MEG Correlates of Learning Novel Objects Properties in Children

Learning the functional properties of objects is a core mechanism in the development of conceptual, cognitive and linguistic knowledge in children. The cerebral processes underlying these learning mechanisms remain unclear in adults and unexplored in children. Here, we investigated the neurophysiological patterns underpinning the learning of functions for novel objects in 10-year-old healthy children. Event-related fields (ERFs) were recorded using magnetoencephalography (MEG) during a picture-definition task. Two MEG sessions were administered, separated by a behavioral verbal learning session during which children learned short definitions about the “magical” function of 50 unknown non-objects. Additionally, 50 familiar real objects and 50 other unknown non-objects for which no functions were taught were presented at both MEG sessions. Children learned at least 75% of the 50 proposed definitions in less than one hour, illustrating children''s powerful ability to rapidly map new functional meanings to novel objects. Pre- and post-learning ERFs differences were analyzed first in sensor then in source space. Results in sensor space disclosed a learning-dependent modulation of ERFs for newly learned non-objects, developing 500–800 msec after stimulus onset. Analyses in the source space windowed over this late temporal component of interest disclosed underlying activity in right parietal, bilateral orbito-frontal and right temporal regions. Altogether, our results suggest that learning-related evolution in late ERF components over those regions may support the challenging task of rapidly creating new semantic representations supporting the processing of the meaning and functions of novel objects in children.     

Characterization of monoclonal antibodies by a fast and easy liquid chromatography–mass spectrometry time-of-flight analysis on culture supernatant

Rapid and efficient structural analysis is key to the development of new monoclonal antibodies. We have developed a fast and easy process to obtain mass spectrometry profiles of antibodies from culture supernatant. Treatment of the supernatant with IdeS generates three fragments of 25 kDa that can be analyzed by liquid chromatography–mass spectrometry time-of-flight (LC–MS TOF) in one run: LC, Fd, and Fc/2. This process gives rapid access to isoform and glycoform profiles. To specifically measure the fucosylation yield, we included a one-pot treatment with EndoS that removes the distal glycan heterogeneity. Our process was successfully compared with high-performance capillary electrophoresis with laser-induced fluorescence detection (HPCE–LIF), currently considered as the “gold standard” method.     

Estimation of Nitrifying Bacterial Activities by Measuring Oxygen Uptake in the Presence of the Metabolic Inhibitors Allylthiourea and Azide

The effects of two metabolic inhibitors on an enriched nitrifying biomass during incubation for short periods of time were investigated by determining respirometric measurements. Allylthiourea (86 μM) and azide (24 μM) were shown to be strong, selective inhibitors of ammonia and nitrite oxidation, respectively. Consequently, a differential respirometry method for estimating nitrifying and heterotrophic bacterial activities within a mixed biomass is proposed.     

Periodic metabolic systems: Oscillations in multiple-loop negative feedback biochemical control networks

Summary For a general multiple loop feedback inhibition system in which the end product can inhibit any or all of the intermediate reactions it is shown that biologically significant behaviour is always confined to a bounded region of reaction space containing a unique equilibrium. By explicit construction of a Liapunov function for the general n dimensional differential equation it is shown that some values of reaction parameters cause the concentration vector to approach the equilibrium asymptotically for all physically realizable initial conditions. As the parameter values change, periodic solutions can appear within the bounded region. Some information about these periodic solutions can be obtained from the Hopf bifurcation theorem. Alternatively, if specific parameter values are known a numerical method can be used to find periodic solutions and determine their stability by locating a zero of the displacement map. The single loop Goodwin oscillator is analysed in detail. The methods are then used to treat an oscillator with two feedback loops and it is found that oscillations are possible even if both Hill coefficients are equal to one.     

Combined glyco- and protein-Fc engineering simultaneously enhance cytotoxicity and half-life of a therapeutic antibody

While glyco-engineered monoclonal antibodies (mAbs) with improved antibody-dependent cell-mediated cytotoxicity (ADCC) are reaching the market, extensive efforts have also been made to improve their pharmacokinetic properties to generate biologically superior molecules. Most therapeutic mAbs are human or humanized IgG molecules whose half-life is dependent on the neonatal Fc receptor FcRn. FcRn reduces IgG catabolism by binding to the Fc domain of endocytosed IgG in acidic lysosomal compartments, allowing them to be recycled into the blood. Fc-engineered mAbs with increased FcRn affinity resulted in longer in vivo half-life in animal models, but also in healthy humans. These Fc-engineered mAbs were obtained by alanine scanning, directed mutagenesis or in silico approach of the FcRn binding site. In our approach, we applied a random mutagenesis technology (MutaGen^TM) to generate mutations evenly distributed over the whole Fc sequence of human IgG1. IgG variants with improved FcRn-binding were then isolated from these Fc-libraries using a pH-dependent phage display selection process. Two successive rounds of mutagenesis and selection were performed to identify several mutations that dramatically improve FcRn binding. Notably, many of these mutations were unpredictable by rational design as they were located distantly from the FcRn binding site, validating our random molecular approach. When produced on the EMABling^® platform allowing effector function increase, our IgG variants retained both higher ADCC and higher FcRn binding. Moreover, these IgG variants exhibited longer half-life in human FcRn transgenic mice. These results clearly demonstrate that glyco-engineering to improve cytotoxicity and protein-engineering to increase half-life can be combined to further optimize therapeutic mAbs.     

TLR4 and Toll-IL-1 receptor domain-containing adapter-inducing IFN-beta, but not MyD88, regulate Escherichia coli-induced dendritic cell maturation and apoptosis in vivo

Dendritic cells (DC) are short-lived, professional APCs that play a central role in the generation of adaptive immune responses. Induction of efficient immune responses is dependent on how long DCs survive in the host. Therefore, the regulation of DC apoptosis in vivo during infection remains an important question that requires further investigation. The impact of Escherichia coli bacteremia on DCs has never been analyzed. We show here that i.v. or i.p. administration of live or heat-killed E. coli in mice induces splenic DC migration, maturation, and apoptosis. We further characterize which TLR and Toll-IL-1R (TIR)-containing adaptor molecules regulate these processes in vivo. In this model, DC maturation is impaired in TLR2(-/-), TLR4(-/-) and TIR domain-containing adapter-inducing IFN-beta (TRIF)(-/-) mice. In contrast, DC apoptosis is reduced only in TLR4(-/-) and TRIF(-/-) mice. As expected, DC apoptosis induced by the TLR4 ligand LPS is also abolished in these mice. Injection of the TLR9 ligand CpG-oligodeoxynucleotide (synthetic bacterial DNA) induces DC migration and maturation, but only modest DC apoptosis when compared with LPS and E. coli. Together, these results suggest that E. coli bacteremia directly impacts on DC maturation and survival in vivo through a TLR4-TRIF-dependent signaling pathway.