Next-generation fungal identification using target enrichment and Nanopore sequencing.

BACKGROUND: Rapid and accurate pathogen identification is required for disease management. Compared to sequencing entire genomes, targeted sequencing may be used to direct sequencing resources to genes of interest for microbe identification and mitigate the low resolution that single-locus molecular identification provides. This work describes a broad-spectrum fungal identification tool developed to focus high-throughput Nanopore sequencing on genes commonly employed for disease diagnostics and phylogenetic inference. RESULTS: Orthologs of targeted genes were extracted from 386 reference genomes of fungal species spanning six phyla to identify homologous regions that were used to design the baits used for enrichment. To reduce the cost of producing probes without diminishing the phylogenetic power, DNA sequences were first clustered, and then consensus sequences within each cluster were identified to produce 26,000 probes that targeted 114 genes. To test the efficacy of our probes, we applied the technique to three species representing Ascomycota and Basidiomycota fungi. The efficiency of enrichment, quantified as mean target coverage over the mean genome-wide coverage, ranged from 200 to 300. Furthermore, enrichment of long reads increased the depth of coverage across the targeted genes and into non-coding flanking sequence. The assemblies generated from enriched samples provided well-resolved phylogenetic trees for taxonomic assignment and molecular identification. CONCLUSIONS: Our work provides data to support the utility of targeted Nanopore sequencing for fungal identification and provides a platform that may be extended for use with other phytopathogens.

Implant-Based Breast Reconstruction in the Elderly: Complications and Patient-Reported Outcomes in Women Older Than 70 Years.

BACKGROUND: Advanced age is considered by many to be a relative contraindication to breast reconstruction. However, despite increased medical comorbidities and a perception that elderly patients are less concerned with body image, more women older than 70 years are choosing to undergo breast reconstruction. There is a paucity of data to guide reconstructive decision-making and counseling in this population. OBJECTIVES: The aim of this study was to evaluate patient satisfaction, complication rates, and long-term outcomes in women older than 70 years undergoing implant-based breast reconstruction. METHODS: A total of 400 patients were identified at the authors' institution and divided into 2 groups: ≥70 and <70 years old. Medical comorbidities, surgical outcomes, and patient-reported outcomes as defined by the BREAST-Q were compared using the χ2 tests for categorical variables and t tests for continuous variables. RESULTS: The cohort of patients older than 70 years was made up of 25 women, with a mean age of 73 years, and the cohort of patients younger than 70 years was made up of 375 women, with a mean age of 50 years. There was no significant difference in body mass index (P = 0.373), smoking status (P = 0.360), or history of prior ipsilateral radiation (P = 0.508) between the 2 cohorts; however, the elderly cohort was significantly more likely to have diabetes (P = 0.026). Although elderly patients were less likely to undergo bilateral mastectomy (P < 0.001), there was no significant difference in the type of mastectomy, pathological diagnosis, or method of reconstruction. There was no significant difference in complication rates when looking at minor infection (P = 0.553) or major infection (P = 0.553). The 2 groups were equally likely to undergo secondary procedures (P = 0.192). Overall satisfaction rates were high in all BREAST-Q categories in the elderly group and not significantly different when compared with the group of patients younger than 70 years. Matched-pair analysis showed a significant difference with the group of patients older than 70 years having higher levels physical well-being (P < 0.001). CONCLUSIONS: Immediate breast reconstruction can be performed safely and with similar high satisfaction rates in the elderly population as their younger counterparts. Age alone should not be used as a reason for excluding women from these life-changing operations.

Close the GAPS: A Standardized Perioperative Protocol Reduces Breast Reconstruction Implant Infections.

BACKGROUND: Implant-based breast reconstruction (IBBR) is a complex process with significant practice variability. Infections after IBBR are associated with higher rates of readmission, reoperation, and reconstructive failure. To reduce process variability and postoperative infections, the authors implemented an evidence-based, standardized protocol for IBBR. METHODS: The protocol was applied to all patients undergoing IBBR at a single institution from December of 2019 to February of 2021. Intraoperative protocol adherence was recorded, and infection events were considered minor (managed with outpatient antibiotics) or major (managed with readmission or reoperation). A historic control group was retrospectively analyzed for comparison. RESULTS: Sixty-nine patients (120 breasts) in the protocol group were compared with 159 patients (269 breasts) in the retrospective group. No differences were found in demographic characteristics, comorbidities, or type of reconstruction (expander versus implant). Intraoperative protocol adherence was 80.5% (SD, 13.9%). Overall infection rate was significantly lower in the protocol group versus controls (8.7% versus 17.0%; P < 0.05). When dichotomized, protocol patients had a lower rate of both minor (2.9% versus 5.7%; P = 0.99) and major (5.8% versus 11.3%; P = 0.09) infections, although this was not statistically significant. Rate of reconstructive failure secondary to infection was significantly lower in the protocol group (4.4% versus 8.8%; P < 0.05). Among protocol patients, those without infection had higher protocol adherence (81.5% versus 72.2%; P < 0.06), which neared statistical significance. CONCLUSION: A standardized perioperative protocol for IBBR reduces process variability and significantly decreases rate of overall infections and reconstructive failure secondary to infection. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, III.

Notch Blockade Specifically in Bone Marrow-Derived FSP-1-Positive Cells Ameliorates Renal Fibrosis.

BACKGROUND: The infiltration of inflammatory cells during a kidney injury stimulates myofibroblast activation leading to kidney fibrosis. Fibroblast-specific protein 1 (FSP-1) positive cells have been reported as either myofibroblasts or monocytes during tissue fibrosis. The functions of FSP-1+ cells that are associated with the development of renal fibrosis and the signaling pathways that regulate FSP-1+ cell activation have not been well defined. METHODS: In mice with unilateral ureteral obstruction (UUO), we characterized FSP-1+ cells and determined the role of the Notch signaling pathway in the activation of bone marrow-derived FSP-1+ cells during kidney fibrosis. RESULTS: In kidneys from mice with UUO, the FSP-1+ cells accumulated significantly in the tubulointerstitial area. By using immunostaining and FSP-1 reporter mice, we found that FSP-1 was co-stained with inflammatory cell markers, but not myofibroblast markers. Results from mice with bone marrow transplantations showed that FSP-1+ cells in obstructed kidneys represent a bone marrow-derived population of inflammatory cells. In cultured FSP-1+ cells, the inhibition of Notch signaling suppressed the activation and cytokine secretion of FSP-1+ cells that were induced by LPS but not by IL-4. The specific KO or blockade of Notch signaling in bone marrow-derived FSP-1+ cells suppressed UUO-induced ECM deposition, the infiltration of FSP-1+ inflammatory cells, and cytokine production. These responses ameliorated myofibroblast accumulation and renal fibrosis in obstructed kidneys. CONCLUSION: Our study reveals that most FSP-1+ cells in obstructed kidneys are activated macrophages that are derived from bone marrow and that Notch signaling activates the production of M1 cytokines in FSP-1+ monocytes/macrophages, which is important for renal inflammation and fibrosis.

Postmitotic accumulation of histone variant H3.3 in new cortical neurons establishes neuronal chromatin, transcriptome, and identity.

Histone variants, which can be expressed outside of S-phase and deposited DNA synthesis-independently, provide long-term histone replacement in postmitotic cells, including neurons. Beyond replenishment, histone variants also play active roles in gene regulation by modulating chromatin states or enabling nucleosome turnover. Here, we uncover crucial roles for the histone H3 variant H3.3 in neuronal development. We find that newborn cortical excitatory neurons, which have only just completed replication-coupled deposition of canonical H3.1 and H3.2, substantially accumulate H3.3 immediately postmitosis. Codeletion of H3.3-encoding genes H3f3a and H3f3b from newly postmitotic neurons abrogates H3.3 accumulation, markedly alters the histone posttranslational modification landscape, and causes widespread disruptions to the establishment of the neuronal transcriptome. These changes coincide with developmental phenotypes in neuronal identities and axon projections. Thus, preexisting, replication-dependent histones are insufficient for establishing neuronal chromatin and transcriptome; de novo H3.3 is required. Stage-dependent deletion of H3f3a and H3f3b from 1) cycling neural progenitor cells, 2) neurons immediately postmitosis, or 3) several days later, reveals the first postmitotic days to be a critical window for de novo H3.3. After H3.3 accumulation within this developmental window, codeletion of H3f3a and H3f3b does not lead to immediate H3.3 loss, but causes progressive H3.3 depletion over several months without widespread transcriptional disruptions or cellular phenotypes. Our study thus uncovers key developmental roles for de novo H3.3 in establishing neuronal chromatin, transcriptome, identity, and connectivity immediately postmitosis that are distinct from its role in maintaining total histone H3 levels over the neuronal lifespan.

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.

Skin Reducing Mastectomy and Immediate Tissue Expander Reconstruction: A Critical Analysis.

BACKGROUND: Complications of implant-based reconstruction have been shown to be related to increasing body mass index (BMI) and breast size. The impact of skin reducing mastectomy (SRM) with a dermal flap is examined. METHODS: A retrospective review of a single surgeon's experience with immediate submuscular tissue expander (TE) reconstruction from 2011 to 2019 was performed. The outcomes of SRM were compared with those of skin sparing mastectomy (SSM). RESULTS: A total of 162 patients (292 breasts) were identified. Mastectomy types were as follows: SRM, 73 (136 breasts) and SSM, 89 (156 breasts). Acellular dermal matrix (ADM) was used to supplement TE coverage in 65.4% of SRM cases. Mean BMI was 29.2 among SRM patients and 25.9 in SSM patients (P < 0.001). Obesity (BMI ≥ 30) was more prevalent in the SRM group (SRM, 38.4% vs SSM, 22.5%; P = 0.03). Mean mastectomy weight was higher in the SRM group (SRM, 833.6 g vs SSM, 425.6 g; P < 0.001). Mean BMI and mastectomy weight were lower in SRM patients who were reconstructed with ADM (ADM, 28.1 vs no ADM, 30.8; P = 0.01; ADM, 746.1 g vs no ADM, 1006.3 g; P < 0.001). Minor complications were more prevalent in the SRM group (SRM, 22.8% vs SSM, 4.5%; P < 0.001). Mastectomy skin flap necrosis (MSFN) was more common in the SRM group (SRM, 22.8% vs SSM, 7.7%; P < 0.001), but MSFN necessitating operative debridement was similarly low in both groups (SRM: 1.9% vs SSM: 4.5%). Major complication rates (SRM 11.0% vs SSM 10.9%) and reconstructive failure rates (SRM 5.9% vs SSM 5.1%) were similar between groups. Mastectomy weight 800 g or higher and BMI of 30 or higher were found to be risk factors for complications on analysis of the SRM cohort (P < 0.05). CONCLUSIONS: Mastectomy weight and BMI were positive predictors of complications after immediate TE reconstruction. Mastectomy skin flap necrosis is more common after SRM than SSM. The use of SRM with a dermal flap has a similar major complication rate as SSM despite its use in obese, large-breasted women. The dermal flap provides soft tissue coverage, which prevents implant exposure and seroma. The use of ADM does not adversely affect the complication rate of SRM.

Behavioral Characteristics and Self-Reported Health Status among 2029 Adults Consuming a "Carnivore Diet".

BACKGROUND: The "carnivore diet," based on animal foods and excluding most or all plant foods, has attracted recent popular attention. However, little is known about the health effects and tolerability of this diet, and concerns for nutrient deficiencies and cardiovascular disease risk have been raised. OBJECTIVES: We obtained descriptive data on the nutritional practices and health status of a large group of carnivore diet consumers. METHODS: A social media survey was conducted 30 March-24 June, 2020 among adults self-identifying as consuming a carnivore diet for ≥6 mo. Survey questions interrogated motivation, dietary intake patterns, symptoms suggestive of nutritional deficiencies or other adverse effects, satisfaction, prior and current health conditions, anthropometrics, and laboratory data. RESULTS: A total of 2029 respondents (median age: 44 y, 67% male) reported consuming a carnivore diet for 14 mo (IQR: 9-20 mo), motivated primarily by health reasons (93%). Red meat consumption was reported as daily or more often by 85%. Under 10% reported consuming vegetables, fruits, or grains more often than monthly, and 37% denied vitamin supplement use. Prevalence of adverse symptoms was low (<1% to 5.5%). Symptoms included gastrointestinal (3.1%-5.5%), muscular (0.3%-4.0%), and dermatologic (0.1%-1.9%). Participants reported high levels of satisfaction and improvements in overall health (95%), well-being (66%-91%), various medical conditions (48%-98%), and median [IQR] BMI (in kg/m2) (from 27.2 [23.5-31.9] to 24.3 [22.1-27.0]). Among a subset reporting current lipids, LDL-cholesterol was markedly elevated (172 mg/dL), whereas HDL-cholesterol (68 mg/dL) and triglycerides (68 mg/dL) were optimal. Participants with diabetes reported benefits including reductions in median [IQR] BMI (4.3 [1.4-7.2]), glycated hemoglobin (0.4% [0%-1.7%]), and diabetes medication use (84%-100%). CONCLUSIONS: Contrary to common expectations, adults consuming a carnivore diet experienced few adverse effects and instead reported health benefits and high satisfaction. Cardiovascular disease risk factors were variably affected. The generalizability of these findings and the long-term effects of this dietary pattern require further study.

Chromatin remodeler Arid1a regulates subplate neuron identity and wiring of cortical connectivity.

Loss-of-function mutations in chromatin remodeler gene ARID1A are a cause of Coffin-Siris syndrome, a developmental disorder characterized by dysgenesis of corpus callosum. Here, we characterize Arid1a function during cortical development and find unexpectedly selective roles for Arid1a in subplate neurons (SPNs). SPNs, strategically positioned at the interface of cortical gray and white matter, orchestrate multiple developmental processes indispensable for neural circuit wiring. We find that pancortical deletion of Arid1a leads to extensive mistargeting of intracortical axons and agenesis of corpus callosum. Sparse Arid1a deletion, however, does not autonomously misroute callosal axons, implicating noncell-autonomous Arid1a functions in axon guidance. Supporting this possibility, the ascending axons of thalamocortical neurons, which are not autonomously affected by cortical Arid1a deletion, are also disrupted in their pathfinding into cortex and innervation of whisker barrels. Coincident with these miswiring phenotypes, which are reminiscent of subplate ablation, we unbiasedly find a selective loss of SPN gene expression following Arid1a deletion. In addition, multiple characteristics of SPNs crucial to their wiring functions, including subplate organization, subplate axon-thalamocortical axon cofasciculation ("handshake"), and extracellular matrix, are severely disrupted. To empirically test Arid1a sufficiency in subplate, we generate a cortical plate deletion of Arid1a that spares SPNs. In this model, subplate Arid1a expression is sufficient for subplate organization, subplate axon-thalamocortical axon cofasciculation, and subplate extracellular matrix. Consistent with these wiring functions, subplate Arid1a sufficiently enables normal callosum formation, thalamocortical axon targeting, and whisker barrel development. Thus, Arid1a is a multifunctional regulator of subplate-dependent guidance mechanisms essential to cortical circuit wiring.

Fat Grafting Subjectively Improves Facial Skin Elasticity and Hand Function of Scleroderma Patients.

Systemic scleroderma is a chronic connective tissue disease characterized by internal organ and skin fibrosis. Unfortunately, there is a lack of efficacious treatments for cutaneous manifestations, and alternative interventions should be considered. Fat grafting has gained significant attention due to its regenerative properties and success in improving skin quality and volume deficits in fibrotic diseases. While some studies have investigated the efficacy of autologous fat grafting, we utilized the Coleman method for harvesting and processing to determine the efficacy of fat grafting to improve skin fibrosis in the hands and face of scleroderma patients without excess processing of adipose tissue. Patients with a diagnosis of scleroderma who underwent fat grafting between March 2015 and March 2019 at the University of Michigan were included. Ten female patients were identified that met inclusion criteria. The mean age at the time of surgery was 48.7 (± 17.6) years. An average of 53.2 (± 15.5) ml of fat was injected into the hands and 26.1 (± 16.4) ml into the face. Patients were treated with 1-4 rounds of grafting depending on the initial severity of skin fibrosis and volume deficiency. Fat grafting subjectively and qualitatively improved perioral skin quality, facial animation, hand range of motion, and hand pain for patients with systemic scleroderma. No complications were identified. Additional studies are necessary to determine the ideal volume, timing of treatments, and type of fat to optimize the efficacy of autologous fat grafting for the treatment of systemic scleroderma.

A Simplified Approach to Myelomeningocele Defect Repair.

BACKGROUND: Repair of the soft tissue defect in myelomeningoceles remains challenging. The literature currently lacks a systematic approach, reporting high rates of complications. We present outcomes from the largest series to date and describe a simplified approach that minimizes morbidity and streamlines decision making. METHODS: Patients 1 year or younger who underwent myelomeningocele repair between 2008 and 2018 were reviewed. Flap types were categorized by tissue composition. Complications were dichotomized into early and late (<30 days and >30 days postoperative, respectively). Logistic regression was used to measure the impact of flap tissue composition and skin closure technique on odds of postoperative complications. RESULTS: Ninety-seven patients met inclusion criteria. Reoperation was required in only 3 (3.0%) patients-1 for wound dehiscence and 2 for surgical site infections. Zero cases of tethered cord or cerebrospinal fluid leak occurred. The most common minor complications were early wound complications (n = 18, 18.6%) and early infection (n = 5, 5.2%). Fascia-only flaps and muscle + other tissue flaps were not associated with higher odds of complications compared with muscle-only flaps (odds ratio [OR], 2.13; 95% confidence interval [CI], 0.53-8.50, P = 0.29; OR = 2.87, 95% CI 0.66-12.51, P = 0.16, respectively). Rhomboid flaps for skin closure were associated with higher odds of complications (OR, 4.47; 95% CI, 1.00-19.97; P = 0.05). CONCLUSIONS: Our approach to myelomeningocele repair demonstrated no cases of secondary tethered cord or cerebrospinal fluid leak, and reoperative rates were extremely low. Because complications were unrelated to flap type, we recommend a simplified approach using any tissue type for dural coverage and 2-layer primary closure of the skin.

Computational modelling of nasal respiratory flow.

CFD has emerged as a promising diagnostic tool for clinical trials, with tremendous potential. However, for real clinical applications to be useful, overall statistical findings from large population samples (e.g., multiple cases and models) are needed. Fully resolved solutions are not a priority, but rather rapid solutions with fast turn-around times are desired. This leads to the issue of what are the minimum modelling criteria for achieving adequate accuracy in respiratory flows for large-scale clinical applications, with a view to rapid turnaround times. This study simulated a highly-resolved solution using the large eddy simulation (LES) method as a reference case for comparison with lower resolution models that included larger time steps and no turbulence modelling. Differences in solutions were quantified by pressure loss, flow resistance, unsteadiness, turbulence intensity, and hysteresis effects from multiple cycles. The results demonstrated that sufficient accuracy could be achieved with lower resolution models if the mean flow was considered. Furthermore, to achieve an established transient result unaffected by the initial start-up quiescent effects, the results need to be taken from at least the second respiration cycle. It was also found that the exhalation phase exhibited strong turbulence. The results are expected to provide guidance for future modelling efforts for clinical and engineering applications requiring large numbers of cases using simplified modelling approaches.

Mean excitation energy determination for Monte Carlo simulations of boron carbide as degrader material for proton therapy.

Boron carbide is a material proposed as an alternative to graphite for use as an energy degrader in proton therapy facilities, and is favoured due to its mechanical robustness and promise to give lower lateral scattering for a given energy loss. However, the mean excitation energy of boron carbide has not yet been directly measured. Here we present a simple method to determine the mean excitation energy by comparison with the relative stopping power in a water phantom, and from a comparison between experimental data and simulations we derive a value for it of 83.1 ± 2.8 eV suitable for use in Monte-Carlo simulation. This is consistent with the existing ICRU estimate (84.7 eV with 10-15% uncertainty) that is based on indirect Bragg additivity calculation, but it has a substantially smaller uncertainty. The method described can be readily applied to predict the ionisation loss of other boron carbide materials in which the atomic constituent ratio may vary, and allows this material to be reliably used as an alternative to graphite, diamond or beryllium.

Essential oil spray reduces clinical signs of insect bite hypersensitivity in horses.

OBJECTIVE: To assess the efficacy of an herbal spray combining various essential oils, with a claim of mast cell stabilisation, antipruritic, anti-inflammatory, and insect repellent effects on the clinical presentation of insect bite hypersensitivity (IBH) in horses. DESIGN: Double-blinded, placebo-controlled, randomised, cross-over clinical trial. METHODS: Twenty adult horses with clinical IBH were treated with a daily application of herbal spray or placebo for 28 days in a randomised, cross-over fashion, separated by a>28-day washout period. Horses were examined and scored prior to and after the completion of each treatment. Histopathology was performed on four horses. Owners kept daily diaries of observations. RESULTS: The herbal spray significantly reduced the severity of all assessed parameters (pruritus, excoriations, lichenification and alopecia; P < 0.05) compared with baseline values (pretreatment) and with placebo. Owners reported improvement of pruritus in 19/20 horses (95%) with complete resolution in 17 horses (85%) following treatment. Skin biopsies showed resolution of orthokeratosis in 4/4 horses, reduced thickness of the stratum spinosum in 2/4 horses and complete resolution of histopathological abnormalities in 1/4 horses after treatment, compared with either no change or deterioration of histopathologic lesions after placebo. No side effects were observed. CONCLUSIONS: The tested herbal spray may be an effective treatment for the management of equine IBH.

Symmetric neural progenitor divisions require chromatin-mediated homologous recombination DNA repair by Ino80.

Chromatin regulates spatiotemporal gene expression during neurodevelopment, but it also mediates DNA damage repair essential to proliferating neural progenitor cells (NPCs). Here, we uncover molecularly dissociable roles for nucleosome remodeler Ino80 in chromatin-mediated transcriptional regulation and genome maintenance in corticogenesis. We find that conditional Ino80 deletion from cortical NPCs impairs DNA double-strand break (DSB) repair, triggering p53-dependent apoptosis and microcephaly. Using an in vivo DSB repair pathway assay, we find that Ino80 is selectively required for homologous recombination (HR) DNA repair, which is mechanistically distinct from Ino80 function in YY1-associated transcription. Unexpectedly, sensitivity to loss of Ino80-mediated HR is dependent on NPC division mode: Ino80 deletion leads to unrepaired DNA breaks and apoptosis in symmetric NPC-NPC divisions, but not in asymmetric neurogenic divisions. This division mode dependence is phenocopied following conditional deletion of HR gene Brca2. Thus, distinct modes of NPC division have divergent requirements for Ino80-dependent HR DNA repair.

Geometry optimisation of graphite energy degrader for proton therapy.

INTRODUCTION: Cyclotron-based proton therapy facilities use an energy degrader of variable thickness to deliver beams of the different energies required by a patient treatment plan; scattering and straggling in the degrader give rise to an inherent emittance increase and subsequent particle loss in the downstream energy-selection system (ESS). Here we study alternative graphite degrader geometries and examine with Monte-Carlo simulations the induced emittance growth and consequent particle transmission. METHODS: We examined the conventional multiple-wedge degrader used in the Paul Scherrer Institute PROSCAN proton therapy system, the equivalent parallel-sided degrader, and a single block degrader of equivalent thickness. G4Beamline Monte-Carlo tracking of protons was benchmarked against measurements of the existing degrader for proton energies from 75 to 230 MeV, and used to validate simulations of the alternative geometries. RESULTS: Using a careful calculation of the beam emittance growth, we determined that a single-block degrader placed close to the collimators of the ESS is expected to deliver significantly larger transmission, up to 17% larger at 150 MeV. At the lowest deliverable of 75 MeV there is still a clear improvement in beam transmission. CONCLUSIONS: Whilst dose rates are not presently limited on the PROSCAN system at higher energies, a single-block degrader offers the ability to access either lower energies for treatment or a larger dose rate at 75 MeV in case transmission optimisation is desired. Single-block degraders should be considered for the delivery of low-energy protons from a cyclotron-based particle therapy system.

Pathological Findings in Koala Retrovirus-positive Koalas (Phascolarctos cinereus) from Northern and Southern Australia.

Koala retrovirus (KoRV) infection shows differences in prevalence and load between northern and southern Australian koala populations; however, the effect of this on diseases such as lymphoma and chlamydial disease is unclear. This study compared clinicopathological findings, haematology and splenic lymphoid area of KoRV-positive koalas from northern (Queensland [Qld], n = 67) and southern (South Australia [SA], n = 92) populations in order to provide further insight into KoRV pathogenesis. Blood was collected for routine haematology and for measurement of KoRV proviral load by quantitative polymerase chain reaction (qPCR). Plasma samples were assessed for KoRV viral load by reverse transcriptase qPCR and conjunctival and cloacal swabs were collected for measurement of the load of Chlamydia pecorum (qPCR). During necropsy examination, spleen was collected for lymphoid area analysis. Lymphoma was morphologically similar between the populations and occurred in koalas with the highest KoRV proviral and viral loads. Severe ocular chlamydial disease was observed in both populations, but urinary tract disease was more severe in Qld, despite similar C. pecorum loads. No associations between KoRV and chlamydial disease severity or load were observed, except in SA where viral load correlated positively with chlamydial disease severity. In both populations, proviral and viral loads correlated positively with lymphocyte and metarubricyte counts and correlated negatively with erythrocyte and neutrophil counts. Splenic lymphoid area was correlated positively with viral load. This study has shown further evidence for KoRV-induced oncogenesis and highlighted that lymphocytes and splenic lymphoid tissue may be key sites for KoRV replication. However, KoRV infection appears to be highly complex and continued investigation is required to fully understand its pathogenesis.

Concentrations in plasma and selected tissues of marbofloxacin after oral and intravenous administration in Bilgorajska geese (Anser anser domesticus).

Aims: To determine the pharmacokinetics and tissue depletion of 2 mg/kg marbofloxacin (MBX) in Bilgorajska geese (Anser anser domesticus) after I/V and oral administration, to calculate the daily dose from experimental data and to compare it with that calculated by allometric scaling.Methods: Eight clinically normal female Bilgorajska geese were used in a three-phase study with a 3-week wash-out period between phases. In the first phase birds received I/V administration of 2 mg/kg MBX; the same dose was given orally in the second and third phases. Blood samples were collected between 0 minutes and 48 hours in the first and second phases, and samples of liver, kidney, lung, muscle and heart were collected following slaughter of birds between 6 and 48 hours in the third phase. Concentrations of MBX in plasma and tissues were analysed using HPLC. Two additional birds served as controls. The optimal dose was calculated based on a minimal inhibitory concentration (MIC) of 0.125 µg/mL using the observed clearance, or using clearance calculated by allometric scaling.Results: Concentrations of MBX in plasma were detectable up to 24 hours following both I/V and oral administration. Mean oral bioavailability was 26.5 (SD 7.7)%. Concentrations of MBX in all tissues were highest at 6 hours and decreased constantly up to 34 hours. The mean optimal daily dose for oral administration of MBX, calculated using the observed clearance was 10.36 (SD 2.18) mg/kg, and using predicted clearance was 5.54 (SD 0.14) mg/kg. The preliminary withdrawal time for a maximum residue limit of 0.15 mg/kg calculated for muscle was 38.4 hours, heart 33.6 hours, kidney 48.3 hours, lung 47.7 hours and liver 49.3 hours.Conclusion and Clinical Relevance: There was insufficient evidence to recommend MBX orally administered to geese at a daily dose of 2 mg/kg for treatment of bacteria with an MIC of 0.125 µg/mL. Further pharmacokinetic/pharmacodynamic studies in geese are recommended to determine the MBX dose regimen and its clinical efficacy in geese.

Pharmacokinetic profiles of 5 mg/kg ibudilast, a phosphodiesterase inhibitor, orally administered to dogs in fasted and non-fasted states. A preliminary study.

Ibudilast (AV-411) is a non-selective inhibitor of cyclic nucleotide phosphodiesterase (PDE). It is currently marketed for human use in Asian countries for the treatment of asthma, cerebrovascular disorders and ocular allergies. Ibudilast has also been found to have an analgesic action for neuropathic pain at doses 5-10 times higher than those used in asthma therapy. Six healthy Labrador dogs were randomly assigned to two treatment groups using an open, single-dose, two-treatment, two-phase, cross-over design (2x2 Latin-square). Dogs in group 1 (n=3) were fasted for at least 10 hours overnight before the beginning of the experiment and 4 h following dosing while dogs in group 2 (n=3) received food ad libitum. During the first phase, each dog in group 1 and 2 received a single dose of 5 mg/kg ibudilast administered orally. After 1-week washout period the groups were rotated and the experiment was repeated. The analytical method, validated for dog plasma, was shown to be linear in the range 0.10-20 µg/mL. The limit of detection (LOD) and quantification (LOQ) were 0.03 and 0.1 µg/mL, respectively. No behavioural or health alterations were observed in the animals during or after the study. Ibudilast was detectable in plasma for up to 24 h showing a wide variability between animals. Although no statistically significant differences were observed in the present study between the fed and fasted states, examination of the raw data suggests that an effect may be present. The wide degree of variation observed in area under the curve (AUC) suggests that the investigation of population pharmacokinetic modelling is warranted.