A CC-NB-LRR gene confers leaf stripe resistance at the Rdg2a locus in barley

Barley leaf stripe, caused by the seed-transmitted hemi-biotrophic fungus Pyrenophora graminea, is a barley disease particularly acute in Nordic countries, during spring sowing, and in the Mediterranean’s winter barley districts where causes severe yield losses. To date, only two P. graminea race-specific resistance genes are known: Rdg1a, identified in cultivar Vada, and Rdg2a, identified in cv. Thibaut. Rdg2a confers immunity to at least three different P. graminea monoconidian isolates, including the most widespread and virulent Italian isolate Dg2, but it is overcome by Dg5. The aim of the present work was to isolate this gene, characterize the Rdg2a locus and its evolution and mine the bases of Rdg2a-mediated resistance. In a previous analysis the map-based cloning and the sequencing of the Rdg2a locus were carried out. Three homolog R genes encoding CC-NB-LRR proteins, that represent the majior class of resistance proteins, were identified at the locus and were named as Nbs1-Rdg2a, Nbs2-Rdg2a and Nbs3-Rdg2a (Bulgarelli et al., 2010). To determine which of the three genes is the Rdg2a gene, we started the research investigating their structure. RACE analyses showed that for the third candidate (Nbs3-Rdg2a) alternative splicing processes determine the synthesis of severly truncated and probably non-functional proteins. This allowed us to excluded Nbs3- Rdg2a from acting in resistance to isolate Dg2. By means RT-PCRs on a pair of Near Isogenic Lines (NIL) that differ only for alleles at the Rdg2a locus (susceptible cv. Mirco and its resistant NIL, NIL3876), we demonstrated that both Nbs1-Rdg2a and Nbs2-Rdg2a are transcribed in embryos (where the resistance takes place) and leaf tissues of the resistant line, but they are not expressed in the susceptible nearisogenic phenotype. Sequencing of the Mirco alleles revealed rearrangements in the putative promoter regions: two insertions, one next to a putative TATA-box element and the other, carrying terminal inverted repeats, in the 5’ UTR, for Nbs1-rdg2a, and a deletion just at the level of a MITE-like element, present in Thibaut Nbs2-Rdg2a, for Nbs2-rdg2a. It is likely that these changes represent the cause of the lacking of expression of these genes in the susceptible genotype. Moreover, qRT-PCRs showed that Nbs1-Rdg2a transcription was un-responsive to P. graminea infection, while Nbs2-Rdg2a transcripts increased during the first stages of infection. However, Nbs2-Rdg2a mRNA abundance was significantly lower than that of Nbs1-Rdg2a. To define which gene is the Rdg2a gene, a complementation assay was conducted. Susceptible cv. Golden Promise was transformed by agoinfiltration with each gene independently, under the control of its native promoter. Interestingly, the rescue of Dg2-resistance was observed only when plants were transformed with Nbs1-Rdg2a (90-100% of resistance), suggesting that this gene is Rdg2a. Comparing the sequences of the three genes at the Rdg2a locus, we found that similarly to other R genes, Rdg2a underwent to diversifying selection, according to a model in which resistance genes co-evolves with pathogen effector(s) gene(s). The fact that the Rdg2a locus contains a gene cluster of highly similar sequences has most likely contributed to significative rearrangements during evolution, probably derived from unequal crossovers resulting in sequence exchange between paralogs and, possibly, in the generation of recombinant genes, as well as to expansion/contraction of gene copy number. Regarding this last case, we have also characterized the rdg2a locus of the susceptible cv. Morex. Morex rdg2a locus carries two deletions and the rdg2a allele might derived from an un-equal crossingover between Rdg2a and Nbs2-Rdg2a ancestors that led to a reduction of the number of the gene family members. Most resistance proteins function through inducing a Programmed Cell Death (PCD) that lead to a Hypersensitive Response (HR) at the level of the infected cells. Histological analyses using the TUNEL method did not reveal any significative difference in PCD between infected embryos of resistant and susceptible varieties and the number of cells undergoing PCD was transcurable. These finding let us to conclude that the Rdg2a-mediated resistance does not involve HR but it is most likely based on the strengthening of physical and chemical barriers in the cell walls and intercellular spaces of the embryo tissues. In conclusion, we identified, cloned and characterized the first resistance gene active against a seed-borne disease; importantly, the gene belongs to the poorly represented class of R genes which does not trigger a hypersensitive responce

Years of life that could be saved from prevention of hepatocellular carcinoma

BACKGROUND: Hepatocellular carcinoma (HCC) causes premature death and loss of life expectancy worldwide. Its primary and secondary prevention can result in a significant number of years of life saved. AIM: To assess how many years of life are lost after HCC diagnosis. METHODS: Data from 5346 patients with first HCC diagnosis were used to estimate lifespan and number of years of life lost after tumour onset, using a semi-parametric extrapolation having as reference an age-, sex- and year-of-onset-matched population derived from national life tables. RESULTS: Between 1986 and 2014, HCC lead to an average of 11.5 years-of-life lost for each patient. The youngest age-quartile group (18-61 years) had the highest number of years-of-life lost, representing approximately 41% of the overall benefit obtainable from prevention. Advancements in HCC management have progressively reduced the number of years-of-life lost from 12.6 years in 1986-1999, to 10.7 in 2000-2006 and 7.4 years in 2007-2014. Currently, an HCC diagnosis when a single tumour <2 cm results in 3.7 years-of-life lost while the diagnosis when a single tumour 65 2 cm or 2/3 nodules still within the Milan criteria, results in 5.0 years-of-life lost, representing the loss of only approximately 5.5% and 7.2%, respectively, of the entire lifespan from birth. CONCLUSIONS: Hepatocellular carcinoma occurrence results in the loss of a considerable number of years-of-life, especially for younger patients. In recent years, the increased possibility of effectively treating this tumour has improved life expectancy, thus reducing years-of-life lost

Rice diversity panel provides accurate genomic predictions for complex traits in the progenies of biparental crosses involving members of the panel

So far, most potential applications of genomic prediction in plant improvement have been explored using cross validation approaches. This is the first empirical study to evaluate the accuracy of genomic prediction of the performances of progenies in a typical rice breeding program. Using a cross validation approach, we first analyzed the effects of marker selection and statistical methods on the accuracy of prediction of three different heritability traits in a reference population (RP) of 284 inbred accessions. Next, we investigated the size and the degree of relatedness with the progeny population (PP) of sub-sets of the RP that maximize the accuracy of prediction of phenotype across generations, i.e., for 97 F5–F7 lines derived from biparental crosses between 31 accessions of the RP. The extent of linkage disequilibrium was high (r2 = 0.2 at 0.80 Mb in RP and at 1.1 Mb in PP). Consequently, average marker density above one per 22 kb did not improve the accuracy of predictions in the RP. The accuracy of progeny prediction varied greatly depending on the composition of the training set, the trait, LD and minor allele frequency. The highest accuracy achieved for each trait exceeded 0.50 and was only slightly below the accuracy achieved by cross validation in the RP. Our results thus show that relatively high accuracy (0.41–0.54) can be achieved using only a rather small share of the RP, most related to the PP, as the training set. The practical implications of these results for rice breeding programs are discussed. (Résumé d'auteur

Development and Validation of a New Prognostic System for Patients with Hepatocellular Carcinoma

Background: Prognostic assessment in patients with hepatocellular carcinoma (HCC) remains controversial. Using the Italian Liver Cancer (ITA.LI.CA) database as a training set, we sought to develop and validate a new prognostic system for patients with HCC. Methods and Findings: Prospective collected databases from Italy (training cohort, n = 3,628; internal validation cohort, n = 1,555) and Taiwan (external validation cohort, n = 2,651) were used to develop the ITA.LI.CA prognostic system. We first defined ITA.LI.CA stages (0, A, B1, B2, B3, C) using only tumor characteristics (largest tumor diameter, number of nodules, intra- and extrahepatic macroscopic vascular invasion, extrahepatic metastases). A parametric multivariable survival model was then used to calculate the relative prognostic value of ITA.LI.CA tumor stage, Eastern Cooperative Oncology Group (ECOG) performance status, Child–Pugh score (CPS), and alpha-fetoprotein (AFP) in predicting individual survival. Based on the model results, an ITA.LI.CA integrated prognostic score (from 0 to 13 points) was constructed, and its prognostic power compared with that of other integrated systems (BCLC, HKLC, MESIAH, CLIP, JIS). Median follow-up was 58 mo for Italian patients (interquartile range, 26–106 mo) and 39 mo for Taiwanese patients (interquartile range, 12–61 mo). The ITA.LI.CA integrated prognostic score showed optimal discrimination and calibration abilities in Italian patients. Observed median survival in the training and internal validation sets was 57 and 61 mo, respectively, in quartile 1 (ITA.LI.CA score ≤ 1), 43 and 38 mo in quartile 2 (ITA.LI.CA score 2–3), 23 and 23 mo in quartile 3 (ITA.LI.CA score 4–5), and 9 and 8 mo in quartile 4 (ITA.LI.CA score > 5). Observed and predicted median survival in the training and internal validation sets largely coincided. Although observed and predicted survival estimations were significantly lower (log-rank test, p < 0.001) in Italian than in Taiwanese patients, the ITA.LI.CA score maintained very high discrimination and calibration features also in the external validation cohort. The concordance index (C index) of the ITA.LI.CA score in the internal and external validation cohorts was 0.71 and 0.78, respectively. The ITA.LI.CA score’s prognostic ability was significantly better (p < 0.001) than that of BCLC stage (respective C indexes of 0.64 and 0.73), CLIP score (0.68 and 0.75), JIS stage (0.67 and 0.70), MESIAH score (0.69 and 0.77), and HKLC stage (0.68 and 0.75). The main limitations of this study are its retrospective nature and the intrinsically significant differences between the Taiwanese and Italian groups. Conclusions: The ITA.LI.CA prognostic system includes both a tumor staging—stratifying patients with HCC into six main stages (0, A, B1, B2, B3, and C)—and a prognostic score—integrating ITA.LI.CA tumor staging, CPS, ECOG performance status, and AFP. The ITA.LI.CA prognostic system shows a strong ability to predict individual survival in European and Asian populations

The CC-NB-LRR-Type Rdg2a Resistance Gene Confers Immunity to the Seed-Borne Barley Leaf Stripe Pathogen in the Absence of Hypersensitive Cell Death

BACKGROUND: Leaf stripe disease on barley (Hordeum vulgare) is caused by the seed-transmitted hemi-biotrophic fungus Pyrenophora graminea. Race-specific resistance to leaf stripe is controlled by two known Rdg (Resistance to Drechslera graminea) genes: the H. spontaneum-derived Rdg1a and Rdg2a, identified in H. vulgare. The aim of the present work was to isolate the Rdg2a leaf stripe resistance gene, to characterize the Rdg2a locus organization and evolution and to elucidate the histological bases of Rdg2a-based leaf stripe resistance. PRINCIPLE FINDINGS: We describe here the positional cloning and functional characterization of the leaf stripe resistance gene Rdg2a. At the Rdg2a locus, three sequence-related coiled-coil, nucleotide-binding site, and leucine-rich repeat (CC-NB-LRR) encoding genes were identified. Sequence comparisons suggested that paralogs of this resistance locus evolved through recent gene duplication, and were subjected to frequent sequence exchange. Transformation of the leaf stripe susceptible cv. Golden Promise with two Rdg2a-candidates under the control of their native 5′ regulatory sequences identified a member of the CC-NB-LRR gene family that conferred resistance against the Dg2 leaf stripe isolate, against which the Rdg2a-gene is effective. Histological analysis demonstrated that Rdg2a-mediated leaf stripe resistance involves autofluorescing cells and prevents pathogen colonization in the embryos without any detectable hypersensitive cell death response, supporting a cell wall reinforcement-based resistance mechanism. CONCLUSIONS: This work reports about the cloning of a resistance gene effective against a seed borne disease. We observed that Rdg2a was subjected to diversifying selection which is consistent with a model in which the R gene co-evolves with a pathogen effector(s) gene. We propose that inducible responses giving rise to physical and chemical barriers to infection in the cell walls and intercellular spaces of the barley embryo tissues represent mechanisms by which the CC-NB-LRR-encoding Rdg2a gene mediates resistance to leaf stripe in the absence of hypersensitive cell death.Davide Bulgarelli, Chiara Biselli, Nicholas C. Collins, Gabriella Consonni, Antonio M. Stanca, Paul Schulze-Lefert and Giampiero Val

Metabolic disorders across hepatocellular carcinoma in Italy

BACKGROUND: Metabolic disorders are well-known risk factors for HCC. Conversely, their impact on the natural history of HCC is not established. This study aimed at evaluating the impact of metabolic disorders on clinical features, treatment and survival of HCC patients regardless of its aetiology. METHODS: We analysed the ITA.LI.CA database regarding 839 HCC patients prospectively collected. The following metabolic features were analysed: BMI, diabetes, arterial hypertension, hypercholesterolaemia and hypertriglyceridaemia. According to these features, patients were divided into 3 groups: 0-1, 2 and 3-5 metabolic features. RESULTS: As compared with patients with 0-1 metabolic features, patients with 3-5 features showed lower percentage of HCC diagnosis on surveillance (P = .021), larger tumours (P = .038), better liver function (higher percentage of Child-Pugh class A [P = .007] and MELD &lt; 10 [P = .003]), higher percentage of metastasis (P = .024) and lower percentage of portal vein thrombosis (P = .010). The BCLC stage and treatment options were similar among the 3 groups, with the exception of a less frequent access to loco-regional therapies for BCLC stage B patients with 3-5 features (P = .012). Overall survival and survival according to BCLC stage and/or treatment did not significantly differ among the 3 groups. Only using a probabilistic sensitivity analysis, diabetic patients showed a lower survival (P = .046). MELD score, HCC morphology, nodule size, BCLC stage, portal vein thrombosis and metastasis were independent predictors of lead-time adjusted survival. CONCLUSIONS: Our "real world" study suggests that metabolic disorders shape the clinical presentation of HCC but do not seem to play a major role in setting patient survival.Background: Metabolic disorders are well-known risk factors for HCC. Conversely, their impact on the natural history of HCC is not established. This study aimed at evaluating the impact of metabolic disorders on clinical features, treatment and survival of HCC patients regardless of its aetiology. Methods: We analysed the ITA.LI.CA database regarding 839 HCC patients prospectively collected. The following metabolic features were analysed: BMI, diabetes, arterial hypertension, hypercholesterolaemia and hypertriglyceridaemia. According to these features, patients were divided into 3 groups: 0-1, 2 and 3-5 metabolic features. Results: As compared with patients with 0-1 metabolic features, patients with 3-5 features showed lower percentage of HCC diagnosis on surveillance (P&nbsp;=.021), larger tumours (P&nbsp;=.038), better liver function (higher percentage of Child-Pugh class A [P&nbsp;=.007] and MELD&nbsp;&lt;&nbsp;10 [P&nbsp;=.003]), higher percentage of metastasis (P&nbsp;=.024) and lower percentage of portal vein thrombosis (P&nbsp;=.010). The BCLC stage and treatment options were similar among the 3 groups, with the exception of a less frequent access to loco-regional therapies for BCLC stage B patients with 3-5 features (P&nbsp;=.012). Overall survival and survival according to BCLC stage and/or treatment did not significantly differ among the 3 groups. Only using a probabilistic sensitivity analysis, diabetic patients showed a lower survival (P&nbsp;=.046). MELD score, HCC morphology, nodule size, BCLC stage, portal vein thrombosis and metastasis were independent predictors of lead-time adjusted survival. Conclusions: Our \u201creal world\u201d study suggests that metabolic disorders shape the clinical presentation of HCC but do not seem to play a major role in setting patient survival