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Error-corrected Duplex Sequencing (DuplexSeq) enables direct quantification of low-frequency mutations and offers tremendous potential for chemical mutagenicity assessment. We investigated the utility of DuplexSeq to quantify induced mutation frequency (MF) and spectrum in human lymphoblastoid TK6 cells exposed to a prototypical DNA alkylating agent,N-ethyl-N-nitrosourea (ENU). Furthermore, we explored appropriate experimental parameters for this application, and assessed inter-laboratory reproducibility. In two independent experiments in two laboratories, TK6 cells were exposed to ENU (25-200 µM) and DNA was sequenced 48, 72, and 96 h post-exposure. A DuplexSeq mutagenicity panel targeting twenty 2.4-kb regions distributed across the genome was used to sample diverse, genome-representative sequence contexts. A robust increase in MF that was unaffected by time was observed in both laboratories. Concentration-response in the MF from the two laboratories was strongly positively correlated (R2=0.95). C:G>T:A, T:A>C:G, T:A>A:T, and T:A>G:C mutations increased in consistent, concentration-dependent manners in both laboratories, with high proportions of C:G>T:A at all time points. The target sites responded similarly between the two laboratories and revealed a higher average MF in intergenic regions. These results, demonstrating remarkable reproducibility across time and laboratory for both MF and spectrum, support the high value of DuplexSeq for characterizing chemical mutagenicity in both research and regulatory evaluation.

The organotypic human air‐liquid‐interface (ALI) airway tissue model has been used as an in vitro cell culture system for evaluating the toxicity of inhaled substances. ALI airway cultures are highly differentiated, which has made it challenging to evaluate genetic toxicology endpoints. In the current study, we assayed DNA damage with the high‐throughput CometChip assay and quantified mutagenesis with Duplex Sequencing, an error‐corrected next‐generation sequencing method capable of detecting a single mutation per 107 base pairs. Fully differentiated human ALI airway cultures were treated from the basolateral side with 6.25 to 100 μg/mL ethyl methanesulfonate (EMS) over a period of 28 days. CometChip assays were conducted after 3 and 28 days of treatment, and Duplex Sequencing after 28 days of treatment. Treating the airway cultures with EMS resulted in time‐ and concentration‐dependent increases in DNA damage and a concentration‐dependent increase in mutant frequency. The mutations observed in the EMS‐treated cultures were predominantly C → T transitions and exhibited a unique trinucleotide signature relative to the negative control. Measurement of physiological endpoints indicated that the EMS treatments had no effect on anti‐p63‐positive basal cell frequency, but produced concentration‐responsive increases in cytotoxicity and perturbations in cell morphology, along with concentration‐responsive decreases in culture viability, goblet cell and anti‐Ki67‐positive proliferating cell frequency, cilia beating frequency, and mucin secretion. The results indicate that a unified 28‐day study can be used to measure several important safety endpoints in physiologically relevant human in vitro ALI airway cultures, including DNA damage, mutagenicity, and tissue‐specific general toxicity.

Sensitive and specific detection of low frequency mutations in acute myeloid leukemia (AML) is critical in research of minimal residual disease (MRD). At variant allele frequencies (VAF) below approximately 1%, PCR and sequencing errors result in prohibitive signal-to-noise ratios with next generation sequencing (NGS). Duplex Sequencing (DS) relates the original top and bottom DNA strands to make double stranded consensus sequences to greatly reduce errors. In two experiments we show further improvement of DS of AML-related genes by the use of enzymatic fragmentation (EF) and an updated gene panel that incorporates 2022 European LeukemiaNet (ELN) recommendations. In the first DS study, 29 AML-related genes (59 kb) were targeted in hybrid capture. Mutant cell line DNA was mixed into DNA from a healthy young donor to simulate MRD. Expected VAFs in mixtures were 1.0-0.003%. Samples comprised a mix of 4 insertions and deletions (indel), a mix of 15 single nucleotide variants (SNV), and 4 serial dilutions of a FLT3 ITD plus an NPM1 insertion. Pure diluent DNA was used as a negative control. Each sample was prepared in quadruplicate with mechanical fragmentation (MF) vs EF. DNA input mass was 1,500 ng per replicate, except for 50-250 ng for the FLT3/NPM1 mixes with expected VAF of 1% and 0.1%, respectively. Input masses were set to ensure >95% probability of detection of all mutations in the combined data. All targeted variants down to 0.003% VAF were detected in spike-in mixtures, with expected vs observed VAF highly correlated whether using MF or EF (R2 >0.98 for all mixtures). Duplex molecular depth was 1.2-2.0x higher with EF vs MF across input masses. Panel-wide mean duplex depth per 1,500 ng replicate was 30,376x for MF and 48,036x for EF, for combined mean depths of 121,508x for MF and 192,144x for EF. In the negative control, background mutational calls at spike-in positions were 4/2,993,429 duplex bases using MF and 0/4,780,491 duplex bases with EF, reflecting increased specificity with EF. Next, a revised panel targeting 36 AML-related genes (80 kb) was used for DS with EF. Here differing mutant cell line DNA was mixed into the same diluent from above (the negative control). This mixture harbors 27 variants with expected VAFs of 0.125-0.006% (4 indels, 21 SNVs, a FLT3 ITD and an NPM1 insertion), 17 of which (2 indels, 14 SNVs and the NPM1 insertion) span VAFs of 0.011-0.009% to establish a limit of detection of 0.01%. With 2,000 ng (+/- 10%) input, for 14 samples, panel-wide mean duplex depth was 56,528x. For variants with expected VAF ≥ 0.009%, the assay had >98% sensitivity, >96% specificity, and >98% accuracy at genomic positions of known true positive variants. In summary, DS with EF yields more data per nanogram of DNA than MF, maximizing the use of precious samples. This method exhibits extremely low background mutation signal and a low limit of detection across targets that are valuable in AML MRD research. Citation Format: Elizabeth Schmidt, Devon M. Fitzgerald, Camila Zanette, Gavin D. Meredith, Mark A. McElwain, Raul Burciaga, Kevin C. Vavra, Thomas H. Smith, Jesse J. Salk, Jake Higgins. Accurate detection of low frequency AML-associated mutations in vitro using Duplex Sequencing with enzymatic fragmentation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB254.

Developing strategies to inflame tumors is critical for increasing response to immunotherapy. Here, we report that low-dose radiotherapy (LDRT) of murine tumors promotes T-cell infiltration and enables responsiveness to combinatorial immunotherapy in an IFN-dependent manner. Treatment efficacy relied upon mobilizing both adaptive and innate immunity and depended on both cytotoxic CD4+ and CD8+ T cells. LDRT elicited predominantly CD4+ cells with features of exhausted effector cytotoxic cells, with a subset expressing NKG2D and exhibiting proliferative capacity, as well as a unique subset of activated dendritic cells expressing the NKG2D ligand RAE1. We translated these findings to a phase I clinical trial administering LDRT, low-dose cyclophosphamide, and immune checkpoint blockade to patients with immune-desert tumors. In responsive patients, the combinatorial treatment triggered T-cell infiltration, predominantly of CD4+ cells with Th1 signatures. Our data support the rational combination of LDRT with immunotherapy for effectively treating low T cell–infiltrated tumors.Significance:Low-dose radiation reprogrammed the tumor microenvironment of tumors with scarce immune infiltration and together with immunotherapy induced simultaneous mobilization of innate and adaptive immunity, predominantly CD4+ effector T cells, to achieve tumor control dependent on NKG2D. The combination induced important responses in patients with metastatic immune-cold tumors.This article is highlighted in the In This Issue feature, p. 1

Common bean (Phaseolus vulgaris L.) is the most important food legume crop worldwide. Canadian beans, especially large seeded cultivars of Andean origin, have relatively narrow genetic diversities. Establishing crops with mixtures of cultivars instead of pure lines is a simple, cost effective way to increase genetic diversity in the field. A number of studies have demonstrated the benefits of mixture cropping over monocropping in controlling disease, increasing water use efficiency and increasing yield stability. The objective of this study was to determine the effects of increasing in-field diversity, by using mixtures of bean cultivars instead of monocultures, on productivity. The feasibility of growing bean cultivar mixtures in southern Ontario environments was confirmed with a small pilot study that was conducted with four bean cultivars and restricted number of mixtures at two locations in 2017. Mixture performance experiments were performed with seven diverse bean genotypes at two Ontario locations [Woodstock and Elora (two planting dates) research stations] as pure stands and all possible binary mixtures (planted in alternate rows or as completely random mixtures) in 2018. Conventional plot-based above ground crop data were collected. Mixing efficiencies were calculated from the yield data using a Relative yield of the mixture (RYM) index. Diallel analysis was used to identify general mixing ability of cultivars and specific mixing abilities of mixtures. Significant differences among seven bean cultivars and their mixtures were identified in all three environments for all analyzed traits. The results indicated multiple benefits of planting mixtures compared to monocultures A number of mixtures overyielded component cultivars grown in pure stands; they had higher yields, RYM index values greater than one and positive specific mixing abilities (for yield) in both types of biblends. The research has the potential to provide a theoretical basis for the use of precision agriculture tools to plant fields with mixtures instead of monocultures. It could lead to greater in-field diversity in the crop and in the above and below ground ecosystems that might provide greater buffering capacity and resiliency to the cropping system as well as increased ecosystem services.

The nitrogen fixing ability of common bean (Phaseolus vulgaris L.) in association with rhizobia is often characterized as poor compared to other legumes, and nitrogen fertilizers are commonly used in bean production to achieve high yields, which in general inhibits nitrogen fixation. In addition, plants cannot take up all the nitrogen applied to the soil as a fertilizer leading to runoff and groundwater contamination. The overall objective of this work is to reduce use of nitrogen fertilizer in common bean production. This would be a major advance in profitability for the common bean industry in Canada and would significantly improve the ecological footprint of the crop. In the current work, 22 bean genotypes [including recombinant inbred lines (RILs) from the Mist × Sanilac population and a non-nodulating mutant (R99)] were screened for their capacity to fix atmospheric nitrogen under four nitrogen regimes. The genotypes were evaluated in replicated field trials on N-poor soils over three years for the percent nitrogen derived from atmosphere (%Ndfa), yield, and a number of yield-related traits. Bean genotypes differed for all analyzed traits, and the level of nitrogen significantly affected most of the traits, including %Ndfa and yield in all three years. In contrast, application of rhizobia significantly affected only few traits, and the effect was inconsistent among the years. Nitrogen application reduced symbiotic nitrogen fixation (SNF) to various degrees in different bean genotypes. This variation suggests that SNF in common bean can be improved through breeding and selection for the ability of bean genotypes to fix nitrogen in the presence of reduced fertilizer levels. Moreover, genotypes like RIL_38, RIL_119, and RIL_131, being both high yielding and good nitrogen fixers, have potential for simultaneous improvement of both traits. However, breeding advancement might be slow due to an inconsistent correlation between these traits.

Patients with locally/regionally advanced melanoma were treated with neoadjuvant combination immunotherapy with high-dose interferon α-2b (HDI) and ipilimumab in a phase I clinical trial. Tumor specimens were obtained prior to the initiation of neoadjuvant therapy, at the time of surgery and progression if available. In this study, gene expression profiles of tumor specimens (N = 27) were investigated using the NanoString nCounter® platform to evaluate associations with clinical outcomes (pathologic response, radiologic response, relapse-free survival (RFS), and overall survival (OS)) and define biomarkers associated with tumor response. The Tumor Inflammation Signature (TIS), an 18-gene signature that enriches for response to Programmed cell death protein 1 (PD-1) checkpoint blockade, was also evaluated for association with clinical response and survival. It was observed that neoadjuvant ipilimumab-HDI therapy demonstrated an upregulation of immune-related genes, chemokines, and transcription regulator genes involved in immune cell activation, function, or cell proliferation. Importantly, increased expression of baseline pro-inflammatory genes CCL19, CD3D, CD8A, CD22, LY9, IL12RB1, C1S, C7, AMICA1, TIAM1, TIGIT, THY1 was associated with longer OS (p < 0.05). In addition, multiple genes that encode a component or a regulator of the extracellular matrix such as MMP2 and COL1A2 were identified post-treatment as being associated with longer RFS and OS. In all baseline tissues, high TIS scores were associated with longer OS (p = 0.0166). Also, downregulated expression of cell proliferation-related genes such as CUL1, CCND1 and AAMP at baseline was associated with pathological and radiological response (unadjusted p < 0.01). In conclusion, we identified numerous genes that play roles in multiple biological pathways involved in immune activation, immune suppression and cell proliferation correlating with pathological/radiological responses following neoadjuvant immunotherapy highlighting the complexity of immune responses modulated by immunotherapy. Our observations suggest that TIS may be a useful biomarker for predicting survival outcomes with combination immunotherapy.

Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous hematological malignancy. Although immunotherapy may be an attractive modality to exploit in patients with AML, the ability to predict the groups of patients and the types of cancer that will respond to immune targeting remains limited. This study dissected the complexity of the immune architecture of AML at high resolution and assessed its influence on therapeutic response. Using 442 primary bone marrow samples from three independent cohorts of children and adults with AML, we defined immune-infiltrated and immune-depleted disease classes and revealed critical differences in immune gene expression across age groups and molecular disease subtypes. Importantly, interferon (IFN)-γ-related mRNA profiles were predictive for both chemotherapy resistance and response of primary refractory/relapsed AML to flotetuzumab immunotherapy. Our compendium of microenvironmental gene and protein profiles provides insights into the immuno-biology of AML and could inform the delivery of personalized immunotherapies to IFN-γ-dominant AML subtypes.

Metaphors appear in scientific theories, guide scientists, teach students and fascinate the public. This chapter sketches a kind of vocation or métier for scientific metaphors in physics and then applies the same outline to three influential conceptual metaphors in social science – dataset, social field, and dynamical system, along with their respective sub-mappings. All three are in continuous use and often reliant on each other. Using corpora derived from recent social science literature I show how metaphors stimulate hypotheses, then are extended to account for results in successive rounds of observation and theory development, tracing the degree to which each metaphor is useful and retained over the years. Of special interest are supplementary metaphors introduced deliberately to summarize complex source domains.

In this introduction, we start by providing an overview of how metaphor makes science accessible (§ 1). The first part describes the intended readership of this book and introduces them to studies of metaphor in science. We then provide the theoretical foundation for the study of metaphor in science that all of the contributions in this volume are based on: Conceptual Metaphor Theory (§ 2). The third part (§ 3) introduces the three interrelated functions or levels of metaphor that are vital for making science accessible: language, thought, and communication. An overview of contributions to this volume concludes this chapter (§ 4).

This final chapter uses the metaphor characteristics set forth in the introductory chapter to comment on the individual studies reported here. Where the introductory chapter describes the principles of modern metaphor research that promise to improve access to science, this chapter highlights the actual application of these principles as found in the chapters of this book. When we focus on key requirements of scientific inquiry – description, explanation, and prediction – metaphor is found to be both very helpful and sometimes to pose difficulties. Such results are reviewed here, with discussions intended to benefit scientists, communicators, and metaphor scholars.

Background: High grade serous ovarian cancer (HGSC), the most common and aggressive type of ovarian cancer, is nearly always driven by TP53 mutations, which can be detected in uterine lavages using ultra-deep duplex sequencing. While this finding supports the development of a uterine lavage assay for early HGSC detection, age-related TP53 mutations compromise specificity. The goal of this study was to thoroughly characterize the nature of TP53 mutations identified in uterine lavage of women with and without HGSC to better understand TP53 clonal evolution during aging and to determine its association to HGSC. Methods: Uterine lavages were collected preoperatively in 54 “average-risk” women undergoing gynecological surgery for suspicious masses (34 women ultimately had HGSC, 20 were cancer free) and in 98 “high-risk” women undergoing risk-reducing salpingo-oopherectomy (17 were found to have HGSC or serous tubal intraepithelial carcinomas (STICs), 81 were cancer-free). Ultra-deep (>10,000x), ultra-accurate sequencing of TP53 was performed on uterine lavage DNA using duplex sequencing. Tumors and STICs were also duplex sequenced to determine TP53 tumor-driving mutations. The frequency of TP53 mutations and large TP53 mutant clones (those found in >2 duplex reads) was compared between women with and without HGSC. Results: The tumor-specific TP53 mutation was detected in 53% of lavages from average-risk women with HGSC and in 22% of lavages from high-risk women with HGSC/STICs. As in prior studies, TP53 biological background mutations were identified in nearly all women and their frequency was age-related. TP53 background mutations, however, were more abundant in women with prior chemotherapy, regardless of their cancer status or age. In women without prior chemotherapy and without high-risk of HGSC, having HGSC was associated with a significantly higher frequency of large TP53 mutant clones (p=0.007) and large TP53 mutant clones containing pathogenic mutations (p=0.007) or TP53 mutations commonly found in cancer (p=0.001). Interestingly, women at high risk of HGSC also carried high frequency of large and pathogenic TP53 mutant clones in lavage, even in those cases in which cancer or STICs were not identified. Conclusions: Ultra-sensitive duplex sequencing enabled detection of a patient’s HGSC or STIC driver mutations, respectively, in about half and a quarter of uterine lavages. However, of even greater interest, we observed a significant excess of large and pathogenic TP53 mutant clones in lavages of women with HGSC that were not present in the tumor. These results indicate that TP53 somatic evolution is associated with HGSC development and suggest that the measurement of TP53 mutant clones in uterine lavages harbors value as a predictor of ovarian cancer risk. Citation Format: Rosana Risques, Thomas H. Smith, Zachary K. Norgaard, Roniz Katz, Fang Yin Lo, Elizabeth K. Schmidt, Jacob E. Higgins, Martin Filipits, Intidhar Labidi-Galy, David Cibula, Lukáš Dostálek, Gabriel Jelenek, Magdalena Plch, Jiří Bouda, Alexander Mustea, Mateja Condic, Sabine Grill, Noreen Gleeson, Peter Oppelt, Gunda Pristauz-Telsnigg, Adriaan Vanderstichele, Siel Obrecht, Adam Rosenthal, Paul Speiser, Jesse Salk. TP53 field defects in uterine fluid are associated with ovarian cancer risk [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr PR005.

This abstract is being presented as a short talk in the scientific program. A full abstract is available in the Proffered Abstracts section (PR005) of the Conference Proceedings. Citation Format: Rosana Risques, Thomas H. Smith, Zachary K. Norgaard, Roniz Katz, Fang Yin Lo, Elizabeth K. Schmidt, Jacob E. Higgins, Martin Filipits, Intidhar Labidi-Galy, David Cibula, Lukáš Dostálek, Gabriel Jelenek, Magdalena Plch, Jiří Bouda, Alexander Mustea, Mateja Condic, Sabine Grill, Noreen Gleeson, Peter Oppelt, Gunda Pristauz-Telsnigg, Adriaan Vanderstichele, Siel Obrecht, Adam Rosenthal, Paul Speiser, Jesse Salk. TP53 field defects in uterine fluid are associated with ovarian cancer risk [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A008.

Rationale: Accurate and sensitive detection of measurable residual disease (MRD) is critical for clinical management and the development of new therapies for pediatric acute myeloid leukemia (AML). The presence of MRD is a well-established predictor of disease relapse and mortality. Ideally, MRD detection would be both broadly applicable across patients and highly specific, thus enabling clinicians to make timely treatment decisions to improve outcomes. Background: In pediatric AML, unlike adult AML, there are few cross-patient recurrent somatic driver mutations. This makes it impractical to use a fixed gene panel for MRD monitoring. One solution is to perform disease tracking with patient-specific mutations that are present in individual AMLs at time of diagnosis (TOD). However, following modern therapies, should residual disease remain, the telltale mutations will be present in only a very small subpopulation of cells–far below the ~1% limit-of-detection of conventional NGS methods. Duplex sequencing (DS) is an ultra-accurate NGS error-correction method which increases the accuracy of base calls by more than 10,000-fold. Here we demonstrate the use of tumor-informed DS to detect patient-specific MRD in pediatric AML samples with unprecedented sensitivity and specificity. Methods: Marrow aspirates were collected from 13 pediatric AML patients at TOD, during treatment, remission, and at relapse. Among the cohort, 9/13 patients ultimately relapsed. Personalized hybrid capture panels were designed to target hundreds of somatic mutations that were present in each patient’s TOD sample, as identified by whole genome sequencing of tumor-normal pairs. DS was used for longitudinal monitoring of these mutations in blood or marrow at up to 5 time points following initial induction. Results: We detected MRD in one or more samples from all 9 patients who went on to relapse. However, we also observed high-confidence TOD variants in the last collected sample from all 5 patients who did not relapse during at least 24 months of clinical follow-up. As a technical control, we tested a subset of patient panels on a blood sample from a healthy subject and found no disease-specific mutations. Interestingly, we observed a marked degree of heterogeneity of response-to-therapy in the cohort. In some cases, all mutations logarithmically decreased uniformly, whereas in others there was clear dispersion of subclones over time, with some declining 100-times more quickly than others. Conclusions: While DS in conjunction with personalized panels represents a powerful approach for MRD tracking, simply the existence of very low frequency TOD mutations in pediatric AML is not specific enough for predicting relapse without additional interpretation. Lingering mutations in non-relapsers might represent preneoplastic field defects, pre-existing somatic mosaicism, or perhaps even true residual disease under immune suppression. Our study highlights an unappreciated degree of complexity that arises when using ultra-accurate mutation detection for cancer monitoring. Citation Format: Thomas H. Smith, Jake E. Higgins, Fang Y. Lo, Michael J. Hipp, Charles C. Valentine, Amanda R. Leonti, Rhonda E. Ries, Soheil Meshinchi, Benjamin J. Huang, Jesse J. Salk. Longitudinal monitoring of pediatric acute myeloid leukemia using duplex sequencing of patient-specific panels reveals ultra-low frequency MRD that marks persistent carcinogenesis and complex clonal evolution [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A009.

Background: The presence of measurable residual disease (MRD) is strongly associated with treatment outcomes in acute myeloid leukemia (AML). However, > 20% of MRD negative cases (as assessed by flow cytometry) subsequently relapse. We sought to determine if ultrasensitive Duplex Sequencing (DS), which relies on double-stranded consensus-making to achieve an error rate below one-in-ten-million, yields better prognostic performance via molecular MRD detection. Methods: Retrospective targeted DNA sequencing of 29 genes recurrently mutated in adult AML was performed on paired diagnostic and remission bone marrow samples from patients enrolled on the SWOG trial S0106 (randomized 7+3 versus 7+3 + gemtuzumab ozogamicin (GO)). Patients were selected if they had remission samples with flow cytometry results (n=67). Non-error corrected sequencing was performed on diagnostic samples (average depth 279x) and DS was performed on remission samples (average duplex molecular depth 27,002x). For each patient, potential germline variants were identified and excluded from the analysis if the variant allele fraction (VAF) was ≥ 35% at both diagnosis and remission, or ≥ 40% at either time point and a gnomAD allele frequency ≥ 0.05. Somatic variants present at diagnosis were classified as potentially deleterious if computationally predicted as such and with a VAF ≥ 5% (≥ 1% for FLT3-ITD/NPM1 insertions). For analysis of residual disease in remission, we evaluated the following outcomes (events): overall survival (OS; death), relapse-free survival (RFS) and time to relapse (TTR; relapse with death a competing event). All outcomes were measured from date of morphologic remission to date of event, with patients without event censored at date of last contact. Associations between residual disease and outcomes were assessed using Cox regression models (cause-specific model for TTR). Results: The median age was 48 years (range 8-60). 32 patients were randomized to 7+3 and 30 to 7+3+GO. A total of 172 potentially deleterious variants were identified in the diagnostic samples. Variants had an average VAF of 31% (range 1.4-91.5%) at diagnosis and were detected in 23 of the 29 genes, with FLT3 being the most frequently mutated. Of the 67 patients analyzed, 93% (n=62) had at least one variant detected at diagnosis (median 2, range 0-9) and 68% (n=42) had at least one residual diagnostic variant also found in the remission sample. We defined the presence of DS MRD as non-DTA (DNMT3A, TET2, ASXL1) time-of-diagnosis mutations identified at the remission time point with a VAF > 0.1% and/or an NPM1 VAF > 0.01% (PMID:31860405). DS MRD was strongly associated with all outcomes, with hazard ratios (and 95% CI) for TTR: 7.1 (2.7-18.9); RFS: 4.9 (2.2-10.9) and OS: 5.1 (2.1-12.3). As a comparator, we correlated treatment outcomes with the results of a flow cytometry MRD assay previously carried out on the same samples during the S0106 trial. The prognostic association of flow MRD with TTR, RFS and OS was less strong (i.e., a smaller hazard ratio) than DS MRD, with TTR: 2.5 (0.9-6.7); RFS: 2.2 (0.9-5.4) and OS: 2.4 (1.0-6.1). RFS and TTR for DS MRD and flow cytometry are plotted below for the 62 patients with a variant detected at diagnosis. Comparing DS MRD with flow cytometry, discordance was found in 20 cases: 15 cases where DS was positive and flow negative, and 5 cases in the opposite direction. Among the 15 discordant cases with DS positive and flow negative, 9 relapsed and 2 died without relapse and 4 were alive without relapse at last contact. Among the 5 discordant cases with DS negative and flow positive, 1 relapsed, 1 died without relapse, and 3 were alive without relapse at last contact. Conclusions: Among the 67 patients evaluated in this prospectively collected study, the presence of MRD defined by DS was strongly associated with adverse disease outcomes. The vast majority of patients had at least one time-of-diagnosis mutation that could be tracked as a measure of MRD. When compared with flow cytometry, DS exhibited superior negative and positive predictive values for foretelling future relapse, although this could potentially reflect the historical version of the flow cytometry assay used. These findings suggest that DS is a powerful tool that could be used in patient management and for early treatment assessment in clinical trials. Figure 1 Figure 1. Disclosures Hourigan: Sellas: Research Funding. Radich: Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees.

Vascular inflammation and thrombosis require the concerted actions of several different agonists, many of which act on G protein-coupled receptors (GPCRs). GPCR dimerization is a well-established phenomenon that can alter protomer function. In platelets and other cell types, protease-activated receptor-4 (PAR4) has been shown to dimerize with the purinergic receptor P2Y12 to coordinate β-arrestin–mediated Akt signaling, an important mediator of integrin activation. However, the mechanism by which the PAR4-P2Y12 dimer controls β-arrestin–dependent Akt signaling is not known. We now report that PAR4 and P2Y12 heterodimer internalization is required for β-arrestin recruitment to endosomes and Akt signaling. Using bioluminescence resonance energy transfer, immunofluorescence microscopy, and co-immunoprecipitation in cells expressing receptors exogenously and endogenously, we demonstrate that PAR4 and P2Y12 specifically interact and form dimers expressed at the cell surface. We also found that activation of PAR4 but not of P2Y12 drives internalization of the PAR4-P2Y12 heterodimer. Remarkably, activated PAR4 internalization was required for recruitment of β-arrestin to endocytic vesicles, which was dependent on co-expression of P2Y12. Interestingly, stimulation of the PAR4-P2Y12 heterodimer promotes β-arrestin and Akt co-localization to intracellular vesicles. Moreover, activated PAR4-P2Y12 internalization is required for sustained Akt activation. Thus, internalization of the PAR4-P2Y12 heterodimer is necessary for β-arrestin recruitment to endosomes and Akt signaling and lays the foundation for examining whether blockade of PAR4 internalization reduces integrin and platelet activation.

Thrombin, a procoagulant protease, cleaves and activates protease-activated receptor-1 (PAR1) to promote inflammatory responses and endothelial dysfunction. In contrast, activated protein C (APC), an anticoagulant protease, activates PAR1 through a distinct cleavage site and promotes anti-inflammatory responses, prosurvival, and endothelial barrier stabilization. The distinct tethered ligands formed through cleavage of PAR1 by thrombin versus APC result in unique active receptor conformations that bias PAR1 signaling. Despite progress in understanding PAR1 biased signaling, the proteins and pathways utilized by thrombin versus APC signaling to induce opposing cellular functions are largely unknown. Here, we report the global phosphoproteome induced by thrombin and APC signaling in endothelial cells with the quantification of 11,266 unique phosphopeptides using multiplexed quantitative mass spectrometry. Our results reveal unique dynamic phosphoproteome profiles of thrombin and APC signaling, an enrichment of associated biological functions, including key modulators of endothelial barrier function, regulators of gene transcription, and specific kinases predicted to mediate PAR1 biased signaling. Using small interfering RNA to deplete a subset of phosphorylated proteins not previously linked to thrombin or APC signaling, a function for afadin and adducin-1 actin binding proteins in thrombin-induced endothelial barrier disruption is unveiled. Afadin depletion resulted in enhanced thrombin-promoted barrier permeability, whereas adducin-1 depletion completely ablated thrombin-induced barrier disruption without compromising p38 signaling. However, loss of adducin-1 blocked APC-induced Akt signaling. These studies define distinct thrombin and APC dynamic signaling profiles and a rich array of proteins and biological pathways that engender PAR1 biased signaling in endothelial cells.

Assisted reproductive technologies are known to alter the developmental environment of gametes and early embryos during the most dynamic period of establishing the epigenome. This may result in the introduction of errors during active DNA methylation reprogramming. Controlled ovarian hyperstimulation, or superovulation, is a ubiquitously used intervention which has been demonstrated to alter the methylation of certain imprinted genes. The objective of this study was to investigate whether ovarian hyperstimulation results in genome-wide DNA methylation changes in mouse early embryos. Ovarian hyperstimulation was induced by treating mice with either low doses (5 IU) or high doses (10 IU) of PMSG and hCG. Natural mating (NM) control mice received no treatment. Zygotes and 8-cell embryos were collected from each group and DNA methylomes were generated by whole-genome bisulfite sequencing. In the NM group, mean CpG methylation levels slightly decreased from zygote to 8-cell stage, whereas a large decrease in mean CpG methylation level was observed in both superovulated groups. A separate analysis of the mean CpG methylation levels within each developmental stage confirmed that significant genome-wide erasure of CpG methylation from the zygote to 8-cell stage only occurred in the superovulation groups. Our results suggest that superovulation alters the genome-wide DNA methylation erasure process in mouse early pre-implantation embryos. It is not clear whether these changes are transient or persistent. Further studies are ongoing to investigate the impact of ovarian hyperstimulation on DNA methylation re-establishment in later stages of embryo development.

Fathom (CFIA registration no. 7544) is a full-season navy bean (Phaseolus vulgaris L.) cultivar with a high yield potential, and resistance to anthracnose (caused by Colletotrichum lindemuthianum ) and common bacterial blight (CBB; caused by Xanthomonas axonopodis pv. phaseoli). Fathom is recommended for the dry bean growing areas in southwestern Ontario.

The breeding line ACUG10-1 named as Bolt (CFIA registration no. 7366) of common bean (Phaseolus vulgaris L.) is resistant to anthracnose [caused by Colletotrichum lindemuthianum (Sacc. & Magnus)] with an upright plant architecture, suitable for direct harvest and high yield potential. Bolt is recommended for the dry bean growing areas in southwestern Ontario.

Dynasty is a dark red kidney bean (Phaseolus vulgaris L.) cultivar with excellent yield potential and resistant to races 17 and 73 of anthracnose.

Yeti is a full-season white kidney bean (Phaseolus vulgaris L.) cultivar with tall bush plant architecture; high yield potential andgood cooking quality. Yeti is adapted to and recommended for the dry bean growing areas in Southwestern Ontario.

The first 23-step total synthesis of the cyclodepsipeptide dolastatin 16 (1) has been achieved. Synthesis of the dolaphenvaline and dolamethylleuine amino acid units using simplified methods improved the overall efficiency. The formation of the 25-membered macrocycle employing lactonization with 2-methyl-6-nitrobenzoic anhydride completed a key step in the synthesis. Regrettably, the synthetic dolastatin 16 (1), while otherwise identical (by X-ray crystal structure and spectral analyses) with the natural product, did not reproduce the powerful (nanomolar) cancer cell growth inhibition displayed by the natural isolate. Presumably this result can be attributed to conformation(s) of the synthetic dolastatin 16 (1) or to a chemically undetected component isolated with the natural product.

AAC Shock is a high-yielding navy bean (Phaseolus vulgaris L.) cultivar with a semi-upright, indeterminate bush growth habit, large seed size, and resistance to common bacterial blight and races 1 and 15 of bean common mosaic virus. AAC Shock is recommended for dry bean growing areas in Ontario, Canada.

AAC Argosy is a high-yielding navy bean (Phaseolus vulgaris L.) cultivar with an upright, indeterminate bush growth habit, large seed size (23 g 100 seeds−1), and resistance to common bacterial blight and races 1 and 15 of bean common mosaic virus. AAC Argosy is recommended for dry bean growing areas with 2600 or more crop heat units in Ontario, Canada.

Navabi A., Rupert, T., Park, S. J, Yu, K., Smith, T. H. and Pauls, K. P. 2013. Apex common bean. Can. J. Plant Sci. 93: 131–135. Apex (CFIA registration no. 7052) is an indeterminate, full-season navy bean (Phaseolus vulgaris L.) cultivar with an upright plant architecture, suitable for direct harvest, high yield potential, moderate resistance to common bacterial blight (CBB; caused by Xanthomonas axonopodis pv. phaseoli) and superior cooking quality. Apex is adapted to and recommended for the dry bean growing areas with higher than 2700 CHU in southwestern Ontario.

Protease-activated receptor-4 (PAR4) is a G protein-coupled receptor (GPCR) for thrombin and is proteolytically activated, similar to the prototypical PAR1. Due to the irreversible activation of PAR1, receptor trafficking is intimately linked to signal regulation. However, unlike PAR1, the mechanisms that control PAR4 trafficking are not known. Here, we sought to define the mechanisms that control PAR4 trafficking and signaling. In HeLa cells depleted of clathrin by siRNA, activated PAR4 failed to internalize. Consistent with clathrin-mediated endocytosis, expression of a dynamin dominant-negative K44A mutant also blocked activated PAR4 internalization. However, unlike most GPCRs, PAR4 internalization occurred independently of β-arrestins and the receptor's C-tail domain. Rather, we discovered a highly conserved tyrosine-based motif in the third intracellular loop of PAR4 and found that the clathrin adaptor protein complex-2 (AP-2) is important for internalization. Depletion of AP-2 inhibited PAR4 internalization induced by agonist. In addition, mutation of the critical residues of the tyrosine-based motif disrupted agonist-induced PAR4 internalization. Using Dami megakaryocytic cells, we confirmed that AP-2 is required for agonist-induced internalization of endogenous PAR4. Moreover, inhibition of activated PAR4 internalization enhanced ERK1/2 signaling, whereas Akt signaling was markedly diminished. These findings indicate that activated PAR4 internalization requires AP-2 and a tyrosine-based motif and occurs independent of β-arrestins, unlike most classical GPCRs. Moreover, these findings are the first to show that internalization of activated PAR4 is linked to proper ERK1/2 and Akt activation.

Mist is an indeterminate navy bean (Phaseolus vulgaris L.) variety with full season maturity, high yield potential, and resistance to common bacterial blight caused by Xanthomonas axonopodis pv. ph...

OAC Spark is a determinate, early maturing navy bean (Phaseolus vulgaris L.) cultivar with an upright plant architecture and high yield potential. OAC Spark is adapted to and recommended for the dry bean growing areas in the Prairie provinces of Canada.

Lighthouse is an indeterminate, full-season navy bean (Phaseolus vulgaris L.) cultivar with an upright plant architecture, suitable for direct harvest, with a high yield potential, and a high level of resistance to common bacterial blight (CBB; caused by Xanthomonas axonopodis pv. phaseoli). Lighthouse is adapted to and recommended for the dry bean growing areas in southwestern Ontario.

Smith, T. H., Michaels, T. E., Navabi, A. and Pauls, K. P. 2012. OAC Inferno common bean. Can. J. Plant Sci. 92: 589–592. OAC Inferno (CFIA registration no. 7020) is dark red kidney bean (Phaseolus vulgaris L.) cultivar with a determinate bushy growth habit, mid to full season maturity and good yield potential. Seed has acceptable cooking and canning quality.

Three advances necessary to bringing dolastatin 16 (1) into full-scale preclinical development as an anticancer drug have been accomplished. The X-ray crystal structure of dolastatin 16 has been solved, which allowed stereoselective syntheses of its two new amino acid units, dolamethylleuine (Dml) and dolaphenvaline (Dpv), to be completed as summarized in reaction Schemes 1 and 3, respectively. The X-ray crystal structures of synthetic Z-Dml and TFA-Dpv have also been completed.

OAC Dublin (CFIA registration no. 6599) is an indeterminate bush white bean (Phaseolus vulgaris L.) cultivar intended for use in areas with greater than 2600 crop heat units (CHU). It has excellent yield potential in either wide or narrow row production. Its seed has acceptable cooking and canning quality.Key words: Phaseolus vulgaris L., white bean, common bean, cultivar description

Total synthesis of the 18-membered ring cyclodepsipeptide believed to be respirantin (1b) has been achieved. The key step in the synthesis is an intramolecular transesterification of the beta-ketoester alcohol 6 to afford the protected macrocycle 5. The synthetic product was shown to be identical to a natural product presumed to be respirantin (1b), and the absolute stereochemistry of six of the seven asymmetric centers of cyclodepsipeptide 1b was unequivocally established. Respirantin (1b) was found to be a remarkable inhibitor of cancer cell growth and related to the antimycin family of antibiotics.

By utilizing a bioassay-guided separation (P388 lymphocytic leukemia and a panel of human cancer cell lines) of fermentation broths from a Kitasatospora sp. collected from a tundra soil sample taken at the shore of the Beaufort Sea, we have isolated three powerful (GI50 to 0.0006 μg/mL) cancer cell growth inhibitors (1−3) and determined their structures to be closely related cyclodepsipeptides. From 380 L fermentations of Kitasatospora sp. were obtained 2.6 mg of a new cyclodepsipeptide designated kitastatin 1 (3), accompanied by the previously known respirantin (1, 10.8 mg) and its valeryl homologue (2, 4.8 mg). The structures were determined by employment of a series of high-resolution mass and 2D NMR spectroscopic analyses. The stereochemical assignments and overall structures were confirmed by subsequent total synthesis of depsipeptide 1, as reported in the accompanying contribution.

K63-linked ubiquitination of GPCRs mediated by the NEDD4-2 E3 ubiquitin ligase regulates recruitment of a TAB1–TAB2 complex on endosomes and stimulates p38 MAPK through a noncanonical pathway, which is critical for endothelial barrier disruption., Protease-activated receptor 1 (PAR1) is a G protein–coupled receptor (GPCR) for thrombin and promotes inflammatory responses through multiple pathways including p38 mitogen-activated protein kinase signaling. The mechanisms that govern PAR1-induced p38 activation remain unclear. Here, we define an atypical ubiquitin-dependent pathway for p38 activation used by PAR1 that regulates endothelial barrier permeability. Activated PAR1 K63-linked ubiquitination is mediated by the NEDD4-2 E3 ubiquitin ligase and initiated recruitment of transforming growth factor-β–activated protein kinase-1 binding protein-2 (TAB2). The ubiquitin-binding domain of TAB2 was essential for recruitment to PAR1-containing endosomes. TAB2 associated with TAB1, which induced p38 activation independent of MKK3 and MKK6. The P2Y1 purinergic GPCR also stimulated p38 activation via NEDD4-2–mediated ubiquitination and TAB1–TAB2. TAB1–TAB2-dependent p38 activation was critical for PAR1-promoted endothelial barrier permeability in vitro, and p38 signaling was required for PAR1-induced vascular leakage in vivo. These studies define an atypical ubiquitin-mediated signaling pathway used by a subset of GPCRs that regulates endosomal p38 signaling and endothelial barrier disruption.

Protease-activated receptor-1 (PAR1) is a G-protein-coupled receptor (GPCR) for the coagulant protease thrombin. Similar to other GPCRs, PAR1 is promiscuous and couples to multiple heterotrimeric G-protein subtypes in the same cell and promotes diverse cellular responses. The molecular mechanism by which activation of a given GPCR with the same ligand permits coupling to multiple G-protein subtypes is unclear. Here, we report that N-linked glycosylation of PAR1 at extracellular loop 2 (ECL2) controls G12/13 versus Gq coupling specificity in response to thrombin stimulation. A PAR1 mutant deficient in glycosylation at ECL2 was more effective at stimulating Gq-mediated phosphoinositide signaling compared with glycosylated wildtype receptor. In contrast, wildtype PAR1 displayed a greater efficacy at G12/13-dependent RhoA activation compared with mutant receptor lacking glycosylation at ECL2. Endogenous PAR1 rendered deficient in glycosylation using tunicamycin, a glycoprotein synthesis inhibitor, also exhibited increased PI signaling and diminished RhoA activation opposite to native receptor. Remarkably, PAR1 wildtype and glycosylation-deficient mutant were equally effective at coupling to Gi and β-arrestin-1. Consistent with preferential G12/13 coupling, thrombin-stimulated PAR1 wildtype strongly induced RhoA-mediated stress fiber formation compared with mutant receptor. In striking contrast, glycosylation-deficient PAR1 was more effective at increasing cellular proliferation, associated with Gq signaling, than wildtype receptor. These studies suggest that N-linked glycosylation at ECL2 contributes to the stabilization of an active PAR1 state that preferentially couples to G12/13 versus Gq and defines a previously unidentified function for N-linked glycosylation of GPCRs in regulating G-protein signaling bias.

OAC Rex is an upright indeterminate bush white bean (Phaseolus vulgaris L.) cultivar intended for use in areas with greater than 2800 crop heat units. It has good yield potential in either wide or narrow row production. It is resistant to races 1 and 15 of bean common mosaic virus and is the first common bean cultivar resistant to common bacterial blight (Xanthomonas axonopodis pv. phaseoli) in Ontario. Seed has acceptable cooking and canning quality. Key words: Phaseolus vulgaris L., white bean, common bean, common bacterial blight, cultivar description