Overcoming polyploidy pitfalls: a user guide for effective SNP conversion into KASP markers in wheat.

KEY MESSAGE: Conversion of SNP chip assays into locus-specific KASP markers requires adapted strategies in polyploid species with high genome homeology. Procedures are exemplified by QTL-associated SNPs in hexaploid wheat. Kompetitive allele-specific PCR (KASP) markers are commonly used in marker-assisted commercial plant breeding due to their cost-effectiveness and throughput for high sample volumes. However, conversion of trait-linked SNP markers from array-based SNP detection technologies into KASP markers is particularly challenging in polyploid crop species, due to the presence of highly similar homeologous and paralogous genome sequences. We evaluated strategies and identified key requirements for successful conversion of Illumina Infinium assays from the wheat 90 K SNP array into robust locus-specific KASP markers. Numerous examples showed that commonly used software for semiautomated KASP primer design frequently fails to achieve locus-specificity of KASP assays in wheat. Instead, alignment of SNP probes with multiple reference genomes and Sanger sequencing of relevant genotypes, followed by visual KASP primer placement, was critical for locus-specificity. To identify KASP assays resulting in false calling of heterozygous individuals, validation of KASP assays using extended reference genotype sets including heterozygous genotypes is strongly advised for polyploid crop species. Applying this strategy, we developed highly reproducible, stable KASP assays that are predictive for root biomass QTL haplotypes from highly homoeologous wheat chromosome regions. Due to their locus-specificity, these assays predicted root biomass considerably better than the original trait-associated markers from the Illumina array.

Meta-analysis of biomarkers predicting risk of malignant progression in Barrett's oesophagus.

BACKGROUND: Barrett's oesophagus is a precursor to the development of oesophageal adenocarcinoma. This study sought to clarify the role of genetic, chromosomal and proliferation biomarkers that have been the subjects of multiple studies through meta-analysis. METHODS: MEDLINE, Embase, PubMed and the Cochrane Library were searched for clinical studies assessing the value of p53, p16, Ki-67 and DNA content abnormalities in Barrett's oesophagus. The main outcome measure was the risk of development of high-grade dysplasia (HGD) or oesophageal adenocarcinoma. RESULTS: Some 102 studies, with 12 353 samples, were identified. Mutation (diagnostic odds ratio (DOR) 10·91, sensitivity 47 per cent, specificity 92 per cent, positive likelihood ratio (PLR) 4·71, negative likelihood ratio (NLR) 0·65, area under the curve (AUC) 0·792) and loss (DOR 16·16, sensitivity 31 per cent, specificity 98 per cent, PLR 6·66, NLR 0·41, AUC 0·923) of p53 were found to be superior to the other p53 abnormalities (loss of heterozygosity (LOH) and overexpression). Ki-67 had high sensitivity in identifying high-risk patients (DOR 5·54, sensitivity 82 per cent, specificity 48 per cent, PLR 1·59, NLR 0·42, AUC 0·761). Aneuploidy (DOR 12·08, sensitivity 53 per cent, specificity 87 per cent, PLR 4·26, NLR 0·42, AUC 0·846), tetraploidy (DOR 5·87, sensitivity 46 per cent, specificity 85 per cent, PLR 3·47, NLR 0·65, AUC 0·793) and loss of Y chromosome (DOR 9·23, sensitivity 68 per cent, specificity 80 per cent, PLR 2·67, NLR 0·49, AUC 0·807) also predicted malignant development, but p16 aberrations (hypermethylation, LOH, mutation and loss) failed to demonstrate any advantage over the other biomarkers studied. CONCLUSION: Loss and mutation of p53, and raised level of Ki-67 predicted malignant progression in Barrett's oesophagus.

Investigating successive Australian barley breeding populations for stable resistance to leaf rust.

KEY MESSAGE: Genome-wide association studies of barley breeding populations identified candidate minor genes for pairing with the adult plant resistance gene Rph20 to provide stable leaf rust resistance across environments. Stable resistance to barley leaf rust (BLR, caused by Puccinia hordei) was evaluated across environments in barley breeding populations (BPs). To identify genomic regions that can be combined with Rph20 to improve adult plant resistance (APR), two BPs genotyped with the Diversity Arrays Technology genotyping-by-sequencing platform (DArT-seq) were examined for reaction to BLR at both seedling and adult growth stages in Australian environments. An integrated consensus map comprising both first- and second-generation DArT platforms was used to integrate QTL information across two additional BPs, providing a total of four interrelated BPs and 15 phenotypic data sets. This enabled identification of key loci underpinning BLR resistance. The APR gene Rph20 was the only active resistance region consistently detected across BPs. Of the QTL identified, RphQ27 on chromosome 6HL was considered the best candidate for pairing with Rph20. RphQ27 did not align or share proximity with known genes and was detected in three of the four BPs. The combination of RphQ27 and Rph20 was of low frequency in the breeding material; however, strong resistance responses were observed for the lines carrying this pairing. This suggests that the candidate minor gene RphQ27 can interact additively with Rph20 to provide stable resistance to BLR across diverse environments.

Do primary care professionals agree about progress with implementation of primary care teams: results from a cross sectional study.

BACKGROUND: Primary care is the cornerstone of healthcare reform with policies across jurisdictions promoting interdisciplinary team working. The effective implementation of such health policies requires understanding the perspectives of all actors. However, there is a lack of research about health professionals' views of this process. This study compares Primary Healthcare Professionals' perceptions of the effectiveness of the Primary Care Strategy and Primary Care Team (PCT) implementation in Ireland. METHODS: Design and Setting: e-survey of (1) General Practitioners (GPs) associated with a Graduate Medical School (N = 100) and (2) Primary Care Professionals in 3 of 4 Health Service Executive (HSE) regions (N = 2309). After piloting, snowball sampling was used to administer the survey. Descriptive analysis was carried out using SPSS. Ratings across groups were compared using non-parametric tests. RESULTS: There were 569 responses. Response rates varied across disciplines (71 % for GPs, 22 % for other Primary Healthcare Professionals (PCPs). Respondents across all disciplines viewed interdisciplinary working as important. Respondents agreed on lack of progress of implementation of formal PCTs (median rating of 2, where 1 is no progress at all and 5 is complete implementation). GPs were more negative about the effectiveness of the Strategy to promote different disciplines to work together (median rating of 2 compared to 3 for clinical therapists and 3.5 for nurses, P = 0.001). Respondents identified resources and GP participation as most important for effective team working. Protected time for meetings and capacity to manage workload for meetings were rated as very important factors for effective team working by GPs, clinical therapists and nurses. A building for co-location of teams was rated as an important factor by nurses and clinical therapists though GPs rated it as less important. Payment to attend meetings and contractual arrangements were considered important factors by GPs but not by nurses or clinical therapists. CONCLUSION: PCPs and GPs agree there is limited PCT implementation. GPs are most negative about this implementation. There is some disagreement about which resources are most important for effective PCT working. These findings provide valuable data for clinicians and policy makers about implementation of interdisciplinary teams in primary care.

Association mapping of resistance to Puccinia hordei in Australian barley breeding germplasm.

KEY MESSAGE: "To find stable resistance using association mapping tools, QTL with major and minor effects on leaf rust reactions were identified in barley breeding lines by assessing seedlings and adult plants." Three hundred and sixty (360) elite barley (Hordeum vulgare L.) breeding lines from the Northern Region Barley Breeding Program in Australia were genotyped with 3,244 polymorphic diversity arrays technology markers and the results used to map quantitative trait loci (QTL) conferring a reaction to leaf rust (Puccinia hordei Otth). The F3:5 (Stage 2) lines were derived or sourced from different geographic origins or hubs of international barley breeding ventures representing two breeding cycles (2009 and 2011 trials) and were evaluated across eight environments for infection type at both seedling and adult plant stages. Association mapping was performed using mean scores for disease reaction, accounting for family effects using the eigenvalues from a matrix of genotype correlations. In this study, 15 QTL were detected; 5 QTL co-located with catalogued leaf rust resistance genes (Rph1, Rph3/19, Rph8/14/15, Rph20, Rph21), 6 QTL aligned with previously reported genomic regions and 4 QTL (3 on chromosome 1H and 1 on 7H) were novel. The adult plant resistance gene Rph20 was identified across the majority of environments and pathotypes. The QTL detected in this study offer opportunities for breeding for more durable resistance to leaf rust through pyramiding multiple genomic regions via marker-assisted selection.

Seasonal influenza vaccine uptake in HSE-funded hospitals and nursing homes during the 2011/2012 influenza season.

Annual seasonal influenza vaccine is recommended for all health care workers (HCWs) in Ireland. For the 2011/2012 influenza season, information was collected on influenza vaccination uptake among HCWs employed in Health Service Executive (HSE)-funded hospitals (primarily acute) and of nursing homes (NHs) and also among NH long-term and short-term respite care residents. Forty-five hospitals (80%) and 120 NHs (75%) provided uptake data. Nationally, influenza vaccine uptake among hospital employed HCWs was estimated to be 18% and 14% among HCWs in NHs; in NHs vaccine uptake among long-term care residents was estimated to 88%. These findings highlight the continued low uptake among HCWs of all categories and demonstrate the need for sustained measures to improve uptake rates.

Study protocol for The GOAL Trial: comprehensive geriatric assessment for frail older people with chronic kidney disease to increase attainment of patient-identified goals-a cluster randomised controlled trial.

BACKGROUND: An increasing number of older people are living with chronic kidney disease (CKD). Many have complex healthcare needs and are at risk of deteriorating health and functional status, which can adversely affect their quality of life. Comprehensive geriatric assessment (CGA) is an effective intervention to improve survival and independence of older people, but its clinical utility and cost-effectiveness in frail older people living with CKD is unknown. METHODS: The GOAL Trial is a pragmatic, multi-centre, open-label, superiority, cluster randomised controlled trial developed by consumers, clinicians, and researchers. It has a two-arm design, CGA compared with standard care, with 1:1 allocation of a total of 16 clusters. Within each cluster, study participants ≥ 65 years of age (or ≥ 55 years if Aboriginal or Torres Strait Islander (First Nations Australians)) with CKD stage 3-5/5D who are frail, measured by a Frailty Index (FI) of > 0.25, are recruited. Participants in intervention clusters receive a CGA by a geriatrician to identify medical, social, and functional needs, optimise medication prescribing, and arrange multidisciplinary referral if required. Those in standard care clusters receive usual care. The primary outcome is attainment of self-identified goals assessed by standardised Goal Attainment Scaling (GAS) at 3 months. Secondary outcomes include GAS at 6 and 12 months, quality of life (EQ-5D-5L), frailty (Frailty Index - Short Form), transfer to residential aged care facilities, cost-effectiveness, and safety (cause-specific hospitalisations, mortality). A process evaluation will be conducted in parallel with the trial including whether the intervention was delivered as intended, any issue or local barriers to intervention delivery, and perceptions of the intervention by participants. The trial has 90% power to detect a clinically meaningful mean difference in GAS of 10 units. DISCUSSION: This trial addresses patient-prioritised outcomes. It will be conducted, disseminated and implemented by clinicians and researchers in partnership with consumers. If CGA is found to have clinical and cost-effectiveness for frail older people with CKD, the intervention framework could be embedded into routine clinical practice. The implementation of the trial's findings will be supported by presentations at conferences and forums with clinicians and consumers at specifically convened workshops, to enable rapid adoption into practice and policy for both nephrology and geriatric disciplines. It has potential to materially advance patient-centred care and improve clinical and patient-reported outcomes (including quality of life) for frail older people living with CKD. TRIAL REGISTRATION: ClinicalTrials.gov NCT04538157. Registered on 3 September 2020.

Mapping Rph20: a gene conferring adult plant resistance to Puccinia hordei in barley.

A doubled haploid (DH) barley (Hordeum vulgare L.) population of 334 lines (ND24260 × Flagship) genotyped with DArT markers was used to map genes for adult plant resistance (APR) to leaf rust (Puccinia hordei Otth) under field conditions in Australia and Uruguay. The Australian barley cultivar Flagship carries an APR gene (qRphFlag) derived from the cultivar Vada. Association analysis and composite interval mapping identified two genes conferring APR in this DH population. qRphFlag was mapped to the short arm of chromosome 5H (5HS), accounting for 64-85% of the phenotypic variation across four field environments and 56% under controlled environmental conditions (CEC). A second quantitative trait locus (QTL) from ND24260 (qRphND) with smaller effect was mapped to chromosome 6HL. In the absence of qRphFlag, qRphND conferred only a low level of resistance. DH lines displaying the highest level of APR carried both genes. Sequence information for the critical DArT marker bPb-0837 (positioned at 21.2 cM on chromosome 5HS) was used to develop bPb-0837-PCR, a simple PCR-based marker for qRphFlag. The 245 bp fragment for bPb-0837-PCR was detected in a range of barley cultivars known to possess APR, which was consistent with previous tests of allelism, demonstrating that the qRphFlag resistant allele is common in leaf rust resistant cultivars derived from Vada and Emir. qRphFlag has been designated Rph20, the first gene conferring APR to P. hordei to be characterised in barley. The PCR marker will likely be effective in marker-assisted selection for Rph20.

Investigation of Pontiac-like illness in office workers during an outbreak of Legionnaires' disease, 2008.

In July 2008, office workers in Dublin complained of influenza-like illness preceding and interspersing two cases of notified Legionnaires' disease. Legionella pneumophila serogroup 1 was identified in both cooling towers supplying the office. A retrospective cohort study was undertaken to investigate possible Pontiac fever (PF). Forty-seven employees (23%) met the clinical case definition for PF but confirmatory testing was negative. Exposure to the smoking area situated beside the cooling towers was associated with an increased risk of PF (RR 2.4, 95% CI 1.5-3.8). The diagnosis of PF should be considered when many persons exposed to a possible reservoir of Legionella spp. present with flu-like symptoms. More sensitive microbiological tests would allow better confirmation and more comprehensive reporting of PF. Early detection is vital to prevent potentially severe illness and outbreaks of PF or Legionnaires' disease.

Lepidopteran leaf mine from the early eocene wind river formation of northwestern wyoming.

A lepidopteran mine, probably of Phyllocnistis, on a leaflet impression of Cedrela (Meliaceae) discovered in late early Eocene strata near Dubois, Wyoming, is the earliest record of leaf mining and of the Phyllocnistidae. Considerable prior evolution of the mining habit, antiquity of the Cedrela-Phyllocnistis relationship, and subtropical climatic conditions are indicated.

An aptian plant with attached leaves and flowers: implications for angiosperm origin.

Recent phylogenetic studies and fossil finds support a new view of the ancestral angiosperm. A diminutive fossil angiosperm from the Aptian of Australia has attached leaves, with intermediate pinnate-palmate, low-rank venation, and lateral axes bearing pistillate organs subtended by bracts and bracteoles that are the oldest direct evidence of flowers. A variety of data suggests a similar morphology for the ancestral angiosperm. This hypothesis explains similarities between rhizomatous to herbaceous Magnoliidae and basal monocots, scarcity of early agniosperm wood, and lack of recognition of earlier remains.

Arctic terrestrial biota: paleomagnetic evidence of age disparity with mid-northern latitudes during the late cretaceous and early tertiary.

Magnetostratigraphic correlation of the Eureka Sound Formation in the Canadian high Arctic reveals profound difference between the time of appearance of fossil land plants and vertebrates in the Arctic and in mid-northern latitudes. Latest Cretaceous plant fossils in the Arctic predate mid-latitude occurrences by as much as 18 million years, while typical Eocene vertebrate fossils appear some 2 to 4 million years early.

Fission-track dating of haughton astrobleme and included biota, devon island, Canada.

Haughton Astrobleme is a major extraterrestrial impact structure located on Devon Island in the Canadian Arctic Archipelago, Northwest Territories. Apatite grains separated from shocked Precambrian gneiss contained in a polymict breccia from the center of the astrobleme yielded a fission-track date of 22.4 million +/- 1.4 million years before the present or early Miocene (Aquitanian). This provides a date for the impact event and an upper limit on the age of crater-filling lake sediments and a flora and vertebrate fauna occurring in them. A geologically precise date for these fossils provides an important biostratigraphic reference point for interpreting the biotic evolution of the Arctic.

The molecular fossil record of oleanane and its relation to angiosperms.

Oleanane has been reported in Upper Cretaceous and Tertiary source rocks and their related oils and has been suggested as a marker for flowering plants. Correspondence of oleanane concentrations relative to the ubiquitous microbial marker 17alpha-hopane with angiosperm diversification (Neocomian to Miocene) suggests that oleanane concentrations in migrated petroleum can be used to identify the maximum age of unknown or unavailable source rock. Rare occurrences of pre-Cretaceous oleanane suggest either that a separate lineage leads to the angiosperms well before the Early Cretaceous or that other plant groups have the rarely expressed ability to synthesize oleanane precursors.

Paleobotanical evidence for near present-day levels of atmospheric Co2 during part of the tertiary.

Understanding the link between the greenhouse gas carbon dioxide (CO(2)) and Earth's temperature underpins much of paleoclimatology and our predictions of future global warming. Here, we use the inverse relationship between leaf stomatal indices and the partial pressure of CO(2) in modern Ginkgo biloba and Metasequoia glyptostroboides to develop a CO(2) reconstruction based on fossil Ginkgo and Metasequoia cuticles for the middle Paleocene to early Eocene and middle Miocene. Our reconstruction indicates that CO(2) remained between 300 and 450 parts per million by volume for these intervals with the exception of a single high estimate near the Paleocene/Eocene boundary. These results suggest that factors in addition to CO(2) are required to explain these past intervals of global warmth.

Laparoscopic transabdominal preperitoneal (TAPP) groin hernia repair using n-butyl-2-cyanoacrylate (Liquiband®Fix8™) for mesh fixation and peritoneal closure: learning experience during introduction into clinical practice.

INTRODUCTION: In a single centre evaluation of a novel hernia repair device, 200 consecutive patients underwent 247 laparoscopic (TAPP) groin hernia repairs (47 bilateral) using n-butyl-2-cyanoacrylate (Liquiband®Fix8™) for mesh fixation and peritoneal closure over a 2-year period by a single experienced laparoscopic surgeon. PATIENTS AND METHODS: All groin hernia patients requiring TAPP repair were included in the study: Inguinal 142, Femoral 14, Spigelian 4, and Inguinal disruption 40. A retrospective review of the data was performed. There were 161 males and 39 females, mean age 55 years (range 20-89 years). Mesh fixation was successful in all 247 TAPP repairs, and 90% of patients had a successful peritoneal closure using the device (20 patients required the use of conventional tacks to complete closure). FOLLOW-UP: Patients were followed up with an out-patient visit at 6 weeks post-op, followed by a Patient Initiated Follow Up programme, and a final Telephone follow-up. To date all patients have completed 1 year of follow-up, and 70% of patients 2 years of follow-up (median 29 months, range 14 to 40 months). RESULTS: There were very few procedure-related adverse events: groin seromas 6 (2.4%), port site bleeding 2 (0.3%), port site hernia 2 (0.3%), and only 1 groin hernia recurrence (0.4%). Prospective surgeon scoring of satisfaction for mesh fixation, peritoneal closure, and device clogging was favourable and increased following the initial learning phase. Changes in the device design during the study period improved the efficacy of the device significantly. CONCLUSION: This retrospective study shows that mesh fixation and peritoneal closure using the Liquiband®Fix8™ device is feasible, safe, practical, and is easy to learn.

The effect of moxifloxacin on QTc and implications for the design of thorough QT studies.

A number of issues have remained unanswered in the design of "thorough QT"(TQT) studies. In this randomized, placebo-controlled, two-period crossover study in 20 healthy subjects, replicate electrocardiograms (ECGs) were recorded on a digital 12-lead Holter recorder, extracted in a core ECG laboratory, and interpreted manually by a cardiologist. The observed within-subject variability was slightly greater when time-matched baselines were employed than when predose baselines were employed, whereas the magnitude of the increase in QTc was similar for both. Moxifloxacin 400 mg was associated with an observed 7.5-12.5 ms increase in the mean placebo- and baseline-corrected QTc interval. A PK-QTc model estimated a 3.9 ms increase in the QTc interval for every 1,000 ng/ml increase in moxifloxacin concentration. The QTc increases associated with moxifloxacin support the appropriateness of its use as a positive control in TQT studies. This crossover study failed to justify the use of time-matched baselines rather than the less resource-intensive predose definition of baseline.

In light of recently published clinical trials and their implication for clinical practice, does a large catchment area acute hospital require 24 hour CT neck and head angiography and/or neuro-interventional services in the setting of acute ischaemic stroke?

BACKGROUND: Recently published clinical trials have resulted in a significant change in the guidelines used to manage patients suffering an acute ischaemic stroke. New neuro-interventional techniques have revolutionised stroke outcomes. Currently, such services are only available in two specialist centres. AIMS: We attempted to evaluate the need for the provision of routine computed tomography (CT) angiography and neuro-interventional services at a university teaching hospital in Limerick. METHODS: A retrospective study was performed based on data collated by the stroke service, University Hospital Limerick (UHL). All patients with a suspected acute ischaemic stroke of anterior circulation and known evolution were included. Baseline clinical and imaging characteristics, thrombolysis data, stroke unit admission rates and discharge destinations were recorded. RESULTS: All 141 patients were suitable for CT angiography and should be performed in accordance with guidelines. Additionally, 165 patients excluded from the study due to an unknown stroke evolution timeframe may have benefitted. Non-contrast CT scan confirmed just 12 anterior circulation strokes. The need for neuro-interventional services proved more difficult to assess, primarily due to the lack of provision of routine CT angiography, employed to confirm anterior circulation occlusion. Secondary results showed a thrombolysis rate of 10.8% and confirmed that time efficiencies result in higher thrombolysis eligibility rates. Stroke unit admissions and discharge destinations were also recorded. CONCLUSION: UHL should provide routine CT angiography to all patients presenting with acute ischaemic stroke in line with current guidelines. The need for provision of neuro-interventional services on-site proved more difficult to assess and requires further analysis.

Benchmarking comparison survey of regional neonatal units.

There are currently eight regional neonatal units in Ireland; three in Dublin and one each in Cork, Limerick, Galway, Drogheda and Waterford. Previous studies have shown a significant variation in the provision of care and services between such units. In July 2005, a postal questionnaire was distributed to a focus group of staff in the eight regional units. The objective was to identify issues of significant concern in current neonatology practice in Ireland. Eighteen people were surveyed in this focus group and we had an overall response rate of almost 78%. All of the respondents felt that there was a difference between practises in the neonatal units and greater than 90% perceived these to be significant. We believe that this study has identified the desire for standardisation of neonatology practises in Ireland and that this may be achieved by the introduction of a Nationwide Newborn Network.