Essential role for IKKβ in production of type 1 interferons by plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs) are characterized by their ability to produce high levels of type 1 interferons in response to ligands that activate TLR7 and TLR9, but the signaling pathways required for IFN production are incompletely understood. Here we exploit the human pDC cell line Gen2.2 and improved pharmacological inhibitors of protein kinases to address this issue. We demonstrate that ligands that activate TLR7 and TLR9 require the TAK1-IKKβ signaling pathway to induce the production of IFNβ via a pathway that is independent of the degradation of IκBα. We also show that IKKβ activity, as well as the subsequent IFNβ-stimulated activation of the JAK-STAT1/2 signaling pathway, are essential for the production of IFNα by TLR9 ligands. We further show that TLR7 ligands CL097 and R848 fail to produce significant amounts of IFNα because the activation of IKKβ is not sustained for a sufficient length of time. The TLR7/9-stimulated production of type 1 IFNs is inhibited by much lower concentrations of IKKβ inhibitors than those needed to suppress the production of NFκB-dependent proinflammatory cytokines, such as IL-6, suggesting that drugs that inhibit IKKβ may have a potential for the treatment of forms of lupus that are driven by self-RNA and self-DNA-induced activation of TLR7 and TLR9, respectively.     

Sublingual administration of bacteria-expressed influenza virus hemagglutinin 1 (HA1) induces protection against infection with 2009 pandemic H1N1 influenza virus

Influenza viruses are respiratory pathogens that continue to pose a significantly high risk of morbidity and mortality of humans worldwide. Vaccination is one of the most effective strategies for minimizing damages by influenza outbreaks. In addition, rapid development and production of efficient vaccine with convenient administration is required in case of influenza pandemic. In this study, we generated recombinant influenza virus hemagglutinin protein 1 (sHA1) of 2009 pandemic influenza virus as a vaccine candidate using a well-established bacterial expression system and administered it into mice via sublingual (s.l.) route. We found that s.l. immunization with the recombinant sHA1 plus cholera toxin (CT) induced mucosal antibodies as well as systemic antibodies including neutralizing Abs and provided complete protection against infection with pandemic influenza virus A/CA/04/09 (H1N1) in mice. Indeed, the protection efficacy was comparable with that induced by intramuscular (i.m.) immunization route utilized as general administration route of influenza vaccine. These results suggest that s.l. vaccination with the recombinant non-glycosylated HA1 protein offers an alternative strategy to control influenza outbreaks including pandemics.     

Hydrogen sulphide-related thiol metabolism and nutrigenetics in relation to hypertension in an elderly population

Hydrogen sulphide (H₂S) is a gaseous signalling molecule that regulates blood flow and pressure. It is synthesised from cysteine via cystathionine β-synthase and cystathionine γ-lyase. We examined whether thiol precursors of H₂S, transsulphuration pathway gene variants (CBS-844ins68 and CTH-G1364T) and key B-vitamin cofactors might be critical determinants of hypertension in an elderly Australian population. An elderly Australian retirement village population (n = 228; age 65–96 years, 91 males and 137 females) was assessed for the prevalence of two transsulphuration pathway–related variant genes associated with cysteine synthesis and hence H₂S production. Thiols were determined by HPLC, genotypes by PCR and dietary intake by food frequency questionnaire. Homocysteine levels were statistically higher in the hypertensive phenotype (p = 0.0399), but there was no difference for cysteine or glutathione. Using nominal logistic regression, cysteine, CTH-G1364T genotype, dietary synthetic folate and vitamin B₆ predicted clinical phenotype (determined as above/below 140/90 mm Hg) and then only in female subjects (p = 0.0239, 0.0178, 0.0249 and 0.0371, respectively). Least-squares regression supports cysteine being highly inversely predictive of diastolic blood pressure: p and r ² values <0.0001 and 0.082; 0.0409 and 0.046; and <0.0001 and 0.113 for all subjects, males and females, respectively. Additionally, CTH-G1364T genotype predicts diastolic blood pressure in males (p = 0.0217; r ² = 0.083), but contrasts with observations for females. Overall, analyses, including stepwise regression, suggest cysteine, dietary natural and synthetic folate, vitamins B₆ and B₁₂, and both genetic variants (CTH-C1364T and CBS-844ins68) are all aetiologically relevant in the regulation of blood pressure. Hydrogen sulphide is a vasorelaxant gasotransmitter with characteristics similar to nitric oxide. Cysteine and the G1364T and 844ins68 variants of the cystathionine γ-lyase and cystathionine β-synthase genes, respectively, are the biological determinants of H₂S synthesis, and all three are shown here to influence the hypertensive phenotype. Additionally, B-vitamin cofactors for these three enzymes may also be important determinants of blood pressure.     

Syntenic relationships between Medicago truncatula and Arabidopsis reveal extensive divergence of genome organization

Arabidopsis and Medicago truncatula represent sister clades within the dicot subclass Rosidae. We used genetic map-based and bacterial artificial chromosome sequence-based approaches to estimate the level of synteny between the genomes of these model plant species. Mapping of 82 tentative orthologous gene pairs reveals a lack of extended macrosynteny between the two genomes, although marker collinearity is frequently observed over small genetic intervals. Divergence estimates based on non-synonymous nucleotide substitutions suggest that a majority of the genes under analysis have experienced duplication in Arabidopsis subsequent to divergence of the two genomes, potentially confounding synteny analysis. Moreover, in cases of localized synteny, genetically linked loci in M. truncatula often share multiple points of synteny with Arabidopsis; this latter observation is consistent with the large number of segmental duplications that compose the Arabidopsis genome. More detailed analysis, based on complete sequencing and annotation of three M. truncatula bacterial artificial chromosome contigs suggests that the two genomes are related by networks of microsynteny that are often highly degenerate. In some cases, the erosion of microsynteny could be ascribed to the selective gene loss from duplicated loci, whereas in other cases, it is due to the absence of close homologs of M. truncatula genes in Arabidopsis.     

Plasmid stability in a recombinant mammalian cell bioprocess

Summary A simple experimental method is devised to determine the fraction of plasmid-harboring cells in a bioprocess employing recombinant mammalian cells. The fraction of plasmid-harboring cells decreased as serum content in the growth medium decreased. The relatively higher increase in the generation time of the plasmid-harboring cell was primarily responsible for this decrease. The mathematical expression obtained for this fraction in terms of the two parameters, i.e. the generation time ratio and the plasmid-loss probability, could represent the experimental data extremely well. The numerical values of these parameters could show the inherent insight of the system. It was found that the data plot against time can draw us to a misleading conclusion of the absence of the effect of serum concentration.     

Minimalistic Predictor of Protein Binding Energy: Contribution of Solvation Factor to Protein Binding

It has long been known that solvation plays an important role in protein-protein interactions. Here, we use a minimalistic solvation-based model for predicting protein binding energy to estimate quantitatively the contribution of the solvation factor in protein binding. The factor is described by a simple linear combination of buried surface areas according to amino-acid types. Even without structural optimization, our minimalistic model demonstrates a predictive power comparable to more complex methods, making the proposed approach the basis for high throughput applications. Application of the model to a proteomic database shows that receptor-substrate complexes involved in signaling have lower affinities than enzyme-inhibitor and antibody-antigen complexes, and they differ by chemical compositions on interfaces. Also, we found that protein complexes with components that come from the same genes generally have lower affinities than complexes formed by proteins from different genes, but in this case the difference originates from different interface areas. The model was implemented in the software PYTHON, and the source code can be found on the Shakhnovich group webpage: http://faculty.chemistry.harvard.edu/shakhnovich/software.     

Identification of gene encoding Plasmodium knowlesi phosphatidylserine decarboxylase by genetic complementation in yeast and characterization of in vitro maturation of encoded enzyme

The 23-megabase genome of Plasmodium falciparum, the causative agent of severe human malaria, contains ～5300 genes, most of unknown function or lacking homologs in other organisms. Identification of these gene functions will help in the discovery of novel targets for the development of antimalarial drugs and vaccines. The P. falciparum genome is unusually A+T-rich, which hampers cloning and expressing these genes in heterologous systems for functional analysis. The large repertoire of genetic tools available for Saccharomyces cerevisiae makes this yeast an ideal system for large scale functional complementation analyses of parasite genes. Here, we report the construction of a cDNA library from P. knowlesi, which has a lower A+T content compared with P. falciparum. This library was applied in a yeast complementation assay to identify malaria genes involved in the decarboxylation of phosphatidylserine. Transformation of a psd1Δpsd2Δdpl1Δ yeast strain, defective in phosphatidylethanolamine synthesis, with the P. knowlesi library led to identification of a new parasite phosphatidylserine decarboxylase (PkPSD). Unlike phosphatidylserine decarboxylase enzymes from other eukaryotes that are tightly associated with membranes, the PkPSD enzyme expressed in yeast was equally distributed between membrane and soluble fractions. In vitro studies reveal that truncated forms of PkPSD are soluble and undergo auto-endoproteolytic maturation in a phosphatidylserine-dependent reaction that is inhibited by other anionic phospholipids. This study defines a new system for probing the function of Plasmodium genes by library-based genetic complementation and its usefulness in revealing new biochemical properties of encoded proteins.