Non-parametric Heat Map Representation of Flow Cytometry Data: Identifying Cellular Changes Associated With Genetic Immunodeficiency Disorders

Genetic primary immunodeficiency diseases are increasingly recognized, with pathogenic mutations changing the composition of circulating leukocyte subsets measured by flow cytometry (FCM). Discerning changes in multiple subpopulations is challenging, and subtle trends might be missed if traditional reference ranges derived from a control population are applied. We developed an algorithm where centiles were allocated using non-parametric comparison to controls, generating multiparameter heat maps to simultaneously represent all leukocyte subpopulations for inspection of trends within a cohort or segregation with a putative genetic mutation. To illustrate this method, we analyzed patients with Primary Antibody Deficiency (PAD) and kindreds harboring mutations in TNFRSF13B (encoding TACI), CTLA4, and CARD11. In PAD, loss of switched memory B cells (B-SM) was readily demonstrated, but as a continuous, not dichotomous, variable. Expansion of CXCR5+/CD45RA- CD4+ T cells (X5-Th cells) was a prominent feature in PAD, particularly in TACI mutants, and patients with expansion in CD21-lo B cells or transitional B cells were readily apparent. We observed differences between unaffected and affected TACI mutants (increased B cells and CD8+ T-effector memory cells, loss of B-SM cells and non-classical monocytes), cellular signatures that distinguished CTLA4 haploinsufficiency itself (expansion of plasmablasts, activated CD4+ T cells, regulatory T cells, and X5-Th cells) from its clinical expression (B-cell depletion), and those that were associated with CARD11 gain-of-function mutation (decreased CD8+ T effector memory cells, B cells, CD21-lo B cells, B-SM cells, and NK cells). Co-efficients of variation exceeded 30% for 36/54 FCM parameters, but by comparing inter-assay variation with disease-related variation, we ranked each parameter in terms of laboratory precision vs. disease variability, identifying X5-Th cells (and derivatives), naïve, activated, and central memory CD8+ T cells, transitional B cells, memory and SM-B cells, plasmablasts, activated CD4 cells, and total T cells as the 10 most useful cellular parameters. Applying these to cluster analysis of our PAD cohort, we could detect subgroups with the potential to reflect underlying genotypes. Heat mapping of normalized FCM data reveals cellular trends missed by standard reference ranges, identifies changes associating with a phenotype or genotype, and could inform hypotheses regarding pathogenesis of genetic immunodeficiency.This study was supported by a National Health and Medical Research Council (NHMRC) of Australia Centre of Research Excellence (APP1079648)

Genome analysis and avirulence gene cloning using a high-density RADseq linkage map of the flax rust fungus, Melampsora lini

Agroinfiltration of avirulence gene constructs. The response of flax cultivars and near-isogenic lines to expression of avirulence gene candidates (AvrM14-A, AvrM14-B and AvrL2-A) using Agrobacterium tumefaciens-mediated transient transformation. (PDF 2637 kb

Flax rust infection transcriptomics reveals a transcriptional profile that may be indicative for rust Avr genes

Secreted effectors of fungal pathogens are essential elements for disease development. However, lack of sequence conservation among identified effectors has long been a problem for predicting effector complements in fungi. Here we have explored the expression characteristics of avirulence (Avr) genes and candidate effectors of the flax rust fungus, Melampsora lini. We performed transcriptome sequencing and real-time quantitative PCR (qPCR) on RNA extracted from ungerminated spores, germinated spores, isolated haustoria and flax seedlings inoculated with M. lini isolate CH5 during plant infection. Genes encoding two categories of M. lini proteins, namely Avr proteins and plant cell wall degrading enzymes (CWDEs), were investigated in detail. Analysis of the expression profiles of 623 genes encoding predicted secreted proteins in the M. lini transcriptome shows that the six known Avr genes (i.e. AvrM (avrM), AvrM14, AvrL2, AvrL567, AvrP123 (AvrP) and AvrP4) fall within a group of 64 similarly expressed genes that are induced in planta and show a peak of expression early in infection with a subsequent decline towards sporulation. Other genes within this group include two paralogues of AvrL2, an AvrL567 virulence allele, and a number of genes encoding putative effector proteins. By contrast, M. lini genes encoding CWDEs fall into different expression clusters with their distribution often unrelated to their catalytic activity or substrate targets. These results suggest that synthesis of M. lini Avr proteins may be regulated in a coordinated fashion and that the expression profiling-based analysis has significant predictive power for the identification of candidate Avr genes.This work was conducted with the support of the Australian Research Council grants DP1093850 (Role of fungal secreted proteins as plant disease effectors. ARH, DAJ, and PND) https://www.arc.gov.au/, DP130104098 (Molecular basis of rust infection and host plant resistance: ARH, DAJ, and PND) https://www.arc.gov.au/, and the China Scholarship Council grant (No.2010630010: WW) https://www.csc.edu.cn

Fungal phytopathogens encode functional homologues of plant rapid alkalinisation factor (RALF) peptides

In this paper we describe the presence of genes encoding close homologues of an endogenous plant peptide, rapid alkalinisation factor (RALF), within the genomes of 26 species of phytopathogenic fungi. Members of the RALF family are key growth factors in plants, and the sequence of the RALF active region is well conserved between the plant and fungal proteins. RALF1-like sequences were observed in most cases; however, RALF27-like sequences were present in the Sphaerulina musiva and Septoria populicola genomes. These two species are pathogens of poplar and interestingly, the closest relative to their respective RALF genes is a poplar RALF27-like sequence. RALF peptides control cellular expansion during plant development, but were originally defined based on their ability to induce rapid alkalinisation in tobacco cell cultures. To test whether the fungal RALF peptides were biologically active in plants, we synthesized RALF peptides corresponding to those encoded by two sequenced genomes of the tomato pathogen Fusarium oxysporum f. sp. lycopersici. One of these peptides inhibited the growth of tomato seedlings and elicited responses in tomato and Nicotiana benthamiana typical of endogenous plant RALF peptides (ROS burst, induced alkalinisation and MAP kinas activation). Gene expression analysis confirmed that a RALF-encoding gene in Fusarium oxysporum f. sp. lycopersici was expressed during infection on tomato. However a subsequent reverse genetics approach revealed that the RALF peptide was not required by Fusarium oxysporum f. sp. lycopersici for infection on tomato roots. This study has demonstrated the presence of functionally active RALF peptides encoded within phytopathogens that harbour an as yet undetermined role in the plant-pathogen interactions. This article is protected by copyright. All rights reserved.The authors would like to thank Dr Markus Albert for technical advice on the alkalinization assay. PSS is an Australian Research Council Future Fellow (FT110100698). BS is supported by an Australian Research Council Discovery Early Career Award (DE150101897). ET is supported by an Australian Postgraduate Award and a Grains Research and Development Corporation Scholarship

Structures of the flax-rust effector AvrM reveal insights into the molecular basis of plant-cell entry and effector-triggered immunity

Fungal and oomycete pathogens cause some of the most devastating diseases in crop plants, and facilitate infection by delivering a large number of effector molecules into the plant cell. AvrM is a secreted effector protein from flax rust (Melampsora lini) that can internalize into plant cells in the absence of the pathogen, binds to phosphoinositides (PIPs), and is recognized directly by the resistance protein M in flax (Linum usitatissimum), resulting in effector-triggered immunity. We determined the crystal structures of two naturally occurring variants of AvrM, AvrM-A and avrM, and both reveal an L-shaped fold consisting of a tandem duplicated four-helix motif, which displays similarity to the WY domain core in oomycete effectors. In the crystals, both AvrM variants form a dimer with an unusual nonglobular shape. Our functional analysis of AvrM reveals that a hydrophobic surface patch conserved between both variants is required for internalization into plant cells, whereas the C-terminal coiled-coil domain mediates interaction with M. AvrM binding to PIPs is dependent on positive surface charges, and mutations that abrogate PIP binding have no significant effect on internalization, suggesting that AvrM binding to PIPs is not essential for transport of AvrM across the plant membrane. The structure of AvrM and the identification of functionally important surface regions advance our understanding of the molecular mechanisms underlying how effectors enter plant cells and how they are detected by the plant immune system

Crystal structure of the Melampsora lini effector AvrP reveals insights into a possible nuclear function and recognition by the flax disease resistance protein P

The effector protein AvrP is secreted by the flax rust fungal pathogen (Melampsora lini) and recognized specifically by the flax (Linum usitatissimum) P disease resistance protein, leading to effector‐triggered immunity. To investigate the biological function of this effector and the mechanisms of specific recognition by the P resistance protein, we determined the crystal structure of AvrP. The structure reveals an elongated zinc‐finger‐like structure with a novel interleaved zinc‐binding topology. The residues responsible for zinc binding are conserved in AvrP effector variants and mutations of these motifs result in a loss of P‐mediated recognition. The first zinc‐coordinating region of the structure displays a positively charged surface and shows some limited similarities to nucleic acid‐binding and chromatin‐associated proteins. We show that the majority of the AvrP protein accumulates in the plant nucleus when transiently expressed in Nicotiana benthamiana cells, suggesting a nuclear pathogenic function. Polymorphic residues in AvrP and its allelic variants map to the protein surface and could be associated with differences in recognition specificity. Several point mutations of residues on the non‐conserved surface patch result in a loss of recognition by P, suggesting that these residues are required for recognition.This research was supported by Australian Research Council (ARC) Discovery Projects DP120100685, DP130104098 and DP160102244. XZ was a recipient of an ANZ Trustees PhD Scholarship for Medical Research in Queensland. BK is a National Health and Medical Research Council (NHMRC) Principal Research Fellow (1003325 and 1110971). MB was a recipient of an ARC Discovery Early Career Research Award (DE130101292)

Medial longitudinal arch development of school children : The College of Podiatry Annual Conference 2015: meeting abstracts

Background Foot structure is often classified into flat foot, neutral and high arch type based on the variability of the Medial Longitudinal Arch (MLA). To date, the literature provided contrasting evidence on the age when MLA development stabilises in children. The influence of footwear on MLA development is also unknown. Aim This study aims to (i) clarify whether the MLA is still changing in children from age 7 to 9 years old and (ii) explore the relationship between footwear usage and MLA development, using a longitudinal approach. Methods We evaluated the MLA of 111 healthy school children [age = 6.9 (0.3) years] using three parameters [arch index (AI), midfoot peak pressure (PP) and maximum force (MF: % of body weight)] extracted from dynamic foot loading measurements at baseline, 10-month and 22-month follow-up. Information on the type of footwear worn was collected using survey question. Linear mixed modelling was used to test for differences in the MLA over time. Results Insignificant changes in all MLA parameters were observed over time [AI: P = .15; PP: P = .84; MF: P = .91]. When gender was considered, the AI of boys decreased with age [P = .02]. Boys also displayed a flatter MLA than girls at age 6.9 years [AI: mean difference = 0.02 (0.01, 0.04); P = .02]. At baseline, subjects who wore close-toe shoes displayed the lowest MLA overall [AI/PP/MF: P < .05]. Subjects who used slippers when commencing footwear use experienced higher PP than those who wore sandals [mean difference = 31.60 (1.44, 61.75) kPa; post-hoc P = .04]. Discussion and conclusion Our findings suggested that the MLA of children remained stable from 7 to 9 years old, while gender and the type of footwear worn during childhood may influence MLA development. Clinicians may choose to commence therapy when a child presents with painful flexible flat foot at age 7 years, and may discourage younger children from wearing slippers when they commence using footwear

Urological cancer care pathways: development and use in the context of systematic reviews and clinical practice guidelines

Background: Making healthcare treatment decisions is a complex process involving a broad stakeholder base including patients, their families, health professionals, clinical practice guideline developers and funders of healthcare. Methods: This paper presents a review of a methodology for the development of urological cancer care pathways (UCAN care pathways), which reflects an appreciation of this broad stakeholder base. The methods section includes an overview of the steps in the development of the UCAN care pathways and engagement with clinical content experts and patient groups. Results: The development process is outlined, the uses of the urological cancer care pathways discussed and the implications for clinical practice highlighted. The full set of UCAN care pathways is published in this paper. These include care pathways on localised prostate cancer, locally advanced prostate cancer, metastatic prostate cancer, hormone-resistant prostate cancer, localised renal cell cancer, advanced renal cell cancer, testicular cancer, penile cancer, muscle invasive and metastatic bladder cancer and non-muscle invasive bladder cancer. Conclusion: The process provides a useful framework for improving urological cancer care through evidence synthesis, research prioritisation, stakeholder involvement and international collaboration. Although the focus of this work is urological cancers, the methodology can be applied to all aspects of urology and is transferable to other clinical specialties.11 page(s