Differential and defective transcription of koala retrovirus indicates the complexity of host and virus evolution.

Koala retrovirus (KoRV) is unique amongst endogenous (inherited) retroviruses in that its incorporation to the host genome is still active, providing an opportunity to study what drives this fundamental process in vertebrate genome evolution. Animals in the southern part of the natural range of koalas were previously thought to be either virus-free or to have only exogenous variants of KoRV with low rates of KoRV-induced disease. In contrast, animals in the northern part of their range universally have both endogenous and exogenous KoRV with very high rates of KoRV-induced disease such as lymphoma. In this study we use a combination of sequencing technologies, Illumina RNA sequencing of 'southern' (south Australian) and 'northern' (SE QLD) koalas and CRISPR enrichment and nanopore sequencing of DNA of 'southern' (South Australian and Victorian animals) to retrieve full-length loci and intregration sites of KoRV variants. We demonstrate that koalas that tested negative to the KoRV pol gene qPCR, used to detect replication-competent KoRV, are not in fact KoRV-free but harbour defective, presumably endogenous, 'RecKoRV' variants that are not fixed between animals. This indicates that these populations have historically been exposed to KoRV and raises questions as to whether these variants have arisen by chance or whether they provide a protective effect from the infectious forms of KoRV. This latter explanation would offer the intriguing prospect of being able to monitor and selectively breed for disease resistance to protect the wild koala population from KoRV-induced disease.

Deafness in Australian Cattle Dogs associated to QTL on chromosome 20 in genome-wide association study analyses.

Pigment-associated deafness is a common hereditary condition in a range of dog breeds. The aim of this study was to perform a genome-wide association analysis to investigate the genetic architecture of deafness in Australian Cattle Dogs. Genotypes for 104 757 polymorphisms in 216 dogs were available for analyses after quality control. A genomic relationship matrix was used in the mixed model analyses to account for polygenic effects, as we tested each polymorphism for its association with deafness, in a case/control experimental design. Three approaches were used to code the genotypes and test for additive, recessive and dominant SNP effects. The genome-wide association study analyses identified a clear association peak on CFA20, with the most significant SNPs on this chromosome (1.29 × 10-4 ) in the vicinity of MITF. Variants in MITF have been associated with white pigmentation in dogs and with deafness in humans and other species, supporting the premise that canine deafness is associated with variants in or near this gene. A recessive inheritance for the peak in CFA20 is possible given the significant results in the recessive model; however, the estimated heritability was low (4.54 × 10-5 ). Further validation, identification of variants and testing in other dog breeds are needed.

The rare C9 P167S risk variant for age-related macular degeneration increases polymerization of the terminal component of the complement cascade.

Age-related macular degeneration (AMD) is a complex neurodegenerative eye disease with behavioral and genetic etiology and is the leading cause of irreversible vision loss among elderly Caucasians. Functionally significant genetic variants in the alternative pathway of complement have been strongly linked to disease. More recently, a rare variant in the terminal pathway of complement has been associated with increased risk, Complement component 9 (C9) P167S. To assess the functional consequence of this variant, C9 levels were measured in two independent cohorts of AMD patients. In both cohorts, it was demonstrated that the P167S variant was associated with low C9 plasma levels. Further analysis showed that patients with advanced AMD had elevated sC5b-9 compared to those with non-advanced AMD, although this was not associated with the P167S polymorphism. Electron microscopy of membrane attack complexes (MACs) generated using recombinantly produced wild type or P167S C9 demonstrated identical MAC ring structures. In functional assays, the P167S variant displayed a higher propensity to polymerize and a small increase in its ability to induce hemolysis of sheep erythrocytes when added to C9-depleted serum. The demonstration that this C9 P167S AMD risk polymorphism displays increased polymerization and functional activity provides a rationale for the gene therapy trials of sCD59 to inhibit the terminal pathway of complement in AMD that are underway.

Identification of stable reference genes for quantitative PCR in koalas.

To better understand host and immune response to diseases, gene expression studies require identification of reference genes with stable expression for accurate normalisation. This study describes the identification and testing of reference genes with stable expression profiles in koala lymph node tissues across two genetically distinct koala populations. From the 25 most stable genes identified in transcriptome analysis, 11 genes were selected for verification using reverse transcription quantitative PCR, in addition to the commonly used ACTB and GAPDH genes. The expression data were analysed using stable genes statistical software - geNorm, BestKeeper, NormFinder, the comparative ΔCt method and RefFinder. All 13 genes showed relative stability in expression in koala lymph node tissues, however Tmem97 and Hmg20a were identified as the most stable genes across the two koala populations.

Studying the effects of asymmetry on the bending rigidity of lipid membranes formed by microfluidics.

In this article we detail a robust high-throughput microfluidic platform capable of fabricating either symmetric or asymmetric giant unilamellar vesicles (GUVs) and characterise the mechanical properties of their membranes.

The effect of hydrostatic pressure on model membrane domain composition and lateral compressibility.

Phase separation in ternary model membranes is known to occur over a range of temperatures and compositions and can be induced by increasing hydrostatic pressure. We have used small angle X-ray scattering (SAXS) to study phase separation along pre-determined tie lines in dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC) and cholesterol (CHOL) mixtures. We can unequivocally distinguish the liquid ordered (Lo) and liquid disordered (Ld) phases in diffraction patterns from biphasic mixtures and compare their lateral compressibility. The variation of tie line endpoints with increasing hydrostatic pressure was determined, at atmospheric pressure and up to 100 MPa. We find an extension and shift of the tie lines towards the DOPC rich region of the phase diagram at increased pressure, this behaviour differs slightly from that reported for decreasing temperature.

Temperature and pressure tuneable swollen bicontinuous cubic phases approaching nature's length scales.

Bicontinuous cubic structures offer enormous potential in applications ranging from protein crystallisation to drug delivery systems and have been observed in cellular membrane structures. One of the current bottlenecks in understanding and exploiting these structures is that cubic scaffolds produced in vitro are considerably smaller in size than those observed in biological systems, differing by almost an order of magnitude in some cases. We have addressed this technological bottleneck and developed a methodology capable of manufacturing highly swollen bicontinuous cubic membranes with length scales approaching those seen in vivo. Crucially, these cubic systems do not require the presence of proteins. We have generated highly swollen Im3m symmetry bicontinuous cubic phases with lattice parameters of up to 480 Å, composed of ternary mixtures of monoolein, cholesterol and negatively charged lipid (DOPS or DOPG) and we have been able to tune their lattice parameters. The swollen cubic phases are highly sensitive to both temperature and pressure; these structural changes are likely to be controlled by a fine balance between lipid headgroup repulsions and lateral pressure in the hydrocarbon chain region.

Preparation and mechanical characterisation of giant unilamellar vesicles by a microfluidic method.

Giant unilamellar vesicles (GUVs) have a wide range of applications in biology and synthetic biology. As a result, new approaches for constructing GUVs using microfluidic techniques are emerging but there are still significant shortcomings in the control of fundamental vesicle structural parameters such as size, lamellarity, membrane composition and internal contents. We have developed a novel microfluidic platform to generate compositionally-controlled GUVs. Water-in-oil (W/O) droplets formed in a lipid-containing oil flow are transferred across an oil-water interface, facilitating the self-assembly of a phospholipid bilayer. In addition, for the first time we have studied the mechanical properties of the resultant lipid bilayers of the microfluidic GUVs. Using fluctuation analysis we were able to calculate the values for bending rigidity of giant vesicles assembled on chip and demonstrate that these correlate strongly with those of traditional low throughput strategies such as electroformation.

A microfluidic platform for size-dependent generation of droplet interface bilayer networks on rails.

Droplet interface bilayer (DIB) networks are emerging as a cornerstone technology for the bottom up construction of cell-like and tissue-like structures and bio-devices. They are an exciting and versatile model-membrane platform, seeing increasing use in the disciplines of synthetic biology, chemical biology, and membrane biophysics. DIBs are formed when lipid-coated water-in-oil droplets are brought together-oil is excluded from the interface, resulting in a bilayer. Perhaps the greatest feature of the DIB platform is the ability to generate bilayer networks by connecting multiple droplets together, which can in turn be used in applications ranging from tissue mimics, multicellular models, and bio-devices. For such applications, the construction and release of DIB networks of defined size and composition on-demand is crucial. We have developed a droplet-based microfluidic method for the generation of different sized DIB networks (300-1500 pl droplets) on-chip. We do this by employing a droplet-on-rails strategy where droplets are guided down designated paths of a chip with the aid of microfabricated grooves or "rails," and droplets of set sizes are selectively directed to specific rails using auxiliary flows. In this way we can uniquely produce parallel bilayer networks of defined sizes. By trapping several droplets in a rail, extended DIB networks containing up to 20 sequential bilayers could be constructed. The trapped DIB arrays can be composed of different lipid types and can be released on-demand and regenerated within seconds. We show that chemical signals can be propagated across the bio-network by transplanting enzymatic reaction cascades for inter-droplet communication.

Microfluidic generation of droplet interface bilayer networks incorporating real-time size sorting in linear and non-linear configurations.

In this study, a novel droplet based microfluidic method for the generation of different sized droplet interface bilayers is reported. A microfluidic platform was designed, which allows the generation and packing of picoliter lipid coated water droplets. Droplets were generated by hydrodynamic focusing coupled with selective transport along grooves according to their size. A trapping structure at the end of the groove and a fine control of the flow pressures allowed for the droplets to be successfully trapped and aligned on demand. This technology facilitates the fine control of droplet size production as well as the generation of extended networks from a variety of lipids including 1,2-diphytanoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphocholine in linear and non-linear configurations, which is vital to the application of Droplet Interface Bilayers to biological network construction on-chip.

Consequences of a screening programme on the prevalence of congenital hereditary sensorineural deafness in the Australian Cattle Dog.

Genetic disease testing programmes are used in domestic animal breeds to guide selective breeding with the aim of reducing disease prevalence. We assessed the change in the prevalence of canine congenital hereditary sensorineural deafness (CHSD) in litters of Australian Cattle Dogs following the introduction of a brainstem auditory evoked response (BAER) testing programme. We studied 608 pups from 122 litters from 10 breeding kennels. Despite 10 years of testing (1998-2008), no substantial reduction in prevalence of CHSD was evident in these 10 breeding kennels. Even for the subset of litters in which both parents were BAER tested as normal hearing (305 pups from 58 litters), there was no evidence of substantial reduction in prevalence. Odds ratios for CHSD in pups for each extra year since testing in the kennel commenced were 1.01 (95% CI, 0.88-1.17) and 1.03 (95% CI, 0.82-1.30) respectively for these populations. Amongst 284 dogs from 54 litters with extended pedigrees and both parents BAER-tested normal hearing, observed prevalences of CHSD were highest in pups with no BAER-tested normal grandparents (17% or 5/29) and lowest in pups with all four grandparents tested normal (0% or 0/9). In pups for which one, two and three grandparents tested negative, prevalences of CHSD were 12% (9/74), 9% (9/101) and 8% (6/71) respectively. Hence, testing programmes based on phenotypic screening may not lead to a substantial reduction in recessive genetic disease prevalence over the medium term, even when only tested normal parents are used. Exclusive breeding of litters in which both parents and all four grandparents are BAER-tested normal is expected to reduce CHSD prevalence in pups to the greatest extent over the long term.

Breast cancer risk after radiotherapy for heritable and non-heritable retinoblastoma: a US-UK study.

BACKGROUND: Retinoblastoma is a rare childhood eye cancer caused by germline or somatic mutations in the RB1 gene. Previous studies observed elevated breast cancer risk among retinoblastoma survivors. However, there has been no research on breast cancer risk in relation to radiation (primarily scatter radiation from the primary treatment) and genetic susceptibility of retinoblastoma survivors. METHODS: Two groups of retinoblastoma survivors from the US and UK were selected, and breast cancer risk analysed using a case-control methodology, nesting within the respective cohorts, matching on heritability (that is to say, having bilateral retinoblastoma or being unilateral cases with at least one relative with retinoblastoma), and using exact statistical methods. There were a total of 31 cases and 77 controls. RESULTS: Overall there was no significant variation of breast cancer risk with dose (P>0.5). However, there was a pronounced and significant (P=0.047) increase in the risk of breast cancer with increasing radiation dose for non-heritable retinoblastoma patients and a slight and borderline significant (P=0.072) decrease in risk of breast cancer with increasing radiation dose for heritable retinoblastoma patients, implying significant (P=0.024) heterogeneity in radiation risk between the heritable and non-heritable retinoblastoma groups; this was unaffected by the blindness status. There was no significant effect of any type of alkylating-agent chemotherapy on breast cancer risk (P>0.5). CONCLUSIONS: There is significant radiation-related risk of breast cancer for non-heritable retinoblastoma survivors but no excess risk for heritable retinoblastoma survivors, and no significant risk overall. However, these results are based on very small numbers of cases; therefore, they must be interpreted with caution.

Acute phase proteins in healthy and sick cats.

Serum acute phase protein concentrations are used as diagnostic, therapeutic and prognostic markers in human and, less frequently, in animal medicine. The aim of this study was to determine how the health status and signalment of the cat are associated with concentrations of acute phase proteins. Generally, medians of the positive acute phase proteins appeared to be higher in sick cats compared to healthy cats. In multivariable regression models, log-transformed serum amyloid A concentration was higher in older cats, in sick and in female cats, while log-transformed α1-acid glycoprotein and haptoglobin concentrations were higher in older cats and were associated with interactions of health status (sick/healthy) and gender (male/female). The data from healthy cats in this study contribute to the limited knowledge of normal reference ranges for this species. This study highlights the potential of acute phase proteins as diagnostic markers in sick cats, but also emphasises that the signalment of the cat needs to be taken into consideration.

Hydrostatic pressure effects on a hydrated lipid inverse micellar Fd3m cubic phase.

Over a range of hydration, unsaturated diacylglycerol/phosphatidylcholine mixtures adopt an inverse micellar cubic phase, of crystallographic space group Fd3m. In this study hydrated DOPC:DOG mixtures with a molar ratio close to 1 : 2 were examined as a function of hydrostatic pressure, using synchrotron X-ray diffraction. The small-angle diffraction pattern at atmospheric pressure was used to calculate 2-D sections through the electron density map. Pressure initially has very little effect on the structure of the Fd3m cubic phase, in contrast to its effect on hydrated inverse bicontinuous cubic phases. At close to 2 kbar, a sharp transition occurs from the Fd3m phase to a pair of coexisting phases, an inverse hexagonal H(II) phase plus an (ordered) lamellar phase. Upon increasing the pressure to 3 kbar, a further sharp transition occurs from the H(II) phase to a (fluid) lamellar phase, in coexistence with the ordered lamellar phase. These transitions are fully reversible, but show hysteresis. Remarkably, the lattice parameter of the Fd3m phase is practically independent of pressure. These results show that these two lipids are miscible at low pressure, adopting a single lyotropic phase (Fd3m); they then become immiscible above a critical pressure, phase separating into DOPC-rich and DOG-rich phases.

Identification of a mutation in the para-sodium channel gene of the cattle tick Rhipicephalus microplus associated with resistance to flumethrin but not to cypermethrin.

A mutation in the domain II S4-5 linker region of the para-sodium channel gene has been associated previously with synthetic pyrethroid (SP) resistance in the cattle tick (Rhipicephalus microplus) in Australia. This is a C→A mutation at nucleotide position 190, which results in a leucine to isoleucine amino acid substitution (L64I). In a survey of 15 cattle tick populations with known SP resistance status, sourced from Queensland and New South Wales in Australia, there was a strong relationship (r=0.98) between the proportion of ticks carrying the L64I homozygous resistant genotype and the survival percentage after exposure to a discriminating concentration of cypermethrin in the bioassay, as expected. However, among populations resistant only to flumethrin, the L64I homozygous genotype was not found. The sequence obtained for a 167 bp region including domain II S4-5 linker in flumethrin-resistant ticks identified a G→T non-synonymous mutation at nucleotide position 214 that results in a glycine to valine substitution (G72V). The frequency of the G72V homozygous genotype in each population was found to be moderately related to the survival percentage at the discriminating concentration of flumethrin in the larval packet test (r=0.74). However, a much stronger relationship between genotype and resistance to flumethrin was observed when the heterozygotes of L64I and G72V were added to the G72V homozygotes (r=0.93). These results suggest that there is an interaction between the two mutations in the same gene, such that flumethrin resistance might be conferred by either two copies of the G72V mutation or by being a L64I and G72V heterozygote.

Evolutionary history of DLA class II haplotypes in canine diabetes mellitus through single nucleotide polymorphism genotyping.

Strong linkage disequilibrium (LD) is a characteristic of the major histocompatibility complex (MHC) region, as well as the genome in general in dogs as a consequence of demographic changes with domestication. Disease association studies of MHC haplotypes may be affected by high LD and the resultant shared genetic backgrounds of haplotypes giving associations with linked but non-causative mutations, and also by convergent haplotypes, in which combinations of alleles have arisen independently. This study provides preliminary tools for dog leukocyte antigen (DLA) class II haplotype analysis with 102 single nucleotide polymorphisms (SNPs) identified in 14.6 kb and genotyping of 20 of these SNPs to tag haplotypes in 60 dogs with diabetes mellitus and in 49 non-diabetic dogs. The pattern of LD and analysis of SNP patterns indicated combinations of exon 2 alleles have arisen through both recombination and convergence. For exon 2 haplotypes associated with susceptibility or protection from diabetes mellitus, a region of fixed differences in SNPs across the DQ region was observed, suggesting a region outside exon 2 may be implicated in disease association. Four new DQB1 promoter alleles restricted to diabetic dogs were identified, as well as a substitution difference in the X1 box of the DQB1 promoter that will potentially modify the effect of the protective haplotypes within diabetic dogs.

Multiple paternity in Rhipicephalus (Boophilus) microplus confirmed by microsatellite analysis.

The aim of this study was to determine if individual ticks among the progeny of a single female Rhipicephalus (Boophilus) microplus tick removed from cattle under natural conditions are the result of mating with one or several males. To this end, simulations were run using an existing dataset of genotypes from 8 microsatellite loci to predict the number of samples required and the best locus. Subsequently, 14-22 progeny from each of 15 engorged female ticks removed from three cows, and the engorged females themselves, were genotyped for the BmM1 locus and the minimum number of potential male parents was determined for each progeny group. Of the 15 progeny groups, 10 must have been sired by more than one male, as indicated by the presence of five unique alleles among the progeny or three unique alleles that could not have been contributed by the female. This finding demonstrates multiple paternity in R. microplus.

Spectral domain optical coherence tomography for quantitative evaluation of drusen and associated structural changes in non-neovascular age-related macular degeneration.

BACKGROUND/AIMS: To demonstrate how spectral domain optical coherence tomography (SDOCT) can better evaluate drusen and associated anatomical changes in eyes with non-neovascular age-related macular degeneration (AMD) compared with time domain optical coherence tomography (TDOCT). METHODS: Images were obtained from three eyes of three patients with AMD using an experimental SDOCT system. Both a titanium-sapphire (Ti:sapphire) laser and a superluminescent diode (SLD) were used as a broadband light source to achieve cross-sectional images of the retina. A qualitative and quantitative analysis was performed for structural changes associated with non-neovascular AMD. An automated algorithm was developed to analyse drusen area and volume from SDOCT images. TDOCT was performed using the fast macular scan (StratusOCT, Carl Zeiss Meditec, Dublin, California). RESULTS: SDOCT images can demonstrate structural changes associated with non-neovascular AMD. A new SDOCT algorithm can determine drusen area, drusen volume and proportion of drusen. CONCLUSIONS: With new algorithms to determine drusen area and volume and its unprecedented simultaneous ultra-high speed ultra-high resolution imaging, SDOCT can improve the evaluation of structural abnormalities in non-neovascular AMD.