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.

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).

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.

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.

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).

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.

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.

Impact of asymptomatic nodavirus carrier state and intraperitoneal viral mimic injection on brain transcript expression in Atlantic cod (Gadus morhua).

Nodaviruses and other RNA viruses have a profoundly negative impact on the global aquaculture industry. Nodaviruses target nervous tissue causing viral nervous necrosis, a disease characterized by neurological damage, swimming abnormalities, and morbidity. This study used functional genomic techniques to study the Atlantic cod (Gadus morhua) brain transcript expression responses to asymptomatic high nodavirus carrier state and intraperitoneal injection of polyriboinosinic polyribocytidylic acid (pIC). Reciprocal suppression subtractive hybridization (SSH) cDNA libraries enriched for virus-responsive brain transcripts were constructed and characterized. We generated 1,938 expressed sequence tags (ESTs) from a forward brain SSH library (enriched for transcripts upregulated by nodavirus and/or pIC) and 1,980 ESTs from a reverse brain SSH library (enriched for transcripts downregulated by nodavirus and/or pIC). To examine the effect of nodavirus carrier state on individual brain gene expression in asymptomatic cod, 27 transcripts of interest were selected for quantitative reverse transcription-polymerase chain reaction (QPCR) studies. Transcripts found to be >10-fold upregulated in individuals with a high nodavirus carrier state relative to those in a no/low nodavirus carrier state were identified as ISG15, IL8, DHX58 (alias LGP2), ZNFX1, RSAD2 (alias viperin), and SACS (sacsin, alias spastic ataxia of Charlevoix-Saguenay). These and other SSH-identified transcripts were also found by QPCR to be significantly (P < 0.05) upregulated by pIC compared with saline-injected controls within 72 h of injection. Several transcripts identified in the reverse SSH library, including two putative ubiquitination pathway members (HERC4 and SUMO2), were found to be significantly (P < 0.05) downregulated in individuals with a high nodavirus carrier state. Our data shows that Atlantic cod brains have a strong interferon pathway response to asymptomatic high nodavirus carrier state and that many interferon pathway and other immune relevant transcripts are significantly induced in brain by both nodavirus and pIC.

Heat-shock responsive genes identified and validated in Atlantic cod (Gadus morhua) liver, head kidney and skeletal muscle using genomic techniques.

BACKGROUND: Daily and seasonal changes in temperature are challenges that fish within aquaculture settings cannot completely avoid, and are known to elicit complex organismal and cellular stress responses. We conducted a large-scale gene discovery and transcript expression study in order to better understand the genes that are potentially involved in the physiological and cellular aspects of stress caused by heat-shock. We used suppression subtractive hybridization (SSH) cDNA library construction and characterization to identify transcripts that were dysregulated by heat-shock in liver, skeletal muscle and head kidney of Atlantic cod. These tissues were selected due to their roles in metabolic regulation, locomotion and growth, and immune function, respectively. Fish were exposed for 3 hours to an 8 degrees C elevation in temperature, and then allowed to recover for 24 hours at the original temperature (i.e. 10 degrees C). Tissue samples obtained before heat-shock (BHS), at the cessation of heat-shock (CS), and 3, 12, and 24 hours after the cessation of heat-shock (ACS), were used for reciprocal SSH library construction and quantitative reverse transcription - polymerase chain reaction (QPCR) analysis of gene expression using samples from a group that was transferred but not heat-shocked (CT) as controls. RESULTS: We sequenced and characterized 4394 ESTs (1524 from liver, 1451 from head kidney and 1419 from skeletal muscle) from three "forward subtracted" libraries (enriched for genes up-regulated by heat-shock) and 1586 from the liver "reverse subtracted" library (enriched for genes down-regulated by heat-shock), for a total of 5980 ESTs. Several cDNAs encoding putative chaperones belonging to the heat-shock protein (HSP) family were found in these libraries, and "protein folding" was among the gene ontology (GO) terms with the highest proportion in the libraries. QPCR analysis of HSP90alpha and HSP70-1 (synonym: HSPA1A) mRNA expression showed significant up-regulation in all three tissues studied. These transcripts were more than 100-fold up-regulated in liver following heat-shock. We also identified HSP47, GRP78 and GRP94-like transcripts, which were significantly up-regulated in all 3 tissues studied. Toll-like receptor 22 (TLR22) transcript, found in the liver reverse SSH library, was shown by QPCR to be significantly down-regulated in the head kidney after heat-shock. CONCLUSION: Chaperones are an important part of the cellular response to stress, and genes identified in this work may play important roles in resistance to thermal-stress. Moreover, the transcript for one key immune response gene (TLR22) was down-regulated by heat-shock, and this down-regulation may be a component of heat-induced immunosuppression.

A universal core genetic map for rice.

To facilitate the creation of easily comparable, low-resolution genetic maps with evenly distributed markers in rice (Oryza sativa L.), we conceived of and developed a Universal Core Genetic Map (UCGM). With this aim, we derived a set of 165 anchors, representing clusters of three microsatellite or simple sequence repeat (SSR) markers arranged into non-recombining groups. Each anchor consists of at least three, closely linked SSRs, located within a distance below the genetic resolution provided by common, segregating populations (<500 individuals). We chose anchors that were evenly distributed across the rice chromosomes, with spacing between 2 and 3.5 Mbp (except in the telomeric regions, where spacing was 1.5 Mbp). Anchor selection was performed using in silico tools and data: the O. sativa cv. Nipponbare rice genome sequence, the CHARM tool, information from the Gramene database and the OrygenesDB database. Sixteen AA-genome accessions of the Oryza genus were used to evaluate polymorphisms for the selected markers, including accessions from O. sativa, O. glaberrima, O. barthii, O. rufipogon, O. glumaepatula and O. meridionalis. High levels of polymorphism were found for the tested O. sativa x O. glaberrima or O. sativa x wild rice combinations. We developed Paddy Map, a simple database that is helpful in selecting optimal sets of polymorphic SSRs for any cross that involves the previously mentioned species. Validation of the UCGM was done by using it to develop three interspecific genetic maps and by comparing genetic SSR locations with their physical positions on the rice pseudomolecules. In this study, we demonstrate that the UCGM is a useful tool for the rice genetics and breeding community, especially in strategies based on interspecific hybridisation.

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.

Identification and analysis of differentially expressed genes in immune tissues of Atlantic cod stimulated with formalin-killed, atypical Aeromonas salmonicida.

Physiological changes, elicited in animal immune tissues by exposure to pathogens, may be studied using functional genomics approaches. We created and characterized reciprocal suppression subtractive hybridization (SSH) cDNA libraries to identify differentially expressed genes in spleen and head kidney tissues of Atlantic cod (Gadus morhua) challenged with intraperitoneal injections of formalin-killed, atypical Aeromonas salmonicida. Of 4,154 ESTs from four cDNA libraries, 10 genes with immune-relevant functional annotations were selected for QPCR studies using individual fish templates to assess biological variability. Genes confirmed by QPCR as upregulated by A. salmonicida included interleukin-1 beta, interleukin-8, a small inducible cytokine, interferon regulatory factor 1 (IRF1), ferritin heavy subunit, cathelicidin, and hepcidin. This study is the first large-scale discovery of bacteria-responsive genes in cod and the first to demonstrate upregulation of IRF1 in fish immune tissues as a result of bacterial antigen stimulation. Given the importance of IRF1 in vertebrate immune responses to viral and bacterial pathogens, the full-length cDNA sequence of Atlantic cod IRF1 was obtained and compared with putative orthologous sequences from other organisms. Functional annotations of assembled SSH library ESTs showed that bacterial antigen stimulation caused changes in many biological processes including chemotaxis, regulation of apoptosis, antimicrobial peptide production, and iron homeostasis. Moreover, differences in spleen and head kidney gene expression responses to the bacterial antigens pointed to a potential role for the cod spleen in blood-borne pathogen clearance. Our data show that Atlantic cod immune tissue responses to bacterial antigens are similar to those seen in other fish species and higher vertebrates.

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.

The genome of Aeromonas salmonicida subsp. salmonicida A449: insights into the evolution of a fish pathogen.

BACKGROUND: Aeromonas salmonicida subsp. salmonicida is a Gram-negative bacterium that is the causative agent of furunculosis, a bacterial septicaemia of salmonid fish. While other species of Aeromonas are opportunistic pathogens or are found in commensal or symbiotic relationships with animal hosts, A. salmonicida subsp. salmonicida causes disease in healthy fish. The genome sequence of A. salmonicida was determined to provide a better understanding of the virulence factors used by this pathogen to infect fish. RESULTS: The nucleotide sequences of the A. salmonicida subsp. salmonicida A449 chromosome and two large plasmids are characterized. The chromosome is 4,702,402 bp and encodes 4388 genes, while the two large plasmids are 166,749 and 155,098 bp with 178 and 164 genes, respectively. Notable features are a large inversion in the chromosome and, in one of the large plasmids, the presence of a Tn21 composite transposon containing mercury resistance genes and an In2 integron encoding genes for resistance to streptomycin/spectinomycin, quaternary ammonia compounds, sulphonamides and chloramphenicol. A large number of genes encoding potential virulence factors were identified; however, many appear to be pseudogenes since they contain insertion sequences, frameshifts or in-frame stop codons. A total of 170 pseudogenes and 88 insertion sequences (of ten different types) are found in the A. salmonicida genome. Comparison with the A. hydrophila ATCC 7966T genome reveals multiple large inversions in the chromosome as well as an approximately 9% difference in gene content indicating instances of single gene or operon loss or gain.A limited number of the pseudogenes found in A. salmonicida A449 were investigated in other Aeromonas strains and species. While nearly all the pseudogenes tested are present in A. salmonicida subsp. salmonicida strains, only about 25% were found in other A. salmonicida subspecies and none were detected in other Aeromonas species. CONCLUSION: Relative to the A. hydrophila ATCC 7966T genome, the A. salmonicida subsp. salmonicida genome has acquired multiple mobile genetic elements, undergone substantial rearrangement and developed a significant number of pseudogenes. These changes appear to be a consequence of adaptation to a specific host, salmonid fish, and provide insights into the mechanisms used by the bacterium for infection and avoidance of host defence systems.

Seasonal expressed sequence tags of rainbow smelt (Osmerus mordax) revealed by subtractive hybridization and the identification of two genes up-regulated during winter.

The rainbow smelt (Osmerus mordax) is freeze-resistant and maintains swimming and feeding activity during winter. In order to identify genes differentially expressed in smelt liver response to winter water temperatures, a large-scale analysis of gene expression using suppression subtractive hybridization was carried out using samples obtained in fall and winter. Forward and reverse subtractions were performed, subtraction-enriched products were cloned, and clones were sequenced from both of the resulting libraries. When 27 of these genes were screened by semi-quantitative RT-PCR to identify candidates for differential expression based generally on 2-fold changes in expression, one encoding FK506-binding protein 5 was classified as up-regulated in response to seasonal change, another encoding the mitochondrial solute carrier 25 member 25 (ATP-Mg/Pi carrier) was similarly classified with seasonal change and low temperature shift, and the one encoding the 78 kDa glucose-regulated protein was provisionally classified as down-regulated with low temperature shift. Analysis of fall (warm) and winter (cold) seasonal samples by quantitative PCR (qPCR) revealed significant up-regulation of genes encoding FK506-binding protein 51 and the mitochondrial solute carrier, whereas the gene encoding the glucose-regulated protein showed no significant change in expression. The mitochondrial solute carrier and FK506-binding protein results may relate to changes in cortisol action, as both are regulated by cortisol in other species.

Functional genomic analysis of the response of Atlantic cod (Gadus morhua) spleen to the viral mimic polyriboinosinic polyribocytidylic acid (pIC).

In order to improve our understanding of how Atlantic cod (Gadus morhua) respond to viruses, we characterized immune-related gene expression in spleen tissues following stimulation with a synthetic double-stranded RNA polyriboinosinic polyribocytidylic acid (pIC). We used reciprocal suppression subtractive hybridization (SSH) cDNA libraries and quantitative RTPCR (QPCR) to identify and quantify pIC-responsive transcripts. A total of 3874 expressed sequence tags (ESTs) were generated from SSH libraries enriched for genes responsive to pIC. Thirteen immune-relevant genes from the libraries were subjected to QPCR. Genes confirmed as up-regulated by pIC included interferon stimulated gene 15, a small inducible cytokine, interferon regulatory factors (1, 7, and 10), MHC class I, viperin, and ATP-dependent helicase LGP2. Alpha-1-microglobulin (bikunin) was down-regulated, suggesting that pIC may suppress the acute phase response. Since the SSH libraries built for this study identified genes involved in the antiviral response, they are important resources for studying the responses of Atlantic cod to viruses. Evidence is provided for the existence of a RIG-I-like RNA helicase viral recognition pathway in Atlantic cod. Taken together, our data show that Atlantic cod can recognize double-stranded RNA and mount a rapid and potent interferon pathway response that is similar to that observed in other fish species and higher vertebrates.