The CARD9 Gene in Koalas (Phascolarctos cinereus): Does It Play a Role in the Cryptococcus-Koala Interaction?

Cryptococcus is a genus of fungal pathogens that can infect and cause disease in a range of host species and is particularly prominent in koalas (Phascolarctos cinerus). Like other host species, koalas display a range of outcomes upon exposure to environmental Cryptococcus, from external nasal colonization to asymptomatic invasive infection and, in rare cases, severe clinical disease resulting in death. Host factors contributing to these varied outcomes are poorly understood. Due to their close relationship with eucalypt trees (a key environmental niche for Cryptococcus gattii) and suspected continual exposure to the pathogen, koalas provide a unique opportunity to examine host susceptibility in natural infections. Caspase recruitment domain-containing protein 9 (CARD9) is a key intracellular signaling protein in the fungal innate immune response. Humans with mutations in CARD9 succumb to several different severe and chronic fungal infections. This study is the first to sequence and explore CARD9 variation in multiple koalas using Sanger sequencing. Four CARD9 exons were successfully sequenced in 22 koalas from a New South Wales, Australia population. We found minimal variation between koalas across all four exons, an observation that was also made when CARD9 sequences were compared between koalas and six other species, including humans and mice. Ten single-nucleotide polymorphisms (SNP) were identified in this study and explored in the context of cryptococcal exposure outcomes. While we did not find any significant association with variation in cryptococcal outcomes, we found a high degree of conservation between species at several SNP loci that requires further investigation. The findings from this study lay the groundwork for further investigations of CARD9 and Cryptococcus both in koalas and other species, and highlight several considerations for future studies.

Bifunctional Au-Sn-SiO2 catalysts promote the direct upgrading of glycerol to methyl lactate.

Valuable alkyl lactates can be obtained from (waste) glycerol, through a two-step process that entails (i) the oxidation of glycerol to dihydroxyacetone (DHA) catalyzed by support Au nanoparticles and (ii) a rearrangement of DHA with an alcohol effectively catalyzed by Sn-based heterogeneous catalysts. To solve selectivity and processing issues we propose to run the process as a cascade reaction, in one step, and with a single bifunctional catalyst. Tackling the challenge associated with the preparation of such bifunctional catalysts, here, an aerosol-assisted sol-gel route is exploited. The catalysts feature small Au nanoparticles (3-4 nm) embedded at the surface of mesoporous Sn-doped silica microspheres. The preparation successfully leads to insert both active sites in their most active forms, and in close proximity. With the bifunctional catalysts, the yield for the final product of the cascade reaction (methyl lactate) is higher than the DHA yield when only the first reaction is carried out. This highlights a beneficial substrate channeling effect which alleviates side reactions. Interestingly, the bifunctional catalysts also markedly outcompeted mechanical mixtures of the corresponding monofunctional Au- and Sn-based catalysts. Thus, the spatial proximity between the two active sites in bifunctional catalysts is identified as a key to stir the cascade reaction towards high lactate yield.

Swapping White for High-Fibre Bread Increases Faecal Abundance of Short-Chain Fatty Acid-Producing Bacteria and Microbiome Diversity: A Randomized, Controlled, Decentralized Trial.

A low-fibre diet leads to gut microbiota imbalance, characterized by low diversity and reduced ability to produce beneficial metabolites, such as short-chain fatty acids (SCFAs). This imbalance is associated with poor gastrointestinal and metabolic health. We aimed to determine whether one dietary change, substitution of white bread with high-fibre bread, improves gut microbiota diversity and SCFA-producing capability. Twenty-two healthy adults completed a two-phase randomized, cross-over trial. The participants consumed three slices of a high-fibre bread (Prebiotic Cape Seed Loaf with BARLEYmax®) or control white bread as part of their usual diet for 2 weeks, with the treatment periods separated by a 4-week washout. High-fibre bread consumption increased total dietary fibre intake to 40 g/d, which was double the amount of fibre consumed at baseline or during the white bread intervention. Compared to white bread, the high-fibre bread intervention resulted in higher faecal alpha diversity (Shannon, p = 0.014) and relative abundance of the Lachnospiracae ND3007 group (p < 0.001, FDR = 0.019) and tended to increase the butyrate-producing capability (p = 0.062). In conclusion, substituting white bread with a high-fibre bread improved the diversity of gut microbiota and specific microbes involved in SCFA production and may enhance the butyrate-producing capability of gut microbiota in healthy adults. These findings suggest that a single dietary change involving high-fibre bread provides a practical way for adults to exceed recommended dietary fibre intake levels that improve gut microbiota composition and support gastrointestinal and metabolic health.

Novel typing scheme reveals emergence and genetic diversity of Chlamydia pecorum at the local management scale across two koala populations.

To overcome shortcomings in discriminating Chlamydia pecorum strains infecting the koala (Phascolarctos cinereus) at the local level, we developed a novel genotyping scheme for this pathogen to inform koala management at a fine-scale subpopulation level. We applied this scheme to two geographically distinct koala populations in New South Wales, Australia: the Liverpool Plains and the Southern Highlands to South-west Sydney (SHSWS). Our method provides greater resolution than traditional multi-locus sequence typing, and can be used to monitor strain emergence, movement, and divergence across a range of fragmented habitats. Within the Liverpool Plains population, suspected recent introduction of a novel strain was confirmed by an absence of genetic diversity at the earliest sampling events and limited diversity at recent sampling events. Across the partially fragmented agricultural landscape of the Liverpool Plains, diversity within a widespread sequence type suggests that this degree of fragmentation may hinder but not prevent spread. In the SHSWS population, our results suggest movement of a strain from the south, where diverse strains exist, into a previously Chlamydia-free area in the north, indicating the risk of expansion towards an adjacent Chlamydia-negative koala population in South-west Sydney. In the south of the SHSWS where koala subpopulations appear segregated, we found evidence of divergent strain evolution. Our tool can be used to infer the risks of strain introduction across fragmented habitats in population management, particularly through practices such as wildlife corridor constructions and translocations.

The influence of the phylum Planctomycetota in the environmental resistome.

Antimicrobial resistance is one of the leading causes of death worldwide and research on this topic has been on the spotlight for a long time. More recently and in agreement with the One Health Approach, the focus has moved towards the environmental resistome. Members of the phylum Planctomycetota are ubiquitously present in the environment including in hotspots for antimicrobial resistance selection and dissemination. Furthermore, phenotypic broad-range resistance has been observed in diverse members of this phylum. Here we review the evidence available on antimicrobial resistance in the underexploited Planctomycetota and highlight key aspects for future studies.

Aspirin Responsiveness in a Cohort of Pediatric Patients with Right Ventricle to Pulmonary Artery Conduits and Transcatheter Valve Replacement Systems.

The aim of this study was to determine the rate of aspirin responsiveness in a cohort of pediatric patients with in situ xenograft valved right ventricle to pulmonary artery (RV-PA) conduits and/or transcatheter valve replacements (TVR). Aspirin is routinely prescribed to these patients. Optimizing anti-platelet therapy could promote valve longevity and reduce the risk of infective endocarditis in this at-risk group. This was a prospective, observational study. Patients were recruited from both ward and outpatient settings. Patients were eligible if under 18 years and taking aspirin. Non-response to aspirin was defined as > 20% platelet aggregation using light transmission platelet aggregometry (LTA) and < 50% platelet inhibition by thromboelastography with platelet mapping (TEGPM). Participants were invited to provide a confirmatory sample in cases of aspirin resistance and dose adjustments were made. Thirty patients participated. Median age was 9 years (2 months to 18 years). The majority (93%) had complex right ventricular outflow tract pathology. 13 (43%) had an RV-PA conduit and 24 (80%) had a TVR, with valve situated in conduit in 7 (23%) cases. Rate of aspirin non-response on initial testing was 23% (n = 7/30) with median LTA 74.55% (60-76%) and TEG 13.25% (0-44%) in non-responders. Non-responders were more likely to be under 1 year. Two patients required dose increases and one patient non-adherence to dose was identified. Four patients on repeat testing were responsive to aspirin by laboratory tests. The rate of aspirin non-response on laboratory testing in this cohort of patients was 23% and resulted in therapeutic intervention in 10%.

A novel multi-variate immunological approach, reveals immune variation associated with environmental conditions, and co-infection in the koala (Phascolarctos cinereus).

External signs of disease are frequently used as indicators of disease susceptibility. However, immune profiling can be a more effective indicator to understand how host responses to infection may be shaped by host, pathogen and environmental factors. To better inform wildlife health assessment and research directions, we investigated the utility of a novel multivariate immunophenotyping approach examining innate and adaptive immune responses in differing climatic, pathogen co-infection and demographic contexts across two koala (Phascolarctos cinereus) populations in New South Wales: the Liverpool Plains (LP), and Southern Highlands to South-west Sydney (SHSWS). Relative to the comparatively healthy SHSWS, the LP had greater and more variable innate immune gene expression (IL-1ß, IL-6), and KoRV transcription. During extreme heat and drought, koalas from the LP displayed upregulation of a stress pathway gene and reduced adaptive immune genes expression, haematocrit and plasma protein, suggesting the possibility of environmental impacts through multiple pathways. In those koalas, KoRV transcription status, Chlamydia pecorum infection loads, and visible urogenital inflammation were not associated with immune variation, suggesting that immune markers were more sensitive indicators of real-time impacts than observed disease outcomes.

Expanding the known distribution of phascolartid gammaherpesvirus 1 in koalas to populations across Queensland and New South Wales.

Koala populations across the east coast of Australia are under threat of extinction with little known about the presence or distribution of a potential pathogen, phascolartid gammaherpesvirus 1 (PhaHV-1) across these threatened populations. Co-infections with PhaHV-1 and Chlamydia pecorum may be common and there is currently a limited understanding of the impact of these co-infections on koala health. To address these knowledge gaps, archived clinical and field-collected koala samples were examined by quantitative polymerase chain reaction to determine the distribution of PhaHV-1 in previously untested populations across New South Wales and Queensland. We detected PhaHV-1 in all regions surveyed with differences in detection rate between clinical samples from rescued koalas (26%) and field-collected samples from free-living koalas (8%). This may reflect increased viral shedding in koalas that have been admitted into care. We have corroborated previous work indicating greater detection of PhaHV-1 with increasing age in koalas and an association between PhaHV-1 and C. pecorum detection. Our work highlights the need for continued surveillance of PhaHV-1 in koala populations to inform management interventions, and targeted research to understand the pathogenesis of PhaHV-1 and determine the impact of infection and co-infection with C. pecorum.

Genetic markers of Chlamydia pecorum virulence in ruminants support short term host-pathogen evolutionary relationships in the koala, Phascolarctos cinereus.

In ruminants infected with Chlamydia pecorum, shorter lengths of coding tandem repeats (CTR) within two genes, the inclusion membrane protein (incA) and Type III secretor protein (ORF663), have been previously associated with pathogenic outcomes. In other chlamydial species, the presence of a chlamydial plasmid has been linked to heightened virulence, and the plasmid is not ubiquitous in C. pecorum across the koala's range. We therefore investigated these three markers: incA, ORF663 and C. pecorum plasmid, as potential indicators of virulence in two koala populations in New South Wales with differing expression of urogenital chlamydiosis; the Liverpool Plains and one across the Southern Highlands and South-west Sydney (SHSWS). We also investigated the diversity of these loci within strains characterised by the national multi-locus sequence typing (MLST) scheme. Although CTR lengths of incA and ORF663 varied across the populations, they occurred only within previously described pathogenic ranges for ruminants. This suggests a relatively short-term host-pathogen co-evolution within koalas and limits the utility of CTR lengths for incA and ORF663 as virulence markers in the species. However, in contrast to reports of evolution of C. pecorum towards lower virulence, as indicated by longer CTR lengths in ruminants and swine, CTR lengths for ORF663 appeared to be diverging towards less common shorter CTR lengths within strains recently introduced to koalas in the Liverpool Plains. We detected the plasmid across 90% and 92% of samples in the Liverpool Plains and SHSWS respectively, limiting its utility as an indicator of virulence. It would be valuable to examine the CTR lengths of these loci across koala populations nationally. Investigation of other hypervariable loci may elucidate the evolutionary trajectory of virulence in C. pecorum induced disease in koalas. Profiling of virulent strains will be important in risk assessments for strain movement to naïve or susceptible populations through translocations and wildlife corridor construction.

Essential gene complement of Planctopirus limnophila from the bacterial phylum Planctomycetes.

Planctopirus limnophila belongs to the bacterial phylum Planctomycetes, a relatively understudied lineage with remarkable cell biology features. Here, we report a genome-wide analysis of essential gene content in P. limnophila. We show that certain genes involved in peptidoglycan synthesis or cell division, which are essential in most other studied bacteria, are not essential for growth under laboratory conditions in this species. We identify essential genes likely involved in lipopolysaccharide biosynthesis, consistent with the view of Planctomycetes as diderm bacteria, and highlight other essential genes of unknown functions. Furthermore, we explore potential stages of evolution of the essential gene repertoire in Planctomycetes and the related phyla Verrucomicrobia and Chlamydiae. Our results provide insights into the divergent molecular and cellular biology of Planctomycetes.

Propylene Metathesis over Molybdenum Silicate Microspheres with Dispersed Active Sites.

In this work, we demonstrate that amorphous and porous molybdenum silicate microspheres are highly active catalysts for heterogeneous propylene metathesis. Homogeneous molybdenum silicate microspheres and aluminum-doped molybdenum silicate microspheres were synthesized via a nonaqueous condensation of a hybrid molybdenum biphenyldicarboxylate-based precursor solution with (3-aminopropyl)triethoxysilane. The as-prepared hybrid metallosilicate products were calcined at 500 °C to obtain amorphous and porous molybdenum silicate and aluminum-doped molybdenum silicate microspheres with highly dispersed molybdate species inserted into the silicate matrix. These catalysts contain mainly highly dispersed MoOx species, which possess high catalytic activity in heterogeneous propylene metathesis to ethylene and butene. Compared to conventional silica-supported MoOx catalysts prepared via incipient wetness impregnation (MoIWI), the microspheres with low Mo content (1.5-3.6 wt %) exhibited nearly 2 orders of magnitude higher steady-state propylene metathesis rates at 200 °C, approaching site time yields of 0.11 s-1.

Multifunctional, Hybrid Materials Design via Spray-Drying: Much more than Just Drying.

Spray-drying is a popular and well-known "drying tool" for engineers. This perspective highlights that, beyond this application, spray-drying is a very interesting and powerful tool for materials chemists to enable the design of multifunctional and hybrid materials. Upon spray-drying, the confined space of a liquid droplet is narrowed down, and its ingredients are forced together upon "falling dry." As  detailed in this article, this enables the following material formation strategies either individually or even in combination: nanoparticles and/or molecules can be assembled; precipitation reactions as well as chemical syntheses can be performed; and templated materials can be designed. Beyond this, fragile moieties can be processed, or "precursor materials" be prepared. Post-treatment of spray-dried objects eventually enables the next level in the design of complex materials. Using spray-drying to design (particulate) materials comes with many advantages-but also with many challenges-all of which are outlined here. It is believed that multifunctional, hybrid materials, made via spray-drying, enable very unique property combinations that are particularly highly promising in myriad applications-of which catalysis, diagnostics, purification, storage, and information are highlighted.

Research Note: Effects of feeding corn naturally contaminated with aflatoxin B1, deoxynivalenol, and zearalenone on reproductive performance of broiler breeders and growth performance of their progeny chicks.

To evaluate the toxic effects of mycotoxin-contaminated corn (MC) on the breeders and their progeny chicks, a total of 480 fifty-wk-old Cobb broiler breeder hens were fed the following dies: 1) a corn-soybean meal diet (Control; containing 70.35% corn), 2) MC substituting for 50% of corn in Control (LM), 3) LM diet plus 2 g/kg 1 mycotoxin sequestrant, Toxy-Nil Plus (TNP) (LMT2.0), 4) MC substituting for 100% of corn in Control (HM), 5) HM diet plus 2 g/kg TN (HMT2.0), and 6) HM diet plus 2.5 g/kg TNP (HMT2.5). The MC contained 69.25 µg aflatoxin B1 (AFB1)/kg, 4,875 µg deoxynivalenol (DON)/kg, and 2,262 µg zearalenone (ZEN)/kg. At wk 4 after MC inclusion, all eggs laid were used for hatch, and all progeny chicks were fed the same mycotoxin-untreated diet for 14 d. Dietary MC inclusion decreased the hatchability of set eggs and increased embryo mortality during d 18 to 21.5. The TNP addition increased these aforementioned indices in MC-included diets. Maternal HM treatment decreased the BW of progeny chicks at age of 14 d and BWG of progeny chicks during d 1 to 14, whereas maternal LM treatment did not affect these indices. In parallel, maternal HM treatment decreased the concentrations of serum IgA, IgG, and lysozyme in the progeny chicks on d 14, but maternal LM treatment did not affect these indices. Overall, maternal dietary TNP treatments increased the growth of progeny chicks and had a trend to increase the concentrations of serum IgA and IgG on d 14 compared to maternal MC treatments. It was concluded that the feeding of relative high ratio of corn contaminated with low level of AFB1, DON, and ZEN negatively affected the reproductive performance of breeders and the growth performance of their progeny chicks, and TNP addition alleviated these toxic effects.

Efficacy of a synthetic peptide Chlamydia pecorum major outer membrane protein vaccine in a wild koala (Phascolarctos cinereus) population.

Chlamydiosis is a significant disease affecting Eastern Australian koala (Phascolarctos cinereus) populations, impacting individual animal welfare and fecundity and therefore influencing population dynamics. The aim of this study was to investigate the effect of a synthetic peptide vaccine based on 4 components of the Chlamydia pecorum major outer membrane protein (MOMP), over an 18-month period in a koala population severely impacted by chlamydiosis. Wild koalas were recruited into a vaccination or a placebo treatment group on a random allocation, then followed through a period of 18 months, with recapture at 6 monthly intervals. Vaccination did not alter clinical disease expression or chlamydial shedding from the ocular or urogenital sites. Vaccination did not stimulate a significant plasma anti-MOMP IgG response, when compared to the placebo group. There was no significant effect of vaccination on IFN-γ and IL-17A mRNA expression of peripheral blood lymphocytes when stimulated with rMOMP. We have demonstrated that a synthetic peptide vaccination against chlamydiosis is not an effective management tool in a koala population with a high prevalence of C. pecorum infection and related disease. The lack of antigenic response found in this study suggests that further research utilising a larger, full-length antigen is an avenue worth investigation if we are to consider vaccination as a part of a management strategy in diseased koala populations.

Health Assessments of Koalas after Wildfire: A Temporal Comparison of Rehabilitated and Non-Rescued Resident Individuals.

Many koalas (Phascolarctos cinereus) required rehabilitation after the 2019/20 Australian megafires. Little is known about how the post-release health of rehabilitated koalas compares to non-rescued resident koalas. We evaluated health parameters in rehabilitated koalas and resident koalas in burnt and unburnt habitat in southern New South Wales, Australia. Health checks were undertaken within six weeks of fire (rehabilitated group), 5-9 months post-fire and 12-16 months post-fire. Body condition improved significantly over time in rehabilitated koalas, with similar condition between all groups at 12-16 months. Rehabilitated koalas therefore gained body condition at similar rates to koalas who remained and survived in the wild. The prevalence of Chlamydia pecorum was also similar between groups and timepoints, suggesting wildfire and rehabilitation did not exacerbate disease in this population. While there was some variation in measured serum biochemistry and haematology parameters between groups and timepoints, most were within normal reference ranges. Our findings show that koalas were generally healthy at the time of release and when recaptured nine months later. Landscapes in the Monaro region exhibiting a mosaic of burn severity can support koalas, and rehabilitated koala health is not compromised by returning them to burnt habitats 4-6 months post-fire.

Evaluation of a biosecurity survey approach for contamination by Chlamydia pecorum in koala rehabilitation, field capture, and captive settings.

Transmission of Chlamydia pecorum between koalas is a potential risk in field capture or rehabilitation settings, where koalas are held in proximity to each other, or equipment is shared between animals. Given the impact of C. pecorum on koala welfare and population viability it is surprising that quarantine and disinfection protocols in a koala rehabilitation facility or capture settings have not previously been evaluated. This study aimed to evaluate an approach, based on the detection of chlamydial DNA and cell viability, to determine the degree of environmental contamination within a koala care facility. Various fomite sites associated with koala care at a koala rehabilitation facility in New South Wales, Australia were identified as potential sources of chlamydial contamination, following exposure to koalas known to be infected with C. pecorum. Fomite sites were swabbed following exposure, and again after decontamination procedures were carried out. Samples were tested for the presence of chlamydial DNA using qPCR and viability using both RT-qPCR and cell culture. From a total of 239 sampling events, 30 tested qPCR positive for chlamydial DNA, with 19 and 11 samples corresponding to pre-decontamination and post-decontamination events respectively. Detection of chlamydial DNA appeared to be most common in the examination room, especially on fomite sites in direct contact with koalas. Physical removal of chlamydial DNA, or its degradation by the elements, appeared to be more common on outdoor enclosures, clothing, and hands. Based on the cell culture assay, of the pre-decontamination samples with chlamydial DNA, eight had viable chlamydial cells, two of these at low levels. Of the post-decontamination samples with chlamydial DNA, one had a moderate number, and one had a very low number of viable chlamydial cells. RT-qPCR was unsuccessful in determining cell viability due to low yields of RNA and high levels of contaminants from the environmental samples. The outcomes of this study provide a knowledge base for the design of future biosecurity evaluation guidelines in captive and koala rehabilitation facilities. The higher incidence of chlamydial DNA detection by qPCR than viable organism highlights the need to use viability assays in similar studies. However, further investment is still needed to optimise these methods and improve sensitivity for complex environmental samples.

Prokaryotic membrane coat - like proteins: An update.

Membrane coat proteins are essential players in the eukaryotic endomembrane traffic system. Previous work identified proteins with the membrane-coat architecture in prokaryotes, specifically in the Planctomycetes, Verrucomicrobia and Chlamydiae (PVC) superphylum, bacteria that display the most developed prokaryotic endomembrane system. Hence, the membrane coat-like (MCL) proteins are predicted to play a central role in this system but their actual function is still unknown. In this work we strengthened previous structure predictions for these prokaryotic MCL proteins. We also detected new putative MCL proteins in the Planctomycete Gemmata obscuriglobus. Structural analysis of these revealed the presence of additional domains apart from the ß-propeller and α-solenoid combination, characteristic of the membrane-coat architecture. Functions associated with these domains include some related to carbohydrate or membrane/lipid binding. Using homology-based methods, we found MCL proteins in other bacterial phyla, but the most abundant hits are still restricted to Planctomycetes and Verrucomicrobia. Detailed inspection of neighbouring genes of MCL in G. obscuriglobus supports the idea that the function of these proteins is related to membrane manipulation. No significant hits were found in Archaea, including Asgard archaea. More than 10 years after their original detection, PVC bacteria are still uniquely linked to eukaryotes through the structure of the MCL proteins sustaining their endomembrane system.

Leveraging Metal Nodes in Metal-Organic Frameworks for Advanced Anodic Hydrazine Oxidation Assisted Seawater Splitting.

Metal-organic frameworks (MOFs) show great promise for electrocatalysis owing to their tunable ligand structures. However, the poor stability of MOFs impedes their practical applications. Unlike the general pathway for engineering ligands, we report herein an innovative strategy for leveraging metal nodes to improve both the catalytic activity and the stability. Our electrolysis cell with a NiRh-MOF||NiRh-MOF configuration exhibited 10 mA cm-2 at an ultralow cell voltage of 0.06 V in alkaline seawater (with 0.3 M N2H4), outperforming its counterpart benchmark Pt/C||Pt/C cell (0.12 V). Impressively, the incorporation of Rh into a MOF secured a robust stability of over 60 h even when working in the seawater electrolyte. Experimental results and theoretical calculations revealed that Rh atoms serve as the active sites for hydrogen evolution while Ni nodes are responsible for the hydrazine oxidation during the hydrazine oxidation assisted seawater splitting. This work provides a paradigm for green hydrogen generation from seawater.

Development of diagnostic and point of care assays for a gammaherpesvirus infecting koalas.

The recent listing of koala populations as endangered across much of their range has highlighted the need for better management interventions. Disease is a key threat to koala populations but currently there is no information across the threatened populations on the distribution or impact of a gammaherpesvirus, phascolarctid gammaherpesvirus 1 (PhaHV-1). PhaHV-1 is known to infect koalas in southern populations which are, at present, not threatened. Current testing for PhaHV-1 involves lengthy laboratory techniques that do not permit quantification of viral load. In order to better understand distribution, prevalence and impacts of PhaHV-1 infections across koala populations, diagnostic and rapid point of care tests are required. We have developed two novel assays, a qPCR assay and an isothermal assay, that will enable researchers, clinicians and wildlife managers to reliably and rapidly test for PhaHV-1 in koalas. The ability to rapidly diagnose and quantify viral load will aid quarantine practices, inform translocation management and guide research into the clinical significance and impacts of PhaHV-1 infection in koalas.

Evolving techniques for reducing phantom limb pain.

At least two million people in the United States of America live with lost limbs, and the number is expected to double by 2050, although the incidence of amputations is significantly greater in other parts of the world. Within days to weeks of the amputation, up to 90% of these individuals develop neuropathic pain, presenting as phantom limb pain (PLP). The pain level increases significantly within one year and remains chronic and severe for about 10%. Amputation-induced changes are considered to underlie the causation of PLP. Techniques applied to the central nervous system (CNS) and peripheral nervous system (PNS) are designed to reverse amputation-induced changes, thereby reducing/eliminating PLP. The primary treatment for PLP is the administration of pharmacological agents, some of which are considered but provide no more than short-term pain relief. Alternative techniques are also discussed, which provide only short-term pain relief. Changes induced by various cells and the factors they release are required to change neurons and their environment to reduce/eliminate PLP. It is concluded that novel techniques that utilize autologous platelet-rich plasma (PRP) may provide long-term PLP reduction/elimination.