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Commercially cultivated Prunus species are commonly grown in adjacent or mixed orchards and can be infected with unique or commonly shared viruses. Apple (Malus domestica), another member of the Rosacea and distantly related to Prunus, can share the same growing regions and common pathogens. Pollen can be a major route for virus transmission, and analysis of the pollen virome in tree fruit orchards can provide insights into these virus pathogen complexes from mixed production sites. Commercial honey bee (Apis mellifera) pollination is essential for improved fruit sets and yields in tree fruit production systems. To better understand the pollen-associated virome in tree fruits, metagenomics-based detection of plant viruses was employed on bee and pollen samples collected at four time points during the peak bloom period of apricot, cherry, peach, and apple trees at one orchard site. Twenty-one unique viruses were detected in samples collected during tree fruit blooms, including prune dwarf virus (PDV) and prunus necrotic ringspot virus (PNRSV) (Genus Ilarvirus, family Bromoviridae), Secoviridae family members tomato ringspot virus (genus Nepovirus), tobacco ringspot virus (genus Nepovirus), prunus virus F (genus Fabavirus), and Betaflexiviridae family member cherry virus A (CVA; genus Capillovirus). Viruses were also identified in composite leaf and flower samples to compare the pollen virome with the virome associated with vegetative tissues. At all four time points, a greater diversity of viruses was detected in the bee and pollen samples. Finally, the nucleotide sequence diversity of the coat protein regions of CVA, PDV, and PNRSV was profiled from this site, demonstrating a wide range of sequence diversity in pollen samples from this site. These results demonstrate the benefits of area-wide monitoring through bee pollination activities and provide new insights into the diversity of viruses in tree fruit pollination ecosystems.

AbstractBackground Burnout in pediatric residents is widespread. Certain factors are associated with decreased burnout, such as empathy, self-compassion, mindfulness, and resilience, while perceived stress is associated with increased burnout. Narrative medicine may reduce burnout by its impact on protective and exacerbating factors and can be an active tool to promote wellness. The objective of this pilot study was to evaluate immediate and delayed benefits of a longitudinal narrative medicine intervention for pediatric residents using qualitative and quantitative measures.Materials and Methods We designed a voluntary longitudinal narrative medicine intervention implemented via Zoom teleconferencing software over five months for pediatric residents at Nationwide Children’s Hospital. It consisted of six one-hour long sessions where residents engaged with literature, responded to a writing prompt, and shared their reflections. It was evaluated using open-ended survey questions and established quantitative assessment tools of well-being with validity evidence. Results were compared before the intervention, immediately after, and six months later using one-way ANOVA and multiple linear regression. Qualitative data was analyzed using thematic analysis.Results Twenty-two (14% of eligible) residents participated in at least one session. After the intervention, the following themes emerged for benefits to resident well-being: the ability to Build Community, have an Outlet for Self-Expression, reap Emotional and Mental Health Benefits, and work on Personal Growth. Benefits were sustained even six months later, which has not been shown previously. While there were significant qualitative findings, between all three time points, there was no change in any quantitative well-being measures.Conclusion Our longitudinal narrative medicine pilot study showed meaningful sustained qualitative benefits, though no quantitative changes, in measured well-being outcomes that have been previously associated with lower resident burnout. While not a panacea, narrative medicine can be a useful strategy for residency programs to improve pediatric resident well-being even after completion of planned interventions.Key MessageWe used a mixed-methods approach to assess the effects of a longitudinal narrative medicine intervention on well-being in pediatric residents.Open-ended responses indicated that residents found utility in and appreciated the intervention and experienced sustained improvements in their mental and emotional health, though the sample size was likely too small to show quantitative changes in well-being measures.Narrative medicine is not a panacea, but it can be a useful tool to provide to pediatric residents to promote sustained improvements in their well-being through the framework of relationship-centered care.

IntroductionHoney bee (Apis mellifera) pollination is widely used in tree fruit production systems to improve fruit set and yield. Many plant viruses can be associated with pollen or transmitted through pollination, and can be detected through bee pollination activities. Honey bees visit multiple plants and flowers in one foraging trip, essentially sampling small amounts of pollen from a wide area. Here we report metagenomics-based area-wide monitoring of plant viruses in cherry (Prunus avium) and apple (Malus domestica) orchards in Creston Valley, British Columbia, Canada, through bee-mediated pollen sampling.MethodsPlant viruses were identified in total RNA extracted from bee and pollen samples, and compared with profiles from double stranded RNA extracted from leaf and flower tissues. CVA, PDV, PNRSV, and PVF coat protein nucleotide sequences were aligned and compared for phylogenetic analysis.ResultsA wide array of plant viruses were identified in both systems, with cherry virus A (CVA), prune dwarf virus (PDV), prunus necrotic ringspot virus (PNRSV), and prunus virus F (PVF) most commonly detected. Citrus concave gum associated virus and apple stem grooving virus were only identified in samples collected during apple bloom, demonstrating changing viral profiles from the same site over time. Different profiles of viruses were identified in bee and pollen samples compared to leaf and flower samples reflective of pollen transmission affinity of individual viruses. Phylogenetic and pairwise analysis of the coat protein regions of the four most commonly detected viruses showed unique patterns of nucleotide sequence diversity, which could have implications in their evolution and management approaches. Coat protein sequences of CVA and PVF were broadly diverse with multiple distinct phylogroups identified, while PNRSV and PDV were more conserved.ConclusionThe pollen virome in fruit production systems is incredibly diverse, with CVA, PDV, PNRSV, and PVF widely prevalent in this region. Bee-mediated monitoring in agricultural systems is a powerful approach to study viral diversity and can be used to guide more targeted management approaches.

BackgroundThe role of Vitamin D in immune function is well reported with a growing evidence base linking low levels to poorer outcomes from infectious disease. Vitamin D deficiency and insufficiency are prevalent worldwide with healthcare workers identified as a known at-risk group. Here we aim to investigate serum Vitamin D levels in a UK population of front line healthcare workers and to promote the occupational risk.MethodsA cross-sectional study of 639 volunteers was conducted to identify the prevalence of Vitamin D deficiency and insufficiency amongst a population of front-line health care workers in the UK. Participant demographics and co-morbid factors were collected at the time of serum sampling for multivariate analysis.ResultsOnly 18.8% of the population had a normal vitamin D level greater than or equal to 75nmol/L. This is compared to Public Health England's (PHE) stipulated normal levels of 60% during winter. 81.2% had a level less than 75nmol/L, with 51.2% less than 50nmol/L and 6.6% less than 25nmol/L. For serum levels less than 25nmol/L, Asian ethnicity was more likely to have a vitamin D deficiency than non-asian (OR (95%CI): 3.81 (1.73-8.39), p = 0.001), whereas white ethnicity was less likely to have a vitamin D deficiency compared to non-white (OR (95%CI: 0.43 (0.20-0.83), p = 0.03). Other factors that contributed to a higher likelihood of lower-than-normal levels within this population included male sex, decreased age and not taking supplementation.ConclusionIt is concluded that our population of healthcare workers have higher rates of abnormal vitamin D levels in comparison with the general UK population reported prevalence. Furthermore, Asian ethnicity and age 30 years and below are more at risk of vitamin D insufficiency and deficiency. This highlights an occupational risk factor for the healthcare community to consider.

Deep Learning models such as Convolutional Neural Networks (CNNs) are very effective at extracting complex image features from medical X-rays. However, the limited interpretability of CNNs has hampered their deployment in medical settings as they failed to gain trust among clinicians. In this work, we propose an interactive framework to allow clinicians to ask what-if questions and intervene in the decisions of a CNN, with the aim of increasing trust in the system. The framework translates a layer of a trained CNN into a measurable and compact set of symbolic rules. Expert interactions with visualizations of the rules promote the use of clinically-relevant CNN kernels and attach meaning to the rules. The definition and relevance of the kernels are supported by radiomics analyses and permutation evaluations, respectively. CNN kernels that do not have a clinically-meaningful interpretation are removed without affecting model performance. By allowing clinicians to evaluate the impact of adding or removing kernels from the rule set, our approach produces an interpretable refinement of the data-driven CNN in alignment with medical best practice.

Healthy agroecosystems are dependent on a complex web of factors and inter-species interactions. Flowers are hubs for pathogen transmission, including the horizontal or vertical transmission of plant-viruses and the horizontal transmission of bee-viruses. Pollination by the European honey bee (Apis mellifera) is critical for industrial fruit production, but bees can also vector viruses and other pathogens between individuals. Here, we utilized commercial honey bee pollination services in blueberry (Vaccinium corymbosum) farms for a metagenomics-based bee and plant virus monitoring system. Following RNA sequencing, viruses were identified by mapping reads to a reference sequence database through the bioinformatics portal Virtool. In total, 29 unique plant viral species were found at two blueberry farms in British Columbia (BC). Nine viruses were identified at one site in Ontario (ON), five of which were not identified in BC. Ilarviruses blueberry shock virus (BlShV) and prune dwarf virus (PDV) were the most frequently detected viruses in BC but absent in ON, while nepoviruses tomato ringspot virus and tobacco ringspot virus were common in ON but absent in BC. BlShV coat protein (CP) nucleotide sequences were nearly identical in all samples, while PDV CP sequences were more diverse, suggesting multiple strains of PDV circulating at this site. Ten bee-infecting viruses were identified, with black queen cell virus frequently detected in ON and BC. Area-wide bee-mediated pathogen monitoring can provide new insights into the diversity of viruses present in, and the health of, bee-pollination ecosystems. This approach can be limited by a short sampling season, biased towards pollen-transmitted viruses, and the plant material collected by bees can be very diverse. This can obscure the origin of some viruses, but bee-mediated virus monitoring can be an effective preliminary monitoring approach.

The liquid biopsy has the potential to improve patient care in the diagnostic and therapeutic setting in non-small cell lung cancer (NSCLC). Consented patients with epidermal growth factor receptor (EGFR) positive disease (n = 21) were stratified into two cohorts: those currently receiving EGFR tyrosine kinase inhibitor (TKI) therapy (n = 9) and newly diagnosed EGFR TKI treatment-naïve patients (n = 12). Plasma genotyping of cell-free DNA was carried out using the FDA-approved cobas® EGFR mutation test v2 and compared to next generation sequencing (NGS) cfDNA panels. Circulating tumor cell (CTC) numbers were correlated with treatment response and EGFR exon 20 p.T790M. The prognostic significance of the neutrophil to lymphocyte ratio (NLR) and lactate dehydrogenase (LDH) was also investigated. Patients in cohort 1 with an EGFR exon 20 p.T790M mutation progressed more rapidly than those with an EGFR sensitizing mutation, while patients in cohort 2 had a significantly longer progression-free survival (p = 0.04). EGFR exon 20 p.T790M was detected by liquid biopsy prior to disease progression indicated by computed tomography (CT) imaging. The cobas® EGFR mutation test detected a significantly greater number of exon 20 p.T790M mutations (p = 0.05). High NLR and derived neutrophil to lymphocyte ratio (dNLR) were associated with shorter time to progression and worse survival outcomes (p < 0.05). High LDH levels were significantly associated with shorter time to disease progression (p = 0.03). These data support the use of liquid biopsy for monitoring EGFR mutations and inflammatory markers as prognostic indicators in NSCLC.

An Interview with James Phelan

Neoadjuvant chemoradiation therapy (CRT) is increasingly the standard of care for locally advanced oesophageal cancer. A complete pathological response to CRT is associated with a favourable outcome. Radiation therapy is important for local tumour control, however, radioresistance remains a substantial clinical problem. We hypothesise that alterations in mitochondrial function and energy metabolism are involved in the radioresistance of oesophageal adenocarcinoma (OAC). To investigate this, we used an established isogenic cell line model of radioresistant OAC. Radioresistant cells (OE33 R) demonstrated significantly increased levels of random mitochondrial mutations, which were coupled with alterations in mitochondrial function, size, morphology and gene expression, supporting a role for mitochondrial dysfunction in the radioresistance of this model. OE33 R cells also demonstrated altered bioenergetics, demonstrating significantly increased intracellular ATP levels, which was attributed to enhanced mitochondrial respiration. Radioresistant cells also demonstrated metabolic plasticity, efficiently switching between the glycolysis and oxidative phosphorylation energy metabolism pathways, which were accompanied by enhanced clonogenic survival. This data was supported in vivo, in pre-treatment OAC tumour tissue. Tumour ATP5B expression, a marker of oxidative phosphorylation, was significantly increased in patients who subsequently had a poor pathological response to neoadjuvant CRT. This suggests for the first time, a role for specific mitochondrial alterations and metabolic remodelling in the radioresistance of OAC.

Cdc42 of the Rho GTPase family has been implicated in cell actin organization, proliferation, survival, and migration but its physiological role is likely cell-type specific. By a T cell-specific deletion of Cdc42 in mouse, we have recently shown that Cdc42 maintains naïve T cell homeostasis through promoting cell survival and suppressing T cell activation. Here we have further investigated the involvement of Cdc42 in multiple stages of T cell differentiation. We found that in Cdc42(-/-) thymus, positive selection of CD4(+)CD8(+) double-positive thymocytes was defective, CD4(+) and CD8(+) single-positive thymocytes were impaired in migration and showed an increase in cell apoptosis triggered by anti-CD3/-CD28 antibodies, and thymocytes were hyporesponsive to anti-CD3/-CD28-induced cell proliferation and hyperresponsive to anti-CD3/-CD28-stimulated MAP kinase activation. At the periphery, Cdc42-deficient naive T cells displayed an impaired actin polymerization and TCR clustering during the formation of mature immunological synapse, and showed an enhanced differentiation to Th1 and CD8(+) effector and memory cells in vitro and in vivo. Finally, Cdc42(-/-) mice exhibited exacerbated liver damage in an induced autoimmune disease model. Collectively, these data establish that Cdc42 is critically involved in thymopoiesis and plays a restrictive role in effector and memory T cell differentiation and autoimmunity.