Design, development and deployment of a web-based patient registry for rare genetic lipid disorders.

Rare genetic lipid disorders comprise all the monogenic disorders of lipoprotein metabolism with the exception of heterozygous familial hypercholesterolaemia (FH). The creation and maintenance of patient registries is critical for disease monitoring, improving clinical best practice, facilitating research and enabling the development of novel therapeutics, but very few disease-specific rare genetic lipid disorder registries currently exist. Our aim was to design, develop and deploy a web-based patient registry for rare genetic lipid disorders. The Rare Genetic Lipid Disorders Registry is based on the FH Australasia Network (FHAN) Registry, which has been operating since 2015. The Rare Genetic Lipid Disorders Registry was deployed utilising the open-source Rare Disease Registry Framework (RDRF), which enables the efficient customisation and sustainable deployment of web-based registries. The Registry has been designed to capture longitudinal data on 13 rare genetic lipid disorders, with the ability to add more if required in the future. Recruitment of volunteers into the Registry is currently through the Royal Perth Hospital Lipid Disorders Clinic in Western Australia. Although in essence a clinic-based patient registry, the web-based design allows for expansion and distribution across Australia and beyond. Data collated by the Registry may ultimately improve the diagnosis, management and treatment of these conditions.

Grapevine virus I, a putative new vitivirus detected in co-infection with grapevine virus G in New Zealand.

A novel virus, with characteristics of viruses classified within the genus Vitivirus, was identified from a sample of Vitis vinifera cv. Chardonnay in New Zealand. The virus was detected with high throughput sequencing (small RNA and total RNA) and its sequence was confirmed by Sanger sequencing. Its genome is 7507 nt long (excluding the polyA tail) with an organisation similar to that described for other classifiable members of the genus Vitivirus. The closest relative of the virus is grapevine virus E (GVE) with 65% aa identity in ORF1 (65% nt identity) and 63% aa identity in the coat protein (66% nt identity). The relationship with GVE was confirmed with phylogenetic analysis, showing the new virus branching with GVE, Agave tequilina leaf virus and grapevine virus G (GVG). A limited survey revealed the presence of this virus in multiple plants from the same location where the newly described GVG was discovered, and in most cases both viruses were detected as co-infections. The genetic characteristics of this virus suggest it represents an isolate of a new species within the genus Vitivirus and following the current nomenclature, we propose the name "Grapevine virus I".

De novo assembly of honey bee RNA viral genomes by tapping into the innate insect antiviral response pathway.

Bee pollination is critical for improving productivity of one third of all plants or plant products consumed by humans. The health of honey bees is in decline in many countries worldwide, and RNA viruses together with other biological, environmental and anthropogenic factors have been identified as the main causes. The rapid genetic variation of viruses represents a challenge for diagnosis. Thus, application of deep sequencing methods for detection and analysis of viruses has increased over the last years. In this study, we leverage from the innate Dicer-2 mediated antiviral response against viruses to reconstruct complete viral genomes using virus-derived small interfering RNAs (vsiRNAs). Symptomatic A. mellifera larvae collected from hives free of Colony Collapse Disorder (CCD) and the parasitic Varroa mite (Varroa destructor) were used to generate more than 107 million small RNA reads. We show that de novo assembly of insect viral sequences is less fragmented using only 22 nt long vsiRNAs rather than a combination of 21-22 nt small RNAs. Our results show that A. mellifera larvae activate the RNAi immune response in the presence of Sacbrood virus (SBV). We assembled three SBV genomes from three individual larvae from different hives in a single apiary, with 1-2% nucleotide sequence variability among them. We found 3-4% variability between SBV genomes generated in this study and earlier published Australian variants suggesting the presence of different SBV quasispecies within the country.

Identification of a novel vitivirus from grapevines in New Zealand.

We report a sequence of a novel vitivirus from Vitis vinifera obtained using two high-throughput sequencing (HTS) strategies on RNA. The initial discovery from small-RNA sequencing was confirmed by HTS of the total RNA and Sanger sequencing. The new virus has a genome structure similar to the one reported for other vitiviruses, with five open reading frames (ORFs) coding for the conserved domains described for members of that genus. Phylogenetic analysis of the complete genome sequence confirmed its affiliation to the genus Vitivirus, with the closest described viruses being grapevine virus E (GVE) and Agave tequilana leaf virus (ATLV). However, the virus we report is distinct and shares only 51% amino acid sequence identity with GVE in the replicase polyprotein and 66.8% amino acid sequence identity with ATLV in the coat protein. This is well below the threshold determined by the ICTV for species demarcation, and we propose that this virus represents a new species. It is provisionally named "grapevine virus G".

Design of a framework for the deployment of collaborative independent rare disease-centric registries: Gaucher disease registry model.

Orphan drug clinical trials often are adversely affected by a lack of high quality treatment efficacy data that can be reliably compared across large patient cohorts derived from multiple governmental and country jurisdictions. It is critical that these patient data be captured with limited corporate involvement. For some time, there have been calls to develop collaborative, non-proprietary, patient-centric registries for post-market surveillance of aspects related to orphan drug efficacy. There is an urgent need for the development and sustainable deployment of these 'independent' registries that can capture comprehensive clinical, genetic and therapeutic information on patients with rare diseases. We therefore extended an open-source registry platform, the Rare Disease Registry Framework (RDRF) to establish an Independent Rare Disease Registry (IRDR). We engaged with an established rare disease community for Gaucher disease to determine system requirements, methods of data capture, consent, and reporting. A non-proprietary IRDR model is presented that can serve as autonomous data repository, but more importantly ensures that the relevant data can be made available to appropriate stakeholders in a secure, timely and efficient manner to improve clinical decision-making and the lives of those with a rare disease.

A web-based, patient driven registry for Angelman syndrome: the global Angelman syndrome registry.

Angelman syndrome (AS) is a rare neurodevelopmental disorder that is characterised by severe global developmental delays, ataxia, loss of speech, epilepsy, sleep disorders, and a happy disposition. There is currently no cure for AS, though several pharmaceutical companies are anticipating drug trials for new therapies to treat AS. The Foundation for Angelman Therapeutics (FAST) Australia therefore identified a need for a global AS patient registry to identify patients for recruitment for clinical trials.The Global AS Registry was deployed in September 2016 utilising the Rare Disease Registry Framework, an open-source tool that enables the efficient creation and management of patient registries. The Global AS Registry is web-based and allows parents and guardians worldwide to register, provide informed consent, and enter data on individuals with AS. 286 patients have registered in the first 8 months since deployment.We demonstrate the successful deployment of the first patient-driven global registry for AS. The data generated from the Global AS Registry will be crucial in identifying patients suitable for clinical trials and in informing research that will identify treatments for AS, and ultimately improve the lives of individuals and their families living with AS.

Design of the Familial Hypercholesterolaemia Australasia Network Registry: Creating Opportunities for Greater International Collaboration.

Familial Hypercholesterolemia (FH) is the most common and serious monogenic disorder of lipoprotein metabolism that leads to premature coronary heart disease. There are over 65,000 people estimated to have FH in Australia, but many remain undiagnosed. Patients with FH are often under-treated, but with early detection, cascade family testing and adequate treatment, patient outcomes can improve. Patient registries are key tools for providing new information on FH and enhancing care worldwide. The development and design of the FH Australasia Network Registry is a crucial component in the comprehensive model of care for FH, which aims to provide a standardized, high-quality and cost-effective system of care that is likely to have the highest impact on patient outcomes. Informed by stakeholder engagement, the FH Australasia Network Registry was collaboratively developed by government, patient and clinical networks and research groups. The open-source, web-based Rare Disease Registry Framework was the architecture chosen for this registry owing to its open-source standards, modular design, interoperability, scalability and security features; all these are key components required to meet the ever changing clinical demands across regions. This paper provides a high level blueprint for other countries and jurisdictions to help inform and map out the critical features of an FH registry to meet their particular health system needs.

An internet-based bioinformatics toolkit for plant biosecurity diagnosis and surveillance of viruses and viroids.

BACKGROUND: Detection and preventing entry of exotic viruses and viroids at the border is critical for protecting plant industries trade worldwide. Existing post entry quarantine screening protocols rely on time-consuming biological indicators and/or molecular assays that require knowledge of infecting viral pathogens. Plants have developed the ability to recognise and respond to viral infections through Dicer-like enzymes that cleave viral sequences into specific small RNA products. Many studies reported the use of a broad range of small RNAs encompassing the product sizes of several Dicer enzymes involved in distinct biological pathways. Here we optimise the assembly of viral sequences by using specific small RNA subsets. RESULTS: We sequenced the small RNA fractions of 21 plants held at quarantine glasshouse facilities in Australia and New Zealand. Benchmarking of several de novo assembler tools yielded SPAdes using a kmer of 19 to produce the best assembly outcomes. We also found that de novo assembly using 21-25 nt small RNAs can result in chimeric assemblies of viral sequences and plant host sequences. Such non-specific assemblies can be resolved by using 21-22 nt or 24 nt small RNAs subsets. Among the 21 selected samples, we identified contigs with sequence similarity to 18 viruses and 3 viroids in 13 samples. Most of the viruses were assembled using only 21-22 nt long virus-derived siRNAs (viRNAs), except for one Citrus endogenous pararetrovirus that was more efficiently assembled using 24 nt long viRNAs. All three viroids found in this study were fully assembled using either 21-22 nt or 24 nt viRNAs. Optimised analysis workflows were customised within the Yabi web-based analytical environment. We present a fully automated viral surveillance and diagnosis web-based bioinformatics toolkit that provides a flexible, user-friendly, robust and scalable interface for the discovery and diagnosis of viral pathogens. CONCLUSIONS: We have implemented an automated viral surveillance and diagnosis (VSD) bioinformatics toolkit that produces improved viruses and viroid sequence assemblies. The VSD toolkit provides several optimised and reusable workflows applicable to distinct viral pathogens. We envisage that this resource will facilitate the surveillance and diagnosis viral pathogens in plants, insects and invertebrates.

A Web-Based Registry for Familial Hypercholesterolaemia.

Familial hypercholesterolaemia (FH) is the most common and serious monogenic disorder of lipoprotein metabolism that leads to premature coronary heart disease. Patients with FH are often under-treated, and many remain undiagnosed. The deployment of the FH Australasia Network Registry is a crucial component of the comprehensive model of care for FH, which aims to provide a standardised, high-quality and cost-effective system of care that is likely to have the highest impact on patient outcomes. The FH Australasia Network Registry was customised using a registry framework that is an open source, interoperable system that enables the efficient customisation and deployment of national and international web-based disease registries that can be modified dynamically as registry requirements evolve. The FH Australasia Network Registry can be employed to improve health services for FH patients across the Australasia-Pacific region, through the collation of data to facilitate clinical service planning, clinical trials, clinical audits, and to inform clinical best practice.

Mistletoebirds and xylose: Australian frugivores differ in their handling of dietary sugars.

Carbohydrate-rich mistletoe fruits are consumed by a wide range of avian species. Small birds absorb a large portion of water-soluble nutrients, such as glucose, via the paracellular pathway. d-xylose, a pentose monosaccharide, is abundant in some nectars and mistletoe fruits consumed by birds, and it has been suggested that it is most likely absorbed via the paracellular pathway in birds. We measured apparent assimilation efficiency ([Formula: see text]) and bioavailability (f) for d-xylose and d- and l-glucose in three frugivorous Australian bird species. Mistletoebirds, silvereyes, and singing honeyeaters showed significantly lower [Formula: see text] for d-xylose than for d-glucose. Across two diet sugar concentrations, silvereyes and singing honeyeaters significantly increased f of both l-glucose (a metabolically inert isomer of d-glucose commonly used to quantify paracellular uptake) and d-xylose on the more concentrated diet, probably because of increased gut processing time. By contrast, mistletoebirds (mistletoe fruit specialists) did not vary f of either sugar with diet concentration. Mistletoebirds also showed higher f for d-xylose than l-glucose and eliminated d-xylose more slowly than silvereyes and singing honeyeaters, demonstrating differences in the handling of dietary xylose between these species. Our results suggest that d-xylose may be absorbed by both mediated and nonmediated mechanisms in mistletoebirds.

Sugar preferences of avian nectarivores are correlated with intestinal sucrase activity.

Nectar-feeding birds generally demonstrate preference for hexose solutions at low sugar concentrations, switching to sucrose/no preference at higher concentrations. Species vary in the concentration at which the switch from hexose preference occurs; this could reflect physiological constraints that would also influence nectar selection when foraging. We recorded concentration-dependent sugar type preferences in three opportunistic/generalist Australian nectarivorous species: Dicaeum hirundinaceum, Zosterops lateralis, and Lichenostomus virescens. All three preferred hexoses up to sugar concentrations of 0.25 mol L(-1) and switched to sucrose/no preference for higher concentrations. Using these and literature records, we investigated physiological mechanisms that may explain the concentration dependence of sugar type preferences and compared diet preference data with foraging records. We measured sucrase activity in Z. lateralis and L. virescens as well as three specialized nectarivorous species (Anthochaera carunculata, Phylidonyris novaehollandiae, and Trichoglossus haematodus) for comparison with published concentration-dependent sugar preference data. Sucrase activity varied between these species ([Formula: see text]). The minimum diet concentration at which birds show no sugar preference was significantly correlated with sucrase activity for the 11 species analyzed ([Formula: see text]). Birds with the lowest sucrase activity showed hexose preference at higher diet concentrations, and birds with the greatest sucrase activity showed either no hexose preference or hexose preference on only the most dilute diets. Foraging data compiled from the literature also support the laboratory analyses; for example, T. haematodus (preference for hexose over a wide range of diet concentrations, low sucrase activity) also feed primarily on hexose nectars in the wild. Intestinal sucrase activity is likely to contribute to diet selectivity in nectarivorous bird species.

Gastrointestinal and renal responses to variable water intake in whitebellied sunbirds and New Holland honeyeaters.

Nectarivores face a constant challenge in terms of water balance, experiencing water loading or dehydration when switching between food plants or between feeding and fasting. To understand how whitebellied sunbirds and New Holland honeyeaters meet the challenges of varying preformed water load, we used the elimination of intramuscular-injected [(14)C]-l-glucose and (3)H2O to quantify intestinal and renal water handling on diets varying in sugar concentration. Both sunbirds and honeyeaters showed significant modulation of intestinal water absorption, allowing excess water to be shunted through the intestine when on dilute diets. Despite reducing their fractional water absorption, both species showed linear increases in water flux and fractional body water turnover as water intake increased (both afternoon and morning), suggesting that the modulation of fractional water absorption was not sufficient to completely offset dietary water loads. In both species, glomerular filtration rate was independent of water gain (but was higher for the afternoon), as was renal fractional water reabsorption (measured in the afternoon). During the natural overnight fast, both sunbirds and honeyeaters arrested whole kidney function. Evaporative water loss in sunbirds was variable but correlated with water gain. Both sunbirds and honeyeaters appear to modulate intestinal water absorption as an important component of water regulation to help deal with massive preformed water loads. Shutting down glomerular filtration rate during the overnight fast is another way of saving energy for osmoregulatory function. Birds maintain osmotic balance on diets varying markedly in preformed water load by varying both intestinal water absorption and excretion through the intestine and kidneys.

A comparison of pharmacokinetic methods for in vivo studies of nonmediated glucose absorption.

Two pharmacokinetic methods are used primarily to assess systematic bioavailability of orally dosed water-soluble compounds in vivo, but there have been no direct comparisons of the estimates obtained. The "area under the curve" (AUC) method employs a single oral dose of probe compound(s) followed by multiple blood sampling to obtain plasma concentration time curves. Separate injection of probe(s) followed by multiple blood sampling is used to calculate fractional elimination rate (K(el)) and distribution pool space (S). The "steady state feeding" method relies on ad lib. feeding of a marked diet, with a single blood sample taken to measure steady state feeding concentration of probe(s); K(el) is estimated from the decline in probe concentration in excreta after injection, with a single blood sample taken to estimate S. We compared these methods directly in the Australian red wattlebird (Anthochaera carnunculata), measuring absorption of (3)H-L-glucose. The K(el) values estimated using the steady state feeding protocol were significantly higher, and estimates of S and bioavailability consequently lower, compared with the AUC protocol. The AUC method relies on fewer assumptions and allows simultaneous comparisons of absorption by mediated and nonmediated (i.e., paracellular) mechanisms but cannot be easily applied to freely feeding animals. The steady state feeding method allows work with smaller species and exploration of the effects of feeding on nutrient uptake but requires careful attention to the validity of assumptions that increase error in the calculations.

Mechanism and rate of glucose absorption differ between an Australian honeyeater (Meliphagidae) and a lorikeet (Loriidae).

Efficient mechanisms of glucose absorption are necessary for volant animals as a means of reducing mass during flight: they speed up gut transit time and require smaller volume and mass of gut tissue. One mechanism that may be important is absorption via paracellular (non-mediated) pathways. This may be particularly true for nectarivorous species which encounter large quantities of sugar in their natural diet. We investigated the extent of mediated and non-mediated glucose absorption in red wattlebirds Anthochaera carunculata (Meliphagidae) and rainbow lorikeets Trichoglossus haematodus (Loriidae) to test the hypothesis that paracellular uptake accounts for a significant proportion of total glucose uptake in these species. We found that routes of glucose absorption are highly dynamic in both species. In lorikeets, absorption of L-glucose (non-mediated uptake) is slower than that of D-glucose (mediated and non-mediated uptake), with as little as 10% of total glucose absorbed by the paracellular pathway initially (contrasting previous indirect estimates of approximately 80%). Over time, however, more glucose may be absorbed via the paracellular route. Glucose absorption by both mediated and non-mediated mechanisms in wattlebirds occurred at a faster rate than in lorikeets, and wattlebirds also rely substantially on paracellular uptake. In wattlebirds, we recorded higher bioavailability of L-glucose (96+/-3%) compared with D-glucose (57+/-2%), suggesting problems with the in vivo use of radiolabeled d-glucose. Further trials with 3-O-methyl-D-glucose revealed high bioavailability in wattlebirds (90+/-5%). This non-metabolisable glucose analogue remains the probe of choice for measuring uptake rates in vivo, especially in birds in which absorption and metabolism occur extremely rapidly.

The sweet life: diet sugar concentration influences paracellular glucose absorption.

Small birds and bats face strong selection pressure to digest food rapidly in order to reduce digesta mass carried during flight. One mechanism is rapid absorption of a high proportion of glucose via the paracellular pathway (transfer between epithelial cells, not mediated by transporter proteins). Intestinal paracellular permeability to glucose was assessed for two nectarivorous passerines, the Australian New Holland honeyeater (Phylidonyris novaehollandiae) and African white-bellied sunbird (Cinnyris talatala) by measuring the bioavailability of radiolabelled, passively absorbed L-glucose. Bioavailability was high in both species and increased with diet sugar concentration (honeyeaters, 37 and 81% and sunbirds, 53 and 71% for 250 and 1,000 mmoll-1 sucrose diets, respectively). We conclude that the relative contribution of paracellular to total glucose absorption increases with greater digesta retention time in the intestine, and paracellular absorption may also be modulated by factors such as intestinal lumen osmolality and interaction with mediated glucose uptake. The dynamic state of paracellular absorption should be taken into account in future studies.