System level analysis of cacao seed ripening reveals a sequential interplay of primary and secondary metabolism leading to polyphenol accumulation and preparation of stress resistance

Theobroma cacao and its popular product, chocolate, are attracting attention due to potential health benefits including antioxidative effects by polyphenols, anti‐depressant effects by high serotonin levels, inhibition of platelet aggregation and prevention of obesity‐dependent insulin resistance. The development of cacao seeds during fruit ripening is the most crucial process for the accumulation of these compounds. In this study, we analyzed the primary and the secondary metabolome as well as the proteome during Theobroma cacao cv. Forastero seed development by applying an integrative extraction protocol. The combination of multivariate statistics and mathematical modelling revealed a complex consecutive coordination of primary and secondary metabolism and corresponding pathways. Tricarboxylic acid (TCA) cycle and aromatic amino acid metabolism dominated during the early developmental stages (stages 1 and 2; cell division and expansion phase). This was accompanied with a significant shift of proteins from phenylpropanoid metabolism to flavonoid biosynthesis. At stage 3 (reserve accumulation phase), metabolism of sucrose switched from hydrolysis into raffinose synthesis. Lipids as well as proteins involved in lipid metabolism increased whereas amino acids and N‐phenylpropenoyl amino acids decreased. Purine alkaloids, polyphenols, and raffinose as well as proteins involved in abiotic and biotic stress accumulated at stage 4 (maturation phase) endowing cacao seeds the characteristic astringent taste and resistance to stress. In summary, metabolic key points of cacao seed development comprise the sequential coordination of primary metabolites, phenylpropanoid, N‐phenylpropenoyl amino acid, serotonin, lipid and polyphenol metabolism thereby covering the major compound classes involved in cacao aroma and health benefits.     

Developmental Variability of Photooxidative Stress Tolerance in Paraquat-Resistant Conyza

Paraquat-resistant hairy fleabane (Conyza bonariensis L. Cronq.) has been extensively studied, with some contention. A single, dominant gene pleiotropically controls levels of oxidant-detoxifying enzymes and tolerance to many photooxidants, to photoinhibition, and possibly to other stresses. The weed forms a rosette on humid short days and flowers in dry long days and, thus, needs plasticity to photooxidant stresses. In a series of four experiments over 20 months, the resistant and susceptible biotypes were cultured in constant 10-h low-light short days at 25[deg]C. Resistance was measured as recovery from paraquat. The concentration required to achieve 50% inhibition of the resistant biotype was about 30 times that of the susceptible one just after germination, increased to >300 times that of the susceptibles at 10 weeks of growth, and then decreased to 20-fold, remaining constant except for a brief increase while bolting. Resistance increased when plants were induced to flower by long days. The levels of plastid superoxide dismutase and of glutathione reductase were generally highest in resistant plants compared to those of the susceptibles at the times of highest paraquat resistance, but they were imperceptibly different from the susceptible type at the times of lower paraquat resistance. Photoinhibition tolerance measured as quantum yield of oxygen evolution at ambient temperatures was highest when the relative amounts of enzymes were highest in the resistant biotype. Resistance to photoinhibition was not detected by chlorophyll a fluorescence. Enzyme levels, photoinhibition tolerance, and paraquat resistance all increased during flowering in both biotypes. Imperceptibly small increases in enzyme levels would be needed for 20-fold resistance, based on the moderate enzyme increases correlated with 300-fold resistance. Thus, it is feasible that either these enzymes play a role in the first line of defense against photooxidants, or another, yet unknown mechanism(s) facilitate(s) the lower level of resistance, or the enzymes and unknown mechanisms act together.     

An ultrastructural approach to feeding behaviour in Philodina roseola and Brachionus calyciflorus (Rotifers) I. The buccal velum

The structure situated in the lumen of the buccal tube, called here buccal velum, separates the buccal space from the pharyngeal space. The velum is a supple membrane-pile. Its structure resembles that of a fish-trap. The food, once ingested, cannot go back into the buccal lumen.     

Preclinical evaluation of two neutralizing human monoclonal antibodies against hepatitis C virus (HCV): a potential treatment to prevent HCV reinfection in liver transplant patients

Passive immunotherapy is potentially effective in preventing reinfection of liver grafts in hepatitis C virus (HCV)-associated liver transplant patients. A combination of monoclonal antibodies directed against different epitopes may be advantageous against a highly mutating virus such as HCV. Two human monoclonal antibodies (HumAbs) against the E2 envelope protein of HCV were developed and tested for the ability to neutralize the virus and prevent human liver infection. These antibodies, designated HCV-AB 68 and HCV-AB 65, recognize different conformational epitopes on E2. They were characterized in vitro biochemically and functionally. Both HumAbs are immunoglobulin G1 and have affinity constants to recombinant E2 constructs in the range of 10(-10) M. They are able to immunoprecipitate HCV particles from infected patients' sera from diverse genotypes and to stain HCV-infected human liver tissue. Both antibodies can fix complement and form immune complexes, but they do not activate complement-dependent or antibody-dependent cytotoxicity. Upon complement fixation, the monoclonal antibodies induce phagocytosis of the immune complexes by neutrophils, suggesting that the mechanism of viral clearance includes endocytosis. In vivo, in the HCV-Trimera model, both HumAbs were capable of inhibiting HCV infection of human liver fragments and of reducing the mean viral load in HCV-positive animals. The demonstrated neutralizing activities of HCV-AB 68 and HCV-AB 65 suggest that they have the potential to prevent reinfection in liver transplant patients and to serve as prophylactic treatment in postexposure events.     

Cubilin,a High Affinity Receptor for Fibroblast Growth Factor 8, Is Required for Cell Survival in the Developing Vertebrate Head

Cubilin (Cubn) is a multiligand endocytic receptor critical for the intestinal absorption of vitamin B12 and renal protein reabsorption. During mouse development, Cubn is expressed in both embryonic and extra-embryonic tissues, and Cubn gene inactivation results in early embryo lethality most likely due to the impairment of the function of extra-embryonic Cubn. Here, we focus on the developmental role of Cubn expressed in the embryonic head. We report that Cubn is a novel, interspecies-conserved Fgf receptor. Epiblast-specific inactivation of Cubn in the mouse embryo as well as Cubn silencing in the anterior head of frog or the cephalic neural crest of chick embryos show that Cubn is required during early somite stages to convey survival signals in the developing vertebrate head. Surface plasmon resonance analysis reveals that fibroblast growth factor 8 (Fgf8), a key mediator of cell survival, migration, proliferation, and patterning in the developing head, is a high affinity ligand for Cubn. Cell uptake studies show that binding to Cubn is necessary for the phosphorylation of the Fgf signaling mediators MAPK and Smad1. Although Cubn may not form stable ternary complexes with Fgf receptors (FgfRs), it acts together with and/or is necessary for optimal FgfR activity. We propose that plasma membrane binding of Fgf8, and most likely of the Fgf8 family members Fgf17 and Fgf18, to Cubn improves Fgf ligand endocytosis and availability to FgfRs, thus modulating Fgf signaling activity.