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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Don't scold smokers.

In response to a recent paper about GPs' difficulties with smokers who are not motivated to quit, points to consider are: (1) nicotine is one of the strongest addictions; (2) smokers hold various beliefs about smoking, and doctors should consider how to communicate risk; (3) confrontation is contraindicated; and (4) some smokers deceive doctors about their smoking.

A systematic review of manic and depressive prodromes.

BACKGROUND: This paper explores whether individuals with a mood disorder can identify the nature and duration of depressive and manic prodromes. METHODS: Seventy-three publications of prodromal symptoms in bipolar and unipolar disorders were identified by computer searches of seven databases (including MEDLINE and PsycLIT) supplemented by hand searches of journals. Seventeen studies (total sample=1191 subjects) met criteria for inclusion in a systematic review. RESULTS: At least 80% of individuals with a mood disorder can identify one or more prodromal symptoms. There are limited data about unipolar disorders. In bipolar disorders, early symptoms of mania are identified more frequently than early symptoms of depression. The most robust early symptom of mania is sleep disturbance (median prevalence 77%). Early symptoms of depression are inconsistent. The mean length of manic prodromes (>20 days) was consistently reported to be longer than depressive prodromes (<19 days). However, depressive prodromes showed greater inter-individual variation (ranging from 2 to 365 days) in duration than manic prodromes (1-120 days). LIMITATIONS: Few prospective studies of bipolar, and particularly unipolar disorders have been reported. CONCLUSIONS: Early symptoms of relapse in affective disorders can be identified. Explanations of the apparent differences in the recognition and length of prodromes between mania and bipolar depression are explored. Further research on duration, sequence of symptom appearance and characteristics of prodromes is warranted to clarify the clinical usefulness of early symptom monitoring.

Beliefs and behavior of deceivers in a randomized, controlled trial of anti-smoking advice at a primary care clinic in Kelantan, Malaysia.

Smoking deception is often ignored, but is important in health care. In this trial it was assessed at both study entry and outcome. At study entry, 1,044 males at a primary care clinic were asked smoking status and tested for breath carbon monoxide (CO). Of self-reported non-smokers, 57/402 (14%) were actually smokers, as were 59/251 (24%) of self-reported ex-smokers. The self-reported smokers (n=387) entered a randomized, controlled trial where the intervention comprised four questions on knowledge and beliefs about smoking, standardized verbal advice against smoking, and a leaflet. At follow-up, subjects were also questioned about beliefs. Follow-up was difficult, but 191/387 (49%) attended at three or six months. Of 27 who claimed to have quit, 6 (22%) were deceivers and 21 were confirmed quitters. Cessation did not differ between intervention and control groups. Overall confirmed cessation at six months was 16/387 (4.1 %). Confirmed quitters were significantly lighter smokers than deceivers and still smokers. There were non-significant trends between the outcome groups whereby deceivers had least knowledge and most lay beliefs, and quitters had most knowledge and fewest lay beliefs. The lay beliefs may prevent some smokers from quitting.

Lay beliefs about smoking in Kelantan, Malaysia.

Studies have shown that smokers rationalize smoking by self-exempting beliefs. This study explored lay beliefs about smoking in Kelantan, Malaysia, using focus groups among outpatients, medical students and staff, and a questionnaire survey of 193 male smokers. In focus groups, patients said they could do something to make smoking safe. When asked, 'Do you think there are any safe ways to smoke?' 132/193 (68%) male smokers described at least one way. The commonest were 'drink water' (69/193, 36%), 'use a filter' (60/193, 31%), 'smoke after food' (27/193, 14%), and 'take sour fruit' (21/193, 11%). At three- or six-month follow-up, numbers agreeing with these beliefs were: for 'drink water' 67/115 (58%), for 'take sour fruit' 61/115 (53%), and for 'smoke after food' 38/115 (33%), with 88/115 (77%) supporting at least one. The main explanations for water were that it cleaned or moistened the lungs or throat. Sour fruit was described as cleaning, and sometimes as 'sharp', able to scrape out the essence of cigarettes. The conclusion is that self-exempting false beliefs about smoking are widespread, and here they probably represent an extension of the traditional humoral system. Anti-smoking campaigns and health workers in smoking cessation services should address these beliefs.

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.

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).