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The use of monoclonal antibodies for the control of drug resistant nosocomial bacteria may alleviate a reliance on broad spectrum antimicrobials for treatment of infection. We identify monoclonal antibodies that may prevent infection caused by carbapenem resistant Acinetobacter baumannii. We use human immune repertoire mice (Kymouse platform mice) as a surrogate for human B cell interrogation to establish an unbiased strategy to probe the antibody-accessible target landscape of clinically relevant A. baumannii. After immunisation of the Kymouse platform mice with A. baumannii derived outer membrane vesicles (OMV) we identify 297 antibodies and analyse 26 of these for functional potential. These antibodies target lipooligosaccharide (OCL1), the Oxa-23 protein, and the KL49 capsular polysaccharide. We identify a single monoclonal antibody (mAb1416) recognising KL49 capsular polysaccharide to demonstrate prophylactic in vivo protection against a carbapenem resistant A. baumannii lineage associated with neonatal sepsis mortality in Asia. Our end-to-end approach identifies functional monoclonal antibodies with prophylactic potential against major lineages of drug resistant bacteria accounting for phylogenetic diversity and clinical relevance without existing knowledge of a specific target antigen. Such an approach might be scaled for a additional clinically important bacterial pathogens in the post-antimicrobial era., (© 2024. The Author(s).)

Background: Gender disparity in authorship broadly persists in medical literature, little is known about female authorship within pulmonary medicine., Methods: A bibliometric analysis of publications from 2012 to 2021 in 12 journals with the highest impact in pulmonary medicine was conducted. Only original research and review articles were included. Names of the first and last authors were extracted and their genders were identified using the Gender-API web. Female authorship was described by overall distribution and distribution by country/region/continent and journal. We compared the article citations by gender combinations, evaluated the trend in female authorship, and forecasted when parity for first and last authorship would be reached. We also conducted a systematic review of female authorship in clinical medicine., Results: 14,875 articles were included, and the overall percentage of female first authors was higher than last authors (37.0% vs 22.2%, p<0.001). Asia had the lowest percentage of female first (27.6%) and last (15.2%) authors. The percentages of female first and last authors increased slightly over time, except for a rapid increase in the COVID-19 pandemic periods. Parity was predicted in 2046 for the first authors and 2059 for the last authors. Articles with male authors were cited more than articles with female authors. However, male-male collaborations significantly decreased, whereas female-female collaborations significantly increased., Conclusions: Despite the slow improvement in female authorship over the past decade, there is still a substantial gender disparity in female first and last authorship in high-impact medical journals in pulmonary medicine., Competing Interests: Conflict of Interest JL discloses research funding from Fisher & Paykel Healthcare Ltd, Aerogen Ltd, and Rice Foundation, and speaker fees from American Association for Respiratory Care, Aerogen Ltd, Heyer Ltd, and Fisher & Paykel Healthcare Ltd. JL also serves as section editor for Respiratory Care. JBS discloses research funding from Teleflex and speaker fees from Aerogen and Medline Industries, LP., (Copyright © 2023 Sociedade Portuguesa de Pneumologia. Published by Elsevier España, S.L.U. All rights reserved.)

Emergence of pathogens with decreased sensitivity to succinate dehydrogenase inhibitor fungicides is a global agronomical issue. Analysis of Didymella tanaceti isolates ( n = 173), which cause tan spot of pyrethrum ( Tanacetum cinerariifolium ), collected prior to (2004 to 2005) and after (2009, 2010, 2012, and 2014) the commercial implementation of boscalid in Tasmanian pyrethrum fields identified that insensitivity developed over time and has become widespread. To evaluate temporal change, isolates were characterized for frequency of mutations in the succinate dehydrogenase (Sdh) B, C, and D subunits associated with boscalid resistance, mating type, and SSR genotype. All isolates from 2004 and 2005 exhibited wild-type (WT) Sdh alleles. Seven known Sdh substitutions were identified in isolates collected from 2009 to 2014. In 2009, 60.7% had Sdh substitutions associated with boscalid resistance in D. tanaceti . The frequency of WT isolates decreased over time, with no WT isolates identified in 2014. The frequency of the SdhB-H277Y genotype increased from 10.7 to 77.8% between 2009 and 2014. Genotypic evidence suggested that a shift in the population structure occurred between 2005 and 2009, with decreases in gene diversity ( uh ; 0.51 to 0.34), genotypic evenness (E 5 ; 0.96 to 0.67), genotypic diversity (G; 9.3 to 6.8), and allele frequencies. No evidence was obtained to support the rapid spread of Sdh genotypes by clonal expansion of the population. Thus, insensitivity to boscalid has developed and become widespread within a diverse population within 4 years of usage. These results suggest that D. tanaceti can disperse insensitivity through repeated frequent mutation, sexual recombination, or a combination of both., Competing Interests: The author(s) declare no conflict of interest.

Introduction. Klebsiella pneumoniae is a major threat to public health worldwide. It is the causative agent for multiple disease presentations including urinary tract infection, septicemia, liver abscess, wound infection and respiratory tract infection. K. pneumoniae causes community- and hospital-acquired pneumonia, which is a devastating disease associated with high mortality rates. Hypothesis. There is a growing concern about the emergence of multidrug-resistant K. pneumoniae strains complicating the treatment with the current available therapeutics; therefore, there is an urgent need for the development of new antimicrobial agents. Aim. K. pneumoniae causes an acute respiratory disease in mice and in the current work we investigated the capability to perform non-invasive monitoring of bioluminescent Klebsiella to monitor therapeutic efficacy. Methodology. We engineered a bioluminescence reporter strain of K. pneumoniae to monitor the impact of antibiotics in a murine respiratory disease model. Results. We demonstrate that bioluminescence correlates with bacterial numbers in host tissues allowing for a non-invasive enumeration of bacterial replication in vivo . Light production is directly linked to bacterial viability, and this novel bioluminescent K. pneumoniae strain enabled monitoring of the efficacy of meropenem therapy in arresting bacterial proliferation in the lung. Conclusion. The use of non-invasive bioluminescent imaging improves preclinical animal model testing to detect study outcome earlier and with higher sensitivity.

Background: During high-flow nasal cannula (HFNC) therapy, flow plays a crucial role in the physiological effects. However, there is no consensus on the initial flow settings and subsequent titration. Thus, we aimed to systematically synthesize the effects of flows during HFNC treatment., Methods: In this systematic review, two investigators independently searched PubMed, Embase, Web of Science, Scopus, and Cochrane for in vitro and in vivo studies investigating the effects of flows in HFNC treatment published in English before July 10, 2022. We excluded studies that investigated the pediatric population (< 18 years) or used only one flow. Two investigators independently extracted the data and assessed the risk of bias. The study protocol was prospectively registered with PROSPERO, CRD42022345419., Results: In total, 32,543 studies were identified, and 44 were included. In vitro studies evaluated the effects of flow settings on the fraction of inspired oxygen (F I O 2 ), positive end-expiratory pressure, and carbon dioxide (CO 2 ) washout. These effects are flow-dependent and are maximized when the flow exceeds the patient peak inspiratory flow, which varies between patients and disease conditions. In vivo studies report that higher flows result in improved oxygenation and dead space washout and can reduce work of breathing. Higher flows also lead to alveolar overdistention in non-dependent lung regions and patient discomfort. The impact of flows on different patients is largely heterogeneous., Interpretation: Individualizing flow settings during HFNC treatment is necessary, and titrating flow based on clinical findings like oxygenation, respiratory rates, ROX index, and patient comfort is a pragmatic way forward., (© 2023. The Author(s).)

This manuscript proposes an electrical definition of therapeutic dose for spinal-cord systems used for the treatment of chronic pain, analogous to the pharmacological definition. Dose-response relationships are fundamental to pharmacology, radio-therapy, and other treatments, but have never been properly established for neuromodulation. This manuscript offers a robust measure of dose, pre-requisite to establishing a reliable and repeatable dose-response relationship. The new definition, enabled by the system transresistance obtained from measurement of evoked action potentials, recognizes the mechanism of action of spinal cord stimulation (SCS), and should improve acceptance of the therapy as compared to pharmacological treatments which are currently used more frequently for the treatment of chronic pain. The new definition suggests methods for personalization and standardization of the dose in SCS, and is potentially generalizable to all neuromodulation therapies in which nervous tissue is excited including sacral nerve stimulation (SNS), vagal nerve stimulation (VNS) and deep-brain stimulation (DBS). Formulas are provided, and applied using patient data. Powerful conclusions are drawn from application of the new measure.

Background: Compassionate extubation (CE) refers to withdrawing mechanical ventilation and allowing a patient to die peacefully at the end of life. The primary objective of this pilot study was to quantify the emotional impact of CE on Respiratory Therapists (RT) and Registered Nurses (RNs)., Methods: This pilot survey was conducted between March and April 2021 at an academic medical center among RTs and RNs. It included questions on participants' demographics, work characteristics, and Impact of Events (IES) scale to assess the subjective stress caused by CE. Data were analyzed using descriptive and χ 2 statistics., Results: Among 20 participants, 18 (90%) were females, 12 (60%) were in the 20-40-year age group, 12 (60%) were RTs, and 8 (40%) RNs. Around 15 (75%) participants worked day shifts with a weekly average of 3-4 shifts, and 14 (70%) performed/observed CE within 1 month before taking this survey. CE performed/observed in a month was ≤2 among 15 (75%) and 3-5 among 4 (20%) participants. Mean total IES score was 16.7 (12.3) among all participants representing 7 (35%) having low, 6 (30%) moderate, and 7 (35%) high emotional impact when performing CE. Risk of developing post-traumatic stress disorder (PTSD) was present in 6 (30%) participants. A significantly higher number of participants in the low impact group were satisfied with the institutional CE process ( p = 0.043) than those in the medium/high impact group., Conclusion: This pilot study findings reveal that RTs and RNs experience moderate to high levels of subjective stress when performing CE. One-third of the survey participants were at risk of developing PTSD., Competing Interests: RK discloses research funding from American Association of Respiratory Care. DV discloses research funding from Teleflex Medical, Inc. and Rice Foundation, and speaker fees from Theravance Biopharma. JBS discloses research funding from Teleflex and speaker fees from Aerogen and Medline Industries, LC. EC, AF and VJL reports no potential conflict of interest.

Introduction: The Transtheoretical Model (TTM) has been used to assess individual readiness for health behavior change. We describe our use of the TTM to assess organizational readiness of African-American churches to participate in the Church Challenge (CC) in Flint, Michigan; the processes of change that moved churches toward readiness for change; and lessons learned., Methods: The CC was a faith-based, multilevel intervention to reduce chronic disease risk. A community-based participatory approach was used to engage and recruit churches. We used the TTM to capture church readiness for change and track church progress through the five stages., Results: We engaged with 70 churches: 35 remained in Stage 1 (precontemplation), 10 remained in Stage 2 (contemplation), 3 remained in Stage 3 (preparation), 5 made it to Stage 4 (action), and 17 finished within Stage 5 (maintenance). Churches engaged in several processes of change as they moved through the various stages of change., Lessons Learned: Utilizing processes of change, establishing rapport, and having previous participants share success stories helped move churches from stage-to-stage. However, certain barriers prevented progression, such as burnout/trauma from the Flint Water Crisis and scheduling conflicts., Discussion: Faith-based organizational readiness greatly impacted participation in the CC. Researchers should utilize established social capital, build rapport, and remain flexible when working with African-American churches., Conclusion: Although traditionally used at the individual level, the TTM works well at the organizational level to assess and monitor church readiness to participate in community-engaged research and health programming to improve health in an African-American faith community.

Background: Optimal flow settings during high-flow nasal cannula (HFNC) therapy are unknown. We investigated the optimal flow settings during HFNC therapy based on breathing pattern and tidal inspiratory flows in patients with acute hypoxemic respiratory failure (AHRF)., Methods: We conducted a prospective clinical study in adult hypoxemic patients treated by HFNC with a fraction of inspired oxygen (F I O 2 ) ≥ 0.4. Patient's peak tidal inspiratory flow (PTIF) was measured and HFNC flows were set to match individual PTIF and then increased by 10 L/min every 5-10 min up to 60 L/min. F I O 2 was titrated to maintain pulse oximetry (SpO 2 ) of 90-97%. SpO 2 /F I O 2 , respiratory rate (RR), ROX index [(SpO 2 /F I O 2 )/RR], and patient comfort were recorded after 5-10 min on each setting. We also conducted an in vitro study to explore the relationship between the HFNC flows and the tracheal F I O 2 , peak inspiratory and expiratory pressures., Results: Forty-nine patients aged 58.0 (SD 14.1) years were enrolled. At enrollment, HFNC flow was set at 45 (38, 50) L/min, with an F I O 2 at 0.62 (0.16) to obtain an SpO 2 /F I O 2 of 160 (40). Mean PTIF was 34 (9) L/min. An increase in HFNC flows up to two times of the individual patient's PTIF, incrementally improved oxygenation but the ROX index plateaued with HFNC flows of 1.34-1.67 times the individual PTIF. In the in vitro study, when the HFNC flow was set higher than PTIF, tracheal peak inspiratory and expiratory pressures increased as HFNC flow increased but the F I O 2 did not change., Conclusion: Mean PTIF values in most patients with AHRF were between 30 and 40 L/min. We observed improvement in oxygenation with HFNC flows set above patient PTIF. Thus, a pragmatic approach to set optimal flows in patients with AHRF would be to initiate HFNC flow at 40 L/min and titrate the flow based on improvement in ROX index and patient tolerance., Trial Registration: ClinicalTrials.gov (NCT03738345). Registered on November 13th, 2018. https://clinicaltrials.gov/ct2/show/NCT03738345?term=NCT03738345&draw=2&rank=1., (© 2021. The Author(s).)

The diversity of the U.S. population is currently not reflected in the genomic workforce and across the greater scientific enterprise. Although diversity and inclusion efforts have focused on increasing the number of individuals from underrepresented groups across scientific fields, structural racism remains. Thus, the cultivation and adoption of diversity as an ethos requires shifting our focus to being intentional about an institution's character, culture, and climate. One way for this ethos to be sustained is by facilitating an intentional anti-racism approach within the field. Adopting a new perspective on diversity utilizing an anti-racism approach will support genomics researchers as we build supportive, collaborative research environments. We seek to expand critical thought in the framing of diversity in the research enterprise and propose an anti-racism approach that informs deliberate actions required to address structural racism., Competing Interests: The authors declare no competing interests.

Background: Awake prone positioning (APP) is widely used in the management of patients with coronavirus disease (COVID-19). The primary objective of this study was to compare the outcome of COVID-19 patients who received early versus late APP., Methods: Post hoc analysis of data collected for a randomized controlled trial (ClinicalTrials.gov NCT04325906). Adult patients with acute hypoxemic respiratory failure secondary to COVID-19 who received APP for at least one hour were included. Early prone positioning was defined as APP initiated within 24 h of high-flow nasal cannula (HFNC) start. Primary outcomes were 28-day mortality and intubation rate., Results: We included 125 patients (79 male) with a mean age of 62 years. Of them, 92 (73.6%) received early APP and 33 (26.4%) received late APP. Median time from HFNC initiation to APP was 2.25 (0.8-12.82) vs 36.35 (30.2-75.23) hours in the early and late APP group (p < 0.0001), respectively. Average APP duration was 5.07 (2.0-9.05) and 3.0 (1.09-5.64) hours per day in early and late APP group (p < 0.0001), respectively. The early APP group had lower mortality compared to the late APP group (26% vs 45%, p = 0.039), but no difference was found in intubation rate. Advanced age (OR 1.12 [95% CI 1.0-1.95], p = 0.001), intubation (OR 10.65 [95% CI 2.77-40.91], p = 0.001), longer time to initiate APP (OR 1.02 [95% CI 1.0-1.04], p = 0.047) and hydrocortisone use (OR 6.2 [95% CI 1.23-31.1], p = 0.027) were associated with increased mortality., Conclusions: Early initiation (< 24 h of HFNC use) of APP in acute hypoxemic respiratory failure secondary to COVID-19 improves 28-day survival. Trial registration ClinicalTrials.gov NCT04325906., (© 2021. The Author(s).)

Background: The role of repeated prone positioning in intubated subjects with acute respiratory distress syndrome caused by COVID-19 remains unclear., Methods: We conducted a retrospective observational cohort study of critically ill intubated patients with COVID-19 who were placed in the prone position between March 18, 2020 and March 31, 2020. Exclusion criteria were pregnancy, reintubation, and previous prone positioning at a referring hospital. Patients were followed up until hospital discharge. The primary outcome was oxygenation assessed by partial pressure of oxygen/fraction of inspired oxygen ratio (Pao 2 /Fio 2 ) ratio. A positive response to proning was defined as an increase in Pao 2 /Fio 2 ratio ≥20%. Treatment failure of prone positioning was defined as death or requirement for extracorporeal membrane oxygenation (ECMO)., Results: Forty-two subjects (29 males; age: 59 [52-69] yr) were eligible for analysis. Nine subjects were placed in the prone position only once, with 25 requiring prone positioning on three or more occasions. A total of 31/42 (74%) subjects survived to discharge, with five requiring ECMO; 11/42 (26%) subjects died. After the first prone positioning session, Pao 2 /Fio 2 (mean (standard deviation)) ratio increased from 17.9 kPa (7.2) to 28.2 kPa (12.2) (P<0.01). After the initial prone positioning session, subjects who were discharged from hospital were more likely to have an improvement in Pao 2 /Fio 2 ratio ≥20%, compared with those requiring ECMO or who died., Conclusion: Patients with COVID-19 acute respiratory distress syndrome frequently responded to initial prone positioning with improved oxygenation. Subsequent prone positioning in subjects discharged from hospital was associated with greater improvements in oxygenation., Competing Interests: Declarations of interest JBS discloses a relationship with Ventec Life Systems and Teleflex. JL discloses research support from Fisher & Paykel Healthcare and Rice Foundation outside the submitted work. All other authors declare that they have no conflicts of interest., (Copyright © 2020 British Journal of Anaesthesia. Published by Elsevier Ltd. All rights reserved.)

The incursion of a plant pathogen into a new geographic area initiates a series of decisions about appropriate control or eradication efforts. Incomplete, erroneous, and/or selective information may be used by diverse stakeholders to support individual goals and positions on how an incursion should be managed. We discuss the complex social, political, and technical factors that shape a biosecurity response prior to reviewing information needs and common stakeholder misunderstandings. Selected examples focus on the rust fungi (order Pucciniales). We then explore how plant pathologists, as technical experts, can interact with biosecurity stakeholders to build empathy and understanding that in turn allows a shift from being a distant subject matter expert to an active participant helping to structure problems and shape knowledge flows for better outcomes.

Colletotrichum tanaceti , the causal agent of anthracnose, is an emerging pathogen of commercially grown pyrethrum ( Tanacetum cinerariifolium ) in Australia. A microsatellite marker library was developed to understand the spatio-genetic structure over three sampled years and across two regions where pyrethrum is cultivated in Australia. Results indicated that C. tanaceti was highly diverse with a mixed reproductive mode; comprising both sexual and clonal reproduction. Sexual reproduction of C. tanaceti was more prevalent in Tasmania than in Victoria. Little differentiation was observed among field populations likely due to isolation by colonization but most of the genetic variation was occurring within populations. C. tanaceti was likely to have had a long-distance gene and genotype flow among distant populations within a state and between states. Anthropogenic transmission of propagules and wind dispersal of ascospores are the most probable mechanisms of long-distance dispersal of C. tanaceti . Evaluation of putative population histories suggested that C. tanaceti most likely originated in Tasmania and expanded from an unidentified host onto pyrethrum. Victoria was later invaded by the Tasmanian population. With the mixed mode of reproduction and possible long-distance gene flow, C. tanaceti is likely to have a high evolutionary potential and thereby has ability to adapt to management practices in the future.

Failures in control of tan spot of pyrethrum, caused by Didymella tanaceti, has been associated with decreased sensitivity within the pathogen population to the succinate dehydrogenase inhibitor (SDHI) fungicide boscalid. Sequencing the SdhB, SdhC, and SdhD subunits of isolates with resistant and sensitive phenotypes identified 15 mutations, resulting in three amino acid substitutions in the SdhB (H277Y/R, I279V), six in the SdhC (S73P, G79R, H134R, H134Q, S135R and combined H134Q/S135R), and two in the SdhD (D112E, H122R). In vitro testing of their boscalid response and estimation of resistance factors (RF) identified isolates with wild-type (WT) Sdh genotypes were sensitive to boscalid. Isolates with SdhB-I279V, SdhC-H134Q and SdhD-D112E exhibited moderate resistance phenotypes (10 ≥ RF < 100) and isolates with SdhC-H134R exhibited very high resistance phenotypes (RF ≥ 1000). All other substitutions were associated with high resistance phenotypes (100 ≥ RF < 1000). High-resolution melt assays were designed and used to estimate the frequencies of substitutions in four field populations (n = 774) collected in August (pre-boscalid application) and November (post-boscalid application) 2012. The SdhB-H277Y, SdhC-H134R and SdhB-H277R genotypes were most frequently observed across populations at 56.7, 19.0, and 10.3%, respectively. In August 92.9% of D. tanaceti contained a substitution associated with decreased sensitivity. Following boscalid application, this increased to 98.9%, with no WT isolates detected in three fields. Overlaying previously obtained microsatellite and mating-type data revealed that all ten recurrent substitutions were associated with multiple genotypes. Thus, boscalid insensitivity in D. tanaceti appears widespread and not associated with clonal spread of a limited pool of individuals., Competing Interests: Funding for this research was received from Botanical Resources Australia Pty. Ltd. This financial support does not alter our adherence to PLOS ONE policies on sharing data and materials.

Colletotrichum tanaceti is an emerging foliar fungal pathogen of commercially grown pyrethrum (Tanacetum cinerariifolium). Despite being reported consistently from field surveys in Australia, the molecular basis of pathogenicity of C. tanaceti on pyrethrum is unknown. Herein, the genome of C. tanaceti (isolate BRIP57314) was assembled de novo and annotated using transcriptomic evidence. The inferred putative pathogenicity gene suite of C. tanaceti comprised a large array of genes encoding secreted effectors, proteases, CAZymes and secondary metabolites. Comparative analysis of its putative pathogenicity gene profiles with those of closely related species suggested that C. tanaceti likely has additional hosts to pyrethrum. The genome of C. tanaceti had a high repeat content and repetitive elements were located significantly closer to genes inferred to influence pathogenicity than other genes. These repeats are likely to have accelerated mutational and transposition rates in the genome, resulting in a rapid evolution of certain CAZyme families in this species. The C. tanaceti genome showed strong signals of Repeat Induced Point (RIP) mutation which likely caused its bipartite nature consisting of distinct gene-sparse, repeat and A-T rich regions. Pathogenicity genes within these RIP affected regions were likely to have a higher evolutionary rate than the rest of the genome. This "two-speed" genome phenomenon in certain Colletotrichum spp. was hypothesized to have caused the clustering of species based on the pathogenicity genes, to deviate from taxonomic relationships. The large repertoire of pathogenicity factors that potentially evolve rapidly due to the plasticity of the genome, indicated that C. tanaceti has a high evolutionary potential. Therefore, C. tanaceti poses a high-risk to the pyrethrum industry. Knowledge of the evolution and diversity of the putative pathogenicity genes will facilitate future research in disease management of C. tanaceti and other Colletotrichum spp., Competing Interests: The authors have declared that no competing interests exist.

Tan spot, caused by Didymella tanaceti, is one of the most important foliar diseases affecting pyrethrum in Tasmania, Australia. Population dynamics, including mating-type ratios and genetic diversity of D. tanaceti, was characterized within four geographically separated fields in both late winter and spring 2012. A set of 10 microsatellite markers was developed and used to genotype 774 D. tanaceti isolates. Isolates were genotypically diverse, with 123 multilocus genotypes (MLG) identified across the four fields. Fifty-eight MLG contained single isolates and P sex analysis estimated that, within many of the recurrent MLG, there were multiple clonal lineages derived from recombination. Isolates of both mating types were at a 1:1 ratio following clone correction in each field at each sampling period, which was suggestive of sexual recombination. No evidence of genetic divergence of isolates of each mating type was identified, indicating admixture within the population. Linkage equilibrium in two of the four field populations sampled in late winter could not be discounted following clone correction. Evaluation of temporal changes in gene and genotypic diversity identified that they were both similar for the two sampling periods despite an increased D. tanaceti isolation frequency in spring. Genetic differentiation was similar in populations sampled between the two sampling periods within fields or between fields. These results indicated that sexual reproduction may have contributed to tan spot epidemics within Australian pyrethrum fields and has contributed to a genetically diverse D. tanaceti population.

Downy mildew is a serious threat to opium poppy production globally. In recent years, two pathogen species, Peronospora somniferi and Peronospora meconopsidis, which induce distinct symptoms, have been confirmed in Australia. In order to manage the spread of these pathogens, identifying the sources of inoculum is essential. In this study, we assessed pathogen presence associated with poppy seed. We developed PCR and qPCR assays targeting the coxI and coxII gene regions, for the detection, differentiation, and quantification of P. somniferi and P. meconopsidis in poppy seed. These results were complemented and compared with direct seed histological examination and a seed washing combined with viability staining for oospore detection. The majority of seed lots from all harvest years contained detectable P. meconopsidis, the earliest (1987) predating the first official record of the disease in Tasmania (1996). In contrast, only seed lots harvested in 2012 or later contained P. somniferi, evidence of its more recent introduction. P. meconopsidis contamination was estimated to be as high as 33.04 pg DNA/g of seed and P. somniferi as high as 35.17 pg DNA/g of seed. Incidence of pathogen contamination of seeds, estimated via a group testing protocol, ranged from 0 to 9% (P. meconopsidis) or 0 to 11% (P. somniferi). Mycelia were predominately found external to the seed coat. Seed washing and viability staining demonstrated that putatively viable oospores were present in the majority of seed lots. Transmission testing confirmed both pathogens can be successfully transmitted from infested seed to infected seedling. PCR and qPCR pathogen assays were found to be reliable and offer a routine test for determining pathogen inoculum in poppy seeds.

Background and Objective: Increasing physical activity (PA) is safe and beneficial in lung cancer (LC) patients. Advanced-stage LC patients are under-studied and have worse symptoms and quality of life (QoL). We evaluated the feasibility of monitoring step count in advanced LC as well as potential correlations between PA and QoL., Methods: This is a prospective, observational study of 39 consecutive patients with advanced-stage LC. Daily step count over 1 week (via Fitbit Zip), QoL, dyspnea, and depression scores were collected. Spearman rank testing was used to assess correlations. Correlation coefficients (ρ) >0.3 or <-0.3 (more and less correlated, respectively) were considered potentially clinically significant., Results: Most (83%) of the patients were interested in participating, and 67% of those enrolled were adherent with the device. Of those using the device (n = 30), the average daily step count was 4877 (range = 504-12 118) steps/d. Higher average daily step count correlated with higher QoL (ρ = 0.46), physical (ρ = 0.61), role (ρ = 0.48), and emotional functioning (ρ = 0.40) scores as well as lower depression (ρ = -0.40), dyspnea (ρ = -0.54), and pain (ρ = -0.37) scores., Conclusion: Remote PA monitoring (Fitbit Zip) is feasible in advanced-stage LC patients. Interest in participating in this PA study was high with comparable adherence to other PA studies. In those utilizing the device, higher step count correlates with higher QoL as well as lower dyspnea, pain, and depression scores. PA monitoring with wearable devices in advanced-stage LC deserves further study.

A complex of foliar diseases can affect pyrethrum in Australia, but those of greatest importance are ray blight, caused by Stagonosporopsis tanaceti, and tan spot, caused primarily by Didymella tanaceti. Isolation of fungi from pyrethrum seed lots produced over 15 years resulted in recovery of six known pathogens: S. tanaceti, D. tanaceti, Alternaria tenuissima, Colletotrichum tanaceti, Stemphylium botryosum, and Botrytis cinerea. The incidence of S. tanaceti and D. tanaceti isolated from seed varied between 0.9 and 19.5% (mean = 7.7%) and 0 and 24.1% (mean = 5.3%) among years, respectively. Commercial heat treatment of pyrethrum seed via steaming reduced the incidence of D. tanaceti from 10.9 to 0.06% and the incidence of S. tanaceti from 24.6% to nondetectable levels (<0.18%). In a second experiment, both species were reduced to nondetectable levels (<0.20%) from their initial incidences of 22.4 and 2.4%, respectively. In a field study in 2013, colonization of pyrethrum foliage by S. tanaceti was reduced from 21.1 to 14.3% in early winter when heat-treated seed was planted. However, isolation frequency of D. tanaceti was not affected significantly by seed treatment in this year. In a related experiment in 2015, the isolation frequency of D. tanaceti in plots planted from heat-treated seed depended on both prior application of an industry-standard fungicide program and proximity to another pyrethrum field in autumn. The fungus was recovered at a similar frequency in fungicide-treated and nontreated plots located near other pyrethrum fields (13.8 versus 16.3%, respectively), whereas recovery of the pathogen was reduced by fungicide applications in geographically remote pyrethrum fields (6.7 versus 1.4%, respectively). However, these differences in isolation frequency of D. tanaceti in autumn did not obviate the need for later fungicide applications to suppress foliar disease intensity in spring or flower yield in summer, independent of the proximity to other pyrethrum fields. This study suggests that steam treatment of seed can delay development of the foliar disease complex on pyrethrum, although an extremely low level of remaining infected seed or exogenous sources of inoculum necessitates the use of foliar fungicide applications in spring.

Volumetric muscle loss (VML) can result from trauma, infection, congenital anomalies, or surgery, and produce permanent functional and cosmetic deficits. There are no effective treatment options for VML injuries, and recent advances toward development of muscle constructs lack the ability to achieve innervation necessary for long-term function. We sought to develop a proof-of-concept biomaterial construct that could achieve acetylcholine receptor (AChR) clustering on muscle-derived cells (MDCs) in vitro . The approach consisted of the presentation of neural (Z+) agrin from the surface of microspheres embedded with a fibrin hydrogel to muscle cells (C2C12 cell line or primary rat MDCs). AChR clustering was spatially restricted to areas of cell (C2C12)-microsphere contact when the microspheres were delivered in suspension or when they were incorporated into a thin (2D) fibrin hydrogel. AChR clusters were observed from 16 to 72 h after treatment when Z+ agrin was adsorbed to the microspheres, and for greater than 120 h when agrin was covalently coupled to the microspheres. Little to no AChR clustering was observed when agrin-coated microspheres were delivered from specially designed 3D fibrin constructs. However, cyclic stretch in combination with agrin-presenting microspheres led to dramatic enhancement of AChR clustering in cells cultured on these 3D fibrin constructs, suggesting a synergistic effect between mechanical strain and agrin stimulation of AChR clustering in vitro . These studies highlight a strategy for maintaining a physiological phenotype characterized by motor endplates of muscle cells used in tissue engineering strategies for muscle regeneration. As such, these observations may provide an important first step toward improving function of tissue-engineered constructs for treatment of VML injuries.

Tan spot of pyrethrum (Tanacetum cinerariifolium) is caused by the ascomycete Didymella tanaceti. To assess the evolutionary role of ascospores in the assumed asexual species, the structure and arrangement of mating-type (MAT) genes were examined. A single MAT1-1 or MAT1-2 idiomorph was identified in all isolates examined, indicating that the species is heterothallic. The idiomorphs were flanked upstream and downstream by regions encoding pyridoxamine phosphate oxidase-like and DNA lyase-like proteins, respectively. A multiplex MAT-specific polymerase chain reaction assay was developed and used to genotype 325 isolates collected within two transects in each of four fields in Tasmania, Australia. The ratio of isolates of each mating-type in each transect was consistent with a 1:1 ratio. The spatial distribution of the isolates of the two mating-types within each transect was random for all except one transect for MAT1-1 isolates, indicating that clonal patterns of each mating-type were absent. However, evidence of a reduced selection pressure on MAT1-1 isolates was observed, with a second haplotype of the MAT1-1-1 gene identified in 4.4% of MAT1-1 isolates. In vitro crosses between isolates with opposite mating-types failed to produce ascospores. Although the sexual morph could not be induced, the occurrence of both mating-types in equal frequencies suggested that a cryptic sexual mode of reproduction may occur within field populations.

In Australia, pyrethrum (Tanacetum cinerariifolium) is affected by a foliar disease complex that can substantially reduce green leaf area and yield. Historically, the most important foliar disease of pyrethrum in Australia has been ray blight, caused by Stagonosporopsis tanaceti, and other fungi generally of minor importance. Temporal fluctuations in the frequency of fungi associated with foliar disease were quantified in each of 83 fields in northern Tasmania, Australia, during 2012 and 2013. Sampling was conducted throughout winter (April to July), spring (August to September), and summer (November) representing different phenological stages. Microsphaeropsis tanaceti, the cause of tan spot, was the pathogen most prevalent and isolated at the highest frequency, irrespective of sampling period. The next most common species was S. tanaceti, whose isolation frequency was low in winter and increased in spring and summer. Known pathogens of pyrethrum, Alternaria tenuissima, Colletotrichum tanaceti, and Stemphylium botryosum were recovered sporadically and at low frequency. Two species of potential importance, Paraphoma chrysanthemicola and Itersonilia perplexans, were also found at low frequency. This finding suggests a substantial shift in the dominant pathogen associated with foliar disease, from S. tanaceti to M. tanaceti, and coincides with an increase in defoliation severity in winter, and control failures of the spring fungicide program. Factors associated with this finding were also investigated. Sensitivity of M. tanaceti and S. tanaceti populations to the fungicides boscalid and cyprodinil collected prior to and following disease control failures in the field were tested under in vitro conditions. A high proportion (60%) of the M. tanaceti isolates obtained from fields in which no response to the spring fungicide program was found were insensitive to 50 µg a.i./ml boscalid. This represented a 4.2-fold increase in the frequency of this phenotype within the M. tanaceti population over 2 years. No shifts in sensitivities to cyprodinil of M. tanaceti and S. tanaceti, or S. tanaceti to boscalid, were observed. Considering the increase in defoliation severity over winter, the benefits of applying fungicides in autumn, in addition to the commercial standard (spring only), were quantified in 14 individual field trials conducted in 2011 and 2012. Mixed-model analysis suggested fungicide application in autumn may improve pyrethrum growth during late winter and early spring, although effects on defoliation and yield were minimal. The increasing prevalence and isolation frequency of M. tanaceti and boscalid resistance within the population is of concern and highlights the urgent need for adoption of nonchemical methods for disease management in Australian pyrethrum fields.

The link between African-Americans' disproportionate rates of diabetes, obesity and vitamin D deficiency may be marked by C-peptide as an indicator of insulin secretion. We hypothesize that vitamin D supplementation will increase C-peptide, a marker of insulin secretion. During 3 winters from 2007-2010, 328 healthy African-Americans (median age, 51 years) living in Boston, MA were randomized into a 4-arm, double-blind trial for 3 months of placebo, 1000, 2000, or 4000 IU of vitamin D3. The differences in non-fasting C-peptide between baseline and 3 months were -0.44 ng/mL for those receiving placebo, -0.10 ng/mL for those receiving 1000 IU/d, 0 ng/mL for those receiving 2000 IU/d, 1.24 ng/mL for those receiving 4000 IU/d (C-peptide increased 0.42 ng/mL for each additional 1000 IU/d of vitamin D3, p < 0.001). Vitamin D supplementation increased C-peptide in overweight African-Americans and may be compatible with other recommendations for diabetes prevention and management including weight loss and increased physical activity.

Lung cancer continues to be a difficult disease frequently diagnosed in late stages with a high mortality and symptom burden. In part because of frequent lung comorbidity, even lung cancer survivors often remain symptomatic and functionally limited. Though targeted therapy continues to increase treatment options for advanced-stage disease, symptom burden remains high with few therapeutic options. In the last several decades, exercise and physical activity have arisen as therapeutic options for obstructive lung disease and lung cancer. To date, exercise has been shown to reduce symptoms, increase exercise tolerance, improve quality of life, and potentially reduce length of stay and postoperative complications. Multiple small trials have been performed in perioperative non-small-cell lung cancer patients, although fewer studies are available for patients with advanced-stage disease. Despite the increased interest in this subject over the last few years, a validated exercise regimen has not been established for perioperative or advanced-stage disease. Clinicians underutilize exercise and pulmonary rehabilitation as a therapy, in part because of the lack of evidence-based consensus as to how and when to implement increasing physical activity. This review summarizes the existing evidence on exercise in lung cancer patients.

Background: Response of parathyroid hormone (PTH) to vitamin D supplementation is determined by the baseline PTH level and change in vitamin D status. Conflicting reports in Blacks exist on the PTH response to vitamin D to supplementation., Methods: During 3 winters from 2007-2010, 328 healthy Blacks (median age, 51 years) living in Boston, MA were randomized into a 4-arm, double-blind trial for 3 months of placebo, 1000, 2000, or 4000 IU of vitamin D3. PTH was measured in 254 participants at baseline and at the end of vitamin D supplementation period., Results: The differences in PTH between baseline and 3 months were 3.93 pg/mL for those receiving placebo, -3.37 pg/mL for those receiving 1000 IU/d, -6.76 pg/mL for those receiving 2000 IU/d, and -8.99 pg/mL for those receiving 4000 IU/d ( -2.98 pg/mL for each additional 1000 IU/d of vitamin D3; p<0.001)., Conclusion: We found a significant decrease in PTH with increasing doses of vitamin D supplementation up to intakes of 4000 IU/d in Blacks. Clinical Trials.gov: NCT00585637.

Klebsiella pneumoniae is a bacterial pathogen of worldwide importance and a significant contributor to multiple disease presentations associated with both nosocomial and community acquired disease. ATCC 43816 is a well-studied K. pneumoniae strain which is capable of causing an acute respiratory disease in surrogate animal models. In this study, we performed sequencing of the ATCC 43816 genome to support future efforts characterizing genetic elements required for disease. Furthermore, we performed comparative genetic analyses to the previously sequenced genomes from NTUH-K2044 and MGH 78578 to gain an understanding of the conservation of known virulence determinants amongst the three strains. We found that ATCC 43816 and NTUH-K2044 both possess the known virulence determinant for yersiniabactin, as well as a Type 4 secretion system (T4SS), CRISPR system, and an acetonin catabolism locus, all absent from MGH 78578. While both NTUH-K2044 and MGH 78578 are clinical isolates, little is known about the disease potential of these strains in cell culture and animal models. Thus, we also performed functional analyses in the murine macrophage cell lines RAW264.7 and J774A.1 and found that MGH 78578 (K52 serotype) was internalized at higher levels than ATCC 43816 (K2) and NTUH-K2044 (K1), consistent with previous characterization of the antiphagocytic properties of K1 and K2 serotype capsules. We also examined the three K. pneumoniae strains in a novel BALB/c respiratory disease model and found that ATCC 43816 and NTUH-K2044 are highly virulent (LD50<100 CFU) while MGH 78578 is relatively avirulent.

Background: Black men exhibit a high prevalence of vitamin D deficiency as well as a higher incidence of prostate cancer and higher mortality rates from prostate cancer than Whites. There are few data about the effect of vitamin D3 (cholecalciferol) supplementation on prostate-specific antigen (PSA) in healthy Black men., Methods: During three winters from 2007 to 2010, 105 Black men (median age, 48.9 years) of Boston, MA were randomized into a four-arm, double-blind trial for 3 months of placebo, 1,000, 2,000, or 4,000 U of vitamin D3. At baseline and 3 months, free and total PSA was measured., Results: With vitamin D supplementation, no significant differences in free and total PSA were observed; free PSA, -0.0004 ng/mL (P = 0.94) and total PSA, -0.004 ng/mL (P = 0.92) for each additional 1,000 U/d of vitamin D3., Conclusion: Within an unselected population of healthy Black men without a cancer diagnosis, we found no effect of vitamin D supplementation on free or total PSA., Impact: These findings support prior findings of no change in PSA with vitamin D supplementation and emphasize the need for new methods to assess the influence of vitamin D supplementation on prostate cancer prevention., (©2014 American Association for Cancer Research.)

Background: Association studies have suggested that lower circulating 25-hydroxyvitamin D [25(OH)D] in African Americans may partially underlie higher rates of cardiovascular disease and cancer in this population. Nonetheless, the relation between vitamin D supplementation and 25(OH)D concentrations in African Americans remains undefined., Objective: Our primary objective was to determine the dose-response relation between vitamin D and plasma 25(OH)D., Design: A total of 328 African Americans in Boston, MA, were enrolled over 3 winters from 2007 to 2010 and randomly assigned to receive a placebo or 1000, 2000, or 4000 IU vitamin D₃/d for 3 mo. Subjects completed sociodemographic and dietary questionnaires, and plasma samples were drawn at baseline and 3 and 6 mo., Results: Median plasma 25(OH)D concentrations at baseline were 15.1, 16.2, 13.9, and 15.7 ng/mL for subjects randomly assigned to receive the placebo or 1000, 2000, or 4000 IU/d, respectively (P = 0.63). The median plasma 25(OH)D concentration at 3 mo differed significantly between supplementation arms at 13.7, 29.7, 34.8, and 45.9 ng/mL, respectively (P < 0.001). An estimated 1640 IU vitamin D₃/d was needed to raise the plasma 25(OH)D concentration to ≥ 20 ng/mL in ≥ 97.5% of participants, whereas a dose of 4000 IU/d was needed to achieve concentrations ≥ 33 ng/mL in ≥ 80% of subjects. No significant hypercalcemia was seen in a subset of participants., Conclusions: Within African Americans, an estimated 1640 IU vitamin D₃/d was required to achieve concentrations of plasma 25(OH)D recommended by the Institute of Medicine, whereas 4000 IU/d was needed to reach concentrations predicted to reduce cancer and cardiovascular disease risk in prospective observational studies. These results may be helpful for informing future trials of disease prevention.

African Americans have a disproportionate burden of inflammation-associated chronic diseases such as cancer and lower circulating levels of 25-hydroxyvitamin D [25(OH)D]. The effect of vitamin D3 (cholecalciferol) supplementation on inflammatory markers is uncertain. We conducted a randomized, double-blind, placebo-controlled trial of supplemental oral vitamin D (placebo, 1,000, 2,000, or 4,000 IU/day of vitamin D3 orally for 3 months) in 328 African Americans (median age, 51 years) of public housing communities in Boston, MA, who were enrolled over three consecutive winter periods (2007-2010). Change from 0 to 3 months of plasma levels of 25(OH)D, high-sensitivity C-reactive protein (CRP), interleukin (IL)-6, IL-10, and soluble TNF-α receptor type 2 (sTNF-R2) in 292 (89%) participants were measured. Overall, no statistically significant changes in CRP, IL-6, IL-10, and sTNF-R2 were observed after the vitamin D supplementation period. Baseline CRP was significantly inversely associated with the baseline 25(OH)D level (P < 0.001) in unadjusted and adjusted models. An interaction between baseline 25(OH)D and vitamin D supplementation was observed for outcome change in log CRP (month 3-month 0; P for interaction = 0.04). Within an unselected population of African Americans, short-term exposure to vitamin D supplementation produced no change in circulating inflammatory markers. This study confirms the strong independent association of CRP with 25(OH)D status even after adjusting for body mass index. Future studies of longer supplemental vitamin D3 duration are necessary to examine the complex influence of vitamin D3 on CRP and other chronic inflammatory cytokines for possible reduction of cancer health disparities in African Americans.

Sclerotinia crown rot, caused by Sclerotinia minor and S. sclerotiorum, is a disease of pyrethrum in Australia that may cause substantial decline in plant density. The spatiotemporal characteristics of the disease were quantified in 14 fields during three growing seasons. Fitting the binary power law to disease incidence provided slope (b = 1.063) and intercept (ln(A p ) = 0.669) estimates significantly (P ≤ 0.0001) greater than 1 and 0, respectively, indicating spatial aggregation at the sampling unit scale that was dependent upon disease incidence. Covariate analyses indicated that application of fungicides did not significantly influence these estimates. Spatial autocorrelation and spatial analysis by distance indices indicated that spatial aggregation above the sampling unit scale was limited to 20 and 17% of transects analyzed, respectively. The range of significant aggregation was limited primarily to neighboring sampling units only. Simple temporal disease models failed to adequately describe disease progress, due to a decline in disease incidence in spring. The relationships between disease incidence at the scales of individual plants within quadrats and quadrats within a field was modeled using four predictors of sample size. The choice of the specific incidence-incidence relationship influenced the classification of disease incidence as greater than or less than 2% of plants, a provisional commercial threshold for fungicide application. Together, these studies indicated that epidemics of Sclerotinia crown rot were dominated by small-scale aggregation of disease. Larger scale patterns of diseased plants, when present, were associated with severe disease outbreaks. The spatial and temporal analyses were suggestive of disease epidemics being associated with localized primary inoculum and other factors that favor disease development at a small scale.

Sclerotinia crown rot, caused by Sclerotinia sclerotiorum and S. minor, is a prevalent disease in pyrethrum fields in Australia. Management involves fungicide applications during the rosette stage of plant development from autumn to early spring in fields approaching first harvest. However, estimates of crop damage and the efficacy of these tactics are poorly understood; therefore, plots were established in 86 pyrethrum fields in Tasmania, Australia during 2010 to 2012 to quantify these and to identify risk factors for disease outbreaks. On average, commercial management for Sclerotinia crown rot reduced disease incidence 43 to 67% compared with nontreated plots. There was a weak but significant relationship between relative increase in flower yield when fungicides were applied and the incidence of crown rot (R 2 = 0.09, P = 0.006), although the mean number of flowers produced was similar regardless of fungicide applications. Flower yield was positively associated with canopy density in spring (S = 0.39, P = 0.001). Moreover, canopy density in spring was linked by both direct and indirect effects to canopy density during autumn and winter which, in turn, were associated with planting date and previous rain events. Modeling canopy density and disease incidence in autumn correctly categorized disease incidence in spring relative to a threshold of 2% in 72% of fields. In a subset of 22 fields monitored over 2 years, canopy density in the autumn following the first harvest had a negative relationship with Sclerotinia crown rot incidence the preceding year (R 2 = 0.23, P = 0.006). On average, however, current commercial management efforts provided only small increases in flower yield in the current season and appear best targeted to fields with well-developed plant canopies and Sclerotinia crown rot present during early autumn.

Blacks have significantly higher rates of hypertension than whites, and lower circulating levels of 25-hydroxyvitamin D. There are few data about the effect of vitamin D3 (cholecalciferol) supplementation on blood pressure in blacks. During 2 winters from 2008 to 2010, 283 blacks (median age, 51 years) were randomized into a 4-arm, double-blind trial for 3 months of placebo, 1000, 2000, or 4000 international units of cholecalciferol per day. At baseline, 3 months, and 6 months, systolic and diastolic pressure and 25-hydroxyvitamin D were measured. The 3-month follow-up was completed in 250 (88%) participants. The difference in systolic pressure between baseline and 3 months was +1.7 mm Hg for those receiving placebo, -0.66 mm Hg for 1000 U/d, -3.4 mm Hg for 2000 U/d, and -4.0 mm Hg for 4000 U/d of cholecalciferol (-1.4 mm Hg for each additional 1000 U/d of cholecalciferol; P=0.04). For each 1-ng/mL increase in plasma 25-hydroxyvitamin D, there was a significant 0.2-mm Hg reduction in systolic pressure (P=0.02). There was no effect of cholecalciferol supplementation on diastolic pressure (P=0.37). Within an unselected population of blacks, 3 months of oral vitamin D3 supplementation significantly, yet modestly, lowered systolic pressure. Future trials of vitamin D supplementation on blood pressure are needed to confirm these promising results, particularly among blacks, a population for whom vitamin D deficiency may play a more specific mechanistic role in the pathogenesis of hypertension.

Potato virus S (PVS) is prevalent within potato (Solanum tuberosum) production worldwide. Traditionally, PVS has been split into two strains, Ordinary (PVS O ) and Andean (PVS A ), based on reaction in herbaceous indicator species such as Chenopodium quinoa. However, recent research has identified further strain designations, such as PVS O-CS (Ordinary and Chenopodium systemic). Forty-four isolates of PVS were collected from potato seed lines in different geographical regions within Tasmania, Australia. Isolates were initially characterized by reactions in C. quinoa. Nineteen isolates were characterized as PVS O , based on the development of local lesions and serological detection in inoculated leaves only. Three isolates were identified as PVS A -like, based on local lesion development in inoculated leaves, mild mottling or chlorotic spots on noninoculated leaves, and serological detection in both inoculated and noninoculated leaves. Thirteen isolates produced no symptoms, and were detected serologically in inoculated leaves only (PVS O -like). Four isolates produced no symptoms but were detected serologically in both inoculated and noninoculated leaves (PVS A -like). Five isolates produced symptoms in inoculated leaves only but were detected serologically in both inoculated and noninoculated leaves (also PVS A -like). The ability of isolates to infect tomato has also been used as a criterion to assist in PVS strain differentiation. A subsample of isolates (n = 16) was unable to infect tomato 'Grosse Lisse'. Seventeen isolates representative of these groupings based on reactions in C. quinoa were also characterized by coat-protein sequencing. Phylogenetic comparisons suggested that all isolates were PVS O rather than PVS A . Therefore, whereas some of these PVS isolates were systemic in C. quinoa, findings from this study suggest that they were not PVS A , and that only PVS O and PVS O-CS isolates are present in Tasmania. The implications of this finding for disease management are discussed.

Ray blight, caused by Phoma ligulicola var. inoxydabilis, causes substantial annual losses in Australian pyrethrum fields. Fifty-nine P. ligulicola var. inoxydabilis isolates were randomly selected from fields in three distinct geographical regions in Tasmania, Australia. Genetic diversity was characterized using random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). Based on genetic similarities of less than 99%, 56 distinct genotypes (putative clones) were observed. Mean haploid gene diversity of clone-corrected populations ranged between 0.05 and 0.31, and 0.11 and 0.32, for the RAPD and AFLP data sets, respectively. Cluster analysis indicated two distinct groups of isolates supported by all bootstrap replicates. The first cluster contained all but four isolates with representatives from all three populations. The second cluster contained two isolates from the Western and Central populations, respectively, while the remaining isolates were not able to be grouped with any distinct cluster. Analysis of the population structure suggested no evidence for spatial autocorrelation at the smallest distance classes. The presence of linkage disequilibrium was indicated regardless of population scale. Collectively, these findings provided further evidence for the absence or minor role of the teleomorph in the epidemiology of ray blight in Australian pyrethrum fields.

Biomaterial nerve cuffs are a clinical alternative to autografts and allografts as a means to repair segmental peripheral nerve defects. However, existing clinical biomaterial constructs lack true incorporation of physical guidance cues into their design. In both two- and three-dimensional systems, it is known that substrate geometry directly affects rates of axon migration. However, the ability to incorporate these cues into biomaterial scaffolds of sufficient porosity to promote robust nerve regeneration in three-dimensional systems is a challenge. We have developed fibrin constructs fabricated by a sacrificial templating approach, yielding scaffolds with multiple 10-250 μm diameter conduits depending on the diameter of the template fibers. The resulting scaffolds contained numerous, highly aligned conduits, had porosity of ∼ 80%, and showed mechanical properties comparable to native nerve (150-300 kPa Young's modulus). We studied the effects of the conduit diameters on the rate of axon migration through the scaffold to investigate if manipulation of this geometry could be used to ultimately promote more rapid bridging of the scaffold. All diameters studied led to axon migration, but in contrast to effects of fiber diameters in other systems, the rate of axon migration was independent of conduit diameter in these templated scaffolds. However, aligned conduits did support more rapid axon migration than non-aligned, tortuous controls., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Pale purple coneflower, Echinacea pallida (Nutt.) Nutt., is an herbaceous perennial cultivated for its ornamental and medicinal properties. In 2005, phytoplasma-like symptoms, including virescence, phyllody, and chlorotic leaves, were first observed in coneflower fields in northern Tasmania, Australia. During the 2010-2011 growing season, the incidence of affected plants was estimated to be 12% within a single field. Total DNA was extracted from symptomatic plants with a DNeasy Plant Mini Kit (QIAGEN Inc., Valencia, CA) according to the manufacturer's instructions. DNA was also extracted, as described above, from asymptomatic coneflower seedlings obtained by germinating surface-sterilized seed on water agar. DNA was amplified via a nested PCR using universal primer pairs P1/P7 followed by R16F2n/R16R2 to detect putative phytoplasmas (2). Amplifications yielded expected products of 1.8 and 1.2 kb, respectively, only from symptomatic samples. Subsequently, PCR products from six arbitrarily selected samples were used for sequencing (Genome Lab Dye Terminator Cycling Sequence with Quick Start Chemistry) and read in a CEQ8000 sequencer (Beckman Coulter Inc., Brea, CA). Sequence homology indicated 100% similarity between isolates designated EWB1 to EWB4 (GenBank Accession Nos. JF340075 and JF340077 to JF340079) and between EWB5 and EWB6 (JF340076 and JF40080). Sequence homology between the two observed groups was 99.7%, resulting from a 4-bp difference in the R16F2n primer region. Blast search revealed isolates EWB1 to EWB4 were 100% homologous with Catharanthus roseus phytoplasma (EU096500.1), Tomato big bud phytoplasma (EF193359.1), Scaevola witches'-broom phytoplasma (AB257291.1), and Mollicutes sp. (Y10097.1 and Y10096.1). Moreover, isolates EWB5 and EWB6 shared 99% sequence identity with the above isolates. iPhyClassifier (4) was used to perform sequence similarity and generate virtual restriction fragment length polymorphism (RFLP) profiles. The 16S rDNA sequence of isolates EWB1 to EWB4 and EWB5 to EWB6 shared 100 and 99.7% similarity, respectively, to the 'Candidatus Phytoplasma australasiae' reference strain (Y10097). RFLP profiles from all isolates suggested that they belonged to the 16SrII-D subgroup. To our knowledge, this is the first report of a 16SrII-D subgroup phytoplasma infecting E. pallida in Australia. Aster yellow phytoplasmas (16SrI-C subgroup) infections of E. purpurea have been recorded in Slovenia (3) and southern Bohemia (1). References: (1) J. Franova et al. Eur. J. Plant Pathol. 123:85, 2009. (2) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (3) S. Radišek et al. Plant Pathol. 58:392, 2009. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

Oxylipins, a class of oxygenase-derived unsaturated fatty acids, are important signal molecules in many biological systems. Recent characterization of an Aspergillus flavus lipoxygenase gene, lox, revealed its importance in maintaining a density-dependent morphology switch from sclerotia to conidia as population density increased. Here, we present evidence for the involvement of four more oxylipin-generating dioxygenases (PpoA, PpoB, PpoC, and PpoD) in A. flavus density-dependent phenomena and the effects of loss of these genes on aflatoxin production and seed colonization. Although several single mutants showed alterations in the sclerotia-to-conidia switch, the major effect was observed in a strain downregulated for all five oxygenases (invert repeat transgene [IRT] strain IRT4 = ppoA, ppoB, ppoC, ppoD, and lox). In strain IRT4, sclerotia production was increased up to 500-fold whereas conidiation was decreased down to 100-fold and the strain was unable to switch into conidial production. Aflatoxin (AF) production for all mutant strains and the wild type was greatest at low population densities and absent in high populations except for strain IRT4, which consistently produced high levels of the mycotoxin. Growth on host seed by both IRT4 and IRT2 (downregulated in ppoA, ppoB, and ppoD) was marked by decreased conidial but increased AF production. We propose that A. flavus oxygenases and the oxylipins they produce act in a highly interdependent network with some redundancy of biological function. These studies provide substantial evidence for oxylipin-based mechanisms in governing fungus-seed interactions and in regulating a coordinated quorum-sensing mechanism in A. flavus.

Downy mildew, caused by Peronospora arborescens, has become the major disease affecting oilseed poppy (Papaver somniferum) since its first record in Tasmania in 1996. Two field trials conducted in 2000 and 2001 studied the progression and spatial distribution of downy mildew epiphytotics. The logistic and exponential models best described the progression of disease incidence and severity, respectively. Incidence and severity increased rapidly following canopy closure. In 2001, incidence increased from 0.16%, prior to canopy closure, to 100% at late flowering (40 days). Spatial analyses of epiphytotics were conducted by fitting the beta-binomial and binomial distributions, median runs analysis, and the spatial analysis by distance indices. All analyses demonstrated that the distribution of incidence and severity was strongly spatially aggregated from canopy closure until at least late flowering. These results suggest that secondary spread from a few primary infections is the major factor in epiphytotics.