A complex receptor locus confers responsiveness to necrosis and ethylene-inducing like peptides in Brassica napus.

Brassica crops are susceptible to diseases which can be mitigated by breeding for resistance. MAMPs (microbe-associated molecular patterns) are conserved molecules of pathogens that elicit host defences known as pattern-triggered immunity (PTI). Necrosis and Ethylene-inducing peptide 1-like proteins (NLPs) are MAMPs found in a wide range of phytopathogens. We studied the response to BcNEP2, a representative NLP from Botrytis cinerea, and showed that it contributes to disease resistance in Brassica napus. To map regions conferring NLP response, we used the production of reactive oxygen species (ROS) induced during PTI across a population of diverse B. napus accessions for associative transcriptomics (AT), and bulk segregant analysis (BSA) on DNA pools created from a cross of NLP-responsive and non-responsive lines. In silico mapping with AT identified two peaks for NLP responsiveness on chromosomes A04 and C05 whereas the BSA identified one peak on A04. BSA delimited the region for NLP-responsiveness to 3 Mbp, containing ~245 genes on the Darmor-bzh reference genome and four co-segregating KASP markers were identified. The same pipeline with the ZS11 genome confirmed the highest-associated region on chromosome A04. Comparative BLAST analysis revealed unannotated clusters of receptor-like protein (RLP) homologues on ZS11 chromosome A04. However, no specific RLP homologue conferring NLP response could be identified. Our results also suggest that BR-SIGNALLING KINASE1 may be involved with modulating the NLP response. Overall, we demonstrate that responsiveness to NLP contributes to disease resistance in B. napus and define the associated genomic location. These results can have practical application in crop improvement.

Pathogen lifestyle determines host genetic signature of quantitative disease resistance loci in oilseed rape (Brassica napus).

KEY MESSAGE: Using associative transcriptomics, our study identifies genes conferring resistance to four diverse fungal pathogens in crops, emphasizing key genetic determinants of multi-pathogen resistance. Crops are affected by several pathogens, but these are rarely studied in parallel to identify common and unique genetic factors controlling diseases. Broad-spectrum quantitative disease resistance (QDR) is desirable for crop breeding as it confers resistance to several pathogen species. Here, we use associative transcriptomics (AT) to identify candidate gene loci associated with Brassica napus constitutive QDR to four contrasting fungal pathogens: Alternaria brassicicola, Botrytis cinerea, Pyrenopeziza brassicae, and Verticillium longisporum. We did not identify any shared loci associated with broad-spectrum QDR to fungal pathogens with contrasting lifestyles. Instead, we observed QDR dependent on the lifestyle of the pathogen-hemibiotrophic and necrotrophic pathogens had distinct QDR responses and associated loci, including some loci associated with early immunity. Furthermore, we identify a genomic deletion associated with resistance to V. longisporum and potentially broad-spectrum QDR. This is the first time AT has been used for several pathosystems simultaneously to identify host genetic loci involved in broad-spectrum QDR. We highlight constitutive expressed candidate loci for broad-spectrum QDR with no antagonistic effects on susceptibility to the other pathogens studies as candidates for crop breeding. In conclusion, this study represents an advancement in our understanding of broad-spectrum QDR in B. napus and is a significant resource for the scientific community.

An optimised CRISPR Cas9 and Cas12a mutagenesis toolkit for Barley and Wheat.

BACKGROUND: CRISPR Cas9 and Cas12a are the two most frequently used programmable nucleases reported in plant systems. There is now a wide range of component parts for both which likely have varying degrees of effectiveness and potentially applicability to different species. Our aim was to develop and optimise Cas9 and Cas12a based systems for highly efficient genome editing in the monocotyledons barley and wheat and produce a user-friendly toolbox facilitating simplex and multiplex editing in the cereal community. RESULTS: We identified a Zea mays codon optimised Cas9 with 13 introns in conjunction with arrayed guides driven by U6 and U3 promoters as the best performer in barley where 100% of T0 plants were simultaneously edited in all three target genes. When this system was used in wheat > 90% of T0 plants were edited in all three subgenome targets. For Cas12a, an Arabidopsis codon optimised sequence with 8 introns gave the best editing efficiency in barley when combined with a tRNA based multiguide array, resulting in 90% mutant alleles in three simultaneously targeted genes. When we applied this Cas12a system in wheat 86% & 93% of T0 plants were mutated in two genes simultaneously targeted. We show that not all introns contribute equally to enhanced mutagenesis when inserted into a Cas12a coding sequence and that there is rationale for including multiple introns. We also show that the combined effect of two features which boost Cas12a mutagenesis efficiency (D156R mutation and introns) is more than the sum of the features applied separately. CONCLUSION: Based on the results of our testing, we describe and provide a GoldenGate modular cloning system for Cas9 and Cas12a use in barley and wheat. Proven Cas nuclease and guide expression cassette options found in the toolkit will facilitate highly efficient simplex and multiplex mutagenesis in both species. We incorporate GRF-GIF transformation boosting cassettes in wheat options to maximise workflow efficiency.

Efficacy and Biological Correlates of Response in a Phase II Study of Venetoclax Monotherapy in Patients with Acute Myelogenous Leukemia.

We present a phase II, single-arm study evaluating 800 mg daily venetoclax, a highly selective, oral small-molecule B-cell leukemia/lymphoma-2 (BCL2) inhibitor in patients with high-risk relapsed/refractory acute myelogenous leukemia (AML) or unfit for intensive chemotherapy. Responses were evaluated following revised International Working Group (IWG) criteria. The overall response rate was 19%; an additional 19% of patients demonstrated antileukemic activity not meeting IWG criteria (partial bone marrow response and incomplete hematologic recovery). Twelve (38%) patients had isocitrate dehydrogenase 1/2 mutations, of whom 4 (33%) achieved complete response or complete response with incomplete blood count recovery. Six (19%) patients had BCL2-sensitive protein index at screening, which correlated with time on study. BH3 profiling was consistent with on-target BCL2 inhibition and identified potential resistance mechanisms. Common adverse events included nausea, diarrhea and vomiting (all grades), and febrile neutropenia and hypokalemia (grade 3/4). Venetoclax demonstrated activity and acceptable tolerability in patients with AML and adverse features. SIGNIFICANCE: Venetoclax monotherapy demonstrated clinical activity in patients with AML (relapsed/refractory or unfit for intensive chemotherapy) with a tolerable safety profile in this phase II study. Predictive markers of response consistent with BCL2 dependence were identified. Clinical and preclinical findings provide a compelling rationale to evaluate venetoclax combined with other agents in AML. Cancer Discov; 6(10); 1106-17. ©2016 AACRSee related commentary by Pullarkat and Newman, p. 1082This article is highlighted in the In This Issue feature, p. 1069.

Quality of life study following cytoreductive surgery and intraperitoneal chemotherapy for pseudomyxoma peritonei including redo procedures.

BACKGROUND: Our aim was to evaluate the quality of life following cytoreductive surgery and intraperitoneal chemotherapy for pseudomyxoma peritonei. We also conducted an analysis of all patients who underwent CRS and HIPEC for pseudomyxoma peritonei from 1997 to 2012. METHODS: We contacted 87 patients using the FACT C (version 4) quality of life questionnaire, and FACIT-TS-G (version 1) was also used. RESULTS: A total of 63 patients (response rate 72%) were available for quality of life interview and analysis. The median time from surgery to questionnaire evaluation was 31 months (range 6-161 months). 62% were females with an average age of 54 years. 22% of the patients had over one cytoreductive surgical procedure. We analysed our patients postoperatively based on physical, functional, social, and emotional well being who reported favourable outcomes in all sections. Patients who had a single procedure had a significantly higher score (P = 0.016) in the additional concerns section of the questionnaire. The patients who had a single procedure had better gastrointestinal digestion in terms of bowel control, appetite, and food digestion and also body appearance scoring. CONCLUSIONS: 79% of the patients stated that they would undergo further cytoreductive surgery and that redo procedures do not result in a significantly worse quality of life.

Outcome measures and scar aesthetics in minimally invasive video-assisted parathyroidectomy.

OBJECTIVES: To compare the scar outcome of video-assisted parathyroidectomy (VAP) with traditional bilateral cervical exploration (BCE) using previously validated scar assessment scales, and to examine the feasibility of introducing VAP into a general otolaryngology-head and neck practice. DESIGN: A retrospective review of medical records from a prospectively obtained database of patients and long-term follow-up of scar analysis. PATIENTS: The records of 60 patients undergoing parathyroidectomy were reviewed: 29 patients underwent VAP and 31 patients underwent an open procedure with BCE. The groups were matched for age and sex. A total of 46 patients were followed up to assess scar outcome. MAIN OUTCOME MEASURES: The primary outcome was a comparison of patient and observer scar satisfaction between VAP and traditional BCE using validated scar assessment tools: the Patient Scar Assessment Scale and the Manchester Scar Scale. The secondary outcomes were to retrospectively evaluate our results with VAP and to assess the suitability of introducing this technique into a general otolaryngology-head and neck practice. RESULTS: The average scar length in the VAP group was 1.7 cm, and the average scar length in the BCE group was 4.3 cm. The patients in the BCE group scored higher than the patients in the VAP group on the Manchester Scar Scale (P < .01) and on the Patient and Observer Scar Scales (P = .02), indicating a worse scar outcome. The mean operative time in the VAP group was 41 minutes compared with 115 minutes in the open procedure BCE group. There was no difference between the 2 groups in terms of postoperative complications. CONCLUSIONS: Video-assisted parathyroidectomy is a safe and feasible procedure in the setting of a general otolaryngology-head and neck practice, with outcomes and complication rates that are comparable to those of traditional bilateral neck exploration. Both patient and observer analysis demonstrated that VAP was associated with a more favorable scar outcome when compared with BCE.