Identifying gene regulatory networks common to multiple plant stress responses

Stress responses in plants can be defined as a change that affects the homeostasis of pathways, resulting in a phenotype that may or may not be visible to the human eye, affecting the fitness of the plant. Crosstalk is believed to be the shared components of pathways of networks, and is widespread in plants, as shown by examples of crosstalk between transcriptional regulation pathways, and hormone signalling. Crosstalk between stress responses is believed to exist, particularly crosstalk within the responses to biotic stress, and within the responses to abiotic stress. Certain hormone pathways are known to be involved in the crosstalk between the responses to both biotic and abiotic stresses, and can confer immunity or tolerance of Arabidopsis thaliana to these stresses. Transcriptional regulation has also been identified as an important factor in controlling tolerance and resistance to stresses. In this thesis, networks of regulation mediating the response tomultiple stresses are studied. Firstly, co-regulation was predicted for genes differentially expressed in two or more stresses by development of a novel multi-clustering approach, Wigwams Identifies Genes Working Across Multiple Stresses (Wigwams). This approach finds groups of genes whose expression is correlated within stresses, but also identifies a strong statistical link between subsets of stresses. Wigwams identifies the known co-expression of genes encoding enzymes of metabolic and flavonoid biosynthesis pathways, and predicts novels clusters of co-expressed genes. By hypothesising that by being coexpressed could also infer that the genes are co-regulated, promoter motif analysis and modelling provides information for potential upstream regulators. The context-free regulation of groups of co-expressed genes, or potential regulons, was explored using models generated by modelling techniques, in order to generate a quantitative model of transcriptional regulation during the response to B. cinerea, P. syringae pv. tomato DC3000 and senescence. This model was subsequently validated and extended by experimental techniques, using Yeast 1-Hybrid to investigate the protein-DNA interactions, and also microarrays. Analysis of mutants and plants overexpressing a predicted regulator, Rap2.6L, by gene expression analysis identified a number of potential regulon members as downstream targets. Rap2.6L was identified as an indirect regulator of the transcription factor members of three potential regulons co-expressed in the stresses B. cinerea, P. syringae pv. tomato DC3000 and long day senescence, allowing the confirmation of a predicted gene regulatory network operating in multiple stress responses

MARDy : Mycology Antifungal Resistance Database

J.R. was supported by an Antimicrobial Research Collaborative (ARC) early career research fellowship, Imperial College London (RSRO_54990). T.S. and J.M.G.S. were supported by a Natural Environment Research Council grant awarded to MCF (NE/P001165/1).Peer reviewedPublisher PD

Illuminating Choices for Library Prep: A Comparison of Library Preparation Methods for Whole Genome Sequencing of Cryptococcus neoformans Using Illumina HiSeq.

The industry of next-generation sequencing is constantly evolving, with novel library preparation methods and new sequencing machines being released by the major sequencing technology companies annually. The Illumina TruSeq v2 library preparation method was the most widely used kit and the market leader; however, it has now been discontinued, and in 2013 was replaced by the TruSeq Nano and TruSeq PCR-free methods, leaving a gap in knowledge regarding which is the most appropriate library preparation method to use. Here, we used isolates from the pathogenic fungi Cryptococcus neoformans var. grubii and sequenced them using the existing TruSeq DNA v2 kit (Illumina), along with two new kits: the TruSeq Nano DNA kit (Illumina) and the NEBNext Ultra DNA kit (New England Biolabs) to provide a comparison. Compared to the original TruSeq DNA v2 kit, both newer kits gave equivalent or better sequencing data, with increased coverage. When comparing the two newer kits, we found little difference in cost and workflow, with the NEBNext Ultra both slightly cheaper and faster than the TruSeq Nano. However, the quality of data generated using the TruSeq Nano DNA kit was superior due to higher coverage at regions of low GC content, and more SNPs identified. Researchers should therefore evaluate their resources and the type of application (and hence data quality) being considered when ultimately deciding on which library prep method to use

Transcriptional Heterogeneity of Cryptococcus gattii VGII Compared with Non-VGII Lineages Underpins Key Pathogenicity Pathways

We thank Jose Munoz for his input on the analysis of the mouse RNA-seq enrichment. R.A.F. was supported by a Wellcome Trust-Massachusetts Institute of Technology (MIT) Postdoctoral Fellowship. M.C.F. and J.R. were supported by Medical Research Council grant MR/K000373/1. R.C.M. is supported by a Wolfson Royal Society Research Merit Award and by funding from the European Research Council under the European Union’s Seventh Framework Program (FP/2007-2013)/ERC (grant agreement no. 614562). This work was funded in part by NIAID grant U19AI110818 to the Broad Institute.Peer reviewedPublisher PD

Architecture and dynamics of the jasmonic acid gene regulatory network

Jasmonic acid (JA) is a critical hormonal regulator of plant growth and defense. To advance our understanding of the architecture and dynamic regulation of the JA gene regulatory network, we performed a high-resolution RNA-seq time series of methyl JA-treated Arabidopsis thaliana at 15 time points over a 16-h period. Computational analysis showed that methyl JA (MeJA) induces a burst of transcriptional activity, generating diverse expression patterns over time that partition into distinct sectors of the JA response targeting specific biological processes. The presence of transcription factor (TF) DNA binding motifs correlated with specific TF activity during temporal MeJA-induced transcriptional reprogramming. Insight into the underlying dynamic transcriptional regulation mechanisms was captured in a chronological model of the JA gene regulatory network. Several TFs, including MYB59 and bHLH27, were uncovered as early network components with a role in pathogen and insect resistance. Analysis of subnetworks surrounding the TFs ORA47, RAP2.6L, MYB59, and ANAC055, using transcriptome profiling of overexpressors and mutants, provided insights into their regulatory role in defined modules of the JA network. Collectively, our work illuminates the complexity of the JA gene regulatory network, pinpoints and validates previously unknown regulators, and provides a valuable resource for functional studies on JA signaling components in plant defense and development

Lensing in the Blue II: Estimating the Sensitivity of Stratospheric Balloons to Weak Gravitational Lensing

The Superpressure Balloon-borne Imaging Telescope (SuperBIT) is a diffraction-limited, wide-field, 0.5 m, near-infrared to near-ultraviolet observatory designed to exploit the stratosphere's space-like conditions. SuperBIT's 2023 science flight will deliver deep, blue imaging of galaxy clusters for gravitational lensing analysis. In preparation, we have developed a weak lensing measurement pipeline with modern algorithms for PSF characterization, shape measurement, and shear calibration. We validate our pipeline and forecast SuperBIT survey properties with simulated galaxy cluster observations in SuperBIT's near-UV and blue bandpasses. We predict imaging depth, galaxy number (source) density, and redshift distribution for observations in SuperBIT's three bluest filters; the effect of lensing sample selections is also considered. We find that in three hours of on-sky integration, SuperBIT can attain a depth of b = 26 mag and a total source density exceeding 40 galaxies per square arcminute. Even with the application of lensing-analysis catalog selections, we find b-band source densities between 25 and 30 galaxies per square arcminute with a median redshift of z = 1.1. Our analysis confirms SuperBIT's capability for weak gravitational lensing measurements in the blue.Comment: Submitted to Astronomical Journa

Dynamic ploidy changes drive fluconazole resistance in human cryptococcal meningitis.

BACKGROUND Cryptococcal meningitis (CM) causes an estimated 180,000 deaths annually, predominantly in sub-Saharan Africa, where most patients receive fluconazole (FLC) monotherapy. While relapse after FLC monotherapy with resistant strains is frequently observed, the mechanisms and impact of emergence of FLC resistance in human CM are poorly understood. Heteroresistance (HetR) - a resistant subpopulation within a susceptible strain - is a recently described phenomenon in Cryptococcus neoformans (Cn) and Cryptococcus gattii (Cg), the significance of which has not previously been studied in humans. METHODS A cohort of 20 patients with HIV-associated CM in Tanzania was prospectively observed during therapy with either FLC monotherapy or in combination with flucytosine (5FC). Total and resistant subpopulations of Cryptococcus spp. were quantified directly from patient cerebrospinal fluid (CSF). Stored isolates underwent whole genome sequencing and phenotypic characterization. RESULTS Heteroresistance was detectable in Cryptococcus spp. in the CSF of all patients at baseline (i.e., prior to initiation of therapy). During FLC monotherapy, the proportion of resistant colonies in the CSF increased during the first 2 weeks of treatment. In contrast, no resistant subpopulation was detectable in CSF by day 14 in those receiving a combination of FLC and 5FC. Genomic analysis revealed high rates of aneuploidy in heteroresistant colonies as well as in relapse isolates, with chromosome 1 (Chr1) disomy predominating. This is apparently due to the presence on Chr1 of ERG11, which is the FLC drug target, and AFR1, which encodes a drug efflux pump. In vitro efflux levels positively correlated with the level of heteroresistance. CONCLUSION Our findings demonstrate for what we believe is the first time the presence and emergence of aneuploidy-driven FLC heteroresistance in human CM, association of efflux levels with heteroresistance, and the successful suppression of heteroresistance with 5FC/FLC combination therapy. FUNDING This work was supported by the Wellcome Trust Strategic Award for Medical Mycology and Fungal Immunology 097377/Z/11/Z and the Daniel Turnberg Travel Fellowship

A realist evaluation of the role of communities of practice in changing healthcare practice

<p>Abstract</p> <p>Background</p> <p>Healthcare organisations seeking to manage knowledge and improve organisational performance are increasingly investing in communities of practice (CoPs). Such investments are being made in the absence of empirical evidence demonstrating the impact of CoPs in improving the delivery of healthcare. A realist evaluation is proposed to address this knowledge gap. Underpinned by the principle that outcomes are determined by the context in which an intervention is implemented, a realist evaluation is well suited to understand the role of CoPs in improving healthcare practice. By applying a realist approach, this study will explore the following questions: What outcomes do CoPs achieve in healthcare? Do these outcomes translate into improved practice in healthcare? What are the contexts and mechanisms by which CoPs improve healthcare?</p> <p>Methods</p> <p>The realist evaluation will be conducted by developing, testing, and refining theories on how, why, and when CoPs improve healthcare practice. When collecting data, context will be defined as the setting in which the CoP operates; mechanisms will be the factors and resources that the community offers to influence a change in behaviour or action; and outcomes will be defined as a change in behaviour or work practice that occurs as a result of accessing resources provided by the CoP.</p> <p>Discussion</p> <p>Realist evaluation is being used increasingly to study social interventions where context plays an important role in determining outcomes. This study further enhances the value of realist evaluations by incorporating a social network analysis component to quantify the structural context associated with CoPs. By identifying key mechanisms and contexts that optimise the effectiveness of CoPs, this study will contribute to creating a framework that will guide future establishment and evaluation of CoPs in healthcare.</p