Medicago truncatula Yellow Stripe-Like7 encodes a peptide transporter participating in symbiotic nitrogen fixation.

Yellow Stripe-Like (YSL) proteins are a family of plant transporters that are typically involved in transition metal homeostasis. Three of the four YSL clades (I, II and IV) transport metals complexed with the non-proteinogenic amino acid nicotianamine or its derivatives. No such capability has been shown for any member of clade III, but the link between these YSLs and metal homeostasis could be masked by functional redundancy. We studied the role of the clade III YSL protein MtSYL7 in Medicago truncatula nodules. MtYSL7, which encodes a plasma membrane-bound protein, is mainly expressed in the pericycle and cortex cells of the root nodules. Yeast complementation assays revealed that MtSYL7 can transport short peptides. M. truncatula transposon insertion mutants with decreased expression of MtYSL7 had lower nitrogen fixation rates and showed reduced plant growth whether grown in symbiosis with rhizobia or not. YSL7 mutants accumulated more copper and iron in the nodules, which is likely to result from the increased expression of iron uptake and delivery genes in roots. Taken together, these data suggest that MtYSL7 plays an important role in the transition metal homeostasis of nodules and symbiotic nitrogen fixation.

Th1/17-Biased Inflammatory Environment Associated with COPD Alters the Response of Airway Epithelial Cells to Viral and Bacterial Stimuli.

Chronic obstructive pulmonary disease (COPD) is characterized by airway inflammation associated with a Th1/17-biased cytokine environment. Acute exacerbations of COPD (AECOPD) are most often triggered by respiratory infections, which elicit an exaggerated inflammatory response in these patients, via poorly defined mechanisms. We investigated the responses of airway epithelial cells (AECs) to infective stimuli in COPD and the effects of the Th1/17-biased environment on these responses. Cytokine expression was assessed following exposure to virus-like stimuli (poly I:C or imiquimod) or bacterial LPS. The effects of pretreatment with Th1/17 cytokines were evaluated in both primary AECs and the Calu-3 AEC cell line. We found that poly I:C induced increased expression of the proinflammatory cytokines IL1ß, IL6, CXCL8, and TNF and IFN-ß1 in AECs from both control subjects and COPD patients. Expression of IL1ß in response to all 3 stimuli was significantly enhanced in COPD AECs. Primary AECs pretreated with Th1/17 cytokines exhibited enhanced expression of mRNA for proinflammatory cytokines in response to poly I:C. Similarly, Calu-3 cells responded to virus-like/bacterial stimuli with increased expression of proinflammatory cytokines, and a Th1/17 environment significantly enhanced their expression. Furthermore, increased expression of pattern recognition receptors for viruses (TLR3, TLR7, IFIH1, and DDX58) was induced by Th1/17 cytokines, in both primary AECs and Calu-3 cells. These findings suggest that the Th1/17-biased environment associated with COPD may enhance the proinflammatory cytokine response of AECs to viral and bacterial infections and that increased signaling via upregulated receptors may contribute to exaggerated inflammation in virus-induced AECOPD.

Knowledge Maps: an Online Tool for Knowledge Mapping with Automated Feedback.

Concept and knowledge maps have been shown to improve students' learning by emphasising meaningful relationships between phenomena. A user-friendly online tool that enables assessment of students' maps with automated feedback might therefore have significant benefits for learning. For that purpose, we developed an online software platform known as Knowledge Maps. Two pilot studies were performed to evaluate the usability and efficacy of Knowledge Maps. Study A demonstrated significantly improved perceptions of learning after using Knowledge Maps to learn pathology. Study B showed significant improvement between pre-test and post-test scores in an anatomy course. These preliminary studies indicate that this software is readily accepted and may have potential benefits for learning.

The role of noncoding RNAs in regulating epithelial responses in COPD.

Chronic obstructive pulmonary disease (COPD), one of the leading causes of death in the world, is a chronic inflammatory disease of the airways usually caused by long-term exposure to inhaled irritants. Airway epithelial cells (AECs) play a key role in initializing COPD and driving the exacerbation of this disease through the release of various cytokines. This AEC-derived cytokine response is tightly regulated possibly through the regulatory effects of noncoding RNAs (ncRNAs). Although the importance of ncRNAs in pulmonary diseases has been increasingly realized, little is known about the role of ncRNA in the regulation of inflammatory responses in COPD. This review outlines the features of AEC-derived cytokine responses in COPD and how ncRNAs regulate these inflammatory responses.

Enhanced Pro-Inflammatory Response of Macrophages to Interleukin-33 in an Allergic Environment.

BACKGROUND: Allergic asthma is common in childhood and is associated with a T-helper type 2 (Th2)-biased immunological response. Exacerbations of asthma are characterised by increased inflammation of the airways, which appears to be driven by interleukin (IL)-33 that can activate pulmonary macrophages. The Th2 cytokine environment of allergic asthma may contribute to exaggerated airway inflammation. OBJECTIVES: To test this, we assessed whether production of pro-inflammatory cytokines by IL-33-stimulated macrophages was enhanced in cells pre-treated with the key Th2 cytokines IL-4 and IL-13. We also investigated whether this was associated with altered expression of regulatory microRNAs (miRNAs). METHODS: RAW264.7 cells cultured with IL-4 and IL-13 for 48 h were stimulated with IL-33 for 4 h. Pro-inflammatory mediators were assessed using quantitative real-time PCR (RT-PCR). Expression of miRNAs was assessed using microarrays and RT-PCR. In further experiments, we examined whether resolvin E1 (RvE1), which promotes the resolution of experimental asthmatic inflammation in vivo, could suppress the enhanced response by treating cells with RvE1 concurrently with IL-33 stimulation. RESULTS: In cells pre-treated with IL-4 and IL-13, expression of mRNA for Ccl3, Ccl5, Ccl17, Ccl24, and Il1b in response to IL-33 stimulation was significantly increased. This was paralleled by up-regulated expression of miR-155-5p, a miRNA that is predicted to regulate several aspects of allergic inflammation. RvE1 suppressed the enhanced production of Ccl3, Ccl5, Ccl24, and Il1b. CONCLUSIONS: We conclude that IL-33-activated macrophages may contribute to the exaggerated airway inflammation in exacerbations of allergic asthma, and that RvE1 has potential as a therapeutic agent that targets macrophages.

Knowledge maps: a tool for online assessment with automated feedback.

In higher education, most assessments or examinations comprise either multiple-choice items or open-ended questions such as modified essay questions (MEQs). Online concept and knowledge maps are potential tools for assessment, which might emphasize meaningful, integrated understanding of phenomena. We developed an online knowledge-mapping assessment tool, which provides automated feedback on student-submitted maps. We conducted a pilot study to investigate the potential utility of online knowledge mapping as a tool for automated assessment by comparing the scores generated by the software with manual grading of a MEQ on the same topic for a cohort of first-year medical students. In addition, an online questionnaire was used to gather students' perceptions of the tool. Map items were highly discriminating between students of differing knowledge of the topic overall. Regression analysis showed a significant correlation between map scores and MEQ scores, and responses to the questionnaire regarding use of knowledge maps for assessment were overwhelmingly positive. These results suggest that knowledge maps provide a similar indication of students' understanding of a topic as a MEQ, with the advantage of instant, consistent computer grading and time savings for educators. Online concept and knowledge maps could be a useful addition to the assessment repertoire in higher education.

A model for the use of blended learning in large group teaching sessions.

BACKGROUND: Although blended learning has the potential to enhance the student experience, both in terms of engagement and flexibility, it can be difficult to effectively restructure existing courses. To achieve these goals for an introductory Pathology course, offered to more than 250 undergraduate students at UNSW Sydney, we devised a novel approach. METHODS: For each topic presented over 2-3 weeks, a single face-to-face overview lecture was retained. The remaining content that had previously been delivered as conventional lectures was converted into short (12-18 min) online modules. These were based on lecture slides with added animations/highlights, plus narration using edited excerpts of previous lecture recordings. The modules also incorporated interactive questions and review quizzes with feedback which used various question types. Modules were developed in PowerPoint and iSpring and uploaded to Moodle as SCORM packages. Each topic concluded with an interactive large-group session focussing on integration of the content, with in-class questions to which students could respond via the Echo360 Active Learning Platform (ALP). Overall, more than 50% of face-to-face lecture time was replaced by online modules and interactive large-group sessions. Quantitative evaluation data included usage statistics from 264 students and feedback via online survey responses from 41 students. Qualitative evaluation data consisted of reflective commentaries from 160 student ePortfolios, which were analysed to identify factors affecting learning benefits and user acceptability. RESULTS: All of the modules were completed by 74% of students and on average, 83.1% of students eventually passed the optional review quizzes. Notably, 88.4% of students responded to in-class questions during the integration and feedback sessions via the ALP. Student reflections emphasised that the modules promoted understanding, which was reinforced through active learning. The modules were described as enjoyable, motivating and were appreciated for their flexibility, which enabled students to work at their own pace. CONCLUSIONS: In transforming this introductory Pathology course, we have demonstrated a model for the use of blended learning in large group teaching sessions, which achieved high levels of completion, satisfaction and value for learning.

Iron-Nicotianamine Transporters Are Required for Proper Long Distance Iron Signaling.

The mechanisms of root iron uptake and the transcriptional networks that control root-level regulation of iron uptake have been well studied, but the mechanisms by which shoots signal iron status to the roots remain opaque. Here, we characterize an Arabidopsis (Arabidopsis thaliana) double mutant, yellow stripe1-like yellow stripe3-like (ysl1ysl3), which has lost the ability to properly regulate iron deficiency-influenced gene expression in both roots and shoots. In spite of markedly low tissue levels of iron, the double mutant does not up- and down-regulate iron deficiency-induced and -repressed genes. We have used grafting experiments to show that wild-type roots grafted to ysl1ysl3 shoots do not initiate iron deficiency-induced gene expression, indicating that the ysl1ysl3 shoots fail to send an appropriate long-distance signal of shoot iron status to the roots. We present a model to explain how impaired iron localization in leaf veins results in incorrect signals of iron sufficiency being sent to roots and affecting gene expression there. Improved understanding of the mechanism of long-distance iron signaling will allow improved strategies for the engineering of staple crops to accumulate additional bioavailable iron in edible parts, thus improving the iron nutrition of the billions of people worldwide whose inadequate diet causes iron deficiency anemia.

Modeling TH 2 responses and airway inflammation to understand fundamental mechanisms regulating the pathogenesis of asthma.

In this review, we highlight experiments conducted in our laboratories that have elucidated functional roles for CD4+ T-helper type-2 lymphocytes (TH 2 cells), their associated cytokines, and eosinophils in the regulation of hallmark features of allergic asthma. Notably, we consider the complexity of type-2 responses and studies that have explored integrated signaling among classical TH 2 cytokines (IL-4, IL-5, and IL-13), which together with CCL11 (eotaxin-1) regulate critical aspects of eosinophil recruitment, allergic inflammation, and airway hyper-responsiveness (AHR). Among our most important findings, we have provided evidence that the initiation of TH 2 responses is regulated by airway epithelial cell-derived factors, including TRAIL and MID1, which promote TH 2 cell development via STAT6-dependent pathways. Further, we highlight studies demonstrating that microRNAs are key regulators of allergic inflammation and potential targets for anti-inflammatory therapy. On the background of TH 2 inflammation, we have demonstrated that innate immune cells (notably, airway macrophages) play essential roles in the generation of steroid-resistant inflammation and AHR secondary to allergen- and pathogen-induced exacerbations. Our work clearly indicates that understanding the diversity and spatiotemporal role of the inflammatory response and its interactions with resident airway cells is critical to advancing knowledge on asthma pathogenesis and the development of new therapeutic approaches.

Allergic environment enhances airway epithelial pro-inflammatory responses to rhinovirus infection.

Airway epithelial cells (AEC) exhibit a pro-inflammatory phenotype in patients with allergic asthma. We examined the effect of an allergic cytokine environment on the response of AEC to rhinovirus (RV), the most common trigger of acute exacerbations of asthma. Calu-3 cells, a well-differentiated human AEC line, were cultured with or without the T-helper type 2 cytokines interleukin (IL)-4 and IL-13, then stimulated with a toll-like receptor (TLR) 3 agonist (poly I:C, dsRNA) or a TLR7 agonist (imiquimod), or infected with RV 16. Expression of pro-inflammatory and antiviral mediators, and of viral pattern-recognition molecules, was assessed using nCounter assays, quantitative real-time PCR (qRT-PCR) and protein immunoassays. Both dsRNA and imiquimod stimulated expression of mRNA for IL6 and IL8 whereas expression of several chemokines and antiviral response genes was induced only by dsRNA. Conversely, expression of other cytokines and growth factors was induced only by imiquimod. RV infection not only stimulated expression of the inflammation-related genes induced by dsRNA, but also of complement factor B and the novel pro-inflammatory cytokine IL-32. In the T helper type 2 (Th2) cytokine environment, several mediators exhibited significantly enhanced expression, whereas expression of interferons was either unchanged or enhanced. The allergic environment also increased expression of pattern-recognition receptors and of intercellular adhesion molecule 1, the cell surface receptor for RV. We conclude that Th2 cytokines promote increased production of pro-inflammatory mediators by AEC following infection with RV. Increased viral entry or enhanced signalling via pattern-recognition receptors could also contribute to the exaggerated inflammatory response to RV observed in allergic asthmatics.

Mouse models of acute exacerbations of allergic asthma.

Most of the healthcare costs associated with asthma relate to emergency department visits and hospitalizations because of acute exacerbations of underlying chronic disease. Development of appropriate animal models of acute exacerbations of asthma is a necessary prerequisite for understanding pathophysiological mechanisms and assessing potential novel therapeutic approaches. Most such models have been developed using mice. Relatively few mouse models attempt to simulate the acute-on-chronic disease that characterizes human asthma exacerbations. Instead, many reported models involve relatively short-term challenge with an antigen to which animals are sensitized, followed closely by an unrelated triggering agent, so are better described as models of potentiation of acute allergic inflammation. Triggers for experimental models of asthma exacerbations include (i) challenge with high levels of the sensitizing allergen (ii) infection by viruses or fungi, or challenge with components of these microorganisms (iii) exposure to environmental pollutants. In this review, we examine the strengths and weaknesses of published mouse models, their application for investigation of novel treatments and potential future developments.

Cytopathology whole slide images and adaptive tutorials for senior medical students: a randomized crossover trial.

BACKGROUND: Diagnostic cytopathology is an essential part of clinical decision-making. However, due to a combination of factors including curriculum reform and shortage of pathologists to teach introductory cytopathology, this area of pathology receives little or no formal attention in most medical school curricula. We have previously described the successful use of efficient and effective digital learning resources, including whole slide images (WSI) and virtual microscopy adaptive tutorials (VMATs), to teach cytopathology to pathology specialist trainees - a group that had prior exposure to cytopathology in their day to day practice. Consequently, in the current study we attempted to demonstrate the efficiency and efficacy of this eLearning resource in a cohort of senior medical students that was completely naïve to the subject matter (cytopathology). METHODS: We evaluated both the quantitative and qualitative impact of these digital educational materials for learning cytopathology compared with existing resources (e-textbooks and online atlases). The senior medical students were recruited from The University of New South Wales Australia for a randomized cross-over trial. Online assessments, administered after each arm of the trial, contained questions which related directly to a whole slide image. Two categories of questions in the assessments (focusing on either diagnosis or identification of cellular features) were utilized to determine efficacy. User experience and perceptions of efficiency were evaluated using online questionnaires containing Likert scale items and open-ended questions. RESULTS: For this cohort of senior medical students, virtual microscopy adaptive tutorials (VMATs) proved to be at least as effective as existing digital resources for learning cytopathology. Importantly, virtual microscopy adaptive tutorials had superior efficacy in facilitating accurate diagnosis on whole slide images. Student perceptions of VMATs were positive, particularly regarding the immediate feedback, interactivity and equity of learning which this learning resource provides. CONCLUSIONS: Virtual microscopy adaptive tutorials have the potential to improve the efficacy of learning microscopic pathology for medical students. The enhanced learning experience provided by these eLearning tools merits further investigation of their utility for other cohorts, including specialist trainees.

Using multiple online databases to help identify microRNAs regulating the airway epithelial cell response to a virus-like stimulus.

BACKGROUND AND OBJECTIVE: Exacerbations of allergic asthma are often triggered by respiratory viral infections. We have previously shown that in a T-helper type 2 (Th2)-biased cytokine environment, mouse and human airway epithelial cells (AEC) exhibit increased expression of pro-inflammatory and anti-viral genes in response to synthetic double-stranded ribonucleic acid (dsRNA), a virus-like stimulus. This implies coordinated regulation of gene expression, suggesting possible involvement of microRNA. To investigate this, we developed a novel approach to identifying candidate microRNA using online databases, then confirmed their expression by quantitative real-time polymerase chain reaction (qRT-PCR). METHODS: Using a list of genes of interest, defined on the basis of the previous study as being up-regulated in a Th2 environment, we searched mouse and human microRNA databases for possible regulatory microRNA, and selected 10 candidates that were conserved across species or predicted by more than one human database. Expression of these microRNA was tested by qRT-PCR, in primary human AEC pre-treated with Th2 cytokines and exposed to dsRNA. RESULTS: Expression of hsa-miR-139-5p, miR-423-5p and miR-542-3p was significantly decreased in Th2 pre-treated AEC, and miR-135a-5p exhibited a trend towards decreased expression. Further database searches confirmed that these microRNA regulated additional pro-inflammatory and anti-viral response genes for which expression had previously been shown to be up-regulated, confirming the validity of this approach. CONCLUSIONS: Our study demonstrates the value of using multiple online databases to identify candidate regulatory microRNA and provides the first evidence that in an allergic environment, microRNA may be important in altering the pro-inflammatory and anti-viral responses of human AEC during exacerbations of asthma.

Adaptive Tutorials Versus Web-Based Resources in Radiology: A Mixed Methods Comparison of Efficacy and Student Engagement.

RATIONALE AND OBJECTIVES: Diagnostic imaging is under-represented in medical curricula globally. Adaptive tutorials, online intelligent tutoring systems that provide a personalized learning experience, have the potential to bridge this gap. However, there is limited evidence of their effectiveness for learning about diagnostic imaging. MATERIALS AND METHODS: We performed a randomized mixed methods crossover trial to determine the impact of adaptive tutorials on perceived engagement and understanding of the appropriate use and interpretation of common diagnostic imaging investigations. Although concurrently engaged in disparate blocks of study, 99 volunteer medical students (from years 1-4 of the 6-year program) were randomly allocated to one of two groups. In the first arm of the trial on chest X-rays, one group received access to an adaptive tutorial, whereas the other received links to an existing peer-reviewed Web resource. These two groups crossed over in the second arm of the trial, which focused on computed tomography scans of the head, chest, and abdomen. At the conclusion of each arm of the trial, both groups completed an examination-style assessment, comprising questions both related and unrelated to the topics covered by the relevant adaptive tutorial. Online questionnaires were used to evaluate student perceptions of both learning resources. RESULTS: In both arms of the trial, the group using adaptive tutorials obtained significantly higher assessment scores than controls. This was because of higher assessment scores by senior students in the adaptive tutorial group when answering questions related to topics covered in those tutorials. Furthermore, students indicated significantly better engagement with adaptive tutorials than the Web resource and rated the tutorials as a significantly more valuable tool for learning. CONCLUSIONS: Medical students overwhelmingly accept adaptive tutorials for diagnostic imaging. The tutorials significantly improve the understanding of diagnostic imaging by senior students.

Cytopathology whole slide images and adaptive tutorials for postgraduate pathology trainees: a randomized crossover trial.

To determine whether cytopathology whole slide images and virtual microscopy adaptive tutorials aid learning by postgraduate trainees, we designed a randomized crossover trial to evaluate the quantitative and qualitative impact of whole slide images and virtual microscopy adaptive tutorials compared with traditional glass slide and textbook methods of learning cytopathology. Forty-three anatomical pathology registrars were recruited from Australia, New Zealand, and Malaysia. Online assessments were used to determine efficacy, whereas user experience and perceptions of efficiency were evaluated using online Likert scales and open-ended questions. Outcomes of online assessments indicated that, with respect to performance, learning with whole slide images and virtual microscopy adaptive tutorials was equivalent to using traditional methods. High-impact learning, efficiency, and equity of learning from virtual microscopy adaptive tutorials were strong themes identified in open-ended responses. Participants raised concern about the lack of z-axis capability in the cytopathology whole slide images, suggesting that delivery of z-stacked whole slide images online may be important for future educational development. In this trial, learning cytopathology with whole slide images and virtual microscopy adaptive tutorials was found to be as effective as and perceived as more efficient than learning from glass slides and textbooks. The use of whole slide images and virtual microscopy adaptive tutorials has the potential to provide equitable access to effective learning from teaching material of consistently high quality. It also has broader implications for continuing professional development and maintenance of competence and quality assurance in specialist practice.

ISU201 enhances the resolution of airway inflammation in a mouse model of an acute exacerbation of asthma.

Glucocorticoids are commonly used for treating asthma and its exacerbations but have well-recognised adverse effects and are not always effective. Few alternative treatments exist. Using a murine model of an acute exacerbation of asthma, we assessed the ability of ISU201, a novel protein drug, to suppress the inflammatory response when administered after induction of an exacerbation. Sensitised mice were chronically challenged with a low mass concentration of aerosolised ovalbumin, and then received a single moderate-level challenge to simulate an allergen-induced exacerbation. ISU201 was administered to mice 2 and 8 hours later, while pulmonary inflammation and expression of mRNA for chemokines and proinflammatory cytokines were assessed after 4, 12, and 24 hours. Relative to vehicle-treated controls, ISU201 suppressed accumulation of pulmonary neutrophils and eosinophils, while accelerating the decline in CXCL1, TNF-α, and IL-6 in lavage fluid and lung tissue. ISU201 significantly reduced peak expression of mRNA for the chemokines Cxcl9 and Cxcl10, the adhesion molecules Icam1 and Vcam1, and the proinflammatory cytokines Il1b, Il12p40, and Csf1. The ability of ISU201 to promote resolution of inflammation suggests that it may have potential as an alternative to glucocorticoids in the management of asthma, including when administered after the onset of an acute exacerbation.

Differential injurious effects of ambient and traffic-derived particulate matter on airway epithelial cells.

BACKGROUND AND OBJECTIVE: Exposure to airborne particulate matter (PM) may promote development of childhood asthma and trigger acute exacerbations of existing asthma via injury to airway epithelial cells (AEC). METHODS: We compared the response of AEC to ambient particulates with median aerodynamic diameters of <10 µm or <2.5 µm from the Sydney metropolitan region (Sydney PM10 or PM2.5), to traffic-derived particulates from the exhaust stack of a motorway tunnel or to inert carbon black as a control. RESULTS: Sydney PM10 strongly stimulated messenger RNA expression and secretion of the pro-inflammatory cytokines interleukin 6 (IL-6) and chemokine (C-X-C motif) ligand 1 (CXCL1) by mouse tracheal AEC. In contrast, traffic-derived particulates did not. Similarly, PM10 stimulated expression of IL6, IL8 and IL1B by human AEC. Mass spectrometric analysis showed that PM10 contained much higher levels of elements associated with dusts of geological origin. In contrast, tunnel soot contained much higher levels of various organic compounds, notably including long straight-chain alkanes and diesel-derived polycyclic aromatic hydrocarbons. Sydney PM2.5, as well as PM10 collected during a period including a major dust storm, both of which contained relatively lower levels of iron but similar levels of other crustal elements, did not stimulate expression or secretion of CXCL1 by mouse AEC. CONCLUSIONS: Ambient PM10 is likely to be more important than traffic-derived PM in causing injury to AEC leading to production of pro-inflammatory cytokines. The injurious effects may be related to the presence of iron in the coarse fraction of airborne PM. These findings are likely to be relevant to the pathogenesis of asthma.