Characterization of proanthocyanidin metabolism in pea (Pisum sativum) seeds

BACKGROUND: Proanthocyanidins (PAs) accumulate in the seeds, fruits and leaves of various plant species including the seed coats of pea (Pisum sativum), an important food crop. PAs have been implicated in human health, but molecular and biochemical characterization of pea PA biosynthesis has not been established to date, and detailed pea PA chemical composition has not been extensively studied. RESULTS: PAs were localized to the ground parenchyma and epidermal cells of pea seed coats. Chemical analyses of PAs from seeds of three pea cultivars demonstrated cultivar variation in PA composition. ‘Courier’ and ‘Solido’ PAs were primarily prodelphinidin-types, whereas the PAs from ‘LAN3017’ were mainly the procyanidin-type. The mean degree of polymerization of ‘LAN3017’ PAs was also higher than those from ‘Courier’ and ‘Solido’. Next-generation sequencing of ‘Courier’ seed coat cDNA produced a seed coat-specific transcriptome. Three cDNAs encoding anthocyanidin reductase (PsANR), leucoanthocyanidin reductase (PsLAR), and dihydroflavonol reductase (PsDFR) were isolated. PsANR and PsLAR transcripts were most abundant earlier in seed coat development. This was followed by maximum PA accumulation in the seed coat. Recombinant PsANR enzyme efficiently synthesized all three cis-flavan-3-ols (gallocatechin, catechin, and afzalechin) with satisfactory kinetic properties. The synthesis rate of trans-flavan-3-ol by co-incubation of PsLAR and PsDFR was comparable to cis-flavan-3-ol synthesis rate by PsANR. Despite the competent PsLAR activity in vitro, expression of PsLAR driven by the Arabidopsis ANR promoter in wild-type and anr knock-out Arabidopsis backgrounds did not result in PA synthesis. CONCLUSION: Significant variation in seed coat PA composition was found within the pea cultivars, making pea an ideal system to explore PA biosynthesis. PsANR and PsLAR transcript profiles, PA localization, and PA accumulation patterns suggest that a pool of PA subunits are produced in specific seed coat cells early in development to be used as substrates for polymerization into PAs. Biochemically competent recombinant PsANR and PsLAR activities were consistent with the pea seed coat PA profile composed of both cis- and trans-flavan-3-ols. Since the expression of PsLAR in Arabidopsis did not alter the PA subunit profile (which is only comprised of cis-flavan-3-ols), it necessitates further investigation of in planta metabolic flux through PsLAR. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12870-014-0238-y) contains supplementary material, which is available to authorized users

Negotiation in strategy making teams : group support systems and the process of cognitive change

This paper reports on the use of a Group Support System (GSS) to explore at a micro level some of the processes manifested when a group is negotiating strategy-processes of social and psychological negotiation. It is based on data from a series of interventions with senior management teams of three operating companies comprising a multi-national organization, and with a joint meeting subsequently involving all of the previous participants. The meetings were concerned with negotiating a new strategy for the global organization. The research involved the analysis of detailed time series data logs that exist as a result of using a GSS that is a reflection of cognitive theory

Age- and Concentration-Dependent Elimination Half-Life of 2,3,7,8-Tetrachlorodibenzo-p-dioxin in Seveso Children

OBJECTIVE: Pharmacokinetic and statistical analyses are reported to elucidate key variables affecting 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) elimination in children and adolescents. DESIGN: We used blood concentrations to calculate TCDD elimination half-life. Variables examined by statistical analysis include age, latency from exposure, sex, TCDD concentration and quantity in the body, severity of chloracne response, body mass index, and body fat mass. PARTICIPANTS: Blood was collected from 1976 to 1993 from residents of Seveso, Italy, who were < 18 years of age at the time of a nearby trichlorophenol reactor explosion in July 1976. RESULTS: TCDD half-life in persons < 18 years of age averaged 1.6 years while those ≥18 years of age averaged 3.2 years. Half-life is strongly associated with age, showing a cohort average increase of 0.12 year half-life per year of age or time since exposure. A significant concentration-dependency is also identified, showing shorter half-lives for TCDD concentrations > 400 ppt for children < 12 years of age and 700 ppt when including adults. Moderate correlations are also observed between half-life and body mass index, body fat mass, TCDD mass, and chloracne response. CONCLUSIONS: Children and adolescents have shorter TCDD half-lives and a slower rate of increase in half-life than adults, and this effect is augmented at higher body burdens. RELEVANCE: Modeling of TCDD blood concentrations or body burden in humans should take into account the markedly shorter elimination half-life observed in children and adolescents and concentration-dependent effects observed in persons > 400–700 ppt

Novel Murine Infection Models Provide Deep Insights into the “Ménage à Trois” of Campylobacter jejuni, Microbiota and Host Innate Immunity

BACKGROUND: Although Campylobacter jejuni-infections have a high prevalence worldwide and represent a significant socioeconomic burden, it is still not well understood how C. jejuni causes intestinal inflammation. Detailed investigation of C. jejuni-mediated intestinal immunopathology is hampered by the lack of appropriate vertebrate models. In particular, mice display colonization resistance against this pathogen. METHODOLOGY/PRINCIPAL FINDINGS: To overcome these limitations we developed a novel C. jejuni-infection model using gnotobiotic mice in which the intestinal flora was eradicated by antibiotic treatment. These animals could then be permanently associated with a complete human (hfa) or murine (mfa) microbiota. After peroral infection C. jejuni colonized the gastrointestinal tract of gnotobiotic and hfa mice for six weeks, whereas mfa mice cleared the pathogen within two days. Strikingly, stable C. jejuni colonization was accompanied by a pro-inflammatory immune response indicated by increased numbers of T- and B-lymphocytes, regulatory T-cells, neutrophils and apoptotic cells, as well as increased concentrations of TNF-α, IL-6, and MCP-1 in the colon mucosa of hfa mice. Analysis of MyD88(-/-), TRIF(-/-), TLR4(-/-), and TLR9(-/-) mice revealed that TLR4- and TLR9-signaling was essential for immunopathology following C. jejuni-infection. Interestingly, C. jejuni-mutant strains deficient in formic acid metabolism and perception induced less intestinal immunopathology compared to the parental strain infection. In summary, the murine gut flora is essential for colonization resistance against C. jejuni and can be overcome by reconstitution of gnotobiotic mice with human flora. Detection of C. jejuni-LPS and -CpG-DNA by host TLR4 and TLR9, respectively, plays a key role in immunopathology. Finally, the host immune response is tightly coupled to bacterial formic acid metabolism and invasion fitness. CONCLUSION/SIGNIFICANCE: We conclude that gnotobiotic and "humanized" mice represent excellent novel C. jejuni-infection and -inflammation models and provide deep insights into the immunological and molecular interplays between C. jejuni, microbiota and innate immunity in human campylobacteriosis

18^{18}F-Fluoride and 18^{18}F-Fluorodeoxyglucose Positron Emission Tomography After Transient Ischemic Attack or Minor Ischemic Stroke: Case-Control Study.

BACKGROUND: Combined positron emission tomography (PET) and computed tomography (CT) can assess both anatomy and biology of carotid atherosclerosis. We sought to assess whether $^{18}$F-fluoride or $^{18}$F-fluorodeoxyglucose can identify culprit and high-risk carotid plaque. METHODS AND RESULTS: We performed $^{18}$F-fluoride and $^{18}$F-fluorodeoxyglucose PET/CT in 26 patients after recent transient ischemic attack or minor ischemic stroke: 18 patients with culprit carotid stenosis awaiting carotid endarterectomy and 8 controls without culprit carotid atheroma. We compared standardized uptake values in the clinically adjudicated culprit to the contralateral asymptomatic artery, and assessed the relationship between radiotracer uptake and plaque phenotype or predicted cardiovascular risk (ASSIGN score [Assessing Cardiovascular Risk Using SIGN Guidelines to Assign Preventive Treatment]). We also performed micro PET/CT and histological analysis of excised plaque. On histological and micro PET/CT analysis, $^{18}$F-fluoride selectively highlighted microcalcification. Carotid $^{18}$F-fluoride uptake was increased in clinically adjudicated culprit plaques compared with asymptomatic contralateral plaques (log$_{10}$ standardized uptake value$_{mean}$ 0.29±0.10 versus 0.23±0.11, P=0.001) and compared with control patients (log$_{10}$ standardized uptake value$_{mean}$ 0.29±0.10 versus 0.12±0.11, P=0.001). $^{18}$F-Fluoride uptake correlated with high-risk plaque features (remodeling index [r=0.53, P=0.003], plaque burden [r=0.51, P=0.004]), and predicted cardiovascular risk [r=0.65, P=0.002]). Carotid $^{18}$F-fluorodeoxyglucose uptake appeared to be increased in 7 of 16 culprit plaques, but no overall differences in uptake were observed in culprit versus contralateral plaques or control patients. However, $^{18}$F-fluorodeoxyglucose did correlate with predicted cardiovascular risk (r=0.53, P=0.019), but not with plaque phenotype. CONCLUSIONS: $^{18}$F-Fluoride PET/CT highlights culprit and phenotypically high-risk carotid plaque. This has the potential to improve risk stratification and selection of patients who may benefit from intervention.Dr Vesey and the study were funded by program grants from the British Heart Foundation (PG12/8/29371) and Chest Heart and Stroke Scotland (R13/A147). Dr Jenkins, Vesey, Dweck, and Newby are supported by the British Heart Foundation (FS/14/78/31020, CH/09/002) and the Wellcome Trust (WT103782AIA). Dr Dweck is the recipient of the Sir Jules Thorn Biomedical Research Award 2015. The Wellcome Trust Clinical Research Facility and the Clinical Research Imaging Centre are supported by National Health Service (NHS) Research Scotland (NRS) through NHS Lothian. Dr Beek is supported by the Scottish Imaging Network—a Platform of Scientific Excellence (SINAPSE). Dr Rudd is part-supported by the National Institute for Health Research Cambridge Biomedical Research Centre, the British Heart Foundation, and the Wellcome Trust

Comparison of the Safety and Pharmacokinetics of ST-246® after IV Infusion or Oral Administration in Mice, Rabbits and Monkeys

ST-246® is an antiviral, orally bioavailable small molecule in clinical development for treatment of orthopoxvirus infections. An intravenous (IV) formulation may be required for some hospitalized patients who are unable to take oral medication. An IV formulation has been evaluated in three species previously used in evaluation of both efficacy and toxicology of the oral formulation. plasma concentrations. These effects were eliminated using slower IV infusions. associated toxicity. Shorter infusions at higher doses in NHP resulted in decreased clearance, suggesting saturated distribution or elimination. Elimination half-lives in all species were similar between oral and IV administration. The administration of ST-246 was well tolerated as a slow IV infusion

Stromal transcriptional profiles reveal hierarchies of anatomical site, serum response and disease and identify disease specific pathways

Synovial fibroblasts in persistent inflammatory arthritis have been suggested to have parallels with cancer growth and wound healing, both of which involve a stereotypical serum response programme. We tested the hypothesis that a serum response programme can be used to classify diseased tissues, and investigated the serum response programme in fibroblasts from multiple anatomical sites and two diseases. To test our hypothesis we utilized a bioinformatics approach to explore a publicly available microarray dataset including rheumatoid arthritis (RA), osteoarthritis (OA) and normal synovial tissue, then extended those findings in a new microarray dataset representing matched synovial, bone marrow and skin fibroblasts cultured from RA and OA patients undergoing arthroplasty. The classical fibroblast serum response programme discretely classified RA, OA and normal synovial tissues. Analysis of low and high serum treated fibroblast microarray data revealed a hierarchy of control, with anatomical site the most powerful classifier followed by response to serum and then disease. In contrast to skin and bone marrow fibroblasts, exposure of synovial fibroblasts to serum led to convergence of RA and OA expression profiles. Pathway analysis revealed three inter-linked gene networks characterising OA synovial fibroblasts: Cell remodelling through insulin-like growth factors, differentiation and angiogenesis through -3 integrin, and regulation of apoptosis through CD44. We have demonstrated that Fibroblast serum response signatures define disease at the tissue level, and that an OA specific, serum dependent repression of genes involved in cell adhesion, extracellular matrix remodelling and apoptosis is a critical discriminator between cultured OA and RA synovial fibroblasts