TLR9 activation induces normal neutrophil responses in a child with IRAK-4 deficiency: involvement of the direct PI3K pathway

Polymorphonuclear neutrophils (PMN) play a key role in innate immunity. Their activation and survival are tightly regulated by microbial products via pattern recognition receptors such as TLRs, which mediate recruitment of the IL-1R-associated kinase (IRAK) complex. We describe a new inherited IRAK-4 deficiency in a child with recurrent pyogenic bacterial infections. Analysis of the IRAK4 gene showed compound heterozygosity with two mutations: a missense mutation in the death domain of the protein (pArg12Cys) associated in cis-with a predicted benign variant (pArg391His); and a splice site mutation in intron 7 that led to the skipping of exon 7. A nontruncated IRAK-4 protein was detected by Western blotting. The patient's functional deficiency of IRAK-4 protein was confirmed by the absence of IRAK-1 phosphorylation after stimulation with all TLR agonists tested. The patient's PMNs showed strongly impaired responses (L-selectin and CD11b expression, oxidative burst, cytokine production, cell survival) to TLR agonists which engage TLR1/2, TLR2/6, TLR4, and TLR7/8; in contrast, the patient's PMN responses to CpG-DNA (TLR9) were normal, except for cytokine production. The surprisingly normal effect of CpG-DNA on PMN functions and apoptosis disappeared after pretreatment with PI3K inhibitors. Together, these results suggest the existence of an IRAK-4-independent TLR9-induced transduction pathway leading to PI3K activation. This alternative pathway may play a key role in PMN control of infections by microorganisms other than pyogenic bacteria in inherited IRAK-4 deficiency.     

Lipidomic analysis of Toxoplasma gondii reveals unusual polar lipids

Analysis of the polar lipids of Toxoplasma gondii by electrospray ionization tandem mass spectrometry provides a detailed picture of the lipid molecular species of this parasitic protozoan. Most notably, T. gondii contains a relatively high level, estimated to about 2% of the total polar lipid, of ceramide phosphoethanolamine. The ceramide phosphoethanolamine has a fatty amide profile with only 16- and 18-carbon species. Compared with the host fibroblasts in which it was grown, T. gondii also has higher levels of phosphatidylcholine but lower levels of sphingomyelin and phosphatidylserine. Analysis at the molecular species level indicated that T. gondii has greater amounts of shorter-chain fatty acid in its polar lipid molecular species than the host fibroblasts. Shorter-chain fatty acids with a combined total of 30 or fewer acyl carbons make up 21% of Toxoplasma's, but only 3% of the host's, diacyl phosphatidylcholine. Furthermore, diacyl phosphatidylcholine with two saturated acyl chains with 12, 14, or 16 carbons make up over 11% of parasite phosphatidylcholine but less than 3% of the host phosphatidylcholine molecular species. The distinctive T. gondii tachyzoite lipid profile may be particularly suited to the function of parasitic membranes and the interaction of the parasite with the host cell and the host's immune system. Combined with T. gondii genomic data, these lipidomic data will assist in elucidation of metabolic pathways for lipid biosynthesis in this important human pathogen.     

High-resolution AFM imaging of single-stranded DNA-binding (SSB) protein—DNA complexes

DNA in living cells is generally processed via the generation and the protection of single-stranded DNA involving the binding of ssDNA-binding proteins (SSBs). The studies of SSB-binding mode transition and cooperativity are therefore critical to many cellular processes like DNA repair and replication. However, only a few atomic force microscopy (AFM) investigations of ssDNA nucleoprotein filaments have been conducted so far. The point is that adsorption of ssDN A–SSB complexes on mica, necessary for AFM imaging, is not an easy task. Here, we addressed this issue by using spermidine as a binding agent. This trivalent cation induces a stronger adsorption on mica than divalent cations, which are commonly used by AFM users but are ineffective in the adsorption of ssDNA–SSB complexes. At low spermidine concentration (<0.3mM), we obtained AFM images of ssDNA–SSB complexes (E. coli SSB, gp32 and yRPA) on mica at both low and high ionic strengths. In addition, partially or fully saturated nucleoprotein filaments were studied at various monovalent salt concentrations thus allowing the observation of SSB-binding mode transition. In association with conventional biochemical techniques, this work should make it possible to study the dynamics of DNA processes involving DNA–SSB complexes as intermediates by AFM.     

Sequence polymorphism-based identification and quantification of Vibrio nigripulchritudo at the species and subspecies level targeting an emerging pathogen for cultured shrimp in New Caledonia

In a previous study, we demonstrated the existence of an emerging cluster of Vibrio nigripulchritudo that proved to be associated with shrimp mortality events in New Caledonia. Using sequence polymorphisms evidenced in this previous MultiLocus Sequence Typing study, we developed two new quantitative PCR assays permitting the detection and quantification of V. nigripulchritudo at the genospecies level using SYBR Green I chemistry and at the emerging cluster level using Fluorescence Resonance Energy Transfer technology with hybridization probes. The use of this molecular diagnostic tool evidenced the colonization of the shrimp pond ecosystem by the pathogenic cluster at least at the onset of the disease. This new tool will allow better investigation of the dynamics of this bacterial pathogen in the shrimp farm ecosystem.     

Less favourable climates constrain demographic strategies in plants

Correlative species distribution models are based on the observed relationship between species’ occurrence and macroclimate or other environmental variables. In climates predicted less favourable populations are expected to decline, and in favourable climates they are expected to persist. However, little comparative empirical support exists for a relationship between predicted climate suitability and population performance. We found that the performance of 93 populations of 34 plant species worldwide – as measured by in situ population growth rate, its temporal variation and extinction risk – was not correlated with climate suitability. However, correlations of demographic processes underpinning population performance with climate suitability indicated both resistance and vulnerability pathways of population responses to climate: in less suitable climates, plants experienced greater retrogression (resistance pathway) and greater variability in some demographic rates (vulnerability pathway). While a range of demographic strategies occur within species’ climatic niches, demographic strategies are more constrained in climates predicted to be less suitable.     

Characterization of the Plasmodium falciparum and P. berghei glycerol 3‐phosphate acyltransferase involved in FASII fatty acid utilization in the malaria parasite apicoplast

Malaria parasites can synthesize fatty acids via a type II fatty acid synthesis (FASII) pathway located in their apicoplast. The FASII pathway has been pursued as an anti‐malarial drug target, but surprisingly little is known about its role in lipid metabolism. Here we characterize the apicoplast glycerol 3‐phosphate acyltransferase that acts immediately downstream of FASII in human (Plasmodium falciparum) and rodent (Plasmodium berghei) malaria parasites and investigate how this enzyme contributes to incorporating FASII fatty acids into precursors for membrane lipid synthesis. Apicoplast targeting of the P. falciparum and P. berghei enzymes are confirmed by fusion of the N‐terminal targeting sequence to GFP and 3′ tagging of the full length protein. Activity of the P. falciparum enzyme is demonstrated by complementation in mutant bacteria, and critical residues in the putative active site identified by site‐directed mutagenesis. Genetic disruption of the P. falciparum enzyme demonstrates it is dispensable in blood stage parasites, even in conditions known to induce FASII activity. Disruption of the P. berghei enzyme demonstrates it is dispensable in blood and mosquito stage parasites, and only essential for development in the late liver stage, consistent with the requirement for FASII in rodent malaria models. However, the P. berghei mutant liver stage phenotype is found to only partially phenocopy loss of FASII, suggesting newly made fatty acids can take multiple pathways out of the apicoplast and so giving new insight into the role of FASII and apicoplast glycerol 3‐phosphate acyltransferase in malaria parasites.     

Tuning the Hydrophilic/Hydrophobic Balance to Control the Structure of Chitosan Films and Their Protein Release Behavior

The control over the crystallinity of chitosan and chitosan/ovalbumin films can be achieved via an appropriate balance of the hydrophilic/hydrophobic interactions during the film formation process, which then controls the release kinetics of ovalbumin. Chitosan films were prepared by solvent casting. The presence of the anhydrous allomorph can be viewed as a probe of the hydrophobic conditions at the neutralization step. The semicrystalline structure, the swelling behavior of the films, the protein/chitosan interactions, and the release behavior of the films were impacted by the DA and the film processing parameters. At low DAs, the chitosan films neutralized in the solid state corresponded to the most hydrophobic environment, inducing the crystallization of the anhydrous allomorph with and without protein. The most hydrophilic conditions, leading to the hydrated allomorph, corresponded to non-neutralized films for the highest DAs. For the non-neutralized chitosan acetate (amorphous) films, the swelling increased when the DA decreased, whereas for the neutralized chitosan films, the swelling decreased. The in vitro release of ovalbumin (model protein) from chitosan films was controlled by their swelling behavior. For fast swelling films (DA?=?45%), a burst effect was observed. On the contrary, a lag time was evidenced for DA?=?2.5% with a limited release of the protein. Furthermore, by blending chitosans (DA?=?2.5% and 45%), the release behavior was improved by reducing the burst effect and the lag time. The secondary structure of ovalbumin was partially maintained in the solid state, and the ovalbumin was released under its native form.     

Nonrandom Distribution of Hepatitis C Virus Quasispecies in Plasma and Peripheral Blood Mononuclear Cell Subsets

The existence of an extrahepatic reservoir of hepatitis C virus (HCV) is suggested by differences in quasispecies composition between the liver, peripheral blood mononuclear cells, and serum. We studied HCV RNA compartmentalization in the plasma of nine patients, in CD19(+), CD8(+), and CD4(+) positively selected cells, and also in the negatively selected cell fraction (NF). HCV RNA was detected in all plasma samples, in seven of nine CD19(+), three of eight CD8(+), and one of nine CD4(+) cell samples, and in seven of eight NF cells. Cloning and sequencing of HVR1 in two patients showed a sequence grouping: quasispecies from a given compartment (all studied compartments for one patient and CD8(+) and NF for the other) were statistically more genetically like each other than like quasispecies from any other compartment. The characteristics of amino acid and nucleotide substitutions suggested the same structural constraints on HVR1, even in very divergent strains from the cellular compartments, and homogeneous selection pressure on the different compartments. These findings demonstrate the compartmental distribution of HCV quasispecies within peripheral blood cell subsets and have important implications for the study of extrahepatic HCV replication and interaction with the immune system.     

N.m.r. studies of synergistic kappa carrageenan—carob galactomannan gels

¹³C-n.m.r. spectroscopy has been used to investigate the carob galactomannan—kappa carrageenan binary gels. Starting from partially depolymerized carob samples, evidence for interaction and intermolecular binding was found by analysis of the spectra obtained in quantitative conditions and in the absolute intensity mode. From these, a reconstitution of the signals corresponding to C-1, C-4, C-5 and C-6 showed the existence of three kinds of galactomannan chains: the first ones with a low galactose content were strongly connected and as much had completely lost their mobility; but the chains with a high galactose content were still detected and could be divided in two groups according to their mobility.