Identification of genetic loci in lettuce mediating quantitative resistance to fungal pathogens.

KEY MESSAGE: We demonstrate genetic variation for quantitative resistance against important fungal pathogens in lettuce and its wild relatives, map loci conferring resistance and predict key molecular mechanisms using transcriptome profiling. Lactuca sativa L. (lettuce) is an important leafy vegetable crop grown and consumed globally. Chemicals are routinely used to control major pathogens, including the causal agents of grey mould (Botrytis cinerea) and lettuce drop (Sclerotinia sclerotiorum). With increasing prevalence of pathogen resistance to fungicides and environmental concerns, there is an urgent need to identify sources of genetic resistance to B. cinerea and S. sclerotiorum in lettuce. We demonstrated genetic variation for quantitative resistance to B. cinerea and S. sclerotiorum in a set of 97 diverse lettuce and wild relative accessions, and between the parents of lettuce mapping populations. Transcriptome profiling across multiple lettuce accessions enabled us to identify genes with expression correlated with resistance, predicting the importance of post-transcriptional gene regulation in the lettuce defence response. We identified five genetic loci influencing quantitative resistance in a F6 mapping population derived from a Lactuca serriola (wild relative) × lettuce cross, which each explained 5-10% of the variation. Differential gene expression analysis between the parent lines, and integration of data on correlation of gene expression and resistance in the diversity set, highlighted potential causal genes underlying the quantitative trait loci.

Identification and Expression of Secreted In Xylem Pathogenicity Genes in Fusarium oxysporum f. sp. pisi.

Fusarium oxysporum is a soilborne fungal plant pathogen responsible for causing disease in many economically important crops with "special forms" (formae speciales) adapted to infect specific plant hosts. F. oxysporum f. sp. pisi (FOP) is the causal agent of Fusarium wilt disease of pea. It has been reported in every country where peas are grown commercially. Disease is generally controlled using resistant cultivars possessing single major gene resistance and therefore there is a constant risk of breakdown. The main aim of this work was to characterise F. oxysporum isolates collected from diseased peas in the United Kingdom as well as FOP isolates obtained from other researchers representing different races through sequencing of a housekeeping gene and the presence of Secreted In Xylem (SIX) genes, which have previously been associated with pathogenicity in other F. oxysporum f. spp. F. oxysporum isolates from diseased United Kingdom pea plants possessed none or just one or two known SIX genes with no consistent pattern of presence/absence, leading to the conclusion that they were foot-rot causing isolates rather than FOP. In contrast, FOP isolates had different complements of SIX genes with all those identified as race 1 containing SIX1, SIX6, SIX7, SIX9, SIX10, SIX11, SIX12, and SIX14. FOP isolates that were identified as belonging to race 2 through testing on differential pea cultivars, contained either SIX1, SIX6, SIX9, SIX13, SIX14 or SIX1, SIX6, SIX13. Significant upregulation of SIX genes was also observed in planta over the early stages of infection by different FOP races in pea roots. Race specific SIX gene profiling may therefore provide potential targets for molecular identification of FOP races but further research is needed to determine whether variation in complement of SIX genes in FOP race 2 isolates results in differences in virulence across a broader set of pea differential cultivars.

Assembly and characterisation of a unique onion diversity set identifies resistance to Fusarium basal rot and improved seedling vigour.

KEY MESSAGE: A unique, global onion diversity set was assembled, genotyped and phenotyped for beneficial traits. Accessions with strong basal rot resistance and increased seedling vigour were identified along with associated markers. Conserving biodiversity is critical for safeguarding future crop production. Onion (Allium cepa L.) is a globally important crop with a very large (16 Gb per 1C) genome which has not been sequenced. While onions are self-fertile, they suffer from severe inbreeding depression and as such are highly heterozygous as a result of out-crossing. Bulb formation is driven by daylength, and accessions are adapted to the local photoperiod. Onion seed is often directly sown in the field, and hence seedling establishment is a critical trait for production. Furthermore, onion yield losses regularly occur worldwide due to Fusarium basal rot caused by Fusarium oxysporum f. sp. cepae. A globally relevant onion diversity set, consisting of 10 half-sib families for each of 95 accessions, was assembled and genotyping carried out using 892 SNP markers. A moderate level of heterozygosity (30-35%) was observed, reflecting the outbreeding nature of the crop. Using inferred phylogenies, population structure and principal component analyses, most accessions grouped according to local daylength. A high level of intra-accession diversity was observed, but this was less than inter-accession diversity. Accessions with strong basal rot resistance and increased seedling vigour were identified along with associated markers, confirming the utility of the diversity set for discovering beneficial traits. The onion diversity set and associated trait data therefore provide a valuable resource for future germplasm selection and onion breeding.

Draft Genome Sequence of an Onion Basal Rot Isolate of Fusarium proliferatum.

Fusarium proliferatum is a component of the onion basal rot disease complex. We present an annotated F. proliferatum draft genome sequence, totaling 45.8 Mb in size, assembled into 597 contigs, with a predicted 15,418 genes. The genome contains 58 secondary metabolite clusters and homologs of the Fusarium oxysporum effector SIX2.

Basal Rot of Narcissus: Understanding Pathogenicity in Fusarium oxysporum f. sp. narcissi.

Fusarium oxysporum is a globally distributed soilborne fungal pathogen causing root rots, bulb rots, crown rots and vascular wilts on a range of horticultural plants. Pathogenic F. oxysporum isolates are highly host specific and are classified as formae speciales. Narcissus is an important ornamental crop and both the quality and yield of flowers and bulbs can be severely affected by a basal rot caused by F. oxysporum f. sp. narcissi (FON); 154 Fusarium isolates were obtained from different locations and Narcissus cultivars in the United Kingdom, representing a valuable resource. A subset of 30 F. oxysporum isolates were all found to be pathogenic and were therefore identified as FON. Molecular characterisation of isolates through sequencing of three housekeeping genes, suggested a monophyletic origin with little divergence. PCR detection of 14 Secreted in Xylem (SIX) genes, previously shown to be associated with pathogenicity in other F. oxysporum f. spp., revealed different complements of SIX7, SIX9, SIX10, SIX12 and SIX13 within FON isolates which may suggest a race structure. SIX gene sequences were unique to FON and SIX10 was present in all isolates, allowing for molecular identification of FON for the first time. The genome of a highly pathogenic isolate was sequenced and lineage specific (LS) regions identified which harboured putative effectors including the SIX genes. Real-time RT-PCR, showed that SIX genes and selected putative effectors were expressed in planta with many significantly upregulated during infection. This is the first study to characterise molecular variation in FON and provide an analysis of the FON genome. Identification of expressed genes potentially associated with virulence provides the basis for future functional studies and new targets for molecular diagnostics.

Pitfalls in diagnosis and management of suspected urinary tract infection in an urban tropical primary care setting.

INTRODUCTION: Diagnosis and management of urinary tract infection (UTI) are complex, and do not always follow guidelines. The aim of this study was to determine adherence to the 2014 Malaysian Ministry of Health guidelines for managing suspected UTI in a Malaysian primary care setting. METHODOLOGY: We retrospectively reviewed computerized medical records of adults with suspected UTI between July-December 2016. Excluded were consultations misclassified by the search engine, duplicated records of the same patient, consultations for follow-up of suspected UTI, patients who were pregnant, catheterised, or who had a renal transplant. Records were reviewed by two primary care physicians and a clinical microbiologist. RESULTS: From 852 records, 366 consultations were a fresh episode of possible UTI. Most subjects were female (78.2%) with median age of 61.5 years. The major co-morbidities were hypertension (37.1%), prostatic enlargement in males (35.5%) and impaired renal function (31.1%). Symptoms were reported in 349 (95.4%) consultations. Antibiotics were prescribed in 307 (83.9%) consultations, which was appropriate in 227/307 (73.9%), where the subject had at least one symptom, and leucocytes were raised in urine full examination and microscopic examination (UFEME). In 73 (23.8%) consultations antibiotics were prescribed inappropriately, as the subjects were asymptomatic (14,4.6%), urine was clear (17,5.5%), or UFEME did not show raised leucocytes (42,13.7%). In 7 (2.3%) consultations appropriateness of antibiotics could not be determined as UFEME was not available. CONCLUSION: Several pitfalls contributed to suboptimal adherence to guidelines for diagnosis and management of suspected UTI. This illustrates the complexity of managing suspected UTI in older subjects with multiple co-morbidities.

Characterisation of pathogen-specific regions and novel effector candidates in Fusarium oxysporum f. sp. cepae.

A reference-quality assembly of Fusarium oxysporum f. sp. cepae (Foc), the causative agent of onion basal rot has been generated along with genomes of additional pathogenic and non-pathogenic isolates of onion. Phylogenetic analysis confirmed a single origin of the Foc pathogenic lineage. Genome alignments with other F. oxysporum ff. spp. and non pathogens revealed high levels of syntenic conservation of core chromosomes but little synteny between lineage specific (LS) chromosomes. Four LS contigs in Foc totaling 3.9 Mb were designated as pathogen-specific (PS). A two-fold increase in segmental duplication events was observed between LS regions of the genome compared to within core regions or from LS regions to the core. RNA-seq expression studies identified candidate effectors expressed in planta, consisting of both known effector homologs and novel candidates. FTF1 and a subset of other transcription factors implicated in regulation of effector expression were found to be expressed in planta.

A study of Attachment Disorders in young offenders attending specialist services.

Attachment disorders, specifically Reactive Attachment Disorder (RAD) and Disinhibited Social Engagement Disorder (DSED) are disorders associated with neglect and abuse in which people have significant difficulties relating to others. This study aims to explore Attachment Disorder symptoms and diagnoses in young offenders and factors that may be associated with them such as mental health problems. A cross-sectional design was used with 29 young people who were known to Intensive Services, aged 12-17 (M=16.2, SD=1.3), 29 carers and 20 teachers. They completed measures investigating symptoms of Attachment Disorders and psychopathology. Eighty-six percent of the young people had experienced some form of maltreatment and the rates of an actual or borderline Attachment Disorder was 52%. A positive correlation between Attachment Disorder symptoms and other mental health problems (as rated by carer-report Strengths and Difficulties Questionnaire Total Difficulties Score), accounting for 36% of the variance was found, with a large effect size (rs=0.60). Attachment Disorder symptoms were associated with hyperactivity and peer relationship problems.

The Drosophila telomere-capping protein Verrocchio binds single-stranded DNA and protects telomeres from DNA damage response.

Drosophila telomeres are sequence-independent structures maintained by transposition to chromosome ends of three specialized retroelements rather than by telomerase activity. Fly telomeres are protected by the terminin complex that includes the HOAP, HipHop, Moi and Ver proteins. These are fast evolving, non-conserved proteins that localize and function exclusively at telomeres, protecting them from fusion events. We have previously suggested that terminin is the functional analogue of shelterin, the multi-protein complex that protects human telomeres. Here, we use electrophoretic mobility shift assay (EMSA) and atomic force microscopy (AFM) to show that Ver preferentially binds single-stranded DNA (ssDNA) with no sequence specificity. We also show that Moi and Ver form a complex in vivo. Although these two proteins are mutually dependent for their localization at telomeres, Moi neither binds ssDNA nor facilitates Ver binding to ssDNA. Consistent with these results, we found that Ver-depleted telomeres form RPA and γH2AX foci, like the human telomeres lacking the ssDNA-binding POT1 protein. Collectively, our findings suggest that Drosophila telomeres possess a ssDNA overhang like the other eukaryotes, and that the terminin complex is architecturally and functionally similar to shelterin.

Identification of pathogenicity-related genes in Fusarium oxysporum f. sp. cepae.

Pathogenic isolates of Fusarium oxysporum, distinguished as formae speciales (f. spp.) on the basis of their host specificity, cause crown rots, root rots and vascular wilts on many important crops worldwide. Fusarium oxysporum f. sp. cepae (FOC) is particularly problematic to onion growers worldwide and is increasing in prevalence in the UK. We characterized 31 F. oxysporum isolates collected from UK onions using pathogenicity tests, sequencing of housekeeping genes and identification of effectors. In onion seedling and bulb tests, 21 isolates were pathogenic and 10 were non-pathogenic. The molecular characterization of these isolates, and 21 additional isolates comprising other f. spp. and different Fusarium species, was carried out by sequencing three housekeeping genes. A concatenated tree separated the F. oxysporum isolates into six clades, but did not distinguish between pathogenic and non-pathogenic isolates. Ten putative effectors were identified within FOC, including seven Secreted In Xylem (SIX) genes first reported in F. oxysporum f. sp. lycopersici. Two highly homologous proteins with signal peptides and RxLR motifs (CRX1/CRX2) and a gene with no previously characterized domains (C5) were also identified. The presence/absence of nine of these genes was strongly related to pathogenicity against onion and all were shown to be expressed in planta. Different SIX gene complements were identified in other f. spp., but none were identified in three other Fusarium species from onion. Although the FOC SIX genes had a high level of homology with other f. spp., there were clear differences in sequences which were unique to FOC, whereas CRX1 and C5 genes appear to be largely FOC specific.

TEMPRANILLO is a regulator of juvenility in plants.

Many plants are incapable of flowering in inductive daylengths during the early juvenile vegetative phase (JVP). Arabidopsis mutants with reduced expression of TEMPRANILLO (TEM), a repressor of flowering locus T (FT) had a shorter JVP than wild-type plants. Reciprocal changes in mRNA expression of TEM and FT were observed in both Arabidopsis and antirrhinum, which correlated with the length of the JVP. FT expression was induced just prior to the end of the JVP and levels of TEM1 mRNA declined rapidly at the time when FT mRNA levels were shown to increase. TEM orthologs were isolated from antirrhinum (AmTEM) and olive (OeTEM) and were expressed most highly during their juvenile phase. AmTEM functionally complemented AtTEM1 in the tem1 mutant and over-expression of AmTEM prolonged the JVP through repression of FT and CONSTANS (CO). We propose that TEM may have a general role in regulating JVP in herbaceous and woody species.

A local regulatory network around three NAC transcription factors in stress responses and senescence in Arabidopsis leaves.

A model is presented describing the gene regulatory network surrounding three similar NAC transcription factors that have roles in Arabidopsis leaf senescence and stress responses. ANAC019, ANAC055 and ANAC072 belong to the same clade of NAC domain genes and have overlapping expression patterns. A combination of promoter DNA/protein interactions identified using yeast 1-hybrid analysis and modelling using gene expression time course data has been applied to predict the regulatory network upstream of these genes. Similarities and divergence in regulation during a variety of stress responses are predicted by different combinations of upstream transcription factors binding and also by the modelling. Mutant analysis with potential upstream genes was used to test and confirm some of the predicted interactions. Gene expression analysis in mutants of ANAC019 and ANAC055 at different times during leaf senescence has revealed a distinctly different role for each of these genes. Yeast 1-hybrid analysis is shown to be a valuable tool that can distinguish clades of binding proteins and be used to test and quantify protein binding to predicted promoter motifs.

Adenotonsillectomy for sleep-disordered breathing in a predominantly obese pediatric population.

OBJECTIVES: To determine the efficacy of adenotonsillectomy (T&A) in the treatment of pediatric sleep-disordered breathing, and to determine the natural history in untreated children. METHODS: The charts of children aged 1-12 who underwent polysomnography (PSG) between 1/2006 and 6/2009 were reviewed to identify children with positive studies. Children not treated by T&A were recruited and matched by age, time since initial PSG, and apnea-hypopnea index (AHI) to children who underwent T&A. All participants were evaluated by a clinical assessment score (CAS-15), follow-up PSG, and the Child Behavior Checklist (CBCL). RESULTS: Sixteen matched pairs completed the study. Ten (63%) T&A patients were overweight or obese compared with 14 (88%) untreated patients. There was a greater median improvement in AHI in the surgical group compared to the nonsurgical group (10.3 vs. 6.5, p=0.044). Although the T&A children were more likely to have a follow-up AHI<5 (81% vs. 69%) and <1 (44% vs. 25%), these results were not significant. The T&A group had significantly lower mean (SD) scores on the CAS-15 [8.9(6.1) vs. 29.4(16.2), p<0.001] and the CBCL total problem score [43.9(8.7) vs. 58.9(13.0), p<0.001]. Younger age at presentation (rho=-0.76, p<0.001), initial AHI (0.87, p<0.001), and initial AI (0.63, p=0.05) were correlated with change in AHI among T&A subjects. CONCLUSIONS: T&A was more effective in reducing AHI than no surgery. Median AHI improved in the nonsurgical group, and 4/16 (25%) untreated patients were cured (AHI<1).

Validation of a clinical assessment score for pediatric sleep-disordered breathing.

OBJECTIVES/HYPOTHESIS: To validate a clinical assessment score for pediatric sleep-disordered breathing. STUDY DESIGN: Prospective instrument validation. METHODS: One hundred children scheduled for overnight polysomnography were evaluated by a standardized history and physical examination and assigned a clinical assessment score. Parents completed the Obstructive Sleep Apnea (OSA)-18, the Pediatric Quality of Life Inventory (PedsQL) 4.0, and the Child Behavior Checklist questionnaires. Children with positive polysomnography underwent adenotonsillectomy or adenoidectomy. The identical assessments were performed at a mean follow-up of 8 months. RESULTS: Item reduction yielded a score of 15 items (Clinical Assessment Score-15 [CAS-15]) that demonstrated the best internal consistency and predictive utility (Cronbach α = .80). Intraclass correlation (ICC) demonstrated good intrarater (ICC, 0.78; 95% confidence interval [CI], 0.58 to 0.89) and inter-rater agreement (ICC, 0.65; 95% CI, 0.26 to 0.84). All change scores were significantly improved after surgery. Effect sizes were large for the CAS-15 (2.6), OSA-18 (2.4), and apnea-hypopnea index (1.4), and moderate for the Child Behavior Checklist (0.7) and PedsQL 4.0 (-0.5). Moderate to strong correlation was found between the initial CAS-15 scores and the external measures (|r| between 0.32 and 0.65). Receiver operating characteristic curves were constructed to determine the optimal initial CAS-15 score for predicting positive polysomnography. The area under the curve was 0.77 (95% CI, 0.67 to 0.87); and a score ≥32 yielded a sensitivity of 77.3% (95% CI, 65.3 to 86.7) and a specificity of 60.7% (95% CI, 40.6 to 78.5). CONCLUSIONS: The CAS-15 proved useful in an office setting and correctly diagnosed 72% of referred children when compared to polysomnography. It correlated well with external measures and demonstrated a good response to clinical change.

Subsurface interactions of Fe(II) with humic acid or landfill leachate do not control subsequent iron(III) (hydr)oxide production at the surface.

At least 93% of Fe(II) remained free, as defined by ferrozine assay under anoxic conditions in the presence of humic acid (HA) and two simulated landfill leachates of different maturities. However, tangential flow ultrafiltration showed a weaker but more extensive interaction of Fe with organic carbon (OC); 90% of Fe associated with the less mature leachate. Despite the existence of this weak interaction under anoxic conditions, there was no difference in iron(III) (hydr)oxide production whether HA was added prior to or coincident with the oxidation of Fe(II) on exposure to oxic conditions. Under oxic conditions ferrozine showed that more Fe(II) bound to OC, up to 50% to HA. However, this occurs via oxidation of Fe(II) to Fe(III), which is bound and then thermally reduced. This affinity for Fe(III) and the ability to carry out thermal reduction both increase with the maturity of the OC. The rate at which ferrozine-defined free Fe(II) was lost on exposure to dissolved oxygen was also enhanced by the more mature OC, while it was slowed by acetogenic leachate. The slowing must be a consequence of the filtration-defined Fe(II)/OC interaction.

A prospective study of the effect of gastroesophageal reflux disease treatment on children with otitis media.

OBJECTIVES: To demonstrate improvements in validated quality-of-life measures for otitis media and gastroesophageal reflux disease (GERD) and an objective score for pediatric reflux obtained by fiberoptic laryngoscopy after treatment with antireflux precautions and therapy in children diagnosed as having either recurrent acute otitis media or otitis media with effusion and GERD. DESIGN: Prospective, before-and-after intervention study. SETTING: Hospital-based pediatric otolaryngology practice. PARTICIPANTS: Population-based sample of 47 patients (mean age, 19.5 months). INTERVENTION: Standard antireflux therapy for 2 consecutive 12-week periods. MAIN OUTCOME MEASURES: Otitis Media 6-Item quality-of-life survey, Infant GERD Questionnaire-Revised, GERD Symptom Questionnaire for Young Children, Pediatric Reflux Finding Score, and speech awareness threshold. RESULTS: Follow-up data were available for 37 patients. Mean (SD) change scores for Otitis Media 6-Item quality-of-life survey were 1.6 (1.1) at visit 2 and 1.5 (1.1) at visit 3 (P < .001 and P = .004, respectively). Change scores were significantly improved for Infant GERD Questionnaire-Revised and GERD Symptom Questionnaire for Young Children at visit 2 and for Infant GERD Questionnaire-Revised at visit 3. Mean (SD) change scores for the Pediatric Reflux Finding Score were 6.4 (4.9) at visit 2 and 8.0 (7.2) at visit 3 (P < .001 and P = .03, respectively). Hearing loss was significantly improved following therapy, as were laryngeal findings of reflux on fiberoptic laryngoscopy. Otitis media was considered by the examining physician to be clinically improved in 28 of 37 children (76%; 95% confidence interval, 60%-87%) at visit 2 and in 6 of 10 children (60%; 95% confidence interval, 31%-83%) at visit 3. Nine children (19.1%) required myringotomy tube placement. CONCLUSIONS: Children with otitis media with effusion or recurrent acute otitis media and GERD have improved quality of life following treatment with antireflux therapy. Control of gastroesophageal reflux may play a role in the management of otitis media and avoidance of tympanostomy.

The CHAPS, SIFTER, and TAPS-R as predictors of (C)AP skills and (C)APD.

PURPOSE: In this study, the authors investigated the relationships between 3 tests used to screen for (central) auditory processing disorder ([C]APD)--the Children's Auditory Performance Scale (CHAPS; W. J. Smoski, M. A. Brunt, & J. C. Tannahill, 1998), the Screening Instrument for Targeting Educational Risk (SIFTER; K. Anderson, 1989), and the Test of Auditory Perceptual Skills--Revised (TAPS-R; M. Y. Gardner, 1997)-and 4 tests used to diagnostically assess for (C)APD: Low-Pass Filtered Speech (LPFS), Competing Sentences (CS), Two-Pair Dichotic Digits (DD), and Frequency Patterns With Linguistic Report (FP). METHOD: The screening and diagnostic (C)APD tests results of 104 children (71 boys, 33 girls) aged 6.9-14.3 years were reviewed following their attendance at a university clinic in Brisbane, Australia. RESULTS: Pearson's product-moment correlation coefficient analysis showed weak (r = .22, p < .05) to moderate (r = .47, p < .01) correlations predominantly between the short-term and working memory test results of the TAPS-R and the DD and FP test results of the (C)AP test battery. Linear and binary logistic regression analyses showed a poor ability of the CHAPS, SIFTER, and TAPS-R test results to predict the individual LPFS, CS, DD, or FP test results or the overall risk for (C)APD. CONCLUSION: The CHAPS, SIFTER, and TAPS-R should be used to highlight concerns about a child but not to determine whether a diagnostic (C)AP assessment is particularly warranted.

The prevalence and incidence of mental ill-health in adults with autism and intellectual disabilities.

The prevalence, and incidence, of mental ill-health in adults with intellectual disabilities and autism were compared with the whole population with intellectual disabilities, and with controls, matched individually for age, gender, ability-level, and Down syndrome. Although the adults with autism had a higher point prevalence of problem behaviours compared with the whole adult population with intellectual disabilities, compared with individually matched controls there was no difference in prevalence, or incidence of either problem behaviours or other mental ill-health. Adults with autism who had problem behaviours were less likely to recover over a two-year period than were their matched controls. Apparent differences in rates of mental ill-health are accounted for by factors other than autism, including Down syndrome and ability level.

Predicting job loss in those off sick.

BACKGROUND: Evidence shows incapacity benefit claimants (those off sick >26 weeks) are at greatest risk of long-term job loss. AIM: To develop a screening tool to select those at risk of job loss, defined as failure to return to work among those off sick. The screening tool was for use in the Job Retention and Rehabilitation Pilot of the Department for Work and Pensions. METHODS: A literature review identified risks for long-term incapacity and job loss as multifactorial. Potential predictors for return to work were then assembled into a set of questions and tested by a prospective study in general practice surgeries and a retrospective study of occupational health records of local authority employees referred for sickness absence management, using univariate and multivariate logistic regression analysis. RESULTS: Univariate logistic regression analysis of the retrospective study produced odds ratios with 95% confidence intervals for each question (where P