Characterisation of the Fusarium graminearum-Wheat Floral Interaction.

PublishedJournal ArticleFusarium Ear Blight is a destructive fungal disease of cereals including wheat and can contaminate the crop with various trichothecene mycotoxins. This investigation has produced a new β-glucuronidase (GUS) reporter strain that facilitates the quick and easy assessment of plant infection. The constitutively expressed gpdA:GUS strain of Fusarium graminearum was used to quantify the overall colonisation pattern. Histochemical and biochemical approaches confirmed, in susceptible wheat ear infections, the presence of a substantial phase of symptomless fungal growth. Separate analyses demonstrated that there was a reduction in the quantity of physiologically active hyphae as the wheat ear infection proceeded. A simplified linear system of rachis infection was then utilised to evaluate the expression of several TRI genes by RT-qPCR. Fungal gene expression at the advancing front of symptomless infection was compared with the origin of infection in the rachis. This revealed that TRI gene expression was maximal at the advancing front and supports the hypothesis that the mycotoxin deoxynivalenol plays a role in inhibiting plant defences in advance of the invading intercellular hyphae. This study has also demonstrated that there are transcriptional differences between the various phases of fungal infection and that these differences are maintained as the infection proceeds.Chinese governmentBritish Society for Plant Pathology (BSPP)BBSRCEU FP 6 Integrated Project BioexploitSyngent

Systemic peptide-mediated oligonucleotide therapy improves long-term survival in spinal muscular atrophy.

The development of antisense oligonucleotide therapy is an important advance in the identification of corrective therapy for neuromuscular diseases, such as spinal muscular atrophy (SMA). Because of difficulties of delivering single-stranded oligonucleotides to the CNS, current approaches have been restricted to using invasive intrathecal single-stranded oligonucleotide delivery. Here, we report an advanced peptide-oligonucleotide, Pip6a-morpholino phosphorodiamidate oligomer (PMO), which demonstrates potent efficacy in both the CNS and peripheral tissues in severe SMA mice following systemic administration. SMA results from reduced levels of the ubiquitously expressed survival motor neuron (SMN) protein because of loss-of-function mutations in the SMN1 gene. Therapeutic splice-switching oligonucleotides (SSOs) modulate exon 7 splicing of the nearly identical SMN2 gene to generate functional SMN protein. Pip6a-PMO yields SMN expression at high efficiency in peripheral and CNS tissues, resulting in profound phenotypic correction at doses an order-of-magnitude lower than required by standard naked SSOs. Survival is dramatically extended from 12 d to a mean of 456 d, with improvement in neuromuscular junction morphology, down-regulation of transcripts related to programmed cell death in the spinal cord, and normalization of circulating insulin-like growth factor 1. The potent systemic efficacy of Pip6a-PMO, targeting both peripheral as well as CNS tissues, demonstrates the high clinical potential of peptide-PMO therapy for SMA

How to deal with uncertainty in prenatal genomics: A systematic review of guidelines and policies

Exome Sequencing (ES) enhanced the diagnostic yield of genetic testing, but has also increased the possibility of uncertain findings. Prenatal ES is increasingly being offered after a fetal abnormality is detected through ultrasound. It is important to know how to handle uncertainty in this particularly stressful period. This systematic review aimed to provide a comprehensive overview of guidelines available for addressing uncertainty related to prenatal chromosomal microarray (CMA) and ES. Ten uncertainty types associated with prenatal ES and CMA were identified and defined by an international multidisciplinary team. Medline (all) and Embase were systematically searched. Laboratory scientists, clinical geneticists, psychologists, and a fetal medicine specialist screened the papers and performed the data extraction. Nineteen papers were included. Recommendations generally emphasized the importance of trio analysis, clinical information, data sharing, validation and re-analysis, protocols, multidisciplinary teams, genetic counselling, whether to limit the possible scope of results, and when to report particular findings. This systematic review helps provide a vocabulary for uncertainties, and a compass to navigate uncertainties. Prenatal CMA and ES guidelines provide a strong starting point for determining how to handle uncertainty. Gaps in guidelines and recommendations were identified and discussed to provide direction for future research and policy making

Assessing women's preferences towards tests that may reveal uncertain results from prenatal genomic testing: Development of attributes for a discrete choice experiment, using a mixed-methods design

Prenatal DNA tests, such as chromosomal microarray analysis or exome sequencing, increase the likelihood of receiving a diagnosis when fetal structural anomalies are identified. However, some parents will receive uncertain results such as variants of uncertain significance and secondary findings. We aimed to develop a set of attributes and associated levels for a discrete-choice experiment (DCE) that will examine parents' preferences for tests that may reveal uncertain test results. A two phase mixed-methods approach was used to develop attributes for the DCE. In Phase 1, a “long list” of candidate attributes were identified via two approaches: 1) a systematic review of the literature around parental experiences of uncertainty following prenatal testing; 2) 16 semi-structured interviews with parents who had experienced uncertainty during pregnancy and 25 health professionals who return uncertain prenatal results. In Phase 2, a quantitative scoring exercise with parents prioritised the candidate attributes. Clinically appropriate levels for each attribute were then developed. A final set of five attributes and levels were identified: likelihood of getting a result, reporting of variants of uncertain significance, reporting of secondary findings, time taken to receive results, and who tells you about your result. These attributes will be used in an international DCE study to investigate preferences and differences across countries. This research will inform best practice for professionals supporting parents to manage uncertainty in the prenatal setting

Dealing with uncertain results from chromosomal microarray and exome sequencing in the prenatal setting: an international cross-sectional study with healthcare professionals.

OBJECTIVES: To conduct qualitative interviews with healthcare providers working in different countries to understand their experiences of dealing with uncertain results from prenatal chromosome microarray analysis (CMA) and exome sequencing (ES). METHODS: Semi-structured interviews with 31 healthcare providers who report or return prenatal CMA and/or ES results (clinicians, genetic counsellors and clinical scientists) in six countries with differing healthcare systems; Australia (4), Denmark (5), Netherlands (6), Singapore (4), Sweden (6) and United Kingdom (6). The topic guide explored the main sources of uncertainty and their management. RESULTS: There was variation in reporting practices both between and across countries for variants of uncertain significance (VUS), however, there was broad agreement on reporting practices for incidental findings. There was also variation in who decides what results are reported (clinical scientists or clinicians). Technical limitations and lack of knowledge (to classify variants and of prenatal phenotypes) were significant challenges, as were turnaround times and lack of guidelines. CONCLUSION: Health professionals around the globe are dealing with similar sources of uncertainty, but managing them in different ways, Continued dialogue with international colleagues on ways of managing uncertain results is important to compare and contrast the benefits and limitations of the different approaches. This article is protected by copyright. All rights reserved

Design and rationale of a multi-center, pragmatic, open-label randomized trial of antimicrobial therapy - the study of clinical efficacy of antimicrobial therapy strategy using pragmatic design in Idiopathic Pulmonary Fibrosis (CleanUP-IPF) clinical trial

Compelling data have linked disease progression in patients with idiopathic pulmonary fibrosis (IPF) with lung dysbiosis and the resulting dysregulated local and systemic immune response. Moreover, prior therapeutic trials have suggested improved outcomes in these patients treated with either sulfamethoxazole/ trimethoprim or doxycycline. These trials have been limited by methodological concerns. This trial addresses the primary hypothesis that long-term treatment with antimicrobial therapy increases the time-to-event endpoint of respiratory hospitalization or all-cause mortality compared to usual care treatment in patients with IPF. We invoke numerous innovative features to achieve this goal, including: 1) utilizing a pragmatic randomized trial design; 2) collecting targeted biological samples to allow future exploration of 'personalized' therapy; and 3) developing a strong partnership between the NHLBI, a broad range of investigators, industry, and philanthropic organizations. The trial will randomize approximately 500 individuals in a 1:1 ratio to either antimicrobial therapy or usual care. The site principal investigator will declare their preferred initial antimicrobial treatment strategy (trimethoprim 160 mg/ sulfamethoxazole 800 mg twice a day plus folic acid 5 mg daily or doxycycline 100 mg once daily if body weight is < 50 kg or 100 mg twice daily if ≥50 kg) for the participant prior to randomization. Participants randomized to antimicrobial therapy will receive a voucher to help cover the additional prescription drug costs. Additionally, those participants will have 4-5 scheduled blood draws over the initial 24 months of therapy for safety monitoring. Blood sampling for DNA sequencing and genome wide transcriptomics will be collected before therapy. Blood sampling for transcriptomics and oral and fecal swabs for determination of the microbiome communities will be collected before and after study completion. As a pragmatic study, participants in both treatment arms will have limited in-person visits with the enrolling clinical center. Visits are limited to assessments of lung function and other clinical parameters at time points prior to randomization and at months 12, 24, and 36. All participants will be followed until the study completion for the assessment of clinical endpoints related to hospitalization and mortality events. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT02759120

The Turkey Ig-like receptor family: identification, expression and function.

The chicken leukocyte receptor complex located on microchromosome 31 encodes the chicken Ig-like receptors (CHIR), a vastly expanded gene family which can be further divided into three subgroups: activating CHIR-A, bifunctional CHIR-AB and inhibitory CHIR-B. Here, we investigated the presence of CHIR homologues in other bird species. The available genome databases of turkey, duck and zebra finch were screened with different strategies including BLAST searches employing various CHIR sequences, and keyword searches. We could not identify CHIR homologues in the distantly related zebra finch and duck, however, several partial and complete sequences of CHIR homologues were identified on chromosome 3 of the turkey genome. They were designated as turkey Ig-like receptors (TILR). Using cDNA derived from turkey blood and spleen RNA, six full length TILR could be amplified and further divided according to the typical sequence features into one activating TILR-A, one inhibitory TILR-B and four bifunctional TILR-AB. Since the TILR-AB sequences all displayed the critical residues shown to be involved in binding to IgY, we next confirmed the IgY binding using a soluble TILR-AB1-huIg fusion protein. This fusion protein reacted with IgY derived from various gallinaceous birds, but not with IgY from other bird species. Finally, we tested various mab directed against CHIR for their crossreactivity with either turkey or duck leukocytes. Whereas no staining was detectable with duck cells, the CHIR-AB1 specific mab 8D12 and the CHIR-A2 specific mab 13E2 both reacted with a leukocyte subpopulation that was further identified as thrombocytes by double immunofluorescence employing B-cell, T-cell and thrombocyte specific reagents. In summary, although the turkey harbors similar LRC genes as the chicken, their distribution seems to be distinct with predominance on thrombocytes rather than lymphocytes

Modeling recursive RNA interference.

An important application of the RNA interference (RNAi) pathway is its use as a small RNA-based regulatory system commonly exploited to suppress expression of target genes to test their function in vivo. In several published experiments, RNAi has been used to inactivate components of the RNAi pathway itself, a procedure termed recursive RNAi in this report. The theoretical basis of recursive RNAi is unclear since the procedure could potentially be self-defeating, and in practice the effectiveness of recursive RNAi in published experiments is highly variable. A mathematical model for recursive RNAi was developed and used to investigate the range of conditions under which the procedure should be effective. The model predicts that the effectiveness of recursive RNAi is strongly dependent on the efficacy of RNAi at knocking down target gene expression. This efficacy is known to vary highly between different cell types, and comparison of the model predictions to published experimental data suggests that variation in RNAi efficacy may be the main cause of discrepancies between published recursive RNAi experiments in different organisms. The model suggests potential ways to optimize the effectiveness of recursive RNAi both for screening of RNAi components as well as for improved temporal control of gene expression in switch off-switch on experiments