The Intestinal Microbiome Restricts Alphavirus Infection and Dissemination through a Bile Acid-Type I IFN Signaling Axis.

Chikungunya virus (CHIKV), an emerging alphavirus, has infected millions of people. However, the factors modulating disease outcome remain poorly understood. Here, we show in germ-free mice or in oral antibiotic-treated conventionally housed mice with depleted intestinal microbiomes that greater CHIKV infection and spread occurs within 1 day of virus inoculation. Alteration of the microbiome alters TLR7-MyD88 signaling in plasmacytoid dendritic cells (pDCs) and blunts systemic production of type I interferon (IFN). Consequently, circulating monocytes express fewer IFN-stimulated genes and become permissive for CHIKV infection. Reconstitution with a single bacterial species, Clostridium scindens, or its derived metabolite, the secondary bile acid deoxycholic acid, can restore pDC- and MyD88-dependent type I IFN responses to restrict systemic CHIKV infection and transmission back to vector mosquitoes. Thus, symbiotic intestinal bacteria modulate antiviral immunity and levels of circulating alphaviruses within hours of infection through a bile acid-pDC-IFN signaling axis, which affects viremia, dissemination, and potentially transmission.

Modulation of flavanone and furocoumarin levels in grapefruits (Citrus paradisi Macfad.) by production and storage conditions.

Grapefruits grown under organic or conventional systems were analyzed for 6,7-dihydroxybergamottin (DHB) and flavanones using HPLC, and DPPH activity and ORAC using a micro-plate reader. Grapefruits harvested in November 2008 (E-1) and February 2010 (E-2) were stored at room temperature (RT) and 9 °C for four weeks. Higher levels of DHB were observed in conventional grapefruits during the second (4.7 ± 0.2 µg/g), third (1.5 ± 0.2 µg/g) and fourth (2.5 ± 0.2 µg/g) week of storage at room temperature in E2. Among flavonoids analyzed, narirutin (666.7 ± 33.9 µg/g), neohesperidin (17.5 ± 1.3 µg/g), didymin (75.5 ± 5.6 µg/g) and poncirin (130.8 ± 10.4 µg/g) levels were significantly higher (P⩽0.05) in organic grapefruits over conventional grapefruits at harvest and storage in E-1. Although DPPH levels were moderately correlated with grapefruit flavanone content, variability in the individual flavanone activity was pronounced, resulting in non-significant differences in antioxidant activity between organic and conventional grapefruits.

A high-throughput, simultaneous analysis of carotenoids, chlorophylls and tocopherol using sub two micron core shell technology columns.

A high-throughput, robust and reliable method for simultaneous analysis of five carotenoids, four chlorophylls and one tocopherol was developed for rapid screening large sample populations to facilitate molecular biology and plant breeding. Separation was achieved for 10 known analytes and four unknown carotenoids in a significantly reduced run time of 10min. Identity of the 10 analytes was confirmed by their UV-Vis absorption spectras. Quantification of tocopherol, carotenoids and chlorophylls was performed at 290nm, 460nm and 650nm respectively. In this report, two sub two micron particle core-shell columns, Kinetex from Phenomenex (1.7µm particle size, 12% carbon load) and Cortecs from Waters (1.6µm particle size, 6.6% carbon load) were investigated and their separation efficiencies were evaluated. The peak resolutions were >1.5 for all analytes except for chlorophyll-a' with Cortecs column. The ruggedness of this method was evaluated in two identical but separate instruments that produced CV<2 in peak retentions for nine out of 10 analytes separated.

Genetic analysis of glucosinolate variability in broccoli florets using genome-anchored single nucleotide polymorphisms.

KEY MESSAGE: The identification of genetic factors influencing the accumulation of individual glucosinolates in broccoli florets provides novel insight into the regulation of glucosinolate levels in Brassica vegetables and will accelerate the development of vegetables with glucosinolate profiles tailored to promote human health. Quantitative trait loci analysis of glucosinolate (GSL) variability was conducted with a B. oleracea (broccoli) mapping population, saturated with single nucleotide polymorphism markers from a high-density array designed for rapeseed (Brassica napus). In 4 years of analysis, 14 QTLs were associated with the accumulation of aliphatic, indolic, or aromatic GSLs in floret tissue. The accumulation of 3-carbon aliphatic GSLs (2-propenyl and 3-methylsulfinylpropyl) was primarily associated with a single QTL on C05, but common regulation of 4-carbon aliphatic GSLs was not observed. A single locus on C09, associated with up to 40 % of the phenotypic variability of 2-hydroxy-3-butenyl GSL over multiple years, was not associated with the variability of precursor compounds. Similarly, QTLs on C02, C04, and C09 were associated with 4-methylsulfinylbutyl GSL concentration over multiple years but were not significantly associated with downstream compounds. Genome-specific SNP markers were used to identify candidate genes that co-localized to marker intervals and previously sequenced Brassica oleracea BAC clones containing known GSL genes (GSL-ALK, GSL-PRO, and GSL-ELONG) were aligned to the genomic sequence, providing support that at least three of our 14 QTLs likely correspond to previously identified GSL loci. The results demonstrate that previously identified loci do not fully explain GSL variation in broccoli. The identification of additional genetic factors influencing the accumulation of GSL in broccoli florets provides novel insight into the regulation of GSL levels in Brassicaceae and will accelerate development of vegetables with modified or enhanced GSL profiles.

High-density single nucleotide polymorphism (SNP) array mapping in Brassica oleracea: identification of QTL associated with carotenoid variation in broccoli florets.

KEY MESSAGE: A high-resolution genetic linkage map of B. oleracea was developed from a B. napus SNP array. The work will facilitate genetic and evolutionary studies in Brassicaceae. A broccoli population, VI-158 × BNC, consisting of 150 F2:3 families was used to create a saturated Brassica oleracea (diploid: CC) linkage map using a recently developed rapeseed (Brassica napus) (tetraploid: AACC) Illumina Infinium single nucleotide polymorphism (SNP) array. The map consisted of 547 non-redundant SNP markers spanning 948.1 cM across nine chromosomes with an average interval size of 1.7 cM. As the SNPs are anchored to the genomic reference sequence of the rapid cycling B. oleracea TO1000, we were able to estimate that the map provides 96 % coverage of the diploid genome. Carotenoid analysis of 2 years data identified 3 QTLs on two chromosomes that are associated with up to half of the phenotypic variation associated with the accumulation of total or individual compounds. By searching the genome sequences of the two related diploid species (B. oleracea and B. rapa), we further identified putative carotenoid candidate genes in the region of these QTLs. This is the first description of the use of a B. napus SNP array to rapidly construct high-density genetic linkage maps of one of the constituent diploid species. The unambiguous nature of these markers with regard to genomic sequences provides evidence to the nature of genes underlying the QTL, and demonstrates the value and impact this resource will have on Brassica research.

Production system and storage temperature influence grapefruit vitamin C, limonoids, and carotenoids.

Concentrations of grapefruit (cv. 'Rio Red'; Citrus paradisi Macf.) bioactives grown under organic and conventional production systems were evaluated after storage at various temperatures. The first experiment was conducted in November 2008 and the second experiment was conducted in February 2011 using commercial production, processing, and packing procedures. The harvested grapefruits were stored at 23 °C (room temperature) or 9 °C for 4 weeks and analyzed for vitamin C, limonoids, and carotenoids at the end of each week using HPLC. Vitamin C levels were higher in organically grown grapefruits (41.8 mg/100 g) compared to conventionally grown grapefruits (39.2 mg/100 g) at 0 days after harvest in the first experiment. However, production system did not significantly affect vitamin C levels in the second experiment. During storage at room temperature, vitamin C degradation losses ranged from 0.5 to 7% for organically produced grapefruits and from 3 to 18% for conventional grapefruits in both experiments. In the first experiment at harvest, organically produced grapefruits had 77% higher (p ≤ 0.05) nomilin than conventionally produced grapefruits, whereas grapefruits grown under the conventional production system had 2-fold higher lycopene levels compared to organic grapefruits. In the second experiment, both ß-carotene and lycopene levels were significantly (p ≤ 0.05) higher in conventionally produced grapefruits than in organic grapefruits. Overall, conventional production significantly increased grapefruit carotenoid levels in both experiments. In general, storage temperature (room temperature and 9 °C) had minimal effects on vitamin C degradation but significant effects on the degradation of carotenoids in the first experiment.

Optimization of flavanones extraction by modulating differential solvent densities and centrifuge temperatures.

Understanding the factors influencing flavonone extraction is critical for the knowledge in sample preparation. The present study was focused on the extraction parameters such as solvent, heat, centrifugal speed, centrifuge temperature, sample to solvent ratio, extraction cycles, sonication time, microwave time and their interactions on sample preparation. Flavanones were analyzed in a high performance liquid chromatography (HPLC) and later identified by liquid chromatography and mass spectrometry (LC-MS). The five flavanones were eluted by a binary mobile phase with 0.03% phosphoric acid and acetonitrile in 20 min and detected at 280 nm, and later identified by mass spectral analysis. Dimethylsulfoxide (DMSO) and dimethyl formamide (DMF) had optimum extraction levels of narirutin, naringin, neohesperidin, didymin and poncirin compared to methanol (MeOH), ethanol (EtOH) and acetonitrile (ACN). Centrifuge temperature had a significant effect on flavanone distribution in the extracts. The DMSO and DMF extracts had homogeneous distribution of flavanones compared to MeOH, EtOH and ACN after centrifugation. Furthermore, ACN showed clear phase separation due to differential densities in the extracts after centrifugation. The number of extraction cycles significantly increased the flavanone levels during extraction. Modulating the sample to solvent ratio increased naringin quantity in the extracts. Current research provides critical information on the role of centrifuge temperature, extraction solvent and their interactions on flavanone distribution in extracts.