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The transcription factor Prdm16 functions as a potent suppressor of transforming growth factor-beta (TGF-β) signaling, whose inactivation is deemed essential to the progression of pancreatic ductal adenocarcinoma (PDAC). Using the KrasG12D-based mouse model of human PDAC, we surprisingly found that ablating Prdm16 did not block but instead accelerated PDAC formation and progression, suggesting that Prdm16 might function as a tumor suppressor in this malignancy. Subsequent genetic experiments showed that ablating Prdm16 along with Smad4 resulted in a shift from a well-differentiated and confined neoplasm to a highly aggressive and metastatic disease, which was associated with a striking deviation in the trajectory of the premalignant lesions. Mechanistically, we found that Smad4 interacted with and recruited Prdm16 to repress its own expression, therefore pinpointing a model in which Prdm16 functions downstream of Smad4 to constrain the PDAC malignant phenotype. Collectively, these findings unveil an unprecedented antagonistic interaction between the tumor suppressors Smad4 and Prdm16 that functions to restrict PDAC progression and metastasis.

A prominent function of TGIF1 is suppression of transforming growth factor beta (TGF‐β) signaling, whose inactivation is deemed instrumental to the progression of pancreatic ductal adenocarcinoma (PDAC), as exemplified by the frequent loss of the tumor suppressor gene SMAD4 in this malignancy. Surprisingly, we found that genetic inactivation of Tgif1 in the context of oncogenic Kras, Kras(G12D), culminated in the development of highly aggressive and metastatic PDAC despite de‐repressing TGF‐β signaling. Mechanistic experiments show that TGIF1 associates with Twist1 and inhibits Twist1 expression and activity, and this function is suppressed in the vast majority of human PDACs by Kras(G12D)/MAPK‐mediated TGIF1 phosphorylation. Ablating Twist1 in Kras (G12D) ;Tgif1 (KO) mice completely blunted PDAC formation, providing the proof‐of‐principle that TGIF1 restrains Kras(G12D)‐driven PDAC through its ability to antagonize Twist1. Collectively, these findings pinpoint TGIF1 as a potential tumor suppressor in PDAC and further suggest that sustained activation of TGF‐β signaling might act to accelerate PDAC progression rather than to suppress its initiation.

Understanding the molecular mechanisms underlying insect compensatory responses to plant defenses could lead to improved plant resistance to herbivores. The Mp708 inbred line of maize produces the maize insect resistant 1-cysteine protease (Mir1-CP) toxin. Reduced feeding and growth of fall armyworm larvae fed on Mp708 was previously linked to impairment of nutrient utilization and degradation of the midgut (MG) peritrophic matrix (PM) by Mir1-CP. Here we examine the biochemical and transcriptional responses of fall armyworm larvae to Mir1-CP. Insect Intestinal Mucin (IIM) was severely depleted from pure PMs treated in vitro with recombinant Mir1-CP. Larvae fed on Mp708 midwhorls excrete frass largely depleted of IIM. Cracks, fissures and increased porosity previously observed in the PM of larvae fed on Mp708 midwhorls could ensue when Mir1-CP degrades the IIM that cross-links chitin fibrils in the PM. Both targeted and global transcriptome analyses were performed to determine how complete dissolution of the structure and function of the PM is prevented, enabling larvae to continue growing in the presence of Mir1-CP. The MGs from fall armyworm fed on Mp708 upregulate expression of genes encoding proteins involved in PM production as an apparent compensation to replace the disrupted PM structure and restore appropriate counter-current MG gradients. Also, several families of digestive enzymes (endopeptidases, aminopeptidases, lipases, amylase) were more highly expressed in MGs from larvae fed on Mp708 than MGs from larvae fed on diets lacking Mir1-CP (artificial diet, midwhorls from Tx601 or B73 maize). Impaired growth of larvae fed on Mp708 probably results from metabolic costs associated with higher production of PM constituents and digestive enzymes in a compensatory attempt to maintain MG function.

Aflatoxin, the most potent carcinogen found in nature, is produced by the fungusAspergillus flavusand occurs naturally in maize,Zea maysL. Growing maize hybrids with genetic resistance to aflatoxin contamination are generally considered a highly desirable way to reduce losses to aflatoxin. Developing resistant hybrids requires reliable inoculation methods for screening maize germplasm for resistance toA. flavusinfection and aflatoxin accumulation. The side-needle technique is a widely used inoculation technique: anA. flavusconidial suspension is injected underneath the husks into the side of the ear. This wounds the ear and limits expression of resistance associated with husk coverage, pericarp thickness, and seed coat integrity. In this investigation, the side-needle technique was compared with a second inoculation method that involved dispensing wheat kernels infected withA. flavusinto plant whorls at 35 and 49 days after planting. Results showed that although the side-needle technique produced higher levels of aflatoxin accumulation, differences inA. flavusbiomass produced by the two inoculation techniques were not significant. Both inoculation techniques were effective in differentiating resistant and susceptible single cross hybrids irrespective of the use ofA. flavusinfection or aflatoxin accumulation as a basis to define resistance.

Aflatoxins, which are produced by Aspergillus flavus, are toxic to humans, livestock, and pets. The value of maize (Zea mays) grain is markedly reduced when contaminated with aflatoxin. Plant resistance and biological control using non-toxin producing strains are considered effective strategies for reducing aflatoxin accumulation in maize grain. Distinguishing between the toxin and non-toxin producing strains is important in determining the effectiveness of bio-control strategies and understanding inter-strain interactions. Using polymorphisms found in the fungal rRNA intergenic spacer region (IGS) between a toxigenic strain of A. flavus (NRRL 3357) and the non-toxigenic strain used in the biological control agent Afla-Guard(®) (NRRL 21882), we developed a set of primers that allows for the identification and quantification of the two strains using quantitative PCR. This primer set has been used to screen maize grain that was inoculated with the two strains individually and co-inoculated with both strains, and it has been shown to be effective in both the identification and quantification of both strains. Screening of co-inoculated ears from multiple resistant and susceptible genotypic crosses revealed no significant differences in fungal biomass accumulation of either strain in the field tests from 2010 and 2011 when compared across the means of all genotypes. Only one genotype/year combination showed significant differences in strain accumulation. Aflatoxin accumulation analysis showed that, as expected, genotypes inoculated with the toxigenic strain accumulated more aflatoxin than when co-inoculated with both strains or inoculated with only the non-toxigenic strain. Furthermore, accumulation of toxigenic fungal mass was significantly correlated with aflatoxin accumulation while non-toxigenic fungal accumulation was not. This primer set will allow researchers to better determine how the two fungal strains compete on the maize ear and investigate the interaction between different maize lines and these A. flavus strains.

Fatty pancreas or pancreatic steatosis has been associated with a variety of debilitating diseases, such as obesity, diabetes, pancreatitis, and pancreatic ductal adenocarcinoma (PDAC). Currently, neither the mechanisms underlying the pathogenesis of fatty pancreas nor the physiopathologic impacts of this disorder are well established. Serendipitously, we found that conditional overexpression of Twist1 in pancreatic progenitor cells in mice resulted in fatty pancreas development, leading to pancreatic insufficiency, severe hyperglycemia, and growth retardation. Molecular and genetic studies demonstrated that Twist1 drives fatty pancreas formation by selectively reprogramming acinar cells towards the adipocyte lineage albeit without affecting islet or ductal cell fate. Moreover, loss-of-function genetic experiments demonstrated that Twist1 plays a causative role in glucose tolerance, pancreatitis genesis, and PDAC progression. Finally, high levels of Twist1 expression correlate with fatty pancreas formation in patients with pancreatitis or PDAC, implicating Twist1 as a prognostic marker and possible target for attenuating several lethal diseases associated with fatty pancreas development. Citation Format: Thien Ly Nguyen, Purba Singh, Parash Parajuli, Lianna Li, Celine Prunier, Santosh Kumar, Sailaja Eragmerdi, Seval Ozkan, Hao Me, Jussara do Carmo, John Hall, Azeddine Atfi. Twist1-driven fatty pancreas formation facilitates pancreatitis and pancreatic ductal adenocarcinoma progression [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A38.

Cancer cachexia is characterized by extreme skeletal muscle loss that results in high morbidity and mortality. The incidence of cachexia varies among tumor types, being lowest in sarcomas, whereas 90% of pancreatic ductal adenocarcinoma (PDAC) patients experience severe weight loss. How these tumors trigger muscle depletion is still unfolding. Serendipitously, we found that overexpression of Twist1 in mouse muscle progenitor cells, either constitutively during development or inducibly in adult animals, caused severe muscle atrophy with features reminiscent of cachexia. Using several genetic mouse models of PDAC, we detected a massive increase in Twist1 expression in muscle undergoing cachexia. In cancer patients, elevated levels of Twist1 are associated with greater degrees of muscle wasting. Both genetic and pharmacologic inactivation of Twist1 in muscle progenitor cells afforded substantial protection against cancer-mediated cachexia, which translated into meaningful survival benefits, implicating Twist1 as a possible target for attenuating muscle cachexia in cancer patients. Citation Format: Parash Parajuli, Santosh Kumar, Audrey Loumaye, Purba Singh, Sailaja Eragamreddy, Thien Ly Nguyen, Seval Ozkan, Mohammed S. Razzaqe, Céline Prunier, Jean-Paul Thissen, Azeddine Atfi. Twist1 activation in muscle progenitor cells during development or adulthood causes severe muscle loss reminiscent of human cancer cachexia [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A40.

Understanding the role of phytohormone auxin in defense responses is one of the vital tools for plant breeders to develop maize germplasm lines that exhibit high resistance to Aspergillus flavus and subsequent aflatoxin accumulation. Besides its critical role in different developmental processes throughout the life cycle of plants, auxin is also involved in the network of plant-pathogen interaction as demonstrated in previous studies. However, the actual mechanism for the auxin signaling pathway leading to resistance is unknown. Therefore, the critical gap in the knowledge base is a lack of understanding of the role of auxin signaling in pathogen resistance in maize. Continuation of this gap is an important problem because fungal resistance is a highly quantitative trait and breeding for resistance is a challenge. A complete understanding of the auxin mechanism in resistance could lead the production of corn hybrids with resistance to A. flavus and aflatoxin accumulation. The focus of this research was to determine the effect of exogenous auxin on A. flavus growth and production of aflatoxin in growth media. In addition, auxin levels, the amount of aflatoxin, and fungal growth in three resistant (Mp313E, Mp715, and Mp719) and one susceptible (B73) germplasm line were determined. As a result, auxin significantly increased mycelium growth and significantly decreased aflatoxin at a high concentration in potato dextrose broth under the lab conditions. Under the field conditions, auxin levels were low in resistant lines but did not change in response to A. flavus infection. Susceptible line had high auxin levels and auxin levels significantly decreased in response to A. flavus infection.

The Gene Ontology (GO) has become the internationally accepted standard for representing function, process, and location aspects of gene products. The wealth of GO annotation data provides a valuable source of implicit knowledge of relationships among these aspects. We describe a new method for association rule mining to discover implicit co-occurrence relationships across the GO sub-ontologies at multiple levels of abstraction. Prior work on association rule mining in the GO has concentrated on mining knowledge at a single level of abstraction and/or between terms from the same sub-ontology. We have developed a bottom-up generalization procedure called Cross-Ontology Data Mining-Level by Level (COLL) that takes into account the structure and semantics of the GO, generates generalized transactions from annotation data and mines interesting multi-level cross-ontology association rules. We applied our method on publicly available chicken and mouse GO annotation datasets and mined 5368 and 3959 multi-level cross ontology rules from the two datasets respectively. We show that our approach discovers more and higher quality association rules from the GO as evaluated by biologists in comparison to previously published methods. Biologically interesting rules discovered by our method reveal unknown and surprising knowledge about co-occurring GO terms.

A public candidate gene testing pipeline for resistance to aflatoxin accumulation or Aspergillus flavus infection in maize is presented here. The pipeline consists of steps for identifying, testing, and verifying the association of selected maize gene sequences with resistance under field conditions. Resources include a database of genetic and protein sequences associated with the reduction in aflatoxin contamination from previous studies, eight diverse inbred maize lines for polymorphism identification within any maize gene sequence, four Quantitative Trait Loci (QTL) mapping populations and one association mapping panel, all phenotyped for aflatoxin accumulation resistance and associated phenotypes, and capacity for Insertion/Deletion (InDel) and SNP genotyping in the population(s) for mapping. To date, ten genes have been identified as possible candidate genes and put through the candidate gene testing pipeline, and results are presented here to demonstrate the utility of the pipeline.

Loblolly pine (LP; Pinus taeda L.) is the most economically important tree in the U.S. and a cornerstone species in southeastern forests. However, genomics research on LP and other conifers has lagged behind studies on flowering plants due, in part, to the large size of conifer genomes. As a means to accelerate conifer genome research, we constructed a BAC library for the LP genotype 7-56. The LP BAC library consists of 1,824,768 individually-archived clones making it the largest single BAC library constructed to date, has a mean insert size of 96 kb, and affords 7.6X coverage of the 21.7 Gb LP genome. To demonstrate the efficacy of the library in gene isolation, we screened macroarrays with overgos designed from a pine EST anchored on LP chromosome 10. A positive BAC was sequenced and found to contain the expected full-length target gene, several gene-like regions, and both known and novel repeats. Macroarray analysis using the retrotransposon IFG-7 (the most abundant repeat in the sequenced BAC) as a probe indicates that IFG-7 is found in roughly 210,557 copies and constitutes about 5.8% or 1.26 Gb of LP nuclear DNA; this DNA quantity is eight times the Arabidopsis genome. In addition to its use in genome characterization and gene isolation as demonstrated herein, the BAC library should hasten whole genome sequencing of LP via next-generation sequencing strategies/technologies and facilitate improvement of trees through molecular breeding and genetic engineering. The library and associated products are distributed by the Clemson University Genomics Institute (www.genome.clemson.edu).

AgBase (http://www.agbase.msstate.edu/) provides resources to facilitate modeling of functional genomics data and structural and functional annotation of agriculturally important animal, plant, microbe and parasite genomes. The website is redesigned to improve accessibility and ease of use, including improved search capabilities. Expanded capabilities include new dedicated pages for horse, cat, dog, cotton, rice and soybean. We currently provide 590 240 Gene Ontology (GO) annotations to 105 454 gene products in 64 different species, including GO annotations linked to transcripts represented on agricultural microarrays. For many of these arrays, this provides the only functional annotation available. GO annotations are available for download and we provide comprehensive, species-specific GO annotation files for 18 different organisms. The tools available at AgBase have been expanded and several existing tools improved based upon user feedback. One of seven new tools available at AgBase, GOModeler, supports hypothesis testing from functional genomics data. We host several associated databases and provide genome browsers for three agricultural pathogens. Moreover, we provide comprehensive training resources (including worked examples and tutorials) via links to Educational Resources at the AgBase website.

Background Aspergillus flavus Link:Fr, an opportunistic fungus that produces aflatoxin, is pathogenic to maize and other oilseed crops. Aflatoxin is a potent carcinogen, and its presence markedly reduces the value of grain. Understanding and enhancing host resistance to A. flavus infection and/or subsequent aflatoxin accumulation is generally considered an efficient means of reducing grain losses to aflatoxin. Different proteomic, genomic and genetic studies of maize (Zea mays L.) have generated large data sets with the goal of identifying genes responsible for conferring resistance to A. flavus, or aflatoxin. Results In order to maximize the usage of different data sets in new studies, including association mapping, we have constructed a relational database with web interface integrating the results of gene expression, proteomic (both gel-based and shotgun), Quantitative Trait Loci (QTL) genetic mapping studies, and sequence data from the literature to facilitate selection of candidate genes for continued investigation. The Corn Fungal Resistance Associated Sequences Database (CFRAS-DB) (http://agbase.msstate.edu/) was created with the main goal of identifying genes important to aflatoxin resistance. CFRAS-DB is implemented using MySQL as the relational database management system running on a Linux server, using an Apache web server, and Perl CGI scripts as the web interface. The database and the associated web-based interface allow researchers to examine many lines of evidence (e.g. microarray, proteomics, QTL studies, SNP data) to assess the potential role of a gene or group of genes in the response of different maize lines to A. flavus infection and subsequent production of aflatoxin by the fungus. Conclusions CFRAS-DB provides the first opportunity to integrate data pertaining to the problem of A. flavus and aflatoxin resistance in maize in one resource and to support queries across different datasets. The web-based interface gives researchers different query options for mining the database across different types of experiments. The database is publically available at http://agbase.msstate.edu.