Interactive transcriptome analyses of Northern Wild Rice (Zizania palustris L.) and Bipolaris oryzae show convoluted communications during the early stages of fungal brown spot development.

Fungal diseases, caused mainly by Bipolaris spp., are past and current threats to Northern Wild Rice (NWR) grain production and germplasm preservation in both natural and cultivated settings. Genetic resistance against the pathogen is scarce. Toward expanding our understanding of the global gene communications of NWR and Bipolaris oryzae interaction, we designed an RNA sequencing study encompassing the first 12 h and 48 h of their encounter. NWR activated numerous plant recognition receptors after pathogen infection, followed by active transcriptional reprogramming of signaling mechanisms driven by Ca2+ and its sensors, mitogen-activated protein kinase cascades, activation of an oxidative burst, and phytohormone signaling-bound mechanisms. Several transcription factors associated with plant defense were found to be expressed. Importantly, evidence of diterpenoid phytoalexins, especially phytocassane biosynthesis, among expression of other defense genes was found. In B. oryzae, predicted genes associated with pathogenicity including secreted effectors that could target plant defense mechanisms were expressed. This study uncovered the early molecular communication between the NWR-B. oryzae pathosystem, which could guide selection for allele-specific genes to boost NWR defenses, and overall aid in the development of more efficient selection methods in NWR breeding through the use of the most virulent fungal isolates.

Community advisory board members' perspectives on their contributions to a large multistate cluster RCT: a mixed methods study.

Background: Community advisory boards (CABs) are an established approach to ensuring research reflects community priorities. This paper examines two CABs that are part of the HEALing Communities Study which aims to reduce overdose mortality. This analysis aimed to understand CAB members' expectations, experiences, and perspectives on CAB structure, communication, facilitation, and effectiveness during the first year of an almost fully remote CAB implementation. Current literature exploring these perspectives is limited. Methods: We collected qualitative and survey data simultaneously from members (n = 53) of two sites' CABs in the first 9 months of CAB development. The survey assessed trust, communication, and relations; we also conducted 32 semi-structured interviews. We analyzed the survey results descriptively. The qualitative data were analyzed using a deductive codebook based on the RE-AIM PRISM framework. Themes were drawn from the combined qualitative data and triangulated with survey results to further enrich the findings. Results: CAB members expressed strong commitment to overall study goals and valued the representation of occupational sectors. The qualitative data described a dissonance between CAB members' commitment to the mission and unmet expectations for influencing the study within an advisory role. Survey results indicated lower satisfaction with the research teams' ability to create a mutually beneficial process, clear communication, and sharing of power. Conclusion: Building a CAB on a remote platform, within a study utilizing a community engagement strategy, still presents challenges to fully realizing the potential of a CAB. These findings can inform more effective operationalizing of community-engaged research through enhanced CAB engagement.

Impact of polyphenol oxidase on the bioavailability of flavan-3-ols in fruit smoothies: a controlled, single blinded, cross-over study.

Flavan-3-ols are bioactive compounds found in a variety of fruits and vegetables (F&V) that have been linked to positive health benefits. Increasing habitual flavan-3-ol intake is challenged by the generally low consumption of F&V. While smoothies are a commonly endorsed, consumer-accepted means to increase the daily intake of these important foods, fruits used for smoothie preparation can have a high polyphenol oxidase (PPO) activity and thus potentially affect the content and bioavailability of flavan-3-ols. To assess whether or not consuming freshly prepared smoothies made with different PPO-containing fruit impacts the bioavailability of the flavan-3-ols, a controlled, single blinded and cross-over study was conducted in healthy men (n = 8) who consumed a flavan-3-ol-containing banana-based smoothie (high-PPO drink), a flavan-3-ol-containing mixed berry smoothie (low-PPO drink) and flavan-3-ols in a capsule format (control). The peak plasma concentration (Cmax) of flavan-3-ol metabolites after capsule intake was 680 ± 78 nmol L-1, which was similar to the levels detected after the intake of the low PPO drink. In contrast, the intake of the high PPO drink resulted in a Cmax of 96 ± 47 nmol L-1, 84% lower than that obtained after capsule intake. In a subsequent study (n = 11), flavan-3-ols were co-ingested with a high-PPO banana drink but contact prior to intake was prevented. In this context, plasma flavan-3-ol levels were still reduced, suggesting an effect possibly related to post-ingestion PPO activity degrading flavan-3-ols in the stomach. There was a substantial range in the PPO activity detected in 18 different fruits, vegetables and plant-derived dietary products. In conclusion, bioavailability of flavan-3-ols, and most likely other dietary polyphenol bioactives, can be reduced substantially by the co-ingestion of high PPO-containing products, the implications of which are of importance for dietary advice and food preparation both at home and in industrial settings.

Flavan-3-ol-methylxanthine interactions: Modulation of flavan-3-ol bioavailability in volunteers with a functional colon and an ileostomy.

Flavan-3-ols, including the flavan-3-ol monomer (-)-epicatechin, are dietary bioactives known to mediate beneficial cardiovascular effects in humans. Recent studies showed that flavan-3-ols could interact with methylxanthines, evidenced by an increase in flavan-3-ol bioavailability with a concomitant increase in flavan-3-ol intake-mediated vascular effects. This study aimed at elucidating flavan-3-ol-methylxanthine interactions in humans in vivo by evaluating the specific contributions of theobromine and caffeine on flavan-3-ol bioavailability. In ileostomists, the effect of methylxanthines on the efflux of flavan-3-ol metabolites in the small intestine was assessed, a parameter important to an understanding of the pharmacokinetics of flavan-3-ols in humans. In a randomized, controlled, triple cross-over study in volunteers with a functional colon (n = 10), co-ingestion of flavan-3-ols and cocoa methylxanthines, mainly represented by theobromine, increased peak circulatory levels (Cmax) of flavan-3-ols metabolites (+21 ± 8%; p < 0.05). Conversely, caffeine did not mediate a statistically significant effect on flavan-3-ol bioavailability (Cmax = +10 ± 8%, p = n.s.). In a subsequent randomized, controlled, double cross-over study in ileostomists (n = 10), cocoa methylxanthines did not affect circulatory levels of flavan-3-ol metabolites, suggesting potential differences in flavan-3-ol bioavailability compared to volunteers with a functional colon. The main metabolite in ileal fluid was (-)-epicatechin-3'-sulfate, however, no differences in flavan-3-ol metabolites in ileal fluid were observed after flavan-3-ol intake with and without cocoa methylxanthines. Taken together, these results demonstrate a differential effect of caffeine and theobromine in modulating flavan-3-ol bioavailability when these bioactives are co-ingested. These findings should be considered when comparing the effects mediated by the intake of flavan-3-ol-containing foods and beverages and the amount and type of methylxanthines present in the ingested matrixes. Ultimately, these insights will be of value to further optimize current dietary recommendations for flavan-3-ol intake. CLINICAL TRIAL REGISTRATION NUMBER: This work was registered at clinicaltrials.gov as NCT03526107 (study part 1, volunteers with functional colon) and NCT03765606 (study part 2, volunteers with an ileostomy).

Phenotypic Variation and the Impact of Admixture in the Oryza rufipogon Species Complex (ORSC).

Crop wild relatives represent valuable reservoirs of variation for breeding, but their populations are threatened in natural habitats, are sparsely represented in genebanks, and most are poorly characterized. The focus of this study is the Oryza rufipogon species complex (ORSC), wild progenitor of Asian rice (Oryza sativa L.). The ORSC comprises perennial, annual and intermediate forms which were historically designated as O. rufipogon, O. nivara, and O. sativa f. spontanea (or Oryza spp., an annual form of mixed O. rufipogon/O. nivara and O. sativa ancestry), respectively, based on non-standardized morphological, geographical, and/or ecologically-based species definitions and boundaries. Here, a collection of 240 diverse ORSC accessions, characterized by genotyping-by-sequencing (113,739 SNPs), was phenotyped for 44 traits associated with plant, panicle, and seed morphology in the screenhouse at the International Rice Research Institute, Philippines. These traits included heritable phenotypes often recorded as characterization data by genebanks. Over 100 of these ORSC accessions were also phenotyped in the greenhouse for 18 traits in Stuttgart, Arkansas, and 16 traits in Ithaca, New York, United States. We implemented a Bayesian Gaussian mixture model to infer accession groups from a subset of these phenotypic data and ascertained three phenotype-based group assignments. We used concordance between the genotypic subpopulations and these phenotype-based groups to identify a suite of phenotypic traits that could reliably differentiate the ORSC populations, whether measured in tropical or temperate regions. The traits provide insight into plant morphology, life history (perenniality versus annuality) and mating habit (self- versus cross-pollinated), and are largely consistent with genebank species designations. One phenotypic group contains predominantly O. rufipogon accessions characterized as perennial and largely out-crossing and one contains predominantly O. nivara accessions characterized as annual and largely inbreeding. From these groups, 42 "core" O. rufipogon and 25 "core" O. nivara accessions were identified for domestication studies. The third group, comprising 20% of our collection, has the most accessions identified as Oryza spp. (51.2%) and levels of O. sativa admixture accounting for more than 50% of the genome. This third group is potentially useful as a "pre-breeding" pool for breeders attempting to incorporate novel variation into elite breeding lines.

Absorption, distribution, metabolism and excretion of apigenin and its glycosides in healthy male adults.

The bioavailability of apigenin and its O-glycosides in humans was investigated with apigenin-4'-glucuronide (Ap-4'-GlcUA), apigenin-7-glucuronide and apigenin-7-sulfate being identified as in vivo metabolites. Apigenin per se was poorly absorbed with metabolites equivalent to 0.5% of intake excreted in urine 0-24 h post-intake. Consumption of a parsley drink containing apigenin-7-O-(2″-O-apiosyl)glucoside resulted in the peak plasma concentration (Cmax) of Ap-4'-GlcUA occurring after 4 h, indicative of absorption in the lower gastrointestinal tract (GIT). Urinary excretion of the three metabolites corresponded to 11.2% of intake. Ingestion of dried powdered parsley leaves with yogurt extended the Cmax of Ap-4'-GlcUA to 6 h. Consumption of chamomile tea containing apigenin-7'-O-glucoside resulted in a 2 h Cmax of Ap-4'-GlcUA, in keeping with absorption in the upper GIT. Urinary excretion was equivalent to 34% of intake. Intake of the parsley drink provided information on intra- and inter-individual variations in the level of excretion of the apigenin metabolites. CLINICAL TRAIL REGISTRATION NUMBER: This trail was registered at clinicaltrials.gov as NCT03526081.

Whole-genome assembly and annotation of northern wild rice, Zizania palustris L., supports a whole-genome duplication in the Zizania genus.

Zizania palustris L. (northern wild rice, NWR) is an aquatic grass native to North America that is notable for its nutritious grain. This is an important species with ecological, cultural and agricultural significance, specifically in the Great Lakes region of the USA. Using flow cytometry, we first estimated the NWR genome size to be 1.8 Gb. Using long- and short-range sequencing, Hi-C scaffolding and RNA-seq data from eight tissues, we generated an annotated whole-genome de novo assembly of NWR. The assembly was 1.29 Gb in length, highly repetitive (approx. 76.0%) and contained 46 421 putative protein-coding genes. The expansion of retrotransposons within the genome and a whole-genome duplication (WGD) after the Zizania-Oryza speciation event have both led to an increase in the genome size of NWR in comparison with Oryza sativa L. and Zizania latifolia. Both events depict a genome rapidly undergoing change over a short evolutionary time. Comparative analyses revealed the conservation of large syntenic blocks between NWR and O. sativa, which were used to identify putative seed-shattering genes. Estimates of divergence times revealed that the Zizania genus diverged from Oryza approximately 26-30 million years ago (26-30 MYA), whereas NWR and Z. latifolia diverged from one another approximately 6-8 MYA. Comparative genomics confirmed evidence of a WGD in the Zizania genus and provided support that the event occurred prior to the NWR-Z. latifolia speciation event. This genome assembly and annotation provides a valuable resource for comparative genomics in the Oryzeae tribe and provides an important resource for future conservation and breeding efforts of NWR.

Genome-wide association analysis of the strength of the MAMP-elicited defense response and resistance to target leaf spot in sorghum.

Plants have the capacity to respond to conserved molecular features known as microbe-associated molecular patterns (MAMPs). The goal of this work was to assess variation in the MAMP response in sorghum, to map loci associated with this variation, and to investigate possible connections with variation in quantitative disease resistance. Using an assay that measures the production of reactive oxygen species, we assessed variation in the MAMP response in a sorghum association mapping population known as the sorghum conversion population (SCP). We identified consistent variation for the response to chitin and flg22-an epitope of flagellin. We identified two SNP loci associated with variation in the flg22 response and one with the chitin response. We also assessed resistance to Target Leaf Spot (TLS) disease caused by the necrotrophic fungus Bipolaris cookei in the SCP. We identified one strong association on chromosome 5 near a previously characterized disease resistance gene. A moderately significant correlation was observed between stronger flg22 response and lower TLS resistance. Possible reasons for this are discussed.

Herbaspirillum rubrisubalbicans as a Phytopathogenic Model to Study the Immune System of Sorghum bicolor.

Herbaspirillum rubrisubalbicans is the causal agent of red stripe disease (RSD) and mottle stripe disease of sorghum and sugarcane, respectively. In all, 63 genotypes of Sorghum bicolor were inoculated with H. rubrisubalbicans, with 59 showing RSD symptoms. Quantitative trait loci (QTL) analysis in a recombinant inbred line (RIL) population identified several QTL associated with variation in resistance to RSD. RNA sequencing analysis identified a number of genes whose transcript levels were differentially regulated during H. rubrisubalbicans infection. Among those genes that responded to H. rubrisubalbicans inoculation were many involved in plant-pathogen interactions such as leucine-rich repeat receptors, mitogen-activated protein kinase 1, calcium-binding proteins, transcriptional factors (ethylene-responsive element binding factor), and callose synthase. Pretreatment of sorghum leaves with the pathogen-associated molecular pattern (PAMP) molecules flg22 and chitooctaose provided protection against subsequent challenge with the pathogen, suggesting that PAMP-triggered immunity plays an important role in the sorghum immunity response. These data present baseline information for the use of the genetically tractable H. rubrisubalbicans-sorghum pathosystem for the study of innate immunity and disease resistance in this important grain and bioenergy crop. Information gained from the use of this system is likely to be informative for other monocots, including those more intractable for experimental study (e.g., sugarcane).

Identification of QTL for Target Leaf Spot resistance in Sorghum bicolor and investigation of relationships between disease resistance and variation in the MAMP response.

Target leaf spot (TLS) of sorghum, a foliar disease caused by the necrotrophic fungus Bipolaris cookei (also known as Bipolaris sorghicola), can affect grain yield in sorghum by causing premature drying of leaves and defoliation. Two sorghum recombinant inbred line (RIL) populations, BTx623/BTx642 and BTx623/SC155-14E, were assessed for TLS resistance in replicated trials. Using least square mean trait data, four TLS resistance QTL were identified, two in each population. Of these, three were previously unidentified while a major QTL on chromosome 5 in the BTx623/BTx642 RIL population corresponded to the previously identified TLS resistance gene ds1. A set of sorghum lines were assessed for production of reactive oxygen species induced by treatment with the microbe-associated molecular pattern (MAMP) flg22 (a derivative of flagellin). Flg22-induced ROS production varied between lines in a consistent fashion. One QTL associated with variation in the flg22 response was detected in the RIL populations. No evidence was found to link variation in the MAMP response to variation in TLS resistance.

Evaluation of (-)-epicatechin metabolites as recovery biomarker of dietary flavan-3-ol intake.

Data from dietary intervention studies suggest that intake of (-)-epicatechin mediates beneficial vascular effects in humans. However, population-based investigations are required to evaluate associations between habitual intake and health and these studies rely on accurate estimates of intake, which nutritional biomarkers can provide. Here, we evaluate a series of structurally related (-)-epicatechin metabolites (SREM), particularly (-)-epicatechin-3'-glucuronide, (-)-epicatechin-3'-sulfate and 3'-O-methyl-(-)-epicatechin-5-sulfate (SREMB), as flavan-3-ol and (-)-epicatechin intake. SREMB in urine proved to be a specific indicator of (-)-epicatechin intake, showing also a strong correlation with the amount of (-)-epicatechin ingested (R2: 0.86 (95% CI 0.8l; 0.92). The median recovery of (-)-epicatechin as SREMB in 24 h urine was 10% (IQR 7-13%) and we found SREMB in the majority of participants of EPIC Norfolk (83% of 24,341) with a mean concentration of 2.4 ± 3.2 µmol/L. Our results show that SREMB are suitable as biomarker of (-)-epicatechin intake. According to evaluation criteria from IARC and the Institute of Medicine, the results obtained support use of SREMB as a recovery biomarker to estimate actual intake of (-)-epicatechin.

High density genetic maps of St. Augustinegrass and applications to comparative genomic analysis and QTL mapping for turf quality traits.

BACKGROUND: St. Augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze] is a warm-season, perennial turfgrass species well adapted for home lawns and commercial landscapes with economic and ecological value. However, a lack of genomic resources in St. Augustinegrass has hindered the full utilization of genetic variance for maximizing genetic gain and limited our understanding of the species' evolution. RESULTS: In this study, we constructed the first high-density linkage map for St. Augustinegrass using a genotyping by sequencing (GBS) approach. The integrated linkage map consists of 2871 single nucleotide polymorphism (SNP) and 81 simple sequence repeat (SSR) markers, spanning 1241.7 cM, with an average distance of 0.4 cM between markers, and thus represents the densest genetic map for St. Augustinegrass to date. Comparative genomic analysis revealed inter-chromosome arrangements and independent nested chromosome fusion events that occurred after St. Augustinegrass, foxtail millet, sorghum, and rice diverged from a common ancestor. Forty-eight candidate quantitative trait loci (QTL) were detected for turf quality-related traits, including overall turf quality, leaf texture, genetic color, and turf density. Three hot spot regions were identified on linkage groups LG3 and LG8, where multi-QTL for different traits overlapped. Several leaf development related genes were contained within these identified QTL regions. CONCLUSIONS: This study developed the first high-density genetic map and identified putative QTL related to turf quality, which provide valuable genetic resources for marker-assisted selection (MAS) in St. Augustinegrass.

Evaluation at scale of microbiome-derived metabolites as biomarker of flavan-3-ol intake in epidemiological studies.

The accurate assessment of dietary intake is crucial to investigate the effect of diet on health. Currently used methods, relying on self-reporting and food composition data, are known to have limitations and might not be suitable to estimate the intake of many bioactive food components. An alternative are nutritional biomarkers, which can allow an unbiased assessment of intake. They require a careful evaluation of their suitability, including: (a) the availability of a precise, accurate and robust analytical method, (b) their specificity (c) a consistent relationship with actual intake. We have evaluated human metabolites of a microbiome-derived flavan-3-ol catabolite, 5-(3',4'-dihydroxyphenyl)-[gamma]-valerolactone (gVL), as biomarker of flavan-3-ol intake in large epidemiological studies. Flavan-3-ols are widely consumed plant bioactives, which have received considerable interest due to their potential ability to reduce CVD risk. The availability of authentic standards allowed the development of a validated high-throughput method suitable for large-scale studies. In dietary intervention studies, we could show that gVL metabolites are specific for flavan-3-ols present in tea, fruits, wine and cocoa-derived products, with a strong correlation between intake and biomarker (Spearman's r = 0.90). This biomarker will allow for the first time to estimate flavan-3-ol intake and further investigation of associations between intake and disease risk.

Population Dynamics Among six Major Groups of the Oryza rufipogon Species Complex, Wild Relative of Cultivated Asian Rice.

BACKGROUND: Understanding population structure of the wild progenitor of Asian cultivated rice (O. sativa), the Oryza rufipogon species complex (ORSC), is of interest to plant breeders and contributes to our understanding of rice domestication. A collection of 286 diverse ORSC accessions was evaluated for nuclear variation using genotyping-by-sequencing (113,739 SNPs) and for chloroplast variation using Sanger sequencing (25 polymorphic sites). RESULTS: Six wild subpopulations were identified, with 25 % of accessions classified as admixed. Three of the wild groups were genetically and geographically closely related to the O. sativa subpopulations, indica, aus and japonica, and carried O. sativa introgressions; the other three wild groups were genetically divergent, had unique chloroplast haplotypes, and were located at the geographical extremes of the species range. The genetic subpopulations were significantly correlated (r 2 = 0.562) with traditional species designations, O. rufipogon (perennial) and O. nivara (annual), differentiated based on morphology and life history. A wild diversity panel of 95 purified (inbred) accessions was developed for future genetic studies. CONCLUSIONS: Our results suggest that the cultivated aus subpopulation is most closely related to an annual wild relative, japonica to a perennial wild relative, and indica to an admixed population of diverse annual and perennial wild ancestors. Gene flow between ORSC and O. sativa is common in regions where rice is cultivated, threatening the identity and diversity of wild ORSC populations. The three geographically isolated ORSC populations harbor variation rarely seen in cultivated rice and provide a unique window into the genetic composition of ancient rice subpopulations.

Risk of HIV Infection in Depot-Medroxyprogesterone Acetate (DMPA) Users: A Systematic Review and Meta-analysis.

OBJECTIVE: As the HIV/AIDS epidemic continues to spread in Africa and Asia, use of the injectable contraceptive steroid DMPA is widespread and has been increasing. Since studies dating back to 1992 have suggested that DMPA may increase the transmission of HIV to women, we endeavored to determine if the extant epidemiological and biological evidence is sufficient to conclude that DMPA use constitutes a definite hazard to women's health. METHODS: We searched Medline using the search terms: contraceptives or contraception AND HIV and searched bibliographies of articles thus identified. We included in the meta-analysis all studies examining the association between use of DMPA (or injectable contraceptives comprising mostly DMPA) and the presence (cross-sectional studies, n = 8) or acquisition (longitudinal studies, n = 16) of HIV+ status in women, using a random effects models to estimate odds ratios (ORs; cross-sectional studies) and hazard ratios (HRs; longitudinal studies). Studies were excluded if the comparison group included women using any form of steroidal contraception. RESULTS: Statistically significant positive associations between DMPA use and HIV positivity were observed both in cross-sectional (OR = 1.41, 95% CI 1.15 - 1.73) and longitudinal studies (HR = 1.49, 95% CI 1.28 - 1.73). The biological plausibility of increased vulnerability to HIV infection due to progestational action (via thinning of the vaginal epithelial barrier and immunosuppression) as well as glucocorticoid agonistic immunosuppression, are discussed. CONCLUSION: The epidemiological and biological evidence now make a compelling case that DMPA adds significantly to the risk of male-to-female HIV transmission.

Safety and efficacy of cocoa flavanol intake in healthy adults: a randomized, controlled, double-masked trial.

BACKGROUND: Evidence from dietary intervention studies shows that the intake of flavanols and procyanidins can be beneficial for cardiovascular health. Nevertheless, there is a clear need for advancing our understanding with regard to safe amounts of intake for these bioactives. OBJECTIVE: The aim was to investigate in healthy adults the effects of cocoa flavanol (CF) intake amount and intake duration on blood pressure, platelet function, metabolic variables, and potential adverse events (AEs). DESIGN: This investigation consisted of 2 parts. Part 1 was an open-label, intake-amount escalation study, in which 34 healthy adults (aged 35-55 y) consumed escalating amounts of CFs, ranging from 1000 to 2000 mg/d over 6 wk. Primary outcomes were blood pressure and platelet function, select metabolic variables, and the occurrence and severity of AEs. Secondary outcomes included plasma concentrations of CF-derived metabolites and methylxanthines. On the basis of the outcomes of study part 1, and assessing the same outcome measures, part 2 of this investigation was a controlled, randomized, double-masked, 2-parallel-arm dietary intervention study in which healthy participants (aged 35-55 y) were asked to consume for 12 consecutive weeks up to 2000 mg CFs/d (n = 46) or a CF-free control (n = 28). RESULTS: Daily intake of up to 2000 mg CFs/d for 12 wk was not associated with significant changes in blood pressure or platelet function compared with CF-free controls in normotensive, healthy individuals who exhibited a very low risk of cardiovascular disease. There were no clinically relevant changes in the metabolic variables assessed in either of the groups. AEs reported were classified as mild in severity and did not significantly differ between study arms. CONCLUSION: The consumption of CFs in amounts up to 2000 mg/d for 12 wk was well tolerated in healthy men and women. This trial was registered at clinicaltrials.gov as NCT02447770 (part 1) and NCT02447783 (part 2).

An integrated approach to gene discovery and marker development in Atlantic cod (Gadus morhua).

Atlantic cod is a species that has been overexploited by the capture fishery. Programs to domesticate this species are underway in several countries, including Canada, to provide an alternative route for production. Selective breeding programs have been successfully applied in the domestication of other species, with genomics-based approaches used to augment conventional methods of animal production in recent years. Genomics tools, such as gene sequences and sets of variable markers, also have the potential to enhance and accelerate selective breeding programs in aquaculture, and to provide better monitoring tools to ensure that wild cod populations are well managed. We describe the generation of significant genomics resources for Atlantic cod through an integrated genomics/selective breeding approach. These include 158,877 expressed sequence tags (ESTs), a set of annotated putative transcripts and several thousand single nucleotide polymorphism markers that were developed from, and have been shown to be highly variable in, fish enrolled in two selective breeding programs. Our EST collection was generated from various tissues and life cycle stages. In some cases, tissues from which libraries were generated were isolated from fish exposed to stressors, including elevated temperature, or antigen stimulation (bacterial and viral) to enrich for transcripts that are involved in these response pathways. The genomics resources described here support the developing aquaculture industry, enabling the application of molecular markers within selective breeding programs. Marker sets should also find widespread application in fisheries management.

Development and experimental validation of a 20K Atlantic cod (Gadus morhua) oligonucleotide microarray based on a collection of over 150,000 ESTs.

The collapse of Atlantic cod (Gadus morhua) wild populations strongly impacted the Atlantic cod fishery and led to the development of cod aquaculture. In order to improve aquaculture and broodstock quality, we need to gain knowledge of genes and pathways involved in Atlantic cod responses to pathogens and other stressors. The Atlantic Cod Genomics and Broodstock Development Project has generated over 150,000 expressed sequence tags from 42 cDNA libraries representing various tissues, developmental stages, and stimuli. We used this resource to develop an Atlantic cod oligonucleotide microarray containing 20,000 unique probes. Selection of sequences from the full range of cDNA libraries enables application of the microarray for a broad spectrum of Atlantic cod functional genomics studies. We included sequences that were highly abundant in suppression subtractive hybridization (SSH) libraries, which were enriched for transcripts responsive to pathogens or other stressors. These sequences represent genes that potentially play an important role in stress and/or immune responses, making the microarray particularly useful for studies of Atlantic cod gene expression responses to immune stimuli and other stressors. To demonstrate its value, we used the microarray to analyze the Atlantic cod spleen response to stimulation with formalin-killed, atypical Aeromonas salmonicida, resulting in a gene expression profile that indicates a strong innate immune response. These results were further validated by quantitative PCR analysis and comparison to results from previous analysis of an SSH library. This study shows that the Atlantic cod 20K oligonucleotide microarray is a valuable new tool for Atlantic cod functional genomics research.

Antitumor activity of hu14.18-IL2 in patients with relapsed/refractory neuroblastoma: a Children's Oncology Group (COG) phase II study.

PURPOSE: The hu14.18-IL2 fusion protein consists of interleukin-2 molecularly linked to a humanized monoclonal antibody that recognizes the GD2 disialoganglioside expressed on neuroblastoma cells. This phase II study assessed the antitumor activity of hu14.18-IL2 in two strata of patients with recurrent or refractory neuroblastoma. PATIENTS AND METHODS: Hu14.18-IL2 was given intravenously (12 mg/m(2)/daily) for 3 days every 4 weeks for patients with disease measurable by standard radiographic criteria (stratum 1) and for patients with disease evaluable only by [(123)I]metaiodobenzylguanidine (MIBG) scintigraphy and/or bone marrow (BM) histology (stratum 2). Response was established by independent radiology review as well as BM histology and immunocytology, and durability was assessed by repeat evaluation after more than 3 weeks. RESULTS: Thirty-nine patients were enrolled (36 evaluable). No responses were seen in stratum 1 (n = 13). Of 23 evaluable patients in stratum 2, five patients (21.7%) responded; all had a complete response (CR) of 9, 13, 20, 30, and 35+ months duration. Grade 3 and 4 nonhematologic toxicities included capillary leak, hypoxia, pain, rash, allergic reaction, elevated transaminases, and hyperbilirubinemia. Two patients required dopamine for hypotension, and one patient required ventilatory support for hypoxia. Most toxicities were reversible within a few days of completing a treatment course and were expected based on phase I results. CONCLUSION: Patients with disease evaluable only by MIBG and/or BM histology had a 21.7% CR rate to hu14.8-IL2, whereas patients with bulky disease did not respond. Hu14.18-IL2 warrants further testing in children with nonbulky high-risk neuroblastoma.