Adaptive selection of a prion strain conformer corresponding to established North American CWD during propagation of novel emergent Norwegian strains in mice expressing elk or deer prion protein.

Prions are infectious proteins causing fatal, transmissible neurodegenerative diseases of animals and humans. Replication involves template-directed refolding of host encoded prion protein, PrPC, by its infectious conformation, PrPSc. Following its discovery in captive Colorado deer in 1967, uncontrollable contagious transmission of chronic wasting disease (CWD) led to an expanded geographic range in increasing numbers of free-ranging and captive North American (NA) cervids. Some five decades later, detection of PrPSc in free-ranging Norwegian (NO) reindeer and moose marked the first indication of CWD in Europe. To assess the properties of these emergent NO prions and compare them with NA CWD we used transgenic (Tg) and gene targeted (Gt) mice expressing PrP with glutamine (Q) or glutamate (E) at residue 226, a variation in wild type cervid PrP which influences prion strain selection in NA deer and elk. Transmissions of NO moose and reindeer prions to Tg and Gt mice recapitulated the characteristic features of CWD in natural hosts, revealing novel prion strains with disease kinetics, neuropathological profiles, and capacities to infect lymphoid tissues and cultured cells that were distinct from those causing NA CWD. In support of strain variation, PrPSc conformers comprising emergent NO moose and reindeer CWD were subject to selective effects imposed by variation at residue 226 that were different from those controlling established NA CWD. Transmission of particular NO moose CWD prions in mice expressing E at 226 resulted in selection of a kinetically optimized conformer, subsequent transmission of which revealed properties consistent with NA CWD. These findings illustrate the potential for adaptive selection of strain conformers with improved fitness during propagation of unstable NO prions. Their potential for contagious transmission has implications for risk analyses and management of emergent European CWD. Finally, we found that Gt mice expressing physiologically controlled PrP levels recapitulated the lymphotropic properties of naturally occurring CWD strains resulting in improved susceptibilities to emergent NO reindeer prions compared with over-expressing Tg counterparts. These findings underscore the refined advantages of Gt models for exploring the mechanisms and impacts of strain selection in peripheral compartments during natural prion transmission.

Application of a bioinformatics training delivery method for reaching dispersed and distant trainees.

Many initiatives have addressed the global need to upskill biologists in bioinformatics tools and techniques. Australia is not unique in its requirement for such training, but due to its large size and relatively small and geographically dispersed population, Australia faces specific challenges. A combined training approach was implemented by the authors to overcome these challenges. The "hybrid" method combines guidance from experienced trainers with the benefits of both webinar-style delivery and concurrent face-to-face hands-on practical exercises in classrooms. Since 2017, the hybrid method has been used to conduct 9 hands-on bioinformatics training sessions at international scale in which over 800 researchers have been trained in diverse topics on a range of software platforms. The method has become a key tool to ensure scalable and more equitable delivery of short-course bioinformatics training across Australia and can be easily adapted to other locations, topics, or settings.

Primary structural differences at residue 226 of deer and elk PrP dictate selection of distinct CWD prion strains in gene-targeted mice.

Although the unifying hallmark of prion diseases is CNS neurodegeneration caused by conformational corruption of host prion protein (PrP) to its infective counterpart, contagious transmission of chronic wasting disease (CWD) results from shedding of prions produced at high titers in the periphery of diseased cervids. While deer and elk PrP primary structures are equivalent except at residue 226, which is glutamate in elk and glutamine in deer, the effect of this difference on CWD pathogenesis is largely unknown. Using a gene-targeting approach where the mouse PrP coding sequence was replaced with elk or deer PrP, we show that the resulting GtE226 and GtQ226 mice had distinct kinetics of disease onset, prion conformations, and distributions of prions in the brains of diseased mice following intracerebral CWD challenge. These findings indicate that amino acid differences at PrP residue 226 dictate the selection and propagation of divergent strains in deer and elk with CWD. Because prion strain properties largely dictate host-range potential, our findings suggest that prion strains from elk and deer pose distinct risks to sympatric species or humans exposed to CWD. GtE226 and GtQ226 mice were also highly susceptible to CWD prions following intraperitoneal and oral exposures, a characteristic that stood in stark contrast to previously produced transgenic models. Remarkably, disease transmission was effective when infected mice were cohoused with naïve cagemates. Our findings indicate that gene-targeted mice provide unprecedented opportunities to accurately investigate CWD peripheral pathogenesis, CWD strains, and mechanisms of horizontal CWD transmission.

Comparative analysis of prions in nervous and lymphoid tissues of chronic wasting disease-infected cervids.

The prevalence, host range and geographical bounds of chronic wasting disease (CWD), the prion disease of cervids, are expanding. Horizontal transmission likely contributes the majority of new CWD cases, but the mechanism by which prions are transmitted among CWD-affected cervids remains unclear. To address the extent to which prion amplification in peripheral tissues contributes to contagious transmission, we assessed the prion levels in central nervous and lymphoreticular system tissues in white-tailed deer (Odocoileus virginianus), red deer (Cervus elaphus elaphus) and elk (Cervus canadensis). Using real-time quaking-induced conversion, cervid prion cell assay and transgenic mouse bioassay, we found that the retropharyngeal lymph nodes of red deer, white-tailed deer and elk contained similar prion titres to brain from the same individuals. We propose that marked lymphotropism is essential for the horizontal transmission of prion diseases and postulate that shed CWD prions are produced in the periphery.

Deficiency of Isoprenylcysteine Carboxyl Methyltransferase (ICMT) Leads to Progressive Loss of Photoreceptor Function.

UNLABELLED: Retinal neurons use multiple strategies to fine-tune visual signal transduction, including post-translational modifications of proteins, such as addition of an isoprenyl lipid to a carboxyl-terminal cysteine in proteins that terminate with a "CAAX motif." We previously showed that RAS converting enzyme 1 (RCE1)-mediated processing of isoprenylated proteins is required for photoreceptor maintenance and function. However, it is not yet known whether the requirement for the RCE1-mediated protein processing is related to the absence of the endoproteolytic processing step, the absence of the subsequent methylation step by isoprenylcysteine methyltransferase (ICMT), or both. To approach this issue and to understand the significance of protein methylation, we generated mice lacking Icmt expression in the retina. In the absence of Icmt expression, rod and cone light-mediated responses diminished progressively. Lack of ICMT-mediated methylation led to defective association of isoprenylated transducin and cone phosphodiesterase 6 (PDE6α') with photoreceptor membranes and resulted in decreased levels of transducin, PDE6α', and cone G-protein coupled receptor kinase-1 (GRK1). In contrast to our earlier findings with retina-specific Rce1 knock-out mice, rod PDE6 in Icmt-deficient mice trafficked normally to the photoreceptor outer segment, suggesting that the failure to remove the -AAX is responsible for blocking the movement of PDE6 to the outer segment. Our findings demonstrate that carboxyl methylation of isoprenylated proteins is crucial for maintenance of photoreceptor function. SIGNIFICANCE STATEMENT: In this report, we show that an absence of isoprenylcysteine methyltransferase-mediated protein methylation leads to progressive loss of vision. Photoreceptors also degenerate, although at a slower pace than the rate of visual loss. The reduction in photoresponses is due to defective association of crucial players in phototransduction cascade. Unlike the situation with RCE1 deficiency, where both methylation and removal of -AAX were affected, the transport of isoprenylated proteins in isoprenylcysteine methyltransferase-deficient retinas was not dependent on methylation. This finding implies that the retention of the -AAX in PDE6 catalytic subunits in Rce1(-/-) mice is responsible for impeding their transport to the rod photoreceptor outer segment. In conclusion, lack of methylation of isoprenylcysteines leads to age-dependent photoreceptor dysfunction.

Structural effects of PrP polymorphisms on intra- and interspecies prion transmission.

Understanding the molecular parameters governing prion propagation is crucial for controlling these lethal, proteinaceous, and infectious neurodegenerative diseases. To explore the effects of prion protein (PrP) sequence and structural variations on intra- and interspecies transmission, we integrated studies in deer, a species naturally susceptible to chronic wasting disease (CWD), a burgeoning, contagious epidemic of uncertain origin and zoonotic potential, with structural and transgenic (Tg) mouse modeling and cell-free prion amplification. CWD properties were faithfully maintained in deer following passage through Tg mice expressing cognate PrP, and the influences of naturally occurring PrP polymorphisms on CWD susceptibility were accurately reproduced in Tg mice or cell-free systems. Although Tg mice also recapitulated susceptibility of deer to sheep prions, polymorphisms that provided protection against CWD had distinct and varied influences. Whereas substitutions at residues 95 and 96 in the unstructured region affected CWD propagation, their protective effects were overridden during replication of sheep prions in Tg mice and, in the case of residue 96, deer. The inhibitory effects on sheep prions of glutamate at residue 226 in elk PrP, compared with glutamine in deer PrP, and the protective effects of the phenylalanine for serine substitution at the adjacent residue 225, coincided with structural rearrangements in the globular domain affecting interaction between α-helix 3 and the loop between ß2 and α-helix 2. These structure-function analyses are consistent with previous structural investigations and confirm a role for plasticity of this tertiary structural epitope in the control of PrP conversion and strain propagation.

RAS-converting enzyme 1-mediated endoproteolysis is required for trafficking of rod phosphodiesterase 6 to photoreceptor outer segments.

Prenylation is the posttranslational modification of a carboxyl-terminal cysteine residue of proteins that terminate with a CAAX motif. Following prenylation, the last three amino acids are cleaved off by the endoprotease, RAS-converting enzyme 1 (RCE1), and the prenylcysteine residue is methylated. Although it is clear that prenylation increases membrane affinity of CAAX proteins, less is known about the importance of the postprenylation processing steps. RCE1 function has been studied in a variety of tissues but not in neuronal cells. To approach this issue, we generated mice lacking Rce1 in the retina. Retinal development proceeded normally in the absence of Rce1, but photoreceptor cells failed to respond to light and subsequently degenerated in a rapid fashion. In contrast, the inner nuclear and ganglion cell layers were unaffected. We found that the multimeric rod phosphodiesterase 6 (PDE6), a prenylated protein and RCE1 substrate, was unable to be transported to the outer segments in Rce1-deficient photoreceptor cells. PDE6 present in the inner segment of Rce1-deficient photoreceptor cells was assembled and functional. Synthesis and transport of transducin, and rhodopsin kinase 1 (GRK1), also prenylated substrates of RCE1, was unaffected by Rce1 deficiency. We conclude that RCE1 is essential for the intracellular trafficking of PDE6 and survival of photoreceptor cells.

The proteome browser web portal.

In 2010, the Human Proteome Organization launched the Human Proteome Project (HPP), aimed at identifying and characterizing the proteome of the human body. To support complete coverage, one arm of the project will take a gene- or chromosomal-centric strategy (C-HPP) aimed at identifying at least one protein product from each protein-coding gene. Despite multiple large international biological databases housing genomic and protein data, there is currently no single system that integrates updated pertinent information from each of these data repositories and assembles the information into a searchable format suitable for the type of global proteomics effort proposed by the C-HPP. We have undertaken the goal of producing a data integration and analysis software system and browser for the C-HPP effort and of making data collections from this resource discoverable through metadata repositories, such as Australian National Data Service's Research Data Australia. Here we present our vision and progress toward the goal of developing a comprehensive data integration and analysis software tool that provides a snapshot of currently available proteomic related knowledge around each gene product, which will ultimately assist in analyzing biological function and the study of human physiology in health and disease.

EMAGE mouse embryo spatial gene expression database: 2010 update.

EMAGE (http://www.emouseatlas.org/emage) is a freely available online database of in situ gene expression patterns in the developing mouse embryo. Gene expression domains from raw images are extracted and integrated spatially into a set of standard 3D virtual mouse embryos at different stages of development, which allows data interrogation by spatial methods. An anatomy ontology is also used to describe sites of expression, which allows data to be queried using text-based methods. Here, we describe recent enhancements to EMAGE including: the release of a completely re-designed website, which offers integration of many different search functions in HTML web pages, improved user feedback and the ability to find similar expression patterns at the click of a button; back-end refactoring from an object oriented to relational architecture, allowing associated SQL access; and the provision of further access by standard formatted URLs and a Java API. We have also increased data coverage by sourcing from a greater selection of journals and developed automated methods for spatial data annotation that are being applied to spatially incorporate the genome-wide (approximately 19,000 gene) 'EURExpress' dataset into EMAGE.

EMAGE--Edinburgh Mouse Atlas of Gene Expression: 2008 update.

EMAGE (http://genex.hgu.mrc.ac.uk/Emage/database) is a database of in situ gene expression patterns in the developing mouse embryo. Domains of expression from raw data images are spatially integrated into a set of standard 3D virtual mouse embryos at different stages of development, allowing data interrogation by spatial methods. Sites of expression are also described using an anatomy ontology and data can be queried using text-based methods. Here we describe recent enhancements to EMAGE which include advances in spatial search methods including: a refined local spatial similarity search algorithm, a method to allow global spatial comparison of patterns in EMAGE and subsequent hierarchical-clustering, and spatial searches across multiple stages of development. In addition, we have extended data access by the introduction of web services and new HTML-based search interfaces, which allow access to data that has not yet been spatially annotated. We have also started incorporating full 3D images of gene expression that have been generated using optical projection tomography (OPT).

Life threatening complication associated with anesthesia in a dog with masticatory muscle myositis.

OBJECTIVE: To report a serious complication in a dog with masticatory muscle myositis (MMM) that occurred during general anesthesia for diagnostic testing. STUDY DESIGN: Case report. ANIMALS: A 2-year-old male Pug. METHODS: MMM was diagnosed in a Pug with a 2-week history of trismus by electrodiagnostics, histopathology, and 2M antibody test. During anesthesia tongue protrusion occurred and because of trismus, an inability to reposition the tongue resulted in venous congestion and severe swelling. Forceful physical attempts and subsequent removal of the rostral digastricus and masseter muscle attachments from the mandible did not increase jaw mobility. Mandibular symphysiotomy was necessary to resolve lingual venous congestion and to reposition the tongue into the oral cavity. RESULTS: Tongue swelling rapidly subsided after symphysiotomy allowing the tongue to be repositioned into the oral cavity. After treatment of MMM with corticosteroids, jaw range of motion improved and at 6 months was approximately 70% normal. CONCLUSIONS: Trismus could not be overcome by detachment of the masseter and digastricus muscle insertions from the mandible, and symphysiotomy was required to reposition the tongue in the oral cavity. CLINICAL RELEVANCE: In dogs with MMM, tongue position should be monitored during anesthesia to avoid inadvertent protrusion and swelling from venous congestion. Use of anesthetic monitoring equipment on the tongue, such as a pulse oximeter probe, should be avoided in these patients.

EMAGE: a spatial database of gene expression patterns during mouse embryo development.

EMAGE (http://genex.hgu.mrc.ac.uk/Emage/database) is a freely available, curated database of gene expression patterns generated by in situ techniques in the developing mouse embryo. It is unique in that it contains standardized spatial representations of the sites of gene expression for each gene, denoted against a set of virtual reference embryo models. As such, the data can be interrogated in a novel and abstract manner by using space to define a query. Accompanying the spatial representations of gene expression patterns are text descriptions of the sites of expression, which also allows searching of the data by more conventional text-based methods.

Best practice data life cycle approaches for the life sciences.

Throughout history, the life sciences have been revolutionised by technological advances; in our era this is manifested by advances in instrumentation for data generation, and consequently researchers now routinely handle large amounts of heterogeneous data in digital formats. The simultaneous transitions towards biology as a data science and towards a 'life cycle' view of research data pose new challenges. Researchers face a bewildering landscape of data management requirements, recommendations and regulations, without necessarily being able to access data management training or possessing a clear understanding of practical approaches that can assist in data management in their particular research domain. Here we provide an overview of best practice data life cycle approaches for researchers in the life sciences/bioinformatics space with a particular focus on 'omics' datasets and computer-based data processing and analysis. We discuss the different stages of the data life cycle and provide practical suggestions for useful tools and resources to improve data management practices.

Severe cholestatic liver disease secondary to liver fluke (Platynosomum concinnum) infection in three cats.

Three middle-aged domestic cats were presented for vomiting, lethargy, anorexia, and jaundice. Complete blood counts, serum biochemical profiles, and abdominal ultrasounds were suggestive of extrahepatic biliary obstruction in all of the cats. Infection with the liver fluke Platynosomum concinnum was confirmed by intraoperative bile cytology in three cases and by histopathology in two cases. All three cats were euthanized in the postoperative period because of complications. These cases illustrate the severity of signs and complications that can occur with liver fluke infection in cats.

Biobank classification in an Australian setting.

In 2011, Watson and Barnes proposed a schema for classifying biobanks into 3 groups (mono-, oligo-, and poly-user), primarily based upon biospecimen access policies. We used results from a recent comprehensive survey of cancer biobanks in New South Wales, Australia to assess the applicability of this biobank classification schema in an Australian setting. Cancer biobanks were identified using publically available data, and by consulting with research managers. A comprehensive survey was developed and administered through a face-to-face setting. Data were analyzed using Microsoft Excel™ 2010 and IBM SPSS Statistics™ version 21.0. The cancer biobank cohort (n=23) represented 5 mono-user biobanks, 7 oligo-user biobanks, and 11 poly-user biobanks, and was analyzed as two groups (mono-/oligo- versus poly-user biobanks). Poly-user biobanks employed significantly more full-time equivalent staff, and were significantly more likely to have a website, share staff between biobanks, access governance support, utilize quality control measures, be aware of biobanking best practice documents, and offer staff training. Mono-/oligo-user biobanks were significantly more likely to seek advice from other biobanks. Our results further delineate a biobank classification system that is primarily based on access policy, and demonstrate its relevance in an Australian setting.

EMAGE: Electronic Mouse Atlas of Gene Expression.

The EMAGE (Electronic Mouse Atlas of Gene Expression) database (http://www.emouseatlas.org/emage) allows users to perform on-line queries of mouse developmental gene expression. EMAGE data are represented spatially using a framework of 3D mouse embryo models, thus allowing uniquely spatial queries to be carried out alongside more traditional text-based queries. This spatial representation of the data also allows a comparison of spatial similarity between the expression patterns. The data are mapped to the models by a team of curators using bespoke mapping software, and the associated meta-data are curated for accuracy and completeness. The data contained in EMAGE are gathered from three main sources: from the published literature, through large-scale screens and collaborations, and via direct submissions from researchers. There are a variety of ways to query the EMAGE database via the on-line search interfaces, as well as via direct computational script-based queries. EMAGE is a free, on-line, community resource funded by the Medical Research Council, UK.

Greasing the protein biosynthesis machinery of photoreceptor neurons: Role for postprenylation processing of proteins.

Daily phagocytosis of outer segments (OS) places extraordinary demands on protein biosynthesis and trafficking in photoreceptor neurons. While the members and roles of the phototransduction pathway in the OS are well characterized, details about protein trafficking are just beginning to emerge. Phosphodiesterase6 (PDE6), the effector enzyme in phototransduction cascade, serves as an example of the steps multimeric proteins must pass through to achieve their functional state in the OS. Genetic model systems have recently provided snapshots of various steps in the pathway, as experimental difficulties such as an inability to maintain ciliated photoreceptor outer segments or express functional PDE6 holoenzyme in vitro necessitate in vivo studies. We will highlight the significant findings, their implications to blinding diseases, as well as discuss the gaps requiring further investigation.

Identification of the hair cell soma-1 antigen, HCS-1, as otoferlin.

Hair cells, the mechanosensitive receptor cells of the inner ear, are critical for our senses of hearing and balance. The small number of these receptor cells in the inner ear has impeded the identification and characterization of proteins important for hair cell function. The binding specificity of monoclonal antibodies provides a means for identifying hair cell-specific proteins and isolating them for further study. We have generated a monoclonal antibody, termed hair cell soma-1 (HCS-1), which specifically immunolabels hair cells in at least five vertebrate classes, including sharks and rays, bony fish, amphibians, birds, and mammals. We used HCS-1 to immunoprecipitate the cognate antigen and identified it as otoferlin, a member of the ferlin protein family. Mutations in otoferlin underlie DFNB9, a recessive, nonsyndromic form of prelingual deafness characterized as an auditory neuropathy. Using immunocytochemistry, we find that otoferlin is associated with the entire basolateral membrane of the hair cells and with vesicular structures distributed throughout most of the hair cell cytoplasm. Biochemical assays indicate that otoferlin is tightly associated with membranes, as it is not solubilized by alterations in calcium or salt concentrations. HCS-1 immunolabeling does not co-localize with ribeye, a constituent of synaptic ribbons, suggesting that otoferlin may, in addition to its proposed function in synaptic vesicle release, play additional roles in hair cells.

Minimum information specification for in situ hybridization and immunohistochemistry experiments (MISFISHIE).

One purpose of the biomedical literature is to report results in sufficient detail that the methods of data collection and analysis can be independently replicated and verified. Here we present reporting guidelines for gene expression localization experiments: the minimum information specification for in situ hybridization and immunohistochemistry experiments (MISFISHIE). MISFISHIE is modeled after the Minimum Information About a Microarray Experiment (MIAME) specification for microarray experiments. Both guidelines define what information should be reported without dictating a format for encoding that information. MISFISHIE describes six types of information to be provided for each experiment: experimental design, biomaterials and treatments, reporters, staining, imaging data and image characterizations. This specification has benefited the consortium within which it was developed and is expected to benefit the wider research community. We welcome feedback from the scientific community to help improve our proposal.