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Osimertinib is an EGFR tyrosine kinase inhibitor (TKI) with proven clinical efficacy; however, acquired resistance presents an obstacle to curing EGFR-driven disease. Recent studies have shown that drug-tolerant persister cells (DTP) have a distinct transcriptional profile that may confer specific vulnerabilities. By definition these cells avoid apoptosis, yet little is known about how their survival is regulated. We found that paradoxically, the proapoptotic gene BIM was upregulated in osimertinib DTPs, and cotreatment with BH3 mimetics could trigger DTP cell death. Furthermore, cIAP proteins, antiapoptotic members of the extrinsic pathway, were significantly elevated in DTPs. cIAP antagonists could block DTP formation as an up-front combination, and could eliminate preformed DTPs. Critically, when treated at the time of maximal osimertinib response, cIAP or MCL1 inhibitor treatment could significantly attenuate the regrowth of EGFRm cell line mouse xenografts. Finally, we show that apoptosis can be maximized in cell lines with acquired osimertinib resistance by combining BH3 or SMAC mimetics with agents that target the resistance driver in these models. Taken together, these data suggest novel therapeutic strategies at the point of minimal residual disease or full osimertinib resistance for patients in this critical area of unmet need. Significance: These studies uncover strategies to use targeted agents that activate apoptosis in non–small cell lung cancer cells that survive initial EGFR TKI treatment.

Osimertinib is an EGFR tyrosine kinase inhibitor (TKI) with proven clinical efficacy; however, acquired resistance presents an obstacle to curing EGFR-driven disease. Recent studies have shown that drug-tolerant persister cells (DTP) have a distinct transcriptional profile that may confer specific vulnerabilities. By definition these cells avoid apoptosis, yet little is known about how their survival is regulated. We found that paradoxically, the proapoptotic gene BIM was upregulated in osimertinib DTPs, and cotreatment with BH3 mimetics could trigger DTP cell death. Furthermore, cIAP proteins, antiapoptotic members of the extrinsic pathway, were significantly elevated in DTPs. cIAP antagonists could block DTP formation as an up-front combination, and could eliminate preformed DTPs. Critically, when treated at the time of maximal osimertinib response, cIAP or MCL1 inhibitor treatment could significantly attenuate the regrowth of EGFRm cell line mouse xenografts. Finally, we show that apoptosis can be maximized in cell lines with acquired osimertinib resistance by combining BH3 or SMAC mimetics with agents that target the resistance driver in these models. Taken together, these data suggest novel therapeutic strategies at the point of minimal residual disease or full osimertinib resistance for patients in this critical area of unmet need.Significance:These studies uncover strategies to use targeted agents that activate apoptosis in non–small cell lung cancer cells that survive initial EGFR TKI treatment.

Supplementary Table 1. Cell lines used. Supplementary Table 2. Antibodies used. Supplementary Figure S1. Osimertinib drug tolerant cells re-aquire apoptotic capacity after drug holiday. Supplementary Figure S2. BH3 mimetics can trigger apoptosis in established DTPs. Supplementary Figure S3. DTPs upregulate cIAP proteins and are sensitive to SMAC mimetic treatment. Supplementary Figure S4. SMAC mimetics inhibit DTP growth in vivo. Supplementary Figure S5. BH3 and SMAC mimetics enhance apoptosis induced by agents targeting resistance drivers. Supplementary Figure S6. Measuring transcript levels for anti-apoptotic genes in osimertinib DTPs.

Resistance to EGFR inhibitors (EGFRi) presents a major obstacle in treating non-small cell lung cancer (NSCLC). One of the most exciting new ways to find potential resistance markers involves running functional genetic screens, such as CRISPR, followed by manual triage of significantly enriched genes. This triage process to identify ‘high value’ hits resulting from the CRISPR screen involves manual curation that requires specialized knowledge and can take even experts several months to comprehensively complete. To find key drivers of resistance faster we build a recommendation system on top of a heterogeneous biomedical knowledge graph integrating pre-clinical, clinical, and literature evidence. The recommender system ranks genes based on trade-offs between diverse types of evidence linking them to potential mechanisms of EGFRi resistance. This unbiased approach identifies 57 resistance markers from >3,000 genes, reducing hit identification time from months to minutes. In addition to reproducing known resistance markers, our method identifies previously unexplored resistance mechanisms that we prospectively validate.

Resistance to EGFR inhibitors (EGFRi) presents a major obstacle in treating non-small cell lung cancer (NSCLC). One of the most exciting new ways to find potential resistance markers involves running functional genetic screens, such as CRISPR, followed by manual triage of significantly enriched genes. This triage process to identify ‘high value’ hits resulting from the CRISPR screen involves significant manual curation that requires specialized knowledge and can take even experts several months to comprehensively complete.To find key drivers of resistance faster we built a hybrid recommendation system on top of a heterogeneous biomedical knowledge graph integrating preclinical, clinical, and literature evidence. Genes were ranked based on trade-offs between diverse types of evidence linking them to potential mechanisms of EGFRi resistance. This unbiased approach identified 36 resistance markers from >3,000 genes, reducing hit identification time from months to minutes. In addition to reproducing known resistance markers, our method identified novel resistance mechanisms that we prospectively validated.

Acquired resistance to tyrosine kinase inhibitors (TKIs) of epidermal growth factor receptor (EGFR) remains a clinical challenge. Especially challenging are cases in which resistance emerges through EGFR-independent mechanisms, such as through pathways that promote epithelial-to-mesenchymal transition (EMT). Through an integrated transcriptomic, proteomic, and drug screening approach, we identified activation of the yes-associated protein (YAP) and forkhead box protein M1 (FOXM1) axis as a driver of EMT-associated EGFR TKI resistance. EGFR inhibitor resistance was associated with broad multidrug resistance that extended across multiple chemotherapeutic and targeted agents, consistent with the difficulty of effectively treating resistant disease. EGFR TKI resistant cells displayed increased abundance of spindle assembly checkpoint (SAC) proteins, including polo-like kinase 1 (PLK1), Aurora kinases, survivin, and kinesin spindle protein (KSP). Moreover, EGFR TKI resistant cells exhibited vulnerability to SAC inhibitors. Increased activation of the YAP/FOXM1 axis mediated an increase in the abundance of SAC components in resistant cells. The clinical relevance of these finding was indicated by evaluation of specimens from patients with EGFR mutant lung cancer, which showed that high FOXM1 expression correlated with expression of genes encoding SAC proteins and was associated with a worse clinical outcome. These data revealed the YAP/FOXM1 axis as a central regulator of EMT-associated EGFR TKI resistance and that this pathway, along with SAC components, are therapeutic vulnerabilities for targeting this multidrug resistant phenotype.

10-20% of lung adenocarcinoma patients harbour activating mutations in EGFR. Although treatment with the EGFR kinase inhibitor osimertinib has improved overall survival in such patients, almost all patients ultimately develop drug resistance. In many cases the molecular resistance mechanisms remain unknown. A systematic identification of the involved genes and pathways is critical to overcome osimertinib resistance. To define the resistance landscape of EGFR kinase inhibition we performed genome-wide gain and loss of function CRISPR screens in EGFR mutant lung cancer cell lines treated with osimertinib. Resistance hits were enriched for genes in previously identified resistance pathways including PI3K (PTEN, TSC2), MAPK (NF1, MET), cell death (BCL2L11, BAX), the mediator complex (MED24, MED19) and ubiquitination (KCTD5, LZTR1). A secondary screen of 63 resistance genes that combined high content microscopy with CRISPR gene knockouts demonstrated that 21% (13/63) of genes were associated with increased nuclear localisation of YAP1/WWTR1, indicating transcriptional activation of the Hippo pathway. According to our screening data, many resistance hits mapped onto the Hippo signaling axis - upstream regulators (NF2, AMOTL2), core signaling genes (LATS1, LATS2), main effectors (WWTR1, YAP1), transcriptional co-effectors (TEAD3, FOSL1, VGLL4) and the SWI/SNF complex (ARID2, SMARCA4, SMARCB1, PBRM1). Hippo signaling is mediated through YAP1 and WWTR1 which bind to TEAD transcription factors and activate transcriptional programs affecting cell proliferation and apoptosis. We confirmed using CRISPR that knockout (NF2) or overexpression (YAP1, WWTR1) of key Hippo genes in the EGFR mutant lung cancer cell lines PC-9, HCC827 and HCC4006 resulted in up to 60-fold increased resistance to osimertinib and elevated activity of a TEAD reporter system, indicating activation of Hippo transcriptional programs. To maintain osimertinib resistance, expression of both YAP1 and WWTR1 was necessary, suggesting non-redundant roles for both Hippo main effectors in mediating osimertinib resistance. The combination of osimertinib and a TEAD inhibitor (MYF-01-37) reversed the resistance phenotype in NF2 KO cell line models in long-term proliferation assays and also substantially repressed the emergence of drug-tolerant persister cells in PC-9, HCC827 and HCC4006 cell lines following osimertinib treatment. These cells exhibited an enhanced apoptotic response when treated with combination of osimertinib and TEAD inhibitor. Consequently, we propose Hippo signaling as an important target mechanism for the prevention of resistance to osimertinib. Citation Format: Matthias Pfeifer, Jonathan S. Brammeld, Stacey Price, Matthew Martin, Hannah Thorpe, Aurelie Bornot, Erica Banks, Nin Guan, Shanade Dunn, Maria Luisa Guerriero, Daniel O'Neill, James Pilling, Davide Gianni, James Brownell, Paul Smith, Ultan McDermott. Gain and loss of function genome-wide CRISPR screens identify Hippo signaling as an important driver of resistance in EGFR mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1100.

BRAF V600E mutations occur in a subset of colon cancers. These are typically resistant to chemotherapy and are associated with a poor outcome. Combination treatment with BRAF and EGFR inhibitors is superior to standard chemotherapy and has recently received FDA approval, however the early emergence of drug resistance is a significant clinical problem. Clinical studies of resistant patients have identified mutations and amplifications in the MAPK pathway as important resistance drivers. To identify novel non-MAPK dependent resistance mechanisms, genome-wide CRISPR/Cas9 knockout screens were performed to identify genes causing resistance to a combination BRAF/MEK/EGFR inhibitor regimen in the BRAF mutant HT29 and LS411N colon cancer cell lines. A number of strong resistance hits were identified but importantly, only 3 genes (CSK, ARID1A and STK11) were detected as significantly enriched in both cell lines screened. Two of these, CSK and ARID1A, have been shown to play a role in activation of the Hippo signaling pathway. The generation of CSK knockout colon cell lines confirmed resistance to BRAF/MEK/EGFR inhibition both in vitro and in vivo using xenograft models. Furthermore, since CSK is a negative regulator of SRC, re-sensitisation of resistant BRAF mutant CSK knockout cells was achieved by adding Src inhibitors (Dasatinib and Saracatinib) to the combination therapy. Nuclear localisation of the transcription factors YAP1 and WWTR1 and binding to TEAD family members are required for Hippo pathway activation and we confirmed significantly increased nuclear YAP1/WWTR1 in CSK knockout cells. Furthermore, YAP1/WWTR1 nuclear localisation in these cells was reversed by treatment with Src inhibitors. Novel pharmacological TEAD inhibitors have recently been developed and will also be used to confirm that resistance can be overcome by specifically targeting the Hippo pathway. Expression profiling of CSK knockout cells revealed significant enrichment of pathways associated with Hippo signaling. Genes involved in regulating Hippo pathway activation were also identified as CRISPR screen resistance hits in both lung and head and neck cancer cell lines, suggesting that this may be an important mechanism of resistance among other tumour types and not limited to the colon. Here we show that activation of the Hippo pathway is a potential MAPK-independent resistance mechanism in BRAF mutant colon cancer, readily reversible by rational pharmacological targeting. Given the development of specific Hippo pathway inhibitors and plans for their use in clinical trials, activation of Hippo signaling should be considered in resistant BRAF mutant colon cancer where alterations in the MAPK pathway are not detected. Citation Format: Jonathan S. Brammeld, Hannah Thorpe, Mar Arias Garcia, Stacey Price, Jamie Young, Matthias Pfeifer, Barbara Lupo, Kosuke Yusa, Livio Trusolino, Mathew Garnett, Andrea Bertotti, Chris Bakal, Ultan McDermott. Genome-wide CRISPR screens reveal Hippo pathway activation as a resistance mechanism in BRAF mutant colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1081.

Der in der zweiten Halfte der 1960er Jahre errichtete „Palast der Kultur” war mit seinen Raumen fur kunstlerische Betatigung sowie seinem grosen Veranstaltungssaal in der Baugeschichte der Stadt Dresden und der ehemaligen DDR ein herausragendes und einzigartiges Gebaude. Nachdem vor allem brandschutztechnische Mangel einen Weiterbetrieb in der bisherigen Form nicht mehr zuliesen, hat sich die Stadt Dresden nach mehreren Studien dazu entschlossen, den Kulturpalast einer neuen multifunktionalen Nutzung zuzufuhren. Das fur diese herausfordernde Baumasnahme mit der Tragwerksplanung beauftragte Ingenieurburo ProfessorPfeiferundPartner untersuchte zuerst das vor rund 50 Jahren errichtete mehrgeschossige Bestandsgebaude umfassend auf Standsicherheit und Durchfuhrbarkeit der geplanten, trotz Denkmalschutz durchaus erheblichen Eingriffe in die Bestandsstruktur zum Zwecke von Umgestaltungen und Nutzungsanderungen. Die weiteren Aufgaben umfassten neben der „behutsamen” Ertuchtigung verbleibender Geschossdecken die Einfugung eines neuen, modernen Konzertsaals fur hochste akustische Anspruche, was umfangreiche Abbruchmasnahmen, schwierige Bauzustande und die Einfugung komplett neuer, weit gespannter Tragwerksteile mit sich brachte.

Die Senckenberg Gesellschaft fur Naturforschung, Trager des renommierten Museums und des nicht minder bedeutenden wissenschaftlichen Instituts, erweitert bis Ende 2018 in Frankfurt/Main ihre Raumlichkeiten fur Forschung, Sammlung, Veranstaltungen und Verwaltung um ca. 19000 m2. Dazu wurden drei historische Gebaude des “Senckenbergareals” nach den Planen des Buros Kulka-Architekten, Koln und Dresden, zusammengefuhrt und so umgestaltet, dass das Ergebnis den Anforderungen eines modernen Forschungsbetriebs sowie zugleich den Belangen des Denkmalschutzes Rechnung tragt. Das fur diese herausfordernde Baumasnahme mit der Tragwerksplanung beauftragte Ingenieurburo ProfessorPfeiferundPartner untersuchte zuerst die vor mehr als 100 Jahren errichteten, mehrgeschossigen Bestandsgebaude umfassend auf Standsicherheit und Durchfuhrbarkeit der geplanten Umgestaltungen und Nutzungsanderungen. Die weiteren Planungsleistungen umfassten neben der “behutsamen” Ertuchtigung einzelner Geschossdecken unterschiedlicher Bauart und dem Einbau neuer Decken zur Herstellung der erforderlichen Barrierefreiheit sowie Schaffung zusatzlicher Flachen gemas den Nutzervorgaben die Erganzung eines Gebaudeteils mit Uberbauung sowie die Zusammenfuhrung der Gebaude durch reprasentative Verbindungsbrucken. Zusatzlich waren komplexe Bauzustande und Abbruchmasnahmen und die Ertuchtigungen hoher belasteter Fundamente durch HD-Injektionen zu beplanen. Redesign of the listed Senckenbergareal in Frankfurt/Main. Using existing qualities and appreciation of historic structures The Senckenberg Gesellschaft fur Naturforschung (Association for Research in Nature), sponsor of the renowned Senckenberg Museum and equally prestigious Senckenberg Institute, aims to increase its facilities for research, collection and administration in Frankfurt/Main by 19,000m2 by the end of 2018. Kulka-Architekten, based in Cologne and Dresden, reconfigured and merged three historic buildings to satisfy the demands of a modern research facility while taking account of the historic building preservation act. The international consultancy ProfessorPfeiferundPartner, which was entrusted with the structural engineering of this challenging project, in a first step assessed the as-built situation of the more than 100 years' old multi-storey buildings for structural stability with a view to establish the feasibility of the envisaged redevelopment. Further design steps included the “cautious” strengthening of floor slabs of various type, the addition of an annex as well as the joining of buildings by means of imposing connection-bridges. Complex demolition and construction sequence analysis, strengthening of foundations by means of HDI injection as well as the restoration of parts of the sandstone facade elements had to be considered as well.

Here we report the draft genome sequence of perennial ryegrass (Lolium perenne), an economically important forage and turf grass species widely cultivated in temperate regions worldwide. It is classified along with wheat, barley, oats and Brachypodium distachyon in the Pooideae sub-family of the grass family (Poaceae). Transcriptome data was used to identify 28,455 gene models, and we utilize macro-co-linearity between perennial ryegrass and barley, and synteny within the grass family to establish a synteny-based linear gene order. The gametophytic self-incompatibility (SI) mechanism enables the pistil of a plant to reject self-pollen and therefore promote outcrossing. We have used the sequence assembly to characterise transcriptional changes in the stigma during pollination with both compatible and incompatible pollen. Characterisation of the pollen transcriptome identified homologs to pollen allergens from a range of species, many of which were expressed to very high levels in mature pollen grains, and potentially involved in the SI mechanism. The genome sequence provides a valuable resource for future breeding efforts based on genomic prediction, and will accelerate the development of varieties for more productive grasslands. Here we report the draft genome sequence of perennial ryegrass (Lolium perenne), an economically important forage and turf grass species that is widely cultivated in temperate regions worldwide. It is classified along with wheat, barley, oats and Brachypodium distachyon in the Pooideae sub-family of the grass family (Poaceae). Transcriptome data was used to identify 28 455 gene models, and we utilized macro-co-linearity between perennial ryegrass and barley, and synteny within the grass family, to establish a synteny-based linear gene order. The gametophytic self-incompatibility mechanism enables the pistil of a plant to reject self-pollen and therefore promote out-crossing. We have used the sequence assembly to characterize transcriptional changes in the stigma during pollination with both compatible and incompatible pollen. Characterization of the pollen transcriptome identified homologs to pollen allergens from a range of species, many of which were expressed to very high levels in mature pollen grains, and are potentially involved in the self-incompatibility mechanism. The genome sequence provides a valuable resource for future breeding efforts based on genomic prediction, and will accelerate the development of new varieties for more productive grasslands.

Viele Jahre leer und ungenutzt fristete das ehemalige Bergheimer Bad in Heidelberg ein unwurdiges Dasein. Niemand war bereit, sich des Bauwerks anzunehmen oder hatte eine gute Idee, wie es sinnvoll weiter genutzt werden konnte. Oft besteht das Hauptproblem historischer Gebaude darin, dass keine Nutzung gefunden wird, die einen finanziellen Anreiz fur Investoren schafft. Glucklicherweise kam es bei diesem Bauwerk dann letztlich doch anders und es fand sich ein der Stadt Heidelberg sehr verbundener Geschaftsmann, der die Vision hatte, man konnte doch etwas aus dem Bauwerk machen, wodurch einerseits das kulturelle Erbe erhalten bliebe, gleichzeitig aber auch ein profitables Geschaft entstehen konnte. Dieser hatte als einziger auf eine europaweite Ausschreibung der Stadt Heidelberg hin ein Angebot abgegeben und erhielt im Oktober 2008 den Zuschlag. Zusammen mit dem namhaften Heidelberger Architekturburo SSV-Architekten und dem im Umgang mit historischer Bausubstanz erfahrenen Ingenieurburo fur Tragwerksplanung ProfessorPfeiferundPartner mit Hauptsitz in Darmstadt wurde das Gebaude in den Jahren 2010 bis 2012 grundlegend saniert, teilweise umgebaut und durch Hinzufugung von zwei Neubauten und einer Tiefgarage, die sich in das Gesamtensemble masstablich einfugen, in ein Marktzentrum mit Hotel und Buros umgewandelt. Besonderes Augenmerk wurde dabei auf die unveranderte Erhaltung der historischen Konstruktion gelegt, die insbesondere in den Kuppelkonstruktionen uber dem ehemaligen Mannerbad und dem ehemaligen Frauenbad sichtbar ist. Mit Ausnahme einer kurzen Beschreibung der Neubauten konzentriert sich dieser Beitrag auf die Statik der Gewolbe. “New old” calculation methods on the example of the historical Bergheimer Bath in Heidelberg Empty and unused for many years, the former Bergheim bath in Heidelberg led an unworthy existence. No one was willing to take care of it or had a good idea of how it could be put to good use. Often the main problem of historical buildings is the fact that no use is found which creates a financial incentive for investors. Fortunately, in this building it was different and there was an astute businessman, highly attached to the town of Heidelberg, who had the vision, that there was the opportunity to create something new out of the building where on the one hand, the cultural heritage could be saved and at the same time a profitable business could arise. This man was the single one who made an offer to a Europe-wide tender of Heidelberg and received the award in October 2008. Together with the renowned Heidelberg architects SSV and the experienced in working with historic buildings engineering office for structural engineering ProfessorPfeiferundPartner, headquartered in Darmstadt, the building in the years 2010–2012 has been thoroughly renovated and partly rebuilt by the addition of two new buildings and an underground parking, well inserted into the entire ensemble and converted into a market center with hotel and offices. Particular attention was paid to the unchanged preservation of the historical structures, especially visible in the vault structures of the former men's and women's baths. Except for a brief description of the new buildings, this paper focuses on the static of the vault.

Diffuse large B-cell lymphoma (DLBCL) represents a heterogeneous diagnostic category with distinct molecular subtypes that can be defined by gene expression profiling. However, even within these defined subtypes, heterogeneity prevails. To further elucidate the pathogenesis of these entities, we determined the expression of the tumor suppressor phosphatase and tensin homolog (PTEN) in 248 primary DLBCL patient samples. These analyses revealed that loss of PTEN was detectable in 55% of germinal center B-cell-like (GCB) DLBCLs, whereas this abnormality was found in only 14% of non-GCB DLBCL patient samples. In GCB DLBCL, the PTEN status was inversely correlated with activation of the oncogenic PI3K/protein kinase B (AKT) pathway in both DLBCL cell lines and primary patient samples. Reexpression of PTEN induced cytotoxicity in PTEN-deficient GCB DLBCL cell line models by inhibiting PI3K/AKT signaling, indicating an addiction to this pathway in this subset of GCB DLBCLs. PI3K/AKT inhibition induced down-regulation of the transcription factor MYC. Reexpression of MYC rescued GCB DLBCL cells from PTEN-induced toxicity, identifying a regulatory mechanism of MYC expression in DLBCL. Finally, pharmacologic PI3K inhibition resulted in toxicity selectively in PTEN-deficient GCB DLBCL lines. Collectively, our results indicate that PTEN loss defines a PI3K/AKT-dependent GCB DLBCL subtype that is addicted to PI3K and MYC signaling and suggest that pharmacologic inhibition of PI3K might represent a promising therapeutic approach in these lymphomas.

About 8,000 years ago in the Fertile Crescent, a spontaneous hybridization of the wild diploid grass Aegilops tauschii (2n = 14; DD) with the cultivated tetraploid wheat Triticum turgidum (2n = 4x = 28; AABB) resulted in hexaploid wheat (T. aestivum; 2n = 6x = 42; AABBDD). Wheat has since become a primary staple crop worldwide as a result of its enhanced adaptability to a wide range of climates and improved grain quality for the production of baker's flour. Here we describe sequencing the Ae. tauschii genome and obtaining a roughly 90-fold depth of short reads from libraries with various insert sizes, to gain a better understanding of this genetically complex plant. The assembled scaffolds represented 83.4% of the genome, of which 65.9% comprised transposable elements. We generated comprehensive RNA-Seq data and used it to identify 43,150 protein-coding genes, of which 30,697 (71.1%) were uniquely anchored to chromosomes with an integrated high-density genetic map. Whole-genome analysis revealed gene family expansion in Ae. tauschii of agronomically relevant gene families that were associated with disease resistance, abiotic stress tolerance and grain quality. This draft genome sequence provides insight into the environmental adaptation of bread wheat and can aid in defining the large and complicated genomes of wheat species.

Activated B-cell-like (ABC) and germinal center B-cell-like diffuse large B-cell lymphoma (DLBCL) represent the 2 major molecular DLBCL subtypes. They are characterized by differences in clinical course and by divergent addiction to oncogenic pathways. To determine activity of novel compounds in these 2 subtypes, we conducted an unbiased pharmacologic in vitro screen. The phosphatidylinositol-3-kinase (PI3K) α/δ (PI3Kα/δ) inhibitor AZD8835 showed marked potency in ABC DLBCL models, whereas the protein kinase B (AKT) inhibitor AZD5363 induced apoptosis in PTEN-deficient DLBCLs irrespective of their molecular subtype. These in vitro results were confirmed in various cell line xenograft and patient-derived xenograft mouse models in vivo. Treatment with AZD8835 induced inhibition of nuclear factor κB signaling, prompting us to combine AZD8835 with the Bruton's tyrosine kinase inhibitor ibrutinib. This combination was synergistic and effective both in vitro and in vivo. In contrast, the AKT inhibitor AZD5363 was effective in PTEN-deficient DLBCLs through downregulation of the oncogenic transcription factor MYC. Collectively, our data suggest that patients should be stratified according to their oncogenic dependencies when treated with PI3K and AKT inhibitors.

PGSB (Plant Genome and Systems Biology; formerly MIPS-Munich Institute for Protein Sequences) has been involved in developing, implementing and maintaining plant genome databases for more than a decade. Genome databases and analysis resources have focused on individual genomes and aim to provide flexible and maintainable datasets for model plant genomes as a backbone against which experimental data, e.g., from high-throughput functional genomics, can be organized and analyzed. In addition, genomes from both model and crop plants form a scaffold for comparative genomics, assisted by specialized tools such as the CrowsNest viewer to explore conserved gene order (synteny) between related species on macro- and micro-levels.The genomes of many economically important Triticeae plants such as wheat, barley, and rye present a great challenge for sequence assembly and bioinformatic analysis due to their enormous complexity and large genome size. Novel concepts and strategies have been developed to deal with these difficulties and have been applied to the genomes of wheat, barley, rye, and other cereals. This includes the GenomeZipper concept, reference-guided exome assembly, and "chromosome genomics" based on flow cytometry sorted chromosomes.

Wheat is the third most important crop for human nutrition in the world. The availability of high-resolution genetic and physical maps and ultimately a complete genome sequence holds great promise for breeding improved varieties to cope with increasing food demand under the conditions of changing global climate. However, the large size of the bread wheat (Triticum aestivum) genome (approximately 17 Gb/1C) and the triplication of genic sequence resulting from its hexaploid status have impeded genome sequencing of this important crop species. Here we describe the use of mitotic chromosome flow sorting to separately purify and then shotgun-sequence a pair of telocentric chromosomes that together form chromosome 4A (856 Mb/1C) of wheat. The isolation of this much reduced template and the consequent avoidance of the problem of sequence duplication, in conjunction with synteny-based comparisons with other grass genomes, have facilitated construction of an ordered gene map of chromosome 4A, embracing ≥85% of its total gene content, and have enabled precise localization of the various translocation and inversion breakpoints on chromosome 4A that differentiate it from its progenitor chromosome in the A genome diploid donor. The gene map of chromosome 4A, together with the emerging sequences of homoeologous wheat chromosome groups 4, 5 and 7, represent unique resources that will allow us to obtain new insights into the evolutionary dynamics between homoeologous chromosomes and syntenic chromosomal regions.

Inhalt dieses Beitrages sind Belastungsversuche an den historischen Treppenhauskonstruktionen des ehemaligen Provinzial-Arresthauses in Mainz. Das zentral im Regierungsviertel von Mainz gelegene Bauwerk hat eine grose architektur- und stadtgeschichtliche Bedeutung. Der im Jahre 1906 realisierte Gesamtkomplex, bestehend aus einem Justizgebaude und dem Arresthaus, Siegerentwurf aus dem Jahre 1903 von Paul Bonatz (1877—1956) wurde bis 2002 als Gefangnis (Justizvollzugsanstalt JVA) genutzt, darauf folgte jahrelanger Leerstand. Mitte des Jahres 2009 begann der Umbau des Gebaudes in ein Buro- und Verwaltungszentrum. Fassaden und Treppenhauser wurden unter Denkmalschutz gestellt. Eine derartige Umnutzung erfordert ggf. Eingriffe in den Bestand in Form von Ertuchtigungen, sofern Nachrechnungen nicht die gewunschte bzw. notwendige Tragfahigkeit nachweisen konnen. Nicht nur bei denkmalgeschutzten Bauwerken, bei denen durch solche Eingriffe der Denkmalwert ggf. beeintrachtigt werden wurde, sondern bei allen historischen Bauten, die weiter genutzt werden sollen, ist schon aus wirtschaftlichen Grunden anzustreben, auf Verstarkungen der Konstruktion moglichst verzichten zu konnen. Fur die ehemalige JVA Mainz war der rechnerische Nachweis nicht moglich, da Material- und Konstruktionsangaben fehlten. Als Alternative zum rechnerischen Nachweis wurden Belastungsversuche durchgefuhrt. Drei von vier Treppenhausern waren denkmalgeschutzt, das Vierte ahnlicher Bauart war zum Abriss bestimmt und konnte fur Traglastversuche verwendet werden. Die Zielstellung war, die vorhandene Konstruktion unverandert belassen zu konnen, wobei die Eingriffe in den historischen Bestand wahrend der Untersuchungen so klein wie moglich gehalten werden sollten. The load carrying capacity of historic sandstone staircase structures in the former ”Provinzial-Arresthaus” in Mainz. Subject of this paper are in-situ-tests of the load carrying capacity of historic staircase structures in the former ”Provinzial-Arresthaus” in Mainz (which was a prison). The building, located in the centre of the governmental area of Mainz, is of prime importance from the architectural and town-historic point of view. The entire complex, built in 1906, consisting of a court house and the prison, the first price of a competition won in 1903 by the famous Architect Paul Bonatz (1877—1956), was in use as such until 2002 and after that it was empty for years. In the middle of the year 2009 the renovation of this building into a modern office and administration building started. The facade and the staircases were listet as historic monuments. It is important to emphasize that such alteration may occasionally cause heavy interventions into the historic stock of the structure in order to ensure the load carrying capacity for the forseen usage by means of strengthening, if the calculations do not prove the existing capacity as sufficient. Not only for listed historic monuments, but rather all historic buildings designated for a further usage it must be the goal in terms of economic efficiency to reduce retrofitting measures on the existing structures as much as possible. For the particular building the calculative proof was not possible since information about material and construction were not available. For this reason the experimental check was a proper alternative method. Three of four staircases were listed, the fourth staircase was designated to be demolished and gave a good chance to determine the real load carrying capacity up to the complete collapse. The goal was altogether to keep the three staircases as they are without any retrofitting measures and to keep them unaffected during the tests as much as possible.

Within the framework of a larger project aimed to assess the potential of second generation hybrid striped bass for German aquaculture the genotypic segregation of five microsatellite loci was analysed in two progeny lots (n = 74 and 76, respectively). There was no consistent correlation between microsatellite genotypes and phenotypic category (white bass, hybrid, or striped bass). None of the individuals expressed neither only white bass nor only striped bass genotypes at all five loci. On the other hand, only hybrid genotypes at all five loci were detected in three individuals of lot 1 and four individuals of lot 2. Single loci tests for conformity of microsatellite genotypic segregation with Mendelian rules revealed significant deviations (P < 0.05) in four cases for lot 1 and in three cases for lot 2. If pooled over all five loci, both lots displayed highly significant deviations (P < 0.01) with an excess of hybrid genotypes and a deficiency of white bass genotypes. It is concluded that stabilizing selection performed on hybrid genotypes might be a suitable approach for practical aquaculture in Europe if the goal is to become independent of first generation hybrid fry supply and/or if establishing domesticated brood stocks of both parental species is impossible. However, more detailed studies on the characteristics and performance of multiple hybrid generations are needed.

Experiments for the production of hybrid striped bass (HSB) in in-pond circulation systems (IPCS) were carried out in 2003 and 2004. The circulation system consisted of two channels with a productive volume of 8.5 cubic meters each. The tanks were installed tightly in a pond, which served for the biological cleaning of the expiry water. In the first year HSB fingerlings with an average weight of 46.4 g were produced. The average yield in the basin was 51.2 kg/m3. The survival rate from stocked 0.44 g advanced fry was 97.8%. The food conversion was 1.16. In 2004 two-year-old HSB were reared in the same IPCS. The tanks were stocked at two different stocking densities, 122 and 244 fingerlings/m3 with a mean weight of 36.5 g. In the tank with the larger stocking density, the yield was almost exactly twice as high as in the other tank (50.0 resp. 24.8 kg), which corresponded to a stocking density of 59.1 or 29.3 kg/m3 at the end of the rearing season. The stocking density had no influence on the increase of the individual body weight. Obviously HSB can therefore still be reared at higher stocking densities.

Kampf-Stegtrager wie die der Eissporthalle in Bad Reichenhall wurden, wenn auch meist in geringeren Dimensionen, in den 1950er und 1960er Jahren vielfach eingesetzt. Sie sind ein Beispiel fur die grosen Anstrengungen, die man in Forschung und Praxis in diesen Jahren unternahm, um die Leimbauweise im Ingenieurholzbau endgultig zu etablieren. Spatestens Ende der 1960er Jahre war dieses Ziel erreicht. Gleichwohl wurden die Ursprunge des Leimbaus bereits ein halbes Jahrhundert fruher durch Otto Hetzer und andere gelegt. Und so ist die Entwicklungsgeschichte des Brettschichtholztragers einmal mehr ein Beispiel dafur, dass der als Diffusion bezeichnete Prozess – von der Innovation bis zur allgemeinen Verbreitung und Anwendung einer Neuerung – sich gerade im Bauwesen oftmals uber ein bis zwei Generationen erstreckt.

PGSB (Plant Genome and Systems Biology; formerly MIPS-Munich Institute for Protein Sequences) has been involved in developing, implementing and maintaining plant genome databases for more than a decade. Genome databases and analysis resources have focused on individual genomes and aim to provide flexible and maintainable datasets for model plant genomes as a backbone against which experimental data, e.g., from high-throughput functional genomics, can be organized and analyzed. In addition, genomes from both model and crop plants form a scaffold for comparative genomics, assisted by specialized tools such as the CrowsNest viewer to explore conserved gene order (synteny) between related species on macro- and micro-levels.The genomes of many economically important Triticeae plants such as wheat, barley, and rye present a great challenge for sequence assembly and bioinformatic analysis due to their enormous complexity and large genome size. Novel concepts and strategies have been developed to deal with these difficulties and have been applied to the genomes of wheat, barley, rye, and other cereals. This includes the GenomeZipper concept, reference-guided exome assembly, and "chromosome genomics" based on flow cytometry sorted chromosomes.

Plants integrate seasonal cues such as temperature and day length to optimally adjust their flowering time to the environment. Compared to the control of flowering before and after winter by the vernalization and day length pathways, mechanisms that delay or promote flowering during a transient cool or warm period, especially during spring, are less well understood. Due to global warming, understanding this ambient temperature pathway has gained increasing importance. In Arabidopsis thaliana, FLOWERING LOCUS M (FLM) is a critical flowering regulator of the ambient temperature pathway. FLM is alternatively spliced in a temperature-dependent manner and the two predominant splice variants, FLM-ß and FLM-δ, can repress and activate flowering in the genetic background of the A. thaliana reference accession Columbia-0. The relevance of this regulatory mechanism for the environmental adaptation across the entire range of the species is, however, unknown. Here, we identify insertion polymorphisms in the first intron of FLM as causative for accelerated flowering in many natural A. thaliana accessions, especially in cool (15°C) temperatures. We present evidence for a potential adaptive role of this structural variation and link it specifically to changes in the abundance of FLM-ß. Our results may allow predicting flowering in response to ambient temperatures in the Brassicaceae.

Arsenic and mercury are frequent contaminants in the environment and care must be taken to limit their entrance into the food chain. The toxicity of both elements strongly depends upon their speciation. Total amounts of As and Hg as well as their species were analyzed in muscle and liver of 26 fishes of seven freshwater fish species caught in the River Elbe. The median concentrations of As were 162 μg kg(-1) w.w. in liver and 92 μg kg(-1) w.w. in muscle. The median concentrations of total Hg were 241 μg kg(-1) w.w. in liver and 256 μg kg(-1) w.w. in muscle. While this level of Hg contamination of the freshwater fish in the River Elbe is significantly lower than 20 years ago, it exceeds the recommended environmental quality standard of 20 μg Hg kg(-1) w.w. by a factor of 5-50. However, the European maximum level of 500 μg Hg kg(-1) for fish for human consumption is rarely exceeded. Arsenic-containing fatty acids and hydrocarbons were determined and partially identified in methanolic extracts of the fish by HPLC coupled in parallel to ICP-MS (element specific detection) and ESI-Q-TOF-MS (molecular structure detection). While arsenobetaine was the dominant As species in the fish, six arsenolipids were detected and identified in the extracts of liver tissue in common bream (Abramis brama), ide (Leuciscus idus), asp (Aspius aspius) and northern pike (Esox lucius). Four arsenic-containing fatty acids (AsFA) and two arsenic-containing hydrocarbons (AsHC) are reported in freshwater fish for the first time. With respect to mercury the more toxic MeHg(+) was the major species in muscle tissue (>90% of total Hg) while in liver Hg(2+) and MeHg(+) were of equal importance. The results show the high relevance of element speciation in addition to the determination of total element concentrations to correctly assess the burden of these two elements in fish.

Vor rund 80 Jahren begann man damit, die Schweistechnik – namentlich die Lichtbogenschweisung – zunachst bei der Reparatur und Ertuchtigung, dann auch beim Neubau von Brucken einzusetzen. Nach anfanglicher Skepsis der Baubehorden und damit verbundener Zuruckhaltung vieler Bauschaffender gegenuber der neuen, im Maschinenbau bereits in den Jahren vor dem 1. Weltkrieg eingefuhrten Verbindungstechnik, nahm die Schweistechnik im Stahlbau Anfang der 30er Jahre eine geradezu sturmische Entwicklung. Diese Euphorie wurde nur wenige Jahre spater durch mehrere spektakulare Schadensfalle an Brucken aus hochwertigem Baustahl der Gute St52 gedampft. Nachdem durch umfangreiche wissenschaftliche Versuche die Schadensursachen geklart werden konnten, etablierte sich das Schweisen spatestens in den 1960er Jahren endgultig als wichtigste Fugetechnik im Stahlbruckenbau. (© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) The first welded steel bridges in Germany – on the rocky beginnings of welding technology. Welding technology – and here primarily arc welding – began to be used about 80 years ago on bridges; first for repairing and strengthening them, and then increasingly, for building new ones. Initially, this new joining technology that had been used since before WWI in mechanical engineering, was met with skepticism on the part of building and code enforcement authorities, and hence, also by many in the building industry. Finally, in the early 1930's, welding technology started its explosive development in steel construction. This euphoric state was dampened a few years later due to several spectacular failures on bridges made of high-quality construction steel. After comprehensive scientific studies were able to determine the causes of these failures, by the 1960's, welding finally established itself as the most important joining technology in steel bridge construction.

Mauerwerk hat in den letzten Jahrzehnten einen rasanten Wandel hin zu einem hochspezialisierten Baustoff vollzogen. Fur einige Disziplinen – z.B. den Warmeschutz – kann man von einem “Hightech”-Produkt sprechen. Auch die Modelle fur die Bemessung von Mauerwerk werden differenzierter. Besonders im Hinblick auf die Vielfalt der angebotenen Steinsorten und Formate wurde in den letzten Jahren versucht, die Bemessung der individuellen Mauerwerkssorte anzupassen. In den Arbeiten [3] und [10] wurde verstarkt die Steingeometrie in die Bemessungsmodelle fur Schubversagen bzw. Stabilitatsversagen einbezogen. Andere Arbeiten, darunter [11], haben das Stabilitatsversagen einachsig gespannter Wande unter Berucksichtigung der unterschiedlichen Spannungs- Dehnungsbeziehungen verschiedener Stein-Mortelkombinationen beleuchtet. Die Plattentragwirkung einer Mauerwerkwand wird wesentlich durch ihre Steifigkeiten in zueinander orthogonalen Richtungen beeinflust. Die Unterschiede in der Fugenausbildung sowie in Steinform, -art und -format fuhren zu unterschiedlichen Steifigkeitsverteilungen, die bisher nur unzureichend bei der Schnittkraftermittlung und Nachweisfuhrung berucksichtigt werden. Im Rahmen von ersten Tastversuchen wurden fur ausgewahlte Steine die Elastizitatsmoduln und die Querdehnzahlen in den beiden wesentlichen zueinander orthogonalen Wandrichtungen bestimmt. Die Stosfuge wird i. allg. als statisch nicht mitwirkend angesetzt, weil sie entweder nicht ausreichend oder gar nicht vermortelt ist. Dies fuhrt zu einer Anisothropie, die sich sowohl bei der Scheibenbeanspruchung als auch bei der Plattenbiegung im Zustand I mit den ermittelten Kennwerten auf einfache Art berucksichtigen last.

Antibody drug conjugates (ADCs), in which cytotoxic drugs are linked to antibodies targeting antigens on tumor cells, represent promising novel agents for the treatment of malignant lymphomas. Pinatuzumab vedotin is an anti-CD22 ADC and polatuzumab vedotin an anti-CD79B ADC that are both linked to the microtubule-disrupting agent monomethyl auristatin E (MMAE). In the present study, we analyzed the activity of these agents in different molecular subtypes of diffuse large B-cell lymphoma (DLBCL) both in vitro and in early clinical trials. Both anti-CD22-MMAE and anti-CD79B-MMAE were highly active and induced cell death in the vast majority of activated B-cell-like (ABC) and germinal center B-cell-like (GCB) DLBCL cell lines. Similarly, both agents induced cytotoxicity in models with and without mutations in the signaling molecule CD79B. In line with these observations, relapsed and refractory DLBCL patients of both subtypes responded to these agents. Importantly, a strong correlation between CD22 and CD79B expression in vitro and in vivo was not detectable, indicating that patients should not be excluded from anti-CD22-MMAE or anti-CD79B-MMAE treatment because of low target expression. In summary, these studies suggest that pinatuzumab vedotin and polatuzumab vedotin are active agents for the treatment of patients with different subtypes of DLBCL.

Mauerwerk wird in Madagaskar nicht als tragender Baustoff verwendet. Obwohl die Grundlagen dafur vorhanden sind und der Einsatz von Mauerwerk fur das Land aus bauklimatischer und auch aus wirtschaftlicher Sicht uberaus sinnvoll ware, wird Massivkonstruktionen aus Stahlbeton, in die Mauerwerk nur als nichttragendes Element integriert wird, der Vorzug gegeben. Der Bericht beschaftigt sich mit der Herstellung und den daraus resultierenden Eigenschaften von Mauerziegeln in Madagaskar, in denen die Hauptursache fur den Nichteinsatz von Mauerwerk als tragendes Bauteil zu suchen ist. Er entstand im Zuge eines groseren Bauvorhabens in Madagaskar, welches von den Autoren betreut wird.

In the restoration or repair of old buildings, the correct estimation of the stress-strain-relationship of the existing structure is an important task. Masonry stiffness is crucial to the load bearing behaviour of a structure. When detailed investigations are lacking, it is common practise to evaluate the masonry stiffness from the compressive strength by standardised empirical relations established for various types of modern masonry. This paper illustrates the danger of this approach in two case studies. The first example is a stone bridge upon the Main at Ochsenfurt. The thickness of the mortar joints in this construction allowed the sampling of mortars suitable for the direct testing of the stress-strain relationship under uniaxial compression. Thus it was possible to calculate the masonry stiffness from the measured elastic modulus of stone and mortar. It became evident that a first rough estimation according to the building standard would not have been safe. In a second case study dealing with masonry pillars it is shown that the quality of mortar samples are influenced by the position of mortar in the masonry section.

Bread wheat (Triticum aestivum) is a globally important crop, accounting for 20 per cent of the calories consumed by humans. Major efforts are underway worldwide to increase wheat production by extending genetic diversity and analysing key traits, and genomic resources can accelerate progress. But so far the very large size and polyploid complexity of the bread wheat genome have been substantial barriers to genome analysis. Here we report the sequencing of its large, 17-gigabase-pair, hexaploid genome using 454 pyrosequencing, and comparison of this with the sequences of diploid ancestral and progenitor genomes. We identified between 94,000 and 96,000 genes, and assigned two-thirds to the three component genomes (A, B and D) of hexaploid wheat. High-resolution synteny maps identified many small disruptions to conserved gene order. We show that the hexaploid genome is highly dynamic, with significant loss of gene family members on polyploidization and domestication, and an abundance of gene fragments. Several classes of genes involved in energy harvesting, metabolism and growth are among expanded gene families that could be associated with crop productivity. Our analyses, coupled with the identification of extensive genetic variation, provide a resource for accelerating gene discovery and improving this major crop.

Sophisticated analysing proceedings have been used in the past to assess the composition of hardened mortars. Chemical and mechanical tests combined with microscopic investigations seem to provide all the information required for the design of repair mortars which physically and visually are appropriate for restoration purposes. Lime putty and hydrated lime of varying quality meanwhile are currently available. Nevertheless, the results are not always convincing. The limit of the analysing techniques has become evident in conservation practise. Knowledge of how mortars have been fabricated, mixed, stored and applied is as important as the chemical composition. The authors have therefore collected information on the fabrication, compositions and use of mortars revealed by an examination of written historic sources.

The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) represents a very aggressive human lymphoma entity. Constitutive NF-κB activation caused by chronic active B-cell receptor (BCR) signaling is common feature of many ABC DLBCL cells; however, the pathways linking BCR signaling to the NF-κB prosurvival network are largely unknown. Here we report that constitutive activity of PI3K and the downstream kinase PDK1 are essential for the viability of two ABC DLBCL cell lines that carry mutations in the BCR proximal signaling adaptor CD79B. In these cells, PI3K inhibition reduces NF-κB activity and decreases the expression of NF-κB target genes. Furthermore, PI3K and PDK1 are required for maintaining MALT1 protease activity, which promotes survival of the affected ABC DLBCL cells. These results demonstrate a critical function of PI3K-PDK1 signaling upstream of MALT1 protease and NF-κB in distinct ABC DLBCL cells and provide a rationale for the pharmacologic use of PI3K inhibitors in DLBCL therapy.

scalasca is a performance toolset that has been specifically designed to analyze parallel application behavior on large-scale systems, but is also well-suited for small- and medium-scale hpc platforms. scalasca offers an incremental performance-analysis process that integrates runtime summaries with in-depth studies of concurrent behavior via event tracing, adopting a strategy of successively refined measurement configurations. A distinctive feature of scalasca is its ability to identify wait states even for very large processor counts. The current version supports the mpi, Openmp and hybrid programming constructs most widely used in highly-scalable hpc applications.

Climate change and air pollution, including ozone is known to affect plants and might also influence the ragweed pollen, known to carry strong allergens. We compared the transcriptome of ragweed pollen produced under ambient and elevated ozone by 454-sequencing. An enzyme-linked immunosorbent assay (ELISA) was carried out for the major ragweed allergen Amb a 1. Pollen surface was examined by scanning electron microscopy and attenuated total reflectanceFourier transform infrared spectroscopy (ATR-FTIR), and phenolics were analysed by high-performance liquid chromatography. Elevated ozone had no influence on the pollen size, shape, surface structure or amount of phenolics. ATR-FTIR indicated increased pectin-like material in the exine. Transcriptomic analyses showed changes in expressed-sequence tags (ESTs), including allergens. However, ELISA indicated no significantly increased amounts of Amb a 1 under elevated ozone concentrations. The data highlight a direct influence of ozone on the exine components and transcript level of allergens. As the total protein amount of Amb a 1 was not altered, a direct correlation to an increased risk to human health could not be derived. Additional, the 454-sequencing contributes to the identification of stress-related transcripts in mature pollen that could be grouped into distinct gene ontology terms. (VLID)1701160

The genomic sequences of many important Triticeae crop species are hard to assemble and analyse due to their large genome sizes, (in part) polyploid genomes and high repeat content. Recently, the draft genomes of barley and bread wheat were reported thanks to cost-efficient and fast NGS technologies. The genome of barley is estimated to be 5 Gb in size whereas the genome of bread wheat accounts for 17 Gb and harbours an allo-hexaploid genome. Direct assembly of the sequence reads and access to the gene content is hampered by the repeat content. As a consequence, novel strategies and data analysis concepts had to be developed to provide much-needed whole genome sequence surveys and access to the gene repertoires. Here we describe some analytical strategies that now enable structuring of massive NGS data generated and pave the way towards structured and ordered sequence data and gene order. Specifically we report on the genomeZipper, a synteny driven approach to order and structure NGS survey sequences of grass genomes that lack a physical map. In addition, to access and analyse the gene repertoire of allo-hexaploid bread wheat from the raw sequence reads, a reference-guided approach was developed utilizing representative genes from rice, Brachypodium distachyon, sorghum and barley. Stringent sub-assembly on the reference genes prevented collapsing of homeologous wheat genes and allowed to estimate gene retention rate and determine gene family sizes. Genomic sequences from the wheat sub-genome progenitors enabled to discriminate a large number of sub-assemblies between the wheat A, B or D sub-genome using machine learning algorithms. Many of the concepts outlined here can readily be applied to other complex plant and non-plant genomes.