The PAS domain-containing histidine kinase RpfS is a second sensor for the diffusible signal factor of <em>Xanthomonas campestris</em>

Summary: A cell-cell signalling system mediated by the fatty acid signal DSF controls the virulence of Xanthomonas campestris pv. campestris (Xcc) to plants. The synthesis and recognition of the DSF signal depends upon different Rpf proteins. DSF signal generation requires RpfF whereas signal perception and transduction depends upon the sensor RpfC and regulator RpfG. Detailed analyses of the regulatory roles of different Rpf proteins have suggested the occurrence of further sensors for DSF. Here we have used a mutagenesis approach coupled with high-resolution transcriptional analysis to identify XC_2579 (RpfS) as a second sensor for DSF in Xcc. RpfS is a complex sensor kinase predicted to have multiple Per/Arnt/Sim (PAS) domains, a histidine kinase domain and a C-terminal receiver (REC) domain. Isothermal calorimetry showed that DSF bound to the isolated N-terminal PAS domain with a Kd of 1.4μM. RpfS controlled expression of a sub-set of genes distinct from those controlled by RpfC to include genes involved in type IV secretion and chemotaxis. Mutation of XC_2579 was associated with a reduction in virulence of Xcc to Chinese Radish when assayed by leaf spraying but not by leaf inoculation, suggesting a role for RpfS-controlled factors in the epiphytic phase of the disease cycle.</p

Resource utilisation and direct costs in patients with recently diagnosed fibromyalgia who are offered one of three different interventions in a randomised pragmatic trial

The purpose of this study is to understand the course of costs over a 2-year period in a cohort of recently diagnosed fibromyalgia (FM) patients receiving different treatment strategies. Following the diagnosis, patients were randomly assigned to a multidisciplinary programme (MD), aerobic exercise (AE) or usual care (UC) without being aware of alternative interventions. Time between diagnosis and start of treatment varied between patients. Resource utilisation, health care costs and costs for patients and families were collected through cost diaries. Mixed linear model analyses (MLM) examined the course of costs over time. Linear regression was used to explore predictors of health care costs in the post-intervention period. Two hundred three participants, 90 % women, mean (SD) age 41.7 (9.8) years, were included in the cohort. Intervention costs per patient varied from €864 to 1392 for MD and were €121 for AE. Health care costs (excluding intervention costs) decreased after diagnosis, but before the intervention in each group, and increased again afterwards to the level close to the diagnostic phase. In contrast, patient and family costs slightly increased over time in all groups without initial decrease immediately after diagnosis. Annualised health care costs post-intervention varied between €1872 and 2310 per patient and were predicted by worse functioning and high health care costs at diagnosis. In patients with FM, health care costs decreased following the diagnosis by a rheumatologist. Offering patients a specific intervention after diagnosis incurred substantial costs while having only marginal effects on cost

Automatic generation of factual questions from video documentaries

Questioning sessions are an essential part of teachers’ daily instructional activities. Questions are used to assess students’ knowledge and comprehension and to promote learning. The manual creation of such learning material is a laborious and time-consuming task. Research in Natural Language Processing (NLP) has shown that Question Generation (QG) systems can be used to efficiently create high-quality learning materials to support teachers in their work and students in their learning process. A number of successful QG applications for education and training have been developed, but these focus mainly on supporting reading materials. However, digital technology is always evolving; there is an ever-growing amount of multimedia content available, and more and more delivery methods for audio-visual content are emerging and easily accessible. At the same time, research provides empirical evidence that multimedia use in the classroom has beneficial effects on student learning. Thus, there is a need to investigate whether QG systems can be used to assist teachers in creating assessment materials from these different types of media that are being employed in classrooms. This thesis serves to explore how NLP tools and techniques can be harnessed to generate questions from non-traditional learning materials, in particular videos. A QG framework which allows the generation of factual questions from video documentaries has been developed and a number of evaluations to analyse the quality of the produced questions have been performed. The developed framework uses several readily available NLP tools to generate questions from the subtitles accompanying a video documentary. The reason for choosing video vii documentaries is two-fold: firstly, they are frequently used by teachers and secondly, their factual nature lends itself well to question generation, as will be explained within the thesis. The questions generated by the framework can be used as a quick way of testing students’ comprehension of what they have learned from the documentary. As part of this research project, the characteristics of documentary videos and their subtitles were analysed and the methodology has been adapted to be able to exploit these characteristics. An evaluation of the system output by domain experts showed promising results but also revealed that generating even shallow questions is a task which is far from trivial. To this end, the evaluation and subsequent error analysis contribute to the literature by highlighting the challenges QG from documentary videos can face. In a user study, it was investigated whether questions generated automatically by the system developed as part of this thesis and a state-of-the-art system can successfully be used to assist multimedia-based learning. Using a novel evaluation methodology, the feasibility of using a QG system’s output as ‘pre-questions’ with different types of prequestions (text-based and with images) used was examined. The psychometric parameters of the automatically generated questions by the two systems and of those generated manually were compared. The results indicate that the presence of pre-questions (preferably with images) improves the performance of test-takers and they highlight that the psychometric parameters of the questions generated by the system are comparable if not better than those of the state-of-the-art system. In another experiment, the productivity of questions in terms of time taken to generate questions manually vs. time taken to post-edit system-generated questions was analysed. A viii post-editing tool which allows for the tracking of several statistics such as edit distance measures, editing time, etc, was used. The quality of questions before and after postediting was also analysed. Not only did the experiments provide quantitative data about automatically and manually generated questions, but qualitative data in the form of user feedback, which provides an insight into how users perceived the quality of questions, was also gathered.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Crystal structure of an HD-GYP domain cyclic-di-GMP phosphodiesterase reveals an enzyme with a novel trinuclear catalytic iron centre

Bis-(3′,5′) cyclic di-guanylate (c-di-GMP) is a key bacterial second messenger that is implicated in the regulation of many crucial processes that include biofilm formation, motility and virulence. Cellular levels of c-di-GMP are controlled through synthesis by GGDEF domain diguanylate cyclases and degradation by two classes of phosphodiesterase with EAL or HD-GYP domains. Here, we have determined the structure of an enzymatically active HD-GYP domain protein from Persephonella marina (PmGH) alone, in complex with substrate (c-di-GMP) and final reaction product (GMP). The structures reveal a novel trinuclear iron binding site, which is implicated in catalysis and identify residues involved in recognition of c-di-GMP. This structure completes the picture of all domains involved in c-di-GMP metabolism and reveals that the HD-GYP family splits into two distinct subgroups containing bi- and trinuclear metal centres.</p

Endothelial Mitogen-Activated Protein Kinase Kinase Kinase Kinase 4 Is Critical for Lymphatic Vascular Development and Function

The molecular mechanisms underlying lymphatic vascular development and function are not well understood. Recent studies have suggested a role for endothelial cell (EC) mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) in developmental angiogenesis and atherosclerosis. Here, we show that constitutive loss of EC Map4k4 in mice causes postnatal lethality due to chylothorax, suggesting that Map4k4 is required for normal lymphatic vascular function. Mice constitutively lacking EC Map4k4 displayed dilated lymphatic capillaries, insufficient lymphatic valves, and impaired lymphatic flow; furthermore, primary ECs derived from these animals displayed enhanced proliferation compared with controls. Yeast 2-hybrid analyses identified the Ras GTPase-activating protein Rasa1, a known regulator of lymphatic development and lymphatic endothelial cell fate, as a direct interacting partner for Map4k4. Map4k4 silencing in ECs enhanced basal Ras and extracellular signal-regulated kinase (Erk) activities, and primary ECs lacking Map4k4 displayed enhanced lymphatic EC marker expression. Taken together, these results reveal that EC Map4k4 is critical for lymphatic vascular development by regulating EC quiescence and lymphatic EC fate

Novel cyclic di-GMP effectors of the YajQ protein family control bacterial virulence

Bis-(3 ',5 ') cyclic di-guanylate (cyclic di-GMP) is a key bacterial second messenger that is implicated in the regulation of many critical processes that include motility, biofilm formation and virulence. Cyclic di-GMP influences diverse functions through interaction with a range of effectors. Our knowledge of these effectors and their different regulatory actions is far from complete, however. Here we have used an affinity pull-down assay using cyclic di-GMP-coupled magnetic beads to identify cyclic di-GMP binding proteins in the plant pathogen Xanthomonas campestris pv. campestris (Xcc). This analysis identified XC_3703, a protein of the YajQ family, as a potential cyclic di-GMP receptor. Isothermal titration calorimetry showed that the purified XC_3703 protein bound cyclic di-GMP with a high affinity (K-d similar to 2 mu M). Mutation of XC_3703 led to reduced virulence of Xcc to plants and alteration in biofilm formation. Yeast two-hybrid and far-western analyses showed that XC_3703 was able to interact with XC_2801, a transcription factor of the LysR family. Mutation of XC_2801 and XC_3703 had partially overlapping effects on the transcriptome of Xcc, and both affected virulence. Electromobility shift assays showed that XC_3703 positively affected the binding of XC_2801 to the promoters of target virulence genes, an effect that was reversed by cyclic di-GMP. Genetic and functional analysis of YajQ family members from the human pathogens Pseudomonas aeruginosa and Stenotrophomonas maltophilia showed that they also specifically bound cyclic di-GMP and contributed to virulence in model systems. The findings thus identify a new class of cyclic di-GMP effector that regulates bacterial virulence

High-resolution transcriptional analysis of the regulatory influence of cell-to-cell signalling reveals novel genes that contribute to Xanthomonas phytopathogenesis

The bacterium Xanthomonas campestris is an economically important pathogen of many crop species and a model for the study of bacterial phytopathogenesis. In X.campestris, a regulatory system mediated by the signal molecule DSF controls virulence to plants. The synthesis and recognition of the DSF signal depends upon different Rpf proteins. DSF signal generation requires RpfF whereas signal perception and transduction depends upon a system comprising the sensor RpfC and regulator RpfG. Here we have addressed the action and role of Rpf/DSF signalling in phytopathogenesis by high-resolution transcriptional analysis coupled to functional genomics. We detected transcripts for many genes that were unidentified by previous computational analysis of the genome sequence. Novel transcribed regions included intergenic transcripts predicted as coding or non-coding as well as those that were antisense to coding sequences. In total, mutation of rpfF, rpfG and rpfC led to alteration in transcript levels (more than fourfold) of approximately 480 genes. The regulatory influence of RpfF and RpfC demonstrated considerable overlap. Contrary to expectation, the regulatory influence of RpfC and RpfG had limited overlap, indicating complexities of the Rpf signalling system. Importantly, functional analysis revealed over 160 new virulence factors within the group of Rpf-regulated genes.</p

Design of small molecule-responsive microRNAs based on structural requirements for Drosha processing

MicroRNAs (miRNAs) are prevalent regulatory RNAs that mediate gene silencing and play key roles in diverse cellular processes. While synthetic RNA-based regulatory systems that integrate regulatory and sensing functions have been demonstrated, the lack of detail on miRNA structure–function relationships has limited the development of integrated control systems based on miRNA silencing. Using an elucidated relationship between Drosha processing and the single-stranded nature of the miRNA basal segments, we developed a strategy for designing ligand-responsive miRNAs. We demonstrate that ligand binding to an aptamer integrated into the miRNA basal segments inhibits Drosha processing, resulting in titratable control over gene silencing. The generality of this control strategy was shown for three aptamer–small molecule ligand pairs. The platform can be extended to the design of synthetic miRNAs clusters, cis-acting miRNAs and self-targeting miRNAs that act both in cis and trans, enabling fine-tuning of the regulatory strength and dynamics. The ability of our ligand-responsive miRNA platform to respond to user-defined inputs, undergo regulatory performance tuning and display scalable combinatorial control schemes will help advance applications in biological research and applied medicine