Organoid cultures derived from patients with advanced prostate cancer.

The lack of in vitro prostate cancer models that recapitulate the diversity of human prostate cancer has hampered progress in understanding disease pathogenesis and therapy response. Using a 3D organoid system, we report success in long-term culture of prostate cancer from biopsy specimens and circulating tumor cells. The first seven fully characterized organoid lines recapitulate the molecular diversity of prostate cancer subtypes, including TMPRSS2-ERG fusion, SPOP mutation, SPINK1 overexpression, and CHD1 loss. Whole-exome sequencing shows a low mutational burden, consistent with genomics studies, but with mutations in FOXA1 and PIK3R1, as well as in DNA repair and chromatin modifier pathways that have been reported in advanced disease. Loss of p53 and RB tumor suppressor pathway function are the most common feature shared across the organoid lines. The methodology described here should enable the generation of a large repertoire of patient-derived prostate cancer lines amenable to genetic and pharmacologic studies.

Phase 1b study of enzalutamide plus CC-115, a dual mTORC1/2 and DNA-PK inhibitor, in men with metastatic castration-resistant prostate cancer (mCRPC).

BACKGROUND: CC-115, a dual mTORC1/2 and DNA-PK inhibitor, has promising antitumour activity when combined with androgen receptor (AR) inhibition in pre-clinical models. METHODS: Phase 1b multicentre trial evaluating enzalutamide with escalating doses of CC-115 in AR inhibitor-naive mCRPC patients (n = 41). Primary endpoints were safety and RP2D. Secondary endpoints included PSA response, time-to-PSA progression, and radiographic progression. RESULTS: Common adverse effects included rash (31.7% Grades 1-2 (Gr); 31.7% Gr 3), pruritis (43.9% Gr 1-2), diarrhoea (37% Gr 1-2), and hypertension (17% Gr 1-2; 9.8% Gr 3). CC-115 RP2D was 5 mg twice a day. In 40 evaluable patients, 80% achieved ≥50% reduction in PSA (PSA50), and 58% achieved ≥90% reduction in PSA (PSA90) by 12 weeks. Median time-to-PSA progression was 14.7 months and median rPFS was 22.1 months. Stratification by PI3K alterations demonstrated a non-statistically significant trend towards improved PSA50 response (PSA50 of 94% vs. 67%, p = 0.08). Exploratory pre-clinical analysis suggested CC-115 inhibited mTOR pathway strongly, but may be insufficient to inhibit DNA-PK at RP2D. CONCLUSIONS: The combination of enzalutamide and CC-115 was well tolerated. A non-statistically significant trend towards improved PSA response was observed in patients harbouring PI3K pathway alterations, suggesting potential predictive biomarkers of response to a PI3K/AKT/mTOR pathway inhibitor. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02833883.

Primary Retroperitoneal Lymph Node Dissection for Seminoma Metastatic to the Retroperitoneum.

PURPOSE: Primary surgical treatment with retroperitoneal lymph node dissection aims to accurately stage and treat patients with node-positive pure seminoma while avoiding long-term risks of chemotherapy or radiation, traditional standard-of-care treatments. MATERIALS AND METHODS: We reported the pathologic and oncologic outcomes of patients with pure seminoma treated with primary retroperitoneal lymph node dissection in a retrospective, single-institution case series over 10 years. The primary outcome was 2-year recurrence-free survival stratified by adjuvant management strategy (surveillance vs adjuvant chemotherapy). RESULTS: Forty-five patients treated with primary retroperitoneal lymph node dissection for pure testicular seminoma metastatic to the retroperitoneum were identified. Median size of largest lymph node before surgery was 1.8 cm. Viable germ cell tumor, all of which was pure seminoma, was found in 96% (n=43) of patients. The median number of positive nodes and nodes removed was 2 and 54, respectively. Median positive pathologic node size was 2 cm (IQR 1.4-2.5 cm, range 0.1-5 cm). Four of 29 patients managed with postoperative surveillance experienced relapse; 2-year recurrence-free survival was 81%. Median follow-up for those managed with surveillance who did not relapse was 18.5 months. There were no relapses in the retroperitoneum, visceral recurrences, or deaths. Among the 16 patients who received adjuvant treatment, 1 patient experienced relapse in the pelvis at 19 months. CONCLUSIONS: Primary retroperitoneal lymph node dissection for pure seminoma with low-volume metastases to the retroperitoneum is safe and effective, allowing most patients to avoid long-term toxicities from chemotherapy or radiation.

Characterization of a new greenbug resistance gene Gb9 in a synthetic hexaploid wheat.

Greenbug [Schizaphis graminum (Rondani)] is a serious insect pest that not only damages cereal crops, but also transmits several destructive viruses. The emergence of new greenbug biotypes in the field makes it urgent to identify novel greenbug resistance genes in wheat. CWI 76364 (PI 703397), a synthetic hexaploid wheat (SHW) line, exhibits greenbug resistance. Evaluation of an F2:3 population from cross OK 14319 × CWI 76364 indicated that a dominant gene, designated Gb9, conditions greenbug resistance in CWI 76364. Selective genotyping of a subset of F2 plants with contrasting phenotypes by genotyping-by-sequencing identified 25 SNPs closely linked to Gb9 on chromosome arm 7DL. Ten of these SNPs were converted to Kompetitive allele-specific polymerase chain reaction (KASP) markers for genotyping the entire F2 population. Genetic analysis delimited Gb9 to a 0.6-Mb interval flanked by KASP markers located at 599,835,668 bp (Stars-KASP872) and 600,471,081 bp (Stars-KASP881) on 7DL. Gb9 was 0.5 cM distal to Stars-KASP872 and 0.5 cM proximal to Stars-KASP881. Allelism tests indicated that Gb9 is a new greenbug resistance gene which confers resistance to greenbug biotypes C, E, H, I, and TX1. TX1 is one of the most widely virulent biotypes and has overcome most known wheat greenbug resistance genes. The introgression of Gb9 into locally adapted wheat cultivars is of economic importance, and the KASP markers developed in this study can be used to tag Gb9 in cultivar development.

Characterization of Quantitative Trait Loci for Leaf Rust Resistance in the Uzbekistani Wheat Landrace Teremai Bugdai.

Leaf rust, caused by Puccinia triticina, is a major cause of wheat yield losses globally, and novel leaf rust resistance genes are needed to enhance wheat leaf rust resistance. Teremai Bugdai is a landrace from Uzebekistan that is highly resistant to many races of P. triticina in the United States. To unravel leaf rust resistance loci in Teremai Bugdai, a recombinant inbred line (RIL) population of Teremai Bugdai × TAM 110 was evaluated for response to P. triticina race Pt54-1 (TNBGJ) and genotyped using single nucleotide polymorphism (SNP) markers generated by genotyping-by-sequencing (GBS). Quantitative trait loci (QTL) analysis using 5,130 high-quality GBS-SNPs revealed three QTLs, QLr-Stars-2DS, QLr-Stars-6BL, and QLr.Stars-7BL, for leaf rust resistance in two experiments. QLr-Stars-2DS, which is either a new Lr2 allele or a new resistance locus, was delimited to an ∼19.47-Mb interval between 46.4 and 65.9 Mb on 2DS and explained 31.3 and 33.2% of the phenotypic variance in the two experiments. QLr-Stars-6BL was mapped in an ∼84.0-kb interval between 719.48 and 719.56 Mb on 6BL, accounting for 33 to 36.8% of the phenotypic variance in two experiments. QLr.Stars-7BL was placed in a 350-kb interval between 762.41 and 762.76 Mb on 7BL and explained 4.4 to 5.3% of the phenotypic variance. Nine GBS-SNPs flanking these QTLs were converted to kompetitive allele specific PCR (KASP) markers, and these markers can be used to facilitate their introgression into locally adapted wheat lines.

Rapid interrogation of cancer cell of origin through CRISPR editing.

The increasing complexity of different cell types revealed by single-cell analysis of tissues presents challenges in efficiently elucidating their functions. Here we show, using prostate as a model tissue, that primary organoids and freshly isolated epithelial cells can be CRISPR edited ex vivo using Cas9-sgRNA (guide RNA) ribotnucleoprotein complex technology, then orthotopically transferred in vivo into immunocompetent or immunodeficient mice to generate cancer models with phenotypes resembling those seen in traditional genetically engineered mouse models. Large intrachromosomal (∼2 Mb) or multigenic deletions can be engineered efficiently without the need for selection, including in isolated subpopulations to address cell-of-origin questions.

Weighted kernels improve multi-environment genomic prediction.

Crucial to variety improvement programs is the reliable and accurate prediction of genotype's performance across environments. However, due to the impactful presence of genotype by environment (G×E) interaction that dictates how changes in expression and function of genes influence target traits in different environments, prediction performance of genomic selection (GS) using single-environment models often falls short. Furthermore, despite the successes of genome-wide association studies (GWAS), the genetic insights derived from genome-to-phenome mapping have not yet been incorporated in predictive analytics, making GS models that use Gaussian kernel primarily an estimator of genomic similarity, instead of the underlying genetics characteristics of the populations. Here, we developed a GS framework that, in addition to capturing the overall genomic relationship, can capitalize on the signal of genetic associations of the phenotypic variation as well as the genetic characteristics of the populations. The capacity of predicting the performance of populations across environments was demonstrated by an overall gain in predictability up to 31% for the winter wheat DH population. Compared to Gaussian kernels, we showed that our multi-environment weighted kernels could better leverage the significance of genetic associations and yielded a marked improvement of 4-33% in prediction accuracy for half-sib families. Furthermore, the flexibility incorporated in our Bayesian implementation provides the generalizable capacity required for predicting multiple highly genetic heterogeneous populations across environments, allowing reliable GS for genetic improvement programs that have no access to genetically uniform material.

Identification of a Novel Pm65 Allele Conferring a Wide Spectrum of Resistance to Powdery Mildew in Wheat Accession PI 351817.

Powdery mildew is caused by the highly adaptive biotrophic fungus Blumeria graminis f. sp. tritici infecting wheat worldwide. Novel powdery mildew resistance genes are urgently needed that can be used rapidly in wheat cultivar development with minimal disruption of trait advances elsewhere. PI 351817 is a German cultivar exhibiting a wide spectrum of resistance to B. graminis f. sp. tritici isolates collected from different wheat-growing regions of the United States. Evaluation of an F2 population and 237 F2:3 lines derived from OK1059060-2C14 × PI 351817 for responses to B. graminis f. sp. tritici isolate OKS(14)-B-3-1 identified a single dominant gene, designated Pm351817, for powdery mildew resistance in PI 351817. Using bulked segregant analysis (BSA) and simple sequence repeat (SSR) markers, Pm351817 was mapped in the terminal region of the long arm of chromosome 2A. Deep sequencing of the genotyping-by-sequencing libraries of the two parental lines identified a set of single-nucleotide polymorphism (SNP) markers in the 2AL candidate gene region. Those SNP markers was subsequently converted to Kompetitive allele-specific PCR (KASP) markers for genotyping the mapping population. Linkage analysis delimited Pm351817 to a 634-kb interval between Stars-KASP656 (771,207,512 bp) and Stars-KASP662 (771,841,609 bp) on 2AL, based on the Chinese Spring reference sequence IWGSC RefSeq v 2.1. Tests of allelism indicated that Pm351817 is located at the Pm65 locus. Pm351817 shows resistance to all B. graminis f. sp. tritici isolates used in this study and can be used to enhance powdery mildew resistance in the United States. KASP markers flanking Pm351817 can be used to select Pm351817 in wheat breeding programs after further tests for polymorphism.

A semi-dominant NLR allele causes whole-seedling necrosis in wheat.

Programmed cell death (PCD) and apoptosis have key functions in development and disease resistance in diverse organisms; however, the induction of necrosis remains poorly understood. Here, we identified a semi-dominant mutant allele that causes the necrotic death of the entire seedling (DES) of wheat (Triticum aestivum L.) in the absence of any pathogen or external stimulus. Positional cloning of the lethal allele mDES1 revealed that this premature death via necrosis was caused by a point mutation from Asp to Asn at amino acid 441 in a nucleotide-binding leucine-rich repeat protein containing nucleotide-binding domain and leucine-rich repeats. The overexpression of mDES1 triggered necrosis and PCD in transgenic plants. However, transgenic wheat harboring truncated wild-type DES1 proteins produced through gene editing that exhibited no significant developmental defects. The point mutation in mDES1 did not cause changes in this protein in the oligomeric state, but mDES1 failed to interact with replication protein A leading to abnormal mitotic cell division. DES1 is an ortholog of Sr35, which recognizes a Puccinia graminis f. sp. tritici stem rust disease effector in wheat, but mDES1 gained function as a direct inducer of plant death. These findings shed light on the intersection of necrosis, apoptosis, and autoimmunity in plants.

Identification and characterization of the novel leaf rust resistance gene Lr81 in wheat.

KEY MESSAGE: The novel, leaf rust seedling resistance gene, Lr81, was identified in a Croatian breeding line and mapped to a genomic region of less than 100 Kb on chromosome 2AS. Leaf rust, caused by Puccinia triticina, is the most common and widespread rust disease in wheat. Races of Puccinia triticina evolve rapidly in the southern Great Plains of the USA, and leaf rust resistance genes often lose effectiveness shortly after deployment in wheat production. PI 470121, a wheat breeding line developed by the University of Zagreb in Croatia, showed high resistance to Puccinia triticina races collected from Oklahoma, suggesting that PI 470121 could be a leaf rust resistance source for the southern Great Plains of the USA. Genetic analysis based on an F2 population and F2:3 families derived from the cross PI 470121 × Stardust indicated that PI 470121 carries a dominant seedling resistance gene, designated as Lr81. Linkage mapping delimited Lr81 to a genomic region of 96,148 bp flanked by newly developed KASP markers Xstars-KASP320 and Xstars-KASP323 on the short arm of chromosome 2A, spanning 67,030,206-67,132,354 bp in the Chinese Spring reference assembly (IWGSC RefSeq v1.0). Deletion bin mapping assigned Lr81 to the terminal bin 2AS-0.78-1.00. Allelism tests indicated that Lr81 is a distinctive leaf rust resistance locus with the physical order Lr65-Lr17-Lr81. Marker-assisted selection based on a set of markers closely linked to leaf rust resistance genes in PI 470121 and Stardust enabled identification of a recombinant inbred line RIL92 carrying Lr81 only. Lr81 is a valuable leaf rust resistance source that can be rapidly introgressed into locally adapted cultivars using KASP markers Xstars-KASP320 and Xstars-KASP323.

Four Cycles of Etoposide plus Cisplatin for Patients with Good-Risk Advanced Germ Cell Tumors.

BACKGROUND: The National Comprehensive Cancer Network recommends either three cycles of bleomycin, etoposide, and cisplatin or four cycles of etoposide and cisplatin (EPx4) as initial chemotherapy for the treatment of good-risk germ cell tumors (GCTs). To assess the response, toxicity, and survival outcomes of EPx4, we analyzed our experience. MATERIAL AND METHODS: Response and survival outcomes, selected toxicities, and adherence to chemotherapy dose and schedule were assessed in patients with good-risk GCT who received EPx4 at Memorial Sloan Kettering Cancer Center between 1982 and 2016. The results were compared with our past results and published data. RESULTS: Between 1982 and 2016, 944 patients with GCT were treated with EPx4, 289 who were previously reported plus 655 treated between January 2000 and August 2016. A favorable response was achieved in 928 of 944 patients (98.3%). Five-year progression-free, disease-specific, and overall survival rates were 93.9%, 98.6%, and 97.9%, respectively. Median follow-up was 7.3 years (range, 2.8 months to 35.5 years). Viable, nonteratomatous malignant GCT was present in 3.5% of 432 postchemotherapy retroperitoneal lymph node dissection specimens from patients with nonseminomatous GCT. Febrile neutropenia and thromboembolic events occurred in 16.0% and 8.9%, respectively, with one treatment-related death. In the more recent 655-patient cohort, full-dose EPx4 was administered to 631 (96.3%), with deviations from planned treatment driven mainly by vascular (n = 13), hematologic (n = 11), renal (n = 7), or infectious (n = 5) events. CONCLUSION: EPx4 is highly effective and well tolerated in patients with good-risk GCTs and remains a standard of care. IMPLICATIONS FOR PRACTICE: Four cycles of etoposide and cisplatin (EPx4) is a standard-of-care regimen for all patients with good-risk germ cell tumors with a favorable response rate and disease-specific survival of 98%. Full-dose administration of etoposide and cisplatin and complete resection of residual disease lead to optimal outcomes. EPx4 should be the recommended regimen in active smokers, patients with reduced or borderline kidney function, and patients aged 50 years or older, which are patient groups at increased risk for bleomycin pulmonary toxicity. Because of a risk of acquired severe pulmonary illness, EPx4 may also be favored for patients who vape or use e-cigarettes and during ongoing transmission of severe acute respiratory syndrome coronavirus 2.

Characterization of wheat curl mite resistance gene Cmc4 in OK05312.

KEY MESSAGE: Cmc4, a wheat curl mite resistance gene, was delimited to a 523 kb region and a diagnostic marker haplotype was identified for selecting Cmc4 in breeding programs. Wheat curl mite (WCM, Aceria tosichella Keifer) is a disastrous wheat pest in many wheat-growing regions worldwide. WCM not only directly affects wheat yield, but also transmits wheat streak mosaic virus. Growing WCM resistant cultivars is the most economical and sustainable method to reduce its damage. A hard winter wheat breeding line OK05312 (PI 670019) carries Cmc4 gene resistance to A. tosichella and has many desirable agronomic traits. To finely map Cmc4 in OK05312, two recombinant inbred line populations were developed from crosses between OK05312 and two susceptible cultivars, SD06165 and Jerry, genotyped using single nucleotide polymorphism (SNP) markers generated from genotyping-by-sequencing (GBS), and phenotyped for WCM resistance. Gene mapping using the two SNP maps confirmed Cmc4 in OK05312 that explained up to 68% of the phenotypic variation. Further analysis delimited Cmc4 to a ~ 523 kb region between SNPs SDOKSNP6314 and SDOKSNP2805 based on the Ae. tauschii reference genome. We developed 18 polymorphic Kompetitive Allele Specific PCR (KASP) markers using the sequences of GBS-SNPs in this region and 23 additional KASP markers based on the SNPs between the parents derived from 90K SNP chips. The KASP markers SDOKSNP6314 and SDOKSNP9699 are closest to Cmc4 and can be used to diagnose the presence of Cmc4 in wheat breeding programs. Haplotype analysis suggested that CmcTAM112 in TAM112 might be the same gene as Cmc4.

Characterization of an Incomplete Leaf Rust Resistance Gene on Chromosome 1RS and Development of KASP Markers for Lr47 in Wheat.

Leaf rust, caused by Puccinia triticina, is one of the most common wheat (Triticum aestivum) diseases in the Great Plains of the United States. A population of recombinant inbred lines from CI 17884 × 'Bainong 418' was evaluated for responses to leaf rust race Pt52-2 and genotyped using single nucleotide polymorphism (SNP) markers. Quantitative trait locus analysis identified a minor gene for resistance to leaf rust, designated QLr.stars-1RS, on the 1BL.1RS translocation segment in 'Bainong 418', and another leaf rust resistance gene, Lr47, on chromosome 7A of CI 17884. Lr47, originally identified in CI 17884 and located in a wheat-T. speltoides translocation segment 7S#1S, remains one of only a few race-specific resistance genes still effective in the Great Plains. A set of 7A-specific simple sequence repeat markers were developed and used to genotype CI 17884 and a pair of near-isogenic lines differing in the presence or absence of 7S#1S, PI 603918, and 'Pavon F76'. Haplotype analysis indicated that the estimated length of 7S#1S was 157.23 to 174.42 Megabases, accounting for ∼23% of the 7A chromosome. Two SNPs on 7S#1S and four SNPs on the 1RS chromosome arm were converted to Kompetitive allele-specific PCR (KASP) markers, which were subsequently validated in a panel of cultivars and elite breeding lines released within the last decade. Of these, one- and two-KASP markers are specific to the 1RS chromosome arm and 7S#1S, respectively, indicating that they can facilitate the introgression of Lr47 and QLr.stars-1RS into locally adapted wheat cultivars and breeding lines.

Gb8, a new gene conferring resistance to economically important greenbug biotypes in wheat.

KEY MESSAGE: A new greenbug resistance gene Gb8 conferring broad resistance to US greenbug biotypes was identified in hard red winter wheat line PI 595379-1 and was mapped to the terminal region of chromosome 7DL. Greenbug [Schizaphis graminum (Rondani)] is a worldwide insect pest that poses a serious threat to wheat production. New greenbug resistance genes that can be readily used in wheat breeding are urgently needed. The objective of this study was to characterize a greenbug resistance gene in PI 595379-1, a single plant selection from PI 595379. Genetic analysis of response to greenbug biotype E in an F2:3 population derived from a cross between PI 595379-1 and PI 243735 indicated that a single gene, designated Gb8, conditioned resistance. Linkage analysis placed Gb8 in a 2.7-Mb interval in the terminal bin of chromosome 7DL (7DL3-082-1.0), spanning 595.6 to 598.3 Mb in the Chinese Spring IWGSC RefSeq version 1.0 reference sequence. Gb8 co-segregated with a newly developed SSR marker Xstars508, positioned at 596.4 Mb in the reference sequence. Allelism tests showed that Gb8 was different from three permanently named genes on the same chromosome arm and the estimated genetic distance between Gb8 and Gb3 was 15.35 ± 1.35 cM. Gb8 can be directly used in wheat breeding to enhance greenbug resistance.

Development and deployment of KASP markers for multiple alleles of Lr34 in wheat.

KEY MESSAGE: Heterogeneous Lr34 genes for leaf rust in winter wheat cultivar 'Duster' and KASP markers for allelic variation in exon 11 and exon 22 of Lr34. Wheat, Triticum aestivum (2n = 6x = 42, AABBDD), is a hexaploid species, and each of three homoeologous genomes A, B, and D should have one copy for a gene in its ancestral form if the gene has no duplication. Previously reported leaf rust resistance gene Lr34 has one copy on the short arm of chromosome 7D in hexaploid wheat, and allelic variation in Lr34 is in intron 4, exon 11, exon 12, or exon 22. In this study, we discovered that Oklahoma hard red winter wheat cultivar 'Duster' (PI 644,016) has two copies of the Lr34 gene, the resistance allele Lr34a and the susceptibility allele Lr34b. Both Lr34a and Lr34b were mapped in the same linkage group on chromosome 7D in a doubled-haploid population generated from a cross between Duster and a winter wheat cultivar 'Billings' which carries the susceptibility allele Lr34c. A chromosomal fragment including Lr34 and at least two neighboring genes on its proximal side but excluding genes on its distal side was duplicated in Duster. The Duster Lr34ab allele was associated with tip necrosis and increased resistance against leaf rust at adult plants in the Duster × Billings DH population tested in the field, demonstrating the function of the Duster Lr34ab allele in wheat. We have developed KASP markers for allelic variation in exon 11 and exon 22 of Lr34 in wheat. These markers can be utilized to accelerate the selection of Lr34 in wheat.

Characterization of Pm63, a powdery mildew resistance gene in Iranian landrace PI 628024.

KEY MESSAGE: A new powdery mildew resistance gene conferring a wide spectrum of resistance to Bgt isolates in the USA, Pm63 , was identified in Iranian wheat landrace PI 628024 and mapped to the terminal region of the long arm of chromosome 2B. Powdery mildew is a globally important wheat disease causing severe yield losses, and host resistance is the preferred strategy for managing this disease. The objective of this study was to characterize a powdery mildew resistance gene in Iranian landrace PI 628024, which exhibited a wide spectrum of resistance to representative Blumeria graminis f. sp. tritici (Bgt) isolates collected from different regions of the USA. An F2 population and F2:3 lines derived from the cross PI 628024 × CItr 11349 were used in this study, and genetic analysis indicated that a single dominant gene, designated Pm63, conferred resistance to Bgt isolate OKS(14)-B-3-1. Linkage analysis located Pm63 to an interval of about 13.1 Mb on the long arm of chromosome 2B, spanning 710.3-723.4 Mb in the Chinese Spring reference sequence. Bin mapping assigned Pm63 to the terminal bin 2BL6-0.89-1.0, 1.1 cM proximal to STS marker Xbcd135-2 and 0.6 cM distal to SSR marker Xstars419. Allelism tests indicated that Pm63 is a new powdery mildew resistance gene, which differs from other genes in the terminal bin by origin, genomic location, and responses to a set of 16 representative US Bgt isolates. Pm63 can be widely used to enhance powdery mildew resistance in the Great Plains, western, and southeastern regions of the USA.

Characterization of Pm65, a new powdery mildew resistance gene on chromosome 2AL of a facultative wheat cultivar.

KEY MESSAGE: A new powdery mildew resistance gene that can be readily used in wheat breeding, Pm65, was identified in the facultative wheat cultivar Xinmai 208 and mapped to the terminal region of chromosome 2AL. Wheat powdery mildew, a widely occurring disease caused by the biotrophic fungus Blumeriagraminis f. sp. tritici (Bgt), poses a serious threat to wheat production. A high breeding priority is to identify powdery mildew resistance genes that can be readily used either alone or in gene complexes involving other disease resistance genes. An F2 population and 227 F2:3 families derived from the cross Xinmai 208 × Stardust were generated to map a powdery mildew resistance gene in Xinmai 208, a high-yielding Chinese wheat cultivar. Genetic analysis indicated that Xinmai 208 carries a single dominant powdery mildew resistance gene, designated herein Pm65, and linkage analysis delimited Pm65 to a 0.5 cM interval covering 531.8 Kb (763,289,667-763,821,463 bp) on chromosome 2AL in the Chinese Spring reference sequence. An allelism test indicated that Pm65 is a new gene about 10.3 cM distal to the Pm4 locus. Pm65 was 0.3 cM proximal to Xstars355 and 0.2 cM distal to Xstars356. It conferred near-immunity to 19 of 20 Bgt isolates collected from different wheat-growing regions of the USA. Coming from a high-yield potential cultivar, Pm65 can be directly used to enhance powdery mildew resistance in wheat. The newly developed SSR markers Xstars355 and Xstars356 have the potential to tag Pm65 for wheat improvement.

The STENOFOLIA gene from Medicago alters leaf width, flowering time and chlorophyll content in transgenic wheat.

Molecular genetic analyses revealed that the WUSCHEL-related homeobox (WOX) gene superfamily regulates several programs in plant development. Many different mechanisms are reported to underlie these alterations. The WOX family member STENOFOLIA (STF) is involved in leaf expansion in the eudicot Medicago truncutula. Here, we report that when this gene was ectopically expressed in a locally adapted hard red winter wheat cultivar (Triticum aestivum), the transgenic plants showed not only widened leaves but also accelerated flowering and increased chlorophyll content. These desirable traits were stably inherited in the progeny plants. STF binds to wheat genes that have the (GA)n /(CT)n DNA cis element, regardless of sequences flanking the DNA repeats, suggesting a mechanism for its pleiotropic effects. However, the amino acids between position 91 and 262 in the STF protein that were found to bind with the (GA)n motif have no conserved domain with any other GAGA-binding proteins in animals or plants. We also found that STF interacted with a variety of proteins in wheat in yeast 2 hybrid assays. We conclude that the eudicot STF gene binds to (GA)n /(CT)n DNA elements and can be used to regulate leaf width, flowering time and chlorophyll content in monocot wheat.

Nitrogen use efficiency is regulated by interacting proteins relevant to development in wheat.

Wheat (Triticum aestivum) has low nitrogen use efficiency (NUE). The genetic mechanisms controlling NUE are unknown. Positional cloning of a major quantitative trait locus for N-related agronomic traits showed that the vernalization gene TaVRN-A1 was tightly linked with TaNUE1, the gene shown to influence NUE in wheat. Because of an Ala180 /Val180 substitution, TaVRN-A1a and TaVRN-A1b proteins interact differentially with TaANR1, a protein encoded by a wheat orthologue of Arabidopsis nitrate regulated 1 (ANR1). The transcripts of both TaVRN-A1 and TaANR1 were down-regulated by nitrogen. TaANR1 was functionally characterized in TaANR1::RNAi transgenic wheat, and in a natural mutant with a 23-bp deletion including 10-bp at the 5' end of intron 5 and 13-bp of exon 6 in gDNA sequence in its gDNA sequence, which produced transcript that lacked the full 84-bp exon 6. Both TaANR1 and TaHOX1 bound to the Ala180 /Val180 position of TaVRN-A1. Genetically incorporating favourable alleles from TaVRN-A1, TaANR1 and TaHOX1 increased grain yield from 9.84% to 11.58% in the field. Molecular markers for allelic variation of the genes that regulate nitrogen can be used in breeding programmes aimed at improving NUE and yield in novel wheat cultivars.

A phase II study of the dual mTOR inhibitor MLN0128 in patients with metastatic castration resistant prostate cancer.

Background MLN0128 is a first-in-class, dual mTOR inhibitor with potential to outperform standard rapalogs through inhibition of TORC1 and TORC2. This phase II study was designed to assess antitumor activity of MLN0128 in metastatic castration-resistant prostate cancer (mCRPC). Methods Eligible patients had mCRPC previously treated with abiraterone acetate and/or enzalutamide. Five patients started MLN0128 at 5 mg once daily, subsequently dose reduced to 4 mg because of toxicity. Four subsequent patients started MLN0128 at 4 mg daily. Primary endpoint was progression-free survival at 6 months. Results Nine patients were enrolled and median time on treatment was 11 weeks (range: 3-30). Best response was stable disease. All patients had a rise in PSA on treatment, with a median 159% increase from baseline (range: 12-620%). Median baseline circulating tumor cell count was 1 cell/mL (range: 0-40); none had a decrease in cell count posttreatment. Grade ≤ 2 adverse events included fatigue, anorexia, and rash. The most common serious adverse events were grade 3 dyspnea and maculopapular rash. Eight patients discontinued treatment early because of radiographic progression (n = 1), grade 3 toxicity (n = 5), or investigator discretion (n = 2). Four patients had immediate PSA decline following drug discontinuation, suggesting MLN0128 could cause compensatory increase of androgen receptor (AR) activity. Correlative studies of pretreatment and posttreatment biopsy specimens revealed limited inhibition of AKT phosphorylation, 4EBP1 phosphorylation, and eIF4E activity. Conclusions Clinical efficacy of MLN0128 in mCRPC was limited likely due to dose reductions secondary to toxicity, PSA kinetics suggesting AR activation resulting from mTOR inhibition, and poor inhibition of mTOR signaling targets.