagonist Binding to Human and Mouse CCR4 Receptors

Background: Adjuvants enhance or modify an immune response that is made to an antigen. An antagonist of the chemokine CCR4 receptor can display adjuvant-like properties by diminishing the ability of CD4+CD25+ regulatory T cells (Tregs) to down-regulate immune responses. Methodology: Here, we have used protein modelling to create a plausible chemokine receptor model with the aim of using virtual screening to identify potential small molecule chemokine antagonists. A combination of homology modelling and molecular docking was used to create a model of the CCR4 receptor in order to investigate potential lead compounds that display antagonistic properties. Three-dimensional structure-based virtual screening of the CCR4 receptor identified 116 small molecules that were calculated to have a high affinity for the receptor; these were tested experimentally for CCR4 antagonism. Fifteen of these small molecules were shown to inhibit specifically CCR4-mediated cellmigration, including that of CCR4(+) Tregs. Significance: Our CCR4 antagonists act as adjuvants augmenting human T cell proliferation in an in vitro immune response model and compound SP50 increases T cell and antibody responses in vivo when combined with vaccine antigens of Mycobacterium tuberculosis and Plasmodium yoelii in mice.